UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Roles of peripheral glia in embryonic nervous system development of drosophilia Sepp, Katharine Julia 2000

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2000-487083.pdf [ 5.91MB ]
Metadata
JSON: 831-1.0089858.json
JSON-LD: 831-1.0089858-ld.json
RDF/XML (Pretty): 831-1.0089858-rdf.xml
RDF/JSON: 831-1.0089858-rdf.json
Turtle: 831-1.0089858-turtle.txt
N-Triples: 831-1.0089858-rdf-ntriples.txt
Original Record: 831-1.0089858-source.json
Full Text
831-1.0089858-fulltext.txt
Citation
831-1.0089858.ris

Full Text

R O L E S O F P E R I P H E R A L G L I A IN E M B R Y O N I C NERVOUS SYSTEM DEVELOPMENT OF DROSOPHILA by KATHARINE JULIA SEPP B.Sc, University o f Toronto, 1992  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F THE REQUREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Zoology  W e a c c e p t this thesis as c o n f o r m i n g to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA February, 2000 © Katharine Julia Sepp, 2000  In  presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or  and study.  his  or  her  Department of  6  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  O^lJl  ZJOOQ  that the  representatives.  may be It  of this thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  is  an  advanced  Library shall make it  that permission for extensive granted  by the  understood be  for  that  allowed without  head  of  my  copying  or  my written  ABSTRACT  P e r i p h e r a l glial cells are m a j o r c o n s t i t u e n t s o f the P N S a n d are r e q u i r e d f o r insulation o f p e r i p h e r a l nerves. T h e i r f u n c t i o n d u r i n g Drosophila n e r v o u s s y s t e m d e v e l o p m e n t h a s not b e e n p r e v i o u s l y s t u d i e d l a r g e l y d u e to a l a c k o f cellular m a r k e r s . T o d e t e r m i n e w h a t r o l e s p e r i p h e r a l g l i a h a v e i n Drosophila e m b r y o n i c n e r v o u s s y s t e m d e v e l o p m e n t , a s e r i e s o f p e r i p h e r a l glial e n h a n c e r trap lines w e r e first g e n e r a t e d b y c o n v e r t i n g p r e v i o u s glial n u c l e a r lacZ e n h a n c e r traps t o the G A L 4 s y s t e m . T h e targeted t r a n s p o s i t i o n t e c h n i q u e , w h i c h i s a direct e x c h a n g e o f o n e P e l e m e n t f o r another, s u c c e s s f u l l y g e n e r a t e d the d e s i r e d p e r i p h e r a l glial G A L 4 lines at a h i g h e f f i c i e n c y . P o t e n t i a l l y , targeted t r a n s p o s i t i o n is a w i d e l y a p p l i c a b l e t e c h n i q u e f o r g e n e r a t i o n o f m a n y other d e s i r a b l e G A L 4 lines i n the future. T h e n e w G A L 4 lines w e r e u s e d to c h a r a c t e r i z e p e r i p h e r a l g l i a i n relation to n e u r o n a l growth. P e r i p h e r a l g l i a l p r o l i f e r a t i o n , m i g r a t i o n , c e l l contacts, m o r p h o l o g i c a l changes, a n d n e r v e w r a p p i n g w e r e d e s c r i b e d . P e r i p h e r a l g l i a are b o r n at the lateral e d g e o f the C N S . M o t o r a n d s e n s o r y n e u r o n s m a k e p h y s i c a l c o n t a c t s w i t h the p e r i p h e r a l g l i a as they migrate a c r o s s the C N S / P N S b o r d e r . T h e p e r i p h e r a l g l i a then m i g r a t e i n t o t h e P N S a l o n g p r e e s t a b l i s h e d n e u r o n a l tracts w h i l e e x t e n d i n g l o n g c y t o p l a s m i c p r o c e s s e s . T h e p e r i p h e r a l g l i a c o m p l e t e l y w r a p all s e n s o r y a x o n tracts b y the e n d o f e m b r y o g e n e s i s , b u t d o n o t w r a p the distal tips o f m o t o r n e u r o n b r a n c h e s . T h e m i g r a t i o n a l patterns a n d n e u r o n a l a s s o c i a t i o n s o f p e r i p h e r a l g l i a d u r i n g d e v e l o p m e n t are v e r y s i m i l a r t o vertebrate g l i a a n d s u g g e s t that Drosophila m a y b e a p o w e r f u l m o d e l s y s t e m t o u n d e r s t a n d m a n y aspects o f glial d e v e l o p m e n t . T h e G A L 4 lines w e r e then u s e d t o ablate p e r i p h e r a l g l i a e a r l y i n d e v e l o p m e n t b y e c t o p i c e x p r e s s i o n o f a p o p t o s i s genes. T h e a n a l y s i s p r o v i d e s i n s i g h t i n t o the m a j o r r o l e s peripheral g l i a p l a y i n g e n e r a t i o n o f the n e r v o u s s y s t e m . P e r i p h e r a l g l i a mediate a x o n m i g r a t i o n o v e r the C N S / P N S b o r d e r a n d g l i a are r e q u i r e d t o prepattern t h e n e u r o n a l C N S exit/entry sites. W h e n the g l i a are ablated, a x o n s p r o j e c t a c r o s s the C N S / P N S b o r d e r at a b n o r m a l p o s i t i o n s a n d p r o p e r n e r v e b u n d l i n g n e v e r f o r m s i n the r e g i o n . P e r i p h e r a l glia also appear t o stimulate a x o g e n e s i s a n d p r o m o t e s e n s o r y n e u r o n s u r v i v a l . T h e g l i a a l s o m a y p r o v i d e contact-mediated c u e s f o r c e n t r a l l y d i r e c t e d s e n s o r y a x o n m i g r a t i o n . P e r i p h e r a l g l i a are a l s o essential f o r maintenance o f tight a x o n f a s c i c u l a t i o n a n d c o m p a c t n e r v e b u n d l i n g . O v e r a l l , the results suggest that p e r i p h e r a l g l i a h a v e i m p o r t a n t f u n c t i o n s i n early a n d late d e v e l o p m e n t o f the n e r v o u s s y s t e m w h i c h a l s o m a y b e c o m m o n t o vertebrate P N S glia. ii  Table of Contents ABSTRACT  ii  List of Figures  iv  Acknowledgements  vi  I.  INTRODUCTION  II.  T A R G E T E D T R A N S P O S I T I O N IS A N E F F E C T I V E M E A N S O F G E N E R A T I N G G A L 4 L I N E S IN D R O S O P H I L A .  21  Introduction  22  Materials and Methods  25  Results  29  Discussion  46  III. D E V E L O P M E N T O F P E R I P H E R A L G L I A IN T H E EMBRYO  IV.  1  DROSOPHILA  Introduction  51  Materials and Methods  54  Results  56  Discussion  74  ABLATION OF PERIPHERAL GLIA  78  Introduction  79  Materials and Methods  81  Results  82  Discussion V.  G E N E R A L DISCUSSION  Drosophila as a PNS glial model system. Peripheral glial roles as guidepost cells. Peripheral glial migration into the PNS. Glial ensheathement of peripheral nerves. Trophic interactions between peripheral glia and peripheral neurons. VI.  50  REFERENCES  108 115  115 117 120 123 124 126  iii  List of  Figures  F i g u r e 1. S c h e m a t i c d i a g r a m o f p e r i p h e r a l g l i a l g r o w t h i n r e l a t i o n to m o t o r a n d s e n s o r y axon development  2  F i g u r e 2. S c h e m a t i c d i a g r a m o f the e n h a n c e r trap P e l e m e n t s i n v o l v e d i n targeted transposition and their surrounding g e n o m i c regions  30  F i g u r e 3. S c h e m a t i c d i a g r a m o f P e l e m e n t r e p l a c e m e n t  32  F i g u r e 4. S c h e m a t i c d i a g r a m o f a c r o s s i n g s c h e m e f o r targeted t r a n s p o s i t i o n  34  F i g u r e 5. G l i a l s p e c i f i c e n h a n c e r trap s t a i n i n g patterns i n D r o s o p h i l a e m b r y o s  36  F i g u r e 6. P C R a n a l y s i s o f t a r g e t e d t r a n s p o s i t i o n l i n e s  41  F i g u r e 7. S o u t h e r n a n a l y s i s o f targeted t r a n s p o s i t i o n l i n e s  44  F i g u r e 8. E a r l y stages o f p e r i p h e r a l g l i a l development...  58  F i g u r e 9. M i g r a t i o n o f p e r i p h e r a l g l i a i n the P N S o c c u r s a l o n g p r e f o r m e d n e u r o n a l pathways 60 F i g u r e 10. N u c l e a r l a b e l i n g o f p e r i p h e r a l g l i a s h o w s p r o l i f e r a t i o n a n d m o v e m e n t o f c e l l bodies 62 F i g u r e 11. M i g r a t i o n o f p e r i p h e r a l g l i a into the P N S f o l l o w s w e l l b e h i n d the g r o w i n g tips of pioneer motorneurons 66 F i g u r e 12. P e r i p h e r a l g l i a p r e f e r e n t i a l l y e x t e n d p r o c e s s e s a l o n g s e n s o r y n e u r o n a l tracts i n r e g i o n s w h e r e s e n s o r y a n d m o t o r f a s c i c l e s d i v e r g e d u r i n g e m b r y o g e n e s i s . ...68 F i g u r e 13. T h e C N S / P N S b o u n d a r y is penetrated b y p e r i p h e r a l g l i a  71  F i g u r e 14. A b l a t i o n o f p e r i p h e r a l g l i a d u r i n g e a r l y n e u r a l d e v e l o p m e n t  84  F i g u r e 15. C N S glial c e l l b o d i e s rearrange i n response to p e r i p h e r a l g l i a l a b l a t i o n  88  F i g u r e 16. E f f e c t s o f p e r i p h e r a l glial a b l a t i o n o n tracheal d e v e l o p m e n t  91 iv  F i g u r e 17. P e r i p h e r a l g l i a p r o m o t e c e n t r a l l y - d i r e c t e d sensory a x o n m i g r a t i o n  94  F i g u r e 18. P e r i p h e r a l g l i a p r o v i d e d i r e c t i o n a l i n f o r m a t i o n to sensory n e u r o n s as they m i g r a t e to the C N S '.  97  F i g u r e 19. A d h e s i o n o f s e n s o r y n e u r o n s to p e r i p h e r a l g l i a m a y b e r e q u i r e d f o r p r o p e r a x o n m i g r a t i o n i n t o the C N S  99  F i g u r e 20. P e r i p h e r a l g l i a are r e q u i r e d f o r m a i n t e n a n c e o f a x o n f a s c i c l u l a t i o n a n d tight n e r v e b u n d l i n g o f p e r i p h e r a l nerves 102 F i g u r e 21. P e r i p h e r a l g l i a are r e q u i r e d to p r e f i g u r e the C N S e x i t p o i n t o f m o t o r a x o n s . 105  v  Acknowledgements I w o u l d l i k e to t h a n k m y s u p e r v i s o r , D r . V a n e s s a A u l d f o r h e r g u i d a n c e t h r o u g h o u t m y w o r k o n this project. V a n e s s a d e s e r v e s a l a r g e a m o u n t o f c r e d i t f o r d e s i g n i n g the m a i n g o a l s o f this thesis a n d their c r a f t y p l a n s o f attack. I was v e r y fortunate to s h o w u p at her o f f i c e d o o r at the r i g h t time, r e a d y a n d w i l l i n g to d o e v e n the m o s t o u t r a g e o u s a n d m o s t t e d i o u s o f e x p e r i m e n t s . W h o w o u l d h a v e k n o w n at that t i m e that o u r s p e c i a l flies, the glial GAL4 d r i v e r lines, w o u l d s o o n be generated a n d these p o w e r f u l t o o l s f o r s t u d y i n g g l i a i n d e v e l o p m e n t w o u l d then be i n h a n d ? I a m a l s o v e r y g r a t e f u l f o r V a n e s s a ' s w i l l i n g n e s s to a l l o w me to w o r k at m y o w n pace w h i c h was p u n c t u a t e d b y m a n y breaks: w e e k d a y sessions f o r p l a y i n g c e l l o , h e l p i n g to raise S y l a the G e r m a n S h e p h e r d p u p p y , a n d time f o r g o i n g h o m e to be w i t h m y f a m i l y i n T o r o n t o . W i t h o u t all o f these extras, I c o u l d not h a v e w o r k e d o n these e x p e r i m e n t s w i t h the s a m e e f f i c i e n c y a n d s p a r k as I d i d . M y thesis committee m e m b e r s h a v e a l s o b e e n i n s t r u m e n t a l i n the p r o g r e s s o f m y w o r k . E a c h h a d a v e r y u s e f u l r o l e to p l a y . D o n M o e r m a n a n d T o m G r i g l i a t t i f o r their Ph.D. committee e x p e r i e n c e a n d genetics e x p e r t i s e , as w e l l as T i m O ' C o n n o r a n d Jane R o s k a m s f o r their k n o w l e d g e o f n e u r o d e v e l o p m e n t . T h e r e h a v e b e e n m a n y i n d i v i d u a l s w h o h a v e w o r k e d i n the A u l d L a b w h o I a l s o thank f o r their c o n s t r u c t i v e c o m m e n t s o v e r the y e a r s at l a b m e e t i n g s a n d i n general c o n v e r s a t i o n . M o s t s i g n i f i c a n t l y , J o o s t S c h u l t e interrupted h i s o w n w o r k m a n y times to v i e w a n d d i s c u s s the p h e n o t y p e s o f c o u n t l e s s m u t a n t e m b r y o s . O n e s u m m e r , w e a l s o m a n a g e d to s t u m b l e t h r o u g h a m u t a n t s c r e e n together w h i c h t u r n e d i n t o a d i s m a l failure but we l e a r n e d a l o t a n d h a d f u n a n y w a y s ! T e c h n i c a l assistance has b e e n e s s e n t i a l to m a k e the e x p e r i m e n t s r u n s m o o t h l y , a n d was p r o v i d e d b y C l a u d i a S a l i n a s w h o i n particular, m a d e sure that the l a b c o m p u t e r q u i c k l y r e c o v e r e d after its m a n y m a j o r c r a s h e s . T h e m i l l i o n s o f fruit flies w h o h a v e p a r t i c i p a t e d i n m y p r o j e c t s a l s o d e s e r v e thanks, but I a m j u s t not sure h o w to tell t h e m ! F i n a l l y , I thank m y parents w h o h a v e g l a d l y p r o v i d e d a l l k i n d s o f s u p p o r t t h r o u g h o u t m y e d u c a t i o n . W i t h o u t their h e l p , m y participation i n these e x p e r i m e n t s w o u l d s i m p l y not h a v e b e e n p o s s i b l e . T h e y a l s o e n s u r e d that m y life as a g r a d student retained s o m e r e s e m b l a n c e o f n o r m a l c y b y r e g u l a r l y s e n d i n g m e E s t o papers to k e e p me u p d a t e d o n what happens outside o f my bubble and by g i v i n g injections o f finance for fancy dinners out o n the t o w n . I t h a n k t h e m f o r their e n c o u r a g e m e n t a n d f o r their c o n f i d e n c e that I c o u l d m a k e this h a p p e n . I d e d i c a t e this thesis to m y f o u r - l e g g e d f r i e n d s M a x , L u l u , a n d N i k k i . vi  I.  INTRODUCTION  P e r i p h e r a l glial cells e n s h e a t h e peripheral nerves o f Drosophila melanogaster. T h e peripheral nerves relay s i g n a l s f r o m p e r i p h e r a l s e n s o r y o r g a n s into the central n e r v o u s s y s t e m ( C N S ) a n d also relay m o t o r i m p u l s e s f r o m the C N S  to the somatic  musculature.  T h u s , the p e r i p h e r a l nerves c o n t a i n s e n s o r y and m o t o r n e u r o n s . T h e p e r i p h e r a l g l i a are essential f o r m a i n t a i n i n g an appropriate e n v i r o n m e n t f o r n e u r o n a l s i g n a l i n g since they insulate n e u r o n s f r o m h a e m o l y m p h ( A u l d et al., 1995). T h e i r f u n c t i o n in d e v e l o p m e n t is p o o r l y understood, o w i n g to a l a c k o f m a r k e r s that label peripheral glial cellular p r o c e s s e s . T h e Drosophila m o d e l s y s t e m has p r o v e n to be a p o w e r f u l m e a n s o f u n d e r s t a n d i n g  what  m o l e c u l e s are essential f o r aspects o f n e u r o d e v e l o p m e n t i n c l u d i n g generation o f neuronal c o n n e c t i v i t y . T h e patterning o f neuronal p a t h w a y s i n Drosophila has m a n y similarities to h i g h e r o r g a n i s m s ( A r e n d t a n d N i i b l e r - J u n g , 1999), yet it is simpler, a l l o w i n g subtle mutant p h e n o t y p e s to be detected w i t h relative ease. T h e p o w e r f u l genetics c o m b i n e d w i t h the s i m p l i c i t y o f the Drosophila n e r v o u s s y s t e m has m a d e i t p o s s i b l e to c l o n e a large n u m b e r o f g e n e s w h i c h also h a v e h o m o l o g u e s in vertebrates ( G o o d m a n , 1994). T h u s m a n y aspects o f neural d e v e l o p m e n t are e v o l u t i o n a r i l y c o n s e r v e d a n d Drosophila is an excellent m o d e l o r g a n i s m i n w h i c h to study this t o p i c . It is unfortunate then, that the c o n t r i b u t i o n s o f p e r i p h e r a l g l i a to neural d e v e l o p m e n t h a v e not b e e n as s t r o n g l y a s s e s s e d s i n c e they constitute a v e r y s i g n i f i c a n t p o r t i o n o f the peripheral n e r v o u s s y s t e m ( P N S ) . T h e m a j o r g o a l o f the current w o r k is to g a i n an u n d e r s t a n d i n g d e v e l o p m e n t o f the e m b r y o n i c  o f the roles p e r i p h e r a l g l i a p l a y in  PNS.  Origin and differentiation of peripheral glia  M o s t p e r i p h e r a l g l i a arise through neuroblast ( N B ) lineages at the lateral e d g e o f the C N S d u r i n g e m b r y o n i c stage 12 ( F i g u r e 1). C l o n a l a n a l y s i s has s h o w n that f o u r to six  1  Figure 1. Schematic diagram of peripheral glial growth in relation to motor and sensory axon development.  ( A ) T h e p e r i p h e r a l g l i a ( n u c l e i i n d i c a t e d as s o l i d b l a c k dots) appear d u r i n g stage 12 at the lateral e d g e o f the C N S . P i o n e e r m o t o r a x o n a C C a p p r o a c h e s the g l i a as they proliferate. A t this time, the first s e n s o r y n e u r o n o f the d o r s a l c l u s t e r is b e i n g g e n e r a t e d ( b r o k e n line o v a l i n P N S r e g i o n ) . T h e Drosophila n e r v o u s s y s t e m is s y m m e t r i c about the m i d l i n e (ML). ( B ) A t e a r l y stage 13, the p i o n e e r m o t o r a x o n a C C e x i t s the C N S . P e r i p h e r a l glia proliferate w h i l e at the lateral e d g e o f the C N S . S e n s o r y n e u r o n s c o n t i n u e to appear i n the PNS. ( C ) T o w a r d s the e n d o f stage 13, the p e r i p h e r a l g l i a h a v e p r o l i f e r a t e d to their final n u m b e r s . T h e a C C p i o n e e r n e u r o n e x t e n d s p e r i p h e r a l l y as s e n s o r y n e u r o n s e x t e n d a x o n s centrally. N o t all a x o n s i n all segments p r o g r e s s at the s a m e rate. In the l o w e r s e g m e n t , the a C C appears to c o n n e c t w i t h s e n s o r y a x o n s e x t e n d i n g f r o m the d o r s a l s e n s o r y c l u s t e r . N e u r o n s c o n t i n u e to a r i s e i n b o t h the C N S a n d P N S . (D) S t a g e 14. P e r i p h e r a l g l i a m i g r a t e into the P N S a l o n g a x o n s . T h e m a i n P N S a x o n tracts, the I S N / a f a n d SN/pf, h a v e f o r m e d at this time. I S N a n d S N g l i a ( o p e n circles) a l s o associate w i t h p e r i p h e r a l n e r v e s , h o w e v e r these g l i a r e m a i n entirely i n the C N S . L o n g i t u d i n a l a x o n c o n n e c t i v e s ( L C ) o f the C N S are n o w apparent. ( A d a p t e d f r o m C a s e y Kopczinsky).  2  3  p e r i p h e r a l g l i a arise f r o m N B 1 - 3  w h i c h a l s o g i v e s rise to intersegmental n e r v e g l i a ( I S N G )  and segmental nerve glia ( S N G )  as w e l l as t w o to three m o t o r n e u r o n s , w h i c h project  contralaterally to exit the C N S  o n the s e g m e n t a l n e r v e ( S N ) and the anterior root o f the  intersegmental n e r v e ( I S N ) . T w o  peripheral g l i a are also generated b y N B 2 - 5 w h i c h is  slightly p o s t e r i o r a n d v e n t r a l to N B 1 - 3 . T h e N B 2 - 5 l i n e a g e generates a v a r i a b l e n u m b e r o f interneurons a n d a s m a l l n u m b e r o f m o t o r n e u r o n s w h i c h p r o j e c t t h r o u g h the p o s t e r i o r root o f the I S N . In a l l , s i x to e i g h t peripheral g l i a are b o r n i n the C N S  ( S c h m i d t et al., 1 9 9 7 ) .  A l l central n e r v o u s s y s t e m ( C N S ) d e r i v e d p e r i p h e r a l g l i a migrate into the p e r i p h e r y shortly after their b i r t h ( H a l t e r et al., 1995; S e p p et al., 2 0 0 0 ) . A s i n g l e p e r i p h e r a l g l i a l c e l l , the dorsolateral p e r i p h e r a l g l i a ( d l P G ) , is a l s o b o r n in the p e r i p h e r y at the d o r s a l s e n s o r y cluster d u r i n g stage 12 a n d migrates slightly ventrally a l o n g s e n s o r y a x o n p r o j e c t i o n s ( N e l s o n a n d L a u g h o n , 1993; H a l t e r et al., 1995; S e p p et al., 2 0 0 0 ) . T h e a p p e a r a n c e o f this cell i n the d o r s a l cluster a n d its s u b s e q u e n t m i g r a t i o n a w a y f r o m it suggests that the glial cell m a y arise f r o m a s e n s o r y o r g a n p r e c u r s o r lineage ( S O P ) . S O P s g i v e rise to the v a r i o u s cell types w h i c h c o m p r i s e Drosophila s e n s e o r g a n s w h i c h f o r the s e n s o r y bristles i n c l u d e n e u r o n s , sheath c e l l s , s o c k e t cells a n d h a i r cells. T h e factors r e s p o n s i b l e f o r cell fate s p e c i f i c a t i o n o f the v a r i o u s S O P been intensively (reviewed by Artavanis-Tsakonas  d a u g h t e r cells has  et al., 1995). R e c e n t l y , the S O P  o f m e c h a n o s e n s o r y bristles o n the n o t u m has b e e n r e v i s e d a n d n o w  lineage  includes a Repo-  p o s i t i v e g l i a l c e l l w h i c h migrates a w a y f r o m the rest o f the o r g a n ( G h o et al., 1999; R e d d y and Rodrigues, 1999b). A n u m b e r o f g e n e s h a v e b e e n isolated w h i c h are r e s p o n s i b l e f o r p e r i p h e r a l glial differentiation. S i n c e p e r i p h e r a l g l i a arise f r o m m i x e d neuronal/glial lineages, it f o l l o w s that the factors w h i c h s p e c i f y N B s  and S O P s , the n e u r o g e n i c and p r o n e u r a l genes, are  r e s p o n s i b l e not o n l y f o r n e u r o g e n e s i s ,  but g l i o g e n e s i s as w e l l .  Indeed, analysis of  p e r i p h e r a l g l i a o f the w i n g has s h o w n that m u t a t i o n s i n the p r o n e u r a l g e n e s o f the achaetescute c o m p l e x , achaete (ac),  scute (sc),  and asense (ase), l e a d to c o n c o m i t a n t l o s s o f both  4  n e u r o n s a n d g l i a ( G i a n g r a n d e , 1995). It is not k n o w n exactly w h i c h p r o n e u r a l g e n e s are r e s p o n s i b l e f o r generation o f the s i n g l e d l P G cell w h i c h is b o r n i n the d o r s a l cluster. H o w e v e r , s p e c i f i c a t i o n o f the peripheral g l i a d e r i v e d f r o m the C N S to the muscle segment homeobox (msh) gene.  Msh is r e s p o n s i b l e  sensory  is i n part due  for  dorsoventral  s p e c i f i c a t i o n o f the n e u r o e c t o d e r m and is p h y l o g e n e t i c a l l y c o n s e r v e d . It is e x p r e s s e d a l o n g the lateral r e g i o n o f the C N S  f r o m stages 8 to 11 ( I s s h i k i et al., 1997). G i v e n that m o s t  glia arise f r o m the lateral e d g e o f the C N S ,  p o s i t i o n a l l y regulated g e n e s o n the  anterior/posterior axis p r o v i d e further N B s p e c i f i c a t i o n . A s a multipotential neuro/glioblast d i v i d e s , the t r a n s c r i p t i o n factor glial cells missing/glide (herein referred to as gem) is asymmetrically ( B e r n a r d o n i et al., 1999). G e m  sequestered  to a g l i o b l a s t  is a transcription factor that is transiently e x p r e s s e d in  early d e v e l o p m e n t a n d activates glial genes. W i t h o u t p r o p e r f u n c t i o n o f gem, glial cells entirely f a i l to differentiate a n d instead a s s u m e neuronal characteristics. E c t o p i c  expression  o f gem i n n e u r o n a l p r e c u r s o r s transforms t h e m into g l i a ( H o s o y a et al., 1995; J o n e s et a l . , 1995; V i n c e n t et al., 1996). A l s o , ectopic e x p r e s s i o n o f gem i n m u s c l e c a n i n d u c e the e x p r e s s i o n o f glial s p e c i f i c g e n e s ( B e r n a r d o n i expression  et al., 1998).  Gem l i k e l y c o n t r o l s the  o f other transcription factors w h i c h are r e q u i r e d  for peripheral  glial  d i f f e r e n t i a t i o n w h i c h i n c l u d e repo ( C a m p b e l l et al., 1994; X i o n g et al., 1994; H a l t e r et a l . , 1995), pointed ( K l a e s et al., 1994), andprospero ( D o e et al., 1991; V a e s s i n et al., 1991). A t the s a m e time, gem is s u g g e s t e d to activate tramtrack, a transcription factor w h i c h represses n e u r o n a l d i f f e r e n t i a t i o n ( G i e s e n et al., 1997). In parallel to gem, the s e g m e n t a t i o n g e n e Krilppel also participates i n the s p e c i f i c a t i o n o f g l i a ( R o m a n i et al., 1996). K r i i p p e l is a z i n c f i n g e r t r a n s c r i p t i o n factor and is e x p r e s s e d e a r l y i n neuroblasts as w e l l as i n g l i a and n e u r o n s i n later stages. T h e Kriippel mutant has m a l f o r m e d a x o n p a t h w a y s i n b o t h the C N S  and P N S  w h i c h is l i k e l y due to a  c o m b i n e d effect o f d e f e c t i v e neuronal and g l i a l d i f f e r e n t i a t i o n ( R o m a n i et al., 1996).  Since  the Kriippel g e n e is e x p r e s s e d i n b o t h g l i a a n d n e u r o n s , it is p o s s i b l e that K r i i p p e l is  5  important f o r a c t i v a t i n g g e n e s w h i c h e n c o d e c o m b i n e d neural/glial p r o t e i n s s u c h as i o n channels, n e u r o t r a n s m i t t e r receptors a n d transporters. T h e pointed g e n e e n c o d e s t w o E t s t r a n s c r i p t i o n f a c t o r s , pnt  a n d pnt .  pl  pnt  pl  is e x p r e s s e d i n p e r i p h e r a l g l i a a n d l o n g i t u d i n a l g l i a w h e r e a s pnt  P2  m i d l i n e g l i a ( K l a e s et al., 1994). L o s s o f pnt  p2  The  is e x p r e s s e d i n the  leads to i n c o m p l e t e d i f f e r e n t i a t i o n o f glia.  C o n v e r s e l y , e c t o p i c pnt e x p r e s s i o n i n b o t h the m i d l i n e and lateral C N S r e g i o n s results i n pl  the generation o f e c t o p i c e x p r e s s i o n o f glial m a r k e r s . Interestingly, the l o n g i t u d i n a l a x o n tracts o f pnt  pl  m u t a n t s are d e f e c t i v e . T h i s is m o s t l i k e l y a s e c o n d a r y e f f e c t o f defective  l o n g i t u d i n a l g l i a l d i f f e r e n t i a t i o n a n d suggests that l o n g i t u d i n a l g l i a m a y  interact w i t h  n e u r o n s that m i g r a t e a l o n g the l o n g i t u d i n a l tracts ( K l a e s et al., 1994). T h e d e v e l o p m e n t o f p e r i p h e r a l g l i a i n the pnt mutant has not b e e n characterized. A s w e l l , the d e v e l o p m e n t o f p e r i p h e r a l n e r v e b u n d l e s i n the pnt has not b e e n d e s c r i b e d . T h e repo h o m e o b o x g e n e i s a l s o essential f o r glial d i f f e r e n t i a t i o n . T h e R e p o protein is e x p r e s s e d in all e c t o d e r m a l l y d e r i v e d g l i a l cells a n d is r e q u i r e d f o r differentiation as w e l l as m a i n t e n a n c e o f glial f u n c t i o n a n d s u r v i v a l ( C a m p b e l l et al., 1994; X i o n g et a l . , 1994; H a l t e r et al., 1995; X i o n g a n d M o n t e l l , 1995). In repo n u l l m u t a n t s , the e f f e c t s o f R e p o l o s s d o not m a n i f e s t until late stages o f e m b r y o n i c d e v e l o p m e n t . e m b r y o s a p p e a r to h a v e n o r m a l g l i o g e n e s i s a n d a x o n e x t e n s i o n . development, Repo-positive  glial n u c l e i are p o o r l y  organized  Initially, these  D u r i n g later stages o f and  axons  appear  d e f a s c i c u l a t e d ( C a m p b e l l et al., 1994; X i o n g et al., 1994; H a l t e r et al., 1995). G i v e n that the g l i a originate i n their correct p o s i t i o n s , the p h e n o t y p e o f late stage e m b r y o s are likely due to an inability o f g l i a to c a r r y out their m i g r a t i o n s p r o p e r l y . A l s o , the l a c k o f p r o p e r f a s c i c u l a t i o n o f a x o n s suggests that the g l i a f a i l to execute nerve w r a p p i n g . T h e r e f o r e , the repo g e n e is m o s t important f o r m a t u r e stages o f e m b r y o n i c g l i a l differentiation.  T h e g e n e s r e s p o n s i b l e f o r glial determination a n d differentiation i n Drosophila are v e r y s i m i l a r to those o f vertebrates. T h e lateral e d g e o f the vertebrate neural plate is s p e c i f i e d b y Msx g e n e s that are h o m o l o g o u s to msh, w h i c h s p e c i f i e s the Drosophila lateral 6  N B c o l u m n ( D a v i d s o n a n d H i l l , 1991; S u et al., 1991; I s s h i k i et al., 1997). T h e n e u r a l plate f o l d s to b e c o m e the n e u r a l tube a n d the c o l u m n o f M s x - s p e c i f i e d cells i s f o u n d o n the m o s t d o r s a l r e g i o n f r o m w h i c h n e u r a l crest i s g e n e r a t e d . T h e Msx g e n e s s p e c i f y d o r s a l n e u r a l fates ( T a k a h a s h i et al., 1992; S h i m e l d et al. 1996; W a n g et al., 1996). A s S c h w a n n cells arise f r o m the n e u r a l crest, they are s p e c i f i e d b y the s a m e n e u r o g e n i c g e n e s as p e r i p h e r a l glia. S c h w a n n cells also e x p r e s s Krox-20, Pax 3, a n d c-Ets-1/2 w h i c h are f u n c t i o n a l l y s i m i l a r to the Drosophila g e n e s Kriippel, repo, a n d pointed r e s p e c t i v e l y ( H e r d e g e n et al., 1993; A l b a g l i et a l . , 1996; S t e w a r t et a l . , 1996).  Krox-20  is e x p r e s s e d d u r i n g  e m b r y o g e n e s i s as S c h w a n n cell p r e c u r s o r s differentiate into i m m a t u r e S c h w a n n cells ( S t e w a r t et al., 1996). I n Krox-20 k n o c k o u t m i c e , S c h w a n n cells f a i l to m y e l i n a t e a n d the e x p r e s s i o n o f m y e l i n p r o t e i n s P a n d M B P is r e d u c e d . H o w e v e r , the S c h w a n n cells s t i l l 0  e x p r e s s the S-100 m a r k e r a n d f o r m 1:1 r e l a t i o n s h i p s w i t h a x o n s ( S t e w a r t et al; 1996). T h e p r e c i s e r o l e o f Krox-20 i n S c h w a n n c e l l d e v e l o p m e n t is n o t k n o w n . Pax 3 i s e x p r e s s e d i n a variety o f tissues d u r i n g e m b r y o n i c d e v e l o p m e n t i n c l u d i n g cranial g a n g l i a a n d cranial facial crest d e r i v a t i v e s . T h e Pax 3 k n o c k o u t m o u s e h a s d i s r u p t e d neural-crest d e r i v e d s p i n a l g a n g l i a a n d heart defects a n d dies i n m i d - e m b r y o g e n e s i s ( G o u l d i n g et al., 1 9 9 1 ) . Pax  3 m u t a t i o n is a l s o a s s o c i a t e d w i t h W a a r d e n b u r g s y n d r o m e i n w h i c h d e a f n e s s a n d  p i g m e n t a t i o n d e f e c t s o c c u r ( T a s s a b e h j i et al., 1994). T h u s Pax 3 m a y h a v e a n i m p o r t a n t role i n n e u r a l crest d e v e l o p m e n t that is n o t l i m i t e d to S c h w a n n cell d e v e l o p m e n t . Vertebrate E t s transcription factors h a v e n o t b e e n i n t e n s i v e l y s t u d i e d i n S c h w a n n cell d e v e l o p m e n t , a l t h o u g h their e x p r e s s i o n has b e e n f o u n d i n m o u s e e m b r y o n i c n e u r a l crest ( M a r o u l a k o u et al., 1994). In vitro studies h a v e s h o w n that Drosophila pointed is c a p a b l e o f b i n d i n g vertebrate c - E t s l / 2 r e s p o n s i v e s e q u e n c e s . C o n v e r s e l y , t a r g e t e d e x p r e s s i o n o f vertebrate c - E t s i n Drosophila pointed mutants i s s u f f i c i e n t to r e s c u e m i d l i n e g l i a a n d p h o t o r e c e p t o r d e f e c t s ( A l b a g l i et al., 1996).  7  Developmental  roles of PNS  Since very few  glia  mutants w h i c h  d i s r u p t p e r i p h e r a l glial f u n c t i o n h a v e b e e n  characterized, it is important to c o n s i d e r other m o d e l s y s t e m s to g a i n i n s i g h t into their developmental  roles. Peripheral glia have a number of morphological and functional  similarities to other g l i a . T h e m o s t a n a l o g o u s glial cells i n vertebrates are S c h w a n n cells, w h i c h w r a p p e r i p h e r a l nerves. P e r i p h e r a l g l i a a n d S c h w a n n cells b o t h appear to prepattern the C N S / P N S a x o n entry/exit z o n e i n early d e v e l o p m e n t . D u r i n g a x o n m i g r a t i o n a c r o s s the C N S / P N S b o r d e r i n Drosophila, neurites contact p e r i p h e r a l "exit" g l i a w h i c h  are  a s s e m b l e d i n c o m p a c t arrays at the lateral e d g e o f the C N S d u r i n g p i o n e e r a x o n p a t h f i n d i n g ( S e p p et al., 2 0 0 0 ) .  T h e p e r i p h e r a l glial structure has a l s o b e e n p r o p o s e d to h a v e a k e y  role i n a x o n g u i d a n c e i n this r e g i o n ( K l a m b t a n d G o o d m a n , 1991).  Similarly, during  m i g r a t i o n o f s e n s o r y a n d m o t o r a x o n s into a n d out o f the vertebrate C N S ,  neurites pass  t h r o u g h c o m p a c t a r r a y s o f astrocytes a n d S c h w a n n cells at the C N S / P N S b o r d e r .  These  structures, c a l l e d the d o r s a l root entry z o n e ( D R E Z ) a n d the transition z o n e ( T Z ) are thought to sort a x o n s a n d p r o v i d e a x o n g u i d a n c e cues ( F r a h e r 1997; G o l d i n g et al., 1997; G o l d i n g a n d C o h e n , 1997; O ' B r i e n et al., 1998). PNS,  A s n e u r o n s migrate f u r t h e r into the  they are a c c o m p a n i e d b y p e r i p h e r a l g l i a i n Drosophila a n d S c h w a n n cells in  vertebrates ( C a r p e n t e r a n d H o l l y d a y , 1992; C a m p b e l l et al., 1994; H a l t e r et al., 1995; S e p p et al., 2000). L a t e r i n d e v e l o p m e n t , p e r i p h e r a l g l i a w r a p p e r i p h e r a l n e r v e s , a l t h o u g h they d o not e x t e n d m u l t i p l e w r a p s a r o u n d a x o n s as m y e l i n a t i n g S c h w a n n cells do.  P e r i p h e r a l glial  w r a p p i n g i n Drosophila a n d m o s t invertebrates is m o r e a k i n to n o n - m y e l i n a t i n g  Schwann  cells, w h e r e b u n d l e s o r s i n g l e s m a l l diameter a x o n s are e n c l o s e d b y s i n g l e w r a p s ( J e s s e n a n d M i r s k y , 1999).  I n t e r e s t i n g l y , not a l l invertebrates l a c k m y e l i n .  The copepod, a  p l a n k t o n i c c r u s t a c e a n , has m y e l i n a t e d a x o n s w h i c h greatly r e s e m b l e vertebrate S c h w a n n c e l l w r a p p i n g ( D a v i s et al., 1999). T h e r e are m a n y m a r k e r s f o r S c h w a n n cells: the c a l c i u m b i n d i n g p r o t e i n S 1 0 0 , a n d the i m m u n o g l o b u l i n  superfamily m e m b e r s myelin associated 8  g l y c o p r o t e i n ( M A G ) a n d Po, the tetraspan p r o t e i n s C D 9 a n d p e r i p h e r a l m y e l i n p r o t e i n ( P M P 2 2 ) , p r o t e o l i p i d p r o t e i n ( P L P ) a n d m y e l i n b a s i c p r o t e i n ( M B P ) ( S p r e y e r et al., 1 9 9 1 ; J e s s e n a n d M i r s k y , 1 9 9 2 , 1999; K a p r i e l i a n et al., 1995; G a r b e r n et al., 1997).  Non-  m y e l i n a t i n g S c h w a n n cells d o n o t e x p r e s s m y e l i n p r o t e i n s a n d i n s t e a d e x p r e s s t h e cell adhesion molecules N C A M  and L l , growth  a s s o c i a t e d p r o t e i n G A P - 4 3 , a n d the  i n t e r m e d i a t e f i l a m e n t p r o t e i n G F A P ( S c h e r e r a n d Salzer, 1996). I n Drosophila, p e r i p h e r a l g l i a e x p r e s s m a r k e r s a s s o c i a t e d w i t h S c h w a n n cells s u c h as G l i o t a c t i n / N e u r o l i g i n III a n d N e u r o g l i a n / L l ( B i e b e r et al., 1989; S c h e r e r a n d S a l z e r , 1996; M . G i l b e r t , J. S m i t h a n d V . A u l d , u n p u b l i s h e d data). It is l i k e l y that i n the m a t u r e o r g a n i s m , p e r i p h e r a l glial a n d vertebrate S c h w a n n cell f u n c t i o n i n g is s i m i l a r , as t h e b a s i c n e e d s o f n e u r o n s s h o u l d n o t c h a n g e drastically o v e r e v o l u t i o n . T h e p r i m a r y r o l e o f g l i a i n the adult is t o m a i n t a i n a n a p p r o p r i a t e e n v i r o n m e n t f o r n e u r o n s to c o n d u c t a c t i o n potentials. W h e n g l i a d o n o t p r o p e r l y ensheathe a x o n s o f b o t h Drosophila a n d vertebrates, p r o p a g a t i o n o f a c t i o n p o t e n t i a l s i n n e u r o n s i s i m p a i r e d ( D e W a e g h et al., 1992; A u l d et al., 1995).  U s i n g an assortment o f i o n channels,  neurotransmitter receptors a n d transporters, glia maintain ionic homeostasis a n d prevent n e u r o t o x i c i t y . I n Drosophila, the g l i a p h y s i c a l l y seal o f f n e u r o n s f r o m t h e h a e m o l y m p h w h i c h w o u l d o t h e r w i s e interfere w i t h n e u r o n a l a c t i o n potentials ( A u l d et al., 1995).  Thus  the o r i g i n a l roots o f g l i a l c e l l r o l e s m a y b e the m a i n t e n a n c e o f the b l o o d n e r v e b a r r i e r w h i c h in turn a l l o w s n e u r o n s to f u n c t i o n i n a n o p t i m a l l y r e g u l a t e d e n v i r o n m e n t . F o r l a r g e o r g a n i s m s , the e v o l u t i o n o f m y e l i n w r a p p i n g w o u l d h a v e b e e n e s s e n t i a l to r e l a y a c t i o n p o t e n t i a l s o v e r v e r y l o n g d i s t a n c e s . P e r i p h e r a l n e r v e s as i n Drosophila w o u l d n o t b e s u f f i c i e n t t o m a i n t a i n a c t i o n potential a m p l i t u d e s i n these c a s e s . R a t h e r , the g e n e r a t i o n o f m y e l i n w r a p p i n g w i t h n o d a l s e p a r a t i o n s b e t w e e n S c h w a n n cells a l l o w s action potential a m p l i t u d e s to b e r e c h a r g e d at e a c h node, s i n c e the i o n c h a n n e l s r e q u i r e d f o r action potential g e n e r a t i o n are c l u s t e r e d i n these r e g i o n s ( J e s s e n a n d M i r s k y ,  1999).  F u r t h e r m o r e , t h e m u l t i p l e S c h w a n n cell m e m b r a n e w r a p s about t h e a x o n s decreases  9  membrane capacitance, allowing for enhanced nerve conduction velocity over internodal r e g i o n s ( J e s s e n a n d M i r s k y , 1999). O v e r the c o u r s e o f e v o l u t i o n , the b l o o d n e r v e b a r r i e r m a y h a v e c h a n g e d to a c c o m o d a t e f o r m y e l i n w r a p p i n g . In Drosophila, G l i o t a c t i n is a p e r i p h e r a l glial t r a n s m e m b r a n e p r o t e i n r e q u i r e d f o r the b l o o d n e r v e b a r r i e r . L o s s o f G l i o t a c t i n f u n c t i o n c a u s e s the glial seal a r o u n d the p e r i p h e r a l n e r v e s to b e faulty, a l l o w i n g h i g h K + h a e m o l y m p h to r e a c h a x o n s a n d interfere w i t h n e r v e c o n d u c t i o n ( A u l d et al., 1995).  G l i o t a c t i n is t h o u g h t to l o c a l i z e to glial-glial  septate j u n c t i o n s a n d thus c o n t r i b u t e to the tight glial seal a r o u n d n e r v e s . N e u r e x i n I V is another Drosophila p r o t e i n w h i c h is r e q u i r e d f o r the b l o o d n e r v e b a r r i e r a n d i s r e q u i r e d f o r septate j u n c t i o n f o r m a t i o n ( B a u m g a r t n e r et al., 1996).  T h e N e u r e x i n I V vertebrate  h o m o l o g u e s , C a s p r a n d C a s p r 2 , are e x p r e s s e d b y n e u r o n s a n d are l o c a l i z e d to j u n c t i o n s at p a r a n o d e s a n d j u x t a p a r a n o d e s o f S c h w a n n cells a n d a x o n s ( E i n h e b e r et al, 1997; P o l i a k et al., 1999).  T h e j u n c t i o n s are v e r y s i m i l a r to septate j u n c t i o n s o f i n v e r t e b r a t e s . It is  p o s s i b l e that p a r a n o d a l j u n c t i o n s arose i n e v o l u t i o n t h r o u g h m o d i f i c a t i o n s to invertebrate septate j u n c t i o n s . T h e transition b e t w e e n glial-glial j u n c t i o n s o f the b l o o d n e r v e barrier i n insects a n d g l i a l - a x o n a l j u n c t i o n s b e t w e e n p a r a n o d a l glial l o o p s a n d a x o n s o f vertebrates is c o m p l e x . S c h w a n n cells m a i n t a i n a s p e c i a l i z e d e n v i r o n m e n t f o r a x o n a l c o n d u c t i o n b y the c l u s t e r i n g o f i o n c h a n n e l s at n o d e s as w e l l as s e a l i n g the p a r a n o d a l l o o p s to a x o n s to insulate the i n t e r n o d a l r e g i o n s as i n i n s e c t s ( r e v i e w e d b y J e s s e n a n d M i r s k y ,  1999).  