UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The genetics and embryopathology of exencephaly in SELH/Bc mice Macdonald, Karen Beth 1988

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

Item Metadata

Download

Media
831-UBC_1988_A6_7 M32.pdf [ 11MB ]
Metadata
JSON: 831-1.0097709.json
JSON-LD: 831-1.0097709-ld.json
RDF/XML (Pretty): 831-1.0097709-rdf.xml
RDF/JSON: 831-1.0097709-rdf.json
Turtle: 831-1.0097709-turtle.txt
N-Triples: 831-1.0097709-rdf-ntriples.txt
Original Record: 831-1.0097709-source.json
Full Text
831-1.0097709-fulltext.txt
Citation
831-1.0097709.ris

Full Text

THE GENETICS AND EMBRYOPATHOLOGY OF EXENCEPHALY IN SELH/BC MICE by KAREN BETH MACDONALD BSc. The U n i v e r s i t y of B r i t i s h Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( G e n e t i c s Programme) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH F e b r u a r y 1988 Karen Beth Macdonald, COLUMBIA 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ri&blcJfrU Gt&MtfKC*^ The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date ftf/Uu M J f g g ii ABSTRACT T h i s p r o j e c t was the f i r s t s t u d y of the g e n e t i c s and embryo-p a t h o l o g y of e x e n c e p h a l y i n a p a r t i a l l y i n b r e d mouse s t o c k , SELH/Bc. E x e n c e p h a l y was found i n 17% of SELH f e t u s e s . A n a l y s i s of day 8-9 g e s t a t i o n embryos i n d i c a t e d t h a t SELH embryos were c o l l e c t i v e l y normal i n g e n e r a l development, but d e l a y e d i n n e u r a l tube c l o s u r e r e l a t i v e t o o v e r a l l or g e n e r a l development compared t o two normal s t r a i n s of mice, ICR/Be and SWV/Bc. Ex e n c e p h a l y was o b s e r v e d t o be caused by a f a i l u r e of f u s i o n of the c r a n i a l n e u r a l f o l d s i n the mesencephalon r e g i o n i n SELH. A l l SELH embryos appeared t o be abnormal i n t h e i r p a t t e r n of c r a n i a l n e u r a l tube c l o s u r e . They f a i l t o make i n i t i a l c o n t a c t a t t h e prosencephalon/mesencephalon j u n c t i o n r e g i o n of t h e c r a n i a l n e u r a l f o l d s ( t h e f i r s t f u s i o n i n the c r a n i a l n e u r a l f o l d s i n normal embryos). SELH embryos, f u s e d t h e i r a n t e r i o r n e u r a l f o l d s v i a an a l t e r n a t e ( p o s s i b l y p a s s i v e ) mechanism compared t o normal s t r a i n s of mice (SWV/Bc, and ICR/Be), by f u s i n g the f o l d s i n a " z i p p e r - l i k e " f a s h i o n from the r o s t r a l base of the p r o s e n c e p h a l o n . T h i s c l o s u r e of the n e u r a l tube i n g e n e t i c a l l y l i a b l e embryos by an abnormal sequence of e v e n t s s u g g e s t s a new model f o r a n t e r i o r n e u r a l tube c l o s u r e f a i l u r e . L i a b i l i t y t o e x e n c e p h a l y appeared t o be f i x e d i n the SELH s t o c k . Of the 53 SELH males t e s t e d , a l l produced e x e n c e p h a l y . SELH a n i m a l s were found t o be heterogeneous i n the f r e q u e n c y of e x e n c e p h a l y t h e y produced, i n d i c a t i n g t h a t t h e r e a r e s t i l l genes i i i s e g r e g a t i n g i n the s t o c k which a f f e c t the a b i l i t y of embryos t o complete a n t e r i o r n e u r a l tube c l o s u r e . E x e n c e p h a l y i n SELH does not appear t o be caused by an autosomal dominant, s e x - l i n k e d dominant or r e c e s s i v e , or s i m p l e autosomal r e c e s s i v e s i n g l e gene, a l t h o u g h F2, B C l , and BC2 e x e n c e p h a l y f r e q u e n c i e s ( a f t e r an o u t c r o s s t o ICR/Be) s u g g e s t t h a t o n l y a s m a l l number of genes a r e i n v o l v e d . A marked e x c e s s of female e x e n c e p h a l i c s was observed i n SELH, F2, B C l , and BC2 f e t u s e s . iv TABLE OF CONTENTS page ABSTRACT 1 i LIST OF TABLES v l LIST OF FIGURES l x AKNOWLEDGEMENTS X 1 i INTRODUCTION I . S t u d i e s of an e n c e p h a l y i n humans 1 I I . Mouse m u t a t i o n s a f f e c t i n g n e u r a l tube c l o s u r e 12 I I I . T e r a t o g e n i c agents p r o d u c i n g n e u r a l tube d e f e c t s 22 IV. N e u r u l a t i o n i n a n i m a l s and man A. D e s c r i p t i o n of the n e u r u l a t i o n p r o c e s s 23 B. Models of n e u r a l tube f o r m a t i o n 27 MATERIALS AND METHODS I . Mice 35 I I . G e n e r a l Care 37 I I I . T e st of s e g r e g a t i o n of e x e n c e p h a l y i n SELH A. Day 14 mouse embryo s t u d y 38 B. I d e n t i f i c a t i o n of e x e n c e p h a l y p r o d u c i n g mice 39 C. C o n t r o l s from the g e n e t i c s t u d y of B C l males 39 IV. Embryology s t u d y A. Developmental s t u d y 40 B. S c a n n i n g e l e c t r o n m i c r o s c o p y (SEM) 41 V. H i s t o l o g y A. P a r a f f i n - e m b e d d e d specimens ( t h i c k s e c t i o n s ) 43 B. P l a s t i c embedded specimens ( t h i n s e c t i o n s ) 44 V I . G e n e t i c s t u d i e s 46 A. R e c i p r o c a l c r o s s e s of SELH and ICR/Be 47 1. ICR/Be females x SELH males 47 2. SELH females x ICR/Be males 47 B. F2 g e n e r a t i o n 48 C. F i r s t b a c k c r o s s t o SELH ( B C l ) 48 D. Second b a c k c r o s s t o SELH (BC2) 49 1. B C l males x SELH females 49 2. SELH females x SELH males 49 V V I I . Enzyme l i n k a g e t e s t i n g 50 V I I I . M i s c e l l a n e o u s s t u d i e s - newborn a d r e n a l g l a n d s 51 RESULTS I . Embryology A. Rate of g e n e r a l development 52 B. N e u r a l tube c l o s u r e and so m i t e development 56 I I . A d d i t i o n a l a n o m a l i e s 83 I I I . H i s t o l o g y A. P a r a f f i n s e c t i o n s 86 B. P l a s t i c s e c t i o n s 87 IV. T e s t of the h y p o t h e s i s of s e g r e g a t i o n of an e x e n c e p h a l y m u t a t i o n i n the SELH s t o c k A. Day 14 mouse embryo s t u d y , summer 1985 100 B. i d e n t i f i c a t i o n of e x e n c e p h a l y - p r o d u c i n g mice 102 C. c o n t r o l s from the g e n e t i c s t u d y of B C l males, 102 s p r i n g 1987 V. T e s t of h e t e r o g e n e i t y i n SELH males 107 V I . E s t i m a t e s of the f r e q u e n c y of e x e n c e p h a l y i n the SELH s t o c k A. Frequency e s t i m a t e s 110 B. Sex r a t i o i n SELH 112 V I I . G e n e t i c o u t c r o s s e s of SELH and ICR/Be A. F l g e n e r a t i o n 114 B. F2 g e n e r a t i o n 115 C. B C l g e n e r a t i o n 123 D. BC2 g e n e r a t i o n 125 V I I I . Enzyme l i n k a g e t e s t i n g 130 IX. M i s c e l l a n e o u s - newborn a d r e n a l g l a n d s 130 DISCUSSION I . The embryopathogeriesis of e x e n c e p h a l y i n SELH mice 132 I I . Sex r a t i o 138 I I I . G e n e t i c s of e x e n c e p h a l y i n SELH and c r o s s e s 140 REFERENCES 146 vi LIST OF TABLES T a b l e I Table I I Tab l e I I I Tab l e IV Tab l e V Tab l e VI Tab l e V I I Ta b l e V I I I T able IX Tab l e X Ta b l e XI Ta b l e X I I Tab l e X I I I page C h r o n o l o g i c a l g e s t a t i o n a l age and mean so m i t e 53 count of ICR/Be embryos c o l l e c t e d C h r o n o l o g i c a l g e s t a t i o n a l age and mean s o m i t e 54 count of SWV/Bc embryos c o l l e c t e d C h r o n o l o g i c a l g e s t a t i o n a l age and mean s o m i t e 55 count of SELH embryos c o l l e c t e d Comparison of mean so m i t e c o u n t s a t 5 57 c h r o n o l o g i c a l g e s t a t i o n a l ages A n t e r i o r n e u r a l tube c l o s u r e i n ICR/Be 59 A n t e r i o r n e u r a l tube c l o s u r e i n SWV/Bc 60 A n t e r i o r n e u r a l tube c l o s u r e i n SELH 61 P r o p o r t i o n of embryos w i t h a n t e r i o r n e u r a l tube 69 c l o s u r e completed a t v a r i o u s s o m i t e c o u n t s Mean so m i t e count i n SELH, ICR/Be, and SWV/Bc 70 embryos d u r i n g a n t e r i o r n e u r a l tube c l o s u r e s t a g e s Comparison between number of SELH, ICR/Be, and 72 SWV/Bc embryos a t s p e c i f i c s t a g e s i n a n t e r i o r n e u r a l tube c l o s u r e A b n o r m a l i t i e s found i n f e t u s e s (day 18 of 84 g e s t a t i o n ) of g e n e t i c c r o s s e s Comparison of mean mesenchyme c e l l d e n s i t y 90 between ICR/BC and SELH specimens of 6 and 8 s o m i t e s Comparison of the number of p y k n o t i c c e l l s per 91 s e c t i o n i n ICR/BC and SELH specimens (a) mean # of p y k n o t i c c e l l s per s e c t i o n (b) d i s t r i b u t i o n of p y k n o t i c s i n the NE of 6 som i t e embryos (c) d i s t r i b u t i o n of p y k n o t i c s i n the NE of 8 somite embryos (d) d i s t r i b u t i o n of p y k n o t i c s i n t h e mesenchyme of 6 so m i t e embryos (e) d i s t r i b u t i o n of p y k n o t i c s i n the mesenchyme of 8 so m i t e embryos ( f ) d i s t r i b u t i o n of p y k n o t i c s i n the ectoderm of 6 s o m i t e embryos vi i T a b l e XIV T a b l e XV T a b l e XVI T a b l e XVII T a b l e X V I I I T a b l e XIX T a b l e XX T a b l e XXI T a b l e XXII T a b l e X X I I I T a b l e XXIV Table XXV T a b l e XXVI Ta b l e XXVII (g) d i s t r i b u t i o n of p y k n o t i c s i n the ectoderm of 6 s o m i t e embryos Comparison of the number of l i p i d d r o p l e t s per 93 s e c t i o n i n the neuroectoderm of ICR/BC and SELH specimens of 6 and 8 s o m i t e s (a) mean # of l i p i d d r o p l e t s per s e c t i o n (b) d i s t r i b u t i o n of l i p i d d r o p l e t s i n the NE of 6 s o m i t e embryos (c) d i s t r i b u t i o n of l i p i d d r o p l e t s i n the NE of 8 s o m i t e embryos I d e n t i f i c a t i o n of e x e n c e p h a l y i n SELH f e t u s e s 101 ( o b s e r v e d on day 14 of g e s t a t i o n - 1985) I d e n t i f i c a t i o n of e x e n c e p h a l y p r o d u c i n g mice 103 i n SELH males Frequency of e x e n c e p h a l y i n SELH f e t u s e s 104 ( o b s e r v e d on day 18 of g e s t a t i o n - 1986) SELH male c o n t r o l s f o r the g e n e t i c s t u d y of 105 B C l males ( o b s e r v e d on day 18 of g e s t a t i o n - 1987) A n a l y s i s of h e t e r o g e n e i t y of e x e n c e p h a l y 108 p r o d u c t i o n among SELH males Anova t e s t f o r h e t e r o g e n e i t y among SELH males 109 i n f r e q u e n c y of e x e n c e p h a l y produced Comparison of number of e x e n c e p h a l i c f e t u s e s 111 found between 3 samples (1985, 1986, and 1987) Sex r a t i o i n day 18 SELH f e t u s e s - female 111 s u s c e p t i b i l i t y F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n 116 g e n e t i c s t u d i e s ( t o t a l f e t u s e s ) F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n the 117 F2 of S.I xS.I F l matings F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n t h e 118 F2 of I .s xI.S F l matings Sex r a t i o s among e x e n c e p h a l i c and normal 121 f e t u s e s F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n 122 g e n e t i c s t u d i e s compared t o e x p e c t a t i o n s based on a 1 l o c u s s i m p l e r e c e s s i v e model w i t h 22.4% p e n e t r a n c e (female f e t u s e s ) v i i i T a b l e X X V I I I F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n 122 g e n e t i c s t u d i e s compared t o e x p e c t a t i o n s based on a 1 l o c u s s i m p l e r e c e s s i v e model w i t h 11.5% p e n e t r a n c e (male f e t u s e s ) T a b l e XXIX F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n t h e 124 the B C l progeny of I.S F l males x SELH females T a b l e XXX F r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n t h e 126 the BC2 progeny of B C l males x SELH females T a b l e XXXI T y p i n g of marker l o c i G p i - 1 , Mod-1, Pgk-2, and Gpt-1 i n B C l males 131 i x LIST OF FIGURES F i g u r e 1. M u l t i f a c t o r i a l t h r e s h o l d model (a) Q u a n t i t a t i v e v a r i a b l e (b) L i a b i l i t y F i g u r e 2. Comparison of the r e l a t i o n s h i p between c h r o n o l o g i c a l age and somite count i n ICR/Be, SWV/Bc, and SELH embryos F i g u r e 3. S t a g e : " f o l d s e v l d e n t " - s l d e v i e w of a 1 s o m i t e ICR/Be embryo F i g u r e 4. s t a g e : " f o l d s c u r l e d " - s i d e v i e w of a 6 s o m i t e ICR/Be embryo F i g u r e 5 S t a g e : " p r o s e n f o l d s < 1 / 2" - a 9 s o m i t e ICR/Be embryo (a) f r o n t a l v i e w (b) back v i e w F i g u r e 6 S t a g e : " p r o s e n f o l d s > 1 / 2" - a 10 s o m i t e ICR/Be embryo (a) f r o n t a l v i e w (b) back v i e w F i g u r e 7 S t a g e : " i n i t i a l c o n t a c t - s i t e A"- f r o n t a l v i e w of a 12 s o m i t e ICR/Be embryo F i g u r e 8 (a) S t a g e : " f u s i o n t o mid-mesen, w i t h anp" - f r o n t a l v i e w of a 13 s o m i t e ICR/Be embryo (b) S t a g e : " f u s i o n t o mid-mesen, no anp" - f r o n t a l v i e w of a 12 s o m i t e SWV/Bc embryo F i g u r e 9 (a) S t a g e : " f u s i o n t o apex, w i t h a n p " - f r o n t a l v i e w of a 15 s o m i t e ICR/Be embryo (b) S t a g e : " f u s i o n t o apex, no a n p " - f r o n t a l v i e w of a 15 s o m i t e SWV/Bc embryo F i g u r e 10 S t a g e : " f u s e d t o rhomb"-back v i e w of a 15 s o m i t e ICR/Be embryo F i g u r e 11 S t a g e : " f u s e d " - b a c k v i e w of a 16 s o m i t e ICR/Be embryo F i g u r e 12 S t a g e : " i n i t i a l c o n t a c t - s i t e B"- f r o n t a l v i e w of a 16 s o m i t e SELH embryo F i g u r e 13 S t a g e : " p r o s e n f u s e d " - f r o n t a l v i e w of an 18 s o m i t e SELH embryo page 8 58 77 77 77 77 78 78 78 79 79 79 79 F i g u r e 14 s t a g e : " f u s e d t o mid-mesen, no anp"-£rontal 80 v i e w of an 18 s o m i t e SELH embryo F i g u r e 15 S t a g e : " f u s e d t o apex, no a n p " - f r o n t a l v i e w 80 of an 18 s o m i t e SELH embryo F i g u r e 16 F r o n t a l v i e w of a 22 s o m i t e SELH embryo 80 F i g u r e 17 S c a n n i n g e l e c t r o n m i c r o s c o p y p i c t u r e s 81 (a) 12 s o m i t e SELH embryo (b) 11 s o m i t e ICR/Be embryo (c) 14 s o m i t e SELH embryo (d) 13 s o m i t e ICR/Be embryo (e) 17 s o m i t e SELH embryo F i g u r e 18 P a t t e r n of c l o s u r e of the a n t e r i o r n e u r a l tube 82 (a) normal mice (b) SELH mice F i g u r e 19 P a r a f f i n s e c t i o n s of ICR/Be and SELH embryos 97 (a) mesencephalon s e c t i o n of a 13 s o m i t e ICR/Be embryo (b) mesencephalon s e c t i o n of a 13 s o m i t e SELH embryo (c) p o s t e r i o r mesencephalon of a 9 s o m i t e ICR/Be embryo (d) p o s t e r i o r mesencephalon of a 10 s o m i t e SELH embryo F i g u r e 20 P l a s t i c s e c t i o n s of ICR/Be and SELH embryos 98 (a) p o s t e r i o r mesencephalon of an 8 s o m i t e ICR/Be embryo (b) p o s t e r i o r mesencephalon of an 8 s o m i t e SELH embryo F i g u r e 21 P l a s t i c s e c t i o n of an SELH embryo 98 (a) a n t e r i o r mesencephalon of an 8 s o m i t e SELH embryo (b) neuroectoderm of the a n t e r i o r mesencephalon of an 8 s o m i t e SELH embryo F i g u r e 22 P l a s t i c s e c t i o n s of ICR/Be and SELH embryos 99 (a) rhombencephalon of a 6 s o m i t e ICR/Be embryo (b) rhombencephalon of a 6 s o m i t e SELH embryo F i g u r e 23 P l a s t i c s e c t i o n s of ICR/Be and SELH embryos 99 (a) p r o s e n c e p h a l o n of a 6 s o m i t e ICR/Be embryo (b) p r o s e n c e p h a l o n of a 6 s o m i t e SELH embryo xi F i g u r e 24 D i s t r i b u t i o n of B C l males i n f r e q u e n c y of 128 e x e n c e p h a l y produced (a) t o t a l f e t u s e s (b) female f e t u s e s (c) male f e t u s e s x i i ACKNOWLEDGEMENTS S p e c i a l thanks to Dr. Diana J u r i l o f f f o r her time, e f f o r t , and e x p e r t i s e . A l s o , the author wishes to thank Dr. M u r i e l H a r r i s f o r her v a l u a b l e a d v i c e , sandy Benson f o r b i o c h e m i c a l t e c h n i c a l a s s i s t a n c e , V a l e r i e Magel f o r animal c a r e , and Dr. V. Diewert, Barb T a l t , Dr. D. Kalousek, and John Leung f o r c o n s u l t a t i o n on the h i s t o l o g y experiments. The author i s a l s o g r a t e f u l of the support and encouragement g i v e n by both f a m i l y and f r i e n d s . 1 INTRODUCTION N e u r a l tube c l o s u r e d e f e c t s a r e a common form of b i r t h d e f e c t , and as y e t t h e i r e t i o l o g y i s p o o r l y u n d e r s t o o d . Both g e n e t i c s and the environment a r e thought t o be i m p o r t a n t t o the development of the s e d e f e c t s , however the e x a c t n a t u r e of the s e f a c t o r s has not been d e t e r m i n e d . The n e u r a l tube I s the p r o g e n i t o r t o the b r a i n and s p i n a l c o r d i n c h o r d a t e s . F a i l u r e of the c r a n i a l n e u r a l f o l d s t o f u s e r e s u l t s i n one of the most common forms of n e u r a l tube d e f e c t s , a n e n c e p h a l y i n humans and ex e n c e p h a l y (the d e v e l o p m e n t a l e q u i v a l e n t of anencephaly) i n mice. The a f f e c t e d f e t u s i s c h a r a c t e r i z e d by an exposed e v e r t e d b r a i n mass, which i n a n e n c e p h a l l c s i s g r o s s l y d e generated ( G i r o u d 1960; L e m i r e , B e c k w i t h , Warkany 1978). The SELH mouse s t o c k d e v e l o p e d i n the M e d i c a l G e n e t i c Mouse U n i t a t the U n i v e r s i t y of B r i t i s h Columbia produces a h i g h f r e q u e n c y of exe n c e p h a l y . C a r e f u l a n a l y s i s of the b a s i s of the c l o s u r e d e f e c t i n t h e s e mice may p r o v i d e h e l p f u l i n s i g h t i n t o a n e n c e p h a l y i n humans, and more g e n e r a l l y i n t o the complex p r o c e s s of n e u r u l a t i o n and n e u r a l tube c l o s u r e f a i l u r e . T h i s p r o j e c t was the f i r s t t o s t u d y the g e n e t i c s and embryo-p a t h o l o g y of e x e n c e p h a l y i n the SELH s t o c k . I . STUDIES OF ANENCEPHALY IN HUMANS Anencephaly i s one of the major forms of n e u r a l tube d e f e c t s i n humans. I t a c c o u n t s f o r al m o s t h a l f the cases of n e u r u l a t i o n d e f e c t s i n B r i t i s h Columbia ( T r i m b l e and B a i r d 1978). E t h n i c 2 d i f f e r e n c e s have been documented (Penrose 1957), and the worldwide i n c i d e n c e of anencephaly v a r i e s . In B r i t i s h C olumbia, a n e n c e p h a l y o c c u r s i n 0.68/1000 t o t a l b i r t h s (McBride 1979), compared t o 3.14/1000 t o t a l b i r t h s i n L i v e r p o o l ( S m i t h e l l s 1968), and 0.98/1000 t o t a l b i r t h s i n New Zealand (Burman, S m i t h , Howard 1986). Great B r i t a i n has b o t h v e r y h i g h i n c i d e n c e of a n e n c e p h a l i c newborn and s t i l l b o r n s and g r e a t v a r i a t i o n between c i t i e s i n f r e q u e n c y ( L i v e r p o o l - 3.14//1000 t o t a l b i r t h s ( S m i t h e l l s 1968); O x f o r d - 1.49/1000 t o t a l b i r t h s ( F e d r i c k 1 9 7 6 ) ) . A number of s t u d i e s have not been i n c l u d e d here because t h e i r r e s u l t s may be b i a s e d due t o s i n g l e s o u r c e a s c e r t a i n m e n t of cases and i n c o m p l e t e i d e n t i f i c a t i o n of a l l or most cases of anencephaly. V a r i a t i o n i n the i n c i d e n c e of a n e n c e p h a l y and o t h e r forms of n e u r a l tube d e f e c t s over time has been documented ( J a n e r i c h 1973, MacMahon and Yen 1971, Leek and Rogers 1967). ^ o w e v e r , d i f f e r e n c e s i n a s c e r t a i n m e n t among s t u d i e s weakens much of t h i s work (Leek and Rogers 1967). In s p i t e of t h e s e c o m p l i c a t i o n s , the d a t a suggest t h a t e n v i r o n m e n t a l and s o c i o -economic c o n d i t i o n s may be f a c t o r s i n f l u e n c i n g the l i a b i l i t y t o a n e n c e p h a l y and o t h e r n e u r a l tube d e f e c t s . There may however be a "base r a t e " of a n e n c e p h a l y i n p o p u l a t i o n s upon which a d v e r s e e n v i r o n m e n t a l f a c t o r s c o n t r i b u t e t o i n c r e a s e the r i s k of the d e f e c t . A number of m a t e r n a l f a c t o r s have been i m p l i c a t e d i n i n c r e a s e d r i s k s of n e u r a l tube d e f e c t s . D e f i c i e n c i e s i n d i e t and n u t r i t i o n , f o r example z i n c and f o l i c a c i d d e f i c i e n c y have been 3 proposed as i n c r e a s i n g r i s k s . A 1981 s t u d y by Laurence e t a l (Lau r e n c e , James, M i l l e r e t a l , 1981) found t h a t f o l a t e s u p p l e m e n t a t i o n reduced t h e r e c u r r e n c e of n e u r a l tube d e f e c t s compared t o p l a c e b o s u p p l e m e n t a t i o n , i n the c h i l d r e n of women who had p r e v i o u s l y had a c h i l d w i t h a n e u r a l tube d e f e c t . However, t h i s d i f f e r e n c e was not s i g n i f i c a n t . A 1981 s t u d y of women who had a l r e a d y had a t l e a s t 1 i n f a n t w i t h a n e u r a l tube d e f e c t by S m i t h e l l s e t a l ( S m i t h e l l s , Sheppard, Schorah e t a l 1981), found t h a t women w i t h m u l t i v i t a m i n supplemented p r e g n a n c i e s had s i g n i f i c a n t l y lower r e c u r r e n c e s of n e u r a l tube d e f e c t s (NTDs) compared w i t h unsupplemented women. A more r e c e n t s t u d y ( S e l l e r 1985) has a l s o i n d i c a t e d v i t a m i n s u p p l e m e n t a t i o n may reduce r e c u r r e n c e r i s k s of n e u r a l tube d e f e c t s , however, both th e s e s t u d i e s i n c l u d e d a b i a s e d sample of t r e a t e d / c o m p l i a n t and c o n t r o l / n o n c o m p l i a n t mothers. A doub l e b l i n d c o o p e r a t i v e s t u d y w i t h the U.K., Canada, and the U.S.A. i s c u r r e n t l y i n p r o g r e s s and may c l a r i f y the v a l u e of m u l t i v i t a m i n s f o r l o w e r i n g NTD r i s k s ( H a l l 1987). I t has been suggested t h a t m a t e r n a l age i n f l u e n c e s