S c h w a n n c e l l s a l s o i n f l u e n c e the o v e r a l l m o r p h o l o g i c a l p r o f i l e o f the a x o n s they w r a p . In the trembler m o u s e w h i c h has d e f e c t i v e P M P 2 2 , p e r i p h e r a l n e r v e s are d e m y e l i n a t e d a n d the a x o n s are r e d u c e d i n diameter. T h e r e are a l s o c h a n g e s i n n e u r o f i l a m e n t d e n s i t y a n d s l o w a x o n t r a n s p o r t rates are d e c r e a s e d ( D e W a e g h et al., 1992).  10  The  "blueprint hypothesis" of glial developmental roles  T h e d e v e l o p m e n t o f C N S g l i a , w h i c h h a s b e e n i n t e n s i v e l y s t u d i e d , m a y also p r o v i d e c l u e s as w h a t r o l e s p e r i p h e r a l g l i a h a v e i n d e v e l o p m e n t . B a s e d o n o b s e r v a t i o n s that g l i a i n the n e w t e m b r y o n i c s p i n a l c o r d prepattern a x o n m i g r a t i o n routes, the " b l u e p r i n t h y p o t h e s i s " w a s generated, s u g g e s t i n g that n e u r o n s r e c o g n i z e g l i a as g u i d e p o s t cells f o r m i g r a t i o n ( S i n g e r et al., 1979). S i n c e then, m a n y e x a m p l e s o f this h a v e b e e n o b s e r v e d . G l i a l cells i n m i c e f o r m a b r i d g e l i k e structure b e t w e e n the t w o c e r e b r a l h e m i s p h e r e s o v e r w h i c h n e u r o n s migrate f r o m o n e h e m i s p h e r e t o another, f o r m i n g the great cerebral c o m m i s s u r e s . W h e n the g l i a are s u r g i c a l l y r e m o v e d , the n e u r o n s c a n n o l o n g e r c r o s s f r o m o n e s i d e t o the other, s u g g e s t i n g that at the v e r y least, g l i a f o r m a p e r m i s s i v e s u r f a c e o v e r w h i c h n e u r o n s c a n m i g r a t e ( S i l v e r et al., 1982). T h e r a d i a l g l i a o f the c e r e b e l l u m a n d the cerebral cortex act as m i g r a t i o n h i g h w a y s f o r n e u r o n s d u r i n g d e v e l o p m e n t ( H a t t e n , 1984; R a k i c , 1 9 9 1 ; N o r r i s a n d K a l i l , 1991). A n u m b e r o f m o l e c u l e s h a v e b e e n i d e n t i f i e d w h i c h are r e s p o n s i b l e f o r steps i n m i g r a t i o n o n r a d i a l g l i a w h i c h i n c l u d e A M O G , astrotactin, a n d e r b B receptors ( A n t o n i c e k et al., 1987; G l o o r et al. 1990; Z h e n g et al., 1996; R i o et a l . , 1997). S t u d i e s o f C N S g l i a i n the invertebrate h a v e b e e n v e r y s u c c e s s f u l i n e v a l u a t i n g the potential f o r glial r o l e s i n g u i d a n c e . I n Drosophila, g l i a a p p e a r e a r l y i n n e u r o d e v e l o p m e n t a n d p r e f i g u r e the a x o n m i g r a t i o n tracts ( J a c o b s a n d G o o d m a n , 1989a). T h e C N S g l i a are c l a s s e d into three s u b g r o u p s b a s e d o n their p o s i t i o n i n g w i t h respect t o a x o n tracts: the m i d l i n e , l o n g i t u d i n a l , a n d s e g m e n t a l b o u n d a r y g l i a . D u r i n g a x o n m i g r a t i o n s , all o f these g l i a h a v e a c o m p a c t , o v a l m o r p h o l o g y a n d are l o c a t e d at the d o r s a l s u r f a c e o f the C N S w h e r e the a x o n tracts later f o r m . D u r i n g their m i g r a t i o n s , n e u r o n s m a k e contacts w i t h the c o m p a c t g l i a a n d o n c e m u c h o f neural m i g r a t i o n i s c o m p l e t e , the g l i a a s s u m e a mature w r a p p i n g m o r p h o l o g y ( J a c o b s a n d G o o d m a n , 1989a,b).  11  T h e m i d l i n e g l i a are essential f o r p r o p e r f o r m a t i o n o f the anterior a n d p o s t e r i o r a x o n c o m m i s s u r e s . I f the g l i a are ablated, the c o m m i s s u r e s fail to f o r m ( Z h o u et al., 1 9 9 7 ) . M i d l i n e g l i a direct a x o n g u i d a n c e a c r o s s a n d a w a y f r o m the m i d l i n e b y e x p r e s s i n g instructive factors, w h i c h i n c l u d e netrin, commissureless, a n d slit ( R o t h b e r g et al., 1990; S e e g e r et al., 1993; H a r r i s et al., 1996; M i t c h e l l et al., 1996). If a n y o f t h e s e m o l e c u l e s are defective, the c o m m i s s u r e s are t h e n m a l f o r m e d . T h e r e f o r e , m i d l i n e g l i a actively mediate a x o n g u i d a n c e at the m i d l i n e . T h e m i d l i n e g l i a also h a v e i m p o r t a n t structural roles as they p h y s i c a l l y separate the t w o c o m m i s s u r e s t h r o u g h a series o f m i g r a t i o n s a r o u n d the a x o n s . In the m i d l i n e glial differentiation m u t a n t s Star, pointed, spitz, a n d rhomboid, these m i g r a t i o n e v e n t s d o n o t o c c u r , r e s u l t i n g i n f u s i o n o f anterior a n d p o s t e r i o r c o m m i s s u r e s ( K l a m b t et al., 1991). T h e r e are a v e r y large n u m b e r o f g e n e s n e c e s s a r y f o r the c o m p l e x f u n c t i o n s o f m i d l i n e g l i a d u r i n g n e r v o u s s y s t e m f o r m a t i o n w h i c h are c u r r e n t l y b e i n g c h a r a c t e r i z e d ( H u m m e l et al., 1999a,b). L o n g i t u d i n a l g l i a are also n e c e s s a r y f o r p r o p e r f o r m a t i o n o f the l o n g i t u d i n a l a x o n tracts that t h e y later ensheathe. W h e n l o n g i t u d i n a l g l i a fail to differentiate n o r m a l l y i n the pointed, prospero, a n d gem mutants, the l o n g i t u d i n a l tracts d o n o t f o r m p r o p e r l y ( C h u et  al., 1991; K l a e s et al., 1994; H o s o y a e t al., 1995; J o n e s et al., 1995; V i n c e n t et al., 1 9 9 6 ) . A s w e l l , w h e n the l o n g i t u d i n a l g l i a are a b l a t e d , there is a w i d e s p r e a d l o s s o f the l o n g i t u d i n a l a x o n tracts ( H i d a l g o et al., 1995; H i d a l g o a n d B o o t h , 2 0 0 0 ) .  U n l i k e the  m i d l i n e g l i a , h o w e v e r , n o g e n e s h a v e b e e n i d e n t i f i e d w h i c h l o n g i t u d i n a l g l i a e x p r e s s to g u i d e n e u r o n s l o n g i t u d i n a l l y . P o s s i b l y , the l o n g i t u d i n a l g l i a s i m p l y p r o v i d e a p e r m i s s i v e s u r f a c e o v e r w h i c h n e u r o n s c a n m i g r a t e a n d their r o l e i n p a t h w a y f o r m a t i o n is passive. T h e s e g m e n t a l b o u n d a r y c e l l ( S B C ) g l i a (Ito et al., 1995) o f Drosophila C N S  have  b e e n s u g g e s t e d to p l a y a n instructive role i n the t u r n i n g o f a x o n s f r o m the l o n g i t u d i n a l c o n n e c t i v e s to a lateral d i r e c t i o n to f o r m the I S N p e r i p h e r a l n e r v e f o r t w o r e a s o n s . F i r s t , the a n a t o m i c a l l o c a t i o n o f the S B C is situated at the v e r y c o r n e r o f w h e r e n e u r o n s m u s t turn to be o n the I S N .  S e c o n d , the S B C i s a n a l o g o u s t o the g r a s s h o p p e r S B C w h i c h w a s  12  s h o w n to h a v e a r o l e i n a x o n t u r n i n g d e c i s i o n s ( J a c o b s a n d G o o d m a n , 1989a). W h e n the g r a s s h o p p e r S B C w a s a b l a t e d , the p i o n e e r g r o w t h c o n e w h i c h w o u l d n o r m a l l y turn laterally w h e n c o n t a c t i n g the S B C c o u l d n o t a c c o m p l i s h this a n d instead c o n t i n u e d to m i g r a t e l o n g i t u d i n a l l y ( B a s t i a n i a n d G o o d m a n , 1986). M o r e recently, a n a t o m i c a l e v i d e n c e f r o m c l o n a l a n a l y s e s o f Drosophila C N S n e u r o b l a s t s m a y s u p p o r t the S B C g u i d a n c e h y p o t h e s i s . T h e N B 1 - 1 a n d N B 7 - 1 g i v e rise t o S B C g l i a as w e l l as the I S N p i o n e e r m o t o r n e u r o n s w h i c h c o n t a c t the S B C . It is a s s u m e d that g l i a often g u i d e n e u r o n s o f the s a m e l i n e a g e ( S c h m i d et al., 1999). T h e S B C g l i a h a v e n e v e r b e e n s p e c i f i c a l l y r e m o v e d d u r i n g d e v e l o p m e n t to s p e c i f i c a l l y assess their c o n t r i b u t i o n t o a x o n p a t h f i n d i n g . P e r i p h e r a l g l i a , d u e t o their p h y s i c a l similarities t o t h e o t h e r C N S g l i a , m a y p r e f i g u r e m i g r a t i o n p a t h w a y s a n d p r o v i d e g u i d a n c e i n f o r m a t i o n t o n e u r o n s as w e l l . F o r this to b e true, o n e m u s t b e a b s o l u t e l y s u r e that the g l i a are p o s i t i o n e d a h e a d o f n e u r o n s i n their r e s p e c t i v e m i g r a t o r y paths. W i t h o u t c y t o p l a s m i c m a r k e r s f o r p e r i p h e r a l g l i a , this c o u l d n o t b e a c c o m p l i s h e d . U s i n g n u c l e a r m a r k e r s t o l a b e l p e r i p h e r a l g l i a , i t i s certain that they a p p e a r at the lateral e d g e o f the C N S b e f o r e p i o n e e r a x o n p a t h f i n d i n g b e g i n s either o u t o f the C N S b y m o t o r n e u r o n s , o r into the C N S b y s e n s o r y n e u r o n s . H o w e v e r , p i o n e e r m o t o r a x o n s s o o n p a s s b y the p e r i p h e r a l glial cell b o d i e s w h i c h a r e s t i l l at the lateral e d g e o f the C N S a n d t h e n later fasciculate w i t h i n c o m i n g s e n s o r y n e u r o n s ( K l a m b t a n d G o o d m a n , 1991; H a l t e r et al.; 1995). B a s e d o n the C N S m o d e l s , i t w o u l d s e e m l i k e l y that there w o u l d b e a n initial g u i d e p o s t cell p h a s e , as p i o n e e r m o t o r n e u r o n s d o m a k e c o n t a c t w i t h the p e r i p h e r a l g l i a . S u b s e q u e n t l y , the g l i a w o u l d n o t h a v e a r o l e i n g u i d a n c e a n d w o u l d actually b e g u i d e d b y m o t o r n e u r o n s s i n c e t h e y f o l l o w t h e m into the P N S .  The  latter statement w o u l d n o t h o l d true i f the g l i a e x t e n d e d l o n g c y t o p l a s m i c p r o c e s s e s p a s t the m i g r a t i n g tips o f p i o n e e r m o t o r n e u r o n g r o w t h c o n e s . T h e r e f o r e , t o u n d e r s t a n d w h a t roles p e r i p h e r a l g l i a m a y h a v e i n d e v e l o p m e n t , a c y t o p l a s m i c m a r k e r i s essential.  It i s also  h e l p f u l to r e v i e w e x p e r i m e n t s o n glial a n d n e u r o n a l m i g r a t i o n i n o t h e r m o d e l s y s t e m s .  13  Interactions between glia and neurons  In b o t h the d e v e l o p i n g w i n g a n d v i s u a l s y s t e m o f Drosophila, g l i a a p p e a r to h a v e a s y m b i o t i c r e l a t i o n s h i p w i t h n e u r o n s . T h e first detectable p e r i p h e r a l glial cells o f the w i n g a p p e a r early, b e f o r e a x o g e n e s i s has o c c u r r e d ( G i a n g r a n d e et al., 1993). T h e early birth o f the g l i a is consistent w i t h the h y p o t h e s i s that g l i a p r o m o t e a x o g e n e s i s ( R o d r i g u e s and R e d d y , 1 9 9 9 b ) . T h e g l i a d e v e l o p i n the w i n g f r o m epithelial cells i n r e g i o n s w h i c h also g i v e rise to s e n s o r y n e u r o n s . In the w i l d type, the w i n g p e r i p h e r a l g l i a a l w a y s m i g r a t e p r o x i m a l l y a l o n g c e n t r a l l y p r o j e c t i n g sensory axons. Infused a n d Notch m u t a t i o n s , w h i c h affect a x o g e n e s i s , glial m i g r a t i o n is a c c o r d i n g l y disrupted. If a x o n s stall, glial cells also stall i n the s a m e r e g i o n ( G i a n g r a n d e , 1994). T h e s e d a t a s u g g e s t that although the g l i a may stimulate a x o g e n e s i s , their o w n m i g r a t i o n c o u l d be d e p e n d e n t o n a x o n extension. T h e m i g r a t i o n o f retinal basal g l i a ( R B G ) , f r o m their birthplace i n the optic stalk to the e y e disc, is a l s o l i n k e d to glial-neuronal interactions. T h e R B G  cells n o r m a l l y m i g r a t e  a l o n g p h o t o r e c e p t o r a x o n s into the disc. T h e p r o l i f e r a t i o n o f the g l i a is l i n k e d to n u m b e r s o f photoreceptors.  The R B G  cells, o n c e i n the eye disc, n e v e r a p p e a r to m i g r a t e a h e a d o f  the e m e r g i n g f r o n t o f photoreceptor clusters. T h u s , they o n l y m i g r a t e to r e g i o n s w h e r e n e u r o n s h a v e b e e n generated. photoreceptor neurons, R B G  In eyes absent and sine oculis m u t a n t s w h i c h lack  cells are incapable o f m i g r a t i n g into the eye d i s c and r e m a i n  i n the o p t i c stalk ( C h o i a n d B e n z e r , 1994). I f photoreceptors  are able to differentiate, but  their a x o g e n e s i s is b l o c k e d b y e x p r e s s i o n o f constitutively activation o f RhoA, R B G  glia  are s t i l l capable o f m i g r a t i n g into the optic stalk, although w i t h m o r e d i f f i c u l t y than in w i l d types. T h u s it is l i k e l y that n e u r o n s secrete a d i f f u s i b l e f a c t o r w h i c h attracts R B G  cells into  the o p t i c stalk. A l s o , it s u g g e s t s that photoreceptor a x o n s are not a b s o l u t e l y r e q u i r e d as a substrate f o r R B G  migration.  O n the other h a n d , i f glial m i g r a t i o n into the optic stalk is  p r e v e n t e d b y e x p r e s s i o n o f d o m i n a n t negative Rasl, photoreceptor  a x o n s are oriented  p r o p e r l y to the o p t i c stalk, h o w e v e r , they are u n a b l e to e x i t the d i s c a n d enter the optic stalk  14  ( R a n g a r a j a n et al., 1999). I n this m o d e l , it is p o s s i b l e that b o t h n e u r o n s a n d g l i a e x p r e s s d i f f u s i b l e f a c t o r s w h i c h g u i d e m i g r a t i o n s t o w a r d s each other. T h e o b s e r v a t i o n that the p h o t o r e c e p t o r n e u r o n s are able t o e x t e n d a x o n s w i t h i n the e y e d i s c b u t c a n n o t m a k e a p r o p e r entry into the o p t i c stalk w i t h o u t g l i a suggests that p e r h a p s the R B G m a y b e r e q u i r e d f o r g r o w t h c o n e s t e e r i n g d e c i s i o n s t h r o u g h p h y s i c a l c o n t a c t as w e l l . A s t r i k i n g e x a m p l e o f g r o w t h c o n e s t e e r i n g i n relation to g l i a has b e e n s t u d i e d i n the o l f a c t o r y s y s t e m o f t h e m o t h Manduca sexta ( R o s s l e r et al., 1999).  O l f a c t o r y receptor  n e u r o n s project f r o m the o l f a c t o r y e p i t h e l i u m to target g l o m e r u l i o f the p r i m a r y o l f a c t o r y centers i n the antennal lobe. I n d i v i d u a l r e c e p t o r n e u r o n s r e c o g n i z e d i f f e r e n t o d o u r a n t s a n d the l o c a t i o n o f the v a r i o u s types o f receptor n e u r o n s is d i s p e r s e d t h r o u g h o u t the o l f a c t o r y e p i t h e l i u m . Y e t as the n e u r o n s project t o w a r d s the g l o m e r u l i , they m a k e s t e e r i n g c h o i c e s so that s i m i l a r r e c e p t o r neurons c o n v e r g e o n the s a m e g l o m e r u l i , e v e n t h o u g h the n e u r o n s c o u l d h a v e b e e n situated i n v e r y d i f f e r e n t l o c i o n the e p i t h e l i u m . T h e r e g i o n w h e r e the neurons alter t h e i r m i g r a t i o n a l p a t h w a y s , the " a x o n s o r t i n g zone", is p o p u l a t e d d e n s e l y b y C N S glial cells w h i c h arise f r o m the antennal l o b e . T h e g l i a appear at the a x o n s o r t i n g z o n e s o o n after the first o l f a c t o r y r e c e p t o r n e u r o n s appear i n this area a n d it is t h o u g h t that p r o l i f e r a t i o n o f the g l i a is d e p e n d e n t o f contact w i t h the s e n s o r y n e u r o n s ( R o s s l e r et a l . , 1999).  T h e c o i n c i d e n c e o f g l i a d e n s e l y p o p u l a t i n g the r e g i o n o f n e u r o n a l p a t h w a y  m o d i f i c a t i o n p r o v i d e s anatomical e v i d e n c e that g l i a m a y h e l p determine g r o w t h c o n e steering d e c i s i o n s o f s e n s o r y n e u r o n s .  T o assess this h y p o t h e s i s , t h e g l i a w e r e ablated  u s i n g C o b a l t g a m m a r a d i a t i o n w h i c h s u b s e q u e n t l y c a u s e d inability o f o l f a c t o r y receptor 6 0  neurons to c o r r e c t l y l o c a t e their target g l o m e r u l i . T h e g u i d a n c e e r r o r s o c c u r r e d i n the gliad e p r i v e d a x o n s o r t i n g z o n e ( R o s s l e r et al., 1999). T h i s m a y b e a n o t h e r e x a m p l e w h e r e glia a n d n e u r o n s are i n t e r d e p e n d e n t f o r their p r o p e r d e v e l o p m e n t .  15  Interactions between PNS glia and neurons  E m b r y o n i c d e v e l o p m e n t o f p e r i p h e r a l g l i a i n Drosophila is l i k e l y m o s t a n a l o g o u s to vertebrate S c h w a n n cell d e v e l o p m e n t s i n c e the p e r i p h e r a l g l i a m u s t a s s o c i a t e w i t h b o t h P N S m o t o r a n d s e n s o r y n e u r o n s as S c h w a n n cells do. T h e e x a m p l e s o f g l i a l - n e u r o n a l s y m b i o s i s i n Drosophila a n d the m o t h o l f a c t o r y s y s t e m m a y s t i l l b e representative o f general p r o p e r t i e s w h i c h m a n y o t h e r g l i a m a y h a v e . S t u d i e s o n the m o u s e erbB2 mutant p r o v i d e s v a l u a b l e i n s i g h t into g l i a - n e u r o n interaction i n the d e v e l o p i n g vertebrate P N S ( W o l d e y e s u s et al., 1999). T h e erbBl g e n e c o d e s f o r a m e m b r a n e - b o u n d  r e c e p t o r o f the  e p i d e r m a l g r o w t h f a c t o r ( E G F ) f a m i l y . T h e e r b B 2 p r o t e i n is f o u n d i n d e v e l o p i n g S c h w a n n cells, s o m a t i c m u s c l e , a n d heart. W h e n the heart d e f e c t i n the erbB2 m u t a n t i s genetically rescued, the d e v e l o p m e n t o f the m o u s e w i t h defective e r b B 2 i n glial a n d m u s c l e tissue c a n be f o l l o w e d . T h e r e is a s e v e r e loss o f S c h w a n n cells i n the erbB2 mutant. T h e S c h w a n n cells w h i c h d o s u r v i v e f a i l to m i g r a t e p a s t the d o r s a l r o o t g a n g l i o n ( D R G ) .  Normally,  m o t o r a n d s e n s o r y a x o n s c a r r y o u t their m i g r a t i o n s into the p e r i p h e r y i n a s s o c i a t i o n w i t h S c h w a n n cells. B u t w i t h S c h w a n n cells s t r a n d e d at the D R G , m o s t a x o n p a t h f i n d i n g m u s t be a c c o m p l i s h e d w i t h o u t s i g n i f i c a n t s u p p o r t f r o m the P N S g l i a . T h e m o t o r a n d s e n s o r y p a t h w a y s d o f o r m i n the e r b B 2 mutant, h o w e v e r t h e y are d i s o r g a n i z e d , d e f a s c i c u l a t e d , a n d h a v e r e d u c e d n e r v e d i a m e t e r . T h e r e f o r e , p a t h f i n d i n g c h o i c e s b e y o n d the p o i n t o f the D R G are not h e a v i l y i n f l u e n c e d b y S c h w a n n cells. T h e S c h w a n n cells h a v e m o r e o f a role i n b u i l d i n g the m a t u r e n e r v e structure ( W o l d e y e s u s et al., 1999). Interestingly, t h e r e is w i d e s p r e a d c e l l d e a t h o f s e n s o r y n e u r o n s i n the erbB2 mutant e m b r y o but o n l y s i g n i f i c a n t d e a t h o f m o t o r n e u r o n s at c e r v i c a l a n d l u m b a r l e v e l s . T h u s it a p p e a r s that s e n s o r y a x o n s are m o r e d e p e n d e n t o n S c h w a n n cells f o r t r o p h i c s u p p o r t d u r i n g d e v e l o p m e n t t h a n m o t o r n e u r o n s are ( W o l d e y e s u s et al., 1999). A g a i n , the s u r v i v a l o f n e u r o n s a n d g l i a i s i n t e r d e p e n d e n t . T h e e r b B 2 r e c e p t o r b i n d s the g r o w t h factor n e u r e g u l i n - 1 , w h i c h is s e c r e t e d b y n e u r o n s a n d i s a m i t o g e n f o r S c h w a n n  cells  ( M a r c h i o n n i et al., 1993; B u r d e n a n d Y a r d e n , 1997). T h u s the l o s s o f S c h w a n n cells i n  16  the e r b B 2 mutant m o s t likely o c c u r r e d b e c a u s e they c o u l d n o t r e c o g n i z e d e v e l o p m e n t a l s i g n a l s f r o m the neurons. A s the S c h w a n n cells c o u l d n o t d e v e l o p , p r e s e n t a t i o n o f t r o p h i c signals to the associated neurons c o u l d not occur leading to the demise o f the neurons t h e m s e l v e s . T r o p h i c interactions b e t w e e n n e u r o n s a n d g l i a h a v e a l s o b e e n r e p o r t e d i n Drosophila.  R e p o is specifically expressed i n glia a n d is required f o r terminal  d i f f e r e n t i a t i o n o f g l i a i n the v i s u a l s y s t e m ( X i o n g et al., 1994). I n the repo mutant, l a m i n a r n e u r o n s o f the o p t i c l o b e u n d e r g o i n c r e a s e d cell death, s u g g e s t i n g that g l i a e x p r e s s f a c t o r s for their s u r v i v a l ( X i o n g a n d M o n t e l l , 1995). M o s t recently, g l i a o f the C N S h a v e b e e n s h o w n t o m a i n t a i n " f o l l o w e r " n e u r o n s u r v i v a l d u r i n g e m b r y o n i c d e v e l o p m e n t ( B o o t h et al., 2 0 0 0 ) . S t u d y o f the erbB2 m u t a n t s t r o n g l y suggests that a x o n g u i d a n c e i n the p e r i p h e r y i s a c c o m p l i s h e d l a r g e l y w i t h o u t the a i d o f P N S g l i a .  H o w e v e r , it c a n n o t a d d r e s s the  p o s s i b i l i t y that the D R E Z a n d T Z m a y h a v e k e y r o l e s as m e d i a t o r s o f s e n s o r y a n d m o t o r a x o n g u i d a n c e i n t o a n d o u t o f the C N S r e s p e c t i v e l y , s i n c e S c h w a n n cells p o p u l a t e these r e g i o n s i n the erbB2 mutant.  T h e s e are the structures w h e r e glial g u i d a n c e o f a x o n  m i g r a t i o n is m o s t l i k e l y t o h a p p e n as a l a r g e a m o u n t o f a x o n s o r t i n g a n d g r o w t h steering d e c i s i o n s o c c u r i n these r e g i o n s ( G o l d i n g a n d C o h e n , 1997). are the o n l y cells i n these r e g i o n s that p h y s i c a l l y contact n e u r o n s .  cone  F u r t h e r m o r e , glia O n e c a n imagine a  s c e n a r i o s i m i l a r t o p h o t o r e c e p t o r a x o n s b e i n g u n a b l e t o enter the o p t i c stalk w i t h the absence o f R B G cells. P o s s i b l y , i f g l i a d i d n o t p o p u l a t e the T Z , m o t o r a x o n s c o u l d n o t turn o u t o f the C N S to enter the P N S . F o r C N S entry, in vitro e v i d e n c e n o w s u g g e s t s that P N S g l i a d o attract s e n s o r y n e u r o n s to the D R E Z ( G o l d i n g a n d C o h e n , 1997). I n rats, the v e r y t i p o f the D R E Z c o n t a i n s b o u n d a r y c a p cells. T h e s e cells e x p r e s s t h e S c h w a n n cell m a r k e r s Po a n d K r o x - 2 0 a n d are the first cells s e n s o r y n e u r o n s c o n t a c t as they enter the C N S f r o m the p e r i p h e r y ( G o l d i n g a n d C o h e n , 1997).  W h e n cultured D R G sensory  n e u r o n s are g r o w n o n c r y o s e c t i o n s o f d o r s a l s p i n a l c o r d w h i c h i n c l u d e n e r v e r o o t s a n d  17  DPvEZ sites, there is a preferential m i g r a t i o n o f the s e n s o r y n e u r o n s o v e r the b o u n d a r y  cap  c e l l s ( G o l d i n g a n d C o h e n , 1997).  Potential developmental roles of peripheral glia  G i v e n the p h y s i c a l similarities o f p e r i p h e r a l glial d e v e l o p m e n t to that o f S c h w a n n cells, a n d k n o w i n g the g e n e r a l d e v e l o p m e n t a l r o l e s o f g l i a i n o t h e r systems, o n e c a n b u i l d a m o d e l o f p e r i p h e r a l glial r o l e s i n d e v e l o p m e n t .  It w o u l d b e l o g i c a l to h y p o t h e s i z e that  peripheral g l i a g u i d e s e n s o r y n e u r o n s i n t o , a n d m o t o r n e u r o n s o u t of, the C N S . m o t o r n e u r o n s h a v e e x i t e d the C N S ,  Once  they w o u l d u s e other substrates as g u i d a n c e  i n f o r m a t i o n to f i n d their a p p r o p r i a t e m u s c l e targets. T h e g l i a w o u l d p r o b a b l y f o l l o w b e h i n d the l e a d i n g e d g e o f g r o w t h c o n e s as they t h e m s e l v e s m i g r a t e to the p e r i p h e r y . D u r i n g the m i g r a t i o n s , i t w o u l d b e p o s s i b l e that b o t h n e u r o n s a n d the g l i a p r o v i d e t r o p h i c s u p p o r t f o r e a c h other. B y the e n d o f m i g r a t i o n , g l i a w o u l d w r a p p e r i p h e r a l n e r v e s to seal the b l o o d n e r v e barrier. T h e m a i n g o a l o f this thesis has b e e n to assess these h y p o t h e s e s a n d d e t e r m i n e the roles o f p e r i p h e r a l g l i a i n e m b r y o n i c d e v e l o p m e n t o f Drosophila.  T o a c c o m p l i s h this,  p e r i p h e r a l m a r k e r l i n e s w e r e g e n e r a t e d b y c o n v e r t i n g p r e v i o u s l y g e n e r a t e d n u c l e a r lacZ e n h a n c e r trap lines to the GAL4 s y s t e m ( B r a n d a n d P e r r i m o n , 1993) v i a targeted transposition. T h e s u c c e s s o f the t e c h n i q u e s u g g e s t e d that targeted t r a n s p o s i t i o n c o u l d be a general m e a n s o f efficiently c o n v e r t i n g e n h a n c e r traps to the GAL4 s y s t e m .  The  new  GAL4 lines w e r e u s e d to l a b e l the c y t o p l a s m i c p r o c e s s e s o f p e r i p h e r a l g l i a w i t h a tau-lacZ  m a r k e r a n d generate a detailed d e v e l o p m e n t a l p r o f i l e o f the g l i a i n relation to m o t o r a n d sensory neuronal development.  D u r i n g e a r l y d e v e l o p m e n t , p e r i p h e r a l g l i a are situated at  the C N S / P N S b o r d e r a n d are c o n t a c t e d b y s e n s o r y a n d m o t o r n e u r o n s m i g r a t i n g into a n d out o f the C N S . T h e p e r i p h e r a l g l i a m i g r a t e i n t o the P N S a l o n g p r e - e s t a b l i s h e d a x o n tracts w i t h n o glial c y t o p l a s m i c p r o c e s s e s e v e r e x t e n d i n g a h e a d o f p i o n e e r m o t o r n e u r o n g r o w t h  18  c o n e s . T h e r e f o r e , i n a x o n g u i d a n c e p e r i p h e r a l g l i a appear to h a v e a potential intermediate target p h a s e f o l l o w e d b y a f o l l o w e r phase.  G l i a l c e l l b o d i e s are e v e n l y s p a c e d a l o n g  p e r i p h e r a l n e r v e s b y the e n d o f e m b r y o g e n e s i s , s u g g e s t i n g that the p e r i p h e r a l g l i a c o m p e t e a m o n g s t e a c h o t h e r f o r t r o p h i c f a c t o r s e x p r e s s e d b y axons. P e r i p h e r a l glial cell c o v e r a g e o f s e n s o r y n e r v e s is c o m p l e t e b y the e n d o f e m b r y o g e n e s i s , h o w e v e r m a n y m o t o r a x o n b r a n c h t e r m i n i are u n e n s h e a t h e d b y p e r i p h e r a l g l i a . T h i s s u g g e s t s that the b l o o d n e r v e barrier m a y i n c l u d e other cells b e s i d e s p e r i p h e r a l g l i a i n the e m b r y o . T h e d e v e l o p m e n t a l p r o f i l e indicates that p e r i p h e r a l g l i a h a v e m a n y similarities to S c h w a n n cells a n d they are a potentially u s e f u l m o d e l s y s t e m f o r P N S g l i a l d e v e l o p m e n t i n g e n e r a l . T h e i n s i g h t g a i n e d f r o m the a n a t o m i c a l data o f the d e v e l o p m e n t a l p r o f i l e w a s then integrated w i t h f i n d i n g s f r o m a p e r i p h e r a l glial a b l a t i o n study. T o ablate g l i a , the G A L 4 lines w e r e u s e d to e c t o p i c a l l y e x p r e s s genes n e c e s s a r y f o r cell death i n Drosophila. W h e n p e r i p h e r a l g l i a are ablated e a r l y i n d e v e l o p m e n t , a c o m p l e x s e r i e s o f e v e n t s o c c u r . In the C N S , other g l i a appear to c o m p e n s a t e f o r the l o s s o f p e r i p h e r a l g l i a w h i c h are n o r m a l l y f o u n d at the lateral e d g e o f the C N S ,  b y m i g r a t i n g to the r e g i o n o f c e l l death.  This  c o m p e n s a t i o n is l i k e l y not altruistic, as g l i a are k n o w n to c o m p e t e f o r a x o n a l contact. H e n c e the other C N S g l i a c o u l d n o r m a l l y be c o m p e t i n g w i t h p e r i p h e r a l g l i a f o r a x o n a l l y d e r i v e d t r o p h i c factor. P e r i p h e r a l glial a b l a t i o n c a u s e s m o t o r n e u r o n s to stall a n d exit the C N S at a b n o r m a l p l a c e s . H o w e v e r , the o v e r a l l glial rearrangements c a u s e d b y p e r i p h e r a l glial l o s s w o u l d h a v e d i s r u p t e d the o v e r a l l a x o n s c a f f o l d w h i c h c o u l d a l s o c a u s e these effects.  F u r t h e r mutant a n a l y s i s w i l l be n e c e s s a r y to d i s t i n g u i s h b e t w e e n  possibilities.  these t w o  P e r i p h e r a l glial a b l a t i o n has v e r y s t r i k i n g e f f e c t s o n s e n s o r y n e u r o n a l  g u i d a n c e . S e n s o r y n e u r o n s stall, m i g r a t e o n aberrant trajectories, a n d are d e f a s c i c u l a t e d as they a p p r o a c h the C N S i n g l i a l - a b l a t e d e m b r y o s . A x o n s m a y f a i l to enter the C N S o r enter the C N S  at i n c o r r e c t p o s i t i o n s . T h e gem mutant, i n w h i c h m o s t g l i a f a i l to p r o p e r l y  differentiate, was o b s e r v e d to h a v e s e r i o u s d e f e c t s i n s e n s o r y a x o n p r o j e c t i o n i n t o the C N S  19  as w e l l . T o g e t h e r , the data s t r o n g l y s u g g e s t that p e r i p h e r a l g l i a are i m p o r t a n t c u e s i n m e d i a t i n g s e n s o r y a x o n g u i d a n c e into the C N S . '  20  T A R G E T E D TRANSPOSITION IS AN E F F E C T I V E MEANS OF GENERATING GAL4 LINES IN DROSOPHILA. ( C h a p t e r c o n t a i n s d a t a p u b l i s h e d b y S e p p , K. a n d A u l d , V . ( 1 9 9 9 ) i n Genetics)  Introduction  F o r the a n a l y s i s o f p e r i p h e r a l glial d e v e l o p m e n t , it is essential that s t r o n g cellular m a r k e r s are available to a n a l y z e the d e v e l o p m e n t o f g l i a i n b o t h w i l d t y p e a n d mutant b a c k g r o u n d s . A s suitable m a r k e r s f o r p e r i p h e r a l g l i a h a v e n o t b e e n a v a i l a b l e , it w a s first n e c e s s a r y t o generate glial m a r k e r s b y g e n e t i c m e a n s . T h e e n h a n c e r trap t e c h n i q u e i s a w i d e l y u s e d m e t h o d f o r s t u d y i n g tissue s p e c i f i c g e n e e x p r e s s i o n i n Drosophila melanogaster. C l a s s i c a l l y , a P e l e m e n t c o n t a i n i n g the lacZ reporter g e n e u n d e r a m i n i m a l  p r o m o t e r is m o b i l i z e d t h r o u g h o u t the g e n o m e u s i n g a t r a n s p o s a s e s o u r c e . T h e P element c o n s t r u c t is s e n s i t i v e t o the r e g u l a t o r y e l e m e n t s o f the r e g i o n w h i c h it inserts. A s a result, detection o f [3-galactosidase activity b y staining reflects the tissue t y p e a n d t i m i n g o f the e n d o g e n o u s g e n e activity ( O ' K a n e a n d G e h r i n g , 1987; B e l l e n , 1990). M a n y libraries o f e n h a n c e r trap s t a i n i n g patterns h a v e b e e n g e n e r a t e d i n this m a n n e r (e.g. B i e r et al., 1989; B e l l e n et al., 1 9 8 9 ; W i l s o n et al., 1 9 8 9 ; K l a m b t a n d G o o d m a n , 1991). A n a l y s e s o f s u c h e n h a n c e r trap s t o c k s u s i n g m o l e c u l a r t e c h n i q u e s h a v e resulted i n the c l o n i n g o f m a n y n e w genes. A major improvement  to the e n h a n c e r trap t e c h n i q u e e m p l o y s  the G A L 4  transcriptional activator ( B r a n d a n d P e r r i m o n , 1993). I n this s y s t e m , the GAL4 g e n e is i n c l u d e d i n the e n h a n c e r trap P e l e m e n t c o n s t r u c t a n d its e x p r e s s i o n i s u n d e r c o n t r o l o f the local r e g u l a t o r y r e g i o n j u s t l i k e the e x p r e s s i o n o f the lacZ g e n e i n earlier style e n h a n c e r traps. T h e G A L 4 s y s t e m is f a r m o r e f l e x i b l e than earlier lacZ r e p o r t e r s y s t e m s .  GAL4  e n h a n c e r traps c a n b e u s e d to d r i v e the e x p r e s s i o n o f a n y o t h e r g e n e p l a c e d d o w n s t r e a m o f the G A L 4 U p s t r e a m A c t i v a t i o n S e q u e n c e ( U A S ) ( B r a n d a n d P e r r i m o n , 1993). T h e G A L 4 s y s t e m i s b e c o m i n g a s t a n d a r d tool i n Drosophila g e n e t i c a n a l y s e s . H o w e v e r , the a v a i l a b i l i t y o f G A L 4 e n h a n c e r traps is l o w s i n c e the m o s t e x t e n s i v e e n h a n c e r trap libraries c o n t a i n the o r i g i n a l n u c l e a r l o c a l i z e d lacZ. O u r o w n f l y s t o c k s are a g o o d example: p r e v i o u s l y , an e n h a n c e r trap s c r e e n u s i n g the ?[lacZ, ry ] c o n s t r u c t g e n e r a t e d +  22  a p p r o x i m a t e l y 1 0 0 lines s h o w i n g glial s p e c i f i c n u c l e a r staining patterns ( K l a m b t a n d G o o d m a n , 1991), s o m e o f w h i c h w e are currently c h a r a c t e r i z i n g i n the p e r i p h e r a l n e r v o u s s y s t e m ( P N S ) . M a n y o f these l i n e s a l l o w e d the c l o n i n g o f n o v e l genes, s u c h as gliotactin ( A u l d et al., 1995), glial cells missing ( J o n e s et al., 1995), a n d pointed ( K l a e s et a l . , 1994).  T h e y h a v e a l s o b e e n u s e f u l to d e s c r i b e the g e n e r a l s t e r e o t y p i c glial n u c l e a r  p a t t e r n i n g i n the e m b r y o n i c n e r v o u s s y s t e m ( K l a m b t a n d G o o d m a n , 1991). H o w e v e r , the n u c l e a r lacZ e n h a n c e r traps are inadequate f o r p r e c i s e characterization o f P N S  glial  m o r p h o l o g y , w h i c h i s the ultimate g o a l . Instead, a GAL4 c o n t a i n i n g P e l e m e n t at the e x i s t i n g e n h a n c e r trap l o c i w o u l d b e f a r m o r e u s e f u l i n that it w o u l d c o n f e r the ability to e x p r e s s a variety o f m a r k e r s t h r o u g h o u t the entire cellular p r o c e s s e s e n a b l i n g m o r p h o l o g i c a l a n a l y s i s . A l s o , the potential to e c t o p i c a l l y e x p r e s s g e n e s i n the p e r i p h e r a l g l i a w o u l d b e g a i n e d w h i c h c o u l d b e u s e d to h e l p u n d e r s t a n d glial b i o l o g i c a l roles. It w o u l d b e f a r m o r e efficient to c o n v e r t o l d lacZ e n h a n c e r trap lines directly to G A L 4 e n h a n c e r trap lines, rather than repeat a n e n h a n c e r trap s c r e e n f o r G A L 4 lines. M a n y o f the o r i g i n a l lacZ e n h a n c e r trap lines are viable a n d i s o l a t e d b a s e d o n their e x p r e s s i o n pattern w h i c h r e p r e s e n t s a c o n s i d e r a b l e a m o u n t o f w o r k . A l s o , the p G a w B (P[GAL4, miniwhite ]) P e l e m e n t m o b i l i t y i s r e p o r t e d to b e s i g n i f i c a n t l y l o w e r than +  p r e v i o u s lacZ c o n s t r u c t s ( B r a n d a n d P e r r i m o n , 1993; G u s t a f s o n a n d B o u l i a n n e , 1996), h e n c e r e p e a t i n g t h e earlier t y p e s c r e e n w o u l d b e far m o r e l a b o r i n t e n s i v e . A t e c h n i q u e w a s d e s i g n e d b a s e d o n targeted g e n e c o n v e r s i o n ( E n g e l s et al. 1990, 1994; G l o o r et al., 1991; J o h n s o n - S c h l i t z a n d E n g e l s 1993; N a s s i f et al., 1 9 9 4 ) to isolate direct r e p l a c e m e n t s o f o r i g i n a l target PflacZ, rosy ] e l e m e n t s w i t h a d o n o r P[GAL4, w ] e l e m e n t . +  +  T h e results o f t w o i n d e p e n d e n t targeted t r a n s p o s i t i o n s c r e e n s w h i c h s u c c e s s f u l l y c o n v e r t e d t w o glial s p e c i f i c lacZ e n h a n c e r traps to the G A L 4 s y s t e m are p r e s e n t e d here. T h e target P e l e m e n t s w e r e o n the s e c o n d a n d t h i r d c h r o m o s o m e s i n p e r i p h e r a l glial selective genes. T h e d o n o r w a s the P[GAL4, w ] l o c a t e d o n the X c h r o m o s o m e . +  The  f r e q u e n c y o f targeted t r a n s p o s i t i o n is r e p o r t e d to b e h i g h l y d e p e n d e n t o n target a n d d o n o r P  23  e l e m e n t l o c a t i o n ( G l o o r et al., 1991). T a r g e t e d t r a n s p o s i t i o n to the p e r i p h e r a l glial and exit glial e n h a n c e r traps o c c u r r e d at 1.3% and 0 . 4 % w] +  r e s p e c t i v e l y , s u g g e s t i n g that the P[GAL4,  d o n o r is at a l o c u s a m e n a b l e to targeted t r a n s p o s i t i o n . M o l e c u l a r a n a l y s i s o f selected  targeted t r a n s p o s i t i o n lines revealed that the d o n o r P e l e m e n t was c o p i e d p r e c i s e l y to the l o c u s o f the target e n h a n c e r traps and that the entire d o n o r P e l e m e n t s e q u e n c e h a d b e e n r e p a i r e d into these sites. T h e s e data suggest that targeted t r a n s p o s i t i o n c a n be an efficient m e a n s o f c o n v e r t i n g p r e v i o u s l y o b t a i n e d lacZ e n h a n c e r traps to the G A L 4 s y s t e m . F o r the p u r p o s e s o f p e r i p h e r a l glial d e v e l o p m e n t a l a n a l y s i s , the technique  successfully  and  e f f i c i e n t l y generated strong glial m a r k e r s .  24  Materials and Methods  Stocks  O r i g i n a l e n h a n c e r trap s t o c k s r L 8 2 , a n d r Q 2 8 6 u s e d f o r targeted t r a n s p o s i t i o n arose f r o m a P[lacZ, rosy ] ( p P [ P Z ] ) e n h a n c e r trap s c r e e n ( K l a m b t a n d G o o d m a n , 1991). T h e P +  element d o n o r s t o c k P[GAL4, white ]/FM7c ( B r a n d a n d P e r r i m o n , 1993) w a s k i n d l y +  d o n a t e d b y G. B o u l i a n n e . T h e Sp/CyO; SbA2-3/TM6 a n d UAS-lacZ s t o c k s w e r e o b t a i n e d f r o m the B l o o m i n g t o n S t o c k C e n t e r . T h e w; Gla/CyO s t o c k w a s d o n a t e d b y H u g h B r o c k . T h e w; GI/TM3 w a s g e n e r a t e d b y c r o s s i n g the w; Gla/CyO s t o c k to Bl/CyO; GI/TM3 which was obtained f r o m B l o o m i n g t o n Stock Center.  Targeted Transposition Screen  A l l genetic c r o s s e s w e r e p e r f o r m e d at 2 5 ° C o n  standard yeast medium.  P r e l i m i n a r y c r o s s e s w e r e c o n d u c t e d to generate s t o c k s c o n t a i n i n g b o t h target a n d d o n o r P elements f o r the r L 8 2 s e c o n d c h r o m o s o m e p e r i p h e r a l glial e n h a n c e r trap a n d the r Q 2 8 6 third c h r o m o s o m e exit glial e n h a n c e r traps. T h e s e P element s t o c k s w e r e u s e d i n the first generations o f the targeted t r a n s p o s i t i o n s c r e e n s .  T h e r L 8 2 screen consisted of six  generations: I. Sp/CyO; SbA2-3/TM6 m a l e s m a t e d to P[GAL4, w+]/FM6; r L 8 2 ; + v i r g i n f e m a l e s en m a s s e ( f e m a l e s w e r e c h o s e n , as the P[GAL4, w ] i n s e r t i o n i s lethal). +  II. I n d i v i d u a l f e m a l e v i r g i n s o f P[GAL4, w+]/+; rL82/Cy<9; SbA2-3/+ w e r e c o l l e c t e d a n d c r o s s e d to w; Gla/CyO.  III.  A l l f e m a l e v i r g i n s c a r r y i n g the CyO b a l a n c e r b u t l a c k i n g the SbA2-3 c h r o m o s o m e  w e r e c o l l e c t e d a n d m a t e d i n d i v i d u a l l y to w; Gla/CyO males: f e m a l e P[GAL4, w ]/(w o r +  +); r L 8 2 * / C y O ; + X m a l e w; Gla/CyO. r L 8 2 * d e n o t e s a p o s s i b l e t r a n s p o s e d P element chromosome.  25  I V . A l l m a l e s c a r r y i n g the CyO balancer w e r e c o l l e c t e d and m a t e d i n d i v i d u a l l y to w~/w~; GlalCyO v i r g i n females.  M a l e g e n o t y p e s f o r these m a t i n g s c o n s i s t e d o f f o u r p o s s i b i l i t i e s  o f w h i c h the first a n d s e c o n d are m o s t desired, but i n d i s t i n g u i s h a b l e f r o m the others: i) +; rL82*/C><9, i i ) w; r L 8 2 * / C y < 9 , iii) P[GAL4, w ] ; vLS2/CyO, i v ) w; rLS2/CyO. +  V. P r o g e n y f r o m generation I V w e r e s c r e e n e d f o r presence o f eye p i g m e n t a t i o n .  Such  p r o g e n y w e r e selected f o r brother-sister m a t i n g s to m a i n t a i n a stable stock: w~; r L 8 2 * / C y O m a l e s c r o s s e d to w'lw; r L 8 2 * / C y O females.  Alternate g e n o t y p e s c a u s e d b y r a n d o m P  e l e m e n t m o b i l i z a t i o n s to a u t o s o m e s w e r e also p o s s i b l e , but i n d i s t i n g u i s h a b l e f r o m the r L 8 2 * c h r o m o s o m e stocks. V I . A l l resultant s t o c k s w e r e c r o s s e d into a UAS-lacZ b a c k g r o u n d c a r r i e d o n the third c h r o m o s o m e and e m b r y o s w e r e stained f o r final s c r e e n i n g f o r the r L 8 2 * c h r o m o s o m e as represented b y s t a i n i n g o f p e r i p h e r a l g l i a l processes. T h e r Q 2 8 6 e n h a n c e r trap targeted t r a n s p o s i t i o n screen c o n s i s t e d o f six generations: I. Sp/CyO; SbA2-3/TM6 m a l e s c r o s s e d to ?[GAL4, w+]; r Q 2 8 6 v i r g i n f e m a l e s i n a bottle. II. P[GAL4, w ]/+ ; SbA2-3/rQ286 v i r g i n f e m a l e s w e r e c o l l e c t e d a n d m a t e d to w~; +  GIITM3 m a l e s e n m a s s e .  III. A l l m a l e s w e r e c o l l e c t e d except f o r SbA2-3/Gl m a l e s a n d m a t e d to w~/w~; GIITM3 v i r g i n f e m a l e s as s i n g l e p a i r m a t i n g s . I V . S i n g l e p a i r m a t i n g s w e r e s c r e e n e d f o r m a l e flies w i t h p i g m e n t e d eyes, i n d i c a t i n g a m o b i l i z a t i o n o f the P[GAL4, w ] t r a n s p o s i t i o n to an a u t o s o m e . T h e s e m a l e s w e r e m a t e d to +  v i r g i n f e m a l e sisters w i t h s i m i l a r e y e p i g m e n t f o r a stable stock: w; rQ286*/TM3 m a l e s X wlw;  vQ2S6*/TM3 females.  V . A l l s u c h s t o c k s w e r e c r o s s e d into a UAS-lacZ  b a c k g r o u n d a n d s c r e e n e d f o r s t a i n i n g o f e x i t g l i a l processes.  Embryo Staining  E x p r e s s i o n o f (3-galactosidase i n e m b r y o s was  detected w i t h  X - G a l staining  ( K l a m b t et al., 1991) a n d H R P i m m u n o h i s t o c h e m i s t r y (Patel et al., 1987). P N S  glia were 26  l a b e l e d w i t h p o l y c l o n a l m o u s e anti-p-galactosidase a n t i b o d y 1:250 ( S i g m a ) a n d s e n s o r y a x o n s w e r e l a b e l e d w i t h m o u s e m o n o c l o n a l a n t i b o d y 2 2 C 1 0 1:10 ( F u j i t a et al., 1 9 8 2 ) . E m b r y o s were cleared in 9 0 % glycerol with P B S , dissected, and photographed.  Southern analysis  G e n o m i c D N A f r o m adult flies w a s p r e p a r e d f r o m t w o c o n t r o l s t o c k s , ii)P[GAL4, w+]/FM7c, t w o parental s t o c k s , iii) r L 8 2  i)white  1118  i v ) r Q 2 8 6 , a n d selected targeted  t r a n s p o s i t i o n l i n e s v ) w ; r L 8 2 # 4 v i ) w"; r L 8 2 # 8 vii) w ; r L 8 2 # 2 9 viii) w"; r Q 2 8 6 # 2 , i x ) w"; r Q 2 8 6 # 5 . A total o f 10 u g o f g e n o m i c D N A w a s d i g e s t e d w i t h Pst 1 a n d b l o t t e d u s i n g s t a n d a r d t e c h n i q u e s . T h e GALA 3 k b c o d i n g s e q u e n c e , a n d the p U A S T v e c t o r ( c o n t a i n i n g the miniwhite* g e n e a n d p l a s m i d s e q u e n c e s ) w e r e u s e d as probes.  PCR analysis  G e n o m i c D N A obtained f o r Southern analysis was also used f o r P C R analysis. O l i g o n u c l e o t i d e p r i m e r s s p e c i f i c f o r the gliotactin g e n e s u r r o u n d i n g the P i n s e r t i o n site w e r e used: r L 8 2 p r i m e r ( 5 ' - C G G G A T C C T G T C T C G C C G A G A G A A G G C a n d R N A II p r i m e r ( 5 ' - C C T G G C C A A C A A T T C T T T C G T T T G T A T T G A  GC-3') GCG-3').  A s w e l l , p r i m e r s internal t o the G A L 4 e n h a n c e r trap c o n s t r u c t w e r e u s e d : G A L 4 p r i m e r ( 5 - G T C A A T C G A T A C A C T C A A C T G T C T T T G A C C - 3 ' ) f o r the G A L 4 g e n e a n d the reverse p r i m e r (5' - T T C A C A C A G G A A A C A G-3') f o r the p B l u e s c r i p t p l a s m i d .  For  analysis o f exit glial e n h a n c e r trap targeted t r a n s p o s i t i o n s the g e n o m i c r e g i o n p r i m e r s w e r e r Q 2 8 6 5' p r i m e r ( 5 ' - G C C G C A G C T G G G  A A A T G C T G A T G G C G C CCG-3') and  r Q 2 8 6 3' p r i m e r ( 5 ' - C A T G T A T G T T T G A A C T T A C C - 3 ' ) . F o r r L 8 2 P C R a n a l y s i s , a m p l i f i c a t i o n c o n d i t i o n s w e r e 3 0 s at 9 4 °C, 1 m i n at 5 5 °C, 2 m i n 3 0 s at 7 2 °C, 3 0 s at 9 4 °C, 1 m i n at 5 5 "C, 2 m i n 3 0 s at 7 2 °C, t o u c h d o w n f r o m 5 5 ° C b y 2 ° C g o i n g to first step for 3 c y c l e s , 3 0 s at 9 4 ° C (step 9), 1 m i n at 4 9 ° C , 2 m i n 3 0 s at 7 2 ° C , c y c l e 2 6 times to step 9, 5 m i n at 7 2 ° C . F o r r Q 2 8 6 P C R a n a l y s i s , a n a l o g o u s t o u c h d o w n p r o g r a m s w e r e 27  used, b u t u s i n g final a n n e a l i n g temperatures o f 5 1 ° C f o r p a i r s w i t h r Q 2 8 6 3' p r i m e r a n d 6 0 ° C f o r p a i r s w i t h r Q 2 8 6 5' p r i m e r w e r e u s e d . s o l u t i o n s ( P h a r m a c i a ) a n d i n c l u d e d 2.5 m M  A l l reactions contained standard P C R  Mg . 2 +  In situ hybridization to polytene chromosomes  Salivary gland polytene c h r o m o s o m e s were prepared using standard techniques. Preparations were probed with a digoxygenin-11-dUTP  labeled 3 kb G A L 4 fragment and  detected w i t h a n a l k a l i n e p h o s p h a t a s e r e a c t i o n a c c o r d i n g to manufacturer's i n s t r u c t i o n s (Boehringer Mannheim).  28  Results Generation and detection of targeted transposition lines  A p r e v i o u s e n h a n c e r trap s c r e e n g e n e r a t e d a n u m b e r o f lines that s p e c i f i c a l l y label glial c e l l s ( K l a m b t a n d G o o d m a n , 1991). O n e s u c h line, r L 8 2 , c o n t a i n s the lacZ e n h a n c e r trap insert at 3 5 F 1 - 2 i n the gliotactin g e n e o n the s e c o n d c h r o m o s o m e a n d s h o w s selective l a b e l i n g o f the p e r i p h e r a l g l i a ( F i g u r e 2 A , 5 A ) ( A u l d et al., 1995). A n o t h e r line, r Q 2 8 6 , c o n t a i n s the s a m e e n h a n c e r trap P e l e m e n t inserted to 6 3 F 1 - 2 o n the t h i r d c h r o m o s o m e a n d results i n the s t a i n i n g o f the exit g l i a ( F i g u r e s 2 C a n d 5 C ) . T h e exit g l i a are a s u b s e t o f the p e r i p h e r a l g l i a that w r a p p e r i p h e r a l n e r v e r o o t s j u s t distal to the C N S .  A targeted  t r a n s p o s i t i o n s c r e e n w a s c a r r i e d out w i t h the g o a l o f efficiently r e p l a c i n g the o r i g i n a l ("target") r L 8 2 a n d r Q 2 8 6 e n h a n c e r trap P e l e m e n t s w i t h a n e w ("donor") e n h a n c e r trap P e l e m e n t c o n t a i n i n g the G A L 4 gene l o c a t e d at p o s i t i o n 12 o n the X c h r o m o s o m e . T h e b a s i s o f the s c r e e n is o u t l i n e d i n F i g u r e 3. F i r s t , the target P e l e m e n t o f the o r i g i n a l e n h a n c e r trap line i s e x c i s e d i n the p r e s e n c e o f a t r a n s p o s a s e s o u r c e . T h i s w i l l leave a b e h i n d a 10-20 b a s e pair footprint f r o m e a c h i n v e r t e d repeat t e r m i n u s ( J o h n s o n S c h l i t z a n d E n g e l s 1993; T a k a s u - I s h i k a w a et al., 1992; S e a r l e s et al., 1982) w i t h a d o u b l e s t r a n d e d g a p ( G l o o r et al., 1991).  G a p repair m a c h i n e r y c o m p a r e s h o m o l o g y o f the  b r o k e n e n d s to o t h e r r e g i o n s o f the g e n o m e . T h e D N A  repair m a c h i n e r y b y p a s s e s the  h o m o l o g o u s c h r o m o s o m e as a template, b e c a u s e i n this s c r e e n it i s r e p r e s e n t e d b y a m u l t i p l y i n v e r t e d b a l a n c e r c h r o m o s o m e w h i c h interferes w i t h h o m o l o g u e p a i r i n g ( E n g e l s et al., 1990). A m a t c h is m a d e w i t h the i n v e r t e d repeats f o u n d o n the d o n o r P e l e m e n t o n the X c h r o m o s o m e , a n d s u b s e q u e n t l y the d o n o r P e l e m e n t s e q u e n c e acts as a template a n d is r e p a i r e d into the target g a p ( G l o o r et al., 1991). I s o l a t i o n o f targeted t r a n s p o s i t i o n repair e v e n t s o c c u r r e d i n a series o f genetic crosses.  A g e n e r a l c r o s s i n g s c h e m e i s s h o w n i n F i g u r e 4.  F o r o n e g e n e r a t i o n the  f o l l o w i n g e l e m e n t s are r e q u i r e d : transposase, the target P e l e m e n t o v e r a b a l a n c e r  29  Figure 2. Schematic diagram of the enhancer trap P elements involved in targeted transposition and their surrounding genomic regions.  P e l e m e n t termini are indicated as b l a c k boxes. (A) rL82  (PflacZ, ry ]) +  insertion into the 5' r e g i o n o f the g l i o t a c t i n gene.  ( B ) T h e P[GAL4, w ] e l e m e n t i n the o r i g i n a l r L 8 2 insertion site. T h e size a n d p o s i t i o n o f PCR p r o d u c t s g e n e r a t e d u s i n g p r i m e r pairs I a n d II a r e s h o w n ( a r r o w s ) . +  (C)  rQ286  (P[lacZ, ry ]) +  insertion site at 6 3 F o n the t h i r d c h r o m o s o m e .  ( D ) T h e P[GAL4, w ] element w h i c h h a s r e p l a c e d the o r i g i n a l r Q 2 8 6 element. T h e size a n d p o s i t i o n o f the PCR p r o d u c t s g e n e r a t e d b y p r i m e r pairs III a n d rv are indicated (arrows). +  30  2_  £  32  _ S.  lkb i—< _ Si  S.  _ _ °= " & i f f ,  B s_ _  CC CC  2 2  2  21  2  -LLfl'  rosy*  *  2  2  '  F^-H  = f  ^"B-L-  1  D £  £  -LL^ggGAL4li|  2 x ,£  miniwhite+ [  III  £  i f f a f  "  P  _^  ^~  IV  31  Figure 3. Schematic diagram of P element replacement.  T a r g e t lacZ P e l e m e n t e x c i s i o n o n the s e c o n d c h r o m o s o m e i s i n d u c e d b y a s o u r c e o f t r a n s p o s a s e (A2-3 c h r o m o s o m e ) . T h i s results i n a d o u b l e s t r a n d e d D N A g a p w i t h remnants o f P element t e r m i n i o n either s i d e . R e p a i r o f the resultant d o u b l e s t r a n d e d g a p u s i n g the sister c h r o m o s o m e i s p r e v e n t e d b y u s e o f a b a l a n c e r c h r o m o s o m e , CyO. Rather, r e c o g n i t i o n o f h o m o l o g o u s s e q u e n c e at the G A L 4 d o n o r P element t e r m i n i o n the X c h r o m o s o m e l e a d s t o r e p a i r o f the g a p w i t h the d o n o r template.  32  |?[GAL4,  |p[lacZ, ry+] |  w+1  A  2-3  A  2-3  A  2-3  CyO  |P[GAL4,w+]|  1 |  | CyO  j>[GAL4,  wTl  _P[GAL4, w + ] | CyO  Figure 4. Schematic diagram of a crossing scheme for targeted transposition.  T o e x c h a n g e a P e l e m e n t o n the t h i r d c h r o m o s o m e , f e m a l e s w i t h d o n o r P[GAL4, w ] a n d target P[lacZ, ry ] e l e m e n t s are c r o s s e d t o m a l e s w i t h a A2-3, TM3 b a l a n c e r . I n the s e c o n d g e n e r a t i o n , a targeted t r a n s p o s i t i o n e v e n t i s p r o m o t e d as the P e l e m e n t s are i n a b a c k g r o u n d o f A2-3 t r a n s p o s a s e . T o r e d u c e d o u b l e - s t r a n d repair f r o m the sister c h r o m o s o m e the target P e l e m e n t i s p l a c e d o v e r a b a l a n c e r c h r o m o s o m e . F r o m s e c o n d g e n e r a t i o n p r o g e n y , m a l e s are s e l e c t e d that h a v e p i g m e n t e d e y e s . T h e y are c r o s s e d i n single-pair m a t i n g s to a b a l a n c e r s t o c k , then c r o s s e d t o their sisters to generate a set o f +  +  stable a u t o s o m a l P[GAL4, w ] s t o c k s . T h e v a r i o u s p i g m e n t e d e y e lines are c r o s s e d later into a UAS-lacZ b a c k g r o u n d a n d s t a i n e d to v e r i f y tissue-specific e x p r e s s i o n o f G A L 4 . A n a l o g o u s c r o s s e s c a n b e u s e d to e x c h a n g e P e l e m e n t s o n the s e c o n d c h r o m o s o m e . +  34  PJGAL4.W+1; PnacZ.rv+l w~ TM3, Ser  y *  PIGAU. w l: PflacZ. ru+1 y wA2-3, TM3, Sb * +  Cf  w" '• PfGAbi.w+1 TMS.Ser  y  ^  QI &2-3,TM3,Sb  c/  w~; QI TM3,Ser  (f  c5 (s.p.m.)  w :  GI  W  TM3, Ser  xif' TM3,Ser +  I TMJ, Ser  ^  +  35  Figure 5. embryos.  Glial specific enhancer trap staining patterns in Drosophila  G l i a w e r e stained w i t h an anti-b-galactosidase p o l y c l o n a l a n t i b o d y (black) a n d m o t o r n e u r o n s w e r e stained w i t h the 2 2 C 1 0 m o n o c l o n a l a n t i b o d y ( b r o w n ) . A n t i b o d y s t a i n i n g was d e t e c t e d u s i n g h r p i m m u n o h i s t o c h e m i s t r y . ( A ) T h e o r i g i n a l l a c Z e n h a n c e r trap s t a i n i n g pattern o f r L 8 2 s p e c i f i c to the p e r i p h e r a l glial nuclei. ( B ) T h e gliotactin ( r L 8 2 # 2 9 ) G A L 4 e n h a n c e r trap s t a i n i n g pattern. T h e p e r i p h e r a l glial c e l l u l a r p r o c e s s e s are l a b e l e d i n a UAS-lacZ ( n o n - n u c l e a r ) b a c k g r o u n d . ( C ) T h e o r i g i n a l l a c Z e n h a n c e r trap s t a i n i n g pattern o f r Q 2 8 6 i n the e x i t glial n u c l e i . ( D ) T h e r Q 2 8 6 # 2 G A L 4 e n h a n c e r trap s t a i n i n g pattern. T h e exit glial cellular p r o c e s s e s are l a b e l e d i n a UAS-lacZ b a c k g r o u n d .  36  37  c h r o m o s o m e , the d o n o r P element, a n d an appropriate b a c k g r o u n d f o r detection o f m o b i l i z a t i o n (white' i n o u r e x p e r i m e n t s ) . event is capable o f o c c u r r i n g .  In this c o m b i n a t i o n , a targeted t r a n s p o s i t i o n  N e x t , the transposase is c r o s s e d out a n d a s u c c e s s f u l  t r a n s p o s i t i o n event is s e l e c t e d for. T h e flies are c r o s s e d to a stock that m a i n t a i n s the white' b a c k g r o u n d a n d that has a b a l a n c e r f o r the target P e l e m e n t c h r o m o s o m e . T h e presence o f the P[GAL4, w ] is d e t e c t a b l e b y f o l l o w i n g the miniwhite (w ) g e n e o f the d o n o r element. +  +  +  A s the o r i g i n a l P[GAL4, vv+] o n the X c h r o m o s o m e is lethal, m a l e s are s e l e c t e d that d o not h a v e the t r a n s p o s a s e c h r o m o s o m e a n d h a v e p i g m e n t e d eyes, i n d i c a t i n g a m o b i l i z a t i o n o f P[GAL4, w ] to an a u t o s o m e . T h e r e are t w o c l a s s e s o f a u t o s o m a l P[GAL4, w ] lines: +  +  d e s i r e d targeted t r a n s p o s i t i o n l i n e s a n d r a n d o m m o b i l i z a t i o n s o f P[GAL4, w ]. T o +  d i s t i n g u i s h w h i c h l i n e s b e l o n g to a p a r t i c u l a r c l a s s , s t o c k s are generated, c r o s s e d w i t h UAS-lacZ c o n t a i n i n g flies a n d the p r o g e n y are stained to determine  the pattern o f lacZ  expression. T h e c r o s s e s u s e d i n o u r targeted t r a n s p o s i t i o n screen are o u t l i n e d i n the m e t h o d s section. In the screen to replace the P[lacZ, r y ] e l e m e n t in the g l i o t a c t i n g e n e ( r L 8 2 ) , 28 +  lines o f flies w e r e r e c o v e r e d f r o m 693  s i n g l e pair matings,  p h e n o t y p e s , i n d i c a t i v e o f a m o b i l i z a t i o n o f the P[GAL4, w ] +  w h i c h had eye color e l e m e n t to an a u t o s o m e .  A f t e r b e i n g c r o s s e d w i t h a UAS-lacZ b a c k g r o u n d , e m b r y o s w e r e c o l l e c t e d a n d s t a i n e d w i t h X g a l . T h e o r i g i n a l s t a i n i n g pattern o f the P[lacZ, ry ] e l e m e n t i n the g l i o t a c t i n g e n e ( r L 8 2 ) +  l a b e l e d the n u c l e i o f m o s t o f the p e r i p h e r a l g l i a ( F i g u r e 5 A ) . O f the 28 lines r e c o v e r e d , 9 s h o w e d s t a i n i n g i n the identical set o f p e r i p h e r a l g l i a as i n the o r i g i n a l r L 8 2 e n h a n c e r trap line, but w i t h entire c e l l u l a r p r o c e s s e s l a b e l e d s u g g e s t i n g that targeted t r a n s p o s i t i o n had o c c u r r e d ( F i g u r e 5 B ) . T h e r e was n o e x p r e s s i o n o f lacZ i n c o n t r o l e m b r y o s that c o n t a i n e d the glial G A L 4 e n h a n c e r trap l i n e alone (data not s h o w n ) . t r a n s p o s i t i o n to this l o c u s was 1.3%.  T h e f r e q u e n c y o f targeted  O u t o f a l l detectable m o b i l i z a t i o n s o f P[GAL4, w ] +  f r o m the X c h r o m o s o m e to a u t o s o m e s , 3 2 % w e r e targeted t r a n s p o s i t i o n events a n d the r e m a i n d e r w e r e r a n d o m P e l e m e n t insertions.  38  It is k n o w n that the l o c a t i o n o f the d o n o r P element h a s a large i n f l u e n c e o n the f r e q u e n c y o f t r a n s p o s i t i o n to a s i n g l e target l o c u s ( E n g e l s et al., 1990; G l o o r et al., 1991; N a s s i f et al., 1994). W e tested the general e f f e c t i v e n e s s o f the P[GAL4, w ] insert as a +  d o n o r b y c o n d u c t i n g an a n a l o g o u s targeted t r a n s p o s i t i o n screen to c o n v e r t the exit glial line, r Q 2 8 6 , to the G A L 4 s y s t e m . O u t o f 8 0 5 s i n g l e p a i r m a t i n g s , 11 p r o g e n y h a d a p i g m e n t e d e y e p h e n o t y p e . T h e s t a i n i n g pattern o f these l i n e s w e r e then c o m p a r e d to the o r i g i n a l lacZ e n h a n c e r trap pattern ( F i g u r e 5 C , 5 D ) . E x i t glial p r o c e s s e s stained i n 3 o f these l i n e s s u g g e s t i n g that targeted t r a n s p o s i t i o n h a d o c c u r r e d at a f r e q u e n c y o f 0.4%.  In  this screen, o u t o f all detectable m o b i l i z a t i o n s o f P[GAL4, w ] to the t h i r d c h r o m o s o m e , +  2 7 % w e r e targeted t r a n s p o s i t i o n events. T h i s indicates that u s i n g targeted t r a n s p o s i t i o n to r e p l a c e an e x i s t i n g P insert w i t h an G A L 4 e l e m e n t i s an efficient m e a n s o f o b t a i n i n g tissue s p e c i f i c G A L 4 lines.  Molecular verification of targeted transposition events.  T o c o n f i r m that the d o n o r P e l e m e n t h a d b e e n r e p a i r e d into the a p p r o p r i a t e l o c a t i o n p o l y t e n e c h r o m o s o m e s q u a s h e s w e r e c a r r i e d out. P r e p a r a t i o n s f r o m s e l e c t e d targeted transposition lines (rL82#4, rL82#8, and rL82#29), were probed with d i g o x y g e n i n - d U T P l a b e l e d GAL4 D N A  to c o n f i r m that the l o c a t i o n o f the P[GAL4, w+] element.  p r e d i c t e d i n s e r t i o n site w a s the l o c a t i o n o f o r i g i n a l r L 8 2 P e l e m e n t .  However,  The there  r e m a i n e d a f o r m a l p o s s i b i l i t y o f a r a n d o m i n s e r t i o n to another c h r o m o s o m a l r e g i o n w h i c h has the s a m e e x p r e s s i o n pattern as gliotactin. I n all cases, the GAL4 p r o b e l a b e l e d o n l y the 3 5 E 1 - 2 r e g i o n o f the s e c o n d c h r o m o s o m e , w h i c h is the l o c a t i o n o f the o r i g i n a l r L 8 2 P[lacZ, ry ] i n s e r t i o n (data n o t s h o w n ) . +  S i m i l a r l y , the G A L 4 insert i n the r Q 2 8 6 # 2 a n d  r Q 2 8 6 # 5 l i n e s w a s m a p p e d to the o r i g i n a l i n s e r t i o n site o f P[lacZ, ry ] at p o s i t i o n 6 3 F 1 - 2 , +  s u g g e s t i n g that the d o n o r P e l e m e n t h a d r e p a i r e d into the target P e l e m e n t l o c u s o f the exit g l i a e n h a n c e r trap as w e l l (data n o t s h o w n ) .  39  T o d e t e r m i n e the p r e c i s e l o c a t i o n o f P[GAL4, w ] i n s e r t i o n i n the new +  lines  obtained, s m a l l r e g i o n s s p a n n i n g the j u n c t i o n b e t w e e n the g e n e a n d i n s e r t e d P e l e m e n t e n d s w e r e a m p l i f i e d u s i n g P C R ( F i g u r e 6).  F o r the r L 8 2 targeted t r a n s p o s i t i o n l i n e s , external  p r i m e r s s p e c i f i c to the gliotactin g e n e and internal p r i m e r s s p e c i f i c to the P[GAL4, w ] +  e l e m e n t w e r e c h o s e n ( F i g u r e 2 B ) . T h e p r e d i c t e d P C R p r o d u c t s o f 7 3 7 b p f o r the 5' e n d and 548 b p f o r the 3' e n d w e r e obtained f r o m all targeted t r a n s p o s i t i o n lines tested, rL82#4, rL82#8, and rL82#29 (Figure 6A).  F u r t h e r m o r e , n o p r o d u c t s w e r e obtained  f r o m either the o r i g i n a l r L 8 2 (P[lacZ, ry ]) e n h a n c e r trap line or the w +  1118  negative  controls. T o test the p o s s i b i l i t y that the d o n o r P[GAL4, w ] e l e m e n t h a d i n s e r t e d to the target +  r L 8 2 e n h a n c e r trap l o c u s i n an alternate 3' to 5' orientation, the P C R  a n a l y s i s was  repeated  u s i n g the o p p o s i t e p r i m e r pairs to the a b o v e e x p e r i m e n t . N o P C R  p r o d u c t was  obtained  ( F i g u r e 6 A ) , s u g g e s t i n g that the P[GAL4, w ] e l e m e n t has inserted to the r L 8 2 e n h a n c e r +  trap l o c u s i n the 5' to 3' o r i e n t a t i o n . T a k e n together, this e v i d e n c e suggests that the d o n o r P[GAL4, w ] e l e m e n t h a d p a i r e d and d o u b l e stranded D N A +  repair h a d o c c u r r e d p r e c i s e l y  at the i n v e r t e d repeats o f the o r i g i n a l r L 8 2 e n h a n c e r trap line. Analogous PCR  a n a l y s i s was p e r f o r m e d o n r Q 2 8 6 # 2 a n d r Q 2 8 6 # 5 t r a n s p o s i t i o n  lines u s i n g external p r i m e r s s p e c i f i c to the r Q 2 8 6 l o c u s and internal p r i m e r s s p e c i f i c to the P[GAL4, w ] e l e m e n t ( F i g u r e 2 D ) . +  T h e expected lengths w e r e 7 3 3 b p a c r o s s the 5'  i n v e r t e d repeat a n d 383 bp across the 3' i n v e r t e d repeat. B o t h lines g a v e P C R p r o d u c t s o f the e x p e c t e d length and w e r e o r i e n t e d i n the o r i g i n a l 5' to 3' d i r e c t i o n as w e l l ( F i g u r e 6 B ) . T h e r e f o r e , as w i t h the r L 8 2 transpositions, P[GAL4, w ] r e p a i r e d p r e c i s e l y to the o r i g i n a l +  target e n h a n c e r trap locus. F o r targeted t r a n s p o s i t i o n to be an e f f e c t i v e m e a n s o f r e p a i r i n g the P[GAL4, w ] to +  a new locus, the r e p a i r i n v o l v e d m u s t be c o m p l e t e s u c h that the entire G A L 4 p r o t e i n c o d i n g r e g i o n p l u s the miniwhite* eye c o l o r m a r k e r is integrated. T h i s is n e c e s s a r y so that the event can be detected as a m o b i l i z a t i o n o f the miniwhite g e n e to an a u t o s o m e , and +  40  Figure 6.  PCR  analysis of targeted transposition lines.  P C R w a s u s e d to d e t e r m i n e the l o c a t i o n a n d orientation o f G A L 4 P elements i n selected targeted t r a n s p o s i t i o n l i n e s ( d i a g r a m m e d i n F i g u r e 1). ( A ) r L 8 2 targeted t r a n s p o s i t i o n l i n e a n a l y s i s . ( L a n e s : 1-10) O d d n u m b e r e d l a n e s are p r o d u c t s o f p r i m e r pair I, e v e n n u m b e r e d lanes are p r o d u c t s o f p r i m e r pair II. (1, 2) n o D N A ; (3, 4) o r i g i n a l r L 8 2 ; (5, 6) r L 8 2 # 4 ; (7, 8) r L 8 2 # 8 ; (9, 10) r L 8 2 # 2 9 ; (11) P C R p o s i t i v e control; (12) w n e g a t i v e c o n t r o l w i t h p r i m e r pair I. P C R p r o d u c t s o f the e x p e c t e d sizes w e r e o b t a i n e d f o r the 5' P e l e m e n t repeat (737 bp, t o p a r r o w ) a n d f o r the 3' P e l e m e n t repeat (548 bp, b o t t o m arrow). ( L a n e s : 13-20) R e v e r s e d p r i m e r p a i r orientation to test the p o s s i b i l i t y o f a 3' to 5' P element i n s e r t i o n . (13, 14) o r i g i n a l r L 8 2 ; (15, 16) r L 8 2 # 4 ; (17, 18) r L 8 2 # 8 ; (19, 20) r L 8 2 # 2 9 . ( B ) r Q 2 8 6 targeted t r a n s p o s i t i o n l i n e a n a l y s i s . ( L a n e s : 1-8) O d d n u m b e r s are pair III p r o d u c t s , e v e n n u m b e r s are p a i r I V p r o d u c t s . (1, 2) n o D N A ; (3, 4) o r i g i n a l r Q 2 8 6 ; (5, 6) r Q 2 8 6 # 2 ; (7, 8) r Q 2 8 6 # 5 . ( L a n e s : 9-14) R e v e r s e d orientation p r i m e r pairs. (9, 10) o r i g i n a l r Q 2 8 6 ; (11, 12) r Q 2 8 6 # 2 ; (13, 14) r Q 2 8 6 # 5 ; (15) P C R p o s i t i v e c o n t r o l ; (16-19) n i 8 n e g a t i v e c o n t r o l w i t h a l l c o m b i n a t i o n s o f p r i m e r pairs u s e d i n p r e c e d i n g reactions. T h e P C R p r o d u c t s o b t a i n e d w e r e 7 3 3 b p f o r the 5' G A L 4 P e l e m e n t (top a r r o w ) a n d 3 8 3 b p l e n g t h f o r the G A L 4 3' P e l e m e n t ( b o t t o m arrow). 1118  W  41  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20  B  1 2 3 4 5 6 7 8 9  10 111213 14 1516 1718 19  subsequent G A L 4 e x p r e s s i o n w i l l l e a d t o p r o p e r activation o f a n y U A S - d r i v e n  constructs.  P r e v i o u s targeted t r a n s p o s i t i o n s o f l a r g e P e l e m e n t s t o s m a l l e r P e l e m e n t l o c i h a v e resulted in c o m p l e t e a n d e x a c t replacements. H o w e v e r , v a r i o u s types o f aberrant repairs h a v e b e e n n o t e d as w e l l i n c l u d i n g large internal deletions ( G e y e r et al., 1988; H e s l i p a n d H o d g e t t s , 1994; S t a v e l e y et al., 1994; G o n z y - T r e b o u l  et al., 1995; K e e l e r a n d G l o o r , 1997). T o  determine the a m o u n t o f G A L 4 P insert that h a d b e e n c o p i e d into o u r o r i g i n a l r L 8 2 a n d r Q 2 8 6 l o c i , w e p r o b e d g e n o m i c S o u t h e r n b l o t s o f the o r i g i n a l lacZ a n d selected t r a n s p o s e d G A L 4 e n h a n c e r trap lines ( F i g u r e 7). U s i n g a G A L 4 D N A probe, a l l targeted t r a n s p o s i t i o n lines tested h a d b o t h the 1.4 a n d 1.8 k b Pst I b a n d s ( F i g u r e 7 A ) w h i c h indicates that the f u l l l e n g t h G A L 4 s e q u e n c e w a s present. T h e targeted t r a n s p o s i t i o n lines w e r e then p r o b e d w i t h a p l a s m i d c o n t a i n i n g the miniwhite g e n e ( F i g u r e 7 B ) . A l l h a d the e x p e c t e d 4.6 k b +  b a n d c o r r e s p o n d i n g to the miniwhite+ g e n e as w e l l as b a n d s c o r r e s p o n d i n g to p l a s m i d s e q u e n c e s that m a t c h those f o u n d i n the o r i g i n a l P[GAL4, w ]/FM7c line. T h i s suggests +  that i n all the targeted t r a n s p o s i t i o n lines tested the entire length o f the F[GAL4, w ] w a s +  present. T h i s is c o m p a r a b l e t o p r e v i o u s w o r k , w h i c h - r e p o r t e d the exact t r a n s p o s i t i o n o f m a n y k i l o b a s e pairs to target l o c i ( H e s l i p a n d H o d g e t t s , 1994; G o n z y - T r e b o u l K e e l e r a n d G l o o r , 1997). O u r results s h o w that e x c h a n g i n g  et al. 1995;  a larger P element f o r a  s m a l l e r o n e i s p o s s i b l e as o u r o r i g i n a l lacZ targets w e r e 5.9 k b l a r g e r than the G A L 4 donor.  T h e r e f o r e , targeted t r a n s p o s i t i o n c o u l d b e a general m e a n s o f e x c h a n g i n g P  e l e m e n t s t h r o u g h o u t the Drosophila g e n o m e a n d is suitable as a m e a n s o f e x c h a n g i n g larger lacZ type e n h a n c e r trap P e l e m e n t s f o r s m a l l e r G A L 4 P elements.  43  Figure 7 . Southern analysis of targeted transposition lines.  ( A ) A G A L 4 D N A p r o b e r e v e a l s the t w o e x p e c t e d Pst I bands: 1.8 k b ( t o p a r r o w ) a n d 1.4 k b ( b o t t o m a r r o w ) i n the o r i g i n a l P[GAL4, w*]IFM7c d o n o r a n d all targeted t r a n s p o s i t i o n lines ( r L 8 2 # 4 , r L 8 2 # 8 , r L 8 2 # 2 9 , r Q 2 8 6 # 2 , r Q 2 8 6 # 5 ) . N o n e o f t h e c o n t r o l o r o r i g i n a l lacZ lines s h o w G A L 4 bands. ( B ) A miniwhite* p l u s p l a s m i d p r o b e r e v e a l s a 4.6 k b Pst I b a n d c o r r e s p o n d i n g to the miniwhite g e n e i n t h e P[GAL4, w*\IFM7c d o n o r s t o c k a n d all targeted t r a n s p o s i t i o n lines (arrow). T h e r e m a i n i n g b a n d s c o r r e s p o n d t o the p l a s m i d s e q u e n c e s a n d are c o n s i s t e n t w i t h a c o m p l e t e r e p a i r o f the P[GAL4, w ] i n t o t h e target site. +  +  44  <  So  a  % fa  ^  00  M  N 00  N N 00 CO  >—1 u •—J u »—1 u  ts in it  **  00 00 ts ts  s  E  < O fi-  sh  00 12,  j Q<  * (O N ****** N N N 00 00 00  j j j  ts in  ** **  00 00 ts ts  45  Discussion  T o l a b e l c e l l s f o r m o r p h o l o g i c a l characterizations i n both w i l d type a n d m u t a n t analysis, the G A L 4 s y s t e m ( B r a n d a n d P e r r i m o n , 1 9 9 3 ) is v e r y d e s i r a b l e . T i s s u e s p e c i f i c e x p r e s s i o n o f G A L 4 c a n d r i v e a v a r i e t y o f c e l l u l a r m a r k e r s p l a c e d d o w n s t r e a m o f the U p s t r e a m A c t i v a t i o n S e q u e n c e s u c h as UAS-lacZ o r UAS-GFP. T h e r e f o r e , as i n the case o f p e r i p h e r a l glia, e v e n t h o u g h r e l i a b l e antibodies s p e c i f i c to p e r i p h e r a l g l i a d o not exist, w e c a n d e s c r i b e the d e v e l o p m e n t o f p e r i p h e r a l g l i a u s i n g p e r i p h e r a l glial G A L 4 e n h a n c e r trap lines. Later, the p e r i p h e r a l glial G A L 4 lines c a n b e u s e d to generate e c t o p i c e x p r e s s i o n o f a variety o f g e n e s o f interest i n mutational studies. R a n d o m m o b i l i z a t i o n o f G A L 4 P e l e m e n t s a b o u t the g e n o m e h a s p r e v i o u s l y b e e n the o n e o f the m o s t w i d e l y u s e d m e t h o d s t o generate d e s i r e d tissue s p e c i f i c G A L 4 e x p r e s s i o n patterns. H o w e v e r , this t e c h n i q u e is very l a b o u r intensive. T o generate p e r i p h e r a l glial G A L 4 lines to u s e as c e l l u l a r m a r k e r s , w e c h o s e to target the G A L 4 P e l e m e n t to glial g e n e s u s i n g targeted t r a n s p o s i t i o n rather than r e l y o n r a n d o m c h a n c e i n a standard P e l e m e n t m o b i l i z a t i o n screen. Efficiency of targeted transposition  W e f i n d that u s i n g targeted t r a n s p o s i t i o n to e x c h a n g e lacZ w i t h G A L 4 e n h a n c e r trap P e l e m e n t s i s a n e f f i c i e n t m e a n s o f a c q u i r i n g tissue s p e c i f i c g e n e e x p r e s s i o n o f G A L 4 . T h i s m e t h o d h a s b e e n s u c c e s s f u l f o r t w o d i f f e r e n t e n h a n c e r trap l i n e s , the r L 8 2 peripheral glial e n h a n c e r trap o n the p r o x i m a l r e g i o n o f the s e c o n d c h r o m o s o m e left a r m a n d the r Q 2 8 6 exit glial e n h a n c e r trap w h i c h is inserted d i s t a l l y o n the t h i r d c h r o m o s o m e left a r m . F r e q u e n c i e s o f targeted t r a n s p o s i t i o n w e r e 1 . 3 % a n d 0 . 4 % r e s p e c t i v e l y a n d are i n the expected r a n g e b a s e d o n p r e v i o u s studies i n g e n e c o n v e r s i o n u s i n g l o w h o m o l o g y ( D r a y and G l o o r , 1997). T h i s supports earlier e v i d e n c e that d o u b l e strand b r e a k h o m o l o g y searches are e f f i c i e n t w i t h f e w e r than 31 nucleotides o f P e l e m e n t s e q u e n c e ( K e e l e r a n d G l o o r , 1997). 46  F o r o u r p u r p o s e s , targeted t r a n s p o s i t i o n h a s b e e n a s u p e r i o r m e t h o d o f o b t a i n i n g G A L 4 e n h a n c e r trap lines f o r P N S g l i a b e c a u s e the r a n d o m t r a n s p o s i t i o n rate o f p G a w B f r o m the X c h r o m o s o m e to a u t o s o m e s is v e r y low. T h e p G a w B e l e m e n t h a s a m o d i f i e d 5' t e r m i n u s w h i c h is t h o u g h t t o r e d u c e its r a n d o m t r a n s p o s i t i o n rate t o o n l y 1 7 % ( G u s t a f s o n a n d B o u l i a n n e , 1996; B r a n d a n d P e r r i m o n , 1993) c o m p a r e d t o 8 7 % r a n d o m t r a n s p o s i t i o n o f l a c Z P e l e m e n t s ( B i e r et al. 1989; B e l l e n et al. 1989). I n d e e d , f o r b o t h p e r i p h e r a l a n d exit glial targeted t r a n s p o s i t i o n s c r e e n s , o f a l l m o b i l i z a t i o n s o f p G a w B t o a n a u t o s o m e detected v i a miniwhite e x p r e s s i o n , a p p r o x i m a t e l y o n e third w e r e targeted t r a n s p o s i t i o n +  e v e n t s as d e t e c t e d b y X g a l s t a i n i n g . O b t a i n i n g G A L 4 e x p r e s s i o n i n P N S g l i a f r o m a classic e n h a n c e r trap s c r e e n w o u l d h a v e a f r e q u e n c y f a r l o w e r t h a n w h a t w e o b s e r v e d u s i n g targeted t r a n s p o s i t i o n . T a r g e t e d t r a n s p o s i t i o n e v e n t s h a v e b e e n d e s c r i b e d at o t h e r l o c i i n t h e Drosophila g e n o m e . A t the yellow l o c u s , a 0.4 k b P e l e m e n t w a s r e p l a c e d b y a 1.1 k b e l e m e n t ( G e y e r et al., 1988). R e p l a c e m e n t s o f a n o r i g i n a l 0.7 k b target P e l e m e n t insert at t h e vestigial g e n e w i t h a 10 k b a n d 11.9 k b d o n o r P e l e m e n t s h a v e b e e n r e p o r t e d ( H e s l i p a n d H o d g e t t s , 1994; S t a v e l e y et al., 1994). A target 0.8 k b P e l e m e n t at t h e Broad-Complex g e n e h a s b e e n r e p l a c e d b y a 10 k b d o n o r P e l e m e n t ( G o n z y - T r e b o u l et al., 1 9 9 5 ) a n d m o r e recently, an 8 k b d o n o r P e l e m e n t w a s targeted to the 0.6 k b P insert i n the white g e n e i n the mutant strain, white ( K e e l e r a n d G l o o r , 1997). O u r results s h o w that a s m a l l e r P e l e m e n t c a n b e hd  targeted to the l o c u s o f a larger one: b o t h c o n v e r s i o n s o f o u r e n h a n c e r trap lines w e r e a r e p l a c e m e n t o f the target p P [ P Z ] (P[lacZ, ry ]) 17.2 k b e l e m e n t w i t h t h e 11.3 k b p G a w B +  d o n o r element. T h e r e f o r e , targeted t r a n s p o s i t i o n c o u l d b e a g e n e r a l m e a n s o f e x c h a n g i n g P e l e m e n t s t h r o u g h o u t the Drosophila g e n o m e a n d is suitable a s a m e a n s o f e x c h a n g i n g larger lacZ t y p e e n h a n c e r trap P e l e m e n t s f o r s m a l l e r G A L 4 P elements.  47  Factors that affect targeted transposition rates  T h e e f f i c i e n c y o f g e n e c o n v e r s i o n / t a r g e t e d t r a n s p o s i t i o n is v a r i a b l e , a n d d e p e n d s o n the l o c a t i o n o f the d o n o r template ( E n g e l s et al., 1990; G l o o r et al., 1991; E n g e l s et al., 1994; N a s s i f et al., 1994). A s w e l l , g e n e c o n v e r s i o n e f f i c i e n c y i s h o m o l o g y d e p e n d e n t ( D r a y a n d G l o o r , 1997).  D u r i n g targeted t r a n s p o s i t i o n , the a m o u n t o f h o m o l o g y  r e m a i n i n g o f the i n v e r t e d repeat P element e n d s i s a p p r o a c h i n g the v e r y l o w e r limit o f that r e q u i r e d to a c h i e v e a s u c c e s s f u l s e a r c h f o r a d o n o r r e p a i r template. W e p r o p o s e that the G A L 4 insert is a m e n a b l e to targeted t r a n s p o s i t i o n s i n g e n e r a l f o r a n u m b e r o f r e a s o n s . F i r s t , the s m a l l a m o u n t o f P element e n d h o m o l o g y has been s u f f i c i e n t f o r the n u m e r o u s l o c i w h i c h h a v e p r e v i o u s l y e x h i b i t e d targeted t r a n s p o s i t i o n s . S e c o n d , the insertion site o f the d o n o r G A L 4 P e l e m e n t o n the X c h r o m o s o m e has lent itself to s u c c e s s f u l targeted t r a n s p o s i t i o n s to l o n g e r P e l e m e n t targets o n b o t h the s e c o n d a n d third c h r o m o s o m e s . It is v e r y l i k e l y that the p G a w B i n s e r t i o n line u s e d i n o u r e x p e r i m e n t s c o u l d b e w i d e l y u s e d to r e p l a c e m a n y o t h e r e x i s t i n g lacZ e n h a n c e r traps. There may  be other considerations w h i c h may  affect targeted t r a n s p o s i t i o n  f r e q u e n c i e s , e s p e c i a l l y f o r r e p l a c e m e n t s w i t h the G A L 4 insert u s e d i n the s c r e e n r e p o r t e d here. First, w e n o t e d that b o t h o f the o r i g i n a l lacZ e n h a n c e r trap lines w e r e i n sites readily a c c e s s i b l e to P e l e m e n t s . T h e o r i g i n a l e n h a n c e r trap s c r e e n that p r o d u c e d the r L 8 2 a n d r Q 2 8 6 lines h a d m u l t i p l e i n d e p e n d e n t insertions o f P e l e m e n t s to e a c h o f the l o c i . P e r h a p s these r e g i o n s are a l s o m o r e s u s c e p t i b l e to P element e x c h a n g e s as w e l l .  