the i n c i d e n c e of a n e n c e p h a l y (Elwood and Elwood, 1980). S t u d i e s i n F r a n c e , A u s t r a l i a , South Wales, and D u b l i n , s u g g e s t a normal d i s t r i b u t i o n of i n c i d e n c e of an e n c e p h a l y w i t h age ( F r e z a l , K e l l e y , G u i l l e m o t e t a l , 1964; Collmann and S t o l l e r 1962; R i c h a r d s , 1973; C o f f e y and J e s s o p , 1957), however, a Canadian s t u d y (Elwood and Elwood 1980) i n d i c a t e s no c l e a r p a t t e r n . A 4 c o n s i s t e n t f i n d i n g i s the i n c r e a s e d i n c i d e n c e of anenc e p h a l y and o t h e r n e u r a l tube d e f e c t s t o low p a r i t y mothers ( F r e z a l , K e l l e y , G u i l l e m o t e t a l 1964, C o f f e y and J e s s o p 1957, R i c h a r d s 1973, Elwood and Elwood 1980). Other m a t e r n a l f a c t o r s w i t h s u g g ested a s s o c i a t i o n s w i t h NTDs a r e : n u t r i t i o n , f e v e r , c l o s e l y spaced p r e g n a n c i e s , b i r t h b e f o r e or a f t e r a m i s c a r r i a g e , a l c o h o l , d r u g s , and d i a b e t e s ( H a l l 1987). A h e a l t h y m a t e r n a l environment may be i m p o r t a n t t o the normal c l o s u r e of the n e u r a l t u b e . N e u r a l tube d e f e c t s a r e a s s o c i a t e d w i t h t r i s o m i e s 13, 14, 15, 18, and t r i p l o i d y i n humans (Elwood and Elwood 1980), but major chromosomal d e f e c t s do not account f o r the l a r g e number of NTDs i n man. G e n e t i c syndromes a s s o c i a t e d w i t h n e u r a l tube d e f e c t s i n c l u d e Meckels syndrome ( F r i e d , L i b a n , L u r i e e t a l 1971), R o b e r t s syndrome (Freeman, W i l l i a m s , Schmike e t a l 1974), b o t h r e c e s s i v e d i s o r d e r s , and the dominant h e r e d i t a r y s p o n d y l o l i s t h e s i s and s p i n a b i f i d a (Amuso and Mankin 1967), median c l e f t f a c e syndrome, the syndrome of a n t e r i o r s a c r a l meningomyelocoele and a n a l s t e n o s i s (Holmes, D r i s c o l l , A t k i n s 1976). Cases of a m n i o t i c band syndrome, and S a c r o c o c c y g e a l teratomas have a l s o been found w i t h NTDs (Elwood and Elwood 1980). Between 1-12% of n e u r a l tube d e f e c t s have r e c o g n i z e d causes (Khoury, E r i c k s o n , James 1982, Holmes, D r i s c o l l , A t k i n s 1976). That i s , between 88 and 99% of NTDs a r e not a s s o c i a t e d w i t h chromosomal imbalance or r e c o g n i z e d syndromes. A marked female e x c e s s of anenc e p h a l y has been demonstrated i n 5 humans. The sex r a t i o of a n e n c e p h a l i c s v a r i e s from 3 t o 7:1 (fe m a l e s : m a l e s ) (Shulman 1979), and 1.3:1 f o r s p i n a b i f i d a (a l e s i o n a t the c a u d a l end of the n e u r a l tube) ( C a r t e r 1974b). The r a t i o l o w e r s i n a r e a s of lower i n c i d e n c e such as i n American negroes and I s r a e l i s ( C a r t e r 1974b). In man i t i s not y e t c l e a r whether male a n e n c e p h a l i c s d i e i n u t e r o , or i f females a r e a c t u a l l y more l i a b l e . Twin s t u d i e s do not h e l p i l l u m i n a t e the e t i o l o g y of NTDs. There i s a g e n e r a l l y r e p o r t e d low concordance r a t e f o r NTDs i n monozygotic t w i n s which i s about e q u a l t o t h a t of d i z y g o t i c t w i n s and lower t h a n r e p o r t e d r e c u r r e n c e r i s k s of 4-5% ( F r e z a l , K e l l e y , G u i l l e m o t e t a l 1964, C a r t e r 1974, L e m i r e , B e c k w i t h , Warkany 1978). Elwood and Elwood (1980) however, found t h e concordance r a t e amongst l i k e - s e x t w i n p a i r s (which would i n c l u d e monozygotic p a i r s ) t o be h i g h e r than t h a t of u n l i k e - s e x ( d i z y g o t i c ) p a i r s . F a m i l i a l r e c u r r e n c e r i s k s of NTDs are g r e a t e r t h a n the g e n e r a l r i s k f o r the p o p u l a t i o n . C a r t e r and Evans (1973) found the p r o p o r t i o n of s i b s a f f e c t e d w i t h s p i n a b i f i d a and/or a n e n c e p h a l y were 3.4% f o r s p i n a b i f i d a i n d e x cases and 5.4% f o r a n e n c e p h a l i c cases i n a s t u d y of 23 Buroughs i n London E n g l a n d . There was a tendency f o r s i b s t o be a f f e c t e d w i t h the same d e f e c t as the in d e x c a s e s , but t h i s was not s i g n i f i c a n t . A s i m i l a r r e c u r r e n c e r i s k of 4.6% f o r anenc e p h a l y and s p i n a b i f i d a was found i n an e a r l i e r s t u d y of 5 h o s p i t a l s on Rhode I s l a n d (USA) (Yen and 6 MacMahon 1968). However, the s e s t u d i e s have not e l i m i n a t e d from t h e i r c a l c u l a t i o n s c a ses of NTDs where t h e r e a r e r e c o g n i z e d causes of the d e f e c t s , f o r example, s i n g l e gene d i s o r d e r s , known syndromes and chromosomal a b n o r m a l i t i e s . R i s k v a l u e s may t h e r e f o r e not be a c c u r a t e f o r the g e n e r a l p o p u l a t i o n . A more r e c e n t s t u d y which r u l e d out cases w i t h known syndromes, found the r e c u r r e n c e r i s k f o r p r i m a r y n e u r u l a t i o n d e f e c t s was 3.5% (4. 5 % f o r a n e n c e p h a l i c probands w i t h o u t a d d i t i o n a l c o n g e n i t a l a n o m a l i e s ) , compared w i t h o n l y 0.8% f o r c a n a l i z a t i o n d e f e c t s i n B r i t i s h Columbia (Keena, p e r s o n a l c ommunication). Two s t u d i e s of p r e c u r r e n c e r a t e s ( r a t e of NTDs i n s i b l i n g s born b e f o r e index c ase) have e x c l u d e d c ases w i t h r e c o g n i z e d c a u s e s . The p r e c u r r e n c e r a t e s f o r s i b l i n g s of i s o l a t e d NTDs were 1.7% (1/54) i n a Boston sample (Holmes, D r i s c o l l , and A t k i n s 1976) and 2.0% (4/200) i n an A t l a n t a p o p u l a t i o n (Khoury, E r i c k s o n , and James, 1982), about l l x the p o p u l a t i o n r a t e . Another s t u d y l o o k e d a t 30 f a m i l i e s i n H o l l a n d w i t h NTDs and found 24 of the f a m i l i e s (more than 3/4 of the f a m i l i e s ) were r e l a t e d . T h i s o b s e r v a t i o n s u g g e s t s a s i g n i f i c a n t g e n e t i c cause, and a s m a l l number of genes r e s p o n s i b l e f o r the d e f e c t (Polman c i t e d by L e m i r e , B e c k w i t h , and Warkany 1978). The presence of a g e n e t i c component i n l i a b i l i t y t o NTDs i s suggested by h i g h r e c u r r e n c e r i s k s , e t h n i c d i f f e r e n c e s i n the i n c i d e n c e of thes e d e f e c t s , and the presence of m u t a t i o n s a f f e c t i n g n e u r a l tube c l o s u r e i n o t h e r s p e c i e s . There i s ev i d e n c e of h e t e r o g e n e i t y ( i d e n t i c a l phenotypes caused by more 7 than one g e n e t i c mechanism) w i t h i n n e u r a l tube d e f e c t s , f o r example the d i f f e r e n t n e u r u l a t i o n p r o c e s s e s of p r i m a r y and se c o n d a r y n e u r u l a t i o n , the a s s o c i a t i o n of NTDs w i t h Meckels syndrome, and NTDs a s s o c i a t e d w i t h chromosomal a n o m a l i e s . Simple m e n d e l i a n s i n g l e gene i n h e r i t a n c e does not account f o r the m a j o r i t y of NTDs however, and t h e r e i s some e v i d e n c e t o suggest t h a t a n e n c e p h a l y and s p i n a b i f i d a a r e m u l t i f a c t o r i a l l y i n h e r i t e d ( C a r t e r and Evans 1973). M u l t i f a c t o r i a l i n h e r i t a n c e means t h a t both g e n e t i c and e n v i r o n m e n t a l f a c t o r s c o n t r i b u t e t o d e t e r m i n e an i n d i v i d u a l ' s l i a b i l i t y f o r the d i s o r d e r ( F r a s e r 1976). There may be a few genes or a number of genes t h a t combined w i t h t h e s p e c i f i c environment can l e a d t o t h e s e d e f e c t s . The m u l t i f a c t o r i a l t h r e s h o l d concept i n v o l v e s a c o n t i n u o u s v a r i a b l e such as "time of e l e v a t i o n of p a l a t a l s h e l v e s " (or time of n e u r a l f o l d a p p r o x i m a t i o n ) , a f r e q u e n c y of d i s t r i b u t i o n of a p o p u l a t i o n , and a t h r e s h o l d p o i n t beyond which i n d i v i d u a l s w i l l be a f f e c t e d . The p o p u l a t i o n mean i s c o n s i d e r e d t o be w e l l away from the t h r e s h o l d ( f i g u r e l a ) . L i a b i l i t y t o a d i s o r d e r would be made up of an i n d i v i d u a l s g e n e t i c and e n v i r o n m e n t a l r i s k f a c t o r s ( f i g u r e l b ) . Assuming a c o n t i n u o u s normal d i s t r i b u t i o n of a m u l t i f a c t o r i a l t r a i t , p r e d i c t i o n s can be made, some of which can be compared w i t h a v a i l a b l e i n f o r m a t i o n about a n e n c e p h a l y ( F r a s e r 1976). The p r e d i c t i o n s a r e : 1. F a m i l i e s w i t h a f f e c t e d i n d i v i d u a l s w i t h i n a 8 F i g u r e 1 (a) MULTIFACTORIAL THRESHOLD MODEL - QUANTITATIVE VARIABLE* TH«.6iHotc> -o-QUANTITATIVE VARIABLE (b) MULTIFACTORIAL THRESHOLD MODEL - LIABILITY+ L I A B I L I T Y n o t e : = f r e q u e n c y w i t h i n a p o p u l a t i o n L i a b i l i t y t o a d i s o r d e r i s c o n s i d e r e d t o be n o r m a l l y d i s t r i b u t e d (as i s the q u a n t i t a t i v e v a r i a b l e ) . The g r e a t e r the l i a b i l i t y , t he g r e a t e r t h e p r o p o r t i o n of a f f e c t e d i n d i v i d u a l s . * : ( F r a s e r 1976) +:(Curnow and Smith 1975) 9 p o p u l a t i o n w i t h a h i g h f r e q u e n c y of the d e f e c t w i l l have a g r e a t e r r e c u r r e n c e r i s k of the d e f e c t t h a n f a m i l i e s of a f f e c t e d i n d i v i d u a l s i n a p o p u l a t i o n w i t h a low f r e q u e n c y of the d e f e c t . However, the i n c r e a s e i n r i s k w i l l not be p r o p o r t i o n a t e l y as g r e a t f o r those of the f i r s t ( h i g h e r ) p o p u l a t i o n compared t o the second ( l o w e r ) p o p u l a t i o n . I n South Wales, the b i r t h f r e q u e n c y of a n e n c e p h a l y and s p i n a b i f i d a i s 0.76% and the p e r c e n t of a f f e c t e d s i b l i n g s i s 5.2%. In London, the f r e q u e n c y i s 0.29% and the p e r c e n t of a f f e c t e d s i b l i n g s i s 4.4% ( C a r t e r 1974). T h i s f i t s w e l l w i t h the p r e d i c t i o n . 2. R e c u r r e n c e r i s k s f o r s i b s i n f a m i l i e s w i t h two a f f e c t e d c h i l d r e n w i l l be g r e a t e r than the r i s k f o r f a m i l i e s w i t h o n l y one a f f e c t e d c h i l d . McBride (1979) found the r e c u r r e n c e r i s k of NTDs was 2.1% f o r a f a m i l y w i t h one a f f e c t e d c h i l d and 4.8% i f t h e r e were 2 a f f e c t e d c h i l d r e n i n the f a m i l y . 3. There s h o u l d be an i n c r e a s e d f r e q u e n c y of the d e f e c t i n c h i l d r e n of consanguineous p a r e n t s . In A l e x a n d r i a , the i n c i d e n c e of NTDs i n o f f s p r i n g of f i r s t c o u s i n s or c l o s e r r e l a t i v e s i s 14.2/1000 v e r s u s the 5.7/1000 i n c i d e n c e i n o f f s p r i n g of u n r e l a t e d p a r e n t s (Stevenson Johnson, S t e w a r t e t a l 1966 see Elwood and Elwood 1980). T h i s i n c r e a s e d f r e q u e n c y would a l s o be ex p e c t e d i f the d e f e c t were due t o a r e c e s s i v e gene, however, i t would not be ex p e c t e d i f n e u r a l tube d e f e c t s were caused by a a d o m i n a n t l y I n h e r i t e d gene. At p r e s e n t , most m u l t i f a c t o r i a l t h r e s h o l d models a r e q u i t e s i m p l i f i e d and work under a number of assumptions which f o r many 10 t r a i t s may not be t r u e . For the most b a s i c m u l t i f a c t o r i a l t h r e s h o l d models, the shape of the d i s t r i b u t i o n of the p o p u l a t i o n i s e s t i m a t e d on the b a s i s of the t a i l of t h e d i s t r i b u t i o n c r o s s i n g the t h r e s h o l d and i s assumed t o be normal, a l t h o u g h i t c o u l d w e l l be skewed i n one d i r e c t i o n or a n o t h e r . T h i s a s s u m p t i o n a f f e c t s e s t i m a t i o n s of p o p u l a t i o n r i s k v a l u e s . Another a s s u m p t i o n i s t h a t a l l f a c t o r s c o n t r i b u t i n g t o the t r a i t have e q u a l and a d d i t i v e e f f e c t s i g n o r i n g the p o s s i b i l i t i e s of dominance, major l o c i , and gene/environment i n t e r a c t i o n s . Models i n v o l v i n g one or a few major gene l o c i may e x p l a i n t r a i t s w i t h "complex" i n h e r i t a n c e e q u a l l y w e l l a s , or b e t t e r t h a n , t h o s e models r e q u i r i n g a l a r g e number of genes. Many v a r i a t i o n s on the m u l t i f a c t o r i a l models can be made. For example, a s m a l l number of genes (2 t o 3 dominant or r e c e s s i v e , a d d i t i v e or e p i s t a t i c genes) may be n e c e s s a r y t o produce a m a l f o r m a t i o n , or a d e f e c t may be the r e s u l t of 1 major gene w i t h reduced pene t r a n c e due t o m o d i f i e r s . These d i s t i n c t i o n s from the most b a s i c a d d i t i v e p o l y g e n i c m u l t i f a c t o r i a l models a r e i m p o r t a n t , s i n c e t o i d e n t i f y the i n d i v i d u a l genes a c t i n g on a t r a i t would be a l m o s t i m p o s s i b l e i n a system w i t h a l a r g e number of genes, but would be p o s s i b l e i f o n l y a few major genes were r e s p o n s i b l e f o r the d e f e c t . These d i s t i n c t i o n s a r e a l s o i m p o r t a n t when c o n s i d e r i n g p r e v e n t i o n of m u l t i f a c t o r i a l c o n g e n i t a l d e f e c t s . I f a d e f e c t were due t o a s m a l l number of major genes i t might be p o s s i b l e t o p r e v e n t the d e f e c t by a l t e r i n g one or a s m a l l number of i n f l u e n c e s . However, i f a l a r g e number of g e n e t i c and 11 e n v i r o n m e n t a l f a c t o r s were e q u a l l y and a d d i t i v e l y c o n t r i b u t i n g t o t he l i a b i l i t y f o r a d e f e c t , a l a r g e number of s m a l l changes might need t o be made t o p u l l the p o p u l a t i o n d i s t r i b u t i o n away from the t h r e s h o l d . The m u l t i f a c t o r i a l t h r e s h o l d models a r e models t h a t can be used t o g e t a t the c a u s a l bases behind d i s o r d e r s w i t h a known g e n e t i c component t h a t do not f i t s i m p l e M e n d e l i a n s e g r e g a t i o n p a t t e r n s . These d i s o r d e r s i n t u r n p r o v i d e us w i t h model systems w i t h which t o b e t t e r e x p l a i n the g e n e t i c mechanisms, t h r e s h o l d s , and u n d e r l y i n g d i s t r i b u t i o n s of l i a b i l i t y of common c o n g e n i t a l m a l f o r m a t i o n s . S m a l l sample s i z e s , b i a s e d a s c e r t a i n m e n t of c a s e s and, the f a i l u r e t o s e p a r a t e g e n e t i c a l l y heterogeneous causes of n e u r a l tube d e f e c t s f l a w much of the work on anenc e p h a l y i n humans. I n human s t u d i e s , i t i s u s u a l l y not p o s s i b l e t o a n a l y s e m u l t i p l e g e n e r a t i o n s , nor can n e u r a l tube c l o s u r e be examined d i r e c t l y . A nimal systems p r o v i d e a means by which the causes of n e u r a l tube d e f e c t s can be d i r e c t l y i n v e s t i g a t e d . There a r e major advantages i n u s i n g mice t o c r e a t e g e n e t i c and d e v e l o p m e n t a l models f o r human n e u r a l tube d e f e c t s . S p e c i f i c g e n e t i c c r o s s e s can be made w i t h mice, t h e i r embryos can be m a n i p u l a t e d , and b e i n g mammals, mice a r e more r e l a t e d t o man than b i r d s or r e p t i l e s . Mice have a r e l a t i v e l y s h o r t g e s t a t i o n p e r i o d and l a r g e l i t t e r s i z e s , and more i s known about the 12 g e n e t i c s and development of the mouse than any o t h e r mammal w i t h t h e p o s s i b l e e x c e p t i o n of man. As w e l l , mutants of n e u r a l tube c l o s u r e e x i s t i n mice and can t h e r e f o r e be s t u d i e d . R esearch on n e u r a l tube d e f e c t s i n mouse mutants may be a b l e t o p r o v i d e some c l e a r e r u n d e r s t a n d i n g of the t i s s u e and c e l l u l a r bases of f a i l u r e of n e u r a l tube c l o s u r e , the g e n e t i c s of NTDs, the r o l e of e n v i r o n m e n t a l f a c t o r s , and p r o v i d e p o s s i b l e p r e v e n t i o n methods. I I . MOUSE MUTATIONS AFFECTING NEURAL TUBE CLOSURE There a r e s e v e r a l m u t a t i o n s i n mice which produce n e u r a l tube d e f e c t s . The m u t a t i o n s " e x e n c e p h a l y " and " c r a n i o r a c h i s c h i s i s " ( f i s s u r e of the c e p h a l i c n e u r a l f o l d s ) b oth produce a h i g h f r e q u e n c y of e x e n c e p h a l y w i t h o u t a s s o c i a t i o n w i t h o t h e r forms of n e u r a l tube d e f e c t s , or o t h e r body d e f e c t s . C r a n i o r a c h i s c h i s i s ( c r n ) i s r e p o r t e d t o be a r e c e s s i v e as y e t unmapped gene m a i n t a i n e d i n a random bred g e n e t i c a l l y heterogenous s t o c k ( K a l t e r 1985). The f r e q u e n c y of the d e f e c t has v a r i e d between 18-21% i n the g e n e t i c a l l y heterogenous s t o c k f o r most y e a r s , and has a sex r a t i o of a f f e c t e d a n i m a l s of about 2:1 ( f e m a l e : m a l e ) . A sex r a t i o of 1.2:1 (female:male) has been r e p o r t e d f o r the s t o c k i n g e n e r a l . I t i s not c l e a r whether more males a r e r e s o r b e d i n u t e r o and/or m u t i l a t e d a t b i r t h , or i f a t r u e sex r a t i o d i f f e r e n c e e x i s t s f o r e x e n c e p h a l i c and normal mice of t h i s type ( K a l t e r 1983). The s t o c k i s d i f f i c u l t t o 13 m a i n t a i n and i s not a v a i l a b l e f o r f u r t h e r s t u d y a t p r e s e n t ( K a l t e r , p e r s o n a l communication). E x e n c e p h a l y (xn) i s a l s o thought t o be a r e c e s s i v e unmapped gene. I t o c c u r r e d i n random bred mice t r e a t e d w i t h d i c h l o r o -d i p h e n y l - t r i c h l o r o t h a n e (DDT) ( W a l l a c e , K n i g h t s , Dye 1976), and shows i n c o m p l e t e and v a r i a b l e p e n e t r a n c e (33-84%) ( W a l l a c e , K n i g h t s , Anderson 1978). The sex r a t i o of a f f e c t e d mice i s about 2 f e m a l e : l male. The t o t a l sex r a t i o i s c o n s i s t e n t between s e g r e g a t i n g and n o n s e g r e g a t i n g l i t t e r s s u g g e s t i n g no p r e n a t a l l o s s of a f f e c t e d males ( W a l l a c e , K n i g h t s , Anderson 1978). The e m b r y o l o g i c a l causes of e x e n c e p h a l y i n c r n and xn are not known. Ex e n c e p h a l y i s a l s o a s s o c i a t e d w i t h the c u r l y t a i l m u t a t i o n i n mice. C u r l y t a i l ( c t ) i s thought t o be a s i n g l e gene r e c e s s i v e m u t a t i o n w i t h i n c o m p l e t e p e n e t r a n c e , a l t h o u g h c o n c l u s i v e e v i d e n c e has not y e t been r e p o r t e d . I t a r o s e s p o n t a n e o u s l y i n the GFF i n b r e d s t r a i n and i s y e t unmapped (Gruneberg 1954). A female mouse ( w i t h a c u r l e d t a i l ) of the GFF s t r a i n was mated t o a male of t h e i n b r e d s t r a i n CBA/Gr t o c r e a t e the c u r l y t a i l s t o c k (Gruneberg 1954). I t i s not c l e a r how many b a c k c r o s s e s t o CBA/Gr were made w i t h the o u t c r o s s a n i m a l s , a l t h o u g h the mice have been a randomly b r e d , c l o s e d c o l o n y s e l e c t e d t o m a i n t a i n t h e c u r l y t a i l phenotype over the y e a r s (Embury, S e l l e r , A d i n o l f i e t a l 1979). About 50-60% of presumed homozygotes a r e abnormal w i t h the m a j o r i t y of c a s e s h a v i n g t a i l d e f o r m i t i e s such 14 as a k i n k e d or c o i l e d t a i l w i t h or w i t h o u t s p i n a b i f i d a . E x e n c e p h a l y o c c u r s i n l e s s than 1% of o f f s p r i n g and has a marked female e x c e s s (7 females:0 males i n t h i s s t u d y ) ( S e l l e r and P e r k i n s - C o l e 1987). The cause of e x e n c e p h a l y i n the s t o c k i s unknown, however p o s t e r i o r neuropore c l o s u r e appears t o be d e l a y e d i n c u r l y t a i l mice r e l a t i v e t o development compared w i t h A/St embryos (Copp, S e l l e r , P o l a n i e t a l 1982). H i s t o l o g i c a l s e c t i o n s of the c u r l e d t a i l of c t embryos showed the n e u r a l tube t o be c l o s e d i n the r e g i o n of the f l e x i o n d e f e c t , but the n e u r a l c a n a l i s overexpanded and p o s i t i o n e d above the mesoderm r a t h e r than embedded w i t h i n the mesoderm as seen i n s t r a i g h t t a i l e d embryos ( I b i d ) . Copp, S e l l e r , P o l a n i e t a l (1982) propose t h a t the d e l a y e d p o s t e r i o r neuropore c l o s u r e produces a m e c h a n i c a l s t r e s s on the d e v e l o p i n g t r u n k and t a i l which causes a f l e x i o n d e f e c t . A comparison of sex and d e v e l o p m e n t a l s t a g e of 10 l i t t e r s (67 embryos) of t h e c t s t r a i n , c o l l e c t e d e a r l y on day 9 of g e s t a t i o n , found females ( i d e n t i f i e d by sex c h r o m a t i n masses) t o be l e s s mature t h a n males ( S e l l e r , P e r k i n s - C o l e 1987). T h i s s t u d y a l s o found t h a t on the morning of day 9 of pregnancy (day of v a g i n a l p l u g = day 1 ) , a t the b e g i n n i n g of n e u r u l a t i o n males averaged 6.2 s o m i t e s compared w