Also, w e  c o n d u c t e d the s c r e e n s at 2 5 ° C , w h i c h m a y p r o m o t e targeted t r a n s p o s i t i o n e v e n t s , as it has b e e n o b s e r v e d that t r a n s p o s a s e has o p t i m a l activity at this temperature ( E n g e l s , 1996). F i n a l l y , w e r e c o g n i z e that m a n y e n h a n c e r traps o f the lacZ n u c l e a r t y p e f r o m earlier s c r e e n s c o n t a i n P e l e m e n t s w h i c h c o n t a i n a P[lacZ, w ] element. In these c a s e s , it w o u l d not be +  p o s s i b l e to s c r e e n f l y s t o c k s initially f o r e y e p i g m e n t a t i o n . R a t h e r , these s c r e e n s c o u l d be c o n d u c t e d u s i n g P C R to detect the d o n o r insert. T h i s a p p r o a c h s h o u l d be f e a s i b l e g i v e n the h i g h rate o f t r a n s p o s i t i o n e v e n t s o b s e r v e d i n this s c r e e n . A l t e r n a t e l y , the c r o s s i n g  48  s c h e m e s c o u l d be c o n d u c t e d u s i n g a P[GAL4, yellow*] insert o n the d o n o r c h r o m o s o m e w h i c h w o u l d b e m o b i l i z e d i n a yellow' b a c k g r o u n d . In c o n c l u s i o n , we h a v e f o u n d targeted t r a n s p o s i t i o n to be an efficient m e a n s o f c o n v e r t i n g o u r o u t m o d e d lacZ e n h a n c e r traps to the GAL4 s y s t e m . W i t h this t e c h n i q u e the GAL4 P e l e m e n t has lent itself to t w o d i f f e r e n t e n h a n c e r trap c o n v e r s i o n s w i t h g o o d e f f i c i e n c y . It is l i k e l y that m a n y other l o c i w i l l efficiently repair target P e l e m e n t s w i t h the GAL4 d o n o r . A s the entire GAL4 d o n o r e l e m e n t r e p a i r e d into b o t h target l o c i , w e  provide  e v i d e n c e that P e l e m e n t e x c h a n g e s m a y be f r o m slightly larger s i z e d P e l e m e n t s to smaller ones. O u r e v i d e n c e suggests that it is m o r e e f f i c i e n t to c o n v e r t e x i s t i n g lacZ e n h a n c e r traps to the GAL4 s y s t e m b y targeted t r a n s p o s i t i o n rather than r e s c r e e n f o r GAL4 e n h a n c e r traps b y r a n d o m m o b i l i z a t i o n . W i t h the new P N S  glial GAL4 e n h a n c e r traps generated i n these  screens, we n o w are a b l e to v i s u a l i z e the m o r p h o l o g y o f these cells u s i n g cellular m a r k e r s s u c h as UAS-lacZ . T h e r e f o r e , a d e v e l o p m e n t a l  p r o f i l e o f w i l d type p e r i p h e r a l glial  d e v e l o p m e n t c a n b e c a r r i e d out. B e y o n d study o f the w i l d type d e v e l o p m e n t , e c t o p i c g e n e e x p r e s s i o n is a l s o p o s s i b l e g i v e n the large n u m b e r o f U A S  s t o c k s that are currently  available. In contrast to o u r o r i g i n a l n u c l e a r s t a i n i n g lacZ e n h a n c e r trap l i n e s , the  new  GAJL4 lines that are d e r i v e d f r o m t h e m c a n l e n d themselves to an i n e x h a u s t i b l e n u m b e r o f future studies as new U A S l i n e s are constructed.  49  III.  D E V E L O P M E N T O F P E R I P H E R A L G L I A IN T H E D R O S O P H I L A EMBRYO  ( C h a p t e r c o n t a i n s d a t a p u b l i s h e d b y S e p p , K., S c h u l t e , J., A u l d , V. ( 2 0 0 0 ) i n Glia)  50  Introduction  In the s t u d y o f d e v e l o p m e n t , a n a t o m i c a l characterizations a r e the f o u n d a t i o n u p o n w h i c h a n u n d e r s t a n d i n g o f b i o l o g i c a l f u n c t i o n s o f v a r i o u s cells is built. B y k n o w i n g the t i m i n g o f cell p r o l i f e r a t i o n s , m i g r a t i o n s , cell-cell c o n t a c t s , a n d the m o r p h o l o g i c a l c h a n g e s cells m a k e as a n o r g a n i s m d e v e l o p s , v a l u a b l e c l u e s about the cells r o l e s c a n b e o b t a i n e d . T h e r e f o r e , b a s e d o n d e s c r i p t i v e s t u d y o f cellular g r o w t h , o n e c a n d e s i g n e x p e r i m e n t s w h i c h w i l l l i k e l y demonstrate the d e v e l o p m e n t a l r o l e s o f a g i v e n cell type. S t u d y o f glial d e v e l o p m e n t i n Drosophila C N S h a s b e e n o n e e x a m p l e o f h o w a n a t o m i c a l characterization c a n l e a d to a n u n d e r s t a n d i n g o f d e v e l o p m e n t a l r o l e s o f a c e l l type. A d e c a d e a g o , the C N S g l i a of Drosophila w e r e first d e s c r i b e d b y J a c o b s a n d G o o d m a n ( 1 9 8 9 ) . S i n c e then, m u c h o f w h a t w a s initially p o s t u l a t e d about glial f u n c t i o n b a s e d o n this w o r k h a s b e e n p r o v e n c o r r e c t b y m u t a n t a n a l y s i s . It w a s o b s e r v e d that glial cells are b o r n e a r l y i n n e u r o g e n e s i s , b e f o r e a x o n o u t g r o w t h o c c u r s . T h e g l i a a p p e a r to f o r m a " s c a f f o l d " o v e r w h i c h a x o n tracts later f o r m . P i o n e e r i n g g r o w t h c o n e s w h i c h e s t a b l i s h the a x o n tracts m a k e c l o s e contact w i t h glia. T h e p r e f e r e n c e o f a x o n s t o migrate stereotypically o v e r c e r t a i n s u b s e t s o f C N S g l i a a l o n g w i t h these other o b s e r v a t i o n s s u g g e s t e d that the g l i a v e r y l i k e l y p r o v i d e g u i d a n c e i n s t r u c t i o n t o g r o w t h cones. Indeed, a n a l y s i s h a s s h o w n that C N S g l i a e x p r e s s i m p o r t a n t m o l e c u l e s s u c h as slit, netrin, a n d commissureless, w h i c h are r e q u i r e d f o r g u i d a n c e o f n e u r o n s to their a p p r o p r i a t e targets ( R o t h b e r g et al., 1990; S e e g e r et al., 1993; H a r r i s et al., 1996; M i t c h e l l et al., 1996).  W h e n a n y o f these m o l e c u l e s are d e f e c t i v e , the a x o n  tracts w h i c h n o r m a l l y t r a v e r s e o v e r the g l i a f a i l to f o r m p r o p e r l y . O v e r a v e r y s h o r t time, Drosophila h a s p r o v e n t o b e a p o w e r f u l m o d e l s y s t e m f o r u n d e r s t a n d i n g the r o l e s o f g l i a i n d e v e l o p m e n t  o f the C N S .  T h e o v e r a l l structural  o r g a n i z a t i o n as w e l l as the m o l e c u l a r cues w h i c h direct m o r p h o g e n e s i s o f the Drosophila C N S h a v e s t r i k i n g similarities to the vertebrate s y s t e m ( r e v i e w e d b y A r e n d t a n d N i i b l e r -  51  Jung, 1999). H o w e v e r , the s i m p l e r g e n e t i c s o f Drosophila w h i c h a l l o w f o r r a p i d isolation o f genes essential f o r d e v e l o p m e n t h a v e not b e e n e x p l o i t e d f o r u n d e r s t a n d i n g glial r o l e s i n the P N S .  T h i s has r e s u l t e d l a r g e l y f r o m a l a c k o f cellular m a r k e r s s p e c i f i c to e m b r y o n i c  p e r i p h e r a l glia.  T h e r e f o r e , m o s t o f w h a t is k n o w n about glial cell f u n c t i o n s i n the  d e v e l o p i n g P N S has been g a i n e d f r o m vertebrate studies. C o u l d Drosophila be a suitable model organism for understanding glia in P N S  development?  F i r s t , it m u s t  be  d e m o n s t r a t e d that there are s i g n i f i c a n t m o r p h o l o g i c a l similarities b e t w e e n Drosophila a n d vertebrates. T o build a foundation for understanding P N S  glial r o l e s i n d e v e l o p m e n t ,  anatomical c h a r a c t e r i z a t i o n o f w i l d type p e r i p h e r a l g l i a o v e r the c o u r s e o f  an  embryogenesis  was p e r f o r m e d . T h e G A L 4 P N S g l i a l e n h a n c e r traps d e s c r i b e d i n C h a p t e r II o f this thesis w e r e u s e d as m a r k e r s w h i c h e n a b l e d v i s u a l i z a t i o n o f the entire glial cellular c y t o p l a s m i c processes. In c o m b i n a t i o n w i t h a n t i b o d y m a r k e r s s p e c i f i c to s e n s o r y a n d m o t o r axons, the g r o w t h o f g l i a i n relation to the n e u r o n s they associate w i t h was o b s e r v e d . developmental  profile  describes  glial  genesis,  developmental  O v e r a l l , the  morphology,  the  e s t a b l i s h m e n t o f transient c e l l u l a r contacts, m i g r a t i o n patterns, a n d the f i n a l extent o f nerve w r a p p i n g i n e m b r y o g e n e s i s . It is s h o w n that e a r l y i n d e v e l o p m e n t i m m a t u r e p e r i p h e r a l glia w h i c h are b o r n i n the C N S  appear to be intermediate targets f o r neurites w h i c h  are  m i g r a t i n g i n t o the p e r i p h e r y . In m i g r a t i o n to the P N S , p e r i p h e r a l g l i a f o l l o w the routes o f these p i o n e e r n e u r o n s .  T h e g l i a preferentially adhere to s e n s o r y a x o n a l p r o j e c t i o n s ,  e x t e n d i n g c y t o p l a s m i c p r o c e s s e s a l o n g t h e m s u c h that b y the e n d o f  embryogenesis  p e r i p h e r a l g l i a l c o v e r a g e o f the s e n s o r y s y s t e m is c o m p l e t e . In contrast, s i g n i f i c a n t lengths o f m o t o r b r a n c h t e r m i n i are u n e n s h e a t h e d  b y p e r i p h e r a l g l i a i n the m a t u r e e m b r y o .  T h r o u g h o u t e m b r y o n i c d e v e l o p m e n t , there appear to be n u m e r o u s similarities b e t w e e n the b e h a v i o u r o f p e r i p h e r a l g l i a a n d vertebrate S c h w a n n c e l l s , w h i c h i n c l u d e structural similarities to the vertebrate C N S / P N S interface a n d the m i g r a t i o n o f g l i a a l o n g n e u r o n a l p i o n e e r e d p a t h w a y s i n t o the P N S .  P e r i p h e r a l g l i a appear to h a v e d y n a m i c a n d d i v e r s e  52  roles a n d their similarities to vertebrate g l i a s u g g e s t that Drosophila m a y i n d e e d serve as a p o w e r f u l t o o l f o r a n a l y s i s o f glial roles i n P N S d e v e l o p m e n t i n the future.  0  53  Materials and Methods Fly Strains  T h e e n h a n c e r trap l i n e s r Q 2 8 6 a n d r L 8 2 w e r e generated i n a standard e n h a n c e r trap s c r e e n ( K l a m b t a n d G o o d m a n , 1991). T h e s e l i n e s express a b - g a l a c t o s i d a s e p r o t e i n that is f u s e d w i t h a n u c l e a r localization s i g n a l . r L 8 2 i s a v i a b l e i n s e r t i o n i n the gliotactin g e n e ( A u l d et al., 1995). T h e insert r Q 2 8 6 is a v i a b l e i n s e r t i o n into a n o v e l p e r i p h e r a l glial g e n e w h i c h is c u r r e n t l y b e i n g characterized. E n h a n c e r trap lines r L 8 2 # 2 9 a n d r Q 2 8 6 # 5 w e r e o b t a i n e d b y c o n v e r t i n g the r L 8 2 a n d r Q 2 8 6 l i n e s to the G A L 4 s y s t e m v i a targeted t r a n s p o s i t i o n ( S e p p a n d A u l d , 1999).  Antibodies  A n t i - b - g a l a c t o s i d a s e (rabbit) w a s p u r c h a s e d f r o m C a p p e l ( I C N P h a r m a c e u t i c a l s , Inc., A u r o r a , O H ) a n d u s e d at 1:1000. T h e anti-repo (rabbit) a n t i b o d y w a s u s e d at 1:200. T h e m o n o c l o n a l a n t i b o d i e s 1 D 4 ( m o u s e a n t i - F a s c i c l i n II) a n d 2 2 C 1 0 ( m o u s e ) ( F u j i t a et a l . , 1982) w e r e u s e d at a 1:2 d i l u t i o n . T h e alkaline p h o s p h a t a s e c o n j u g a t e d goat anti-rabbit (1:1500 d i l u t i o n ) a n d h o r s e r a d i s h p e r o x i d a s e ( h r p ) c o n j u g a t e d goat a n t i - m o u s e (1:300 dilution) w e r e b o t h p u r c h a s e d f r o m J a c k s o n I m m u n o R e s e a r c h L a b o r a t o r i e s , Inc. ( W e s t Grove, PA).  Immunohistochemistry  For brightfield microscopy,  e m b r y o s w e r e f i x e d a n d stained as d e s c r i b e d  p r e v i o u s l y ( K l a m b t et al., 1991; Ito et al., 1995). E m b r y o s o f the n u c l e a r lacZ e n h a n c e r trap l i n e s w e r e s t a i n e d w i t h the anti-b-galactosidase a n t i b o d y . T h e anti-repo a n t i b o d y w a s u s e d to label glial n u c l e i i n w i l d type e m b r y o s . T h e G A L 4 e n h a n c e r trap l i n e s w e r e c r o s s e d to the UAS-tau-lacZ line, e m b r y o s c o l l e c t e d a n d stained u s i n g the anti-bg a l a c t o s i d a s e antibody. A l l e m b r y o s w e r e also d o u b l e stained u s i n g either 1 D 4 o r 2 2 C 1 0 54  m o n o c l o n a l . E m b r y o s w e r e c l e a r e d i n a series o f 5 0 % , 7 0 % , a n d 9 0 % g l y c e r o l ( i n P B S ) and dissected. A l l dissections were mounted with 9 0 % glycerol and v i e w e d with a Zeiss A x i o s k o p microscope. 35 m m slides were m a d e using K o d a c h r o m e I S O 64 f i l m , digitized a n d a s s e m b l e d into f i g u r e s u s i n g A d o b e P h o t o s h o p 4.0.  55  Results  T o g a i n an u n d e r s t a n d i n g o f w h a t f u n c t i o n a l roles P N S g l i a h a v e d u r i n g e m b r y o n i c d e v e l o p m e n t , a d e v e l o p m e n t a l p r o f i l e s p a n n i n g the first a p p e a r a n c e o f g l i a o n w a r d s to the w r a p p i n g o f mature e m b r y o n i c n e r v e s w a s p e r f o r m e d . P e r i p h e r a l glial G A L 4 e n h a n c e r trap lines w e r e u s e d to d r i v e the e x p r e s s i o n o f a UAS-tau-lacZ m a r k e r w h i c h p r o v i d e s detailed l a b e l i n g o f g l i a l c y t o p l a s m i c processes. B y d o u b l e l a b e l i n g g l i a a l o n g w i t h s e n s o r y or m o t o r axons, the selectivity a n d extent o f c e l l c o n t a c t s that p e r i p h e r a l g l i a m a k e o v e r the c o u r s e o f d e v e l o p m e n t w e r e a l s o d i s c e r n e d . F o r s i m p l i c i t y , the s t e r e o t y p i c a l l y patterned a b d o m i n a l s e g m e n t s ( A 2 - A 7 ) are d e s c r i b e d here. P e r i p h e r a l g l i a o f t h o r a c i c s e g m e n t s d e v e l o p i n a h i g h l y a n a l o g o u s m a n n e r , the m a i n d i f f e r e n c e b e i n g that there are f e w e r t h o r a c i c p e r i p h e r a l g l i a c o m p a r e d to a b d o m i n a l s e g m e n t a l n u m b e r s ( d a t a n o t s h o w n ) .  Early Embryonic Peripheral Glial Development  S i n c e glial cells o f the Drosophila C N S are k n o w n to h a v e integral r o l e s i n a x o n g u i d a n c e d u r i n g early n e u r o d e v e l o p m e n t , w e w a n t e d to e x a m i n e w h e t h e r p e r i p h e r a l glia m a y h a v e a n a l o g o u s roles i n the P N S . P r e v i o u s l i n e a g e a n a l y s i s has s h o w n that a l m o s t all p e r i p h e r a l g l i a are b o r n i n the e a r l y e m b r y o n i c C N S . T h e C N S - d e r i v e d p e r i p h e r a l g l i a v a r y in n u m b e r f r o m 6 to 8 a n d arise f r o m n e u r o b l a s t s 1-3 a n d 2-5 ( S c h m i d t et al., 1997). I n the P N S , a s i n g l e i n t e r s e g m e n t a l n e r v e ( I S N ) a s s o c i a t e d p e r i p h e r a l g l i a l c e l l i s b o r n a n d r e m a i n s r o u g h l y i n its initial p o s i t i o n t h r o u g h o u t e m b r y o n i c d e v e l o p m e n t ( H a l t e r et a l . , 1995; C a m p b e l l et al., 1994). G C M  a n d R e p o , the earliest glial s p e c i f i c m a r k e r s , are  d e t e c t e d i n all p e r i p h e r a l g l i a l n u c l e i b y stage 11 (Jones et al., 1995). T w o o f the e n h a n c e r trap lines u s e d i n this s t u d y l a b e l p e r i p h e r a l g l i a b e g i n n i n g at stage 11, h e n c e w e are able to f o l l o w the d e v e l o p m e n t o f p e r i p h e r a l g l i a f r o m their o r i g i n .  O n e s u c h e n h a n c e r trap,  r Q 2 8 6 , is a P[lacZ, ry ] i n s e r t i o n w h i c h labels p e r i p h e r a l glial n u c l e i . T h e o t h e r e n h a n c e r +  56  trap, r Q 2 8 6 # 5 , i s a P[GAL4, w ] +  i n s e r t i o n to the s a m e p o s i t i o n a n d a l l o w s the s t a i n i n g o f  p e r i p h e r a l g l i a l c y t o p l a s m i c processes. D u r i n g stage 12, p e r i p h e r a l g l i a are situated at their b i r t h p l a c e at the n e r v e exit/entry r e g i o n o f the C N S / P N S b o r d e r , w i t h n o a s s o c i a t e d m o t o r o r s e n s o r y n e u r o n s . S o o n after its birth, the a C C n e u r o n , w h i c h p i o n e e r s the intersegmental n e r v e ( I S N ) tract, p r o j e c t s a g r o w t h c o n e directly t o w a r d s the p e r i p h e r a l g l i a a n d m a k e s contact w i t h t h e m . A f t e r this contact a C C s u b s e q u e n t l y m i g r a t e s f u r t h e r i n t o the P N S  ( F i g u r e s 8, 9 A , B ) .  This  a p p r o a c h , t o u c h a n d p a s s o f the n e r v e tract p i o n e e r is stereotypical f o r a l l h e m i s e g m e n t s a n d suggests that the p e r i p h e r a l g l i a act as i n t e r m e d i a t e targets f o r p i o n e e r i n g g r o w t h c o n e s . T h e nature o f the m o l e c u l a r signals c o n t r o l l i n g this interaction has y e t to b e established. A s stage 12 p r o g r e s s e s , p e r i p h e r a l g l i a proliferate u n t i l stage 13 at w h i c h p o i n t 8 to 10 glial cells p e r a b d o m i n a l h e m i s e g m e n t c a n b e c o u n t e d . T h e p e r i p h e r a l glial e n h a n c e r traps a l s o label the s u b p e r i n e u r i a l g l i a (Ito et al., 1995), w h i c h w o u l d a c c o u n t f o r the e v i d e n c e that o n l y u p to 8 C N S - d e r i v e d p e r i p h e r a l glial n u c l e i are e v e r s e e n b y l i n e a g e a n a l y s i s ( S c h m i d t et al., 1997). A t all stages, the l o c a t i o n o f p e r i p h e r a l glial cell b o d i e s (as detected b y the n u c l e a r e n h a n c e r traps) c o r r e s p o n d to R e p o s t a i n i n g patterns, c o n f i r m i n g that the e n h a n c e r traps are i n d e e d glial ( e x a m p l e s h o w n i n F i g u r e 1 0 B , C ) .  D u r i n g the  p r o l i f e r a t i o n stage, the g l i a arrange i n t o a c o n e s h a p e d array at the C N S / P N S b o r d e r . D u r i n g e a r l y n e u r o d e v e l o p m e n t , the p e r i p h e r a l g l i a h a v e a r o u n d e d , c o m p a c t m o r p h o l o g y the a p p e a r a n c e o f n u c l e a r a n d c y t o p l a s m i c l a b e l i n g p r e p a r a t i o n s are a l m o s t identical ( F i g u r e 8A,B).  In i n s t a n c e s w h e r e g l i a f u n c t i o n as g u i d e p o s t cells, the r o u n d e d  immature  m o r p h o l o g y is o f t e n o b s e r v e d i n b o t h vertebrates a n d invertebrates (e.g. S i l v e r et al., 1982; J a c o b s a n d G o o d m a n , 1989). It is d u r i n g stage 13 that the p e r i p h e r a l glial c y t o p l a s m a n d m e m b r a n e p r o c e s s e s b e g i n to e x p a n d ( c o m p a r e F i g u r e s 8 C , 9 A ) .  A x o n s migrating across  the C N S / P N S b o r d e r m a k e e x t e n s i v e g r o w t h c o n e contacts w i t h the g l i a as they a r r i v e at the c o n e array a n d o f t e n their m i g r a t i o n paths appear to a c c o m m o d a t e the glial matrix (Figure 8C). T h e compact glial conduit through which neurites project bears striking  57  Figure 8 .  Early stages of peripheral glial development.  C N S - d e r i v e d p e r i p h e r a l g l i a m a y f u n c t i o n as intermediate targets f o r a x o n o u t g r o w t h into the P N S . ( A ) A t stage 12, glial cells appear o n the lateral e d g e o f the C N S w i t h n o a s s o c i a t e d n e u r o n s ( a r r o w h e a d s ) . S o o n after its birth, the a C C p i o n e e r n e u r o n extends a g r o w t h c o n e ( a r r o w s ) t o w a r d s the glial c e l l s . ( B ) a C C m a k e s g r o w t h c o n e contacts w i t h p e r i p h e r a l g l i a b e f o r e e x i t i n g the C N S ( h i g h e r m a g n i f i c a t i o n at e a r l y stage 13). C y t o p l a s m i c l a b e l i n g o f p e r i p h e r a l g l i a is v e r y s i m i l a r to n u c l e a r l a b e l i n g at the s a m e stage in ( A ) i n d i c a t i n g c o m p a c t cell m o r p h o l o g y . ( C ) T o reach the P N S , m o t o r n e u r o n g r o w t h c o n e s n a v i g a t e b e t w e e n p e r i p h e r a l g l i a w h i c h h a v e p r o l i f e r a t e d a n d a r r a n g e d into a c o n e s h a p e d m a t r i x b y stage 13. W h o l e m o u n t e m b r y o s w e r e s t a i n e d w i t h anti-b-galactosidase e x p r e s s i o n to l a b e l g l i a (blue). m A b 2 2 C 1 0 was u s e d to l a b e l n e u r o n s ( b r o w n ) . P e r i p h e r a l glial n u c l e i w e r e l a b l e d u s i n g the r Q 2 8 6 n u c l e a r e n h a n c e r trap ( A , C ) , glial c y t o p l a s m w a s l a b e l e d u s i n g r Q 2 8 6 # 5 ( B ) . A n t e r i o r is at the top, C N S m i d l i n e is i n the center ( A ) a n d at the l e f t ( B . C ) .  58  59  Figure 9. Migration of peripheral glia in the PNS occurs along preformed neuronal pathways.  G l i a l cells e x t e n d c y t o p l a s m i c p r o c e s s e s w h i l e this m i g r a t i o n o c c u r s . C o n c a v e a r r o w s s h o w d i r e c t i o n o f glial m i g r a t i o n . ( A ) Stage 13. P e r i p h e r a l glia ( a r r o w ) b e g i n to migrate a n d e x t e n d c y t o p l a s m i c p r o c e s s e s a l o n g the I S N m o t o r root. ( B ) L a t e stage 13. P e r i p h e r a l glia e x t e n d a l o n g the I S N , a p p r o a c h i n g the d e v e l o p i n g lateral c h o r d o t o n a l (Ich) o r g a n cell bodies (lower focal plane). The I S N motor axons meet with centrally migrating dorsal s e n s o r y c l u s t e r a x o n s w h i c h c o n t r i b u t e to the anterior fascicle (af). ( C ) Stage 14. V e n t r a l p e r i p h e r a l glia ( v P G , a r r o w h e a d ) migrate into the p e r i p h e r y o n the p o s t e r i o r f a s c i c l e (pf) w h i c h is c o m b i n e d w i t h the S N m o t o r r o o t i n this r e g i o n . ( D ) Stage 15. P N S - d e r i v e d dorsolateral p e r i p h e r a l glia ( d l P G ) e x t e n d a l o n g p r o j e c t i o n s o f the d o r s a l s e n s o r y c l u s t e r . V e n t r a l p e r i p h e r a l glial cell b o d y m i g r a t i o n o n the p o s t e r i o r f a s c i c l e is c o m p l e t e ( a r r o w h e a d , l o w e r f o c a l p l a n e ) . ( E ) E a r l y stage 16. C N S - d e r i v e d p e r i p h e r a l glial c y t o p l a s m i c p r o c e s s e s (arrow) s t i l l r e a c h o n l y as f a r as the lateral r e g i o n o f the P N S . ( F ) Stage 16. G l i a m i g r a t i n g p e r i p h e r a l l y e x t e n d u p to the lateral c h o r d o t o n a l o r g a n (Ich). T h e length o f the u n e n s h e a t h e d n e r v e b e t w e e n p e r i p h e r a l l y the c e n t r a l l y e x t e n d i n g glia is d e c r e a s e d ( c o m p a r e to E ) . T h e v P G cell extends p r o c e s s e s a l o n g the v e n t r a l (v) a n d ventral' (v') s e n s o r y c l u s t e r p r o j e c t i o n s . ( G ) L a t e stage 16. G l i a l c o v e r a g e o f anterior f a s c i c l e / I S N is c o n t i g u o u s as C N S a n d P N S d e r i v e d glial p r o c e s s e s m e e t i n Ich r e g i o n . ( H ) Stage 17. G l i a l w r a p p i n g appears m o r e c o m p a c t i n the late e m b r y o . T h e ventral p e r i p h e r a l g l i a l c e l l ( v P G , a r r o w h e a d ) w r a p s all p r o j e c t i o n s o f v e n t r a l a n d ventral' s e n s o r y clusters. P e r i p h e r a l g l i a (blue) w e r e l a b e l e d w i t h anti-b-galactosidase u s i n g e n h a n c e r traps r Q 2 8 6 # 5 ( A to C ) a n d r L 8 2 # 2 9 ( D to H ) . S e n s o r y s y s t e m n e u r o n s w e r e l a b e l e d w i t h m A b 2 2 C 1 0 ( b r o w n ) . T h e C N S m i d l i n e is to the left, a n t e r i o r is at the top.  60  Figure 10. Nuclear labeling of peripheral glia shows proliferation and movement of cell bodies.  ( A ) S t a g e 13. A glial cell b o d y ( a r r o w ) b e g i n s to migrate into the P N S a l o n g a pree s t a b l i s h e d n e u r o n a l p a t h w a y . (B) S t a g e 13. v P G c e l l b o d y ( a r r o w h e a d ) b e g i n s to migrate into the P N S a l o n g the p o s t e r i o r f a s c i c l e / S N p a t h w a y . C o n c a v e a r r o w indicates d i r e c t i o n o f m i g r a t i o n . ( C ) R e p o a n t i b o d y s t a i n i n g labels almost all glial n u c l e i . Part o f its pattern m a t c h e s p e r i p h e r a l glial e n h a n c e r trap s t a i n i n g as peripheral g l i a are a s m a l l e r subset o f all P N S glial cells ( c o m p a r e to panel B ) . (D) F i n a l l o c a t i o n o f p e r i p h e r a l glial cell b o d i e s in the stage 17 e m b r y o . P e r i p h e r a l g l i a tightly associate w i t h the anterior f a s c i c l e (af) and the p o s t e r i o r f a s c i c l e (pf). T h e p o s i t i o n o f the v P G n u c l e u s ( a r r o w h e a d ) is o n l y slightly variable, as are the other p e r i p h e r a l glial cell b o d i e s . W h o l e m o u n t e m b r y o s w e r e stained w i t h anti-b-galactosidase u s i n g nuclear e n h a n c e r trap lines r Q 2 8 6 ( A , B ) a n d r L 8 2 ( D ) . A n t i - R e p o a n t i b o d y was u s e d to label n u c l e i o f w i l d type e m b r y o s ( C ) . S e n s o r y s y s t e m n e u r o n s are l a b e l e d w i t h m A b 2 2 C 1 0 .  62  s i m i l a r i t y to the vertebrate C N S / P N S v e n t r a l t r a n s i t i o n z o n e ( T Z ) a n d the d o r s a l r o o t entry z o n e ( D R E Z ) ( F r a h e r , 1997; O ' B r i e n et al., 1998; G o l d i n g et al., 1997).  Glial Migration into the  PNS  A n o t h e r m a j o r q u e s t i o n we s o u g h t to a n s w e r in o u r s t u d y was to e x a m i n e p e r i p h e r a l glia migrate into the P N S .  how  D o glial p r o c e s s e s precede m i g r a t i n g g r o w t h c o n e  fronts? W h a t are their p r e f e r r e d m i g r a t i o n a l substrates? C y t o p l a s m i c l a b e l i n g o f p e r i p h e r a l glia has p r o v i d e d great i n s i g h t into glial m i g r a t i o n patterns. W e o b s e r v e that t o w a r d s the e n d o f stage 13, the p e r i p h e r a l glial c o n e s h a p e d f o r m a t i o n at the C N S / P N S b o r d e r l o o s e n s as p e r i p h e r a l glia start their m i g r a t i o n into the P N S .  T h e m i g r a t i o n b e g i n s after the first  m o t o r n e u r o n p i o n e e r s h a v e e x t e n d e d past the g l i a i n t o the p e r i p h e r y . T h e glial cell b o d i e s m o v e p e r i p h e r a l l y w h i l e their c y t o p l a s m i c p r o c e s s e s elongate i n a d v a n c e o f the cell b o d i e s ( c o m p a r e F i g u r e s 9 a n d 10).  P e r i p h e r a l glial p r o c e s s e s are n e v e r o b s e r v e d to extend  b e y o n d the l e a d i n g tip o f a p i o n e e r g r o w t h cone. P N S g l i a are a l s o o b s e r v e d to trail b e h i n d (and not precede) p i o n e e r g r o w t h c o n e s p r o j e c t i n g p e r i p h e r a l l y i n vertebrates ( C a r p e n t e r a n d H o l l y d a y , 1992). It is therefore likely that glia r e q u i r e a x o n s as substrates to migrate into the P N S . B y e x a m i n i n g p e r i p h e r a l glial m i g r a t i o n i n r e l a t i o n to m o t o r o r s e n s o r y a x o n d e v e l o p m e n t , we are able to g a i n i n s i g h t into the p r e f e r r e d m i g r a t i o n a l substrates and p o s s i b l e t r o p h i c d e p e n d e n c i e s o f glia. P e r i p h e r a l glia associate w i t h m A b a x o n a l tracts t h r o u g h o u t all m i g r a t i o n i n e m b r y o g e n e s i s  22C10-positive  ( F i g u r e 9). T h e 2 2 C 1 0 antigen is  e x p r e s s e d o n all s e n s o r y n e u r o n s as w e l l as s o m e m o t o r n e u r o n s a n d its s t a i n i n g pattern in the e m b r y o has b e e n c h a r a c t e r i z e d in detail p r e v i o u s l y ( G h y s e n et al., 1986).  Briefly,  s e n s o r y cell c l u s t e r s in d o r s a l (d) a n d lateral c h o r d o t o n a l (Ich) r e g i o n s p r o j e c t a x o n s centrally i n a c o m m o n anterior f a s c i c l e (af) i n the m a t u r e e m b r y o ( F i g u r e 10D).  In  a d d i t i o n , v e n t r a l a n d ventral' (v a n d v') s e n s o r y c e l l c l u s t e r s p r o j e c t a x o n s c e n t r a l l y a l o n g a c o m m o n p o s t e r i o r f a s c i c l e (pf) ( F i g u r e 10D).  F a s II m o t o r a x o n s t a i n i n g o v e r l a p s w i t h 64  sensory a x o n l a b e l i n g i n the ventral P N S  r e g i o n ( K r u e g e r et al., 1996; D a v i s et al.,  1997)  but is v e r y d i v e r g e n t f o r a l l m o t o r b r a n c h e s t o w a r d s their t e r m i n i . W e f i n d that this o v e r l a p and d i v e r g e n c e is o f s i g n i f i c a n c e i n e m b r y o n i c glial d e v e l o p m e n t . H e n c e w e use the terms anterior f a s c i c l e / I S N a n d p o s t e r i o r f a s c i c l e / S N to refer to c o m b i n e d s e n s o r y / m o t o r tracts, anterior f a s c i c l e a n d p o s t e r i o r f a s c i c l e to refer to p u r e sensory tracts, as w e l l as I S N , I S N b , I S N d , S N a a n d S N c to r e f e r to p u r e m o t o r b r a n c h e s .  Ensheathment of the posterior fascicle/SN.  M i g r a t i o n o f a l l centrally d e r i v e d  peripheral  g l i a d u r i n g stages 13 a n d 14 o c c u r s a l o n g c o m b i n e d sensory a n d m o t o r a x o n p r o j e c t i o n s i n the ventral r e g i o n o f the e m b r y o . T h e g l i a initially associate w i t h the anterior f a s c i c l e / I S N and p o s t e r i o r f a s c i c l e / S N ( F i g u r e 9B,C,  1 1 A , B ) . O n e c e l l , w h i c h w e designate ventral  p e r i p h e r a l glial c e l l ( v P G ) , migrates o n the S N a / p o s t e r i o r f a s c i c l e w h i l e a l l other peripheral g l i a migrate a l o n g the I S N / a n t e r i o r f a s c i c l e ( F i g u r e s 9C-H, 1 0 B , D ) . B y stage 15, the  vPG  s o m a arrives at its final e m b r y o n i c destination ( F i g u r e 9D, 10D) w h i c h is u s u a l l y at the j u n c t i o n o f the ventral a n d ventral' (v and v') s e n s o r y cluster p r o j e c t i o n s .  The  vPG  e x t e n d s p r o c e s s e s a l o n g the v a n d v' c l u s t e r p r o j e c t i o n s and a p p e a r s to h a v e f u l l y w r a p p e d t h e m b y stage 17 ( F i g u r e 12C). T h i s c e l l o n l y reaches part w a y a l o n g the S N a n e r v e b y the e n d o f e m b r y o g e n e s i s ( F i g u r e 11D, 12B).  T h e v P G d o e s not associate w i t h the I S N b ,  I S N d , or S N c m o t o r tracts to any s i g n i f i c a n t extent i n the e m b r y o ( F i g u r e 1 2 A , B ) . Ensheathement of the Anterior Fascicle/ISN. T o establish e n s h e a t h m e n t o f the anterior  f a s c i c l e / I S N , p e r i p h e r a l g l i a l m i g r a t i o n a n d w r a p p i n g o c c u r s i n an a n a l o g o u s m a n n e r to the v P G . F o l l o w i n g m i g r a t i o n o f the a l o n g c o m b i n e d s e n s o r y / m o t o r tracts ( F i g u r e s 9 A to C, 11 A, B ) d u r i n g stages 13 to 15, C N S - d e r i v e d p e r i p h e r a l g l i a a p p e a r to p r e f e r e n t i a l l y e x t e n d their p r o c e s s e s a l o n g s e n s o r y tracts i n r e g i o n s w h e r e the sensory a n d m o t o r b u n d l e s d i v e r g e ( F i g u r e s 9 E to H, 11C,D, 12). T h i s is apparent w h e r e p e r i p h e r a l g l i a e x t e n d a l o n g p r o j e c t i o n s o f the lateral c h o r d o t o n a l o r g a n as w e l l as the d o r s a l s e n s o r y c l u s t e r  65  Figure 11. Migration of peripheral glia into the PNS follows well behind the growing tips of pioneer motorneurons.  ( A ) S t a g e 14. A f t e r its initial contacts w i t h p e r i p h e r a l g l i a i n the C N S at stage 12, the I S N p i o n e e r m i g r a t e s into the P N S w e l l ahead o f the glial cell b o d i e s . ( B ) C y t o p l a s m i c e x t e n s i o n o f g l i a l p r o c e s s e s ( a r r o w ) l a g s b e h i n d the I S N p i o n e e r g r o w t h c o n e s (stage 14). T h e v P G cell ( a r r o w h e a d ) m i g r a t e s a l o n g S N a n e r v e r o o t w h i c h c o m b i n e s w i t h the p o s t e r i o r s e n s o r y f a s c i c l e in this r e g i o n . ( C ) E a r l y stage 16. T h e d l P G cell b e g i n s to e x t e n d c y t o p l a s m i c p r o c e s s e s a l o n g the I S N w h i c h is c o m b i n e d w i t h the a n t e r i o r fascicle s e n s o r y n e u r o n s i n m o r e lateral r e g i o n s o f the h e m i s e g m e n t . T h e v P G cell f u r t h e r extends p r o c e s s e s a l o n g the S N a m o t o r b r a n c h . ( D ) Stage 17. G l i a l c o v e r a g e o f m o t o r tracts is c o n t i g u o u s but n o t c o m p l e t e as s i g n i f i c a n t l e n g t h s o f b r a n c h t e r m i n i are n o t sheathed. T h e f o c a l p l a n e s h o w s S N c a n d S N a m o t o r b r a n c h t e r m i n i n o t w r a p p e d b y p e r i p h e r a l glia. G l i a are s t a i n e d w i t h anti-b-galactosidase ( b l u e ) u s i n g n u c l e a r e n h a n c e r traps r Q 2 8 6 ( A ) a n d c y t o p l a s m i c e n h a n c e r traps r Q 2 8 6 # 5 ( B ) a n d r L 8 2 # 2 9 ( C , D ) . M o t o r n e u r o n s are l a b e l e d u s i n g m A b 1 D 4 ( b r o w n ) . C N S m i d l i n e is to the left, anterior is at the top. C o n c a v e a r r o w s i n d i c a t e d i r e c t i o n s o f glial m i g r a t i o n .  66  67  Figure 12. Peripheral glia preferentially extend processes along sensory neuronal tracts in regions where sensory and motor fascicles diverge during embryogenesis.  ( A ) L a t e stage 16. T h e I S N b m o t o r b r a n c h has n o associated g l i a i n its target m u s c l e r e g i o n (indicated b y asterisk). ( B ) S t a g e 17. T h e m o t o r b r a n c h e s S N a , S N c , a n d I S N d are largely u n e n s h e a t h e d b y peripheral glia. ( C ) L a t e stage 16. T h e v P G cell w r a p s all p r o j e c t i o n s o f the ventral a n d ventral' s e n s o r y o r g a n s . ( D ) S t a g e 16. P e r i p h e r a l glia e x t e n d p r o c e s s e s a l o n g lateral c h o r d o t o n a l o r g a n s e n s o r y p r o j e c t i o n s ( a r r o w ) . ( E ) T h e d l P G tightly associates w i t h the p r o j e c t i o n s e m a n a t i n g f r o m the d o r s a l s e n s o r y o r g a n cluster (d). (F) T h e d l P G has l i t t l e affinity f o r m o t o r tracts i n the d o r s a l r e g i o n . T h i s particular c e l l has e x t e n d e d its centrally directed p r o c e s s parallel to the I S N m o t o r b r a n c h , a l o n g the s e n s o r y n e u r o n s (not labeled). G l i a are l a b e l e d w i t h anti-b-galactosidase u s i n g c y t o p l a s m i c e n h a n c e r trap r L 8 2 # 2 9 . S e n s o r y a n d m o t o r s y s t e m s are l a b e l e d w i t h m A b s 2 2 C 1 0 ( C to E ) a n d 1 D 4 ( A , B , E ) r e s p e c t i v e l y .  68  69  ( F i g u r e 12D,E).  P e r i p h e r a l g l i a e x t e n d f u r t h e r d o r s a l l y until late stage 16 w h e n their  p r o c e s s e s m e e t the ventrally e x t e n d i n g p r o c e s s o f the o n l y P N S - d e r i v e d glial c e l l , w h i c h w e designate the dorsolateral p e r i p h e r a l glial cell ( d l P G ) . T h e d l P G s o m a m o v e s v e r y slightly ventrally d u r i n g e m b r y o n i c d e v e l o p m e n t .  Its c y t o p l a s m i c p r o c e s s e s e x t e n d f r o m  stage 15 o n w a r d b o t h d o r s a l l y a l o n g the anterior fascicle a n d ventrally a l o n g the c o m b i n e d anterior f a s c i c l e / I S N ( F i g u r e 9 D , 1 1 C , D ) . T h e v e n t r a l e x t e n s i o n reaches at least as f a r as the d o r s a l l y m i g r a t i n g C N S d e r i v e d glial p r o c e s s e s ( F i g 9 D - H ) . T h e d l P G is m o s t likely not i n v o l v e d i n e m b r y o n i c m o t o r a x o n g u i d a n c e . M o t o r a x o n s o f t e n r u n parallel to the d l P G a n d its a s s o c i a t e d s e n s o r y n e r v e i n s t e a d o f a l o n g w i t h t h e m ( F i g u r e 1 2 E , F ) .  This  suggests a p r e f e r e n t i a l a f f i n i t y o f p e r i p h e r a l g l i a f o r s e n s o r y a x o n substrates i n the e m b r y o . B y the e n d o f e m b r y o g e n e s i s at stage 17, anterior f a s c i c l e c o v e r a g e b y p e r i p h e r a l glial cells is c o m p l e t e ( F i g u r e 9 H ) . A t this stage, a s i g n i f i c a n t distal p o r t i o n o f the I S N is not c o v e r e d b y these glial cells. H o w e v e r it is p o s s i b l e that n o n R e p o - p o s i t i v e cells m a y p r o v i d e c o v e r a g e o f the d o r s a l m o t o r n e r v e r e g i o n . F o r e x a m p l e , w i t h N o m a r s k i o p t i c s w e are able to detect a persistent t w i s t cell a s s o c i a t i n g w i t h the I S N j u s t d o r s a l to the d l P G (data n o t s h o w n ) . In contrast, f o r a x o n b r a n c h t e r m i n i o f S N a , S N c , I S N b a n d I S N d that are not s h e a t h e d b y p e r i p h e r a l g l i a w e c a n not detect any o t h e r w r a p p i n g cells w i t h N o m a r s k i i n the m a t u r e e m b r y o . S i n c e m o t o r p r o j e c t i o n s h a v e f o r m e d f u n c t i o n a l s y n a p s e s b y the e n d o f e m b r y o g e n e s i s ( D a v i s et al., 1997), o u r o b s e r v a t i o n s s u g g e s t that p e r i p h e r a l g l i a are not directly r e q u i r e d f o r initial g e n e r a t i o n a n d c o n s o l i d a t i o n o f the n e u r o m u s c u l a r junction. F i n a l l y , i n the mature stage 17 e m b r y o , a p o r t i o n o f p e r i p h e r a l glial c y t o p l a s m i c p r o c e s s e s r e m a i n s p a n n i n g the C N S / P N S b o r d e r .  T h i s is s h o w n b y c o m p a r i n g  the  gliotactin n u c l e a r e n h a n c e r trap w i t h the c o r r e s p o n d i n g gliotactin G A L 4 e n h a n c e r trap  staining patterns ( F i g u r e 13). A l t h o u g h p e r i p h e r a l glial cell b o d i e s are l o c a t e d i n the P N S , their p r o c e s s e s e x t e n d i n t o the C N S . T h e g l i a l p r o c e s s e s w r a p the I S N / a f a n d S N / p f n e r v e roots as t h e y c r o s s the C N S / P N S b o u n d a r y . H e n c e p e r i p h e r a l glia, w h i c h b e a r  70  Figure 13.  The CNS/PNS boundary is penetrated by peripheral glia.  ( A ) Stage 17. P e r i p h e r a l glial cell b o d i e s ( a r r o w h e a d s ) are situated i n the P N S and associate w i t h c o m b i n e d I S N a n d S N roots. ( B ) Stage 17. T h e c y t o p l a s m i c p r o c e s s e s o f p e r i p h e r a l glia penetrate into the C N S a l o n g the p e r i p h e r a l nerve roots. G l i a are l a b e l e d w i t h anti-b-galactosidase (blue) u s i n g nuclear e n h a n c e r trap r L 8 2 ( A ) a n d c y t o p l a s m i c e n h a n c e r trap r L 8 2 # 2 9 ( B ) . M o t o r n e u r o n s are l a b e l e d w i t h m A b 1D4.  71  72  m o r p h o l o g i c a l s i m i l a r i t y to the vertebrate D R E Z a n d T Z d u r i n g s e n s o r y a n d m o t o r n e u r o n a l m i g r a t i o n a c r o s s the C N S / P N S b o r d e r , r e m a i n l i n k i n g the C N S a n d P N S i n the m a t u r e embryo.  73  Discussion P e r i p h e r a l g l i a , s e n s o r y n e u r o n s , a n d m o t o r n e u r o n s f o r m the m a j o r cellular c o m p o n e n t s o f the P N S .  Unfortunately, peripheral glia have been omitted f r o m most  p h e n o t y p i c a n a l y s e s o f mutants i n P N S d e v e l o p m e n t b e c a u s e s t r o n g c y t o p l a s m i c m a r k e r s h a v e n o t b e e n a v a i l a b l e . T h u s the a d v a n t a g e s o f Drosophila as a m o d e l o r g a n i s m t o s t u d y p e r i p h e r a l g l i a l roles i n d e v e l o p m e n t h a v e b e e n u n d e r u t i l i z e d . T o o v e r c o m e this, p e r i p h e r a l glial G A L 4 e n h a n c e r traps w e r e g e n e r a t e d a n d u s e d as m a r k e r s t o o b s e r v e glial d e v e l o p m e n t i n the e m b r y o . H e r e , the w i l d t y p e d e v e l o p m e n t o f p e r i p h e r a l g l i a as w e l l as the a s s o c i a t i o n s that the g l i a m a k e w i t h s e n s o r y a n d m o t o r a x o n s a r e d e s c r i b e d . T h i s d e v e l o p m e n t a l p r o f i l e e x a m i n e s g l i a f r o m their o r i g i n i n early n e u r o g e n e s i s t h r o u g h t o the mature e m b r y o where neuronal pathfinding has been established a n d expands o n previous investigations o f p e r i p h e r a l g l i a ( F r e d i e u a n d M a h o w a l d , 1989; K l a m b t a n d G o o d m a n , 1991; N e l s o n a n d L a u g h o n , 1993). T h e results s u g g e s t that there are n u m e r o u s f u n c t i o n a l p h a s e s f o r p e r i p h e r a l glia during embryonic nervous system development.  PNS  g l i a a p p e a r v e r y early i n  n e u r o g e n e s i s a n d are c o n t a c t e d b y p i o n e e r m o t o r n e u r o n g r o w t h c o n e s b e f o r e e x i t i n g the C N S . T h e r e f o r e , p e r i p h e r a l g l i a h a v e the potential to p r o v i d e c o n t a c t m e d i a t e d g u i d a n c e c u e s a n d act as a n i n t e r m e d i a t e target f o r m o t o r a x o n p i o n e e r s . G l i a l cells h a v e b e e n o b s e r v e d t o b e critical i n t e r m e d i a t e targets i n a x o n p a t h f i n d i n g i n m a n y i n s t a n c e s , m o s t n o t a b l y the m i d l i n e g l i a ( K l a m b t et al., 1 9 9 1 ) a n d the m e s o d e r m a l l y d e r i v e d t r a n s v e r s e n e r v e exit g l i a o f Drosophila ( G o r c z y c a et al., 1994), the s e g m e n t b o u n d a r y cell i n g r a s s h o p p e r s ( B a s t i a n i a n d G o o d m a n , 1 9 8 6 ) a n d the great cerebral c o m m i s s u r e s i n m i c e ( S i l v e r et al., 1982). Interestingly d u r i n g this p h a s e , p e r i p h e r a l g l i a are c l u s t e r e d together in a c o n e s h a p e d a r r a y at the C N S / P N S b o r d e r w h i c h b e a r s structural similarity to the vertebrate T Z a n d D R E Z r e g i o n s that are s u g g e s t e d to b e i m p o r t a n t i n a x o n s o r t i n g . T h e T Z i s a d e n s e , f u n n e l s h a p e d c o l l a r c o m p o s e d o f astrocytes, e x t e n d i n g d e e p l y into the 74  s p i n a l c o r d t h r o u g h w h i c h m o t o r a x o n b u n d l e s migrate ( F r a h e r , 1997; O ' B r i e n et a l . , 1998).  T h e D R E Z is a s i m i l a r glial interface o f S c h w a n n cells a n d a s t r o c y t e s t h r o u g h  w h i c h s e n s o r y a x o n s p r o j e c t into the s p i n a l c o r d ( G o l d i n g et al., 1997).  S i m i l a r to  vertebrates, the a n a t o m i c a l o b s e r v a t i o n that a x o n s pass t h r o u g h a glial array a n d b e c o m e r o u t e d a l o n g a particular n e r v e fascicle p r o v i d e s e v i d e n c e that p e r i p h e r a l glia a l s o m a y  be  i n v o l v e d i n a x o n sorting. A f t e r n e r v e tract p i o n e e r s h a v e c r o s s e d the C N S / P N S b o r d e r , the array o f p e r i p h e r a l g l i a b e g i n s to disperse. T h e p e r i p h e r a l glia b e g i n to m i g r a t e i n t o the P N S  along  a x o n a l tracts w h i l e e x t e n d i n g c y t o p l a s m i c p r o c e s s e s b e g i n to w r a p the n e r v e b u n d l e s . G l i a l p r o c e s s e s n e v e r e x t e n d f u r t h e r than the m i g r a t i n g f r o n t o f the p e r i p h e r a l n e r v e g r o w t h c o n e s , p r e s u m a b l y a l l o w i n g n e u r o n a l g r o w t h c o n e s t e e r i n g to o c c u r w i t h o u t i n t e r f e r e n c e . T h i s p h e n o m e n o n is s i m i l a r to o b s e r v a t i o n s that S c h w a n n cell p r e c u r s o r s m i g r a t e into the PNS  a l o n g a x o n a l tracts ( C a r p e n t e r a n d H o l l y d a y ; 1992).  T h e p e r i p h e r a l glial  m o r p h o l o g i c a l c h a n g e s at this time are a n a l o g o u s to the c h a n g e s seen i n s u b s e t s o f  CNS  glia w h i c h initially p r e f i g u r e p a t h w a y s as " u n d i f f e r e n t i a t e d " c o m p a c t g l i a a n d then mature after n e r v e tract g e n e r a t i o n i n t o glial cells that ensheathe a x o n s ( J a c o b s a n d G o o d m a n , 1989). Initially, p e r i p h e r a l glial m i g r a t i o n into the P N S o c c u r s a l o n g m o t o r a x o n b u n d l e s in the v e n t r a l r e g i o n o f e a c h h e m i s e g m e n t . T h e v e n t r a l m o t o r a x o n r o o t s s o o n m e e t centrally p r o j e c t i n g s e n s o r y n e u r o n s c r e a t i n g c o m b i n e d s e n s o r y / m o t o r tracts a l o n g w h i c h the glia c o n t i n u e to migrate. In r e g i o n s w h e r e the m o t o r a x o n tracts d i v e r g e f r o m s e n s o r y tracts, p e r i p h e r a l g l i a p r e f e r e n t i a l l y e x t e n d their p r o c e s s e s a l o n g s e n s o r y tracts. M o t o r a n d s e n s o r y a x o n s e x p r e s s d i f f e r e n t a x o n a l m a r k e r s w h i c h c o u l d c o n f e r s u c h different glial cell affinities.  P e r h a p s s e n s o r y n e u r o n s p r o v i d e t r o p h i c s u p p o r t f o r p e r i p h e r a l glia w h i c h  e m b r y o n i c m o t o r n e u r o n s l a c k . P e r i p h e r a l glial cells are i n c o n s t a n t c o n t a c t w i t h s e n s o r y n e u r o n s as they m i g r a t e as a c h a i n i n t o the P N S , a n d n e v e r leap a h e a d o f o t h e r glia. A l s o , p e r i p h e r a l glial cell b o d i e s are a l w a y s w e l l s p a c e d f r o m each other a l o n g the P N S  nerve  75  tracts b y the e n d o f glial m i g r a t i o n . T h e s e o b s e r v a t i o n s suggest t r o p h i c interactions o c c u r b e t w e e n p e r i p h e r a l g l i a and n e u r o n s i n the e m b r y o to l i m i t the n u m b e r s o f glial cells as has b e e n s h o w n i n the C N S ( S o n n e n f e l d a n d Jacobs, 1995b). In the m a t u r e stage 17 e m b r y o , g l i a extend a n d w r a p all s e n s o r y a x o n a l tracts entirely but leave m a n y m o t o r a x o n b r a n c h e s u n t o u c h e d . P e r h a p s g l i a d o not reach u p to the tips o f m o t o r a x o n s d u r i n g this time as they c o u l d interfere w i t h m u s c l e target r e c o g n i t i o n a n d s y n a p s e c o n s o l i d a t i o n . H o w e v e r , d u r i n g first instar l a r v a l d e v e l o p m e n t , p e r i p h e r a l g l i a c o n t i n u e to extend their c y t o p l a s m i c processes so that they r e a c h t o w a r d s the distal reaches o f m o t o r a x o n s ( S e p p et al., 2 0 0 0 ) .  It w i l l be interesting to d e t e r m i n e  w h e t h e r there is a c h a n g e i n glial t r o p h i c d e p e n d e n c e s u c h that g l i a are h i g h l y attracted to m o t o r n e u r o n s after e m b r y o hatching. In the c o u r s e o f characterization o f peripheral glial d e v e l o p m e n t , it was f o u n d that all peripheral g l i a a p p e a r to d e v e l o p a c c o r d i n g to a s i m i l a r p r o g r a m w i t h the e x c e p t i o n o f two glia, the v e n t r a l a n d lateral p e r i p h e r a l g l i a . T h e d l P G cell is d i s t i n c t i v e i n that it is the o n l y p e r i p h e r a l glial cell b o r n i n the P N S . p e r i p h e r a l glial cell b o r n i n the C N S  T h e v P G cell is u n i q u e i n that it is the o n l y to associate w i t h a n e r v e tract other than the  I S N / a n t e r i o r f a s c i c l e . It is u n n e c e s s a r y to n u m b e r the p e r i p h e r a l g l i a , as their n u m b e r s are slightly v a r i a b l e f r o m s e g m e n t to s e g m e n t a n d since there are no d i s t i n g u i s h i n g qualities a m o n g s t the p e r i p h e r a l glial cells. It is also agreed w i t h H a l t e r et al. ( 1 9 9 5 ) that " e x i t " g l i a are s i m p l y a transient c o n f i g u r a t i o n o f peripheral g l i a b o r n i n the C N S  a n d so a s i m p l e r  n o m e n c l a t u r e w h i c h m a y reflect m o r e the d i f f e r e n c e s i n d i f f e r e n t i a t i o n and/or determination o f these cells is suggested. H e n c e , a l l p e r i p h e r a l g l i a are r e f e r r e d to h e r e b y the s a m e n a m e except f o r the m o r e d i s t i n c t i v e glia, v P G a n d d l P G , w h i c h h a v e p r e v i o u s l y b e e n c a l l e d PG1 and P G 3 respectively. In s u m m a r y , the c h a n g i n g m o r p h o l o g i e s , c e l l n u m b e r s , a n d c e l l - c e l l a s s o c i a t i o n s o f p e r i p h e r a l g l i a d u r i n g e m b r y o n i c a n d l a r v a l d e v e l o p m e n t are n o w  characterized.  o b s e r v a t i o n s indicate that g l i a m a y act as intermediate targets d u r i n g a x o n a l  The  pathfinding  76  early i n n e r v o u s s y s t e m d e v e l o p m e n t .  It is c o n c l u d e d that g l i a m i g r a t e i n t o the P N S v i a  p r e e x i s t i n g n e r v e tracts, a n d preferentially associate w i t h s e n s o r y n e u r o n f a s c i c l e s .  Glia  e x t e n d their c y t o p l a s m i c p r o c e s s e s d u r i n g their m i g r a t i o n s a n d b y the e n d o f e m b r y o g e n e s i s , glial w r a p p i n g o f s e n s o r y a x o n tracts i s c o m p l e t e , h o w e v e r m a n y m o t o r n e u r o n b r a n c h e s are n o t w r a p p e d b y p e r i p h e r a l glia. T h e s t u d y s h o w s that the p e r i p h e r a l g l i a h a v e m a n y qualities s i m i l a r t o vertebrate S c h w a n n cells a n d s u g g e s t s that Drosophila m a y b e a g o o d m o d e l o r g a n i s m to g a i n i n s i g h t i n t o h o w glial cells h e l p t o f o r m the P N S . L a s t l y , a b a s i s f r o m w h i c h m o d e l s o f a x o n g u i d a n c e , t r o p h i c s u p p o r t , a n d glial maturation c a n b e better u n d e r s t o o d i n m e c h a n i s t i c a n a l y s e s o f mutants h a s n o w b e e n p r o v i d e d .  77  ABLATION OF PERIPHERAL  GLIA  Introduction  G l i a l cells h a v e i m p o r t a n t instructive r o l e s i n n e r v o u s s y s t e m d e v e l o p m e n t .  I n the  C N S , g l i a o f t e n prepattern the routes w h i c h neurites s u b s e q u e n t l y travel d u r i n g n e u r o n a l p a t h f i n d i n g p h a s e s . E a r l y g l i a l cell a b l a t i o n s t u d i e s i n b o t h vertebrates a n d invertebrates p r o v i d e d k e y e v i d e n c e w h i c h s u p p o r t e d the glial " b l u e p r i n t " h y p o t h e s i s w h i c h a r o s e f r o m a n a t o m i c a l study o f n e w t e m b r y o g e n e s i s ( S i n g e r et al., 1979). A c o m m o n effect o f early glial a b l a t i o n d u r i n g d e v e l o p m e n t i s a d i s r u p t i o n o f n e u r o n a l m i g r a t i o n w h i c h  would  n o r m a l l y o c c u r o v e r the g l i a ( S i l v e r et al., 1982; B a s t i a n i a n d G o o d m a n , 1986; H i d a l g o et al., 1995; Z h o u et al., 1997; H i d a l g o a n d B o o t h , 2 0 0 0 ) . T h e r e f o r e , these s t u d i e s h a v e all s u p p o r t e d t h e i d e a that g l i a p r o v i d e a n i n s t r u c t i v e b l u e p r i n t n e c e s s a r y f o r n e u r o n a l m i g r a t i o n . A l s o , f u r t h e r m u t a t i o n a l a n a l y s i s h a s n o w p r o v i d e d e v i d e n c e that g l i a e x p r e s s m o l e c u l e s w h i c h n e u r o n s r e c o g n i z e a n d u s e as g u i d a n c e i n f o r m a t i o n ( r e v i e w e d b y A u l d , 1999). In c o n t r a s t t o w h a t is k n o w n about glial r o l e s i n C N S d e v e l o p m e n t , t h e r o l e s o f P N S g l i a i n n e r v o u s s y s t e m d e v e l o p m e n t are p o o r l y u n d e r s t o o d . H o w e v e r , a n u m b e r o f mutant s t u d i e s s u g g e s t that g l i a c o u l d a l s o h a v e i m p o r t a n t r o l e s i n P N S  neuronal  p a t h f i n d i n g as their c o u n t e r p a r t s h a v e i n the C N S . I n Drosophila, m e s o d e r m a l l y d e r i v e d transverse n e r v e exit g l i a p r e f i g u r e the future m i g r a t o r y path o f the t r a n s v e r s e nerve. W h e n the g l i a fail to differentiate i n the tinman m e s o d e r m a l mutant, t r a n s v e r s e n e r v e f o r m a t i o n is s u b s e q u e n t l y d i s r u p t e d ( G o r c z y c a et al., 1994). T h e m e s o d e r m a l t r a n s v e r s e n e r v e exit glia are u n r e l a t e d to the l a r g e set o f a x o n - w r a p p i n g g l i a i n the P N S , the p e r i p h e r a l g l i a , w h i c h are e c t o d e r m a l l y d e r i v e d . F u r t h e r m o r e , p e r i p h e r a l g l i a d o n o t p r e f i g u r e n e u r o n a l m i g r a t o r y pathways i n the periphery.  T h u s the roles o f peripheral glia i n nervous  system  d e v e l o p m e n t are l i k e l y n o t a n a l o g o u s to t r a n s v e r s e n e r v e exit glia. T h e gem m u t a n t o f Drosophila p r o v i d e s s t r o n g e r e v i d e n c e that p e r i p h e r a l g l i a m a y have roles in P N S development.  T h e gem g e n e is e x p r e s s e d i n a l l e c t o d e r m a l l y d e r i v e d 79  g l i a a n d n u l l m u t a n t s s h o w a f a i l u r e o f g l i a t o differentiate. A s a result, p e r i p h e r a l n e r v e p a t h w a y s a p p e a r d i s r u p t e d ( H o s o y a et al., 1995; J o n e s et al., 1995; V i n c e n t et al., 1 9 9 6 ) . It is unclear, h o w e v e r , w h e t h e r the p e r i p h e r a l n e r v e defects are s p e c i f i c a l l y d u e t o a l o s s o f peripheral g l i a s i n c e m o s t other g l i a o f the C N S are lost i n the m u t a n t as w e l l .  From  d e t a i l e d a n a t o m i c a l study ( C h a p t e r III o f this thesis), the p o s s i b i l i t y that p e r i p h e r a l g l i a m a y act as intermediate targets f o r neuronal p a t h f i n d i n g is r e i n f o r c e d . T o d e t e r m i n e roles o f p e r i p h e r a l g l i a i n e m b r y o n i c n e r v o u s s y s t e m d e v e l o p m e n t , an ablation o f p e r i p h e r a l g l i a w a s p e r f o r m e d . A b l a t i o n w a s a c h i e v e d b y u s i n g the G A L 4 7 U A S s y s t e m to d r i v e ectopic e x p r e s s i o n o f g e n e s w h i c h stimulate a p o p t o s i s s e l e c t i v e l y i n peripheral g l i a ( B r a n d a n d P e r r i m o n , 1993; S h i g e n a g a et al., 1997; Z h o u et al., 1 9 9 7 ) . T h e e m b r y o n i c p h e n o t y p e s r e s u l t i n g f r o m glial ablation w e r e c o m p a r e d t o t h e gem to d e t e r m i n e w h i c h aspects o f the gem  mutant  m u t a n t p h e n o t y p e a r e a result o f p e r i p h e r a l glial  loss. In the a b s e n c e o f p e r i p h e r a l glia, n e u r o n a l patterning is i n d e e d d e f e c t i v e . S e n s o r y n e u r o n s h a v e d i f f i c u l t y i n p r o j e c t i n g a x o n s t o the C N S : stalls, aberrant n e r v e trajectories, and f a i l u r e to project a x o n s are all o b s e r v e d .  T h e a x o n patterning at the C N S / P N S  border  is a b n o r m a l i n all peripheral g l i a l - a b l a t e d a n d gem e m b r y o s , s u g g e s t i n g that p e r i p h e r a l glia mediate s e n s o r y a x o n g u i d a n c e across the C N S / P N S border.  A b s e n c e o f p e r i p h e r a l glia  results i n n e r v e d e f a s c i c u l a t i o n as w e l l as a b n o r m a l separation o f the p e r i p h e r a l nerves. T h u s p e r i p h e r a l g l i a are also r e q u i r e d to t i g h t l y b u n d l e p e r i p h e r a l n e r v e fibres. M o t o r a x o n p a t h f i n d i n g is a l s o d i s r u p t e d w h e n peripheral g l i a are lost. M o t o r a x o n s exit t h e C N S at irregular p o s i t i o n s a n d their m i g r a t i o n s a p p e a r delayed. T h e m o t o r a x o n f a s c i c l e s i n mature e m b r y o s are d e f a s c i c u l a t e d a n d m o t o r a x o n patterning is disrupted. T h e s e data s u p p o r t the h y p o t h e s i s that p e r i p h e r a l g l i a are intermediate targets f o r m o t o r a x o n p a t h f i n d i n g into the P N S . O v e r a l l , the o b s e r v a t i o n s presented here suggest that p e r i p h e r a l g l i a are important c o m p o n e n t s o f the d e v e l o p i n g n e r v o u s s y s t e m w h i c h p r o v i d e e n v i r o n m e n t a l  cues which  enable m i g r a t i n g n e u r o n s to m a k e correct p a t h f i n d i n g d e c i s i o n s .  80  Materials and Methods  Fly stocks  G A L 4 e n h a n c e r trap s t o c k s r Q 1 4 # 7 a n d r Q 2 8 6 # 5 w e r e g e n e r a t e d i n targeted t r a n s p o s i t i o n s c r e e n s ( c h a p t e r II o f this thesis).  E n h a n c e r trap lines 2 2 7 - G A L 4 , 3 3 1 -  G A L 4 , 3 5 3 - G A L 4 , 3 5 4 - G A L 4 , and 3 6 9 - G A L 4 were donated by Cahir O'Kane. T r a n s g e n i c UAS-ced-3 s t o c k s ( S h i g e n a g a e t al., 1 9 9 7 ) w e r e a gift f r o m T e i i c h i T a n i m u r a , a n d the UAS-grim s t o c k ( Z h o u et al., 1 9 9 7 ) w a s k i n d l y d o n a t e d b y J o h n N a m b u . glial cells missing  AP]  The  (gem) m u t a n t ( J o n e s et al., 1 9 9 5 ) w a s a gift f r o m C o r e y G o o d m a n .  Antibodies  M o u s e m o n o c l o n a l a n t i b o d i e s 1 D 4 ( G r e n n i n g l o h et al., 1 9 9 1 ) a n d 2 2 C 1 0 ( F u j i t a et al., 1 9 8 2 ) w h i c h r e c o g n i z e e p i t o p e s o n m o t o r n e u r o n s a n d s e n s o r y n e u r o n s r e s p e c t i v e l y w e r e d o n a t e d b y C o r e y G o o d m a n a n d r a i s e d b y M a y D a n g a n d L i n d a M a t s u u c h i . T o label glial n u c l e i , a rabbit a n t i - R e p o a n t i b o d y ( H a l t e r et al., 1 9 9 5 ) d o n a t e d b y S a r b N e r w a s u s e d . T h e m A b 4 9 C 4 ( K o l o d z i e j et al., 1 9 9 5 ) u s e d to label lateral c h o r d o t o n a l o r g a n neurons was donated b y Peter Kolodziej.  A l l p r i m a r y a n t i b o d i e s w e r e u s e d at a 1:2  dilution, e x c e p t f o r the a n t i - R e p o a n t i b o d y w h i c h w a s u s e d at 1:200. A b i o t i n y l a t e d antim o u s e s e c o n d a r y w a s u s e d at 1:300 f o r h r p i m m u n o h i s t o c h e m i s t r y ( V e c t a s t a i n K i t , V e c t o r L a b o r a t o r i e s ) a n d a n alkaline p h o s p h a t a s e anti-rabbit s e c o n d a r y w a s u s e d at 1:1500 (Jackson).  Embryo Staining  E m b r y o s r a i s e d at 2 5 ° C w e r e f i x e d a n d s t a i n e d as d e s c r i b e d p r e v i o u s l y (Ito et al., 1991). E m b r y o s w e r e c l e a r e d i n a series o f 5 0 % , 7 0 % , a n d 9 0 % g l y c e r o l i n P B S , dissected, and photographed o n a Zeiss A x i o s k o p microscope u s i n g F u j i c h r o m e I S O 6 4 T f i l m . S l i d e s w e r e d i g i t i z e d a n d i m a g e s w e r e p r o c e s s e d u s i n g A d o b e P h o t o s h o p 4.0. 81  Results  T o ablate p e r i p h e r a l glia, e c t o p i c e x p r e s s i o n o f genes w h i c h activate p r o g r a m m e d cell death w e r e targeted to the g l i a u s i n g the G A L 4 7 U A S s y s t e m ( B r a n d a n d P e r r i m o n , 1993).  T h e r Q 2 8 6 # 5 e n h a n c e r trap line, generated i n C h a p t e r II a n d c h a r a c t e r i z e d i n  C h a p t e r III, was c h o s e n as the best G A L 4 d r i v e r f o r this p u r p o s e as it is active selectively in the p e r i p h e r a l glial s u b s e t as s o o n as the cells are b o r n ( F i g u r e 4 B ) . F l i e s that c a r r i e d genes w h i c h i n d u c e a p o p t o s i s i n Drosophila, e a c h d o w n s t r e a m o f a U p s t r e a m A c t i v a t i o n S e q u e n c e ( U A S ) , w e r e c r o s s e d w i t h flies c a r r y i n g the r Q 2 8 6 # 5 G A L 4 d r i v e r .  The  resultant e m b r y o s w e r e a n a l y z e d f o r e v i d e n c e o f a p o p t o s i s i n p e r i p h e r a l glia.  The  a p o p t o s i s genes d r i v e n e c t o p i c a l l y i n the p e r i p h e r a l g l i a w e r e UAS-grim a n d UAS-ced-3. Grim is a n o v e l g e n e i n Drosophila w h i c h is t h o u g h t to cooperate w i t h head involution defective (hid) a n d reaper to stimulate a p o p t o s i s ( Z h o u et al., 1997) a n d ced-3 is a C. elegans g e n e w h i c h is s i m i l a r to the c y s t e i n e protease, caspase-1 ( S h i g e n a g a et al., (1997).  L o s s o f p e r i p h e r a l g l i a i n g l i a l - a b l a t e d e m b r y o s was d e t e r m i n e d b y s t a i n i n g w i t h the anti-Repo a n t i b o d y , w h i c h r e c o g n i z e s all e m b r y o n i c glial cells e x c e p t f o r the m i d l i n e glial subset o f the C N S  ( X i o n g et al., 1994; C a m p b e l l et al., 1994; H a l t e r et al., 1995). I n  c o n t r o l e m b r y o s w h i c h c a r r i e d the r Q 2 8 6 # 5 d r i v e r , UAS-grim, o r UAS-ced-3 alone, n e r v o u s s y s t e m p a t t e r n i n g was w i l d type, as d e t e r m i n e d b y l a b e l i n g s e n s o r y m o t o r n e u r o n s , a n d g l i a w i t h 2 2 C 1 0 , 1D4, a n d  neurons,  anti-Repo antibodies respectively. T o  c o n t r o l f o r e x t r a n e o u s G A L 4 e x p r e s s i o n i n other t i s s u e s b e s i d e s p e r i p h e r a l glia, a c o l l e c t i o n o f G A L 4 l i n e s e x p r e s s e d i n p e r i p h e r a l g l i a as w e l l as a v a r i e t y o f other t i s s u e s i n c l u d i n g m u s c l e , n e u r o n s , tracheae, a n d e p i d e r m i s w e r e c r o s s e d to the U A S -  apoptosis  transgene s t o c k s . T h e s e c o n t r o l l i n e s i n c l u d e d l i n e s 2 2 7 - G A L 4 , 3 3 1 - G A L 4 , 3 5 3 - G A L 4 , 3 5 4 - G A L 4 , 3 6 9 - G A L 4 , a n d the r Q 1 4 # 7 G A L 4 w h i c h w a s g e n e r a t e d i n a targeted t r a n s p o s i t i o n s c r e e n ( S e p p a n d A u l d , 1999; C. O ' K a n e , u n p u b l i s h e d data). T h e r Q 1 4 # 7 is a G A L 4 P element i n s e r t i o n i n t o the s a m e g e n e 5 0 0 base p a i r s 3' to r Q 2 8 6 # 5 a n d has a  82  s i m i l a r pattern o f e x p r e s s i o n as r Q 2 8 6 # 5 except it has v e r y l i t t l e e x p r e s s i o n i n p e r i p h e r a l glia. T h e r Q 1 4 # 7 line was u s e d to c o n t r o l f o r potential G A L 4 e x p r e s s i o n p a r t i c u l a r l y i n tracheae and m u s c l e .  C o n t r o l r Q 1 4 # 7 UAS-grim e m b r y o s s h o w e d no i n d i c a t i o n o f  p e r i p h e r a l g l i a l loss, s u g g e s t i n g that the G A L 4 e x p r e s s i o n i n p e r i p h e r a l g l i a was not s t r o n g e n o u g h to c a u s e ablation. In experimental e m b r y o s w h i c h c a r r i e d both the r Q 2 8 6 # 5 p e r i p h e r a l glial G A L 4 d r i v e r p l u s UAS-grim or UAS-ced-3, a s p e c t r u m o f n e r v o u s s y s t e m p h e n o t y p e s observed.  These ranged f r o m disrupted C N S  and P N S  was  n e r v e p a t h w a y s to essentially  n o r m a l . T h e s e v e r i t y o f n e u r o n a l defects m i r r o r e d the extent o f glial ablation w h i c h v a r i e d f r o m c o m p l e t e r e m o v a l to w i l d type. In " n o r m a l " e m b r y o s i n w h i c h R e p o glial s t a i n i n g patterns m a t c h e d w i l d type ( F i g u r e 14A), p e r i p h e r a l glial p r o c e s s e s w e r e a l s o v i s i b l e u s i n g b r i g h t f i e l d m i c r o s c o p y w i t h N o m a r s k i o p t i c s . It was c o n c l u d e d that cell death d i d not o c c u r early e n o u g h to s i g n i f i c a n t l y d i s r u p t the p e r i p h e r a l glial d e v e l o p m e n t i n those embryos.  T h e r Q 2 8 6 # 5 e n h a n c e r trap line has transient e x p r e s s i o n i n d e v e l o p m e n t  b e t w e e n stages 11 to 14.  T h u s i f the delay i n G A L 4 a n d U A S -  apoptosis  gene  transcription is too l o n g , the w i n d o w o f o p p o r t u n i t y f o r ablation is m i s s e d a n d the glial cells w i l l s u r v i v e . G A L 4 - i n d u c e d g e n e e x p r e s s i o n i n a g i v e n tissue c a n be h e t e r o g e n e o u s ( L e e and L u o , 1999), a n d therefore it is l i k e l y that not a l l g l i a e x p e r i e n c e d a h i g h e n o u g h l e v e l o f a p o p t o t i c g e n e e x p r e s s i o n to c a u s e death. In contrast, a l l e m b r y o s w i t h aberrant C N S  and P N S  structures h a d d i s r u p t e d glial  patterns at early e m b r y o n i c stages. In c o n t r o l and w i l d type e m b r y o s the p e r i p h e r a l glia migrated into the P N S  as expected starting at stage 13 ( F i g u r e 4 B ) .  e m b r y o s , the n u c l e i o f the p e r i p h e r a l g l i a r e m a i n e d i n s i d e the C N S  In glial-ablated  (Figure 14C).  A t these  p o s i t i o n s , m u l t i p l e p y k n o t i c n u c l e i c o u l d be o b s e r v e d w h i c h h a d patchy a n d faint R e p o s t a i n i n g . In late stage e m b r y o s , p e r i p h e r a l glial cells w e r e not v i s i b l e w i t h R e p o staining nor w e r e o u t l i n e s o f these c e l l s o b s e r v e d u s i n g N o m a r s k i optics. It was c o n c l u d e d that i n  83  Figure 14.  Ablation of peripheral glia during early neural development.  A b l a t i o n o f p e r i p h e r a l g l i a was a s s e s s e d b y s t a i n i n g e m b r y o s w i t h the a n t i - R e p o glial m a r k e r w h i c h detects all e c t o d e r m a l l y d e r i v e d g l i a l cells. ( A ) W i l d type R e p o p a t t e r n i n g o f glial n u c l e i (blue) i n a stage 14 e m b r y o . ( B ) N u c l e a r lacZ r Q 2 8 6 e n h a n c e r trap l a b e l i n g o f p e r i p h e r a l g l i a (blue) at stage 14. T h e p e r i p h e r a l g l i a are b o r n at the lateral e d g e o f the C N S a n d m i g r a t e i n t o the P N S a l o n g d e v e l o p i n g p e r i p h e r a l n e r v e s ( a r r o w s , l o w e r f o c a l p l a n e i n b r o w n ) . ( C ) A b l a t i o n o f p e r i p h e r a l g l i a u s i n g the r Q 2 8 6 G A L 4 e n h a n c e r trap a n d U A S g r i m transgene r e s u l t s i n e a r l y glial death. N o p e r i p h e r a l g l i a are s e e n i n the P N S a n d d e n s e l y l a b e l e d c l u m p s o f R e p o - p o s i t i v e g l i a are o b s e r v e d at the lateral e d g e o f the C N S ( a p p r o x i m a t e l y stage 14). T h e o v e r a l l repeated cone-shape C N S p r o f i l e is n o l o n g e r u n i f o r m as i n the w i l d type ( c o m p a r e to A ) . S e n s o r y n e u r o n s w e r e l a b e l e d w i t h m A b 2 2 C 1 0 . A n t e r i o r is to the top, C N S is o n the left.  84  85  this p o p u l a t i o n o f the e m b r y o s , a p o p t o t i c c e l l death o c c u r r e d i n the p e r i p h e r a l g l i a a n d  was  not r e s c u e d b y a n y other g l i a l cells. T h e f r e q u e n c y o f glial-ablated e m b r y o s c o u l d be i n c r e a s e d i n t w o w a y s .  First,  c h r o m o s o m a l p o s i t i o n a l effects cause v a r i a t i o n i n p o t e n c y a m o n g s t d i f f e r e n t i n s e r t i o n s o f a s i n g l e UAS- c o n s t r u c t . F o u r d i f f e r e n t i n s e r t i o n s o f UAS-ced-3 w e r e each c r o s s e d to the r Q 2 8 6 # 5 G A L 4 d r i v e r . T h e UAS-ced-3#6-6 i n s e r t i o n p r o d u c e d the h i g h e s t f r e q u e n c y o f g l i a l - a b l a t e d e m b r y o s a n d indeed, this i n s e r t i o n has b e e n r e p o r t e d to generate the strongest a b l a t i o n e f f e c t s u n d e r other G A L 4 d r i v e r s as w e l l ( S h i g e n a g a et al., 1997). S e c o n d , s i n c e the G A L 4 s y s t e m is y e a s t - d e r i v e d , it is m o r e active at h i g h e r temperatures (Ito et a l . , 1995).  In these e x p e r i m e n t s , the f r e q u e n c y o f glial-ablated e m b r y o s i n c r e a s e d as the  temperature the e m b r y o s w e r e i n c u b a t e d at i n c r e a s e d . O n l y e m b r y o s w h i c h w e r e r a i s e d at 2 5 ° C a n d s h o w e d a s i g n i f i c a n t l o s s o f g l i a (to be r e f e r r e d to as "glial-ablated") are d i s c u s s e d h e r e i n . A l s o , s i n c e the ablated g l i a p h e n o t y p e s generated b y grim e x p r e s s i o n w e r e i d e n t i c a l to those generated b y ced-3 e x p r e s s i o n (data not s h o w n ) , o n l y targeted ablation e m b r y o s generated w i t h the UAS-grim s t o c k are d i s c u s s e d f u r t h e r . E f f e c t s o n glial ablation i n r Q 2 8 6 # 5 , UAS-grim e m b r y o s w e r e a l s o c o m p a r e d to the p h e n o t y p e s gcm^F! n u l l m u t a n t e m b r y o s , w h i c h l a c k all e m b r y o n i c  of  g l i a except the m i d l i n e g l i a o f the  C N S ( H o s o y a et al., 1995; J o n e s et al., 1995; V i n c e n t et al., 1996). CNS shortening and dorsal closure are delayed in glial-ablated embryos.  D u r i n g e m b r y o n i c d e v e l o p m e n t , the C N S  shortens as it matures.  e m b r y o s are o f t e n v e r y s i g n i f i c a n t l y d e f e c t i v e i n this C N S w i t h the greatest d e l a y s i n C N S  Glial-ablated  shortening process.  Embryos  s h o r t e n i n g correlate w i t h the m o s t severe p e r i p h e r a l glial  loss. In s u c h e m b r y o s , g l i a entirely f a i l to migrate into the P N S a n d w r a p any p e r i p h e r a l nerves. In m o r e m i l d l y g l i a l - a b l a t e d e m b r y o s , w h e r e o n l y o n e o r t w o s e g m e n t s h a v e glial loss, C N S  s h o r t e n i n g rates appear to be relatively n o r m a l (data not s h o w n ) . I n the glial  gem, repo, a n d prospero m u t a n t s , C N S  c o n d e n s a t i o n is a l s o d i s r u p t e d ( D o e et al., 1991;  86  C a m p b e l l et al., 1994; H o s o y a et al, 1995).  S i n c e glial-ablated e m b r y o s a n d other glial  mutants all s h o w a s i m i l a r C N S s h o r t e n i n g p h e n o m e n o n , a h y p o t h e s i s that g l i a contribute to C N S c o n d e n s a t i o n d u r i n g e m b r y o g e n e s i s is s u p p o r t e d . G l i a l - a b l a t e d e m b r y o s a l s o h a v e d e l a y e d d o r s a l c l o s u r e . In e a r l y n e u r o g e n e s i s (stage 12), the C N S  is adjacent to the e p i d e r m i s w h i c h is f o l d e d o v e r the y o l k . A s the  C N S shortens, s u c h that it rests o n l y o n the v e n t r a l s i d e o f the e m b r y o , the e p i d e r m i s m u s t migrate d o r s a l l y to c o v e r the space p r e v i o u s l y o c c u p i e d b y the f o l d e d - o v e r g e r m b a n d . E v e n t u a l l y , the e p i d e r m i s o n b o t h the left a n d r i g h t s i d e s o f the e m b r y o m i g r a t e s f a r e n o u g h to seal together a l o n g the d o r s a l anterior/posterior a x i s , thus f o r m i n g the d o r s a l c l o s u r e (at stage 15). T h e C N S  m u s t s h o r t e n b e f o r e the e p i d e r m i s c a n migrate to f o r m the d o r s a l  c l o s u r e . T h u s it is v e r y l i k e l y that d o r s a l c l o s u r e d e l a y s i n glial-ablated e m b r y o s are a secondary effect of retarded C N S shortening.  Glial cells compensate for absence ofperipheral glia in glial-ablated embryos.  W h e n p e r i p h e r a l glial cells are ablated e a r l y at a p p r o x i m a t e l y stage 12, a dramatic rearrangement o f all other R e p o - p o s i t i v e g l i a i n the C N S  o c c u r s ( F i g u r e 15). O t h e r glia  w h i c h w e r e not targeted f o r a b l a t i o n appear t o m i g r a t e to the lateral e d g e o f the C N S  where  the p e r i p h e r a l g l i a w o u l d n o r m a l l y be. U p o n c l o s e i n s p e c t i o n , the i m m i g r a n t glial n u c l e i w h i c h stain d a r k l y w i t h a n t i - R e p o i n t e r m i n g l e w i t h p a t c h y a n d f a d e d p y k n o t i c n u c l e i , s u g g e s t i n g that other g l i a l cells o f the C N S m i g r a t e d t o w a r d s the r e g i o n o f a p o p t o t i c g l i a to r e p l a c e the p e r i p h e r a l glia. A s a result, the lateral e d g e o f the C N S  appears to be  expanded  in these e m b r y o s ( F i g u r e 1 5 B , D ) . F u r t h e r m o r e , the l o s s o f o v e r a l l C N S structure appears to be irreparable, as o l d e r e m b r y o s w h i c h h a v e e x p e r i e n c e d severe glial a b l a t i o n d o not r e c o v e r f r o m the r e a r r a n g e m e n t ( F i g u r e 15D). R e p o - p o s i t i v e g l i a appear t o m i g r a t e out o f p o s i t i o n a c c o r d i n g to w h i c h other g l i a h a v e been ablated. T h e c o n t r o l 3 3 1 - G A L 4 UASgrim w h i c h is a c t i v e e a r l y i n p e r i p h e r a l a n d m i d l i n e glia, there is less o f an o v e r a l l f l u x o f  87  Figure 15. ablation.  CNS  glial cell bodies rearrange in response to peripheral glial  R e p o - p o s i t i v e g l i a m i g r a t e f r o m m e d i a l C N S p o s i t i o n s to the lateral e d g e o f the C N S after ablation. ( A ) W i l d type stage 14 R e p o s t a i n i n g pattern (blue) a l o n g w i t h s e n s o r y n e u r o n s ( b r o w n ) . T h e R e p o s t a i n i n g pattern is o r d e r l y and w e l l d i s p e r s e d t h o u g h o u t the C N S . (B) A glial-ablated e m b r y o at a p p r o x i m a t e l y stage 14 s h o w s a lack o f R e p o s t a i n i n g i n m i d l i n e r e g i o n ( c o m p a r e asterisk to A ) a n d v e r y d e n s e l a b e l i n g o f glial n u c l e i at the lateral e d g e o f the C N S c o m p a r e d to w i l d types. C N S g l i a a p p e a r to h a v e m i g r a t e d f r o m m o r e m e d i a l p o s i t i o n s to the lateral r e g i o n s w h e r e cell death was stimulated. ( C ) A 3 3 1 - G A L 4 UASgrim e m b r y o i n w h i c h a p o p t o s i s was stimulated i n b o t h the m i d l i n e a n d p e r i p h e r a l g l i a . T h e r e is less o f a f l u x o f R e p o p o s i t i v e g l i a f r o m the m e d i a l r e g i o n o f l o n g i t u d i n a l g l i a ( a r r o w ) to the lateral C N S e d g e . (D) G A L 4 e x p r e s s i o n is not a l w a y s h i g h e n o u g h to stimulate the apoptotic c e l l death p r o g r a m . S o m e e m b r y o s a p p e a r as m o s a i c s i n w h i c h o n l y s o m e h e m i s e g m e n t s e x p e r i e n c e p e r i p h e r a l glial loss. A n e x a m p l e is s h o w n w h e r e glial loss o n l y o c c u r r e d o n the right h e m i s e g m e n t s . O n the left, l o n g i t u d i n a l g l i a are o n their correct p o s i t i o n s i n a s s o c i a t i o n w i t h the l o n g i t u d i n a l a x o n tracts, o n the right, l o n g i t u d i n a l g l i a a p p e a r to h a v e m i g r a t e d t o w a r d s the lateral e d g e o f the C N S .  88  89  glia to the lateral e d g e o f the C N S .  Instead, s o m e l o n g i t u d i n a l g l i a r e m a i n p r o p e r l y  a s s o c i a t e d w i t h the l o n g i t u d i n a l c o n n e c t i v e s ( F i g u r e 15C).  Tracheal defects occur as a result of glial loss.  T r a c h e a e are a b r a n c h e d n e t w o r k o f h o l l o w epithelial p a s s a g e w a y s t h r o u g h w h i c h o x y g e n f r o m the external e n v i r o n m e n t enters the b o d y c a v i t y i n i n s e c t s .  During  e m b r y o g e n e s i s , the tracheal s y s t e m is generated i n a s e r i e s o f cell m i g r a t i o n s o r i g i n a t i n g f r o m the d o r s o l a t e r a l r e g i o n o f the e m b r y o . In e a c h e m b r y o n i c h e m i s e g m e n t , o n e m a i n tracheal b r a n c h , the g a n g l i o n i c b r a n c h ( G B ) m i g r a t e s a l o n g a c o m p l e x s e r i e s o f n e u r o n a l a n d glial s u b s t r a t a ( E n g l u n d et al., 1999).  A t stage 13, the tracheae initially migrate  ventrally a l o n g s e n s o r y n e u r o n s a r i s i n g f r o m the d o r s a l s e n s o r y cluster. A t stage 14, the m i g r a t i n g tracheae s w i t c h n e u r o n a l m i g r a t i o n substrates b y m o v i n g f r o m the anterior fascicle/intersegmental n e r v e ( a f / I S N ) to the p o s t e r i o r f a s c i c l e / s e g m e n t a l n e r v e ( p f / S N ) . S i n c e the m i g r a t i n g tracheae m a k e contact w i t h p e r i p h e r a l g l i a as they s w i t c h n e r v e p a t h w a y s , it has b e e n p r o p o s e d that p e r i p h e r a l g l i a m a y h a v e a r o l e i n this p r o c e s s ( E n g l u n d et a l , 1999). In g l i a l - a b l a t e d e m b r y o s , the tracheae are d i s o r g a n i z e d as s o o n as they first appear as tracheal p l a c o d e s d u r i n g stage 12. L a t e r , tracheae appear to b e s e v e r e l y s t u n t e d a n d d o not enter the C N S ( F i g u r e 16B). T h e gem m u t a n t has tracheal d e f e c t s , but f a r m o r e subtle than the glial-ablated e m b r y o . In gem  mutants, all m a i n tracheal b r a n c h e s f o r m , a n d the  G B a l w a y s m i g r a t e s i n t o the C N S . H o w e v e r , w i t h their n o r m a l p e r i p h e r a l g l i a l m i g r a t i o n a l substrates m i s s i n g , the G B migrate a l o n g a s s o r t e d aberrant p a t h w a y s s u c h as h a l f w a y b e t w e e n the I S N a n d the S N . O f t e n , the G B f a i l to c r o s s f r o m the a f / I S N to the p f / S N as they w o u l d i n the w i l d t y p e ( F i g u r e 1 6 C ) .  S i n c e the gem  mutant s h o w s tracheal  p a t h f i n d i n g d e f e c t s i n the r e g i o n s w h e r e g l i a w o u l d n o r m a l l y o c c u r , it is c o n c l u d e d that p e r i p h e r a l g l i a participate i n the g u i d a n c e o f tracheal cells to the C N S .  90  Figure 16.  Effects of peripheral glial ablation on tracheal development.  P e r i p h e r a l g l i a p o s s i b l y g u i d e tracheal cells (blue) as they migrate a l o n g p e r i p h e r a l n e r v e tracts ( b r o w n ) into the C N S . ( A ) T h e tracheae o r i g i n a t e i n the d o r s a l r e g i o n o f the e m b r y o and migrate first a l o n g the I S N tract (top s e g m e n t , arrow). In the ventral r e g i o n o f the e m b r y o , the tracheae contact the p e r i p h e r a l g l i a as they turn p o s t e r i o r l y ( l o w e r s e g m e n t , a r r o w ) a n d then r e s u m e c e n t r a l l y d i r e c t e d m i g r a t i o n o n the S N tract. T h e tracheae penetrate the C N S i n c o n j u n c t i o n w i t h the S N tract. (B) T r a c h e a e d o not g r o w into the C N S i n glialablated e m b r y o s ( a r r o w ) a n d a p p e a r a b n o r m a l l y w i d e . ( C ) In the gem mutant, tracheae often neglect to turn p o s t e r i o r l y f r o m the I S N to the S N ( f o l l o w a r r o w s ) a n d as a result, enter the C N S w i t h o u t a s s o c i a t i o n to the peripheral nerves. A n t e r i o r is to the top, C N S is o n the left. T r a c h e a e w e r e l a b e l e d w i t h anti-twist (blue), m o t o r n e u r o n s w e r e l a b e l e d w i t h a n t i - F a s c i c l i n II ( b r o w n ) . S t a g e 16 e m b r y o s .  91  92  Sensory neurons stall during migrations to the CNS.  S e n s o r y a x o n s contact p e r i p h e r a l g l i a as they migrate i n the ventrolateral a n d ventral r e g i o n s a n d i n t o the C N S ( S e p p et al., 2 0 0 0 ) . It is thus p h y s i c a l l y p o s s i b l e that p e r i p h e r a l g l i a may assist s e n s o r y a x o n m i g r a t i o n into the C N S .  In b o t h g l i a l - a b l a t e d a n d gem  mutants, s e n s o r y a x o n s stall d u r i n g their m i g r a t i o n s to the C N S  In gem  ( F i g u r e 17).  e m b r y o s , a l t h o u g h a x o n s w e r e s i g n i f i c a n t l y stalled at early stages ( F i g u r e 17D), e v e n t u a l l y p r o j e c t e d into the C N S  in mature embryos.  they  In the m o s t s e v e r e l y glial-ablated  cases, s o m e s e n s o r y a x o n s d i d not p r o j e c t i n t o the C N S b y the e n d o f e m b r y o g e n e s i s .  As  w e l l , m a n y cases w e r e o b s e r v e d i n w h i c h s e n s o r y n e u r o n s c o u l d not project a x o n s at a l l . In e m b r y o s that l a c k e d a x o n s , the p o s i t i o n i n g o f s e n s o r y n e u r o n a l cell b o d i e s was p o o r l y o r g a n i z e d a n d o f t e n s e n s o r y n e u r o n s w e r e m i s s i n g altogether ( F i g u r e 17C). T h e greater effects o n sensory a x o n p r o j e c t i o n i n g l i a l - a b l a t e d e m b r y o s c o m p a r e d to gem mutants c o u l d be an artifact o f G A L 4 e x p r e s s i o n d r i v i n g UAS-grim i n m u s c l e .  