i t h 3.7 s o m i t e s i n f e m a l e s . I t remains t o be d e t e r m i n e d whether females d e v e l o p a t the same r a t e as males t h r o u g h n e u r a l tube c l o s u r e i n the c u r l y t a i l s t r a i n . I t i s a l s o not known a t what d e v e l o p m e n t a l s t a g e female 15 and male c t embryos c l o s e the n e u r a l t u b e , and whether females l a g b ehind males i n n e u r a l tube c l o s u r e . I f male and female development i n c u r l y t a i l a r e o f f s e t , but d e v e l o p i n g a t the same r a t e f o r both n e u r a l tube c l o s u r e and o v e r a l l development, t h e n t h i s t e m p o r a l d i f f e r e n c e i n m a t u r i t i e s l i k e l y does not c o n t r i b u t e t o the p r o d u c t i o n of n e u r a l tube c l o s u r e d e f e c t s . M u t a t i o n s a t the S p l o t c h l o c u s on chromosome 1, S p l o t c h ( S p J ) and S p l o t c h - d e l a y e d ( S p d ) a r o s e i n d e p e n d e n t l y i n the i n b r e d C57BL/6J s t r a i n and both a r e semidominant homozyogous l e t h a l m u t a t i o n s ( D i c k i e 1964). H e t e r o z y g o t e s e x p r e s s a w h i t e b e l l y s p o t , Sp J homozygote embryos d i e on about day 13 of g e s t a t i o n w i t h l u m b o s a c r a l r a c h i s c h i s i s w i t h or w i t h o u t e x e n c e p h a l y and t a i l a n o m a l i e s (Dempsey and T r a s l e r 1983). The g e n e t i c background i n f l u e n c e s the e x p r e s s i o n of t h e Sp m u t a t i o n . Sp i n a b a l a n c e d l e t h a l o t o c e p h a l y s t o c k of mixed background (DBA/2J, C57BL/10J, C57L/J, and C57BL/6J) produced e x e n c e p h a l y i n about 1/2 of Sp homozygotes ( J u r i l o f f p e r s o n a l communication, J u r i l o f f , S u l i k , R o d e r i c k e t a l 1985). S p d homozygotes d i e a t b i r t h w i t h c a u d a l r a c h i s c h i s i s . N e u r a l tube c l o s u r e of b o t h the a n t e r i o r and p o s t e r i o r neuropore i s d e l a y e d i n Sp/+ l i t t e r s (Dempsey and T r a s l e r 1983). An overabundance of gap j u n c t i o n a l v e s i c l e s found i n the n e u r a l groove of day 9 Sp/Sp embryos may r e p r e s e n t a l o s s of c e l l c o n t a c t and/or communication i n the n e u r o e p i t h e l i u m ( W i l s o n and F i n t a 1979). Both S p l o t c h and S p l o t c h - d e l a y e d have n e u r a l c r e s t c e l l 16 d e r i v a t i v e d e f e c t s , f o r example pigment and s p i n a l g a n g l i o n d e f e c t s . Homozygous S p d embryos have s i g n i f i c a n t r e d u c t i o n of s p i n a l g a n g l i a volume compared t o h e t e r o z y g o u s or homozygous normal l i t t e r m a t e s , r e g a r d l e s s of whether S p d / S p d embryos were m o r p h o l o g i c a l l y normal or not (Moase and T r a s l e r 1987). The u n d e r l y i n g d e f e c t i n S p l o t c h - d e l a y e d mice may be t o i n h i b i t n e u r a l c r e s t c e l l m i g r a t i o n which s e c o n d a r i l y causes n e u r a l tube d e f e c t s (Moase and T r a s l e r 1987). A l t e r n a t i v e l y , n e u r a l . c r e s t c e l l s may be d e f i c i e n t i n the n e u r a l ectoderm as a r e s u l t of some more p r i m a r y d e f e c t i n n e u r a l t i s s u e i n d u c t i o n which d i s r u p t s normal n e u r a l tube,, c l o s u r e . C u r l y t a i l , C r a n i o r a c h i s c h i s i s , and S p l o t c h a r e a l l s e n s i t v e t o V i t a m i n A induced n e u r a l tube d e f e c t s ( S e l l e r , P e r k i n s , A d i n o l f i 1983, K a l t e r 1983, Dempsey, T r a s l e r 1983). Dempsey and T r a s l e r (1983) found a s i g n i f i c a n t i n c r e a s e i n s p i n a b i f i d a w i t h or w i t h o u t e x e n c e p h a l y i n Sp/+ v i t a m i n A (day 8- 50 mg/kg) t r e a t e d l i t t e r s compared t o +/ + l i t t e r s . K a l t e r t r e a t e d w i t h 5-25 mg/kg on day 8 and found l a r g e f r e q u e n c i e s (72-87%) of e x e n c e p h a l y i n crn/+ l i t t e r s , but found l e s s than 1% e x e n c e p h a l y i n S p d / + l i t t e r s w i t h the same t r e a t m e n t ( K a l t e r 1983). I t i s not known i f the r e s p o n s i v e n e s s of crn/+ l i t t e r s i s due t o the c r n gene or t o g e n e t i c background. C u r l y t a i l mice have been shown t o be more s u s c e p t i b l e t o v i t a m i n A Induced n e u r a l tube d e f e c t s t h a n A/St mice ( S e l l e r , Embury, P o l a n i e t a l 1979). There a l s o appears t o be a m a t e r n a l e f f e c t of A/St and c u r l y t a i l mothers on the i n c i d e n c e of n e u r a l tube d e f e c t s i n female embryos 17 ( S e l l e r , P e r k i n s , A d i n o l f i 1983). A r e d u c t i o n of the i n c i d e n c e of n e u r a l tube d e f e c t s w i t h the day 9 a d m i n i s t r a t i o n of low doses of v i t a m i n A has a l s o been found ( S e l l e r and P e r k i n s 1982) . L o o p - t a i l (Lp) a semidominant m u t a t i o n a l s o on chromosome 1 produces t a i l t w i s t i n g i n h e t e r o z y g o t e s (Green 1981). In homozygotes i t produces open n e u r a l f o l d s i n the m i d b r a i n -h i n d b r a i n r e g i o n w i t h or w i t h o u t open f o l d s i n the r e s t of the n e u r a l tube ( S t r o n g and H o l l a n d e r 1949). H i s t o l o g i c a l s e c t i o n s of Lp/Lp embryos found no a b n o r m a l i t i e s of 9-18 day g e s t a t i o n a l embryos o t h e r t h a n an i n c r e a s e i n m i t o t i c f i g u r e s i n the b r a i n r e g i o n of Lp/Lp embryos ( S t e i n and Rudin 1953). A l a t e r s t u d y ( W i l s o n and Center 1974), found the i n c r e a s e i n m i t o t i c f i g u r e s t o be due t o p r o l o n g e d c e l l c y c l e r a t h e r than i n c r e a s e d p r o l i f e r a t i o n . H i s t o c h e m i c a l s t u d i e s i n d i c a t e the presence of more s u l f a t e d g l y c o s a m i n o g l y c a n s and l e s s h y a l u r o n i c a c i d i n Lp/Lp embryos compared w i t h normal l i t t e r mates on day 9 ( W i l s o n 1985). Lp/Lp embryos of 9 - 9 1/2 days g e s t a t i o n a l s o show a s h o r t e r n e u r a l p l a t e and n o t o c h o r d t h a n normal and a r e t a r d e d r e g r e s s i o n of the p r i m i t i v e s t r e a k (Smith and S t e i n 1962). I t has been s u g g e s t e d t h a t a f a i l u r e i n e l o n g a t i o n of the n e u r a l tube and n o t o c h o r d may be r e s p o n s i b l e f o r the d e f e c t s . However, the o r i g i n of t h e n e u r a l tube d e f e c t i n L o o p - t a i l i s s t i l l not known (Smith and S t e i n 1962). 18 The autosomal semidominant gene r i b f u s i o n ( R f ) produces s k e l e t a l a n o m a l i e s (such as f u s e d r i b s ) i n h e t e r o z y g o t e s . T h e i l e r and Stevens (1960) r e p o r t e d i r r e g u l a r waves i n t h e n e u r a l p l a t e and n e u r a l tube of homozygotes, and t h a t about 2/3 of R f / R f embryos were e x e n c e p h a l i c . Homozygotes f a i l t o form s o m i t e s and d i e a t or b e f o r e b i r t h w i t h e x t e n s i v e s k e l e t a l problems. C o l e and T r a s l e r (1980) found Rf/+ and Cd/+ (crooked) embryos t o be more s e n s i t i v e t o I n s u l i n i n d u ced e x e n c e p h a l y t h a n t h e i r +/+ l i t t e r m a t e s , when h e t e r o z y g o t e s were mated t o A/J or SWV mice. T h i s i n d i c a t e s a gene - t e r a t o g e n i n t e r a c t i o n i n the p r o d u c t i o n of e x e n c e p h a l y i n t h i s system. Crooked (Cd) i s a semidominant m u t a t i o n on chromosome 6 which o c c u r r e d i n the i n b r e d A/_ s t r a i n . H e t e r o z y g o t e s have c r o o k e d t a i l s and abnormal v e r t e b r a e , a l t h o u g h some f e t u s e s do not appear t o be a f f e c t e d . Crooked t a i l s a l o n g w i t h growth r e t a r d a t i o n , I n c i s o r d e f e c t s , nervous head movements, and e x e n c e p h a l y can be found i n the more s e v e r e l y a f f e c t e d homozygotes. E x e n c e p h a l y has been found i n 4.8% (26/544) of f e t u s e s o b s e r v e d i n Cd/_ x Cd/_ matings (Morgan 1954). E x t r a t o e s ( X t ) and e x t r a t o e s - b r a c h y p h a l a n g y ( X t b p h ) a r e semidominant l e t h a l m u t a t i o n s mapped t o chromosome 13. Xt a r o s e s p o n t a n e o u s l y i n a c o n t r o l s e r i e s of r a d i a t i o n e x p e r i m e n t s on the background of e i t h e r (101 x C3H/He)Fl or the CBA/H mouse (Lyon, M o r r i s , S e a r l e e t a l 1967). X t b p h a r o s e i n a n e u t r o n 19 i r r a d i a t i o n exper iment on the background genome of (C3H/HeH x 101) F l (Johnson 1969) . The two a l l e l e s a re s i m i l a r In phenotype , ye t d i s t i n c t . H e t e r o z y g o t e s have l a r g e i n t e r f r o n t a l b o n e s , and whi te b e l l y s p o t s on some g e n e t i c b a c k g r o u n d s . Xt h e t e r o z y g o t e s a l s o have e x t r a d i g i t s on the h i n d f e e t , whereas X t b p h / + embryos have t h i c k e n e d f i r s t d i g i t s on a l l f e e t and some s y n d a c t y l i s m (Johnson 1969, Johnson 1967) . Xt homozygotes have edema, h e m l m e l l a , h y d r o c e p h a l y , and open p o r t i o n s of the h l n d b r a i n r e g i o n of the n e u r a l t u b e , as w e l l as a number of o ther anomal ies (Johnson 1967) . The r o o f of the rhombencephalon i f c l o s e d i n X t / x t embryos i s a b n o r m a l l y s h a p e d , x t b p n homozygotes a re more s e v e r e l y a f f e c t e d w i th f a c i a l b l e b s , edema, u n i l a t e r a l or b i l a t e r a l h a i r l i p s , and a h i g h f r e q u e n c y ( p e r c e n t u n s t a t e d ) of e x e n c e p h a l y (Johnson 1967) . A number of muta t ions i n the "T" complex a l s o have d e f e c t s i n the n e u r a l t u b e . Two of these mutants have an a c t u a l c l o s u r e f a i l u r e . The T c ( c u r t a i l e d ) a l l e l e was found i n the o f f s p r i n g of i r r a d i a t e d (C3H/He x 101/H) F l males mated to a female of a m u l t i r e c e s s i v e marker s t o c k ( B a t c h e l o r , P h i l l i p s , S e a r l e 1966) . H e t e r o z y g o t e s have v e r t e b r a l and r i b f u s i o n , w i th most or a l l of the t a i l a b s e n t . Homozygotes d i e around day 10 of g e s t a t i o n w i t h l o s s of f o r e l imb buds and n o t o c h o r d , open n e u r a l f o l d s i n the t r u n k , and k i n k s i n the s p i n a l c o r d ( S e a r l e 1966) . S e a r l e (1966) proposed t h a t the a b n o r m a l i t i e s of n o t o c h o r d and p a r a x i a l mesoderm d e r i v a t i v e s i n T c embryos s u g g e s t s t h a t the p r i m a r y d e f e c t may r e s i d e i n the p r i m i t i v e s t r e a k but t h i s has not been 20 t e s t e d . T n, T - H a r w e l l , i s a n o t h e r "T" complex mutant w i t h n e u r a l tube c l o s u r e problems (Lyon 1959). T n was found i n a c o n t r o l s e r i e s of i r r a d i a t i o n e x p e r i m e n t s and i s i n d i s t i n g u i s h a b l e from T c. E x e n c e p h a l y has been observed i n 100% of t r i s o m y 12 embryos and i n 50% of t r i s o m y 14 embryos on a MFl ( o u t b r e d s t r a i n ) , and mixed w i l d t y pe background (M. p o s h i a v i n u s and M. musculus) ( P u t z and M o r r i s s - K a y 1981). Both t r i s o m i e s have been used t o s t u d y e x e n c e p h a l y and n e u r a l tube c l o s u r e f a i l u r e . The t r i s o m i e s a r e g e n e r a t e d by mating males d o u b l y h e t e r o z y g o u s f o r 2 p a r t i a l l y homologous R o b e r t s o n i a n chromosomes ( t h a t i s , c o n t a i n i n g chromosome 12 or 14 and a n o t h e r autosome) on a mixed w i l d type background t o female mice of the o u t b r e d MFl s t r a i n . The f r e q u e n c y of e x e n c e p h a l y can be m o d i f i e d u s i n g d i f f e r e n t m a t e r n a l s t r a i n backgrounds (Gropp and Grohe 1981). For both t r i s o m i e s , e x e n c e p h a l y appears t o be the r e s u l t of a f a i l u r e of c l o s u r e of the n e u r a l f o l d s ( P u t z and M o r r i s - K a y 1981). F l a r e d a n t e r i o r n e u r a l f o l d s f a l l t o e l e v a t e and f u s e . In t r i s o m y - 1 2 , the f o l d s are open back t o the o t i c p i t s compared w i t h t r i s o m y - 1 4 embryos which a r e open o n l y t o the m i d b r a i n -h i n d b r a i n j u n c t i o n ( i b i d ) . Tfisomy-12 and t r l s o m y - 1 4 embryos demonstrate h y p o p l a s i a , s l o w e r somite development, and r e t a r d a t i o n of n e u r a l tube development r e l a t i v e t o s o m i t e development compared w i t h e u p l o l d l i t t e r m a t e s ( I b i d ) . E a r l y d e f e c t s of t h e c r a n i a l mesenchyme such as abnormal c e l l d i s t r i b u t i o n and c e l l c o n t a c t s , low c e l l number, and a b n o r m a l l y low s y n t h e s i s of g l y c o s a m i n o g l y c a n s were found i n l a t e p r e s o m i t i c t r i s o m y - 1 2 and t r i s o m y - 1 4 embryos ( M o r r i s s - K a y and P u t z 1986). S h o r t e r a p i c a l neuroectoderm m i c r o v i l l i as w e l l as p y k n o t i c c e l l s and dense c e l l i n c l u s i o n s i n the n e u r o e p i t h e l i u m were found i n t r i s o m y - 1 2 and t r i s o m y - 1 4 embryos compared w i t h MF1 c o n t r o l s of 9-20 s o m i t e s ( i b i d ) . P u t z and M o r r i s s - K a y (1986) propose t h a t the e a r l y mesenchymal a b n o r m a l i t i e s a r e the p r i m a r y e f f e c t of the t r i s o m y which e v e n t u a l l y l e a d t o i n s u f f i c i e n t c r a n i a l n e u r a l f o l d c u r v a t u r e and e x e n c e p h a l y . The advantage of u s i n g t r i s o m i c mice i s t h a t the t r i s o m i e s can be e a s i l y k a r y o t y p e d and i n the case of Ts-12, 100% of embryos w i l l be e x e n c e p h a l i c , t h u s a n a l y s i s of abnormal c l o s u r e can be f o l l o w e d w i t h r e l i a b i l i t y . The g r e a t d i s a d v a n t a g e of t h i s s ystem i s the unknown c o n f o u n d i n g f a c t o r of the a n e u p l o i d y i t s e l f . The presence of an e x t r a chromosome can g r o s s l y a f f e c t the e n t i r e embryo and t h e r e f o r e a b n o r m a l i t i e s d e t e c t e d may not be d i r e c t l y c a u s a l of the c l o s u r e d e f e c t o b s e r v e d . However, i f a p a r t i c u l a r gene or r e g i o n of chromosome 12 or 14 c o u l d be c a u s a l l y r e l a t e d t o NTDs we may be a b l e t o i d e n t i f y the f u n c t i o n of the g e n e ( s ) . A l t h o u g h a number of n e u r a l tube d e f e c t i v e mutants e x i s t i n mice, we do not have a c l e a r u n d e r s t a n d i n g of the morphogenesis of n e u r a l tube f o r m a t i o n and the r e a s o n s f o r c l o s u r e f a i l u r e . E x c ept f o r xn, c r n , and c t , the n e u r a l tube d e f e c t s d e s c r i b e d i n 22 mice occur as p a r t of syndromes due to the mutant genes or chromosomal imbalances. These may prove to be homologues of some human s i n g l e gene syndromes t h a t i n c l u d e tube d e f e c t s . However, the m a j o r i t y of human ne u r a l tube d e f e c t s are not of simple mendelian o r i g i n , nor syndromic. I t i s t h e r e f o r e u s e f u l to study n e u r a l tube d e f e c t s t h a t are non-syndromic and of apparent m u l t i f a c t o r i a l o r i g i n i n mice. The SELH mouse stock, developed i n the Medical Genetics mouse u n i t a t UBC produces a high frequency of exencephaly (14-17%) which does not occur as p a r t of a syndrome. No s p i n a b i f i d a or t a i l anomalies are seen. SELH i s a new stock and n e i t h e r the g e n e t i c s nor the embryology of i t s n e u r a l tube d e f e c t has been s t u d i e d p r e v i o u s l y . I t i s con s i d e r e d t h a t these mice c o u l d provide a u s e f u l , much needed model f o r ne u r a l tube c l o s u r e d e f e c t s . I I I . TERATOGENIC AGENTS PRODUCING NEURAL TUBE DEFECTS A wide v a r i e t y of chemicals and drugs i s known to a f f e c t the c l o s u r e of the n e u r a l tube. Mi c r o t u b u l e i n h i b i t o r s ( c o l c h i c i n e , nocodazole i n c h i c k ) , m i c r o f i l a m e n t i n h i b i t o r s ( c a f f e i n e , diazepam, c y t o c h a l a s i n B i n c h i c k ) , and i n h i b i t o r s of c e l l d i v i s i o n (phleomycin i n c h i c k ) , c a l c i u m b l o c k e r s (papaverine i n ch i c k and mouse), d i s r u p t o r s of e x t r a c e l l u l a r matrix (streptomyces hyaluronidase i n c h i c k ) , f o l i c a c i d analogues (methotrexate i n mouse), and many teratogens f o r which the 23 mechanism of a c t i o n i s unknown ( t r y p a n b l u e i n c h i c k and r a t , r e t i n o i c a c i d i n c h i c k , mouse and r a t , heat i n mouse, p h e n y t o i n i n mouse, i n s u l i n i n mouse and c h i c k , e t h a n o l i n mouse) a r e a l l c a p a b l e of c a u s i n g n e u r a l tube d e f e c t s i f a d m i n i s t e r e d a t the s e n s i t i v e p e r i o d of n e u r a l tube c l o s u r e ( C a m p b e l l , Dayton, S o h o l 1986, Brun and Garson 1983, Schoenwolf and F i s h e r 1983, F i n n e l l , Moon, Abbott e t a l 1986, S u l i k and J o h n s t o n 1983). Many of t h e s e c h e m i c a l s tend t o g r o s s l y d i s t u r b growth and development of the embryo and do not s p e c i f i c a l l y a f f e c t n e u r a l tube c l o s u r e . T e r a t o g e n s t u d i e s of n e u r a l tube d e f e c t s a r e u s e f u l i n d e t e r m i n i n g the s e n s i t i v e p e r i o d of n e u r a l tube c l o s u r e t o t e r a t o g e n i c a g e n t s , but have not proven v e r y u s e f u l i n i d e n t i f y i n g the u n d e r l y i n g causes of spontaneous n e u r a l tube c l o s u r e d e f e c t s i n humans or mice. The environment has been su g g e s t e d t o i n f l u e n c e the r i s k of n e u r a l tube d e f e c t s ( C o l e and T r a s l e r 1980; Khoury, E r i c k s o n , James 1982), but t e r a t o g e n s do not appear t o be a major cause of spontaneous NTDs i n humans or mice ( C a r t e r 1974; Yen and MacMahon 1968). IV. NEURULATION IN ANIMALS AND MAN A. D e s c r i p t i o n of the n e u r u l a t i o n p r o c e s s To u n d e r s t a n d why the n e u r a l tube f a i l s t o c l o s e i n some embryos, i t would h e l p t o u n d e r s t a n d how the n e u r a l tube c l o s e s n o r m a l l y . N e u r u l a t i o n , the p r o c e s s of n e u r a l p l a t e and n e u r a l tube f o r m a t i o n , i n v o l v e s the morphogenetic movement of a f l a t 24 s h e e t of c e l l s i n t o a h o l l o w tube. N e u r a l tube c l o s u r e i s the e a r l i e s t event i n o r g a n o g e n e s i s . The n e u r a l tube i t s e l f i s the p r o g e n i t o r t o the b r a i n and s p i n a l c h o r d of c h o r d a t e s . N e u r a l t i s s u e g i v e s r i s e t o the n e u r a l c r e s t c e l l s which m i g r a t e from the n e u r a l tube t o form pigment c e l l s , a wide v a r i e t y of n e u r a l g a n g l i a , and s k e l e t a l and c o n n e c t i v e t i s s u e elements (Karp and B e r r i l l 1981). A l t h o u g h n e u r u l a t i o n has been c h a r a c t e r i z e d d e s c r i p t i v e l y i n a g r e a t number of organisms, the u n d e r l y i n g mechanisms of t h i s p r o c e s s a r e s t i l l u n c l e a r . T h i s s e c t i o n w i l l l o o k a t what i s known about n e u r u l a t i o n and c o n s i d e r t h e hypotheses proposed f o r n e u r a l tube c l o s u r e . S p i n a l n e u r a l tube c l o s u r e may be q u i t e s i m i l a r between mammals and o t h e r a m n i o t e s , a l t h o u g h both b i r d s and mammals grow d u r i n g n e u r u l a t i o n w h i l e amphibian embryos do not (Jacobson and Tarn 1982). The i n t e r a c t i o n between embryo growth and n e u r a l tube development i s not u n d e r s t o o d . C r a n i a l n e u r u l a t i o n i n mammals i s much more c o m p l i c a t e d t h a n i n a l l nonmammalian v e r t e b r a t e s . C r a n i a l s e c t i o n s of r a t , c h i c k and f r o g show a number of d i f f e r e n c e s i n the o r g a n i z a t i o n , s i z e , appearance and number of n e u r o e p i t h e l i a l c e l l s , and i n p o s i t i o n i n g of n e u r a l c r e s t c e l l s , n o t o c h o r d s i z e , and mesenchyme, as w e l l as an o v e r a l l shape d i f f e r e n c e between s p e c i e s ( M o r r i s s - K a y 1981). Mammalian embryos a r e unique i n the f o r m a t i o n of a c r a n i a l f l e x u r e e a r l y i n n e u r u l a t i o n ( K a r f u n k e l 1974) . 25 N e u r u l a t i o n i n mice c o r r e l a t e s w e l l w i t h what i s known about c l o s u r e i n r a t s ( Jacobson and Tarn 1982, M o r r i s s - K a y 1981). Both r o d e n t s a r e cupshaped p r e s o m i t i c embryos b e f o r e n e u r u l a t i o n ( f i g u r e 3 ) . Humans and o t h e r nonrodent mammalian embryos are s i m i l a r a t t h i s p o i n t , a l t h o u g h wider and f l a t t e r . The v e n t r a l f l e x u r e of the a n t e r i o r n e u r a l t i s s u e b e g i n s by the one somite s t a g e i n the mouse and becomes more pronounced w i t h f u r t h e r development (Jacobson and Tarn 1982). The n e u r a l ectoderm p o s t e r i o r t o the c r a n i a l f l e x u r e ( p r e s u m p t i v e mesencephalon and rhombencephalon t i s s u e ) becomes "V" shaped and the f o l d s i n c r e a s e i n s i z e and t h i c k n e s s . By about the 6 somite s t a g e , t h e s e e l e v a t e d f o l d s t h i n out and e l o n g a t e ( f i g u r e 4 ) . In the p r o s e n c e p h a l o n (the n e u r a l t i s s u e a n t e r i o r t o the c r a n i a l f l e x u r e ) , t h e two h a l v e s of the n e u r a l f o l d s expand i n s i z e and c u r v e inward towards each o t h e r . O p t i c e v a g i n a t i o n s b e g i n f o r m a t i o n by 5-6 s o m i t e s . In both r a t and mouse, i n i t i a l f u s i o n of the n e u r a l tube i s i n the c e r v i c a l (or m i d d l e back) r e g i o n and b e g i n s a t about the 7 s o m i t e s t a g e ( M o r r i s s - K a y 1981, Jacobson and Tam 1982). The s h a r p m e d i a l c u r v a t u r e of the l a t e r a l edges p r i o r t o c o n t a c t i s thought t o be augmented by ongoing n e u r a l c r e s t c e l l e m i g r a t i o n a t t h i s time ( M o r r i s s - K a y 1981). F u s i o n spreads b i d i r e c t i o n a l l y over the n e u r a l tube i n the s p i n a l r e g i o n ( c l o s u r e 1 ) , but c l o s u r e of the c r a n i a l f o l d s i n v o l v e s a complex p a t t e r n of f u s i o n zones. F u s i o n i n the m i d / f o r e b r a i n r e g i o n o c c u r s i n the p r o s e n c e p h a l o n , 26 near the m e s e n c e p h a l i c border i m m e d i a t e l y above the d e v e l o p i n g c r a n i a l f l e x u r e and proceeds b i d i r e c t i o n a l l y ( c l o s u r e 2, f i g u r e 18a). At t h i s t i m e , t h e r e a r e two openings i n the a n t e r i o r n e u r a l t u b e , the r o s t r a l a n t e r i o r neuropore and the opening over the mesencephalon- a n t e r i o r rhombencephalon r e g i o n . T h i s c l o s u r e p a t t e r n i s a l s o seen i n human embryos ( M o r r i s s - K a y 1981). Golden and C h e r n o f f (1983) r e p o r t t h a t a u n i d i r e c t i o n a l c l o s u r e o c c u r s over the r o s t r a l end of the p r o s e n c e p h a l o n and proceeds c a u d a l l y towards the f u s i o n of the p o s t e r i o r p r o s e n c e p h a l o n ( c l o s u r e 3 ) . The c l o s u r e of the rhombencephalon i s not a f u s i o n of n e u r a l f o l d s , but a growth of e p i t h e l i a l membrane over the rhombencephalon gap ( c l o s u r e 4) (Golden and C h e r n o f f 1983) ( F i g u r e 18a). By 16 s o m i t e s i n r a t and 17-19 s o m i t e s i n mouse, c l o s u r e of the c r a n i a l n e u r a l tube i s completed ( M o r r i s s - K a y 1981, Jacobson and Tarn 1982). C e l l shape changes i n the neuroectoderm of r o d e n t s are s i m i l a r t o those d e s c r i b e d f o r the c h i c k , t h a t i s , c e l l e l o n g a t i o n , and a p i c a l c o n s t r i c t i o n s accompanied by a p i c a l m i c r o f i l a m e n t bundles ( K a r f u n k e l 1974). Recent work on S y r i a n hamster i n d i c a t e s a l a r g e number of c y t o p l a s m i c p r o j e c t i o n s between columnar, t i g h t l y packed c e l l s of the n e u r o e p i t h e l i a a t the s t a r t of n e u r u l a t i o n ( H o l l o w a y , Hornby, Muntz 1984). As n e u r u l a t i o n p r o c e e d s , c y t o p l a s m i c p r o j e c t i o n s a r e reduced and c e l l s become f l a s k shaped and p s e u d o s t r a t i f i e d . The m a j o r i t y of m i t o t i c s p i n d l e s i n the mouse and r a t n e u r a l p l a t e a r e o r i e n t e d t o p l a c e new c e l l s i n t o the l o n g a x i s of the p l a t e . The s p i n d l e s i n the f o r e b r a i n however, a r e o r i e n t e d t r a n s v e r s a l l y ( M o r r i s s - k a y 1981, Jacobson and Tam 1982). C e l l d i v i s i o n i n the m i d b r a i n and r o s t r a l h i n d b r a i n c o n t r i b u t e t o e l o n g a t i o n of the m i d l i n e and edge of the n e u r a l p l a t e . C r o s s -s e c t i o n a l a r e a remains c o n s t a n t i n t h i s r e g i o n of the r a t neuro-e p i t h e l i u m . The i n c r e a s e d volume of the neuroectoderm i s a t t r i b u t e d t o changes i n the c e l l shape and volume d i s t r i b u t i o n of the neuroectoderm and not t o an i n c r e a s e d c e l l number. An i n c r e a s e i n c e l l number i s found i n the f o r e b r a i n ( p r o s e n c e p h a l o n ) f o l d s . T u c k e t t and M o r r i s s - K a y (1985) propose t h a t the p r e o t i c s u l c u s a c t s as a b a r r i e r t o caudate movement of n e u r o e p i t h e l i a l c e l l s , and t h a t t h e r e i s a p a s s i v e m i g r a t i o n of the whole s h e e t of c e l l s g e n e r a t e d i n the m i d b r a i n and r o s t r a l h i n d b r a i n a n t e r i o r l y t o p o p u l a t e and expand the f o r e b r a i n . There has been no t e s t of t h i s h y p o t h e s i s . Changes i n c e l l shape i n the n e u r a l e p i t h e l i u m , c o n t r a c t i l e e l e m e n t s , and the s u r r o u n d i n g mesenchyme a r e a l l thought t o be i m p o r t a n t i n the r o l l i n g up of the n e u r a l p l a t e i n t o a t u b e . B. Models of n e u r a l tube f o r m a t i o n A number of mechanisms have been proposed t o e x p l a i n n e u r a l tube c l o s u r e , many of which seem p l a u s i b l e , and most of which have been i n a d e q u a t e l y t e s t e d . An o l d p r o p o s a l s u g g e s t s t h a t the n e u r a l f o l d s a r e pushed up t h r o u g h a c t i v e e x p a n s i o n of the 28 n o n - n e u r a l ectoderm, an e f f e c t which changes the shape of the n e u r a l p l a t e and causes i t t o b u c k l e ( K a r f u n k e l 1974). R e s u l t s of e x c i s i o n e x p e r i m e n t s performed on the n e u r a l p l a t e and f o l d s i n Ambystoma (a u r o d e l e ) seem t o e l i m a n t e n o n - n e u r a l ectoderm as a major f a c t o r i n n e u r a l tube c l o s u r e . S e p a r a t i o n of the n e u r a l p l a t e from l a t e r a l p a r t s s t i l l r e s u l t e d i n a p p a r e n t l y normal n e u r a l tube f o r m a t i o n . An experiment by Lewis i n 1947 a l s o p r o v i d e s e v i d e n c e a g a i n s t an a c t i v e r o l e of the n o n - n e u r a l ectoderm (Lewis 1947). H i s experiment i n v o l v e d making a l a t e r a l s l i t i n the ectoderm of an Ambystoma ( u r o d e l e ) embryo t o one s i d e of the n e u r a l f o l d s . The r e s u l t was a d e v i a t i o n of the n e u r a l tube toward the n o n - s l i t s i d e , f a s t e r c l o s u r e , and a w i d e n i n g of the s l i t i n t o a gap. T h i s s u g g ested t h a t l a t e r a l ectoderm p l a c e s a t e n s i o n on n e u r a l tube c l o s u r e and does not push up the n e u r a l f o l d s . An Ambystoma embryo whose n e u r a l p l a t e i s removed i s s t i l l a b l e t o c l o s e i t s n e u r a l f o l d s (Gordon 1985). T h i s o b s e r v a t i o n has prompted the s u g g e s t i o n t h a t a b a s i c m i g r a t i o n of the n e u r a l f o l d s i s r e s p o n s i b l e f o r n e u r u l a t i o n . K a r f u n k e l (1974) showed however t h a t the n e u r a l f o l d s d i d not have an i n n a t e tendency' t o m i g r a t e s i n c e m e d i a l m i g r a t i o n w i t h n e u r a l p l a t e e x c i s i o n c o n s t i t u t e d wound h e a l i n g and not normal n e u r u l a t i o n ( i n Ambystoma). The movements of the u n d e r l y i n g p r e s u m p t i v e mesoderm (mesenchyme) have been f r e q u e n t l y s u g g e s t e d as a f o r c e i n n e u r u l a t i o n . M a r i n - P a d i l l a (1966), In s t u d y i n g the mechanism of n e u r a l tube d e f e c t s caused by h y p e r v i t a m i n o s i s A i n hamster 29 embryos found t h a t a l t e r a t i o n s appeared i n the c e p h a l i c and s o m i t i c mesoderm, and i n v o l v e d enlargement of i n t e r c e l l u l a r s p a c e s , c e l l c y t o p l a s m s h r i n k a g e and a reduced m i t o t i c r a t e of t h i s t i s s u e . An i n c r e a s e i n mesenchyme c e l l s and the a c c u m u l a t i o n of h y a l u r o n a t e r i c h e x t r a c e l l u l a r m a t r i x has been observed i n normal n e u r a l f o l d c l o s u r e ( M o r r i s s and S o l u r s h 1978). I n c r e a s e s i n the volume of the e x t r a c e l l u l a r m a t r i x (ECM) of the mesenchyme may be i m p o r t a n t i n n e u r a l tube f o r m a t i o n . H y a l u r o n i c a c i d (HA) the major component of the ECM can h y d r a t e g r e a t l y and i s thus c a p a b l e of c r e a t i n g l a r g e volume i n c r e a s e s . Streptomyces h y a l u r o n i d a s e , an enzyme which b r e a k s down HA causes f a i l u r e of the n e u r a l f o l d s t o converge and c o n s e q u e n t l y produces n e u r a l tube d e f e c t s i n c h i c k (Schoenwolf and F i s h e r 1983). In c o n t r a s t t o the mesenchyme h y p o t h e s i s , T h e o d o s i s and F r a s e r (1978) found t h a t h y p e r v i t a m i n o s i s A a f f e c t e d mouse n e u r o e p i t h e l i u m ( p r o d u c i n g a u t o p h a g o c y t o s i s , rounded c e l l s w i t h l o s s of i n t e r c o n n e c t i o n s , and a d i s o r g a n i z e d n e u r o e p i t h e l i u m ) not mesenchyme c e l l s . As w e l l , c o l c h i c i n e e x p e r i m e n t s showing c o l l a p s e of c h i c k n e u r a l f o l d s and l o s s of e l o n g a t e d neuroectoderm c e l l shape are c o n s i d e r e d t o demonstrate c o l c h i c i n e ' s d i s r u p t i v e e f f e c t on m i c r o t u b u l e s of neuroectoderm c e l l s i n the absence of a d i s r u p t i o n of u n d e r l y i n g mesoderm ( K a r f u n k e l 1974). The c o l c h i c i n e t e s t r e s u l t s a r e d i f f i c u l t t o i n t e r p r e t however, as the drug has been found t o p r e v e n t l e n s e p i t h e l i a l c e l l e l o n g a t i o n not by m i c r o t u b u l e d i s r u p t i o n , but by 30 b l o c k i n g normal i n c r e a s e s i n c e l l volume (Schoenwolf 1982). The r o l e of the mesenchyme i n n e u r u l a t i o n remains u n c l e a r . Jacobson and Gordon ( c i t e d by Gordon 1985) propose t h a t the n o t o c h o r d i s e s s e n t i a l f o r n e u r a l f o l d c l o s u r e , c i t i n g i t s f i r m a t t a c h m e n t , and i d e n t i c a l movements t o the n e u r a l p l a t e . When newt n e u r a l p l a t e i s e x c i s e d w i t h the n o t o c h o r d i n t a c t , the f o l d s form a k e y h o l e shape, y e t when the n o t o c h o r d i s e x c i s e d a l o n e , the f o l d s do not converge. T h i s o b s e r v a t i o n may be an a r t i f a c t of the wound h e a l i n g p r o c e s s . U l t r a v i o l e t i r r a d i a t i o n of Xenopus eggs has a l l o w e d the p r o d u c t i o n of n o t o c h o r d l e s s embryos, which appear t o n e u r u l a t e n o r m a l l y (Gordon 1985). S i n c e n e u r u l a t i o n can occur i n i t s absence, the n o t o c h o r d ' s f u n c t i o n i n c l o s u r e i s l i k e l y t o be of a s e c o n d a r y n a t u r e . D i f f e r e n t i a l p r o l i f e r a t i o n i n the n e u r a l p l a t e has been p o s t u l a t e d t o g e n e r a t e the shape changes observed i n the n e u r a l f o l d s . Salamander and newt n e u r o e p i t h e l i u m does not change i n m i t o t i c i n d e x d u r i n g n e u r u l a t i o n , a l t h o u g h c h i c k n e u r o e p i t h e l i u m does. C e l l g e n e r a t i o n time i n the c h i c k i s a p p r o x i m a t e l y 8 h o u r s , and the n e u r a l p l a t e shape changes from f l a t t o "V" shaped and from "V" shaped t o "C" shaped f o l d s t a k e about 4 hours each (Bush, Marchander, Nguyen e t a l 1986). UV i r r a d i a t i o n of e a r l y c h i c k embryo a l s o showed t h a t n e u r a l tube c l o s u r e c o u l d be i n h i b i t e d w i t h o u t a f f e c t i n g the c e l l d i v i s i o n of n e u r a l p l a t e c e l l s (Gordon 1985). The shape change o c c u r s most l i k e l y t h e n , w i t h o u t an I n c r e a s e i n c e l l number and more 31 l i k e l y by changes i n c e l l shape. A l e s s e r known model proposes n o n u n i f o r m s w e l l i n g of n e u r a l p l a t e c e l l s due t o g r e a t e r water uptake by the b a s a l ends t h a n the a p i c a l ends (Gordon 1985). T h i s would p o t e n t i a l l y l e a d t o n e u r a l p l a t e shape changes and f o l d c l o s u r e . Measurements of a p i c a l v e r s u s b a s a l n e u r o e p i t h e l i a l c e l l water uptake have not been performed and c o u l d p r o v i d e a t e s t of t h i s h y p o t h e s i s . I n t e r c e l l u l a r a d h e s i v e n e s s has been put f o r w a r d as a mechanism t h r o u g h which the shape of n e u r a l p l a t e c e l l s can be a l t e r e d ( J a c o b s o n , O d e l l , O s t e r 1985). I f t h e n e u r a l p l a t e c e l l s had i n c r e a s e d l a t e r a l a d h e s i o n , t h i s c o u l d i n c r e a s e the l a t e r a l membrane a r e a and d e c r e a s e the a p i c a l and b a s a l a r e a s . A d h e s i v e n e s s measured by d i s s o c i a t i o n k i n e t i c s i n Xenopus and Ambystoma embryos was found t o i n c r e a s e between n e u r o e p i t h e l i a l c e l l s d u r i n g the s t a r t of n e u r u l a t i o n ( K a r f u n k e l , Hoffman, P h i l l i p s e t a t 1978). G r a d i e n t s of i n t e r c e l l u l a r a d h e s i o n from a p i c a l t o b a s a l end of n e u r o e p i t h e l i a l c e l l s c o u l d e x i s t ( T u c k e t t , M o r r i s s - K a y 1985). T h i s i d e a c o u l d be t e s t e d t h r o u g h s t u d y of n e u r o e p i t h e l i a l membrane complexes by t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . T e n s i o n g e n e r a t e d by c e l l s of the n e u r a l p l a t e has a l s o been put f o r w a r d as an e x p l a n a t i o n f o r a l i g n m e n t of s p i n d l e f i b e r s . T h i s r e l a t e s t o t h e o b s e r v a t i o n t h a t s t r e t c h i n g an e l a s t i c s h e e t a l o n g a l i n e causes the s h e e t t o b u c k l e and r o l l i n t o a tu b e . 32 In c h i c k , a s p u r t of e l o n g a t i o n has been observed i n the r e g i o n of c l o s u r e (Gordon 1985). The q u e s t i o n i s - what would be the o r i g i n of such a t e n s i o n ? One of the c u r r e n t l y p o p u l a r t h e o r i e s of n e u r u l a t i o n i n v o l v e s c e l l e l o n g a t i o n p e r p e n d i c u l a r t o the n e u r a l p l a t e ( i e . d o r s o -v e n t r a l e l o n g a t i o n ) combined w i t h a p i c a l c o n s t r i c t i o n of n e u r a l p l a t e c e l l s t o a c h i e v e n e u r a l f o l d e l e v a t i o n . Both phenomena are observed i n the n e u r u l a t i o n of mammals, c h i c k , and a m p h i b i a . M i c r o t u b u l e s (MTs) have been proposed i n m a i n t a i n i n g c e l l e l o n g a t i o n , and m i c r o f i l a m e n t s (MFs) i n a p i c a l c o n s t r i c t i o n s ( K a r f u n k e l 1974, Gordon 1985). N e u r a l p l a t e c e l l s w i t h d i s r u p t e d MTs round up and the f o l d s c o l l a p s e i n r a t (Smedley, S t a n i s t r e e t 1986). D i s r u p t i o n of MFs w i t h c y t o c h a l a s i n B a l s o l e a d s t o c o l l a p s e of n e u r a l f o l d s , a l t h o u g h columnar c e l l shape i s m a i n t a i n e d (Smedley, S t a n i s t r e e t 1986). The a gents used t o a c h i e v e MT and MF d i s r u p t i o n however, are not s p e c i f i c t o t h e s e elements a l o n e , t h e r e f o r e as Schoenwolf mentions t h e s e r e s u l t s a r e d i f f i c u l t t o e v a l u a t e (Schoenwolf 1982). Brun and Garson (1983) t r e a t e d salamander embryos w i t h nocodazole (an MT i n h i b i t o r ) , and found t h a t embryos n e u r u l a t e i n the absence of wedge shaped n e u r a l p l a t e c e l l s . C e l l shape change does not appear t o be a c r i t i c a l f a c t o r i n salamander n e u r a l f o l d movement. C a l c i u m a n t a g o n i s t s have a l s o been used t o s t u d y c e l l c o n c e n t r a t i o n s of c a l c i u m d u r i n g n e u r a l tube c l o s u r e . P a p a v e r i n e 33 d e c r e a s e s the e n t r y of c a l c i u m i n t o c e l l s and can i n h i b i t n e u r u l a t i o n i n a m p h i b i a , c h i c k , and mouse (Smedley, S t a n i s t r e e t 1986). R e v e r s a l can be a c h i e v e d w i t h c a l c i u m ionophore A23187. Nagele, P i e t r o l u n g o , and Lee (1981) found t h a t c o a t e d v e s i c l e s accumulated c a l c i u m i n neuroectoderm c e l l s and may p l a y a r o l e i n r e g u l a t i n g c o n t r a c t i o n of a p i c a l MF bundles i n n e u r u l a t i o n . I t i s e v i d e n t t h a t e x t r a c e l l u l a r c a l c i u m i s r e q u i r e d f o r the maintenance of e l e v a t e d n e u r a l f o l d s and normal n e u r u l a t i o n . I n d i r e c t immunofluorescence p a t t e r n s of both a c t i n and myosin a r e s i m i l a r t o each o t h e r , w i t h c o n c e n t r a t i o n of b o t h i n the a p i c a l r e g i o n s of neuroectoderm c e l l s , c o i n c i d i n g w i t h a dense p a c k i n g of m i c r o f i l a m e n t s d u r i n g n e u r u l a t i o n (Lee, K o s c i u k , Nagele 1983, Lee and Nagele 1985). F l u o r e s c e n c e i s most i n t e n s e i n those a r e a s of the n e u r a l f o l d s w i t h the g r e a t e s t bend (eg. the m i d d l e of the concave h a l v e s of the n e u r a l f o l d s ) (Lee, Nagele 1985). A c t i n d i s t r i b u t i o n p a t t e r n s i n mouse c o r r e s p o n d t o shape changes i n the n e u r o e p i t h e l i u m ( S a d l e r , Greenberg, C o u g h l i n 1982). High c o n c e n t r a t i o n s of a c t i n a r e found f i r s t i n the b a s a l r e g i o n s of neuroectoderm c e l l s and l a t e r i n the a p i c a l a r e a s of t h e s e c e l l s , c o r r e s p o n d i n g t o the convex and concave c u r v a t u r e movements of the f o l d s ( S a d l e r , Greenberg, C o u g h l i n 1982). The mechanism of m i c r o f i l a m e n t c o n t r a c t i o n i s not w e l l u n d e r s t o o d , a l t h o u g h i t i s thought t o be s i m i l a r t o the s l i d i n g f i l a m e n t method of c o n t r a c t i o n i n muscle c e l l s which r e q u i r e s a c t i n and myosin. There a r e a number of models put f o r t h t o e x p l a i n n e u r a l tube f o r m a t i o n . I t i s apparent however, t h a t the p r o c e s s w h i l e w e l l d e s c r i b e d , i s s t i l l not w e l l u n d e r s t o o d . N e u r a l tube c l o s u r e i s an e v o l u t i o n a r i l y a n c i e n t p r o c e s s . A l l c h o r d a t e s form a h o l l o w d o r s a l n e u r a l tube ( i e . a l l v e r t e b r a t e s and some i n v e r t e b r a t e s ) . F o s s i l r e c o r d s of c h o r d a t e s date t o the Cambrian p e r i o d of the P a l e o z o i c e r a , a p p r o x i m a t e l y 570 m i l l i o n y e a r s ago (Keeton 1980). I t i s q u i t e l i k e l y t h a t mammals, a m p h i b i a n s , and b i r d s s h a r e a common b a s i c mechanism f o r c l o s u r e of the n e u r a l t u b e . M o r p h o g e n e t i c a l l y a t l e a s t , t h e s e a n i m a l s do have some d i f f e r e n c e s i n c e l l shape, number and growth d u r i n g n e u r a l tube c l o s u r e , however, the p r o c e s s we see i n the mouse i s p r o b a b l y v e r y r e l e v a n t t o human n e u r u l a t i o n . R e s e a r c h i n d i c a t e s t h a t t h e r e i s no one mechanism t o cause f a i l u r e of n e u r a l tube e l e v a t i o n and c l o s u r e i n a n i m a l systems. The n e u r o e p i t h e l i u m , and mesenchyme t i s s u e s a r e fundamental t o n e u r u l a t i o n as a r e such elements as m i c r o t u b u l e s , m i c r o f i l a m e n t s , and the e x t r a c e l l u l a r m a t r i x . We do not know i f the b u l k of human n e u r a l tube d e f e c t s ( e x c l u d i n g n e u r a l tube d e f e c t s of known cause) a r e due t o the f a i l u r e of one b a s i c mechanism of c l o s u r e or i f the cases a r e r e a l l y a heterogenous group of d i f f e r e n t problems l e a d i n g t o the same d e f e c t . However, u n d e r s t a n d i n g some of the causes of t h e s e d e f e c t s i n the mouse w i l l p r o v i d e u s e f u l c o n c e p t s t o a p p l y t o human n e u r a l tube d e f e c t s . 35 MATERIALS AND METHODS: I . MICE The ICR/Be s t r a i n i s an i n b r e d s t r a i n m a i n t a i n e d i n the M e d i c a l G e n e t i c s Mouse U n i t a t the U n i v e r s i t y of B r i t i s h Columbia (UBC). I t was devel o p e d from BLU:Ha(ICR) "random-bred" mice o b t a i n e d from Arbor S c i e n t i f i c i n 1977. These mice have been used f o r o t h e r s t u d i e s of development ( H a r r i s and McLeod 1982; J u r i l o f f , H a r r i s , F r o s t e r - I s k e n i u s 1987) and have no un u s u a l i n c i d e n c e of m a l f o r m a t i o n s . Mice from F29-F32 of b r o t h e r s i s t e r i n b r e e d i n g a t UBC were used. They were aged 2-10 months a t b r e e d i n g . U n t r e a t e d ICR/Be f e t u s e s c o l l e c t e d day 15-17 of g e s t a t i o n f o r an u n p u b l i s h e d s t u d y of e y e l i d c l o s u r e by D.M. J u r i l o f f , observed 0 e x e n c e p h a l i c s i n 420 f e t u s e s ( 0 % ) . The SWV/Bc s t r a i n i s an i n b r e d s t r a i n d e v e l o p e d and m a i n t a i n e d i n t he M e d i c a l G e n e t i c s Mouse U n i t a t UBC. The s t r a i n was deve l o p e d from s t o c k o b t a i n e d from a c l o s e d c o l o n y of mice a t the C e n t r a l Animal Depot a t UBC, which was d e r i v e d from s t o c k o b t a i n e d from the Defense Research b u i l d i n g , S u f f i e l d , A l b e r t a , Canada i n 1949. I n b r e e d i n g began i n 1959 ( S t a a t s 1985). These mice have been w i d e l y used i n s t u d i e s of development ( F i n n e l l , Moon, Abbott e t a l 1986; J u r i l o f f 1987; B i d d l e 1975) and have no unus u a l i n c i d e n c e of m a l f o r m a t i o n s . Mice from F77-F81 of b r o t h e r s i s t e r i n b r e e d i n g were used. They were aged 2-7 months a t b r e e d i n g . O b s e r v a t i o n of u n t r e a t e d SWV/Bc f e t u s e s c o l l e c t e d on days 15 and 16 of g e s t a t i o n f o r an u n p u b l i s h e d s t u d y of 36 e y e l i d c l o s u r e by D.M. J u r i l o f f , found e x e n c e p h a l y i n 2/401 f e t u s e s ( 0 . 