I n d e e d , u s i n g N o m a r s k i optics to o b s e r v e m u s c l e , glial-ablated e m b r y o s a p p e a r to h a v e r o u n d e d m u s c l e s that are p o o r l y o r g a n i z e d . S e n s o r y n e u r o n s use m u s c l e as a m i g r a t i o n a l substrate f o r m i g r a t i o n i n the lateral to d o r s a l r e g i o n o f the e m b r y o . In the twist mutant, w h i c h entirely l a c k s m u s c l e s ,  sensory  n e u r o n s are s i g n i f i c a n t l y s t a l l e d ( Y o u n o s s i -  H a r t e n s t e i n a n d H a r t e n s t e i n , 1993). T o c o n t r o l f o r G A L 4 e x p r e s s i o n i n m u s c l e i n glialablated e m b r y o s , a series o f G A L 4 e n h a n c e r trap l i n e s w e r e u s e d to d r i v e UAS-grim. T h e s e lines, r Q 1 4 # 7 , 2 2 7 - G A L 4 , 3 5 3 - G A L 4 , 3 5 4 - G A L 4 , a n d 3 6 9 - G A L 4 all h a v e w e a k e r e x p r e s s i o n in the p e r i p h e r a l glia, but m o r e r o b u s t m u s c l e e x p r e s s i o n c o m p a r e d to the r Q 2 8 6 # 5 line. In these e m b r y o s , G A L 4 e x p r e s s i o n was not s t r o n g e n o u g h to ablate p e r i p h e r a l g l i a , h o w e v e r , m u s c l e s w e r e m o r e d i s o r g a n i z e d than r Q 2 8 6 # 5 UAS-grim e m b r y o s . In all c o n t r o l l i n e s , s e n s o r y n e u r o n a l patterning was not as d i s r u p t e d as i n the r Q 2 8 6 # 5 UAS-grim e m b r y o ( F i g u r e 17B)  e v e n i n the p r e s e n c e  o f greater m u s c l e  d i s r u p t i o n . T h e r e f o r e , an a d d i t i v e effect o n s e n s o r y a x o n p r o j e c t i o n p r o b a b l y w h e r e b o t h loss o f p e r i p h e r a l g l i a a n d m u s c l e l e a d to greater stalling than i n the  occurs,  gem 93  Figure 17. migration.  Peripheral glia promote centrally-directed sensory axon  P e r i p h e r a l g l i a assist s e n s o r y a x o n m i g r a t i o n i n the ventral r e g i o n o f the e m b r y o . ( A ) A w i l d type stage 15 e m b r y o has w e l l e s t a b l i s h e d s e n s o r y n e r v e tracts ( b r o w n ) e x t e n d i n g w e l l into the the C N S ( a r r o w ) . ( B ) C o n t r o l r Q 1 4 # 7 UAS-grim e m b y o s h a v e w i l d type s e n s o r y a x o n p a t t e r n i n g (stage 16). ( C ) I n the glial-ablated mutant (stage 16), s e n s o r y n e u r o n s stall ( a r r o w ) a n d there is a v a r i a b l e l o s s o f n e u r o n s , particularly i n v e n t r a l r e g i o n s ( c o m p a r e a s t e r i s k s to A ) . ( D ) T h e r e is a l s o s e n s o r y a x o n stalling i n the stage 15 gem mutant ( c o m p a r e a r r o w s i n u p p e r a n d l o w e r s e g m e n t s a n d to A ) , a l t h o u g h not as p r o n o u n c e d as i n the g l i a l - a b l a t e d m u t a n t ( c o m p a r e to C ) . A n t e r i o r is to the top, C N S is o n the left. S e n s o r y a x o n s w e r e l a b e l e d w i t h m A b 2 2 C 1 0 .  94  mutant, w h o s e m u s c l e s are p r o p e r l y f o r m e d .  T o e x t e n d a x o n s i n t o the C N S ,  sensory  a x o n s a p p e a r to d e p e n d o n n u m e r o u s tissues w h i c h i n c l u d e p e r i p h e r a l glia.  Sensory axons make pathfinding errors in absence of peripheral glia.  P e r i p h e r a l g l i a p r e f i g u r e the C N S  n e r v e entry p o i n t s f o r s e n s o r y a x o n s .  During  d e v e l o p m e n t , s e n s o r y a x o n s m i g r a t e i n c o n t a c t w i t h p e r i p h e r a l g l i a as they m i g r a t e f r o m the v e n t r a l r e g i o n o f the P N S i n t o the C N S . S e n s o r y a x o n s n o r m a l l y enter the C N S a l o n g t w o m a i n n e r v e s , the anterior a n d p o s t e r i o r f a s c i c l e s w h i c h are distinct yet b u n d l e d c l o s e together b y glia. In the ventral r e g i o n , i n a d d i t i o n to stalling, the s e n s o r y a x o n s o f b o t h glial-ablated a n d gem e m b r y o s migrate a l o n g aberrant trajectories w h i l e c r o s s i n g the C N S / P N S b o u n d a r y . T h e s e n s o r y n e r v e s i n the v e n t r a l r e g i o n o f the e m b r y o n e v e r appear w i l d type i n glial-ablated a n d gem  embryos.  In b o t h o f these e m b r y o s , s e n s o r y a x o n s  m a k e a w i d e r a n g e o f p a t h f i n d i n g e r r o r s as they a p p r o a c h a n d c r o s s the C N S / P N S border. M o s t often, s e n s o r y n e r v e s p r o j e c t i n t o the C N S at i n c o r r e c t p o s i t i o n s ( F i g u r e 18). T h e anterior f a s c i c l e is u s u a l l y the m o s t affected, d i s p l a y i n g t w o extremes. S e n s o r y n e r v e s stray a n t e r i o r l y a n d p r o j e c t into the C N S  at a n e i g h b o u r i n g s e g m e n t ( F i g u r e 1 8 B ) .  A l t e r n a t e l y , the a n t e r i o r fascicle c a n f u s e w i t h the p o s t e r i o r f a s c i c l e a n d enter the C N S  as  o n e n e r v e rather than t w o c l o s e l y a s s o c i a t e d but distinct f a s c i c l e s as i n w i l d t y p e ( F i g u r e 18C).  A s w e l l , n e u r o n s c r o s s incorrectly f r o m o n e fascicle to a n o t h e r w h i l e m i g r a t i n g  v e n t r a l l y ( F i g u r e 18C). A n o t h e r g e n e r a l p a t h f i n d i n g defect seen i n b o t h glial-ablated a n d gem mutant e m b r y o s is n e r v e b i f u r c a t i o n . S e n s o r y n e r v e b i f u r c a t i o n s c o m m o n l y o c c u r j u s t o u t s i d e the C N S ( F i g u r e 1 9 C , D , E ) . W h e n s e n s o r y n e u r o n s first a p p r o a c h this r e g i o n , p e r i p h e r a l glia n o r m a l l y w i l l h a v e a l r e a d y m i g r a t e d a n d e x t e n d e d p r o c e s s e s a c r o s s to the lateral r e g i o n o f the P N S .  T h e r e f o r e , g l i a s u r r o u n d i n g the s e n s o r y n e u r o n s w o u l d c o n f i n e the directional  c h o i c e s o f m i g r a t i n g s e n s o r y axons. W i t h p e r i p h e r a l g l i a m i s s i n g , s e n s o r y a x o n s h a v e a m u c h larger c a p a c i t y f o r e r r o r i n m i g r a t i n g to the C N S , as the e n v i r o n m e n t g r o w t h c o n e s 96  Figure 18. Peripheral glia provide directional information to sensory neurons as they migrate to the C N S .  P e r i p h e r a l g l i a are r e q u i r e d to p r e f i g u r e the C N S entry p o i n t s o f s e n s o r y a x o n s d u r i n g e m b r y o g e n e s i s . ( A ) In the w i l d type stage 16 e m b r y o , s e n s o r y a x o n s ( b r o w n ) enter the C N S a l o n g t w o m a i n n e r v e roots a n d enter the C N S at regular p o s i t i o n s i n e a c h h e m i s e g m e n t . T h e t w o m a i n n e r v e roots are a l w a y s distinct, b u t penetrate the C N S i n c l o s e p r o x i m i t y to o n e another (asterisk). ( B ) S e n s o r y a x o n s migrate o n aberrant tangents, often s t r a y i n g to n e i g h b o u r i n g s e g m e n t s i n the g l i a l - a b l a t e d e m b r y o (stage 16). T h e a x o n s also h a v e d i f f i c u l t y l o c a t i n g the C N S entry p o i n t a n d m a y stall i n the ventral r e g i o n (arrows). T h e a x o n s enter the C N S at aberrant p o s i t i o n s ( c o m p a r e asterisk to A ) . ( C ) I n the gem mutant, s e n s o r y a x o n s migrate into the C N S at i r r e g u l a r p o s i t i o n s i n e a c h h e m i s e g m e n t (stage 16). T h e errors are h i g h l y v a r i a b l e . S o m e t i m e s , the anterior f a s c i c l e c a n b e f u s e d w i t h the p o s t e r i o r f a s c i c l e at the C N S / P N S b o r d e r ( t o p s e g m e n t , c o m p a r e to * ) . A s w e l l , s e n s o r y a x o n s w h i c h n o r m a l l y enter the C N S o n the anterior f a s c i c l e migrate incorrectly a l o n g the p o s t e r i o r f a s c i c l e ( c o n c a v e a r r o w ) o r s p r e a d apart a n d a p p e a r h i g h l y d e f a s c i c u l a t e d ( s o l i d arrow). A n t e r i o r is to the top, C N S i s o n the left. S e n s o r y a x o n s labeled with m A b 22C10.  97  98  Figure 19. Adhesion of sensory neurons to peripheral glia may be required for proper axon migration into the C N S .  P e r i p h e r a l g l i a m a y p r o v i d e a b r i d g e f o r s e n s o r y n e u r o n s to m i g r a t e f r o m the s o m a t i c m u s c l e f i e l d i n t o the C N S n e u r o m e r e , s i n c e i n the a b s e n c e o f g l i a s e n s o r y n e u r o n s h a v e d i f f i c u l t y i n e n t e r i n g the C N S i n this r e g i o n . ( A ) In the w i l d t y p e stage 16 e m b r y o , s e n s o r y n e u r o n a f a n d p f tracts ( b r o w n ) are r o b u s t a n d e x t e n d w e l l i n t o the C N S . R e p o p o s i t i v e glial cells s u r r o u n d the n e r v e s i n the P N S ( b l u e ) . ( C ) In the glial ablated mutant (stage 16), s e n s o r y a x o n s stall a n d b i f u r c a t e o u t s i d e the C N S / P N S b o r d e r ( a r r o w ) s u g g e s t i n g i n c r e a s e d d i f f i c u l t y i n m i g r a t i n g f r o m the P N S i n t o the C N S . ( E ) S e n s o r y a x o n s h a v e s i m i l a r b i f u r c a t i o n s ( a r r o w s ) i n the g e m mutant (stage 16). ( B ) L a t e r a l c h o r d o t o n a l n e u r o n s enter the C N S a l o n g the anterior f a s c i c l e (af) (stage 16). ( D ) In the gem mutant, the c h o r d o t o n a l s d i d not c h o o s e to m i g r a t e to the C N S as a s i n g l e fascicle ( a r r o w ) , i n d i c a t i n g that like s e n s o r y a x o n s are not s u f f i c i e n t to g u i d e e a c h o t h e r centrally (stage 16). ( F ) A t the v e n t r a l e d g e o f the s o m a t i c m u s c l e f i e l d , s e n s o r y a x o n s w h i c h initially m i g r a t e d o n a c o r r e c t c o u r s e c e n t r a l l y m a d e an abrupt t u r n to f o l l o w the ventral o b l i q u e m u s c l e s i n the gem mutant (stage 15). Interestingly, the s e n s o r y a x o n s d i d not fasciculate o n p r e e x i s t i n g n e r v e f a s c i c l e s ( v i s i b l e w i t h N o m a r s k i o p t i c s , a r r o w ) w h i c h c r o s s e d the C N S / P N S b o r d e r a l o n g the c h o r d o t o n a l s o r i g i n a l trajectory. S e n s o r y a x o n s w e r e l a b e l e d w i t h m A b 2 2 C 1 0 ( A , C , E ) a n d m A b 4 9 C 4 (B,D,F). G l i a l n u c l e i w e r e l a b e l e d w i t h a n t i - R e p o ( A , C ) . A n t e r i o r is to the top, C N S i s to the left.  99  100  see is m o r e v a r i e d . F o r e x a m p l e , s e n s o r y a x o n s w o u l d not n o r m a l l y contact m u s c l e o r tracheae i n the ventral r e g i o n o f the e m b r y o . W i t h peripheral g l i a lost, the s e n s o r y a x o n s m u s t c h o s e to m i g r a t e o n either m o t o r axons, m u s c l e , tracheae, o r o t h e r s e n s o r y axons. In the a b s e n c e o f g l i a , s e n s o r y n e u r o n s d o not n e c e s s a r i l y f o l l o w e x i s t i n g n e u r o n into the C N S  ( F i g u r e 19D,F).  pathways  Instead, w h e n the ventral-most e d g e o f the a b d o m i n a l  m u s c l e f i e l d is r e a c h e d , s o m e s e n s o r y a x o n s m a y turn p o s t e r i o r l y a n d migrate further t o w a r d s the C N S  a l o n g ventral o b l i q u e m u s c l e s ( F i g u r e 19F).  Since glia normally  p h y s i c a l l y adhere to s e n s o r y a x o n s as they c r o s s the C N S / P N S a n d i n the a b s e n c e o f peripheral g l i a , s e n s o r y a x o n s h a v e s u c h a penetrant p h e n o t y p e o f a b n o r m a l trajectories a c r o s s this r e g i o n , it is c o n c l u d e d that peripheral g l i a mediate s e n s o r y a x o n g u i d a n c e into the C N S . Loss of tight peripheral nerve bundling in mutants which lack peripheral glia.  In m a t u r e w i l d t y p e e m b r y o s , w h e n glial w r a p p i n g o f s e n s o r y n e r v e s is c o m p l e t e , the anterior a n d p o s t e r i o r f a s c i c l e s are b o t h v e r y tightly f a s c i c u l a t e d . A s w e l l , the anterior a n d p o s t e r i o r f a s c i c l e s are c l o s e l y a p p o s e d t o o n e another. In m a t u r e g l i a l - a b l a t e d a n d  gem  mutant e m b r y o s , neither the peripheral n e r v e s are tightly f a s c i c u l a t e d n o r are they h e l d c l o s e but s e p a r a t e d f r o m o n e a n o t h e r ( F i g u r e 2 0 B , C ) .  Defasciculation o f peripheral nerves  also has b e e n o b s e r v e d p r e v i o u s l y i n repo e m b r y o s i n w h i c h g l i a f a i l to differentiate p r o p e r l y ( C a m p b e l l et al., 1994; X i o n g et al., 1994; H a l t e r et al., 1995).  These  o b s e r v a t i o n s s u g g e s t that b y w r a p p i n g o f a x o n s , peripheral g l i a refine the o v e r a l l a p p e a r a n c e o f s e n s o r y n e r v e structure o f the e m b r y o .  Loss ofmAb 22C10 sensory neuronal antigenicity in glial-ablated mutants.  W h e n s e n s o r y a x o n s w e r e l a b e l e d u s i n g the 2 2 C 1 0 a n t i b o d y , s t a i n i n g w a s w e a k i n b o t h g l i a l - a b l a t e d a n d gem e m b r y o s c o m p a r e d to w i l d type. It has b e e n o b s e r v e d  101  Figure 20. Peripheral glia are required for maintenance of axon fasciclulation and tight nerve bundling of peripheral nerves.  E n s h e a t h e m e n t o f p e r i p h e r a l n e r v e s is the final stage o f glial m o r p h o l o g i c a l d e v e l o p m e n t . T h i s p r o c e s s is essential f o r the f o r m a t i o n o f the b l o o d - n e r v e - b a r r i e r . ( A ) M a t u r e w i l d t y p e e m b r y o s h a v e tightly fasciclulated a x o n tracts (stage 16). T h e t w o m a i n s e n s o r y f a s c i c l e s ( b r o w n ) are b u n d l e d c l o s e l y together b y p e r i p h e r a l glia o u t s i d e the C N S / P N S b o u n d a r y . ( B ) In the glial-ablated mutant (stage 16), s e n s o r y a x o n s are d e f a s c i c u l a t e d ( s o l i d a r r o w s ) a n d the m a j o r n e r v e r o o t s are not g r o u p e d together c l o s e l y o u t s i d e the C N S ( c o n c a v e a r r o w ) . ( C ) S e n s o r y a x o n s are d e f a s c i c u l a t e d ( a r r o w ) i n the stage 16 gem e m b r y o . F u r t h e r m o r e , the m a i n n e r v e r o o t s are not b u n d l e d p r o p e r l y together as i n w i l d type ( c o n c a v e arrows, c o m p a r e to A ) . S e n s o r y a x o n s w e r e l a b e l e d w i t h m A b 2 2 C 1 0 . A n t e r i o r is to the top, C N S is o n the left.  102  103  p r e v i o u s l y that g l i a affect the l e v e l o f 2 2 C 1 0 antigen e x p r e s s i o n i n n e u r o n s . I n the pointed mutant, loss o f glial differentiation i n the C N S  l e a d s to a l o s s o f 2 2 C 1 0 staining i n  n e u r o n s . C o n v e r s e l y , e c t o p i c glial cells c a u s e adjacent n e u r o n s to e x p r e s s e c t o p i c 2 2 C 1 0 ( K l a e s et al., 1 9 9 4 ) . T h e l o s s o f 2 2 C 1 0 staining i n glial-ablated e m b r y o s s u g g e s t s that peripheral g l i a also interact w i t h s e n s o r y n e u r o n s i n w a y s w h i c h s u b s e q u e n t l y alter neuronal gene expression.  Peripheral glia may be intermediate targets for motor axon pathfinding into the periphery.  P i o n e e r m o t o r n e u r o n s n o r m a l l y m a k e g r o w t h c o n e contacts w i t h p e r i p h e r a l g l i a as they m i g r a t e to the P N S ( S e p p et al., 2 0 0 0 ) . G i v e n this o b s e r v a t i o n , it is r e a s o n a b l e to s u g g e s t that p e r i p h e r a l g l i a c o u l d act as s t e p p i n g s t o n e s f o r m i g r a t i n g m o t o r a x o n s . I n d e e d , m o t o r n e u r o n s stall a n d exit the C N S at irregular p o s i t i o n s i n the glial-ablated e m b r y o as w e l l as the gem  e m b r y o ( F i g u r e 2 1 C , E ) . H o w e v e r , ablation o f p e r i p h e r a l glia  c a u s e s r e a r r a n g e m e n t s o f o t h e r r e p o - p o s i t i v e g l i a i n the C N S ( F i g u r e 15). T h e m i g r a t i o n p a t h w a y s o f m o t o r n e u r o n s i n the C N S  w o u l d t h u s b e d e f e c t i v e d u e to a n o v e r a l l  d i s r u p t i o n o f the C N S glial s c a f f o l d w h i c h i s r e q u i r e d f o r m o t o r a x o n p a t h f i n d i n g i n s i d e the C N S ( H i d a l g o et al., 1995). In m o r e m a t u r e e m b r y o s , it i s e v i d e n t that m o t o r a x o n defects i n the P N S o f the glial-ablated a n d gem o r i g i n a t e d i n the C N S .  mutants likely s t e m f r o m p r o b l e m s w h i c h  S e g m e n t s w h e r e the C N S m o t o r a x o n pattern i s v e r y s e v e r e l y  d i s r u p t e d also h a v e h i g h l y d i s r u p t e d P N S m o t o r a x o n patterns c o m p a r e d to o t h e r s e g m e n t s ( F i g u r e 2 I F ) . T h e h y p o t h e s i s that p e r i p h e r a l g l i a act as i n t e r m e d i a t e targets f o r m o t o r a x o n p i o n e e r s is s u p p o r t e d b y these data, h o w e v e r , o n l y future a n a l y s i s o f m u t a n t s w h i c h l a c k putative m o t o r a x o n g u i d a n c e m o l e c u l e s i n p e r i p h e r a l g l i a w o u l d p r o v e it. K n o w i n g that f o l l o w e r m o t o r a x o n s n o r m a l l y u s e the p a t h w a y s l a i d o u t b y p i o n e e r m o t o r n e u r o n s ( L i n et al., 1995; H i d a l g o a n d B r a n d , 1997), w o u l d m o t o r n e u r o n p a t h w a y s a r i s i n g f r o m the C N S b e u s e d b y s e n s o r y a x o n s to m i g r a t e centrally?  In glial-ablated  e m b r y o s , m o t o r a x o n s a p p e a r to b e a b l e to exit the C N S . If s e n s o r y a x o n s s i m p l y u s e d 104  Figure 21. Peripheral glia are required to prefigure the C N S exit point of motor axons.  M o t o r a x o n s n o r m a l l y contact p e r i p h e r a l g l i a at regular p o s i t i o n s a l o n g the anterior/posterior e m b r y o n i c axis as t h e y e x i t the C N S . L o s s o f p e r i p h e r a l g l i a d i s r u p t s the f o r m a t i o n o f these r e g u l a r m o t o r a x o n e x i t points. ( A ) A stage 13 w i l d t y p e e m b r y o s h o w s p i o n e e r m o t o r a x o n s ( b r o w n ) e x i t i n g the C N S at s t e r e o t y p e d l o c a t i o n s . ( C ) A stage 13 glial-ablated e m b r y o h a s p r o n o u n c e d irregularities i n m o t o r a x o n p e r i p h e r a l trajectories. A x o n s a p p e a r to h a v e stalled i n m a n y s e g m e n t s , w h e n c o m p a r e d to e a c h o t h e r s p r o g r e s s ( c o m p a r e a r r o w s ) . ( E ) I n the gem stage 13 mutant, m o t o r a x o n s also f a i l to exit the C N S i n r e g u l a r p o s i t i o n s as i n the w i l d t y p e ( c o m p a r e t o A ) . ( B ) M o t o r a x o n patterning o f stage m a t u r e w i l d t y p e e m b r y o s i s v e r y s t e r e o t y p e d a c r o s s a l l P N S s e g m e n t s as w e l l as i n the C N S . ( D ) I n the glial-ablated mutant (stage 16), p e r i p h e r a l n e r v e s h a v e w e a k o v e r a l l structural o r g a n i z a t i o n . F u r t h e r m o r e , the l o n g i t u d i n a l tracts o f the C N S are d i s r u p t e d ( a r r o w , c o m p a r e to B ) . ( F ) T h e stage 16 e m b r y o has d i s o r g a n i z a t i o n o f p e r i p h e r a l n e r v e tracts ( c o m p a r e to B ) . T h e m o s t severely affected segment, w h i c h i s m i s s i n g the I N S b n e r v e ( c o m p a r e s o l i d a r r o w to B ) c o i n c i d e n t a l l y h a s s e v e r e d i s r u p t i o n o f C N S l o n g i t u d i n a l a x o n tracts ( c o m p a r e c o n c a v e a r r o w to B ) . A n t e r i o r i s to the top, C N S i s i n the m i d d l e i n ( A , C , E ) a n d o n the left i n ( B , D , F ) . M o t o r n e u r o n s w e r e l a b e l e d w i t h a n t i - F a s c i c l i n II.  105  106  m o t o r a x o n s as a m i g r a t i o n a l substrate, o n e w o u l d expect that s e n s o r y a x o n s w o u l d n o t stall at the C N S / P N S b o r d e r g i v e n that m o t o r a x o n s h a v e a l r e a d y e n t e r e d the P N S . T h e r e f o r e , m o t o r a x o n s a l o n e are m o s t l i k e l y n o t sufficient f o r p r o p e r s e n s o r y a x o n m i g r a t i o n into the C N S .  107  Discussion  A n a t o m i c a l study o f p e r i p h e r a l glial d e v e l o p m e n t has s u g g e s t e d that p e r i p h e r a l glia m a y h a v e a r o l e i n a x o n a l guidance.  H e r e , u s i n g targeted ablation, it is d e m o n s t r a t e d that  peripheral g l i a m e d i a t e s e n s o r y a x o n m i g r a t i o n into the C N S .  A s w e l l , e v i d e n c e is  p r o v i d e d w h i c h s u p p o r t s the h y p o t h e s i s that peripheral g l i a are intermediate targets f o r m o t o r a x o n g u i d a n c e into the P N S .  It is also s h o w n that p e r i p h e r a l g l i a are r e q u i r e d f o r  tight b u n d l i n g and f a s c i c u l a t i o n o f p e r i p h e r a l nerves. P e r i p h e r a l g l i a a l s o a p p e a r to h a v e roles i n g u i d i n g the m i g r a t i o n o f non-neural cells, as tracheal m i g r a t i o n d u r i n g e m b r y o n i c d e v e l o p m e n t is also d i s r u p t e d b y a l o s s o f g l i a . disruptions in C N S  P e r i p h e r a l glial ablation also c a u s e s  structure, illustrating h o w the m o r p h o g e n e s i s and m i g r a t i o n s o f these  cells m a y c o n t r i b u t e the general b o d y p l a n o f the e m b r y o .  Peripheral glial loss in the CNS.  P e r i p h e r a l g l i a are b o r n d u r i n g early n e u r o g e n e s i s at the lateral e d g e o f the  CNS.  W h e n p e r i p h e r a l g l i a are ablated v e r y early, other C N S g l i a not targeted f o r ablation appear to migrate to the lateral r e g i o n p o s s i b l y to c o m p e n s a t e f o r the l o s s .  It has  d e m o n s t r a t e d p r e v i o u s l y that g l i a to n e u r o n ratios are d e v e l o p m e n t a l l y regulated.  been  Survival  o f C N S g l i a is d e p e n d e n t o n a x o n a l contact, s u g g e s t i n g that g l i a h a v e a t r o p h i c d e p e n d e n c e o n n e u r o n s d u r i n g d e v e l o p m e n t . F o r e x a m p l e , i n the commissureless m u t a n t s w h i c h lack c o m m i s s u r a l a x o n tracts, the majority o f the c o m m i s s u r e - w r a p p i n g m i d l i n e g l i a die.  The  f e w g l i a that s u r v i v e d o so b y adjusting their p o s i t i o n s laterally to g a i n a x o n contact o n l o n g i t u d i n a l a x o n tracts ( S o n n e n f e l d a n d J a c o b s , 1 9 9 5 b ) . S i m i l a r l y , the m i d l i n e g l i a are able to migrate to and w r a p the c o m m i s s u r e s o f adjacent s e g m e n t s w h i c h l a c k m i d l i n e glia as a result o f f a i l u r e to d i f f e r e n t i a t e ( C o n d r o n et al., 1994). In the current study, m i g r a t i o n of C N S  g l i a to the r e g i o n o f p e r i p h e r a l glial apoptotic death p r e s u m a b l y o c c u r s to receive  t r o p h i c s i g n a l s f r o m a x o n s w h i c h n o r m a l l y w o u l d h a v e b e e n taken up b y p e r i p h e r a l glia. 108  T h e resultant d i s o r g a n i z a t i o n o f C N S glia likely c a u s e s other s u b s e q u e n t defects. D e l a y e d C N S s h o r t e n i n g is a d i s r u p t i o n f o u n d i n the g l i a l m u t a n t s gem,  repo, a n d prospero  ( D o e et al., 1991; C a m p b e l l et al., 1994; H o s o y a et al., 1995). L i k e w i s e , i n the glialablated e m b r y o , C N S s h o r t e n i n g is d e l a y e d . T h i s effect is likely n o t c a u s e d b y a simple loss o f p e r i p h e r a l glia, b u t the rather the c a s c a d e o f C N S glial cell r e a r r a n g e m e n t s w h i c h result f r o m p e r i p h e r a l glial ablation.  A l l Repo-positive  C N S glia likely s u f f e r f r o m  p e r i p h e r a l glial loss s i n c e they all a p p e a r to lose their n o r m a l p o s i t i o n i n g . G i v e n that C N S s h o r t e n i n g o c c u r s o v e r a p e r i o d o f m a j o r glial cell r e a r r a n g e m e n t s i n the C N S ( J a c o b s et al., 1989), a n d that glial cells also b e g i n t o b u n d l e a n d w r a p n e r v e tracts at this time ( K l a m b t et al., 1991), it is likely that C N S glia c o l l e c t i v e l y contribute t o C N S  shortening  b y t i g h t e n i n g their w r a p s a r o u n d n e r v e bundles. D i s r u p t i o n o f C N S s h o r t e n i n g m a y also c a u s e d e l a y e d d o r s a l c l o s u r e w h i c h w a s o b s e r v e d i n g l i a l - a b l a t e d e m b r y o s , s i n c e the C N S m u s t c o n d e n s e b e f o r e the e p i d e r m i s c a n migrate o v e r the d o r s a l h a l f o f the e m b r y o . T h i s is n o t a c a u s e f o r c o n c e r n i n interpreting the effects o f glial ablation o n neuronal p a t h f i n d i n g .  M a n y embryos were observed in  w h i c h o n l y a f e w s e g m e n t s e x p e r i e n c e d s u c c e s s f u l g l i a l ablation, a n d i n these cases, d o r s a l c l o s u r e d i d occur. T h e n e u r o n a l defects i n glial-ablated h e m i s e g m e n t s o f s u c h e m b r y o s w e r e identical t o those w h i c h h a d w i d e s p r e a d ablation a n d n o d o r s a l c l o s u r e .  Therefore,  the n e u r o n a l p h e n o t y p e s i n g l i a l - a b l a t e d e m b r y o s are not a result o f d o r s a l c l o s u r e defects. T h e d i s o r g a n i z a t i o n o f C N S g l i a is h o w e v e r , p r o b l e m a t i c i n interpreting the effects o f p e r i p h e r a l g l i a l a b l a t i o n o n m o t o r a x o n p a t h f i n d i n g . M o t o r n e u r o n s are b o r n i n the C N S and u s e a variety o f C N S glial substrates, i n c l u d i n g the m i d l i n e a n d l o n g i t u d i n a l glia to migrate to their final destinations (Jacobs a n d G o o d m a n , 1989; H i d a l g o et al., 1995; Z h o u et al., 1997). I n the g l i a l - a b l a t e d e m b r y o , d a m a g e to the glial s c a f f o l d likely c a u s e s m o t o r a x o n stalling a n d m i s r o u t i n g b e f o r e the m o t o r a x o n s reach the lateral e d g e o f the C N S w h e r e the p e r i p h e r a l g l i a are situated. T h u s m o t o r a x o n defects i n the glial-ablated mutant m a y s t e m f r o m p r o b l e m s w h i c h o c c u r u p s t r e a m o f the p e r i p h e r a l glia. A n a l y s i s o f the  gem  109  mutant c a n n o t r e s o l v e this issue, s i n c e m o s t C N S g l i a f a i l t o differentiate, c a u s i n g d i s r u p t i o n t o the C N S g l i a l s c a f f o l d as w e l l . I n glial-ablated e m b r y o s , m o t o r a x o n s are d e l a y e d i n their m i g r a t i o n to the P N S a n d they exit the C N S at a b n o r m a l p o s i t i o n s , w h i c h w o u l d b e a l i k e l y effect i f peripheral g l i a act as intermediate targets f o r these cells. T h e r e f o r e , s u p p o r t f o r the p e r i p h e r a l glial intermediate target h y p o t h e s i s is p r o v i d e d f r o m these  experiments.  Tracheal pathfinding in the absence of peripheral glia.  T r a c h e a l cells o f the g a n g l i o n i c b r a n c h contact a variety o f substrates as they migrate f r o m the p e r i p h e r y into the C N S .  A s they a p p r o a c h the ventral r e g i o n o f the  e m b r y o , the g a n g l i o n i c b r a n c h i n i t i a l l y migrates a l o n g the I S N but switches to the S N o n c e it contacts the p e r i p h e r a l " e x i t " g l i a ( E n g l u n d et al., 1999).  F r o m these o b s e r v a t i o n s , it  w a s p r o p o s e d that p e r i p h e r a l g l i a m a y p l a y a r o l e i n this d e c i s i o n . P e r i p h e r a l glial ablation w o u l d b e a n ideal m e a n s o f a d d r e s s i n g this h y p o t h e s i s .  I n the glial-ablated e m b r y o ,  tracheae are h i g h l y d i s r u p t e d , h o w e v e r this d i s r u p t i o n o c c u r s b e f o r e the tracheae  make  p h y s i c a l contact w i t h the glia. It is p o s s i b l e that tracheal d i s r u p t i o n is d u e t o a l o w l e v e l o f G A L 4 e x p r e s s i o n i n the tracheae. T h e e x p r e s s i o n o f gem is e x c l u s i v e l y g l i a l ( H o s o y a et al., 1995; J o n e s et al., 1995; V i n c e n t et al., 1 9 9 6 ) a n d s o it is a s s u m e d that a n y n o n - g l i a l defects s e e n i n the n u l l m u t a n t is a s e c o n d a r y effect o f glial l o s s . T h e gem  mutant does  h a v e d i s r u p t i o n s i n tracheal f o r m a t i o n , h o w e v e r the defects a r e m i l d e r a n d the g a n g l i o n i c b r a n c h i s capable o f m i g r a t i n g into the C N S .  S i n c e g l i a i n the gem  m u t a n t f a i l to  differentiate, it i s a l s o p o s s i b l e that the undifferentiated cells still e x p r e s s factors that contribute to tracheal g r o w t h a n d i n that case, o n e w o u l d expect f e w e r tracheal d i s r u p t i o n s i f g l i a w e r e r e q u i r e d f o r tracheal m o r p h o g e n e s i s . A s G B tracheae contact p e r i p h e r a l glia w h i l e they migrate t o the C N S , it is p o s s i b l e that the peripheral g l i a d o e x p r e s s  molecules  w h i c h p r o m o t e their m i g r a t i o n a n d s u r v i v a l .  110  Sensory axon pathfinding into the CNS in the absence of peripheral glia.  S e n s o r y a x o n s n o r m a l l y c o n t a c t p e r i p h e r a l g l i a i n the ventral to ventrolateral r e g i o n o f the e m b r y o as they migrate i n t o the C N S .  In b o t h glial-ablated a n d gem e m b r y o s ,  s e n s o r y a x o n s o f t e n stall, migrate a l o n g aberrant trajectories t o w a r d s the C N S , the C N S  a n d enter  at a b n o r m a l p o s i t i o n s a l o n g the C N S / P N S b o r d e r . A s w e l l , s e n s o r y n e r v e s are  d e f a s c i c u l a t e d a n d are not p r o p e r l y b u n d l e d together i n the ventral r e g i o n o f the e m b r y o . T h e data as a w h o l e s u g g e s t that p e r i p h e r a l g l i a mediate s e n s o r y a x o n g u i d a n c e into the CNS. gem  T h e glial-ablated e m b r y o , h o w e v e r , s h o w s greater s e n s o r y a x o n stalling than the m u t a n t a n d as w e l l as l o s s o f n e u r o n s a n d f a i l u r e to p r o j e c t s e n s o r y a x o n s w h i c h is not  seen e i t h e r i n gem In the gem  e m b r y o s . T h e r e are a n u m b e r o f p o s s i b l e c a u s e s f o r this d i f f e r e n c e . mutant, m o s t glial cells fail to differentiate a n d i n d e e d , there are n o  R e p o - p o s i t i v e g l i a w h i c h c a n be detected i n gem to differentiate into n e u r o n s i n the gem V i n c e n t et a l . , 1996).  e m b r y o s . Instead, the g l i a are s u g g e s t e d  mutant ( H o s o y a et al., 1995; J o n e s et al., 1995;  In the s e n s o r y s y s t e m w h i c h has p l a i n l y v i s i b l e cell b o d i e s , one  w o u l d e x p e c t to see an e x a c t d o u b l i n g o f n e u r o n s , as s e n s o r y n e u r o n s h a v e R e p o - p o s i t i v e cells a s s o c i a t e d w i t h t h e m . In the c u r r e n t study, the n u m b e r s o f s e n s o r y n e u r o n s i n the gem  n u l l m u t a n t w e r e c o u n t e d a n d r a r e l y was a p e r f e c t d o u b l i n g o f n e u r o n s apparent.  For  e x a m p l e , the f i v e lateral c h o r d o t o n a l o r g a n s are p l a i n l y v i s i b l e a n d o n l y v e r y r a r e l y the c h o r d o t o n a l s a p p e a r e d to be d o u b l e d .  M o s t o f t e n , the c h o r d o t o n a l s h a d o n e o r three  ectopic n e u r o n s . F u r t h e r m o r e , o n e p h e n o t y p i c a n a l y s i s o f gem  m u t a n t alleles has s h o w n  that not all g l i a a c q u i r e a n e u r o n a l - l i k e fate as s o m e glial p r e c u r s o r s fail to e x p r e s s the n e u r o n a l s p e c i f i c E l a v m a r k e r a n d there is n o dramatic increase i n o v e r a l l E l a v l a b e l i n g o f the m u t a n t ( V i n c e n t et a l . , 1996). T h e r e f o r e , gem  mutant e m b r y o s w h i c h d o not appear to  h a v e a true d o u b l i n g o f s e n s o r y n e u r o n s likely h a v e u n d i f f e r e n t i a t e d g l i a a s s o c i a t e d w i t h the s e n s o r y cell clusters. T h e u n d i f f e r e n t i a t e d g l i a c o u l d e x p r e s s f a c t o r s w h i c h e n h a n c e n e u r o n a l s u r v i v a l a n d f u n c t i o n . T h e r e f o r e , the glial-ablated e m b r y o s l i k e l y h a v e a greater loss o f g l i a l c e l l s than gem  embryos.  Ill  In the glial-ablated e m b r y o , m u s c l e defects c a u s e d b y m u s c l e G A L 4 activity m a y c o n t r i b u t e to loss o f s e n s o r y axons. In the r Q 2 8 6 # 5 line, G A L 4 activity i n m u s c l e d o e s n o t a p p e a r to be v e r y s i g n i f i c a n t w h e n a s s e s s e d w i t h UAS-lacZ s t a i n i n g . H o w e v e r , e v e n a l o w level o f G A L 4 e x p r e s s i o n m a y be e n o u g h to d i s r u p t n o r m a l m u s c l e f o r m a t i o n . A s m u s c l e is o n e substrate w h i c h s e n s o r y a x o n s use i n m i g r a t i o n t o the C N S , t h e r e is l i k e l y an a d d i t i v e effect o f p e r i p h e r a l glial a n d m u s c l e loss c a u s i n g i n c r e a s e d a x o n p r o j e c t i o n d e f e c t s c o m p a r e d to the gem  mutant.  T h i s k i n d o f effect o n s e n s o r y a x o n m i g r a t i o n h a s b e e n  n o t e d p r e v i o u s l y . In the twist mutant, a l l m u s c l e s are a b s e n t y e t m o s t s e n s o r y n e r v e s f o r m n o r m a l l y . T h i s is also true f o r the trachealess mutant, w h i c h l a c k s tracheae. H o w e v e r , the twist, trachealess d o u b l e m u t a n t h a s a s i g n i f i c a n t l y h i g h e r rate o f m i s r o u t e d a x o n s as w e l l  as s e n s o r y n e u r o n s w h i c h f a i l entirely to project an a x o n ( Y o u n o s s i - H a r t e n s t e i n a n d H a r t e n s t e i n , 1993). It a p p e a r s that as m o r e structures w h i c h s e n s o r y n e u r o n s u s e s o m i g r a t e are r e m o v e d , the health o f the n e u r o n s d e c r e a s e s a n d their c a p a c i t y f o r p a t h f i n d i n g errors increases. T h e c o m p o u n d effects o n s e n s o r y n e u r o n s i n the g l i a l - a b l a t e d e m b r y o c a n be r e s o l v e d b y c o m p a r i n g the p h e n o t y p e to the gem e m b r y o w h i c h h a s w i l d type m u s c u l a t u r e . A s w e l l , the r Q 1 4 # 7 UAS-grim c o n t r o l e m b r y o s h a v e w o r s e m u s c l e d e f e c t s t h a n r Q 2 8 6 # 5 UAS-grim e m b r y o s , y e t t h e y h a v e entirely n o r m a l s e n s o r y a x o n p r o j e c t i o n s a n d n o p e r i p h e r a l glial l o s s . T h e r e f o r e , defects that are i n b o t h the glial-ablated a n d gem e m b r y o c a n b e attributed to the l o s s o f p e r i p h e r a l g l i a . It i s certain that p e r i p h e r a l glia mediate s e n s o r y a x o n g u i d a n c e into the C N S  as loss o f the g l i a c a u s e s s e n s o r y a x o n  stalling a n d m i s g u i d a n c e .  Survival of sensory neurons - dependence on glia.  T h e loss o f a x o n s i n the g l i a l - a b l a t e d e m b r y o raises the q u e s t i o n w h e t h e r p e r i p h e r a l glia are i n v o l v e d i n a x o n p r o j e c t i o n . T h e failure o f a s e n s o r y n e u r o n to project a n a x o n m a y r e n d e r i t u n a b l e t o r e c e i v e t r o p h i c factors a n d thus die. T h i s w o u l d e x p l a i n w h y s o m e s e n s o r y n e u r o n s w e r e lost. I n Drosophila recent e v i d e n c e s u g g e s t s that glial cells d o h a v e  112  the c a p a c i t y to s t i m u l a t e a x o n g r o w t h a n d m a i n t a i n s u r v i v a l .  T h e m e c h a n o s e n s o r y lineage  of the m i c r o c h a e t e o n the n o t u m has b e e n r e v i s e d s u c h that a R e p o - p o s i t i v e glial cell a n d a sister P H I p r e c u r s o r c e l l arise f r o m the d i v i s i o n o f the P l l b cell. T h e P H I cell s u b s e q u e n t l y d i v i d e s to g i v e rise to a n e u r o n a n d sheath cell ( R e d d y a n d R o d r i g u e s , 1999b).  Originally,  n o R e p o - p o s i t i v e glial cell h a d b e e n detected i n the l i n e a g e . T h i s o v e r s i g h t is t h o u g h t to have h a p p e n e d s i n c e the R e p o - p o s i t i v e glial c e l l m i g r a t e s a w a y f r o m the rest o f the cells o f its l i n e a g e s o o n after its birth. T h e glial cell, w h i c h is a l s o P r o s p e r o - p o s i t i v e , is t h o u g h t to g u i d e s e n s o r y a x o n a l p r o j e c t i o n s i n c e i n P r o p s e r o - p o s i t i v e mutant c l o n e s , n e u r o n s are lost o r f a i l to p r o j e c t a x o n s ( G h o et a l . , 1999; M a n n i n g a n d D o e , 1999; R e d d y a n d R o d r i g u e s , 1999a,b). T h e g l i a a b l a t e d i n the c u r r e n t s t u d y w e r e a l l d e r i v e d f r o m the C N S  a n d arise  f r o m different l i n e a g e s than the s e n s o r y o r g a n p r e c u r s o r l i n e a g e . H o w e v e r , the p r o p e r t y o f g l i a p r o v i d i n g t r o p h i c s u p p o r t to n e u r o n s d u r i n g d e v e l o p m e n t m a y b e a w i d e s p r e a d p h e n o m e n o n o f g l i a t h r o u g h o u t the o r g a n i s m . Indeed, t r o p h i c d e p e n d e n c e o f n e u r o n s o n g l i a h a s a l s o b e e n o b s e r v e d e l s e w h e r e i n Drosophila. I n the eye, l a m i n a r n e u r o n s are u n a b l e to s u r v i v e w h e n the R e p o - p o s i t i v e l a m i n a r g l i a w h i c h a s s o c i a t e w i t h these cells f a i l t o differentiate i n the repo mutant ( X i o n g a n d M o n t e l l , 1995). Drosophila C N S ,  F u r t h e r m o r e , i n the  g l i a are n e e d e d to s u p p o r t the a x o n s w h i c h m i g r a t e a l o n g  pathways  f o u n d e d b y p i o n e e r n e u r o n s ( B o o t h et a l . , 2 0 0 0 ) .  Sensory axons do not necessarily use motor axons as migrational substrates.  S e n s o r y a x o n s a n d p e r i p h e r a l g l i a m a k e c l o s e p h y s i c a l contacts w i t h e a c h other during embryonic development.  W h e n p e r i p h e r a l g l i a are lost, the c o n s e q u e n c e s  s e n s o r y a x o n m i g r a t i o n into the C N S are p r o n o u n c e d .  on  A s m o t o r a x o n s fasciculate w i t h  o n e another a n d u s e p r e - e s t a b l i s h e d n e u r o n a l m i g r a t i o n r o u t e s f o r " f o l l o w e r n e u r o n " m i g r a t i o n ( L i n et a l . , 1995; H i d a l g o a n d B r a n d , 1997), i t w o u l d b e interesting to determine i f s e n s o r y a x o n s c a n use the p r e - e s t a b l i s h e d routes o f m o t o r a x o n s to m i g r a t e i n t o the CNS.  