5 % ) . The SELH s t o c k was d e v e l o p e d i n the Department of M e d i c a l G e n e t i c s a t UBC. I t s g e n e t i c h i s t o r y i s as f o l l o w s . A p a r t i a l l y i n b r e d s t o c k of mixed g e n e t i c background (BALB/cGa, 129/-, CBA/-) and homozygous f o r the m u t a t i o n l i d g a p - G a t e s l g was c r o s s e d t o "random b r e d " ICR mice a t UBC i n 1977. The l g G a m u t a t i o n was b a c k c r o s s e d i n t o t h i s ICR background by the l n t e r c r o s s - b a c k c r o s s method (Green 1981). A new r e c e s s i v e m u t a t i o n , sph (Unger, H a r r i s , B e r n s t e i n e t a l 1983) appeared In the second i n t e r c r o s s g e n e r a t i o n , and a s u b l i n e was d e r i v e d from the p a r e n t s of the a f f e c t e d pups. I t was m a i n t a i n e d by b r o t h e r - s i s t e r i n b r e e d i n g w i t h s e l e c t i o n f o r s p h 2 B c and a g a i n s t l g G a . In 1981 e x e n c e p h a l y appeared i n the F5 newborns of b r e e d i n g p a i r s t h a t a l l t r a c e d back t o one F3 b r e e d i n g p a i r . E x e n c e p h a l y - p r o d u c i n g p a r e n t s were s e l e c t e d a t t h a t time and a l l SELH a n i m a l s t r a c e back t o one F6 b r e e d i n g p a i r . At F10-12 t h r e e s u b l i n e s were i n t e r c r o s s e d and t h e i r progeny were used t o r e - e s t a b l i s h the c o l o n y i n a new a n i m a l f a c i l i t y . A l l of the mice used i n the p r e s e n t s t u d y a r e d e s c e n d a n t s of 8 female and 5 male mice from t h i s t r a n s f e r g e n e r a t i o n . P r i o r t o the b e g i n n i n g of t h i s s t u d y , the mice i n the new f a c i l i t y were m a i n t a i n e d f o r one t o t h r e e g e n e r a t i o n s by random mating w i t h s e l e c t i o n f o r e x e n c e p h a l y . D u r i n g the c o u r s e of t h i s s t u d y , i n b r e e d i n g was resumed and up t o 5 a d d i t i o n a l g e n e r a t i o n s of b r o t h e r - s i s t e r i n b r e e d i n g have t a k e n p l a c e . 37 As a consequence of t h i s b r e e d i n g h i s t o r y , the SELH a n i m a l s used a r e e x p e c t e d t o be c o n s t i t u t e d of a p p r o x i m a t e l y 88% genes of BLU:Ha (ICR) o r i g i n and 12% mixed BALB/cGa, 129/-, and CBA/-o r i g i n . The SELH s t o c k s e g r e g a t e s f o r s p h 2 B c , and a l s o f o r n u B c , a n o t h e r spontaneous r e c e s s i v e m u t a t i o n t h a t was d e t e c t e d i n the s t o c k a t about F7. Homozygotes f o r t h e s e m u t a t i o n s were not used i n t h i s s t u d y . Twenty f o u r enzyme l o c i , most of which a r e p o l y m o r p h i c among s t r a i n s of mice, have been t y p e d i n 2-8 mice each i n the SELH s t o c k (mean=4.5). No v a r i a t i o n and no h e t e r o z y g o t e s have been found a t any l o c u s . I I . GENERAL CARE The mice were housed i n the M e d i c a l G e n e t i c s A n i m a l U n i t a t the U n i v e r s i t y of B r i t i s h Columbia (Vancouver, B.C.). Mice were f e d P u r i n a Lab Chow and a c i d i f i e d water (pH 3.1, HCl) ad l i b i t u m and housed i n p o l y c a r b o n a t e cages w i t h S a n i c e l b e d d i n g . A l l mice were kept on the same twenty f o u r hour l i g h t c y c l e w i t h l i g h t s on from 6am to 6pm d a i l y . A n i mals were housed t o g e t h e r w i t h no more th a n f i v e a n i m a l s per cage. Males used f o r matings were housed i n s e p a r a t e cages w i t h a maximum of 3 females per n i g h t . Timed p r e g n a n c i e s were o b t a i n e d as f o l l o w s : females were p l a c e d i n cages w i t h s i n g l e males from a p r o x i m a t e l y 5pm u n t i l 9am the f o l l o w i n g morning, when females were checked f o r v a g i n a l p l u g s . 38 Females w i t h p l u g s were caged s e p a r a t e l y , w i t h the date of v a g i n a l p l u g and p e d i g r e e number of the male mated t o her r e c o r d e d . O v u l a t i o n i n mice t a k e s p l a c e on average near t h e m i d p o i n t of the dark c y c l e , and under t h i s m ating system, f e r t i l i z a t i o n o c c u r s a few hours l a t e r (Bronson, Dagg and S n e l l 1966). Mice were k i l l e d by c e r v i c a l d i s l o c a t i o n . The whole u t e r u s of the female was i m m e d i a t e l y d i s s e c t e d out i n t a c t . I I I . TEST OF SEGREGATION OF EXENCEPHALY IN SELH At the b e g i n n i n g of t h i s s t u d y , i t was not known whether or not e x e n c e p h a l y was due t o the s e g r e g a t i o n of a r e c e s s i v e l e t h a l m u t a t i o n i n the SELH s t o c k . T h i s q u e s t i o n was a d d r e s s e d d i r e c t l y i n an i n i t i a l s t u d y ( a ; b e l o w ) , and was a l s o d e a l t w i t h i n d i r e c t l y i n two o t h e r s e t s of o b s e r v a t i o n s (b, c; b e l o w ) . They were: a. day 14 mouse embryo s t u d y b. i d e n t i f i c a t i o n of e x e n c e p h a l y p r o d u c i n g mice c. c o n t r o l s from the g e n e t i c s t u d y of B C l males A. Day 14 mouse embryo s t u d y T h i s s t u d y was performed t o b e g i n t o d e t e r m i n e whether a s i n g l e l e t h a l m u t a t i o n c a u s i n g e x e n c e p h a l y had a r i s e n i n the s t o c k and was s e g r e g a t i n g , or whether a m u t a t i o n of low p e n e t r a n c e had been f i x e d i n the s t o c k . E i g h t SELH male mice were time mated t o between 3-5 SELH v i r g i n 39 females each. Females were a l l 2 1/2 t o 4 months o l d . On day 14 of g e s t a t i o n , females were k i l l e d by c e r v i c a l d i s l o c a t i o n and the u t e r u s was removed and p l a c e d i n a 0.85% s a l i n e s o l u t i o n , where the embryos were d i s s e c t e d o u t . Embryos were s c o r e d f o r n e u r a l tube c l o s u r e , v i t a l i t y , and any un u s u a l f e a t u r e s . R e s o r p t i o n s i t e s were a l s o r e c o r d e d . Embryos were s t o r e d i n Bouin's f i x a t i v e . B. I d e n t i f i c a t i o n of e x e n c e p h a l y p r o d u c i n g mice The SELH s t o c k i s not y e t f u l l y i n b r e d . To de t e r m i n e t o what e x t e n t the gene or genes c a u s i n g e x e n c e p h a l y were s p r e a d t h r o u g h o u t the s t o c k , and t o ensure g e n e t i c s t u d i e s were performed on a n i m a l s c a r r y i n g the mutant g e n e ( s ) , c r o s s e s were s e t up t o i d e n t i f y " c a r r i e r s " of e x e n c e p h a l y . Male SELH mice were mated w i t h one t o f i v e female SELH mice i n timed m a t i n g s . The females were t h r e e t o t e n months i n age, and ranged from v i r g i n s t o h a v i n g had 3 p r e v i o u s l i t t e r s . A f t e r b i r t h on day 19 or 20 of g e s t a t i o n , the newborn l i t t e r s were s c o r e d f o r e x e n c e p h a l y and sex. S t i l l b o r n s and m u t i l a t e d newborns were a l s o r e c o r d e d . Some male c a r r i e r s were i d e n t i f i e d t h r o u g h o b t a i n i n g the female's l i t t e r on day 18 and s c o r i n g the f e t u s e s f o r e x e n c e p h a l y and sex. R e s o r p t i o n s i t e s were a l s o r e c o r d e d . C. C o n t r o l s from the g e n e t i c s t u d y of B C l males In o r d e r t o p r o v i d e d a t a f o r the SELH s t r a i n t o compare w i t h t h a t of the B C l male t e s t c r o s s e s t o SELH (page 4 9 ) , f i v e randomly chosen SELH males were time mated t o 8 n u l l i p a r o u s SELH females each. Females were k i l l e d on day 18 of g e s t a t i o n , and t h e i r l i t t e r s c o l l e c t e d . L i t t e r s were s c o r e d f o r e x e n c e p h a l y , p a l a t e c l o s u r e , e x t e r n a l s e x , and r e s o r p t i o n s . I n t e r n a l sex was det e r m i n e d a f t e r f e t u s e s were f i x e d i n Bouin's f l u i d f o r 1-6 weeks. IV. EMBRYOLOGY STUDY T h i s s t u d y was done t o o b t a i n a scheme of the morphology and t i m i n g of the sequence of d e v e l o p m e n t a l e v e n t s d u r i n g b o t h normal n e u r a l tube c l o s u r e i n the mouse and n e u r u l a t i o n i n the ex e n c e p h a l y p r o d u c i n g s t o c k (SELH). The work w i t h normal s t r a i n s of mice p r o v i d e d a s e r i e s of landmarks t o which n e u r a l tube c l o s u r e i n the SELH s t o c k c o u l d be compared. A. Developmental s t u d y Mice of the SWV/Bc and ICR/Be s t r a i n s were used a l o n g w i t h the SELH s t o c k . Timed p r e g n a n c i e s were used t o o b t a i n l i t t e r s a t v a r i o u s g e s t a t i o n a l ages, between day 8 a t noon t o day 9 a t 2pm. The time a t which the female was k i l l e d and the embryos removed was r e c o r d e d . Specimens were f i x e d i n one of two ways, e i t h e r the i n t a c t u t e r u s was p l a c e d i n t o Bouins f i x a t i v e , and f i x e d f o r a minimum of twenty f o u r hours b e f o r e embryos were d i s s e c t e d out and s c o r e d , or embryos were f i x e d as d e s c r i b e d i n s e c t i o n IV.B. A l l females used were between t h r e e and t e n months o l d . F or 41 the SELH s t u d y , a l l females and males used had been proven t o t r a n s m i t the e x e n c e p h a l y t r a i t , thus females used i n the day 8/9 embryo c o l l e c t i o n s t u d y had had one p r e v i o u s l i t t e r . ICR/Be and SWV/Bc l i t t e r s were from v i r g i n females t h r e e t o t e n months o l d . Embryos were s c o r e d under a Z e i s s d i s s e c t i o n m i c r o s c o p e f o r som i t e c o u n t , n e u r a l tube (or n e u r a l f o l d ) development, and any unus u a l f e a t u r e s . R e s o r p t i o n s i t e s were a l s o r e c o r d e d . A system of c l a s s i f i c a t i o n ("stages") of n e u r a l tube c l o s u r e was de v e l o p e d from e x a m i n a t i o n of 675 embryos of two normal s t r a i n s of mice (333 SWV/Bc, 342 ICR/Be), and 279 SELH embryos a t v a r i o u s phases of a n t e r i o r n e u r a l tube f o r m a t i o n . 29 SWV/Bc, 19 ICR/Be, and 3 SELH embryos had z e r o s o m i t e s and were e x c l u d e d from the n e u r a l tube c l o s u r e c h a r t by b e i n g " o f f s c a l e " . 342 of the ICR/Be, 280 of the SWV/Bc, and 228 of the SELH embryos c o l l e c t e d were used i n the c h r o n o l o g i c a l t i m e / s o m i t e count s t u d y . B. Scanning e l e c t r o n m i c r o s c o p y (SEM) Timed matings were used t o ge n e r a t e SELH and ICR pregnant f e m a l e s . SELH females were mated t o proven e x e n c e p h a l y -p r o d u c i n g SELH males. V i r g i n ICR/Be females were mated t o ICR/Be males. Pregnant females were k i l l e d by c e r v i c a l d i s l o c a t i o n . S i x SELH and 5 ICR/Be l i t t e r s were o b t a i n e d a t v a r i o u s t i m e s from day 9 a t 4am, t o day 9 a t 9:30am. The i n t a c t u t e r u s was d i s s e c t e d from the mother and immersed i n 42 c o l d (4 degrees C e l c i u s ) Sorensen's phosphate b u f f e r (pH 7.3). I n d i v i d u a l I m p l a n t a t i o n s i t e s were d i s s e c t e d out and s e p a r a t e d i n t o i n d i v i d u a l l a b e l l e d v i a l s . W i t h i n t e n minutes of m a t e r n a l d e a t h , a l l embryos were submerged In 2.5% g l u t a r a l d e h y d e In Sorensen's b u f f e r . Specimens were f i x e d f o r 4 t o 8 hours a t 4 degrees C e l c i u s b e f o r e b e i n g t r a n s f e r e d i n t o Sorensen's phosphate b u f f e r (2 r i n s e s of 2 t o 8 hours) and m a i n t a i n e d a t 4 degrees C e l c i u s . Embryos were t h e n d i s s e c t e d out of t h e i r e x t r a embryonic membranes under Sorensen's b u f f e r and s c o r e d f o r s o m i t e c o u n t , n e u r a l tube development, and t a i l p o s i t i o n . Embryos were t r a n s f e r e d t o f r e s h b u f f e r and m a i n t a i n e d a t 4 degrees C e l c i u s f o r 1-5 days. Specimens were p o s t f i x e d i n 2% osmium t e t r o x l d e i n Sorensen's phosphate b u f f e r f o r 90 m i n u t e s , r i n s e d 3 t i m e s i n Sorensen's phosphate b u f f e r f o r 20 minutes each, and d e h y d r a t e d t h r o u g h a s e r i e s of a l c o h o l s ( a t room t e m p e r a t u r e ) from 25% t o 100% f o r 15 minutes each. The p r o c e s s ended w i t h t h r e e r i n s e s i n 100% a l c o h o l f o r 20 m i n u t e s . Specimens were c r i t i c a l p o i n t d r i e d i n i n d i v i d u a l b a s k e t s from l i q u i d C 0 2 u s i n g a Ladd c r i t i c a l p o i n t d r y e r and mounted on s t u b s w i t h c o l l o i d a l s i l v e r u s i n g a m i c r o s c o p e a t 40x t o o r i e n t embryos. Specimens were s t o r e d i n a vacuum d e s s i c a t o r o v e r n i g h t . A Hummer IV was used t o s p u t t e r c o a t the specimens i n vacuo w i t h g o l d - p a l l a d i u m . Specimens were t h e n viewed i m m e d i a t e l y In a Cambridge S t e r e o s c a n 100 s c a n n i n g e l e c t r o n m i c r o s c o p e a t 15kv. The t a i l s of some embryos were removed e i t h e r a f t e r d e h y d r a t i o n or c r i t i c a l p o i n t d r y i n g t o a l l o w an 43 u n o b s t r u c t e d f r o n t a l v i e w of the a n t e r i o r n e u r a l f o l d s . V. HISTOLOGY A. P a r a f f i n - e m b e d d e d Specimens ( t h i c k s e c t i o n s ) : SELH (from proven e x e n c e p h a l y - p r o d u c i n g males and f e m a l e s ) , and ICR/Be embryos were c o l l e c t e d a t v a r i o u s t i m e s on days e i g h t and n i n e of g e s t a t i o n as d e s c r i b e d p r e v i o u s l y (page 40) and p l a c e d i n t o B ouin's f i x a t i v e f o r a minimum of twenty f o u r hours b e f o r e s c o r i n g . Embryos were s c o r e d f o r s t a g e of n e u r a l tube c l o s u r e , s o m ite c o u n t , t a i l p o s i t i o n , and p o s t e r i o r neuropore c l o s u r e . They were photographed b e f o r e embedding u s i n g a W i l d d i s s e c t i o n m i c r o s c o p e a t 25x w i t h a camera attachment. For the embedding p r o c e s s , the f i x e d embryos were i n d i v i d u a l l y wrapped i n t i s s u e paper and p l a c e d i n t o l a b e l l e d p r o c e s s i n g b a s k e t s . They were then p r o c e s s e d i n an A u t o t e c h n i c a n t h r o u g h a graded s e r i e s of a l c o h o l s (70%, 95%, 10 0 % ) , c h l o r o f o r m , and molten wax b a t h s . The i n f i l t r a t e d specimens were p l a c e d i n t o embedding d i s h e s , o r i e n t e d , and embedded. Specimen b l o c k s were s t o r e d a t z e r o degrees C e l s i u s f o r a minimum of 12 hours b e f o r e s e c t i o n i n g . An American O p t i c a l Spencer "820" microtome was used t o c u t 7um s e r i a l wax s e c t i o n s which were p l a c e d on g l a s s s l i d e s . S l i d e s were then i n c u b a t e d a t 56 degrees C e l s i u s o v e r n i g h t . S l i d e s were soaked i n two baths of x y l e n e f o r a t o t a l of s i x m i n u t e s , 44 the n h y d r a t e d t h r o u g h a s e r i e s of a l c o h o l s , from 100% t o 95% t o 70% and down t o water . The y e l l o w s t a i n i n g of Bou i n ' s f i x a t i v e was removed i n a r i n s e i n 1% l i t h i u m c a r b o n a t e ( i n d i s t i l l e d w a t e r ) . The s e c t i o n s were s t a i n e d w i t h H a r r i s ' s H a e m o t o x y l i n f o r f i v e m i n u t e s . H a e m o t o x y l i n i s a mordant-dye n u c l e a r s t a i n w i t h a p o s i t i v e l y charged dye l a k e t h a t a c t s as a b a s i c dye and produces a deep r e d s t a i n (Sheenan and Hrapchk 1980). S e c t i o n s were t h e n b l u e d i n 1% l i t h i u m c a r b o n a t e . The samples were immersed i n 1% E o s i n Y ( i n 70% a l c o h o l w i t h 0.5ml a c e t i c a c i d ) f o r 90 seconds, then q u i c k l y passed t h r o u g h baths of 100% a l c o h o l i n t o x y l e n e . E o s i n i s an a c i d dye and i s used here as a background or c o u n t e r s t a i n t o s t a i n plasma p i n k . S l i d e s were mounted w i t h e i t h e r DPX or Permount, and s t o r e d f l a t f o r one month b e f o r e v e r t i c a l f i l i n g . G l a s s s l i d e s were viewed under a L e i t z m i c r o s c o p e a t 25x, lOOx, and 400x ( o i l i m m e r s i o n ) . B. P l a s t i c - e m b e d d e d Specimens ( t h i n s e c t i o n s ) SELH (from proven e x e n c e p h a l y - p r o d u c i n g males and females) and ICR/Be embryos were c o l l e c t e d on day 9 between 3:30 and 5:30 am. The c o l l e c t i o n p rocedure was as d e s c r i b e d f o r the SEM s t u d y . I n d i v i d u a l i m p l a n t a t i o n s were p l a c e d i n t o 4.0% g l u t a r a l d e h y d e i n Sorensen's b u f f e r (pH 7.3) a f t e r d i s s e c t i o n from the u t e r u s and s t o r e d a t 4 degrees C e l s i u s f o r 2 ho u r s . I m p l a n t s were t h e n t r a n s f e r e d i n t o Sorensen's phosphate b u f f e r f o r two r i n s e s of 2-8 hours each, and m a i n t a i n e d a t 4 degrees C e l s i u s . Embryos were then d i s s e c t e d from t h e i r membranes as o u t l i n e d f o r the s c a n n i n g e l e c t r o n m i c r o s c o p y methods (page 4 1 ) . Embryos were 45 photographed ( a t 25x) ( u s i n g a W i l d d i s s e c t i o n m i c r o s c o p e w i t h camera att a c h m e n t ) b e f o r e embedding. To p r e p a r e p l a s t i c b l o c k s , specimens were i n f i l t r a t e d and embedded i n S o r v a l embedding medium f o r p l a s t i c h i s t o l o g y . S o r v a l P l a s t i c i s an embedding medium c o n t a i n i n g hydroxy e t h y l m e t h a c r y l a t e , 2- Butoxy E t h a n o l and N,N1-Dimethyl A n i l i n e . I t c o n t a i n s a water s o l u b l e monomer, p r e s e r v e s b i o l o g i c a l s t r u c t u r e s w e l l , and a l l o w s t h i n s e c t i o n h i s t o l o g y . Embryos were o r i e n t e d i n the p l a s t i c and the b l o c k s were kept a t room t e m p e r a t u r e . A S o r v a l JB-4A microtome was used t o o b t a i n 2um t h i n s e c t i o n s of samples. S e c t i o n s were p l a c e d i n two rows of 4 s e c t i o n s on s l i d e s and i n c u b a t e d a t 37 degrees C e l s i u s u n t i l d r y . S e c t i o n s were p l a c e d as f o l l o w s : 4 s e q u e n t i a l s e c t i o n s were p l a c e d on the f i r s t row of s l i d e 1, w i t h the next 4 i n the f i r s t row of s l i d e 2, the t h i r d s e t i n the second row of s l i d e 1, and the f o u r t h s e t i n the second row of s l i d e 2, and so on. S l i d e s were s t o r e d a t 56 degrees C e l s i u s o v e r n i g h t b e f o r e s t a i n i n g . P l a s t i c s e c t i o n s were s t a i n e d w i t h H a e m o t o x y l i n and E o s i n as f o l l o w s : S e c t i o n s were s t a i n e d i n G i l l ' s H a e m o t o x y l i n f o r 2 h o u r s , t h e n r i n s e d i n d i s t i l l e d water and s t a i n e d i n 1% E o s i n Y f o r 5 m i n u t e s . S e c t i o n s were then q u i c k l y passed t h r o u g h two baths of 100% e t h y l a l c o h o l and i n t o a t h i r d 100% e t h y l a l c o h o l b a t h f o r 1.5 m i n u t e s . S l i d e s were then r i n s e d i n two baths of x y l e n e and mounted w i t h F i s h e r Permount. Of each embryo 46 s e c t i o n e d , a l t e r n a t e s l i d e s were a n a l y s e d and 1 s e c t i o n from each of 2 rows on a s l i d e were s t u d i e d , t h a t i s , 1 s l i d e s e c t i o n i n 8 was s t u d i e d . I t s h o u l d be n o t e d , t h a t p l a s t i c s e c t i o n s cannot be c o n s i d e r e d s e r i a l s e c t i o n s due t o the n a t u r e of t h e t e c h n i q u e and thus the l o s s of some s e c t i o n s . I n t h i s s t u d y , i t i s e s t i m a t e d t h a t over 70% of a l l s e c t i o n s were mounted, w i t h no more t h a n 4 s e q u e n t i a l s e c t i o n s b e i n g l o s t a t any p o i n t . An e q u a l number of s e c t i o n s from SELH and ICR/Be embryos of 6 s o m i t e s (two embryos of 12 s e c t i o n s each) and 8 s o m i t e s (two embryos of 12 s e c t i o n s each) were compared e x t e n s i v e l y . X 2 t e s t s of independance were used t o compare the number of p y k n o t i c c e l l s and " l i p i d d r o p l e t s " between ICR/Be and SELH embryos. VI. GENETIC STUDIES The purpose of t h e g e n e t i c s t u d i e s was t o d e t e r m i n e t h e mode of i n h e r i t a n c e of l i a b i l i t y t o the e x e n c e p h a l y t r a i t of the SELH s t o c k . C l a s s i c a l g e n e t i c c r o s s e s were used. As t h e t a r g e t of the i n v e s t i g a t i o n was the major e x e n c e p h a l y - c a u s i n g f a c t o r ( s ) of SELH, and not the range of p o s s i b l e m o d i f y i n g f a c t o r s i n mice, i t was d e c i d e d t h a t the most a p p r o p r i a t e normal s t r a i n t o use would be ICR/Be. As d e s c r i b e d p r e v i o u s l y , SELH and ICR/Be have common a n c e s t r y , and s h o u l d s hare the m a j o r i t y of t h e i r a l l e l e s i n common. U n l i k e SELH, ICR/Be has not produced e x e n c e p h a l y . C l a s s i c a l g e n e t i c a n a l y s i s of c r o s s e s between t h e s e two s t o c k s s h o u l d i d e n t i f y t he n a t u r e and number of f a c t o r s d i f f e r i n g between them t h a t cause the h i g h f r e q u e n c y of e x e n c e p h a l y i n 47 SELH. A. R e c i p r o c a l C r o s s e s of SELH and ICR/Be R e c i p r o c a l c r o s s e s were made t o t e s t a v a r i e t y of g e n e t i c hypotheses and a l s o t o generate F l a n i m a l s f o r use i n f u r t h e r g e n e t i c s t u d i e s . 