T h e c u r r e n t s t u d y s u g g e s t s that s e n s o r y a x o n s d o n o t rely u p o n m o t o r  axon  113  p r o j e c t i o n s f r o m the C N S to g u i d e t h e i r entry across the C N S / P N S b o r d e r . I n vertebrates, s e n s o r y p r o j e c t i o n s d o n o t a p p e a r to u s e m o t o r n e u r o n s t o g u i d e their m i g r a t i o n s either ( W a n g a n d Scott, 1999).  Boundary cap cells of the vertebrate DREZ may be analogous to peripheral glia.  In vertebrates, s e n s o r y n e u r o n s enter the s p i n a l c o r d at the d o r s a l r o o t entry z o n e ( D R E Z ) , a s p e c i a l i z e d structure at the C N S / P N S b o u n d a r y . I n rats, n e u r a l c r e s t - d e r i v e d b o u n d a r y c a p c e l l s , w h i c h e x p r e s s S c h w a n n cell m a r k e r s , r e s i d e at the D R E Z a x o n exit /entry p o i n t w h i l e s e n s o r y a x o n s p r o j e c t i n t o the C N S . C e n t r a l l y p r o j e c t i n g s e n s o r y a x o n s are o b s e r v e d to m i g r a t e d i r e c t l y t o w a r d s the b o u n d a r y c a p cells, p a u s e b r i e f l y as they t o u c h t h e m , a n d then p r o c e e d into the C N S . I n a c r y o c u l t u r e m o d e l , c u l t u r e d s e n s o r y n e u r o n s f r o m the d o r s a l root g a n g l i o n g r o w n o n a c r y o s e c t i o n o f d o r s a l s p i n a l c o r d ( w i t h nerve roots a n d s p i n a l c o r d entry sites i n c l u d e d ) p r e f e r e n t i a l l y migrate o v e r the b o u n d a r y c a p cells ( G o l d i n g a n d C o h e n , 1997). T h i s study suggests that b o u n d a r y c a p cells attract s e n s o r y a x o n s into the C N S . T h e r e f o r e , it is p o s s i b l e that vertebrates h a v e a n a n a l o g o u s r o l e f o r g l i a m e d i a t i n g s e n s o r y a x o n g u i d a n c e across the C N S / P N S border. It w i l l b e interesting i n the future t o k n o w w h e t h e r l o s s o f f u n c t i o n studies o n b o u n d a r y c a p cells i n vertebrates cause aberrant s e n s o r y a x o n entry into the C N S . A s correct C N S exit/entry o f n e u r o n s is imperative f o r f u n c t i o n a l l i n k i n g o f the C N S a n d P N S , p e r i p h e r a l g l i a p l a y k e y r o l e s i n o r g a n i z i n g a m a j o r aspect o f n e u r o d e v e l o p m e n t .  114  V.  GENERAL  DISCUSSION  In the past, Drosophila has b e e n u n d e r u t i l i z e d as a m o d e l s y s t e m f o r s t u d y i n g P N S glial d e v e l o p m e n t . N o w ,  p e r i p h e r a l glial G A L 4 lines h a v e b e e n c r e a t e d w h i c h m a y be  u s e f u l i n m a n y m o r e m u t a n t studies i n the future. U s i n g the G A L 4 lines as a glial m a r k e r , a detailed p r o f i l e o f w i l d t y p e p e r i p h e r a l glial d e v e l o p m e n t h a s b e e n g e n e r a t e d w h i c h p r o v i d e s i n f o r m a t i o n o n m o r p h o l o g y , m i g r a t i o n , a n d cell-cell contacts. T h e d e v e l o p m e n t of p e r i p h e r a l g l i a is v e r y s i m i l a r to vertebrate S c h w a n n cells a n d p o s s i b l y , t h e y m a y h a v e s i m i l a r f u n c t i o n s i n the d e v e l o p i n g n e r v o u s s y s t e m . T o e x p e r i m e n t a l l y a s s e s s the roles o f p e r i p h e r a l glia i n d e v e l o p m e n t , cell ablation e x p e r i m e n t s w h i c h f o l l o w e d s u g g e s t e d that peripheral glia are n e e d e d f o r s e n s o r y a x o n g u i d a n c e into the C N S  and motor axon  g u i d a n c e into the p e r i p h e r y . W h e n p e r i p h e r a l glia are lost, m i g r a t i n g a x o n s m a k e n a v i g a t i o n a l errors, particularly w h e n c r o s s i n g the C N S / P N S b o u n d a r y w h i c h is p r e f i g u r e d b y the glia. T h e o b s e r v a t i o n s b r i n g insight into the c o m m u n i c a t i o n s b e t w e e n glia a n d n e u r o n s a n d the m o d e s o f interaction b e t w e e n these t w o cell t y p e s c a n n o w  be  addressed.  D r o s o p h i l a as a P N S  glial m o d e l system.  In c h a r a c t e r i z a t i o n o f p e r i p h e r a l glial d e v e l o p m e n t , a n u m b e r o f interesting parallels w i t h S c h w a n n c e l l d e v e l o p m e n t w e r e o b s e r v e d . B o t h cell t y p e s arise f r o m the lateral e d g e o f central n e u r a l e c t o d e r m a n d s u b s e q u e n t l y m i g r a t e p e r i p h e r a l l y a l o n g the r o u t e s o f m i g r a t i n g axons. B o t h p e r i p h e r a l g l i a a n d S c h w a n n cells f o r m the p r i m a r y glial s h e a t h that w r a p s p e r i p h e r a l n e r v e a x o n s a n d thus l i k e l y h a v e s i m i l a r roles i n m a i n t e n a n c e o f n e u r o n a l health.  O n e v e r y m a r k e d similarity is the p o s i t i o n i n g o f P N S  glia at C N S n e u r o n a l  exit/entry p o i n t s . I n the vertebrate D R E Z , p r i m i t i v e b o u n d a r y c a p cells w h i c h e x p r e s s S c h w a n n cell m a r k e r s a p p e a r to b e v e r y s i m i l a r to Drosophila p e r i p h e r a l glia.  The 115  b o u n d a r y c a p cells are thought t o h a v e d u a l f u n c t i o n s i n d e f i n i n g the C N S exit/entry p o i n t as w e l l as d i r e c t i n g s e n s o r y a x o n s t o w a r d this l o c a t i o n . Drosophila d e v e l o p m e n t d i f f e r s f r o m the vertebrate D R E Z i n that the p e r i p h e r a l g l i a r e m a i n s p a n n i n g t h e C N S / P N S  border  e v e n w h e n a l l n e u r o n a l p a t h f i n d i n g h a s f i n i s h e d at the e n d o f e m b r y o g e n e s i s .  The  vertebrate b o u n d a r y c a p cells a p p e a r t o attract initial a x o n m i g r a t i o n t o t h e C N S a n d prepattern the later f o r m i n g a s t r o c y t i c - S c h w a n n cell interface. B o u n d a r y c a p cells d e c l i n e in n u m b e r t o w a r d s the e n d o f rat e m b r y o g e n e s i s a n d c a n n o t b e o b s e r v e d  beyond  p o s t e m b r y o n i c d a y 6 ( G o l d i n g a n d C o h e n , 1997). T h e m o s t o b v i o u s d i f f e r e n c e b e t w e e n Drosophila a n d vertebrates i s the l a c k o f p e r i p h e r a l glial m y e l i n a t i o n . Drosophila g l i a w r a p o n l y o n e c i r c u m f e r e n c e a r o u n d a x o n s a n d seal v i a glial-glial septate j u n c t i o n s ( A u l d et al., 1995). I n contrast, S c h w a n n cell m e m b r a n e s b i n d e x t e n s i v e l y to each other as they w r a p m a n y c i r c u m f e r e n c e s o f a n a x o n . S c h w a n n cells express m a n y m y e l i n - a s s o c i a t e d m o l e c u l e s s u c h as PQ, m y e l i n b a s i c p r o t e i n , a n d P M P 2 2 w h i c h h a v e n o t b e e n f o u n d i n Drosophila (Stewart et al., 1996). It c a n b e e n v i s i o n e d that Drosophila l a c k s a h o m o p h i l i c m e m b r a n e b o u n d cell a d h e s i o n p r o t e i n . I f the g l i a h a d s u c h a m o l e c u l e , the a d h e s i v i t y o f b o t h sides o f the m e m b r a n e c o u l d p o s s i b l y generate e n o u g h affinity f o r glial-glial m e m b r a n e w r a p p i n g s u c h as i n m y e l i n a t i o n . I n the future, e c t o p i c e x p r e s s i o n o f m y e l i n - a s s o c i a t e d p r o t e i n s i n Drosophila g l i a m a y address this q u e s t i o n . A l s o , as the invertebrate c o p e p o d h a s m y e l i n a t e d a x o n s ( D a v i s et al., 1999), it w o u l d b e interesting to c o m p a r e its glial g e n e e x p r e s s i o n w i t h that o f Drosophila to u n d e r s t a n d w h a t m o l e c u l e s h a v e first b e e n g a i n e d i n e v o l u t i o n t o a c q u i r e m y e l i n a t i o n . G i v e n the similarities a n d d i f f e r e n c e s b e t w e e n p e r i p h e r a l glial cells a n d S c h w a n n cells, o n e c a n estimate h o w studies i n Drosophila c a n contribute to k n o w l e d g e o f P N S glial d e v e l o p m e n t i n general. T h e m i g r a t i o n o f P N S g l i a into the p e r i p h e r y f o l l o w i n g p i o n e e r i n g a x o n s suggests that b o t h c e l l types m a y b e d e p e n d e n t o n s i m i l a r a x o n a l c u e s f o r m i g r a t i o n . A s Drosophila h a s v e r y s i m p l e patterning o f p e r i p h e r a l glial c e l l s , o n e c a n easily detect aberrations i n glial m i g r a t i o n s u c h that large scale p h e n o t y p i c screens c o u l d turn u p m a n y  116  genes i n v o l v e d i n glial m i g r a t i o n d e c i s i o n s . B o t h vertebrate a n d invertebrate g l i a are initially c o m p a c t a n d e x t e n d l o n g c y t o p l a s m i c p r o c e s s e s i n their m o r e mature f o r m s . Drosophila,  In  peripheral glial membrane processes extend over a longer proportion o f nerves  than S c h w a n n cells do. T h e r e f o r e , inabilities o f mutant p e r i p h e r a l g l i a to p r o p e r l y generate m e m b r a n e e x t e n s i o n to c o v e r the n e r v e s c o u l d b e e a s i l y d e t e c t e d i n s c r e e n s as w e l l . G i v e n that p e r i p h e r a l g l i a c a n be v i s u a l i z e d i n live e m b r y o s u s i n g G F P f l u o r e s c e n c e ( S e p p a n d A u l d , 1999), s c r e e n i n g f o r s u c h mutants c o u l d b e h i g h l y efficient.  G i v e n that s i m i l a r  m o l e c u l e s are u s e d i n m a n y different c e l l t y p e s o f b o t h invertebrates a n d vertebrates f o r cell m i g r a t i o n a n d g r o w t h ( V a n A e l s t a n d D ' S o u z a - S c h o r e y , 1997; M o n t e l l , 1999), it w o u l d be c o n c e i v a b l e that S c h w a n n cells a n d p e r i p h e r a l g l i a p r o b a b l y u s e the s a m e m e a n s o f initial process extension. A s  Drosophila  has p o w e r f u l g e n e t i c s a n d s i m p l e m o r p h o l o g y , it w o u l d  be a v e r y u s e f u l m o d e l o r g a n i s m to a d d r e s s these q u e s t i o n s .  Peripheral glial roles as guidepost cells.  T h i s thesis p r o v i d e s b o t h a n a t o m i c a l a n d  in vivo  l o s s o f f u n c t i o n e v i d e n c e that  peripheral g l i a p r o v i d e a x o n g u i d a n c e at the C N S / P N S b o r d e r . F i r s t , it is o b s e r v e d that b o t h m o t o r a x o n s p h y s i c a l l y contact the p e r i p h e r a l g l i a as they m i g r a t e a c r o s s this r e g i o n . S e c o n d , i n b o t h g l i a l - a b l a t e d a n d gem positions.  e m b r y o s , a x o n s enter a n d exit the C N S at incorrect  A l s o , m o t o r a n d s e n s o r y n e u r o n s stall as they exit a n d enter the  W i d e s p r e a d r e a r r a n g e m e n t s o f the C N S  glial s c a f f o l d r e s u l t s f r o m c o m p e n s a t i o n  CNS. of  p e r i p h e r a l g l i a l loss. G i v e n the c o m p l e x i t i e s o f the m o t o r a x o n p h e n o t y p e interpretation i n this case, f u r t h e r a n a l y s i s i n other glial mutant b a c k g r o u n d s s h o u l d p r o v i d e definitive e v i d e n c e o f a r o l e f o r p e r i p h e r a l g l i a i n m o t o r a x o n g u i d a n c e . M u t a n t s w h i c h maintain peripheral glial cell p r e s e n c e , yet l a c k m o l e c u l e s w h i c h m a y g u i d e m o t o r a x o n s to the p e r i p h e r y w o u l d p r o v i d e the m o s t c o n c l u s i v e e v i d e n c e .  H e r e , the m a i n t e n a n c e  of  117  p e r i p h e r a l glial p r e s e n c e w o u l d not affect other glial cell p o s i t i o n i n g i n the C N S s c a f f o l d , yet i f the p e r i p h e r a l g l i a l a c k m o t o r a x o n g u i d a n c e m o l e c u l e s the m o t o r a x o n s s h o u l d stall i n the v i c i n i t y o f the p e r i p h e r a l glia. T h e r o l e o f p e r i p h e r a l g l i a g u i d i n g s e n s o r y a x o n s i n t o the C N S  is s i m i l a r to the  D R G s e n s o r y a x o n g u i d a n c e r o l e p r o p o s e d f o r b o u n d a r y c a p cells i n vertebrates. It is important to note that glial cells are a m o n g s t m a n y o t h e r structures, s u c h as m u s c l e a n d tracheae, w h i c h g u i d e s e n s o r y a x o n s to their a p p r o p r i a t e targets. T h i s is apparent i n the glial-ablated a n d gem  e m b r y o s , w h e r e s e n s o r y a x o n s are s t i l l attracted to the C N S ,  even  t h o u g h they m a y enter the C N S at a b n o r m a l p o s i t i o n s . T h e r e f o r e , l o n g r a n g e d i f f u s i b l e factors s e c r e t e d i n the C N S m a y attract s e n s o r y a x o n s centrally. T h e r o l e o f p e r i p h e r a l glia appears to be m o r e o f a f i n e - t u n i n g o f s e n s o r y a x o n m i g r a t i o n . S e n s o r y a x o n s m u s t all penetrate the C N S at the p r o p e r C N S entry p o i n t i f they are to f o r m tight p e r i p h e r a l n e r v e r o o t b u n d l e s that c a n b e e n s h e a t h e d together b y o n l y o n e o r t w o p e r i p h e r a l g l i a , as they are in w i l d types. F u r t h e r m o r e , i f they are to p a t h f i n d i n the C N S to their a p p r o p r i a t e s y n a p s e targets, t h e y m u s t b e g i n their C N S m i g r a t i o n s at the c o r r e c t l o c a t i o n . F o r instance, i n cases o f p e r i p h e r a l glial loss, s e n s o r y a x o n s entered the C N S  too f a r anteriorly s u c h that they  e x t e n d e d to C N S targets o n e s e g m e n t m o r e anterior than the o n e they o r i g i n a t e d f r o m . T h e r e are a n u m b e r o f w a y s b y w h i c h the g l i a c o u l d g u i d e a x o n s to their appropriate entry p o i n t i n the C N S / P N S b o u n d a r y .  In Drosophila, a x o n  guidance  m o l e c u l e s k n o w n to be e x p r e s s e d b y g l i a i n c l u d e netrin a n d slit ( R o t h b e r g et al., 1990; H a r r i s et al., 1996; M i t c h e l l et al., 1996). T h e s e m o l e c u l e s are a l l e x p r e s s e d b y the m i d l i n e g l i a w h i c h are a v e r y s p e c i a l i z e d g l i a d e r i v e d f r o m the m e s e c t o d e r m a n d t h e r e f o r e m a y not b e a v e r y a n a l o g o u s c e l l to the p e r i p h e r a l g l i a ( K l a m b t et al., 1991). Interestingly, n o a x o n g u i d a n c e m o l e c u l e s s p e c i f i c to any other subset o f g l i a i n Drosophila h a v e b e e n f o u n d . P e r h a p s p e r i p h e r a l g l i a d o not e x p r e s s a n y m o l e c u l e s w h i c h alter s e n s o r y n e u r o n a l g r o w t h c o n e d e c i s i o n s at a l l . T h i s c o u l d easily b e the case, as s e n s o r y a x o n s i n g e n e r a l d o not m a k e alterations i n their s t e e r i n g t o w a r d s the C N S w h e n they contact the p e r i p h e r a l glia.  118  Instead, p e r i p h e r a l glial cells m a y s i m p l y e x p r e s s a d h e s i o n m o l e c u l e s o n their s u r f a c e s w h i c h b i n d m o l e c u l e s o n the s u r f a c e s o f s e n s o r y axons. In s u c h a s c e n a r i o , the centrally m i g r a t i n g s e n s o r y a x o n s w o u l d h a v e affinity f o r the p e r i p h e r a l l y m i g r a t i n g glia, w h o s e initial m i g r a t o r y path has i n turn b e e n d e t e r m i n e d b y m o t o r axons. A s the p e r i p h e r a l glia migrate as an u n b r o k e n c h a i n o f cells into the p e r i p h e r y , the entire route o f s e n s o r y a x o n m i g r a t i o n to the C N S  w o u l d be l a b e l e d w i t h p e r i p h e r a l glial p r o c e s s e s e x p r e s s i n g the  sensory axon-recognizing adhesion molecule. T h e r e are a variety o f cell a d h e s i o n m o l e c u l e s identified i n Drosophila w h i c h c o u l d w o r k in such a model.  T h e h o m o p h i l i c a d h e s i o n m o l e c u l e , C o n n e c t i n , is a v e r y likely  c a n d i d a t e f o r m e d i a t i n g p e r i p h e r a l glial m i g r a t i o n into the p e r i p h e r y . expressed on motor neurons and peripheral glia during embryogenesis.  C o n n e c t i n is T h r o u g h ectopic  C o n n e c t i n e x p r e s s i o n s t u d i e s , it is k n o w n that C o n n e c t i n e x p r e s s i o n c a n c a u s e  axon  m i s g u i d a n c e a n d has an important r o l e i n cell-cell a d h e s i o n ( R a g h a v a n a n d W h i t e , 1997). A s p e r i p h e r a l g l i a f o l l o w m o t o r a x o n s i n t o the p e r i p h e r y w h i l e they e x p r e s s C o n n e c t i n , this m o l e c u l e c o u l d c o n f e r affinity f o r the P N S  pathways  w h i c h the g l i a s u b s e q u e n t l y  ensheathe. S h o r t l y after glial m i g r a t i o n i n t o the P N S , s e n s o r y a x o n s c o n t a c t p e r i p h e r a l glial cells d u r i n g their m i g r a t i o n s to the C N S .  T h e s e n s o r y a x o n s m u s t c o r r e c t l y migrate  w i t h i n the i n n e r sheathes o f the g l i a i n o r d e r to enter the C N S  s u c c e s s f u l l y . A s the  C o n n e c t i n n u l l mutant is viable, it is l i k e l y that other m o l e c u l e s a l s o e x i s t w h i c h o v e r l a p i n f u n c t i o n w i t h C o n n e c t i n ( R a g h a v a n a n d W h i t e , 1997). T o a s s o c i a t e w i t h p e r i p h e r a l glia, s e n s o r y a x o n s m i g h t r e c o g n i z e the N e u r o g l i a n a d h e s i o n m o l e c u l e , w h i c h is e x p r e s s e d b y b o t h s e n s o r y n e u r o n s a n d p e r i p h e r a l g l i a a n d is t h o u g h t to h a v e i m p o r t a n t r o l e s i n n e u r o n a l - g l i a l a d h e s i o n ( B i e b e r et al., 1989). neuroglian mutant  The  s h o w s a d i s o r g a n i z a t i o n o f s e n s o r y n e u r o n a l cell b o d i e s , as d o  p e r i p h e r a l g l i a l - a b l a t e d e m b r y o s . U n f o r t u n a t e l y , the s e n s o r y a x o n p a t t e r n i n g o f neuroglian n u l l mutants has n e v e r been p r e s e n t e d . A detailed e x a m i n a t i o n o f s e n s o r y a x o n m i g r a t i o n in the neuroglian mutant s h o u l d be d o n e to assess i f this m o l e c u l e is i m p o r t a n t i n s e n s o r y  119  a x o n r e c o g n i t i o n o f g l i a l p r o c e s s e s . It is p o s s i b l e t h o u g h , that there are o v e r l a p p i n g roles for cell r e c o g n i t i o n i n Drosophila. F o r instance, both neuroglias, a n d connectin a p p e a r to be regulated b y engrailed e x p r e s s i o n . C e l l s w h i c h s t r o n g l y e x p r e s s connectin a l s o h a v e h i g h neuroglian e x p r e s s i o n a n d v i c e versa. W h e n connectin a n d neuroglian e x p r e s s i o n is repressed b y u b i q u i t o u s engrailed e x p r e s s i o n i n the C N S , s e n s o r y a x o n s s o m e t i m e s f a i l to c r o s s the C N S / P N S b o r d e r c o r r e c t l y as i n the glial-ablated a n d gem e m b r y o s ( S i e g l e r a n d Jia, 1999). T h e r e f o r e , altering e x p r e s s i o n b o t h o f these g e n e s s i m u l t a n e o u s l y causes a w o r s e p h e n o t y p e than i n the s i n g l e m u t a n t s a n d suggests that the t w o g e n e s c o u l d cooperate i n t h e i r d e v e l o p m e n t a l roles.  Peripheral glial migration into the PNS.  P e r i p h e r a l g l i a migrate a l o n g a x o n a l p r o c e s s e s into the P N S . P e r h a p s p e r i p h e r a l g l i a require a x o n s as a m i g r a t i o n a l substrate. C o u l d p e r i p h e r a l g l i a migrate t o places w h e r e there are n o a x o n s ? U n l e s s a x o n s w o u l d s u b s e q u e n t l y f o l l o w p e r i p h e r a l g l i a to r e g i o n s w h e r e there a r e n o n e u r o n s , there w o u l d b e n o p r a c t i c a l u s e f o r g l i a t o e x i s t a n y w h e r e alone.  T h e r e are n u m e r o u s reasons w h y glial d e p e n d e n c e o n n e u r o n s f o r u s e as a  m i g r a t i o n a l substrate i s b e n e f i c i a l . G l i a w o u l d a l w a y s b e p h y s i c a l l y a s s o c i a t e d w i t h n e u r o n s w h i c h is important i f g l i a act as n e u r o n a l s u p p o r t cells. A l s o , i f g l i a f o l l o w rather than lead n e u r o n s into the P N S , then n e u r o n a l g r o w t h c o n e s w o u l d b e able t o steer a n d p r o b e p o t e n t i a l m u s c l e targets without a n y interference f r o m g l i a . F i n a l l y , glial cells m i g h t not require a n y extra g u i d a n c e i n f o r m a t i o n to p a t h f i n d to the P N S i f they are s i m p l y to f o l l o w routes p i o n e e r e d b y a x o n s . T h i s w o u l d reduce the potential f o r glial m i g r a t i o n a l errors. T h e r e is m o s t l i k e l y a large n e t w o r k o f s i g n a l i n g p a t h w a y s w h i c h c o n t r o l p e r i p h e r a l glial m i g r a t i o n into the P N S . F i r s t , p e r i p h e r a l g l i a m o s t l i k e l y r e c e i v e t r o p h i c s u p p o r t  120  signals f r o m neurons.  P r e v i o u s w o r k has s h o w n that i n the Drosophila C N S  the g l i a to  n e u r o n ratios are tightly regulated. In m u t a n t s w h i c h l a c k axons, glial cells w h i c h w o u l d n o r m a l l y h a v e a s s o c i a t e d w i t h the a x o n s decrease i n n u m b e r v i a apoptotic  death  ( S o n n e n f e l d a n d Jacobs, 1995b). It is c o n c e i v a b l e that p e r i p h e r a l g l i a are attracted into the PNS  to g a i n t r o p h i c s u p p o r t s i g n a l s f r o m the s u r f a c e s o f e x t e n d i n g axons.  Trophic  s u p p o r t c a n be essential f o r cell m i g r a t i o n i n Drosophila. F o r e x a m p l e , mutation o f the b a s i c f i b r o b l a s t g r o w t h f a c t o r ( b F G F ) receptor breathless causes d i s r u p t i o n o f tracheal cell m i g r a t i o n n e c e s s a r y f o r f o r m a t i o n o f the tracheal system. T h e inability o f c e l l s to m i g r a t e is likely due to a l o s s o f s t i m u l a t i o n o f the R a s / R a f / M A P K s i g n a l i n g c a s c a d e w h i c h is d o w n s t r e a m o f the breathless receptor. T h i s c a s c a d e w o u l d l i k e l y d r i v e e x p r e s s i o n o f g e n e s that are d i r e c t l y r e q u i r e d f o r tracheal cell m i g r a t i o n ( K l a m b t et al., 1992). R a s s i g n a l i n g is a l s o v e r y l i k e l y to be i n v o l v e d i n p e r i p h e r a l glial m i g r a t i o n into the PNS.  P e r i p h e r a l glial cells e x p r e s s the E t s t r a n s c r i p t i o n f a c t o r pointed d u r i n g their  m i g r a t i o n into the P N S  ( K l a e s et a l . , 1994).  T h e activity o f pointed is generated v i a  phosphorylation by M A P  k i n a s e w h i c h is e n c o d e d b y rolled ( B r u n n e r et a l . , 1994).  Since  pointed m u t a n t s fail to differentiate p r o p e r l y , it is l i k e l y that g e n e s r e q u i r e d f o r the later  m i g r a t i o n a l p h a s e o f glial d e v e l o p m e n t are not p r o p e r l y r e g u l a t e d ( K l a e s et a l . , 1994). T h i s c o u l d be s i m i l a r to the d e v e l o p i n g Drosophila eye, w h e r e retinal b a s a l g l i a r e q u i r e p r o p e r R a s s i g n a l i n g f o r their m i g r a t i o n .  I f a d o m i n a n t negative f o r m o f R a s is e c t o p i c a l l y  e x p r e s s e d i n the glia, they fail to migrate into the e y e d i s c ( R a n g a r a j a n et al., 1999). It is u n c l e a r w h a t f a c t o r s m a y initially stimulate the R a s s i g n a l i n g c a s c a d e .  T h e Drosophila  e p i d e r m a l g r o w t h f a c t o r ( E G F ) receptor t y r o s i n e k i n a s e is o n e v e r y l i k e l y candidate. EGF  The  receptor is e x p r e s s e d w i d e l y i n the d e v e l o p i n g e m b r y o a n d is i m p l i c a t e d in  e s t a b l i s h i n g cell fates a l o n g w i t h m a n y other d e v e l o p m e n t a l p r o c e s s e s . T h e s p e c i f i c i t y o f E G F receptor f u n c t i o n i n a g i v e n c e l l i s d e t e r m i n e d b y the p a r t i c u l a r l i g a n d s u c h as spitz, gurken, o r vein, w h i c h b i n d s the receptor. T h e r e are a l s o other r e g u l a t o r y p r o t e i n s s u c h as  121  star, rho, a n d argos w h i c h m o d i f y activity o f the r e c e p t o r i n a t i s s u e s p e c i f i c m a n n e r  ( r e v i e w e d b y P e r r i m o n a n d P e r k i n s , 1997). Interestingly, vertebrate S c h w a n n cells e x p r e s s erbB E G F r e c e p t o r s w h i c h are r e q u i r e d f o r their m i g r a t i o n i n t o the p e r i p h e r y ( W o l d e y e s u s et al., 1999). T h e i r l i g a n d , N e u r e g u l i n , is e x p r e s s e d b y n e u r o n s a n d is v e r y s i m i l a r t o the Drosophila E G F receptor l i g a n d , V e i n ( S c h n e p p et al., 1996). H o w e v e r , i n Drosophila, V e i n p r o t e i n e x p r e s s i o n h a s o n l y b e e n d e t e c t e d i n m y o t u b e s . It is secreted a n d accumulates at m u s c l e - t e n d o n j u n c t i o n s w h e r e it l i k e l y b i n d s E G F receptors o n m u s c l e s , thereby m e d i a t i n g the attachment o f m u s c l e to t e n d o n ( Y a r n i t z k y et al., 1997). T h e r e f o r e , it is u n c e r t a i n at this time whether m i g r a t i o n s o f p e r i p h e r a l g l i a into the P N S c o u l d b e a n a l o g o u s l y d e p e n d e n t  on E G F  r e c e p t o r s i g n a l i n g as S c h w a n n c e l l s are. D u r i n g m i g r a t i o n into the P N S , large s c a l e rearrangements o f the p e r i p h e r a l glial cytoskeleton must occur.  P e r i p h e r a l glial cell b o d i e s m o v e p e r i p h e r a l l y as the cell  processes elongate. I n m a n y different types o f cells, the r h o family G T P a s e signaling m o l e c u l e s c o n t r o l m a n y aspects o f actin c y t o s k e l e t a l rearrangements i n o r d e r t o a c h i e v e motility. T h e s e p r o t e i n s i n c l u d e rho, rac, a n d c d c 4 2 a n d they regulate t h e f o r m a t i o n o f actin stress fibres, l a m e l l i p o d i a , a n d f i l o p o d i a r e s p e c t i v e l y ( N o b e s a n d H a l l , 1995). W i t h i n the Drosophila n e r v o u s s y s t e m , the R a c h o m o l o g u e D r a c l i s r e q u i r e d f o r a x o n a l outgrowth.  T h e c d c 4 2 h o m o l o g u e D c d c 4 2 a l s o i s t h o u g h t t o a f f e c t cell p o s i t i o n i n g a n d  aspects o f a x o n a l o u t g r o w t h ( L u o et al., 1994).  D i s r u p t i o n o f the actin d y n a m i c s v i a  e c t o p i c e x p r e s s i o n o f d o m i n a n t n e g a t i v e D r a c l leads to d e f e c t i v e m o t o r a x o n p a t h f i n d i n g as w e l l ( K a u f m a n n et al., 1998). T h e r o l e s o f r h o G T P a s e s h a v e n o t b e e n a s s e s s e d i n glial cells, h o w e v e r , i t w o u l d b e l i k e l y that g l i a w o u l d a l s o u s e these p r o t e i n s f o r m i g r a t i o n . Rho  G T P a s e s c o u l d enable glial d i r e c t i o n a l m i g r a t i o n t o o c c u r a l o n g a x o n a l  p r o c e s s e s b y s t a b i l i z i n g the l e a d i n g p o r t i o n o f the c y t o p l a s m i c p r o c e s s e s v i a actin cytoskeletal a s s e m b l y a n d d i s a s s e m b l i n g the actin n e t w o r k o n the trailing p r o c e s s e s . T h e p r i m a r y s o u r c e o f glial m i g r a t i o n a l directionality c o u l d b e i m p a r t e d b y bare p e r i p h e r a l nerve  122  a x o n s w h i c h m a y e x p r e s s t r o p h i c s u p p o r t f a c t o r s f o r glia. S u p p o r t f o r this h y p o t h e s i s c o m e s f r o m o b s e r v a t i o n that C N S - d e r i v e d p e r i p h e r a l glial c e l l s m i g r a t e p e r i p h e r a l l y a l o n g a x o n s o n l y until t h e y m e e t u p w i t h the g l i a l c e l l p r o c e s s e s o f the dorsolateral p e r i p h e r a l glia w h i c h i s b o r n i n the P N S ( C h a p t e r III).  Glial ensheathement of peripheral nerves.  . T h e final m o r p h o g e n i c p r o c e s s f o r p e r i p h e r a l glial cells i s the e n s h e a t h e m e n t  of  p e r i p h e r a l n e r v e s to f o r m the b l o o d - n e r v e - b a r r i e r . P r o p e r e n s h e a t h e m e n t o f n e r v e s is o n e o f the m o s t i m p o r t a n t f u n c t i o n s o f p e r i p h e r a l g l i a l cells. If there is e v e n j u s t a slight l e a k i n the barrier, n e r v e s get e x p o s e d to the h a e m o l y m p h w h i c h is h i g h i n K  +  i o n s . A s a result,  the n e u r o n s s u f f e r f r o m deficient a x o n p o t e n t i a l p r o p a g a t i o n l e a d i n g to p a r a l y s i s a n d e v e n t u a l death o f the e m b r y o ( A u l d et al.,1995). T h e r e are l i k e l y a n u m b e r o f p r o c e s s e s that c o o p e r a t e to f o r m a glial sheath. F i r s t l y , the p e r i p h e r a l g l i a m u s t e x t e n d their cell p r o c e s s e s a l o n g the entire l e n g t h s o f bare peripheral n e r v e s . I f p e r i p h e r a l n e r v e s s u p p l y t r o p h i c s u p p o r t to g l i a d u r i n g d e v e l o p m e n t , t h e n g l i a w o u l d naturally be i n d u c e d to contact a l l a v a i l a b l e a x o n s u r f a c e s . A t the s a m e time, p e r i p h e r a l g l i a m u s t tightly c o n t a c t each other.  G l i a - g l i a c o n t a c t s c o u l d b e m e d i a t e d t h r o u g h cell a d h e s i o n m o l e c u l e s s u c h as  N e u r o g l i a n , G l i o t a c t i n , a n d N e u r e x i n ( B i e b e r et al., 1989; A u l d et al., 1995; B a u m g a r t n e r et al., 1996). In b o t h vertebrates a n d invertebrates, i m m u n o g l o b u l i n (Ig) s u p e r f a m i l y p r o t e i n s s u c h as L I , M A G ,  W r a p p e r , a n d R E G A - 1 are n o t e d f o r their r o l e s i n glial ensheathement  ( O w e n s et al., 1990; W o o d et al., 1990; S e a v e r et al., 1996; N o o r d e r m e e r et al., 1998). As  N e u r o g l i a n i s a l s o a m e m b e r o f the I g f a m i l y a n d i s v e r y s i m i l a r to vertebrate L I  ( B i e b e r et al., 1989), it is h i g h l y likely that it p l a y s a s i m i l a r r o l e i n  Drosophila.  Its  e x p r e s s i o n o n b o t h n e u r o n s a n d p e r i p h e r a l g l i a s u g g e s t s that it c o u l d m e d i a t e the b i n d i n g o f glial cells a r o u n d the s u r f a c e s o f n e r v e b u n d l e s . G l i o t a c t i n a n d N e u r e x i n are b o t h t h o u g h t  123  to c o n t r i b u t e to the p e r i p h e r a l glial sheath b y h e l p i n g to f o r m septate j u n c t i o n s w h i c h tightly seal glial p r o c e s s e s together ( A u l d et al., 1995; B a u m g a r t n e r  et a l . , 1996).  These  structures p r o b a b l y f o r m as the f i n a l step i n glial ensheathement, s i n c e glial cells m u s t first s u r r o u n d the n e r v e b u n d l e w i t h their p r o c e s s e s b e f o r e they c a n seal together w i t h septate j u n c t i o n s . T h e r e f o r e , there are l i k e l y m u l t i p l e different aspects o f a d h e s i v i t y w h i c h m u s t b e r e g u l a t e d d u r i n g n e r v e ensheathement. Blood-nerve-barrier morphogenesis.  formation  i s the ultimate g o a l  o f p e r i p h e r a l glial  H o w e v e r , f o r this t o o c c u r , p r o p e r ensheathement o f n e r v e s m u s t b e g i n  relatively e a r l y d u r i n g p e r i p h e r a l glial p r o c e s s e x t e n s i o n . T h e r o l e o f p e r i p h e r a l g l i a as m e d i a t o r s o f n e u r o n a l m i g r a t i o n a c r o s s the C N S / P N S b o r d e r ties i n w i t h o v e r a l l glial e n s h e a t h e m e n t o f p e r i p h e r a l nerves. P e r i p h e r a l g l i a a b s o l u t e l y m u s t g u i d e all a x o n s a c r o s s a d e s i g n a t e d r e g i o n o f the C N S b o u n d a r y i f they are later t o b e able t o w r a p the c i r c u m f e r e n c e o f the n e r v e s w i t h o n l y a s i n g l e glial c e l l . T h i s m a y b e the m a i n r e a s o n w h y peripheral glia have axonal pathfinding roles during embryogenesis.  Trophic interactions between peripheral glia and peripheral neurons.  Neurons  a n d g l i a are o b s e r v e d t o h a v e  trophic interdependence  during  d e v e l o p m e n t . I n d e v e l o p m e n t , l a r g e r n u m b e r s o f n e u r o n s a n d g l i a are g e n e r a t e d i n greater n u m b e r s than are n e c e s s a r y .  O v e r time, a f r a c t i o n o f the n e u r o n s a n d g l i a d i e v i a  a p o p t o s i s . T h e cells w h i c h d o n o t s u r v i v e are o n e s w h i c h h a v e b e e n d e p r i v e d o f t r o p h i c s u p p o r t ( R a f f et al., 1993; B e r g e r o n a n d Y u a n , 1998).  I f glia depend o n neurons f o r  s u r v i v a l a n d v i c e v e r s a , there w o u l d n e v e r b e r e g i o n s w h e r e either o f these cell types w o u l d e x i s t a l o n e . T h u s t r o p h i c i n t e r d e p e n d e n c e i s a n e x c e l l e n t m e a n s o f e n s u r i n g that n e u r o n s a l w a y s h a v e glial w r a p p i n g a n d that glial cells are a l w a y s p u t t o g o o d u s e .  124  Competition amongst different glia for neuronal-derived trophic support w o u l d also ensure that e v e n s p a c i n g o f g l i a is a c h i e v e d w h i c h is o p t i m a l f o r m a t u r e n e u r o n a l f u n c t i o n . In the current study, there are a n u m b e r o f lines o f e v i d e n c e w h i c h suggest that peripheral g l i a r e c e i v e n e u r o n a l - d e r i v e d t r o p h i c support.  P e r i p h e r a l g l i a migrate into the  P N S in constant c o n t a c t w i t h peripheral nerves. B y the e n d o f e m b r y o g e n e s i s , peripheral glial cells are e v e n l y s p a c e d a l o n g p e r i p h e r a l nerves.  F i n a l l y , p e r i p h e r a l g l i a are v e r y  robust cells d u r i n g their d e v e l o p m e n t , as the apoptotic cell death p r o g r a m is d i f f i c u l t to initiate b y ectopic e x p r e s s i o n o f cell death genes. O n l y ectopic e x p r e s s i o n o f the m o s t potent apoptosis g e n e constructs c o u l d i n d u c e a c o m p l e t e l o s s o f p e r i p h e r a l g l i a . It also is l i k e l y that p e r i p h e r a l g l i a i n turn o f f e r t r o p h i c s u p p o r t f o r m i g r a t i n g n e u r o n s , s i n c e n e u r o n s p h y s i c a l l y contact the g l i a d u r i n g their m i g r a t i o n a n d w o u l d benefit f r o m p o s i t i v e r e i n f o r c e m e n t o f their correct m i g r a t i o n a l p a t h w a y c h o i c e . A l s o , i n p e r i p h e r a l glial-ablated e m b r y o s , n e u r o n s stall, h a v e d i f f i c u l t y p r o j e c t i n g a x o n s , or are lost altogether. T h e r e are no t r o p h i c factors i d e n t i f i e d so f a r i n Drosophila w h i c h m a y be r e s p o n s i b l e f o r the effects o b s e r v e d here. In the future, large scale screens to isolate m u t a n t lines w h o s e e m b r y o s s h o w d i f f i c u l t y i n a x o n e x t e n s i o n m a y result i n the c l o n i n g o f t r o p h i c f a c t o r genes.  Conclusion P e r i p h e r a l g l i a are essential f o r the p r o p e r f o r m a t i o n o f the e m b r y o n i c peripheral nervous system.  T h r o u g h anatomical a n d l o s s o f f u n c t i o n studies, it is s h o w n that  peripheral g l i a p r o v i d e g u i d a n c e f o r n e u r o n a l m i g r a t i o n a c r o s s the C N S / P N S border. I n addition, p e r i p h e r a l g l i a l i k e l y express factors w h i c h p r o m o t e s e n s o r y a x o n e x t e n s i o n  and  s u r v i v a l . P e r i p h e r a l g l i a use p r e f o r m e d neuronal p a t h w a y s to migrate into the P N S  and  likely d e p e n d o n these substrates f o r peripheral m i g r a t i o n s .  The physical and functional  similarities b e t w e e n p e r i p h e r a l g l i a a n d S c h w a n n cells suggests that Drosophila m a y be an e x c e l l e n t m o d e l o r g a n i s m to study m a n y aspects o f P N S glial d e v e l o p m e n t i n the future.  125  VI.  REFERENCES  A l b a g l i , O., K l a e s , A., F e r r e i r a , E., L e p r i n c e , D., K l a m b t , C. ( 1 9 9 6 ) F u n c t i o n o f ets g e n e s is c o n s e r v e d b e t w e e n vertebrates a n d Drosophila. Mech. Dev. 59:29-40. A n t o n i c e k , H., P e r s o h n , E., S c h a c h n e r , M . ( 1 9 8 7 ) B i o c h e m i c a l a n d f u n c t i o n a l c h a r a c t e r i z a t i o n o f a n o v e l n e u r o n - g l i a a d h e s i o n m o l e c u l e that is i n v o l v e d i n n e u r o n a l m i g r a t i o n . J. C e l l B i o l . 104:1587-1595.  A r e n d t , D., N u b l e r - J u n g , K. ( 1 9 9 9 ) C o m p a r i s o n o f e a r l y n e r v e c o r d d e v e l o p m e n t i n insects a n d vertebrates. Development 126:2309-2325.  A r t a v a n i s - T s a k o n a s , S., M a t s u n o , K., F o r t i n i , M.E.  ( 1 9 9 5 ) N o t c h s i g n a l i n g . Science  268:225-232  A u l d , V.J., Fetter, R.D.,  B r o a d i e , K., G o o d m a n , C S .  (1995) Gliotactin, a novel  t r a n s m e m b r a n e p r o t e i n o n p e r i p h e r a l g l i a , is r e q u i r e d to f o r m the b l o o d - n e r v e b a r r i e r i n D r o s o p h i l a . Cell 81:757-767.  Auld, V.J.  ( 1 9 9 9 ) G l i a as mediators o f g r o w t h c o n e guidance: s t u d i e s f r o m insect  n e r v o u s systems. Cell. Mol. Life Sci. 5 5 : 1 3 7 7 - 1 3 8 5 .  B a s t i a n i , M.J.,  Goodman, CS.  ( 1 9 8 6 ) G u i d a n c e o f n e u r o n a l g r o w t h c o n e s i n the  g r a s s h o p p e r e m b r y o . III. R e c o g n i t i o n o f s p e c i f i c glial p a t h w a y s . J. Neurosci. 6:35423551.  126  B a u m g a r t n e r , S., L i t t l e t o n , J.T., B r o a d i e , K., B h a t , M.A., H a r b e c k e , R., L e n g y e l , J.A., C h i q u e t - E h r i s m a n n , R., P r o k o p , A . B e l l e n , H. ( 1 9 9 6 ) A D r o s o p h i l a n e u r e x i n is r e q u i r e d f o r septate j u n c t i o n a n d b l o o d - n e r v e b a r r i e r f o r m a t i o n a n d f u n c t i o n . Cell 87:1059-1068.  B e l l e n , H.J., O ' K a n e , C.J., W i l s o n , C , G r o s s n i k l a u s , U., P e a r s o n , R.K., W.J.  ( 1 9 8 9 ) P-element  m e d i a t e d e n h a n c e r detection:  Gehring,  a versatile m e t h o d to study  d e v e l o p m e n t i n Drosophila. Genes Dev. 3:1288-1300.  B e l l e n , H.J., W i l s o n , C., G e h r i n g , W.J.  ( 1 9 9 0 ) D i s s e c t i n g the c o m p l e x i t y o f the  n e r v o u s s y s t e m b y e n h a n c e r d e t e c t i o n . BioEssays 12:199-204.  B e r g e r o n , L., Y u a n , J. ( 1 9 9 8 ) S e a l i n g one's fate: c o n t r o l o f cell death i n n e u r o n s . Curr. Opin. Neurobiol. 8:55-63.  B e r n a r d o n i , R., M i l l e r , A.A.,  Giangrande, A.  ( 1 9 9 8 ) G l i a l differentiation d o e s not  r e q u i r e a n e u r a l g r o u n d state. Development 125:3189-3200.  B e r n a r d o n i , R., K a m m e r e r , M., V o n e s c h , J-L., G i a n g r a n d e , A . d e p e n d s o n glide/gem t h r o u g h a s y m m e t r i c  (1999)  division of neuroglioblasts.  Gliogenesis Dev.  Biol.  F.R. J a c o b s , T r a q u i n a ,  Z.R.,  216:265-275.  