1. ICR/Be females x SELH males F i v e proven e x e n c e p h a l y - p r o d u c i n g male SELH mice were time mated t o ICR f e m a l e s . F i v e of the p r e g n a n c i e s per s i r e were t e r m i n a t e d on day 18 of g e s t a t i o n . These l i t t e r s were p l a c e d i n t o a 0.85% s a l i n e s o l u t i o n and s c o r e d i m m e d i a t e l y f o r n e u r a l tube c l o s u r e , p a l a t e c l o s u r e , and sex ( e x t e r n a l s c o r i n g by examining t h e d i s t a n c e between the anus and g e n i t a l p a p i l l a ) . R e s o r p t i o n s i t e s were a l s o n o t e d . F e t u s e s were then s t o r e d i n Bouin's f i x a t i v e . U s i n g s t a n d a r d mouse g e n e t i c n o m e n c l a t u r e , t h e s e F l a n i m a l s are denoted as ICR.SELH (or I . S ) . 2. SELH females x ICR/Be males To c o l l e c t F l day 18 f e t u s e s , proven e x e n c e p h a l y p r o d u c i n g SELH females were time mated t o ICR males. L i t t e r s were c o l l e c t e d on day 18 of g e s t a t i o n and s c o r e d under 0.85% s a l i n e s o l u t i o n . F e t u s e s were s c o r e d f o r n e u r a l tube c l o s u r e , p a l a t e c l o s u r e , and e x t e r n a l s e x . R e s o r p t i o n s i t e s were a l s o r e c o r d e d . F e t u s e s were f i x e d and s t o r e d i n Bouin's f i x a t i v e . F o l l o w i n g s t a n d a r d n o m e n c l a t u r e , t h e s e F l a n i m a l s a r e denoted as SELH.ICR (or S . I ) . 48 B. F2 G e n e r a t i o n Two s e t s of F2 f e t u s e s were examined - one from each r e c i p r o c a l c r o s s . From the c r o s s of SELH males by ICR/Be f e m a l e s , 3 F l l i t t e r s (ICR.SELH) from each of the f i v e males were r a i s e d . F i v e t o e i g h t (mean=7) F l n u l l i p a r o u s females from each of the SELH s i r e s were mated t o each of two of t h e i r F l male s i b s . The p r e g n a n c i e s were t e r m i n a t e d on day 18 of g e s t a t i o n and the F2 l i t t e r s c o l l e c t e d , s c o r e d and s t o r e d i n Bouin's f i x a t i v e . Some weeks l a t e r t h e y were examined f o r i n t e r n a l s e x . For the c r o s s of SELH females by ICR/Be males, 8 proven e x e n c e p h a l y - p r o d u c i n g SELH females were used t o produce s e v e r a l r a i s e d F l l i t t e r s (SELH.ICR) each. N u l l i p a r o u s F l females w i t h i n each m a t e r n a l l i n e were bred w i t h one or o t h e r of two male F l s i b s . The F l females were k i l l e d on day 18 of g e s t a t i o n and t h e i r l i t t e r s were c o l l e c t e d and s c o r e d i n the same way as d e s c r i b e d above f o r the o t h e r F2 s e t . C. F i r s t B a c k c r o s s t o SELH ( B C l ) The f i r s t b a c k c r o s s ( B Cl) of F l males by SELH females was made ( B C l ) . These F l males were the same i n d i v i d u a l s as had been used t o produce the F2, and were a l l from the o r i g i n a l c r o s s of SELH males w i t h ICR/Be females (ICR.SELH). Ten ICR.SELH F l males were each mated w i t h 5 n u l l i p a r o u s SELH f e m a l e s . B C l f e t u s e s were c o l l e c t e d on day 18 of g e s t a t i o n , c o l l e c t e d and s c o r e d as p r e v i o u s l y d e s c r i b e d f o r the F2. They were s c o r e d f o r i n t e r n a l 49 sex a f t e r f i x a t i o n . One of the 10 ICR.SELH F l males was a l s o mated w i t h a s e r i e s of 5 n u l l i p a r o u s SELH females and the l i t t e r s r a i s e d t o produce a d u l t B C l g e n e r a t i o n a n i m a l s . D. Second B a c k c r o s s t o SELH (BC2) As S e w a l l Wright p o i n t e d out (Wright 1934), i t i s n e c e s s a r y t o c a r r y c l a s s i c a l b r e e d i n g t e s t s t o a t e s t c r o s s g e n e r a t i o n i n o r d e r t o c o r r e c t l y d i s t i n g u i s h between s i n g l e l o c u s and m u l t i p l e l o c u s i n h e r i t a n c e , because F2 and B C l p h e n o t y p i c p r o p o r t i o n s due t o m u l t i p l e l o c i can mimick those due t o s i n g l e l o c i . The d i s t r i b u t i o n of i n d i v i d u a l v a l u e s of t e s t c r o s s d a t a from a s e g r e g a t i n g g e n e r a t i o n however do d i s t i n g u i s h between d i f f e r e n t modes of i n h e r i t a n c e . 1. B C l males x SELH females 26 B C l males ( d e s c r i b e d above) were each mated t o 7-9 SELH n u l l i p a r i o u s f e m a l e s . Females were k i l l e d on day 18 of g e s t a t i o n and the l i t t e r s were c o l l e c t e d and s c o r e d as d e s c r i b e d p r e v i o u s l y f o r B C l and F2 f e t u s e s . 2. SELH females x SELH males In o r d e r t o p r o v i d e d a t a f o r the SELH s t r a i n t o compare w i t h t h a t of the B C l males, f i v e randomly chosen SELH males were mated t o 8 v i r g i n SELH f e m a l e s . The females were k i l l e d on day 18 of g e s t a t i o n and t h e i r l i t t e r s c o l l e c t e d . F e t u s e s were h a n d l e d , s c o r e d and f i x e d and d e s c r i b e d f o r F2 l i t t e r s of the 50 above two e x p e r i m e n t s . V I I . ENZYME LINKAGE TESTING The SELH s t o c k and the ICR s t r a i n a r e known t o d i f f e r a t f o u r p o l y m o r p h i c enzyme l o c i - Mod-1, Gpt-1, Pgk-2, and G p i - 1 . The mice a re known not t o d i f f e r a t 20 o t h e r l o c i t h a t have been t y p e d i n our l a b o r a t o r y . The o p p o r t u n i t y t o t e s t f o r a s s o c i a t i o n between t r a n s m i s s i o n of h i g h l i a b i l i t y t o e x e n c e p h a l y and t h e s e 4 marker l o c i on chromosomes 9, 15, 17, and 7 r e s p e c t i v e l y was a v a i l a b l e i n 25 B C l males t h a t had been t e s t e d f o r e x e n c e p h a l y p r o d u c t i o n w i t h SELH f e m a l e s . Any a s s o c i a t i o n s , i f found, would suggest t h a t a l o c u s near one of th e s e markers, or the polymorphism i t s e l f , i n f l u e n c e d l i a b i l i t y t o e x e n c e p h a l y . T h e r e f o r e the B C l males were typed f o r t h e i r genotype a t t h e s e 4 l o c i , u s i n g p u b l i s h e d methods (Mod-1, Shows and Ruddle, 1968; Gpt-1, E i c h e r and Womach, 1977; Pgk-2, E i c h e r , C h e r r y , F l a h e r t y 1978; G p i - 1 , DeLorezo and Ruddle , 1969) adapted t o T i t a n I I I c e l l u l o s e a c e t a t e i n common usage i n our l a b o r a t o r y . Each l o c u s was the n t e s t e d f o r a s s o c i a t i o n w i t h e x e n c e p h a l y f r e q u e n c y w i t h a one way Mann-Whitney U t e s t ( S i e g e l 1956). The t o t a l e x e n c e p h a l y f r e q u e n c y ( t r a n s f o r m e d i n t o a p r o b i t s c o r e ) f o r each B C l male, as w e l l as the e x e n c e p h a l y s c o r e i n female and male progeny a l o n e were compared w i t h the B C l male's enzyme l o c u s genotype. In t h i s way, sex l i m i t e d m o d i f i e r s of 51 e x e n c e p h a l y c o u l d a l s o be mapped. V I I I . MISCELLANEOUS STUDIES - Newborn A d r e n a l Glands A common anomaly a s s o c i a t e d w i t h a n e n c e p h a l y i n humans i s h y p o p l a s t i c a d r e n a l g l a n d s (Warkany 1971). Normal and e x e n c e p h a l i c newborn SELH mice, and normal SWV/Bc mice were c o l l e c t e d a t b i r t h . They were t h e n k i l l e d w i t h C 0 2 and p l a c e d i n t o B ouin's f i x a t i v e f o r a minlmun of 3 day s , t h e n d i s s e c t e d and t h e i r a d r e n a l g l a n d s o b s e r v e d . 52 RESULTS I . Embryology A. Rate of G e n e r a l Development A comparison between normal s t r a i n s and the SELH s t o c k i n the t i m i n g of g e n e r a l development was made t o d e t e r m i n e whether SELH embryos d e v e l o p a t the same r a t e as normal s t r a i n s . C h r o n o l o g i c a l age was t o o broad a measure of development t o be used, as t h e r e i s a r e l a t i v e l y l a r g e v a r i a t i o n i n embryonic development w i t h i n and between l i t t e r s a t a g i v e n c h r o n o l o g i c a l t i m e . An i n t e r n a l measure of g e n e r a l development was needed t h e n , t o compare n e u r a l tube c l o s u r e d u r i n g development between SELH embryos and normal s t r a i n s . As i s common i n s t u d i e s of n e u r a l tube development (eg. Dempsey and T r a s l e r 1983), we used s o m i t e number as the i n t e r n a l measure of development. We wanted t o know i f SELH embryos p r o g r e s s e d t h r o u g h n e u r a l tube c l o s u r e d u r i n g the same s t a g e s of g e n e r a l development as normal mice. For s o m i t e number t o be u s e f u l f o r such a c o m p a r i s o n , we needed t o know whether SELH and the normal s t r a i n s i n c r e a s e d s o mite number a t the same r a t e over t i m e . 280, 342, and 228 embryos from ICR/Be, SWV/Bc, and SELH females r e s p e c t i v e l y , were c o l l e c t e d from day 8-4pm t o day 9-12pm of g e s t a t i o n d u r i n g the p e r i o d of a n t e r i o r n e u r a l tube c l o s u r e . The mean number of s o m i t e s f o r each l i t t e r was c a l c u l a t e d , and l i t t e r s were grouped i n t o 4 hour c o l l e c t i o n p e r i o d s w i t h c e n t r e s around 4pm, 8pm, 12am, 4am, 8am, and 12pm ( t a b l e s I - I I I ) . The r e g r e s s i o n l i n e s f o r SELH, ICR/Bc, and SWV/Bc f o r c h r o n o l o g i c a l age v e r s u s mean 53 Table I . CHRONOLOGICAL GESTATIONAL AGE AND MEAN SOMITE COUNT OF ICR/BC EMBRYOS COLLECTED C h r o n o l o g i c a l G e s t a t i o n a l Age Number of L i t t e r s c o l l e c t e d Number of Embryos Examined L i t t e r Means Mean number of Somites per embryo* Day 8:1600 h r s Day 8:2000 h r s 5 Day 9:0000 h r s 2 Day 9:0400 h r s 12 Day 9:0800 h r s 10 40 7.4, 1.4, 9.1 5.0 4.0, 3.3 25 7.2, 5.7 6.4 123 9.6, 3.8,11.8 9.2 8.0, 8.6, 13.4 9.6, 4.7, 10.3 10.1, 10.5, 9.7 108 13.2, 12.5 13.4 12.2, 17.1, 6.5 14.4, 13.5, 11.9 14.7, 18.0 Day 9:1200 h r s 46 13.8, 15.7 19.3, 16.5 16.3 * c a l c u l a t e d as mean of l i t t e r means 54 Tab l e I I . CHRONOLOGICAL GESTATIONAL AGE AND MEAN SOMITE COUNT OF SWV/BC EMBRYOS COLLECTED C h r o n o l o g i c a l G e s t a t i o n a l Age Number of L i t t e r s C o l l e c t e d Number of Embryos Examined L i t t e r Means Mean number of Somites per embryo* Day 8:1600 h r s Day 8:2000 h r s Day 9:0000 h r s Day 9:0400 h r s Day 9:0800 h r s Day 9:1200 h r s 1 2 8 58 5.8, 2.8, 3.6 4.3 5.0, 4.2 38 4.7, 5.2 5.7 7.1 9 8.2 8.2 24 13.5, 13.2 13.4 96 13.8, 15.3, 9.4 12.6 12.2, 15.5, 9.9 13.2, 11.7 55 16.8, 25.5 .17.5 20.5, 7.2 * c a l c u l a t e d as mean of l i t t e r means 55 T a b l e I I I . CHRONOLOGICAL GESTATIONAL AGE AND MEAN SOMITE COUNT OF SELH EMBRYOS COLLECTED C h r o n o l o g i c a l Number of Number of L i t t e r Mean number G e s t a t i o n a l L i t t e r s Embryos Means of Somites Age C o l l e c t e d Examined per embryo* Day 8:1600 h r s 1 Day 8:2000 h r s 2 Day 9:0000 h r s Day 9:0400 h r s 6 Day 9:0800 h r s 10 Day 9:1200 h r s 3 12 2.4 2.4 24 5.2, 4.2 4.7 70 15.0, 12.5, 16.4 13.6 14.3, 10.8, 12.5 87 11.6, 15.9, 14.7 15.4 15.7, 20.9, 19.0 22.0, 18.1, 5.2 11.0 35 18.3, 19.6 18.5 17.5 * c a l c u l a t e d as mean of l i t t e r means n o t e : r e g r e s s i o n c a l c u l a t i o n s a r e based on l i t t e r s c o l l e c t e d from day 8:20hrs - day 9:12hrs. 56 somite c o u n t , were compared as demo n s t r a t e d In Snedecor and Cochran (1967, pages 432-436) ( t a b l e IV, f i g u r e 2 ) . The c h r o n o l g i c a l ages used were from day 8 a t 8pm t o day 9 a t 12pm as shown i n t a b l e IV. They were c o n v e r t e d t o v a l u e s of 1, 5, 9, 13, and 17 f o r use i n the r e g r e s s i o n a n a l y s i s . No s i g n i f i c a n t d i f f e r e n c e between s t r a i n s was found f o r s l o p e of the r e g r e s s i o n l i n e s ( F 2 8 = 1.29; p>0.05). These r e s u l t s mean t h a t the SELH s t o c k d i d not appear t o d i f f e r i n r a t e of change of number of s o m i t e s over time from ICR/Be or SWV/Bc s t r a i n s d u r i n g the p e r i o d of n e u r a l tube c l o s u r e , and t h a t s o m i t e count can be used as an i n d i c a t o r of d e v e l o p m e n t a l age f o r the t h r e e s t r a i n s . Comparison of e l e v a t i o n of the l i n e s however I n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e between the s t r a i n s ( F 2 1 Q =4.41; p<0.05). The d i f f e r e n c e s i n i n t e r c e p t mean t h a t t h e r e d i d appear t o be a d i f f e r e n c e i n the mean number of s o m i t e s found i n each s t r a i n a t any g i v e n t i m e . SELH embryos were about 1 so m i t e ahead of SWV/Bc and 2 s o m i t e s ahead of ICR/Be embryos a t each c h r o n o l o g i c a l age. I t i s c l e a r t h a t t h e r e i s no d e l a y i n g e n e r a l development of SELH embryos. B. N e u r a l Tube C l o s u r e and Somite Development A d e s c r i p t i o n of normal a n t e r i o r n e u r a l tube c l o s u r e was de v e l o p e d t h r o u g h o b s e r v a t i o n s of 323 and 304 embryos from timed p r e g n a n c i e s of the two normal s t r a i n s of mice, ICR/Be and SWV/Bc r e s p e c t i v e l y (19 ICR/Be and 29 SWV/Bc embyros had no s o m i t e s and were e x c l u d e d from s o m i t e count c o m p a r i s o n s ) ( f i g u r e 1 8a). The p r o c e s s was d i v i d e d i n t o a s e r i e s of r e c o g n i z a b l e s t a g e s . 57 Table IV. COMPARISON OF MEAN SOMITE COUNTS AT 5 CHRONOLOGICAL GESTATIONAL AGES CHRONOLOGICIAL TIME (*) STRAIN AGE 1 SWV/Bc ICR/Bc SELH 8:20 1 5.7 5.0 4.7 9:00 1 8.2 6.4 -9:04 1 13.4 9 . 2 13.6 9 :08 1 12.6 13.4 15.4 9:12 1 17.5 16.3 18.5 *: time = da y : h r s Comparison of s l o p e s : F 2 Q =1.29; p>0.05 Comparison of e l e v a t i o n s : F 9 1 f. =4.41; p<0.05 58 F i g u r e 2 COMPARISON OF THE RELATIONSHIP BETWEEN CHRONOLOGICAL AGE AND SOMITE COUNT IN ICR/BC, SWV/BC, AND SELH EMBRYOS 3 2 1 1 5 9 13 17 (8:20) (9:00) (9:04) (9:08) (9:12) G e s t a t i o n a l Time (0AY:H0UR) regression l i n e s : (++): f i t to the a=intercept SELH Y = 5.45 SWV/Bc Y - 4.64 ICK/llc Y » 3.22 li n e a r regression , b=slope + 0.76X (++) + 0.76X + 0.76X Y=a + bx, where T a b l e V. ANTERIOR NEURAL TUBE CLOSURE IN I C R / B c + Somite 1 Folds Evident Folds Cur led Prosen F o l d s * J Prosen Folds >} I n i t i a l Contact A 1 B * Prosen Fused * Fusion to H1d-Hesen With ANP . No ANP Fused to Apex With ANP No ANP Fused to Rhomb. Fused 1 II 1 2 1 I I 1 | 1 3 1 m I 4 « n j « 1 1 5 unjwiHI il l 1 | 1 6 wri | 7 JW1H1 1 I I 1 8 • 111 Jwrjwr-Hff 1 1 | 1 9 tutelar mi mt I I I 10 HI W J X T H 1 11 IMf j«r>n 1 1 1 | 1 12 M llll tHi HO | «l f | 1 13 X Mf ' tut in 1 14 '1 1 llll | VKMfijrf 1 II 15 j |l Jttf Wl 1 16 1 1 won JMWfM 17 I | 1 Mil J»T H I 18 | 1 inOtrt mi 19 1 j * r II 20 1 | 1 mr 21 1 j LwC 22 1 HI 23 1 | | 24 1 I 25 1 26 1 | | 27 1 I 2B 1 1 * = o c c u r s o n l y i n SELH + = each s t r o k e r e p r e s e n t s 1 embryo Tab le VI. ANTERIOR NEURAL TUBE CLOSURE IN SWV/Bc + Somite 1 F o l d s Ev ident Folds Cur led Prosen F o l d s i J Prosen Folds >J I n i t i a l Contact A 1 B * Prosen Fused * Fusion to Hid-Mesen With ANP , No ANP Fused to Apex With ANP ( No ANP Fused to Rhomb. Fused 1 J K f II . 1 2 mi 1 1 | 1 3 I 4 iMUftjrtwniu 1 5 w w w " i I | 1 6 Hll W in" I 7 mr j«r mi 1 8 mr j»i mi Mil I | j 9 jut mi -MiT 1 | 1 10 II 1 1 11 1 1 1 it | 1 12 " 1 IMfMII I f 13 1 II « ' III 1 l l 14 1 Jtfl 1 | l l III 15 I 1 I'll 11 JtffJKf in 16 1 1 I'll nil i 17 1 | 1 | m m m i 18 1 I JIK mi 19 1 J W | 20 1 | | JWH 21 1 | WT 22 1 w 23 1 | 1 24 1 | u 25 1 II 26 1 | | Mil 27 I 1 II 28 1 * = o c c u r s o n l y i n SELH + = e a c h s t r o k e r e p r e s e n t s 1 embryo T a b l e VII. ANTERIOR NEURAL TUBE CLOSURE IN SELH + Somite f Fo lds Evident Folds Cur led Prosen F o l d s * } Prosen Folds >} I n i t i a l Contact A I B * Prosen Fused * Fusion to Mld-Mesen WHh ANP . No ANP Fused to Apex With ANP No ANP Fused to Rhomb. Fused 1 (11 1 2 1 ill 1 | 1 3 mi | 1 4 «fl,»n n 1 5 I | 1 6 1 I 1 7 1 8 1 | 1 9 i 1 i 10 UM 1 I 1 11 iur i 1 | 1 12 mi W i 1 1 13 J-KT HI 1 14 i Wi- I | 1 15 ll ' lm I I 1" I f il •16 I I / / w III 17 llll J* 1 1 I I I 11 18 | l pur n I 19 J W mi il i i 1 20 III Jtff | lllll iW I 21 II I I 1 . I I I I'll 22 m 1 1 i jurnii 23 1 | 1 m 24 1 1 . 1 iin 25 1 in 26 1 | 1 i 27 1 I . 28 1 1 * = o c c u r s o n l y i n SELH *+ = e a c h s t r o k e r e p r e s e n t s 1 embryo 62 F o o t n o t e s f o r t a b l e s V, V I , and V I I A n t e r i o r n e u r a l tube c l o s u r e s t a g e s : F o l d s e v i d e n t - A n t e r i o r n e u r a l f o l d s v i s i b l e as t h i c k e n e d t i s s u e i n t h e a n t e r i o r h a l f of the cup shaped embryo, ( f i g u r e 3) F o l d s c u r l e d - A n t e r i o r n e u r a l f o l d s have e n l a r g e d . The c r a n i a l f l e x u r e i n the f o l d s p l a c e s the pre s u m p t i v e p r o s e n c e p h a l o n t i s s u e r o s t r a l t o the f l e x u r e , ( f i g u r e 4) P r o s e n f o l d s c u r v e d < l/2- A n t e r i o r n e u r a l f o l d s have g r e a t l y expanded. O p t i c s u l c i a r e w e l l d e v e l o p e d and the cup shaped h a l v e s of the p r o s e n c e p h a l o n a r e b e g i n n i n g t o cu r v e inward toward the m i d l i n e . The mesencephalon/rhombencephalon t i s s u e p r o g r e s s e s from t h i c k e n e d f o l d s t o t h i n e l o n g a t e d f l a r e d f o l d s , ( f i g u r e 5a,b) P r o s e n f o l d s c u r v e d >l/2- P r o s e n c e p h a l o n f o l d s a re n e a r i n g t h e m i d l i n e and i n i t i a l c o n t a c t . Mesencephalon/rhombencephalon t i s s u e i s f l a r e d out but s l o w l y c h a n g i n g c u r v a t u r e t o concave f o l d s , ( f i g u r e 6a,b) I n i t i t a l c o n t a c t - Denotes embryos i n f i r s t c o n t a c t between the edges of the a n t e r i o r n e u r a l w a l l s . S i t e A i n d i c a t e s c o n t a c t a t a band of t i s s u e between the m i d / p o s t e r i o r p r o s e n c e p h a l o n and the a n t e r i o r mesencephalon. S i t e B i n d i c a t e s c o n t a c t a t the r o s t r a l base of the p r o s e n c e p h a l o n and f u s i o n of up t o 1/4 of the most r o s t r a l p r o s e n c e p h a l o n t i s s u e , ( f i g u r e s 7 and 12) P r o s e n f u s e d - Embryos have f u s e d 1/2 t o a l l of the p r o s e n c e p h a l o n w i t h no f u s i o n of the mesencephalon t i s s u e . Mesencephalon/rhombencephalon t i s s u e may or may not be f l a r e d . Some f u s i o n of the s i n g l e c e l l membrane c o v e r i n g the rhombencephalon may have o c c u r r e d , ( f i g u r e 13) F u s i o n t o mid-mesen w i t h anp or no anp- F u s i o n of the a n t e r i o r n e u r a l tube t o the m i d d l e of the mesencephalon. Embryos may or may not have an a n t e r i o r neuropore gap or t h i n s l i t , ( f i g u r e s 8a,b, and 14) Fused t o apex w i t h anp or no anp- F u s i o n t o the apex of the embryo ( i e . t o the p o s t e r i o r mesencephalon). Embryos may or may not have an a n t e r i o r neuropore s l i t s t i l l v i s i b l e , ( f i g u r e s 9a,b, and 15) Fused t o rhomb- F u s i o n of the n e u r a l tube t o the a n t e r i o r rhombencephalon, ( f i g u r e 10) Fused- F u s i o n of the a n t e r i o r n e u r a l tube i s complete. The s i n g l e c e l l membrane c o v e r i n g the rhombencephalon gap i s comp l e t e d , ( f i g u r e 11) 63 A n t e r i o r n e u r a l tube c l o s u r e s t a g e s were then c h a r t e d a g a i n s t s o m ite count of the embryos t o c r e a t e a scheme of the sequence of e v e n t s i n c l o s u r e (see t a b l e s V and V I , and f i g u r e s 3 t o 1 1 ) . I n d i v i d u a l embryos were r e c o r d e d on the c h a r t a c c o r d i n g t o t h e i r s o m i te count and n e u r a l tube c l o s u r e s t a g e . The r e s u l t was a continuum from the s t a g e when the a n t e r i o r n e u r a l f o l d s a r e f i r s t e v i d e n t t o the s t a g e when the a n t e r i o r n e u r a l tube i s c o m p l e t e l y c l o s e d . The sequence of events i n c l o s u r e was v e r y s i m i l a r i n both normal s t r a i n s as was the r a t e of n e u r a l tube development i n r e l a t i o n t o somite count ( t a b l e s V I I I and I X ) . The n e u r a l tube c l o s u r e p r o c e s s appears t o be q u i t e s i m i l a r among d i f f e r e n t normal s t r a i n s of mice. Golden and C h e r n o f f s d e s c r i p t i o n of n e u r a l tube c l o s u r e i n "C3H" mice agrees w e l l w i t h the c l o s u r e p a t t e r n observed i n t h i s s t u d y (Golden and C h e r n o f f 1983) . The normal p r o c e s s of n e u r a l tube c l o s u r e as seen i n ICR/Be and SWV/Bc embryos and the s t a g e s d e s c r i b e d by the headings of t a b l e s V and VI a r e as f o l l o w s : By the one or two somite s t a g e i n development (about 8 3/4 days of g e s t a t i o n ) , a n t e r i o r n e u r a l f o l d t i s s u e i s v i s i b l e ( " f o l d s e v i d e n t " ; f i g u r e 3 ) . These f o l d s expand and e n l a r g e and form a c r a n i a l f l e x u r e u s u a l l y a t about 4-7 s o m i t e s ( " f o l d s c u r l e d " ; f i g u r e 4). O p t i c s u l c i become e v i d e n t i n the two h a l v e s of the f u t u r e p r o s e n c e p h a l o n (the r e g i o n a n t e r i o r t o the c r a n i a l f l e x u r e ) . O p t i c s u l c i can be seen i n f i g u r e 5a. At about 6-9 s o m i t e s , the two concave h a l v e s of the p r o s e n c e p h a l o n s t a r t t o 64 converge ("prosen f o l d s c u r v e d 11/2"; f i g u r e 5a, w h i l e the f u t u r e