B i e b e r , A.J., S n o w , P.M.,  H o r t s c h , M., P a t e l , N.H.,  S c h i l l i n g , J., G o o d m a n , C.S. Immunoglobulin  (1989)  D r o s o p h i l a N e u r o g l i a n : A m e m b e r o f the  s u p e r f a m i l y w i t h e x t e n s i v e h o m o l o g y to the vertebrate n e r u a l a d h e s i o n  m o l e c u l e L l . Cell 59:447-460.  127  B i e r , E., V a e s s i n , H., S h e p h e r d , S., L e e , K., M c C a l l , K., B a r b e l , S., A c k e r m a n , L., R. Carretto, U e m u r a , T., G r e l l , E., Jan, L., Jan, Y . N .  ( 1 9 8 9 ) S e a r c h i n g f o r pattern a n d  m u t a t i o n i n the Drosophila g e n o m e w i t h a P-LacZ vector. Genes Dev. 3:1273-1287.  Birling M.C,  P r i c e , J. ( 1 9 9 5 ) I n f l u e n c e o f g r o w t h f a c t o r s o n n e u r o n a l differentiation.  Curr. Opin. Cell Biol. 7:878-884.  B o d m e r , R., Jan, Y.N.  ( 1 9 8 7 ) M o r p h o l o g i c a l differentiation o f the e m b r y o n i c p e r i p h e r a l  n e u r o n s i n D r o s o p h i l a . Roux' Arch. Dev. Biol. 196:69-77.  B o o t h , G.E.,  K i n r a d e , E.F.V., H i d a l g o , A .  (2000) G l i a maintain follower neuron  s u r v i v a l d u r i n g Drosophila C N S d e v e l o p m e n t . Development 127:127-244.  B r a n d , A.H., P e r r i m o n , N . ( 1 9 9 3 ) T a r g e t e d g e n e e x p r e s s i o n as a m e a n s o f altering cell fates a n d g e n e r a t i n g d o m i n a n t p h e n o t y p e s . Development 118:401-415.  B r u n n e r , D., D u c k e r , K., O e l l e r s , N., H a f e n , E., S c h o l z , H., K l a m b t , C. ( 1 9 9 4 ) T h e E T S d o m a i n p r o t e i n p o i n t e d - P 2 is a target o f M A P  k i n a s e i n the s e v e n l e s s s i g n a l  t r a n s d u c t i o n p a t h w a y . Nature 370:386-389.  B u r d e n , S., Y a r d e n , Y.  ( 1 9 9 7 ) N e u r e g u l i n s a n d their receptors: A v e r s a t i l e s i g n a l i n g  m o d u l e i n o r g a n o g e n e s i s a n d o n c o g e n e s i s . Neuron 18:847-855.  C a m p b e l l , G., G o r i n g , H., L i n , T., S p a n a , E., A n d e r s s o n , S., D o e , C.Q., T o m l i n s o n , A . (1994) R K 2 , a glial-specific h o m e o d o m a i n protein required f o r embryonic nerve c o r d c o n d e n s a t i o n a n d v i a b i l i t y i n Drosophila. Development 120:2957-2966.  128  C a r p e n t e r , E.M.,  Hollyday, M.  ( 1 9 9 2 ) T h e l o c a t i o n a n d d i s t r i b u t i o n o f n e u r a l crest-  d e r i v e d S c h w a n n cells i n d e v e l o p i n g p e r i p h e r a l nerves in the c h i c k f o r e l i m b . Dev. Biol. 150:144-159.  C h o i , K-W.,  B e n z e r , S.  ( 1 9 9 4 ) M i g r a t i o n o f g l i a a l o n g p h o t o r e c e p t o r a x o n s i n the  d e v e l o p i n g D r o s o p h i l a eye. Neuron 12:423-431.  C h u , L.Q., W r i g h t , D.M.,  M c N e i l , L.K., D o e , C.Q.  ( 1 9 9 1 ) T h e prospero g e n e e n c o d e s  a d i v e r g e n t h o m e o d o m a i n p r o t e i n that c o n t r o l s n e u r o n a l  identity i n Drosophila.  Development 2:79-85.  C o n d r o n , B.G.,  P a t e l , N.H.,  Z i n n , K.  ( 1 9 9 4 ) engrailed c o n t r o l s g l i a l / n e u r o n a l cell fate  d e c i s i o n s at the m i d l i n e o f the c e n t r a l n e r v o u s system. Neuron 13:541.  D a v i d s o n , D.R.,  H i l l , R.E.  ( 1 9 9 1 ) Msh-Wke genes: a f a m i l y o f h o m e o b o x g e n e s w i t h  w i d e - r a n g i n g e x p r e s s i o n d u r i n g v e r t e b r a t e d e v e l o p m e n t . Sem. Dev. Biol. 2:405-412.  D a v i s , A.D., W e a t h e r b y , T.M.,  H a r t l i n e , D.K.,  L e n z , P.H.  ( 1 9 9 9 ) M y e l i n - l i k e sheaths  i n c o p e p o d axons. Nature 398:571.  D a v i s , G.W.,  Schuster, CM.,  Goodman, CS.  (1997)  G e n e t i c a n a l y s i s o f the  m e c h a n i s m s c o n t r o l l i n g target selection: target-derived F a s c i c l i n II regulates the pattern o f s y n a p s e f o r m a t i o n . Neuron 19:561-573.  D e W a e g h , S.M.,  L e e , V.M-Y., B r a d y , S.T.  (1992) L o c a l modulation of neurofilament  p h o s p h o r y l a t i o n , a x o n a l caliber, a n d s l o w a x o n a l transport b y m y e l i n a t i n g S c h w a n n cells. Cell 6 8 : 4 5 1 - 4 6 3 .  129  D o e , C.Q.,  C h u - L a G r a f f , Z., W r i g h t , D.M.,  S c o t t , M.P.  ( 1 9 9 1 ) T h e prospero g e n e  s p e c i f i e s c e l l fates i n the Drosophila c e n t r a l n e r v o u s system. C e l l 65:451-464.  D r a y , T. G l o o r , G.B.  (1997)  H o m o l o g y requirements for targeting heterologous  s e q u e n c e s d u r i n g P - i n d u c e d g a p r e p a i r i n Drosophila melanogaster. Genetics 147:689699.  E i n h e b e r , S., Z a n a z z i , G., C h i n g , W., S c h e r e r , S., M i l n e r , T.A., P e l e s , E., S a l z e r , J . L . (1997) The axonal m e m b r a n e protein Caspr, a.homologue of Neurexin IV, is a component o f the septate-like p a r a n o d a l j u n c t i o n s that a s s e m b l e d u r i n g m y e l i n a t i o n . J. C e l l B i o l . 139:1495-1506.  E n g e l s , W.R.,  J o h n s o n - S c h l i t z , D.M.,  E g g l e s t o n , W.B.,  S v e d , J.  (1990)  High-  f r e q u e n c y P e l e m e n t l o s s i n Drosophila is h o m o l o g dependent. Cell 62:515-525.  E n g e l s , W.R.,  P r e s t o n , C.R., J o h n s o n - S c h l i t z , D.M.  (1994) Long-range cis preference  i n D N A h o m o l o g y s e a r c h o v e r the l e n g t h o f a. Drosophila c h r o m o s o m e . Science 2 6 3 : 1 6 2 3 1625.  E n g e l s , W.R.  ( 1 9 9 6 ) Transposable Elements (ed. H. S a e d l e r a n d A . G i e r l . ) , pp.  103-  123. S p r i n g e r - V e r l a g , B e r l i n .  E n g l u n d , C , U v , A.E., C a n t e r a , R., M a t h i e s , L.D., K r a s n o w , M.A.,  S a m a k o v l i s , C.  ( 1 9 9 9 ) adrift, a n o v e l b n l - i n d u c e d Drosophila gene, r e q u i r e d f o r t r a c h e a l p a t h f i n d i n g i n t o the C N S . Development 126:1505-1514.  130  F r e d i e u , J.R., M a h o w a l d , A . P . ( 1 9 8 9 ) G l i a l i n t e r a c t i o n s w i t h n e u r o n s d u r i n g Drosophila e m b r y o g e n e s i s . Development 106:749-757.  F r a h e r , J.P.  (1997)  Axon-glial relationships i n early C N S - P N S  transitional z o n e  d e v e l o p m e n t : a n ultrastructural study. /. Neurocytol. 26:41-52.  F u j i t a , S. C , Z i p u r s k y , S.L., B e n z e r , S., F e r r u s , A., S h o t w e l l , S.L. ( 1 9 8 2 ) M o n o c l o n a l a n t i b o d i e s against the Drosophila n e r v o u s s y s t e m . Proc. Natl. Acad. Sci. USA 79:79297933.  G a r b e r n , J.Y., C a m b i , F., T a n g , X-M., S i m a , A.A.F., V a l l a t , J.M., B o s c h , E.P., L e w i s , R. et al. ( 1 9 9 7 ) P r o t e o l i p i d p r o t e i n is n e c e s s a r y i n p e r i p h e r a l as w e l l a s central m y e l i n . Neuron 19:205-218.  G e y e r , P.K., R i c h a r d s o n , K.L., C o r c e s , V.G., G r e e n , M . M .  ( 1 9 8 8 ) G e n e t i c instability  i n Drosophila melanogaster. P-element m u t a g e n e s i s b y g e n e c o n v e r s i o n .  Proc. Natl.  Acad. Sci. USA 8 5 : 6 4 5 5 - 6 4 5 9 .  G h o , M., B e l l a c h i e , Y., S c h w e i s g u t h , F. ( 1 9 9 9 ) R e v i s i t i n g the Drosophila m i c r o c h a e t e lineage: a n o v e l intrinsically a s y m m e t r i c cell d i v i s i o n generates a glial c e l l . Development 126:3573-3584.  G h y s e n , A., D a m b l y - C h a u d i e r e , C., A c e v e s , E., J a n , L., J a n , Y . n e u r o n s a n d p e r i p h e r a l p a t h w a y s i n Drosophila e m b r y o s .  (1986)  Sensory  Roux's Arch. Dev. Biol.  195:281-289.  131  G i a n g r a n d e , A., M u r r a y , M.A., P a l k a , J. ( 1 9 9 3 ) D e v e l o p m e n t a n d o r g a n i z a t i o n o f glial cells i n the p e r i p h e r a l n e r v o u s s y s t e m o f Development 117:895-904.  G i a n g r a n d e , A . ( 1 9 9 4 ) G l i a i n the f l y w i n g are c l o n a l l y related t o epithelial cells a n d u s e the n e r v e as a p a t h w a y for m i g r a t i o n . Development 120:523-534.  Giangrande, A.  (1995)  P r o n e u r a l g e n e s i n f l u e n c e g l i o g e n e s i s i n Drosophila.  Development 121:429-438.  G i e s e n , K., H u m m e l , T., S t o l l e w e r k , A., H a r r i s o n , S., T r a v e r s , A., K l a m b t , C . ( 1 9 9 7 ) G l i a l d e v e l o p m e n t i n the Drosophila C N S r e q u i r e s c o n c o m i t a n t activation o f glial a n d r e p r e s s i o n o f n e u r o n a l d i f f e r e n t i a t i o n genes. Development 124:2307-2311.  G l o o r , G.B., N a s s i f , N . A., J o h n s o n - S c h l i t z , D.M., P r e s t o n , C.R., E n g e l s , W.R. (1991)  T a r g e t e d g e n e r e p l a c e m e n t i n Drosophila v i a P e l e m e n t - i n d u c e d g a p repair.  Science 2 5 3 : 1 1 1 0 - 1 1 1 7 .  G l o o r , S., A n t o n i c e k , H., S w e a d n e r , K.J., P a g l i u s i , S., F r a n k , R., M o o s ,  M.,  S c h a c h n e r , M . ( 1 9 9 0 ) T h e a d h e s i o n m o l e c u l e o n g l i a ( A M O G ) is a h o m o l o g u e o f the beta s u b u n i t o f the N a , K - A T P a s e . J. C e l l B i o l . 110:165-174.  G o l d i n g , J., S h e w a n , D., C o h e n , J. ( 1 9 9 7 ) M a t u r a t i o n o f t h e m a m m a l i a n d o r s a l root entry z o n e - f r o m e n t r y t o n o entry. Trends Neurosci. 20:303-308.  G o l d i n g , J.P., C o h e n , J. ( 1 9 9 7 ) B o r d e r c o n t r o l s at the m a m m a l i a n s p i n a l c o r d : late s u r v i v i n g n e u r a l crest b o u n d a r y c a p cells at d o r s a l r o o t entry sites m a y r e g u l a t e s e n s o r y afferent i n g r o w t h a n d e n t r y z o n e m o r p h o g e n e s i s . Mol. Cell. Neurosci. 9:381-396.  132  G o n z y - T r e b o u l , G., L e p e s a n t , J., D e u t s c h , J. ( 1 9 9 5 ) E n h a n c e r - t r a p targeting at the B r o a d - C o m p l e x l o c u s o f Drosophila melanogaster. Genes Dev. 9:1137-1148.  Goodman, C S .  (1994) T h e likeness o f being: phylogenetically c o n s e r v e d molecular  m e c h a n i s m s o f g r o w t h c o n e g u i d a n c e . Cell 78:353-356.  G o r c z y c a , M.G., A u g a r t , C , B u d n i k , V . ( 1 9 9 3 ) I n s u l i n - l i k e r e c e p t o r a n d i n s u l i n - l i k e peptide are l o c a l i z e d at n e u r o m u s c u l a r j u n c t i o n s i n Drosophila.  J. Neurosci.  13:3692-  3704.  G o r c z y c a , M.G., P h i l l i s , R.W.,  B u d n i k , V . ( 1 9 9 4 ) T h e r o l e o f tinman, a m e s o d e r m a l  cell fate gene, i n a x o n p a t h f i n d i n g d u r i n g the d e v e l o p m e n t o f the t r a n s v e r s e n e r v e i n Drosophila. Development 120:2143-2152.  G o u l d i n g , M.D., C h a l e p a k i s , G., D e u t s c h , U., E r s e l i u s , J.R., G r u s s , P. ( 1 9 9 1 ) P a x - 3 , a n o v e l m u r i n e D N A b i n d i n g p r o t e i n e x p r e s s e d d u r i n g early n e u r o g e n e s i s . EMBO J. 10:1135-1147.  G r a n d e r a t h , S., S t o l l e w e r k , A., G r e i g , S., G o o d m a n , C.S., O ' K a n e , C.J., K l a m b t , C . (1999)  loco e n c o d e s an R G S  p r o t e i n r e q u i r e d f o r Drosophila glial differentiation.  Development 126:1781-1791.  G r e n n i n g l o h , G., R e h m , E.J., G o o d m a n , C S .  (1991) Genetic analysis o f growth cone  g u i d a n c e i n Drosophila: f a s c i c l i n II f u n c t i o n s as a n e u r o n a l r e c o g n i t i o n m o l e c u l e . Cell 67:45-57.  133  Gustafson,  K., B o u l i a n n e , G .  (1996)  D i s t i n c t e x p r e s s i o n patterns detected  within  i n d i v i d u a l tissues b y t h e G A L 4 e n h a n c e r trap technique. Genome 39:174-182. H a l t e r , D.A., U r b a n , J., R i c k e r t , C , N e r , S.S., Ito, K., T r a v e r s , A.A., T e c h n a u , G . M . ( 1 9 9 5 ) T h e h o m e o b o x g e n e r e p o is r e q u i r e d f o r the d i f f e r e n t i a t i o n a n d m a i n t e n a n c e o f glia in the e m b r y o n i c n e r v o u s s y s t e m o f D r o s o p h i l a m e l a n o g a s t e r . Development 121:317-332.  H a r r i s , R., S a b a t e l l y , L.M., S e e g e r , M . A . ( 1 9 9 6 ) G u i d a n c e c u e s at the Drosophila C N S midline: i d e n t i f i c a t i o n a n d characterization o f t w o Drosophila netrin/unc-6 h o m o l o g s . Neuron 17:217-228.  H a t t e n , M . E . ( 1 9 8 4 ) E m b r y o n i c c e r e b e l l a r a s t r o g l i a i n vitro. Brain Res. 315:309-313.  H e r d e g e n , T., K i e s s l i n g , M., B e l e , S., B r a v o , R., Z i m m e r m a n n , M., G a s s , P.  (1993)  T h e K R O X - 2 0 t r a n s c r i p t i o n f a c t o r i n the rat central a n d p e r i p h e r a l n e r v o u s s y s t e m s : n o v e l e x p r e s s i o n pattern o f a n i m m e d i a t e e a r l y g e n e - e n c o d e d protein. Neuroscience 57:41-52.  H e s l i p , T.R., W i l l i a m s , J.A., B e l l , J.B., H o d g e t t s , R.B. ( 1 9 9 2 ) A P e l e m e n t c h i m e r a c o n t a i n i n g c a p t u r e d g e n o m i c s e q u e n c e s w a s r e c o v e r e d at the v e s t i g i a l l o c u s i n Drosophila f o l l o w i n g targeted t r a n s p o s i t i o n . Genetics 131:917-927.  H e s l i p , T.R., H o d g e t t s , R . B .  ( 1 9 9 4 ) T a r g e t e d t r a n s p o s i t i o n at the v e s t i g i a l l o c u s o f  Drosophila melanogaster. Genetics 138:1127-1135.  H i d a l g o , A., U r b a n , J., B r a n d , A . ( 1 9 9 5 ) T a r g e t e d ablation o f g l i a d i s r u p t s a x o n tract f o r m a t i o n i n the Drosophila C N S . Development 121:3703-3712.  134  H i d a l g o , A., B r a n d , A . ( 1 9 9 7 ) T a r g e t e d n e u r o n a l ablation: t h e role o f p i o n e e r n e u r o n s i n g u i d a n c e a n d f a s c i c u l a t i o n i n the C N S o f D r o s o p h i l a . Development 124:3253-3262.  H i d a l g o , A., B o o t h , G.E. ( 2 0 0 0 ) G l i a dictate p i o n e e r a x o n trajectories i n t h e Drosophila e m b r y o n i c C N S . Development 127:393-402. H o s o y a , T., T a k i z a w a , K., N i t t a , K., H o t t a , Y . ( 1 9 9 5 ) glial cells missing: A b i n a r y s w i t c h b e t w e e n n e u r o n a l a n d g l i a l d e t e r m i n a t i o n i n D r o s o p h i l a . Cell 82:1025-1036.  H u m m e l , T., S c h i m m e l p h e n g , K., K l a m b t , C . ( 1 9 9 9 a ) C o m m i s s u r e f o r m a t i o n i n the e m b r y o n i c C N S o f Drosophila. I. I d e n t i f i c a t i o n o f the r e q u i r e d g e n e f u n c t i o n s . Dev. Biol. 209:381-398.  H u m m e l , T., S c h i m m e l p f e n g , K., K l a m b t , C . ( 1 9 9 9 b ) C o m m i s s u r e f o r m a t i o n i n the e m b r y o n i c C N S o f Drosophila II. F u n c t i o n o f the different m i d l i n e c e l l s . Development 126:771-779.  I s s h i k i , T., T a k e i c h i , M., N o s e , A . ( 1 9 9 7 ) the role o f the m s h h o m e o b o x g e n e d u r i n g Drosophila n e u r o g e n e s i s :  implication  f o r the d o r s o v e n t r a l  specification  o f the  n e u r o e c t o d e r m . Development 124:3099-3109.  Ito, K., U r b a n , J., T e c h n a u , G.M.  (1995) Distribution, classification, a n d development  o f Drosophila glial c e l l s i n the late e m b r y o n i c a n d early larval ventral n e r v e c o r d . Roux 's Arch. Dev. Biol. 2 0 4 : 2 8 4 - 3 0 7 .  135  J a c o b s , J.R., G o o d m a n , C.S.  (1989a) E m b r y o n i c d e v e l o p m e n t o f a x o n p a t h w a y s i n the  Drosophila C N S . I. A glial s c a f f o l d appears b e f o r e the first g r o w t h c o n e s . J. Neurosci.  9:2402-2411.  J a c o b s , J.R., G o o d m a n , C.S. ( 1 9 8 9 b )  E m b r y o n i c d e v e l o p m e n t o f a x o n p a t h w a y s i n the  D r o s o p h i l a C N S . II. B e h a v i o u r o f p i o n e e r g r o w t h c o n e s . /. Neurosci. 9:2412-2422.  J a c o b s , J.R.,  H i r o m i , Y.,  G o o d m a n , C.S.  (1989)  Lineage, migration, and  m o r p h o g e n e s i s o f l o n g i t u d i n a l glia i n the D r o s o p h i l a C N S  as r e v e a l e d b y a m o l e c u l a r  l i n e a g e marker. Neuron 2:1625-1611.  J a c o b s , J.R. ( 1 9 9 3 ) P e r t u r b e d glial s c a f f o l d f o r m a t i o n p r e c e d e s a x o n tract m a l f o r m a t i o n in Drosophila m u t a n t s . J. Neurobiol. 24:611-626.  Jessen, K.R., M i r s k y , R. ( 1 9 9 2 ) S c h w a n n cells: e a r l y l i n e a g e , r e g u l a t i o n o f p r o l i f e r a t i o n a n d c o n t r o l o f m y e l i n f o r m a t i o n . Current Opin. Neurobiol. 2:575-581.  J e s s e n , K.R.,  M i r s k y , R. (1999) S c h w a n n cells a n d their p r e c u r s o r s e m e r g e as m a j o r  r e g u l a t o r s o f n e r v e d e v e l o p m e n t . Trends Neurosci. 22:402-410.  J o h n s o n - S c h l i t z , D. M.,  E n g e l s , W.R.  (1993)  P-element-induced  interallelic g e n e  c o n v e r s i o n o f i n s e r t i o n s a n d d e l e t i o n s i n Drosophila melanogaster. Mol. Cell. Biol. 13: 7006-7018.  Jones, B.W.,  F e t t e r , R.D,.  T e a r , G., G o o d m a n , C.S.  (1995)  glial cells missing: a  g e n e t i c s w i t c h that c o n t r o l s glial v e r s u s n e u r o n a l fate. Cell 82:1013-1023.  136  K a p r i e l i a n , Z., C h o , K.O., H a d j i a r g y r o u , M., P a t t e r s o n , P.H.  (1995) C D 9 , a major  platelet cell s u r f a c e g l y c o p r o t e i n , i s a R O C A antigen a n d i s e x p r e s s e d i n the n e r v o u s s y s t e m . J. Neurosci. 15:562-573.  K a u f m a n n , N., W i l l s , Z.P., V a n V a c t o r , D . ( 1 9 9 8 ) Drosophila R a c l c o n t r o l s m o t o r a x o n g u i d a n c e . Development 125:453-461.  K e e l e r , K.J., G l o o r , G.B.  (1997)  E f f i c i e n t g a p repair i n Drosophila melanogaster  r e q u i r e s a m a x i m u m o f 31 n u c l e o t i d e s o f h o m o l o g o u s s e q u e n c e at the s e a r c h i n g e n d s . Mol. Cell. Biol.  17:627-634.  K l a e s , A., M e n n e , T., S t o l l e w e r k , A., S c h o l z , H., K l a m b t , C . ( 1 9 9 4 ) T h e E t s transcription factors e n c o d e d b y the Drosophila g e n e pointed direct glial c e l l differentiation i n the e m b r y o n i c C N S . Cell 78:149-160.  K l a m b t , C . J a c o b s , J.R., G o o d m a n , C S . ( 1 9 9 1 ) T h e m i d l i n e o f the Drosophila central n e r v o u s s y s t e m : a m o d e l f o r the genetic a n a l y s i s o f cell fate, cell m i g r a t i o n , a n d g r o w t h c o n e g u i d a n c e . Cell 64:801-815.  Klambt, C , Goodman, C S .  (1991)  T h e d i v e r s i t y a n d pattern o f glia d u r i n g a x o n  p a t h w a y f o r m a t i o n i n the Drosophila e m b r y o . Glia 4:205-213.  K l a m b t , C , G l a z e r , L., S h i l o , B . Z .  (1992)  breathless, a Drosophila F G F  h o m o l o g , i s e s s e n t i a l f o r m i g r a t i o n o f tracheal a n d s p e c i f i c m i d l i n e glial  receptor cells.  Development 117:163-176.  137  K o l o d z i e j , P.A., J a n , L.Y., J a n , Y . N .  (1995)  M u t a t i o n s that affect the length,  f a s c i c u l a t i o n , o r ventral orientation o f s p e c i f i c s e n s o r y a x o n s i n the D r o s o p h i l a  embryo.  Neuron 15:273-286.  K r u e g e r , N.X., V a n V a c t o r , D., W a n , H.I., G e l b a r t , W.M.,  G o o d m a n , C.S., S a i t o , H .  (1996) T h e transmembrane tyrosine phosphatase D L A R controls motor axon guidance in D r o s o p h i l a . Cell 84:611-622.  Lee, T., L u o , L . ( 1 9 9 9 ) . M o s a i c a n a l y s i s w i t h a r e p r e s s i b l e c e l l m a r k e r f o r studies o f g e n e f u n c t i o n i n n e u r o n a l m o r p h o g e n e s i s . Neuron 22: 451-461.  L i n d s l e y , D . L . a n d G.G. Z i m m . 1 9 9 2 . The Genome of Drosophila. A c a d e m i c P r e s s , San Diego.  L u o , L., L i a o , Y.J., J a n , L.Y., J a n , Y . N . ( 1 9 9 4 ) D i s t i n c t m o r p h o g e n e t i c f u n c t i o n s o f s i m i l a r s m a l l G T P a s e s : Drosophila D r a c l i s i n v o l v e d i n a x o n a l o u t g r o w t h a n d m y o b l a s t f u s i o n . Genes Dev. 8:1787-1802.  M a n n i n g , L., D o e , C.Q.  (1999)  P r o s p e r o d i s t i n g u i s h e s s i b l i n g cell fate w i t h o u t  a s y m m e t r i c l o c a l i z a t i o n i n the Drosophila adult external e n s e o r g a n lineage. Development 126:2063-2071.  M a r c h i o n n i , M.A.,  G o o d e a r l , A.D., C h e n , M.S., B e r m i n h a m ,  M.O.,  Kirk, C ,  H e n d r i c k s , M., e t a l . ( 1 9 9 3 ) G l i a l g r o w t h factors are alternatively s p l i c e d e r b B 2 l i g a n d s e x p r e s s e d i n the n e r v o u s s y s t e m . Nature 362:312-318.  138  M a r o l a k o u , I.G., P a p a s , T.S., G r e e n J.E. ( 1 9 9 4 ) D i f f e r e n t i a l e x p r e s s i o n o f ets-1 a n d ets2 p r o t o - o n c o g e n e s d u r i n g m u r i n e e m b r y o g e n e s i s . Oncogene 9:1551-1565.  M i t c h e l l , K.J., D o y l e , J.L., S e r a f i n i , T., K e n n e d y , T.E., T e s s i e r - L a v i g n e , M., G o o d m a n , C.S.  (1996)  G e n e t i c a n a l y s i s o f netrin g e n e s i n Drosophila: netrins g u i d e C N S  c o m m i s s u r a l a x o n s a n d p e r i p h e r a l m o t o r axons. Neuron 17:203-215.  M o n t e l l , D.J. ( 1 9 9 9 ) T h e genetics o f cell m i g r a t i o n i n Drosophila melanogaster a n d Caenorhabditis elegans d e v e l o p m e n t . Development 126:3035-3046.  N a s s i f , N., P e n n e y , J., P a l , S., E n g e l s , W.R., G l o o r , G.B. ( 1 9 9 4 ) E f f i c i e n t c o p y i n g o f n o n h o m o l o g o u s s e q u e n c e s f r o m e c t o p i c sites v i a P - e l e m e n t - i n d u c e d g a p repair. Mol. Cell. Biol. 14:1613-1625.  N e l s o n , H.B. L a u g h o n , A . ( 1 9 9 3 ) D r o s o p h i l a g l i a l a r c h i t e c t u r e a n d d e v e l o p m e n t a n a l y s i s u s i n g a c o l l e c t i o n o f n e w c e l l - s p e c i f i c m a r k e r s . Roux's Arch. Dev. Biol. 202:341-354.  N o b e s , C D . , H a l l , A . ( 1 9 9 5 ) R h o , R a c , a n d C d c 4 2 G T P a s e s regulate the a s s e m b l y o f m u l t i m o l e c u l a r focal c o m p l e x e s a s s o c i a t e d w i t h actin stress f i b e r s , l a m e l l i p o d i a , a n d f i l o p o d i a . Cell 81:53-62.  N o o r d e r m e e r , J.N., K o p c z y n s k i , C C , Fetter, R.D., B l a n d , K.S., C h e n , W., G o o d m a n , C.S. ( 1 9 9 8 ) W r a p p e r , a n o v e l m e m b e r o f the I g s u p e r f a m i l y , is e x p r e s s e d b y m i d l i n e glia a n d i s r e q u i r e d f o r t h e m t o e n s h e a t h c o m m i s s u r a l a x o n s i n Drosophila. Neuron 21:9911001.  139  N o r r i s , C.R., K a l i l , K. ( 1 9 9 1 ) G u i d a n c e o f c a l l o s a l a x o n s b y r a d i a l g l i a i n the d e v e l o p i n g c e r e b r a l cortex. J. Neurosci, 11:3481-3492.  O ' B r i e n , D., D o c k e r y , P., M c D e r m o t t , K., F r a h e r , J.P. ( 1 9 9 8 ) T h e ventral m o t o n e u r o n e a x o n b u n d l e i n the C N S - a c o r d o n e s y s t e m ? J. Neurocytol. 27:247-258.  O ' K a n e , C.J., G e h r i n g , W.J. ( 1 9 8 7 ) D e t e c t i o n i n situ o f g e n o m i c r e g u l a t o r y e l e m e n t s i n Drosophila. Proc. Natl. Acad. Sci. USA 84:9123-9127.  O w e n s , G.C., B o y d , C.J., B u n g e , R.P., S a l z e r , J.L. ( 1 9 9 0 ) E x p r e s s i o n o f r e c o m b i n a n t m y e l i n - a s s o c i a t e d g l y c o p r o t e i n i n p r i m a r y S c h w a n n cells p r o m o t e s the initial i n v e s t m e n t o f a x o n s b y m y e l i n a t i n g S c h w a n n cells. J. C e l l B i o l .  Patel, N.H.,  S n o w , P.M.,  G o o d m a n , C.S.  111:1171-1182.  (1987)  Characterization and cloning o f  f a s c i c l i n III: a g l y c o p r o t e i n e x p r e s s e d o n a s u b s e t o f n e u r o n s a n d a x o n p a t h w a y s i n Drosophila. Cell 48: 9 7 5 - 9 8 8 .  P e r r i m o n , N., P e r k i n s , L . A . ( 1 9 9 7 ) T h e r e m u s t b e 5 0 w a y s to r u l e the s i g n a l : T h e c a s e o f the D r o s o p h i l a E G F receptor. Cell 89:13-16.  P f r i e g e r , F.W.,  B a r r e s , B.A.  ( 1 9 9 7 ) S y n a p t i c e f f i c a c y e n h a n c e r b y glial cells i n vitro.  Science 2 7 7 : 1 6 8 4 - 1 6 8 7 .  P o l i a k , S., G o l l a n , L., M a r t i n e z , R., C u s t e r , A., E i n h e b e r , S., S a l z e r , J.L., T r i m m e r , J.S., S h r a g e r , P., P e l e s , E . ( 1 9 9 9 ) C a s p r 2 , a n e w m e m b e r o f the N e u r e x i n s u p e r f a m i l y , is l o c a l i z e d at the j u x t a p a r a n o d e s o f m y e l i n a t e d a x o n s a n d associates w i t h K c h a n n e l s . +  Neuron 2 4 : 1 0 3 7 - 1 0 4 7 .  140  R a f f , M., B a r r e s , B.A., B u r n e , J.F., C o l e s , H.S., I s h i z a k i , Y., J a c o b s o n , M . D .  (1993)  P r o g r a m m e d c e l l d e a t h a n d the c o n t r o l o f c e l l s u r v i v a l : l e s s o n s f r o m the n e r v o u s system. Science 2 6 2 : 6 9 5 - 7 0 0 .  R a g h a v a n , S., W h i t e , R.A.H. ( 1 9 9 7 ) C o n n e c t i n m e d i a t e s a d h e s i o n i n D r o s o p h i l a . Neuron 18:873-880.  R a k i c , P. ( 1 9 9 1 ) G l i a l c e l l s i n d e v e l o p m e n t : i n v i v o a n d i n v i t r o a p p r o a c h e s . Ann.  NY  Acad. Sci. 6 3 3 : 9 6 - 9 9 .  R a n g a r a j a n , R., G o n g , Q., G a u l , U . ( 1 9 9 9 ) M i g r a t i o n a n d f u n c t i o n o f g l i a i n t h e d e v e l o p i n g Drosophila eye. Development 126:3285-3292.  R e d d y , G.V., R o d r i g u e s , V . (1999a) S i b l i n g c e l l fate i n the Drosophila a d u l t e x t e r n a l sense o r g a n l i n e a g e is s p e c i f i e d b y P r o s p e r o f u n c t i o n w h i c h is r e g u l a t e d b y N u m b a n d N o t c h . Development 126:2083-2092.  R e d d y , G.V., R o d r i g u e s , V . ( 1 9 9 9 b ) . A g l i a l c e l l arises f r o m a n a d d i t i o n a l d i v i s i o n w i t h i n the m e c h a n o s e n s o r y lieage d u r i n g d e v e l o p m e n t o f the m i c r o c h a e t e o n the Drosophila n o t u m . Development 126:4617-4622.  Rio, C , R i e f f , H.I., Q i , P., C o r f a s , G. ( 1 9 9 7 ) N e u r e g u l i n a n d e r b B r e c e p t o r s p l a y a critical role i n n e u r o n a l m i g r a t i o n . Neuron 19:39-50.  141  R o m a n i , S., J i m e n e z , F., H o c h , M., P a t e l , N.H., T a u b e r t , H., J a c k i e , H. ( 1 9 9 6 ) Kruppel, a Drosophila s e g m e n t a t i o n gene, participates i n the s p e c i f i c a t i o n o f n e u r o n s a n d  g l i a l c e l l s . Mech. Dev. 60:95-107.  R o t h b e r g , J.M., J a c o b s , J.R., G o o d m a n , C.S., A r t a v a n i s , T.S.  (1990)  Slit: an  extracellular p r o t e i n n e c e s s a r y f o r d e v e l o p m e n t o f m i d l i n e g l i a a n d c o m m i s s u r a l a x o n p a t h w a y s c o n t a i n s b o t h E G F a n d L R R d o m a i n s . Genes Dev. 4:2169-2187.  R o s s l e r , W., O l a n d , L.A., H i g g i n s , M.R., H e l d e b r a n d , J.G., T o l b e r t , L.P. ( 1 9 9 9 ) . D e v e l o p m e n t o f a g l i a - r i c h a x o n - s o r t i n g z o n e i n the o l f a t o r y p a t h w a y o f the m o t h Manduca sexta. J. Neurosci. 19:9865-9877.  S c h e r e r , S.S., S a l z e r , J.L. ( 1 9 9 6 ) Glial Cell Development: Basic principles and clinical relevance C h . 9 Axon-Schwann cell interactions during peripheral nerve degeneration and regeneration, pp. 1 6 5 - 1 9 6 B i o s ed. K.R. J e s s e n a n d W.D. R i c h a r d s o n .  S c h m i d , A., C h i b a , A., D o e , C Q .  ( 1 9 9 9 ) C l o n a l a n a l y s i s o f Drosophila e m b r y o n i c  neuroblasts: n e u r a l cell t y p e s , a x o n p r o j e c t i o n s a n d m u s c l e targets.  Development  126:4653-4689/  S c h m i d t , H., R i c k e r t , C , B o s s i n g , T., V e f , O., U r b a n , J., T e c h n a u , G.M.  (1997) The  e m b r y o n i c central n e r v o u s s y s t e m lineages o f Drosophila melanogaster. II. N e u r o b l a s t l i n e a g e s d e r i v e d f r o m the d o r s a l part o f the n e u r o e c t o d e r m . Dev. Biol. 189:186-204.  S c h n e p p , B., G r u m b l i n g , G., D o n a l d s o n , T., S i m c o x , A .  (1996)  V e i n is a novel  c o m p o n e n t i n the D r o s o p h i l a e p i d e r m a l g r o w t h factor receptor p a t h w a y w i t h similarity to the n e u r e g u l i n s . G e n e s D e v . 10:2302-2313.  142  S e a r l e s , L . L., J o k e r s t , R.S., B i n g h a m , P.M.,  V o e l k e r , R.A., G r e e n l e a f , A . L .  (1982)  M o l e c u l a r c l o n i n g o f s e q u e n c e s f r o m a Drosophila R N A p o l y m e r a s e II l o c u s b y P element t r a n s p o s o n t a g g i n g . Cell 31:585-592. S e a v e r , E.C., C a r p e n t e r , E.M., B a s t i a n i , M.J.  (1996) R E G A - 1 is a G P I - l i n k e d m e m b e r  of the i m m u n o g l o b u l i n s u p e r f a m i l y p r e s e n t o n restricted r e g i o n s o f sheath cell p r o c e s s e s i n g r a s s h o p p e r . Development 122:567-578.  Seeger, M., T e a r , G., F e r r e s - M a r c o , D., G o o d m a n , C.S.  (1993)  M u t a t i o n s affecting  g r o w t h c o n e g u i d a n c e i n D r o s o p h i l a : genes n e c e s s a r y f o r g u i d a n c e t o w a r d o r a w a y f r o m the m i d l i n e . Neuron 10:409-426.  S e p p , K.J., A u l d , V . J . ( 1 9 9 9 )  C o n v e r s i o n o f lacZ e n h a n c e r trap lines to GAL4 lines  u s i n g t a r g e t e d t r a n s p o s i t i o n i n Drosophila melanogaster. Genetics 151:1093-1101.  Sepp, K.J., S c h u l t e , J., A u l d , V . J . ( 2 0 0 0 )  The developmental dynamics o f peripheral  g l i a i n Drosophila melanogaster. Glia ( i n press)  S h i g e n a g a , A., K i m u r a , K., K o b a y a k a w a , Y., T s u j i m o t o , Y., T a n i m u r a , T. ( 1 9 9 7 ) C e l l a b l a t i o n b y e x p r e s s i o n o f c e l l death genes, ced-3 a n d Ice, i n Drosophila. Develop. Growth Differ. 39: 4 2 9 - 4 3 6 .  S h i m e l d , S., M c K a y , I.J., S h a r p e , P.T.  (1996)  T h e m u r i n e h o m e o b o x g e n e Msx-3  s h o w s h i g h l y restricted e x p r e s s i o n i n the d e v e l o p i n g n e u r a l tube. Mech. Dev. 55:201-210.  143  S i e g l e r , M.V.S., J i a , X . X .  ( 1 9 9 9 ) E n g r a i l e d n e g a t i v e l y regulates the e x p r e s s i o n o f cell  a d h e s i o n m o l e c u l e s C o n n e c t i n a n d N e u r o g l i a n i n e m b r y o n i c Drosophila n e r v o u s  system.  Neuron 2 2 : 2 6 5 - 2 7 6 .  S i l v e r , J., L o r e n z , S.E., W a h l s t e i n , D, C o u g h l i n , J. ( 1 9 8 2 ) A x o n a l g u i d a n c e  during  d e v e l o p m e n t o f the great cerebral c o m m i s s u r e s : d e s c r i p t i v e a n d e x p e r i m e n t a l studies i n v i v o o n the r o l e o f p r e f o r m e d g l i a l p a t h w a y s . J. Comp. Neurol. 210:10-29.  S i l v e r , J. ( 1 9 9 3 ) G l i a - n e u r o n interactions at the m i d l i n e o f the d e v e l o p i n g m a m m a l i a n b r a i n a n d s p i n a l c o r d . Perspect. Dev. Neurobiol. 1:227-236.  S i n g e r , M., N o r d l a n d e r , R.H., E g a r , M .  (1979) Axonal guidance during  embryogenesis  a n d r e g e n r a t i o n i n the s p i n a l c o r d o f the newt: T h e b l u e p r i n t h y p o t h e s i s o f n e u r o n a l p a t h w a y p a t t e r n i n g . J. Comp. Neurol. 185:1-22.  S o n n e n f e l d , M.J., J a c o b s , J.R. ( 1 9 9 5 a ) M a c r o p h a g e s a n d g l i a participate i n the r e m o v a l o f apoptotic n e u r o n s f r o m the Drosophila e m b r y o n i c n e r v o u s s y s t e m . J. Comp. Neurol. 359:644-652.  S o n n e n f e l d , M.J., J a c o b s , J.R. ( 1 9 9 5 b ) A p o p t o s i s o f the m i d l i n e g l i a d u r i n g Drosophila e m b r y o g e n e s i s : a c o r r e l a t i o n w i t h a x o n contact. Development 121:569-578.  S p r e y e r , P., K u h n , G., H a n e m a n n , C O . , H.W.  G i l l e n , C P . , K u h n , R., L e m k e , G., M u l l e r ,  ( 1 9 9 1 ) A x o n regulated e x p r e s s i o n o f a S c h w a n n cell m R N N A h o m o l o g o u s to a  g r o w t h arrest s p e c i f i c gene. EMBOJ. 10:3661-3668.  144  S t a v e l e y , B.E., H o d g e t t s , R.B.,  O ' K e e f e , S.L., B e l l , J.B.  ( 1 9 9 4 ) T a r g e t i n g o f an  e n h a n c e r trap to vestigial. Dev. Biol. 165:290-293.  S t a v e l y , B.E., H e s l i p , T.R.,  H o d g e t t s , R.B.,  B e l l , J.B.  ( 1 9 9 5 ) P r o t e c t e d P-element  t e r m i n i suggest a r o l e f o r i n v e r t e d - r e p e a t - b i n d i n g p r o t e i n i n t r a n s p o s a s e - i n d u c e d gap repair in Drosophila melanogaster. Genetics 139:1321-1329.  Stewart, H.J.S., M i r s k y , R., Jessen, K.R. principles and clinical relevance C h .  (1996)  Glial Cell Development: Basic  1: The Schwann cell lineage: embryonic and early  postnatal development, pp. 1-30 B i o s ed. K.R. J e s s e n a n d W.D.  Su, M-W.,  S u z u k i , H.R.,  S o l u r s h , M., R a m i r e z , F.  expression of a new homeobox-containing  Richardson.  ( 1 9 9 1 ) P r o g r e s s i v e l y restricted  g e n e d u r i n g Xenopus laevis e m b r y o g e n e s i s .  Development 111:1179-1187.  S u t h e r l a n d , D.,  Samakovlis,  C,  K r a s n o w , M.A.  (1996)  branchless e n c o d e s a  Drosophila F G F h o m o l o g that c o n t r o l s tracheal c e l l m i g r a t i o n a n d the pattern o f b r a n c h i n g .  Cell 87: 1 0 9 1 - 1 1 0 1 .  T a k a h a s h i , Y., M o n s o r o - B u r q , A-H., B o n t o u x , M . L e D o u a r i n , N.M.  ( 1 9 9 2 ) A role f o r  Quox-8 i n e s t a b l i s h m e n t o f the d o r s o v e n t r a l pattern d u r i n g vertebrate Proc.Natl.Acad.Sci.  Takasu-Ishikawa,  development.  USA 8 9 : 1 0 2 3 7 - 1 0 2 4 1 .  E., Y o s h i h a r a , M., H o t t a , Y.  ( 1 9 9 2 ) E x t r a s e q u e n c e s f o u n d at P  e l e m e n t e x c i s i o n sites i n Drosophila melanogaster. Mol. Gen. Genet. 232:17-23.  145  T a s s a b e h j i , M., N e w t o n , V.E., L e v e r t o n , K., T u r n b u l l , K., S e e m a n o v a , E., K u n z e , J . , S p e r l i n g , K., S t r a c h a n , T., R e a d , A . ( 1 9 9 4 ) PAX3 g e n e structure a n d mutations: c l o s e a n a l o g i e s b e t w e e n W a a r d e n b u r g s y n d r o m e a n d the Splotch m o u s e . Hum. Mol. Genet. 3:1069-1074.  T a v s a n l i , B., M a r d o n , G.  (1999)  S t u d y o f retinal d e t e r m i n a t i o n g e n e s a n d their  d o w n s t r e a m targets. 4 0 t h A n n u a l D r o s o p h i l a R e s e a r c h C o n f e r e n c e A b s t r a c t .  T e a r , G., H a r r i s , R., Sutaria, S., K i l o m a n s k i , K., G o o d m a n , C.S., .Seeger, (1996)  commissureless c o n t r o l s g r o w t h c o n e g u i d a n c e a c r o s s the C N S  M.A.  midline in  D r o s o p h i l a a n d e n c o d e s a n o v e l m e m b r a n e protein. Neuron 16:501-514.  T r e h e r n e , J.E., S c h o f i e l d , P.K.  ( 1 9 8 1 ) M e c h a n i s m s o f i o n i c h o m e o s t a s i s i n the central  n e r v o u s s y s t e m o f a n insect. J. Exp. Biol. 95:61-73.  V a e s s i n , H., G r e l l , E., W o l f f , E., B i e r , E., J a n , L.Y., J a n , Y . N .  ( 1 9 9 1 ) prospero is  e x p r e s s e d i n n e u r o n a l p r e c u r s o r s a n d e n c o d e s a n u c l e a r p r o t e i n that i s i n v o l v e d i n the c o n t r o l o f a x o n a l o u t g r o w t h i n Drosophila. Cell 67:941-953.  V a n A e l s t , L., D ' S o u z a - S c h o r e y , C.  (1997) R h o G T P a s e s and signaling networks.  Genes Dev. 11:2295-2322.  V a n V a c t o r , D., S i n k , H., F a m b r o u g h , D., T s o o , R., G o o d m a n , C.S. ( 1 9 9 3 ) G e n e s that c o n t r o l n e u r o m u s c u l a r s p e c i f i c i t y in Drosophila. Cell 73:1137-1153.  V i n c e n t , S., V o n e s c h , J-L., G i a n g r a n d e , A . ( 1 9 9 6 ) glide directs g l i a l fate c o m m i t m e n t and c e l l fate s w i t c h b e t w e e n n e u r o n e s a n d glia. Development 122:131-139.  146  W a n g , G., Scott, S.A. ( 1 9 9 9 ) I n d e p e n d e n t d e v e l o p m e n t o f s e n s o r y a n d m o t o r i n n e r v a t i o n patterns i n e m b r y o n i c c h i c k h i n d l i m b s . Dev. Biol. 208:324-336.  W a n g , W., C h e n , X., X u , H., L u f k i n , T . ( 1 9 9 6 ) M s x 3 : a n o v e l m u r i n e h o m o l o g u e o f the Drosophila msh h o m e o b o x g e n e restricted to the d o r s a l e m b r y o n i c central n e r v o u s s y s t e m . Mech.Dev. 58:203-215.  W i l s o n , C , P e a r s o n , R.K., B e l l e n , H.J., O ' K a n e , C.J., G r o s s n i k l a u s , U., G e h r i n g , W.J. ( 1 9 8 9 ) P - e l e m e n t m e d i a t e d e n h a n c e r trap detection: a n e f f i c i e n t m e t h o d f o r isolating a n d c h a r a c t e r i z i n g d e v e l o p m e n t a l l y r e g u l a t e d g e n e s i n Drosophila. Genes Dev. 3:13011313.  W o l d e y e s u s , M.T., B r i t s c h , S., R i e t h m a c h e r , D., X u , L., S o n n e n b e r g - R i e t h m a c h e r , E., A b o u - R e b y e h , F., H a r v e y , R., C a r o n i , P., B i e r c h m e i e r , C . ( 1 9 9 9 ) P e r i p h e r a l n e r v o u s s y s t e m d e f e c t s i n erbB2 m u t a n t s f o l l o w i n g g e n e t i c r e s c u e o f heart d e v e l o p m e n t . Genes Dev.  13:2538-2548.  W o o d , P.M., S c h a c h n e r , M., B u n g e , R.P.  (1990)  I n h i b i t i o n o f S c h w a n n cell  m y e l i n a t i o n i n v i t r o b y a n t i b o d y to the L I a d h e s i o n m o l e c u l e . /. Neurosci. 10:3635-3645.  X i o n g , W - C , O k a n o , H., P a t e l , N.H., B l e n d y , J.A., M o n t e l l , C . ( 1 9 9 4 ) repo e n c o d e s a g l i a l - s p e c i f i c h o m e o d o m a i n p r o t e i n r e q u i r e d i n the Drosophila n e r v o u s s y s t e m . Genes Dev.  8:981-994.  X i o n g , W - C , M o n t e l l , C . ( 1 9 9 5 ) D e f e c t i v e g l i a i n d u c e n e u r o n a l a p o p t o s i s i n the repo v i s u a l s y s t e m o f D r o s o p h i l a . Neuron 14:581-590.  147  1  Y a r n i t z k y , T., M i n , L., V o l k , T.  ( 1 9 9 7 ) T h e Drosophila n e u r e g u l i n h o m o l o g V e i n  mediates i n d u c t i v e interactions b e t w e e n m y o t u b e s a n d their e p i d e r m a l attachment  cells.  Genes Dev. 11:2691-2700.  Y o u n o s s i - H a r t e n s t e i n , A., H a r t e n s t e i n , V . ( 1 9 9 3 ) T h e r o l e o f tracheae a n d m u s c u l a t u r e d u r i n g p a t h f i n d i n g o f Drosophila e m b r y o n i c s e n s o r y axons. Dev. Biol. 158:430-447.  Z h e n g , T., H e i n t z , N., H a t t e n , M.E.  ( 1 9 9 6 ) C N S g e n e e n c o d i n g astrotactin, w h i c h  supports n e u r o n a l m i g r a t i o n a l o n g g l i a l fibers. Science 272:417-419.  Z h o u , L., S c h n i t z l e r , A., A g a p i t e , J., S c h w a r t z , L.M., Steller, H., N a m b u , J.R.  (1997).  C o o p e r a t i v e f u n c t i o n s o f the reaper a n d head involution defective g e n e s i n the p r o g r a m m e d c e l l death o f Drosophila c e n t r a l n e r v o u s s y s t e m m i d l i n e cells. Proc. Natl. Acad. Sci. USA 94:5131-5136.  148  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089858/manifest

Comment

Related Items