mesencephalon and rhombencephalon t i s s u e p o s t e r i o r t o t h i s i s f o r m i n g convex, f l a r e d f o l d s ( f i g u r e 5 b ). The c u r v i n g i n of the p r o s e n c e p h a l o n f o l d s p r o g r e s s e s d u r i n g the 8-13 somite s t a g e s , c l o s i n g the gap from h a l f of i t s former w i d t h t o near c o n t a c t ("prosen f o l d s c u r v e d > l / 2 " ; f i g u r e 6 a ) . At t h i s t i m e , the mesencephalon and rhombencephalon f l a r e d f o l d s a r e c h a n g i n g c u r v a t u r e from convex t o concave ( f i g u r e 6 b ). When the embryo has 10-14 s o m i t e s , the two h a l v e s of the a n t e r i o r n e u r a l f o l d s make t h e i r i n i t i a l c o n t a c t and f u s i o n i n a s m a l l band of t i s s u e i n the middl e or p o s t e r i o r p r o s e n c e p h a l o n / mesencephalon r e g i o n ( c l o s u r e 2) ( " i n i t i a l c o n t a c t A"; f i g u r e 7 ) . C l o s u r e 2 expands b i d i r e c t i o n a l l y . When t h i s c l o s u r e p r o g r e s s e s t o h a v i n g f u s e d 1/2 of the mesencephalon (12-15 s o m i t e s ) , some embryos s t i l l have an a n t e r i o r neuropore (anp) w h i l e o t h e r s do not ( " f u s i o n t o mid-mesen w i t h anp"; f i g u r e 8a/ " f u s i o n t o mid-mesen, no anp"; f i g u r e 8 b ) . The c a u d a l e x p a n s i o n of c l o s u r e 2 completes f u s i o n over the mesencephalon by 13-15 so m i t e s ("fused t o apex w i t h or w i t h o u t a n p " ; f i g u r e s 9a and 9 b ) . D u r i n g the p e r i o d when c l o s u r e 2 f u s e s the mesencephalon and a n t e r i o r rhombencephalon f o l d s , the r o s t r a l gap i n the a n t e r i o r n e u r a l f o l d s ( the anp) i s f u s e d from two d i r e c t i o n s , v i a the c o n t i n u a t i o n of c l o s u r e 2 and from a f u s i o n o r i g i n a t i n g a t t h e most r o s t r a l end of the f o l d s , a t the base of the p r o s e n c e p h a l o n ( c l o s u r e 3) ( f i g u r e 18a). C l o s u r e 3 i s e v i d e n t by about 14-15 65 s o m i t e s . F u s i o n up t o the p o s t e r i o r end of the rhombencephalon proceeds as the a n t e r i o r e x t e n s i o n of f u s i o n from the middl e back of the embryo. The l a s t c l o s u r e t o occur over the a n t e r i o r n e u r a l f o l d s i s t h a t of the s i n g l e c e l l l a y e r membrane which c o v e r s the rhombencephalon gap ( c l o s u r e 4 ) . A n t e r i o r n e u r a l tube c l o s u r e t o t h i s gap i s completed by 14-17 s o m i t e s ("fused t o rhomb"; f i g u r e 1 0 ) . T h i s l a s t c l o s u r e d i f f e r s from the o t h e r a r e a s of the a n t e r i o r n e u r a l tube i n t h a t the n e u r a l f o l d s approach each o t h e r but do not c o n t a c t . I n s t e a d , an e p i t h e l i a l l a y e r of c e l l s c o v e r s the a r e a (Golden and C h e r n o f f 1983). T h i s c e l l l a y e r appears f i r s t a t the p o s t e r i o r end of the rhombencephalon and grows i n an a n t e r i o r d i r e c t i o n over the e n t i r e gap. T h i s membrane c o v e r i n g i s the f i n a l s t a g e i n a n t e r i o r n e u r a l tube c l o s u r e , and i s completed i n both ICR/Be and SWV/Bc embryos by 15-18 s o m i t e s ("fused"; f i g u r e 1 1 ) . While t h i s a n t e r i o r development i s t a k i n g p l a c e , n e u r a l tube development i s a l s o p r o g r e s s i n g a t the p o s t e r i o r a r e a of the embryo. P o s t e r i o r n e u r a l f o l d s a r e v i s i b l e by about 4-5 s o m i t e s i n development. The f i r s t f u s i o n a l o n g the l e n g t h of the n e u r a l tube i s i n the mid back r e g i o n of the 6 somite embryo a t the l e v e l of about s o m i t e s 2-3 ( c l o s u r e 1 ) . T h i s f u s i o n proceeds b i d i r e c t i o n a l l y c a u d a l l y t o the t a i l and r o s t r a l l y t o t h e p o s t e r i o r rhombencepalon r e g i o n . A s m a l l d i f f e r e n c e was found between ICR/Bc and SWV/Bc embryos i n the l o c a t i o n of the i n i t i a l c o n t a c t p o i n t of the a n t e r i o r n e u r a l f o l d s ( c l o s u r e 2 ) . ICR/Bc a n t e r i o r n e u r a l f o l d s f i r s t c o n t a c t a t the p o s t e r i o r end of the pr e s u m p t i v e p r o s e n c e p h a l o n , or a t the j u n c t i o n between the p r o s e n c e p h a l o n and mesencephalon. T h i s f u s i o n l e a v e s a l a r g e gap r o s t r a l t o the c o n t a c t ( the a n t e r i o r n e u r o p o r e ) . In SWV/Bc embryos, the f i r s t c o n t a c t between the two h a l v e s of the a n t e r i o r n e u r a l f o l d s ( c l o s u r e 2) was found t o be s l i g h t l y more r o s t r a l , t h a t i s , f u s i o n was f i r s t n o ted i n t h e middl e of the p r o s e n c e p h a l o n and i n v o l v e d l i t t l e of the mesencephalon f o l d s . The gap r o s t r a l t o t h i s c o n t a c t was a l s o g e n e r a l l y s h o r t e r and more narrow i n SWV/Bc tha n the gap seen i n ICR/Bc embryos. T h i s d i f f e r e n c e i n s i z e of the a n t e r i o r neuropore (anp) i s c o n s i s t e n t w i t h the number of SWV/Bc and ICR/Bc embryos w i t h or w i t h o u t f u s i o n of the anp i n l a t e r s t a g e s of development ( t a b l e s V and V I ) . For example, v e r y few ICR/Bc embryos (1/16 ( 6 % ) ) w i t h f u s i o n t o the middl e of the mesencephalon had f u s e d the anp, w h i l e 12/38 (32%) of SWV/Bc embryos had f u s e d t h i s anp gap a t the same s t a g e . T h i s v a r i a t i o n i n p o s i t i o n of f i r s t f u s i o n d i d not however t r a n s l a t e i n t o an o b s e r v a b l e d i f f e r e n c e i n the t i m i n g of t h e c o m p l e t i o n of a n t e r i o r n e u r a l tube c l o s u r e between the s t r a i n s . I t does i n d i c a t e though, t h a t normal n e u r a l tube c l o s u r e may encompass a range of s m a l l v a r i a t i o n s i n c o n t a c t l o c a t i o n s and 67 t i s s u e i n v o l v e m e n t s w h i l e f o l l o w i n g a b a s i c normal p a t t e r n of c l o s u r e . A n t e r i o r n e u r a l tube c l o s u r e was a l s o examined i n 276 SELH embryos, w i t h a s c o r i n g of s t a g e of c l o s u r e and somite count ( t a b l e V I I , f i g u r e 18b). SELH embryos p r o g r e s s e d t h r o u g h the same c l o s u r e s t a g e s as normal s t r a i n embryos up t o the p o i n t of " i n i t i a l c o n t a c t " . However m o r p h o l o g i c a l l y , from as e a r l y as 7 s o m i t e s , the p r o s e n c e p h a l o n f o l d s were wider a p a r t i n SELH th a n normal, and the mesencephalon/rhombencephalon f o l d s remained f l a r e d open i n s t e a d of r i s i n g up t o meet i n the m i d l i n e (SEM p l a t e , f i g u r e 17a,b). SELH embryos appeared t o have a b a s i c problem i n e l e v a t i n g the two h a l v e s of the a n t e r i o r n e u r a l f o l d s i n t o the m i d l i n e . I n i t i a l c o n t a c t between the a n t e r i o r n e u r a l f o l d s took p l a c e i n SELH embryos between about 14-21 s o m i t e s , and o c c u r r e d a t a d i f f e r e n t s i t e t h a n i n normal s t r a i n embryos ( t a b l e V I I , SEM p l a t e , f i g u r e 1 7 c , d ) . The i n i t i a l f u s i o n a t the base of the p r o s e n c e p h a l o n i n SELH ( c l o s u r e 3, f i g u r e 18b) ( " i n i t i a l c o n t a c t B"; f i g u r e s 12 and 17d), proceeded c a u d a l l y , f u s i n g the p r o s e n c e p h a l o n by 15-22 s o m i t e s ("prosen f u s e d " ; f i g u r e s 13 and SEM p l a t e , f i g u r e 17e). F u s i o n p r o g r e s s e d t h r o u g h the m i d d l e of t h e mesencephalon by 15-19 s o m i t e s ("fused t o mid-mesen"; f i g u r e 1 4 ) , and completed f u s i o n of the mesencephalon by 16-24 s o m i t e s ("fused t o apex"; f i g u r e 1 5 ) . F u s i o n t o the rhombencephalon ("fused t o rhomb") o c c u r r e d i n 18-23 somite embryos, w i t h 68 c o m p l e t i o n of a n t e r i o r n e u r a l tube c l o s u r e i n the range of 18-24 s o m i t e s ( " f u s e d " ) . The SELH c l o s u r e p a t t e r n d i f f e r e d from n e u r a l tube c l o s u r e i n the normal s t r a i n s i n a number of i m p o r t a n t ways. F i r s t l y , c ompleted c l o s u r e was d e l a y e d r e l a t i v e t o somite number. At 17 s o m i t e s , when 67% (8/12) of ICR/Be and 70% (14/20) of SWV/Bc embryos had complete c l o s u r e of t h e i r a n t e r i o r n e u r a l t u b e , no SELH embryos 0% (0/13) had f i n i s h e d c l o s u r e ( t a b l e V I I I ) . The f i r s t embryos found t o be c o m p l e t e l y f u s e d were a t 15 s o m i t e s ' d e v e l o p m e n t a l age i n both SWV/Bc and ICR/Be. In c o n t r a s t , the e a r l i e s t d e v e l o p m e n t a l age a t which an SELH embryo was c o m p l e t e l y f u s e d was 18 s o m i t e s . By 18 s o m i t e s i n SWV/Bc and ICR/Be s t r a i n s , a l l embryos had c o m p l e t e l y f u s e d t h e i r a n t e r i o r n e u r a l tube (Table V I I I ) . S e c o n d l y , the t i m i n g of i n i t i a l c o n t a c t i s a l s o q u i t e d i f f e r e n t i n SELH from t h a t d e s c r i b e d f o r ICR/Be and SWV/Bc embryos. C o n t a c t was seen i n SELH embryos by 14 s o m i t e s a t the e a r l i e s t , and 21 s o m i t e s a t the l a t e s t , compared w i t h 12-14 and 10-13 som i t e s t a g e s i n ICR/Be and SWV/Bc ( t a b l e s V - V I I ) . Comparison of mean somite count i n SELH, ICR/Be, and SWV/Bc embryos d u r i n g a n t e r i o r n e u r a l tube c l o s u r e s t a g e s i n d i c a t e d t h a t SELH embryos appeared t o f o l l o w the normal t i m i n g of a n t e r i o r n e u r a l tube c l o s u r e up t o the s t a g e of c u r l i n g of the c r a n i a l n e u r a l f o l d s i n the p r o s e n c e p h a l i c r e g i o n ("prosen f o l d s c u r v e d <.l/2") ( t a b l e I X ) . T h i s s t a g e n o r m a l l y took p l a c e i n 7-8 somite embryos Table V I I I . PROPORTION OF EMBRYOS WITH ANTERIOR NEURAL TUBE CLOSURE COMPLETED AT VARIOUS SOMITE COUNTS STRAINS Somite count I SELH ICR/Bc SWV/Bc 15 s o m i t e s I 0/24 (0%) 1/28 (4%) 3/19 (16%) 16 1 0/22 (0%) 12/23 (52%) 1/10 (10%) 17 1 0/13 (0%) 8/12 (67%) 14/20 (70%) 18 1 1/21 (5%) 14/14 (100%) 9/9 (100%) 19 1 0/13 (0%) 7/7 (100%) 6/6 (100%) 20 1 6/18 (33%) 5/5 (100%) 7/7 (100%) Table IX. MEAN SOMITE COUNT IN SELH, ICR/Be, AND SWV/Bc EMBRYOS DURING ANTERIOR NEURAL TUBE CLOSURE STAGES f o l d s prosen prosen i n i t i a l fused fused fused f o l d s evident c u r l e d (cran. f l e x ) f o l d s curved^l/2 f o l d s curved>l/2 contact A B prosen f used to mid mesen to apex to rhomb. SWV/Bc 1.4 4.8 7.3 9.4 11.2 - 13.0 14.9 16.6 ICR/Be 1.8 5.4 8.2 10.8 12.6 - 12.9 14.2 15.6 SELH 1.2 4.8 10.2 13.0 15.0 15.6 18.5 17.0 18.7 20.5 Folds curled: F = 4.44; p<0.05; Prosen folds < h : F = 28.71; p<0.001; 2 , 198 <i, I J J Prosen folds > h : F_ = 40.53; p<0.001; Fused to mid-mesen: F = 71.69; p<0.001; 2,81 2,69 Fused to apex: F = 65.68; p<0.001; Fused to rhomb: F = 42.73; p<0.001 2,85 2,37 and was d e l a y e d i n SELH t o the 10 somite embryo ( t a b l e I X ) . From the "prosen f o l d s c u r v e d £ . 1 / 2 " s t a g e onward, the SELH s t o c k lagged f u r t h e r b e h i n d both ICR/Bc and SWV/Bc c l o s u r e s t a g e s r e l a t i v e t o somite c o u n t , so t h a t i n any g i v e n s t a g e of n e u r a l tube c l o s u r e , the SELH embryos had more s o m i t e s , t h a t i s , t h e y were d e v e l o p m e n t a l l y o l d e r ( t a b l e I X ) . I n the more advanced c l o s u r e s t a g e s , SELH embryos were c o n s i s t e n t l y 2.5 t o 4 s o m i t e s b e h i n d both SWV/Bc and ICR/Bc embryos a t each c l o s u r e s t a g e ( t a b l e I X ) . For example, the average SELH embryo i n t h e c l o s u r e s t a g e " f u s e d t o apex", had about 4 s o m i t e s more th a n the average SWV/Bc embryo and about 4 and 1/2 s o m i t e s more than the average ICR/Bc embryo (18.7, 14.9, and 14.2 s o m i t e s r e s p e c t i v e l y ) ( t a b l e I X ) . The d i f f e r e n c e between somite c o u n t s a t each n e u r a l tube c l o s u r e s t a g e f o r the 2 normal s t r a i n s and the SELH s t o c k was s i g n i f i c a n t a t a l l but the f i r s t s t a g e , " f o l d s e v i d e n t " , which was not compared u s i n g the a n a l y s i s of v a r i a n c e as i t i s a s t a g e w i t h a t r u n c a t e d d i s t r i b u t i o n ( t a b l e IX) ( n o t e : the d i f f e r e n c e found i n the " f o l d s c u r l e d " s t a g e was due t o ICR/Bc compared t o SELH and SWV/Bc embryos). T h i r d l y , the l o c a t i o n of i n i t i a l c o n t a c t i n SELH was not a t the prosencephalon/mesencephalon r e g i o n as i n the normal s t r a i n s ( " i n i t i a l c o n t a c t - A", t a b l e X ) . I n s t e a d , most SELH embryos (43/44) w i t h i n i t i a l c o n t a c t between n e u r a l f o l d s had t h a t c o n t a c t a t the r o s t r a l base of the p r o s e n c e p h a l o n ( " i n i t i a l c o n t a c t - B"; SEM p l a t e , f i g u r e 17a,b) and had 14-21 s o m i t e s . T h i s c o n t a c t and f u s i o n p o i n t ( c l o s u r e 3) was a l s o observed i n 72 T a b l e X. C 0 M > ™ Z f 2 " B E T W E E N DUMBER OF SELH, ICR/BC, AND SWV/BC EMBRYOS AT SPECIFIC STAGES IN ANTERIOR NEURAL TUBE CLOSURE CLOSURE STAGE ( ) * INITIAL CONTACT S i t e A S i t e B PROSEN FUSED MESEN NOT FUSED ICR/Bc 17/17(100) - | 0/26 (0) 1 26/26(100) SWV/Bc 8/8(100) _ | 0/37 (0) 1 37/37(100) SELH 1/44(02) 43/44(98)1 37/81(46) 1 44/81 (54) PROSEN FUSED MESEN FUSED *: (#) = p e r c e n t 73 ICR/Bc and SWV/Bc embryos w i t h 14-18 so m i t e s as d e s c r i b e d e a r l i e r , however, i t was never the i n i t i a l c o n t a c t p o i n t of t h e c r a n i a l f o l d s , but occu r e d a f t e r c l o s u r e 2. S e v e r a l SELH embryos were found t o have f u s e d o n l y the p r o s e n c e p h a l o n ( p r o s e n f u s e d , mesen not f u s e d , t a b l e X, SEM p l a t e 1 7 e ) , whereas a l l ICR/Bc and SWV/Bc embryos w i t h f u s i o n i n t h e p r o s e n c e p h a l o n a l s o had fu s e d p a r t or a l l of the mesencephalon (pr o s e n f u s e d , mesen f u s e d [ i n c l u d e s embryos w i t h f u s i o n t o the rhombencephalon]; t a b l e X ) . The l a r g e number of SELH embryos h a v i n g i n i t i a l c o n t a c t a t the pr o s e n c e p h a l o n base, the l a r g e number of SELH embryos h a v i n g o n l y t h e " p r o s e n c e p h a l o n f u s e d " , and the l a c k of SELH embryos w i t h f u s i o n i n the mesencephalon and an a n t e r i o r neuropore combine t o suggest t h a t n e a r l y a l l SELH embryos fu s e t h e i r a n t e r i o r n e u r a l tubes by a d i f f e r e n t p a t h t h a n normal ICR/Bc and SWV/Bc s t r a i n s ( t a b l e X, f i g u r e 18a-b). In normal s t r a i n s , t he f u s i o n of the base of the p r o s e n c e p h a l o n ( c l o s u r e 3) h e l p s t o c l o s e the a n t e r i o r n europore, but most of the p r o s e n c e p h a l o n , mesencephalon, and a n t e r i o r rhombencephalon i s f u s e d by a c o n t i n u a t i o n of the c l o s u r e i n i t i a t e d i n the m i d d l e - t o - p o s t e r i o r p r o s e n c e p h a l o n r e g i o n ( c l o s u r e 2 ) . The f u s i o n of the r o s t r a l base of the p r o s e n c e p h a l o n b e g i n s a t s i m i l a r t i m e s i n SELH and normal SWV/Bc and ICR/Bc s t r a i n s (14 s o m i t e s ) . However, n e a r l y a l l SELH embryos l a c k c l o s u r e 2 and r e l y on c l o s u r e 3 t o p u l l the f o l d s t o g e t h e r i n a z i p p e r - l i k e manner over the c r a n i a l a n t e r i o r n e u r a l t u b e . T h i s a l t e r n a t e f u s i o n mechanism, (a p a s s i v e mechanisim i n the sense t h a t the a c t i v e i n c u r l i n g of the f o l d s t h a t presumably f a c i l i t a t e s normal c l o s u r e , i s a p p a r e n t l y a b s e n t ) seems t o be the mechanism by which the m a j o r i t y of SELH embryos ( a p p r o x i m a t e l y 80%) manage t o c l o s e the a n t e r i o r n e u r a l tube even though n e a r l y a l l l a c k c l o s u r e 2, and f a i l t o make an i n i t i a l c r a n i a l f o l d c o n t a c t i n the mesencephalon/prosencephalon j u n c t i o n of the n e u r a l f o l d s . E i g h t day 9 SELH embryos of 20 - 22 s o m i t e s (8/42 19%) were found t o have o b v i o u s l y abnormal c r a n i a l n e u r a l tube development w i t h w i d e l y f l a r e d mesencephalon/rhombencephalon r e g i o n and o n l y some f u s i o n of the p r o s e n c e p h a l o n ( f i g u r e 1 6 ) . These embryos presumably r e p r e s e n t f u t u r e e x e n c e p h a l i c s . The b a s i c mechanism c a u s i n g e x e n c e p h a l y i n SELH mice i s thus seen t o be a f a i l u r e of e l e v a t i o n of the n e u r a l f o l d s and the consequent f a i l u r e of c o n t a c t and c l o s u r e of the a n t e r i o r n e u r a l tube i n the mesencephalon/prosencephalon j u n c t i o n ( c l o s u r e 2 ) . More s p e c i f i c a l l y , the mechanism t h a t causes some SELH t o d e v e l o p e x e n c e p h a l y w h i l e the m a j o r i t y do n o t , appears t o be the f a i l u r e of the compensatory c l o s u r e a r i s i n g from the normal c l o s u r e of the a n t e r i o r neuropore ( c l o s u r e 3 ) . By the time the e x t e n s i o n of c l o s u r e 3 has reached the mesencephalon, the embryos a r e w e l l p a s t the u s u a l time of a n t e r i o r n e u r a l tube c l o s u r e . Perhaps i n some, the f o l d s a r e wider a p a r t , or the a b i l i t y of the f o l d s t o be p u l l e d t o the m i d l i n e has been l o s t . 75 FIGURE DESCRIPTIONS FIGURE KEY: anp= a n t e r i o r neuropore; m/m= middl e mesencephalon; apex= head apex; t= t a i l ; i= i n i t i a l c o n t a c t ; p/m= prosencephalon/mesencephalon j u n c t i o n ; p= p r o s e n c e p h a l o n f o l d s ; m/r= mesencephalon/rhombencephalon t i s s u e ; os= o p t i c s u l c i ; op= o t i c p i t s ; s= s o m i t e s ; h= h e a r t ; m= mesencephalon; cf= c r a n i a l f o l d s . F i g u r e 3: S t a g e : " f o l d s e v i d e n t " - S i d e v i e w of a 1 somite cup shaped ICR/Bc embryo w i t h a n t e r i o r n e u r a l t i s s u e e v i d e n t . F i g u r e 4: S t a g e : " f o l d s c u r l e d " - S i d e v i e w of a 6 somite ICR/Bc embryo. The c r a n i a l f l e x u r e i s v i s i b l e . F i g u r e 5a: St a g e : " p r o s e n f o l d s < l / 2 " - F r o n t a l v i e w of a 9 som i t e ICR/Bc embryo w i t h o p t i c s u l c i v i s i b l e and p r o s e n c e p h a l o n f o l d s <l/2 c l o s e t o g e t h e r . The t a i l i s a t the back. F i g u r e 5b: Back v i e w of embryo i n f i g u r e 5a. The mesencephalon/ rhombencephalon f o l d s a r e f l a r e d wide. F i g u r e 6a: St a g e : " p r o s e n f o l d s > l / 2 " - F r o n t a l v i e w of a 10 somite ICR/Bc embryo w i t h p r o s e n c e p h a l o n f o l d s >l/2 c l o s e and mesencephalon f o l d s r i s i n g from f l a r e d t o p a r a l l e l . F i g u r e 6b: Back v i e w of the 10 somite embryo i n f i g u r e 6a. The back of the n e u r a l tube i s fu s e d up t o the o t i c p i t s . Rhombencephalon f o l d s a r e "V" shaped and no l o n g e r f l a r e d . F i g u r e 7: S t a g e : " i n i t i a l c o n t a c t - s i t e A"- F r o n t a l v i e w of a 12 som i t e ICR/Bc embryo. I n i t i a l c o n t a c t has. been made between n e u r a l t i s s u e a t the j u n c t i o n between the p r o s e n c e p h a l o n and mesencephalon. A l a r g e a n t e r i o r neuropore (anp) i s e v i d e n t , and the t a i l i s t w i s t i n g from back t o f r o n t . F i g u r e 8a: S t a g e : " f u s i o n t o mid-mesen w i t h anp"- F r o n t a l v i e w of a 13 somite ICR/Bc embryo. F u s i o n of the a n t e r i o r n e u r a l tube has reached t h e mi d d l e of the mesencephalon; a l a r g e anp i s v i s i b l e . F i g u r e 8b: S t a g e : " f u s i o n t o mid-mesen, no anp"- F r o n t a l v i e w of a 12 somite SWV/Bc embryo w i t h f u s i o n t o the mi d d l e of the mesencephalon, the anp i s f u s e d . F i g u r e 9a: S t a g e : " f u s i o n t o apex w i t h anp"- F r o n t a l v i e w of a 15 so m i t e ICR/Bc embryo w i t h f u s i o n t o the head apex ( p o s t e r i o r mesencephalon), the anp i s v i s i b l e . F i g u r e 9b: S t a g e : " f u s i o n t o apex, no anp"- F r o n t a l v i e w of a 15 som i t e SWV/Bc embryo w i t h f u s i o n t o the head apex. The anp i s f u s e d . 76 F i g u r e 10: S t a g e : " f u s e d t o rhomb"- Back v i e w of a 15 s o m i t e ICR/Be embryo w i t h f u s i o n completed t o the rhombencephalon. The rhombencephalon web has formed over the lower 2/3 of the gap. F i g u r e 11: S t a g e : " f u s e d " - Back v i e w of a 16 somite ICR/Be embryo w i t h a c o m p l e t e l y f u s e d a n t e r i o r n e u r a l tube. F u s i o n of the s i n g l e c e l l membrane over the rhombencephalon i s v i s i b l e . . F i g u r e 12: S t a g e : " i n i t i a l c o n t a c t - s i t e B": F r o n t a l v i e w of a 16 so m i t e SELH embryo. I n i t i a l c o n t a c t i s a t the base of the p r o s e n c e p h a l o n . The mesencephalon f o l d s a r e f l a r e d wide. F i g u r e 13: S t a g e : " p r o s e n f u s e d " : F r o n t a l v i e w of an 18 s o m i t e SELH embryo w i t h f u s i o n of the p r o s e n c e p h a l o n . The mesencephalon f o l d s a r e s t i l l m i l d l y f l a r e d a p a r t . F i g u r e 14: S t a g e : " f u s e d t o mid-mesen": F r o n t a l v i e w of an 18 so m i t e SELH embryo w i t h f u s i o n t o t h e m i d d l e of t h e mesencephalon. The mesencephalon and rhombencephalon t i s s u e s t i l l u n fused remains wide a p a r t . F i g u r e 15: S t a g e : " f u s e d t o apex": F r o n t a l v i e w of an 18 s o m i t e SELH embryo w i t h f u s i o n t o the head apex. The anp i s f u s e d . F i g u r e 16: F r o n t a l v i e w of a 22 somite SELH embryo. F u s i o n of the p r o s e n c e p h a l o n has o c c u r r e d , but the mesencephalon f o l d s a r e f l a r e d wide a p a r t - p o s s i b l y a f u t u r e e x e n c e p h a l i c embryo. F i g u r e 17a: Scan n i n g e l e c t r o n m i c r o s c o p y (SEM) p i c t u r e of an 11 so m i t e ICR/Be embryo n e a r i n g c o n t a c t a t the p r o s e n c e p h a l o n / mesencephalon j u n c t i o n . The mesencephalon/rhombencephalon f o l d s havae r i s e n from f l a r e d t o "V" shaped. F i g u r e 17b: SEM p i c t u r e of a 12 somite SELH embryo. The p r o s e n c e p h a l o n f o l d s a r e 1/2 c l o s e and mesencephalon/ rhombencephalon f o l d s a r e f l a r e d . F i g u r e 17c: SEM p i c t u r e of a 13 somite ICR/Be embryo. I n i t i a l c o n t a c t has o c c u r r e d a t the j u n c t i o n of the p r o s e n c e p h a l o n and the mesencephalon. A l a r g e anp i s v i s i b l e . F i g u r e 17d: SEM p i c t u r e of a 14 somite SELH embryo. I n i t i a l c o n t a c t has o c c u r r e d a t the base of the p r o s e n c e p h a l o n . The r e s t the c r a n i a l n e u r a l f o l d s a r e f a r a p a r t . F i g u r e 17e: SEM p i c t u r e of a 17 somite SELH embryo. F u s i o n has o c c u r r e d over the p r o s e n c e p h a l o n w i t h no f u s i o n i n the mesencephalon. 77 F i g u r e 18 PATTERN OF CLOSURE OF THE ANTERIOR NEURAL TUBE 82 a) NORMAL MICE* b) SELH MICE * Golden and C h e r n o f f (1983) 83 I I . ADDITIONAL ANOMALIES Any a b n o r m a l i t i e s of f e t u s e s i n a l l of the g e n e t i c c r o s s e s s c o r e d on day 18 of g e s t a t i o n were r e c o r d e d f o r a l l s t u d i e s ( t a b l e X I ) . Ten f e t u s e s w i t h e x e n c e p h a l y had another anomaly as w e l l . F i v e e x e n c e p h a l i c f e t u s e s ( a l l female) were found t o have e x c e s s i v e subcutaneous f l u i d , or g e n e r a l i z e d edema. Two o t h e r e x e n c e p h a l i c s (female) were m i s s i n g 1 or both e y e s , and one female e x e n c e p h a l i c had a g r o s s l y d e f i c i e n t f a c e w i t h what appeared t o be o n l y m a x i l l a r y t i s s u e p r e s e n t . These f a c i a l a b n o r m a l i t i e s may r e f l e c t d i s r u p t i o n s of n e u r a l c r e s t c e l l m i g r a t i o n and/or o t h e r nerve c e l l s , d i s t u r b a n c e s of mesodermal t i s s u e , or a b n o r m a l i t i e s of the n e u r a l p l a t e . One dead e x e n c e p h a l i c f e t u s had an omphalocele ( d e a t h o c c u r r e d on a p p r o x i m a t e l y day 16 of g e s t a t i o n ) , as d i d one male e x e n c e p h a l i c . The v a s t m a j o r i t y of e x e n c e p h a l i c f e t u s e s , 96.7%, (291/301) d i d not have any ano m a l i e s a s s o c i a t e d w i t h e x e n c e p h a l y , and no o t h e r n e u r a l tube d e f e c t such as s p i n a b i f i d a or c r a n i o r a c h i s c i s i s t o t a l i s was found i n any f e t u s . A b n o r m a l i t i e s found i n n o n e x e n c e p h a l i c f e t u s e s i n c l u d e 1 male w i t h an omphalocele, 2 f e t u s e s (1 f e m a l e r l male) w i t h m i c r o g n a t h i a , 1 f e t u s e (male) w i t h an e n c e p h a l o c e l e , and 22 f e t u s e s (3 female:19 male) w i t h c l e f t p a l a t e i n a t o t a l of 5364 f e t u s e s (2570 F:2794 M). The omphalocele and m i c r o g n a t h i a may r e p r e s e n t background f r e q u e n c i e s of th e s e d e f e c t s i n ICR/Be and SELH, or t h e y may be r e p r e s e n t a t i v e of some i n s t a b i l i t y w i t h i n t h e SELH s t o c k . C l e f t p a l a t e (CP) appears t o occur a t a 84 Tab l e X I . ABNORMALITIES FOUND IN FETUSES (DAY 18 OF GESTATION) OF GENETIC CROSSES Genotype A b n o r m a l i t i e s * SELH F l I.S F2 S.I F2 B C l BC2 3 CP (3 M); 2 m i c r o g n a t h i a (1 F : l M); 70 Ex (46 F: 24 M) 1 CP (1 F ) ; 1 Ex (F) 3 CP (1 F:2 M); 1 omphalocele (1 M); 12 Ex (10 F:2 M) 2 CP (1 F : l M); 4 Ex w i t h edema (4 F ) ; 30 Ex (23 F:7 M) 6 CP (6 M); 2 Ex w i t h omphalocele (1 M + 1 day 16 f e t u s sex ? ) ; 21 Ex (16 F:4 M + 1 sex ?) 7 CP (7 M); 1 Ex w i t h edema ( I F ) ; 2 Ex m i s s i n g 1 or both eyes ( 2 F ) ; 1 Ex w i t h g r o s s l y abnormal f a c e (1 F ) ; 1 e n c e p h a l o c e l e (1 M); 167 Ex (123 F:44 M) * n o t e : CP= c l e f t p a l a t e ; Ex= ex e n c e p h a l y ; F= female; M= male 85 f r e q u e n c y of about 0.7% (3/413) In SELH f e t u s e s . CP was r e c o v e r e d i n 1.0% of B C l f e t u s e s , and 0.3% of BC2 f e t u s e s . The appearance of CP d i d not c o r r e l a t e w i t h h i g h or low f r e q u e n c i e s of e x e n c e p h a l y i n l i t t e r s . There i s a s i g n i f i c a n t d i f f e r e n c e i n the sex of a f f e c t e d c l e f t p a l a t e f e t u s e s , and many more males t h a n females had c l e f t p a l a t e ( c o n t i n g e n c y X 2 1 d f = 8.97; p<0.01). There seems t o be a sex I n f l u e n c e on c l e f t p a l a t e i n t h e s e mice. 86 I I I . HISTOLOGY The embryology s t u d i e s of m o r p h o l o g i c a l development i n d i c a t e d t h a t the cause of e x e n c e p h a l y i n SELH mice i s a f a i l u r e of e l e v a t i o n and subsequent c o n t a c t and f u s i o n of the a n t e r i o r n e u r a l f o l d s . H i s t o l o g i c a l s e c t i o n s were made t o attempt t o det e r m i n e the b a s i s of the d e f e c t a t the t i s s u e and c e l l u l a r l e v e l . A. P a r a f f i n s e c t i o n s ICR/Bc and SELH embryos were embedded i n p a r a f f i n . T r a n s v e r s e s e r i a l s e c t i o n s were made t h r o u g h the a n t e r i o r n e u r a l tube of embryos of 8-20 s o m i t e s . The s e c t i o n s were s t a i n e d i n h a e m o t o x y l i n and e o s i n and comparisons were made between embryos of e q u a l somite c o u n t s . 32 ICR/Bc and 21 SELH embryos were s e c t i o n e d . A f t e r specimens w i t h u n s a t i s f a c t o r y s e c t i o n i n g a n g l e s , l o s s of s e c t i o n s , and 15 s o m i t e s or more were e x c l u d e d , 14 SELH and 16 ICR/Bc embryos of 8-13 s o m i t e s were examined. Of t h o s e , s e c t i o n s of 8 SELH and 10 ICR/Bc embryos were photographed and compared c l o s e l y . Embryos of 15 s o m i t e s or g r e a t e r were not s t u d i e d because the aim of t h i s s t u d y was t o i d e n t i f y d i f f e r e n c e s i n SELH embryos a t the t i s s u e and c e l l u l a r l e v e l p r i o r t o the c l o s u r e of the a n t e r i o r n e u r a l tube i n normal mice ( t h a t i s , j u s t b e f o r e SELH embryos a r e n o t i c e a b l y l a g g i n g b e h i n d ICR/Bc mice i n n e u r a l tube c l o s u r e ) . The " f l a r e d " f o l d s whole SELH embryos d e s c r i b e d were a l s o from e x a m i n a t i o n of morphology of observed i n h i s t o l o g i c a l s e c t i o n s . 87 Comparison of 8-13 somite ICR/Be and SELH embryos demonstrated a c o n s i s t e n t l a c k of e l e v a t i o n of the n e u r o e p i t h e l i u m i n t r a n s v e r s e s e c t i o n s from the mid- f o r e b r a i n t o the h i n d b r a i n r e g i o n of SELH embryos ( F i g u r e 19 a - d ) . Most but not a l l SELH (13/14 embryos) had wide s e p a r a t i o n of the a n t e r i o r n e u r a l f o l d s compared w i t h 16 ICR/Be embryos. However, some SELH f o l d s were " f l a r e d o u t " t o a g r e a t e r e x t e n t than o t h e r s . One u n u s u a l f e a t u r e was i d e n t i f i e d i n the mesenchyme and s u r f a c e e p i t h e l i u m u n d e r l y i n g the f l a r e d f o l d s . The mesenchyme of a l l SELH embryos s e c t i o n e d was c o l l a p s e d , and the s u r f a c e e p i t h e l i u m a t the s i d e s and underneath the f l a r e d m i d b r a i n neuroectoderm appeared r i p p l e d i n some SELH embryos ( F i g u r e 19d). Both t h e mesenchyme and s u r f a c e ectoderm were normal i n c e l l shape. The "squashed" mesenchyme may i n d i c a t e e i t h e r a f a i l u r e of t h i s t i s s u e t o f o r c e the n e u r a l f o l d s t o g e t h e r , or ex c e s s p r e s s u r e of the neuroectoderm t i s s u e on the mesenchyme. No l a c k of any c e l l type was observed i n SELH s e c t i o n s . Nor was any change i n f r e q u e n c y of c e l l d e a t h or c e l l d i v i s i o n observed i n t h e s e s e c t i o n s . C e l l shapes of mesenchyme, n e u r o e p i t h e l i a and s u r f a c e e p i t h e l i a c o u l d not be de t e r m i n e d i n wax s e c t i o n s due t o the c e l l o v e r l a p i n 7um s e c t i o n s . B. P l a s t i c s e c t i o n s To compare c e l l shape, number and d e n s i t y , t h i n p l a s t i c (2um) s e c t i o n i n g was used. Embryos of 6 and 8 s o m i t e s were s t u d i e d i n 88 an a t t e m p t t o d e t e r m i n e the e a r l y c e l l u l a r e v e n t s l e a d i n g t o the d e l a y of c l o s u r e of the a n t e r i o r n e u r a l tube and e x e n c e p h a l y found i n the SELH s t o c k . Four SELH and f o u r ICR/Bc embryos were embedded i n p l a s t i c and s e c t i o n e d t r a n s v e r s e l y a t 2um. S e c t i o n s were a n a l y s e d f o r : shape of t h e i r a n t e r i o r n e u r a l f o l d s , the t o t a l a r e a of n e u r o e p i t h e l i u m and mesenchyme, the c e l l d e n s i t y and c e l l shape w i t h i n r e g i o n s of the neuroectoderm (NE) and mesenchyme, the number of p y k n o t i c c e l l s i n each t i s s u e , and f i n a l l y f o r any u n u s u a l f e a t u r e s of the s e c t i o n s . In embryos of 6 s o m i t e s (2-ICR/Bc, and 2-SELH) and 8 s o m i t e s (2-ICR/Bc and 2-SELH), t h e r e were no o b s e r v a b l e d i f f e r e n c e s i n mesenchymal or n e u r o e p i t h e l i a l c e l l shape. N e u r o e p i t h e l i a l c e l l s were g e n e r a l l y b o t t l e - a n d wedge- shaped, a l t h o u g h t h o s e c e l l s of the lower h a l f of the NE rounded up i n the p o s t e r i o r mesencephalon and rhombencephalon s e c t i o n s ( F i g u r e 20a,b). Mesenchyme c e l l shape d i d not d i f f e r between ICR/Bc and SELH embryos and the c e l l s had a g l o b u l a r shape w i t h l o n g f i l o p o d l a e x t e n d i n g outwards ( F i g u r e 20a,b). 2 Area was measured u s i n g a g r a d i c u l e g r i d w i t h s q u a r e s of 1mm a t a m a g n i f i c a t i o n of lOOx. The t o t a l a r e a of n e u r o e p i t h e l i u m and mesenchyme t i s s u e d i d not v a r y g r e a t l y between SELH and ICR/Bc embryos, and no d e f i c i e n c y of e i t h e r t i s s u e was found i n SELH. For example, i n the m i d d l e mesencephalon r e g i o n of 6 89 so m i t e specimens, t o t a l NE a r e a averaged 1.75 u n i t s +0.3 and 1.62 u n i t s +0.1 f o r the two ICR/Bc embryos and two SELH embryos r e s p e c t i v e l y , w i t h mesenchyme a r e a s of 3.25 +0.2 and 3.25 +0.5 u n i t s f o r ICR/Bc and SELH r e s p e c t i v e l y . C e l l d e n s i t y t h r o u g h o u t the c r a n i a l f o l d s was d e t e r m i n e d t h r o u g h c e l l c o u n t s i n 1 mm sq u a r e s under a m a g n i f i c a t i o n of lOOx (where p o s s i b l e , c e l l # w i t h i n 4-6 a d j a c e n t s q u a r e s were counted and c e l l d e n s i t y a v e r a g e d ) . C e l l s w i t h g r e a t e r t h a n h a l f t h e i r a r e a w i t h i n a square were c o u n t e d . The c e l l d e n s i t y w i t h i n b o t h the n e u r o e p i t h e l i u m and mesenchyme t i s s u e s d i d not d i f f e r s t a t i s t i c a l l y between ICR/Bc and SELH 6 or 8 s o m i t e embryos. Mesenchyme c e l l d e n s i t i e s were q u i t e s i m i l a r t h r o u g h o u t a l l s e c t i o n s , a v e r a g i n g 2.4 ±1.2 and 2.2 +0.6 c e l l s / u n i t i n the two 6 s o m i t e ICR/Bc and SELH embryos r e s p e c t i v e l y , and 2.0 +0.8, and 2.2 +0.8 c e l l s / u n i t i n 8 s o m i t e ICR/Bc and SELH embryos ( T a b l e X I I ) . C e l l d e n s i t y v a r i e d w i t h i n s e c t i o n s of the neuroectoderm and between r e g i o n s of the c r a n i a l f o l d s . Comparison of c e l l number i n s e c t i o n s of the mesencephalon of the 6 and 8 s o m i t e embryos (4-ICR/Bc and 4-SELH embryos i n t o t a l ) , d i d not i n d i c a t e any c o n s i s t e n t d i f f e r e n c e s i n c e l l number between embryos ( p a i r e d t 3 d f = 3 . 0 4 ; p>.05). C e l l d e a t h was e s t i m a t e d by c o u n t i n g the number of p y k n o t i c c e l l i n c l u s i o n s w i t h i n each s e c t i o n ( T a ble X l l l a ) . The d i f f e r e n c e between ICR/Bc and SELH i n the number of p y k n o t i c c e l l s i n the neuroectoderm was s i g n i f i c a n t f o r embryos of b o t h 6 s o m i t e s 90 Ta b l e X I I . COMPARISON OF MEAN MESENCHYME CELL DENSITY BETWEEN ICR/BC AND SELH SPECIMENS OF 6 AND 8 SOMITES X mesenchyme c e l l d e n s i t y * ( c e l l s / m m 2 ) 6 somites+ 8 somites+ SELH | 2 .2 +0.6 2 .5 +0.8 ICR/BC | 2 .4 +1.2 2 .0 +0.8 * : o b s e r v a t i o n s were made u s i n g a m a g n i f i c a t i o n of lOOx and a g r i d of 1mm s q u a r e s . + : 2 ICR/Be and 2 SELH embryos were used t o g e n e r a t e mean v a l u e s . 91 T a b l e X I I I . ( a ) COMPARISON OF THE NUMBER OF PYKNOTIC CELLS PER SECTION IN SELH AND ICR/BC SPECIMENS X number of p y k n o t i c c e l l s / s e c t i o n Neuroectoderm Mesenchyme Ectoderm ( t o t a l #) ( t o t a l #) ( t o t a l #) 6 somi tes SELH 1 1.1(27) 1.0(25) 0.3 (8) ICR/Bc 1 0.1 (2) 0.1 (2) 0.1 (3) 8 somi tes SELH I 1 .9(45) 1.3(32) 1.8(44) ICR/Bc I 1 .0(24) 0.1 (2) 0 .8(20) DISTRIBUTION OF PYKNOTIC CELLS ( b ) . NE (6 s o m i t e ) ( c ) . NE (8 s o m i t e ) # p y k n o t i c s per s e c t i o n # p y k n o t i c s per s e c t i o n I 0-1 1 2 - 4 1 T I 0-1 I 2-4 I 5-718-101 T ICR/Bc I 24 | 0 I 24 ICR/Bc I 19 I 3 I 1 1 1 124 I I I I I I I I SELH I 16 I 8 I 24 SELH I 11 I 11 I 1 1 1 124 X 2 i d f = 7 . 3 5 * X 2 1 ( J f =4.36* ( d ) . Mesenchyme (6 s o m i t e ) ( e ) . Mesenchyme (8 s o m i t e ) # p y k n o t i c s per s e c t i o n # p y k n o t i c s per s e c t i o n I 0-1 I 2-4 I 5-6 I T I 0-1 I 2-4 I T ICR/Bc I 23 I 1 I 0 1 2 4 ICR/Bc 1 2 4 1 0 I 24 l l l l I I I SELH I 18 I 4 I 2 | 24 SELH I 15 I 9 I 24 X 2 1 d f =2.68 X 2 1 d f = 8 . 7 5 * ( f ) . Ectoderm (6 s o m i t e ) ( g ) . Ectoderm (8 s o m i t e ) # p y k n o t i c s per s e c t i o n # p y k n o t i c s per s e c t i o n I 0 I 1-3 | T I 0-1 I 2-4 I 5-718-101 T ICR/Bc| 22 I 2 I 24 ICR/Bc I 19 I 4 I 1 1 0 124 I I I I I I I I SELH I 19 | 5 I 24 SELH I 15 I 5 I 3 1 1 124 X 2 1 d f = 0 . 6 7 X 2 1 d f = 0 . 9 1 X 2 g r o u p i n g s : 6 s o m i t e embryos: # p y k n o t i c s / s e c t i o n : NE ( 0 - 1 , 2 - 4 ) / Mesenchyme ( 0 - 1 , 2 - 6 ) / Ectoderm (0 , 1 - 3 ) . 8 s o m i t e embryos: # p y k n o t i c s / s e c t i o n : NE ( 0 - 1 , 2 - 1 0 ) / Mesenchyme ( 0 - 1 , 2 - 4 ) / Ectoderm ( 0 - 1 , 2 - 1 0 ) . 92 ( i n d e p . X 2 1 ( j f=7.35; p<0.01) and 8 s o m i t e s ( i n d e p . X 2 1 d f=4.36; p<0.05) ( t a b l e X I I I b , c ) . The d i f f e r e n c e between the number of p y k n o t i c s i n the mesenchyme was s i g n i f i c a n t i n the 8 s o m i t e embryo comparison ( i n d e p . X 2 1 d f = 8.75; p<0.01), but was not s i g n i f i c a n t between the 6 somite ICR/Bc and SELH embryos ( i n d e p . X 2 1 d f = 2 . 6 8 ; p>0.05) ( t a b l e X I I I d , e ) . There was no s i g n i f i c a n t d i f f e r e n c e between ICR/Bc and SELH i n the number of p y k n o t i c s w i t h i n the ectoderm of e i t h e r 6 or 8 so m i t e embryos (6 s o m i t e s : i n d e p . X 2 1 d f=0.67; p>0.05; 8 s o m i t e s : i n d e p . X 2 1 d f = 0 . 9 l ; p>0.05) ( t a b l e X I I I f , g ) . P y k n o t i c c e l l s w i t h i n SELH were not found t o c l u s t e r w i t h i n r e g i o n s of the p r e s u m p t i v e b r a i n t i s s u e . The i n c r e a s e i n c e l l d e a t h i n bo t h the mesenchyme and neuro-e p i t h e l i u m of SELH embryos i s r e f l e c t e d i n the number of s e c t i o n s w i t h 2-4 p y k n o t i c s compared t o ICR/Bc ( t a b l e X I I I b - e ) . T h i s low f r e q u e n c y of c e l l d e a t h a l o n e would not appear t o be s u f f i c i e n t t o e x p l a i n the observed d e l a y i n c l o s u r e of SELH embryos. S m a l l u n s t a i n e d round i n c l u s i o n s were noted t h r o u g h o u t the c r a n i a l f o l d s i n the neuroectoderm of the SELH embryos ( f i g u r e 21a,b). B o d i e s l i k e t h e s e o f t e n c o n t a i n l i p i d s , but u n t i l l i p i d s a r e t e s t e d f o r d i r e c t l y , t h e i r c o n t e n t s have not been p r o v e n . These b o d i e s w i l l be r e f e r r e d t o as l i p i d d r o p l e t s f o r c o n v e n i e n c e . In both 6 and 8 somite embryos, the number of th e s e i n c l u s i o n s i s i n c r e a s e d by a p p r o x i m a t e l y 9 i n each s e c t i o n compared w i t h the ICR/Bc s e c t i o n s ( t a b l e X i v a ) . T h i s d i f f e r e n c e i s s i g n i f i c a n t (6 s o m i t e s : i n d e p . X 2 ? .f= 28.80; p<0.01; 8 93 Ta b l e XIV.(a) COMPARISON OF THE NUMBER OF L I P I D DROPLETS PER SECTION IN THE NEUROECTODERM OF SELH AND ICR/BC SPECIMENS OF 6 AND 8 SOMITES X number of l i p i d d r o p l e t s / s e c t i o n 6 s o m i t e s 8 s o m i t e s ( t o t a l #) ( t o t a l #) SELH | 9.0 (215) 12.5 (300) ICR/BC | 0.5 (11) 3.0 (73) ( b ) . 6 so m i t e embryos+ # l i p i d d r o p l e t s per s e c t i o n 1 0-1 2-3 1 4-6 7-10 11-15 16-22 ICR/Bc| 20 4 1 0 0 0 0 24 SELH | 3 3 1 2 5 8 3 24 +1X 2 g r o u p i n g s : 6 so m i t e embryos: 0-3, 4-10, and 11-22 X 2 2 d f =28.80* ": t o t a l # s e c t i o n s ( c ) . 8 somite embryos+ # l i p i d d r o p l e t s per s e c t i o n 1 0-1 2-3 1 4-6 7-10 11-15 16-22 23-28 1 T ICR/BC| 14 4 1 4 0 0 1 1 24 SELH | 3 1 1 4 4 1 7 4 24 +: X^ g r o u p i n g s : 8 s o m i t e embryos: 0-3, 4-10, and 11-28 x 2df=17.60* ~: t o t a l # s e c t i o n s s o m i t e s : i n d e p . X 2 2 d f = 1 7 . 6 0 ; p<0 .01 ) ( t a b l e XIV b , c ) . The i n c r e a s e i n l i p i d d r o p l e t s i n SELH i s found i n the number of s e c t i o n s of n e u r a l ectoderm w i t h 7 t o 22 or 28 d r o p l e t s ( t a b l e XIV b / c ) . The s i g n i f i c a n c e of an i n c r e a s e i n t h e number of l i p i d d r o p l e t s i s not c l e a r . One 6 s o m i t e SELH embryo had many l a r g e gaps between c e l l s of the n e u r o e p i t h e l i u m and c e l l s of t h e s u r f a c e e p i t h e l i u m ( f i g u r e 22a,b). T h i s was not seen i n any ICR/Be embryos nor i n the o t h e r SELH embryos. T h i s l a c k of c e l l c o n t a c t was found o n l y i n t h i s one SELH embryo, and may r e p r e s e n t an a n o m a l l y , or a s t a g e l e a d i n g t o e x e n c e p h a l y . The n e u r a l f o l d shape of 6 s o m i t e SELH embryos was d i f f e r e n t from t h a t of ICR/Be embryos, SELH n e u r a l f o l d s were f l a r e d w i d e r t h a n ICR/Be n e u r a l f o l d s ( f i g u r e 22a,b). A d i f f e r e n c e was a l s o o bserved between ICR/Be and SELH embryos i n the shape of the n e u r a l f o l d s i n the most f r o n t a l r e g i o n of the embryos ( f i g u r e 23a,b). SELH a n t e r i o r n e u r a l f o l d s were w i d e r a p a r t t h a n the ICR f o l d s . T h i s o b s e r v a t i o n s u g g e s t s t h a t a l a g i n e l e v a t i o n of the n e u r a l f o l d s may be ap p a r e n t as e a r l y as 6 s o m i t e s i n SELH embryos. I n summary, compared w i t h ICR/Be embryos, SELH embryos have more w i d e l y f l a r e d n e u r a l f o l d s , w i t h l a t e r a l u n d e r l y i n g " c o l l a p s e d " a r e a s i n the mesenchyme, but no d e t e c t a b l e changes i n c e l l d e n s i t y ; p o s s i b l y more c e l l d e a t h i n the n e u r o e p i t h e l i u m and mesenchyme, and more f a t d r o p l e t s i n the n e u r o e p i t h e l i u m . The f l a r i n g of the n e u r a l f o l d s i s v i s i b l e from the a n t e r i o r 95 mesencephalon back t o the rhombencephalon. In some SELH embryos, the p r o s e n c e p h a l o n f o l d s were a l s o more w i d e l y s e p a r a t e d t h a n i n ICR/Be. No c o n s i s t e n t d i f f e r e n c e s i n c e l l d e n s i t y , or c e l l - s h a p e were observed between SELH and ICR/Be specimens. I t i s not c l e a r whether the d i f f e r e n c e s i n c e l l d e a t h , and l i p i d d r o p l e t s , a r e c a u s a l l y r e l a t e d t o the f a i l u r e of c l o s u r e 2 i n a n t e r i o r n e u r a l tube c l o s u r e i n SELH, or i f t h e s e d i f f e r e n c e s a re r e p r e s e n t a t i v e of normal v a r i a t i o n s between the s t r a i n s . A l s o , i t i s not known i f the c o l l a p s e d mesenchyme, and f l a r e d n e u r a l f o l d s a r e c a u s a l l y r e l a t e d t o the c l o s u r e f a i l u r e , or a consequence of a more c e n t r a l d e f e c t . 96 FIGURE DESCRIPTION FIGURE KEY: M= mesenchyme, N= neuroectoderm, py= p y k n o t i c c e l l , ld= l i p i d d r o p l e t , g = gaps. F i g u r e 19a: P a r a f f i n s e c t i o n of a 13 s o m i t e ICR/Bc embryo i n t h e mesencephalon r e g i o n . The c r a n i a l n e u r a l f o l d s a r e c u r v i n g i n t o t h e m i d l i n e ( x l O O ) . F i g u r e 19b: P a r a f f i n s e c t i o n of a 13 s o m i t e SELH embryo i n the mesencephalon r e g i o n . Note the w i d e l y f l a r e d c r a n i a l n e u r a l f o l d s and the "squashed" u n d e r l y i n g mesenchyme ( x l O O ) . F i g u r e 19c: P a r a f f i n s e c t i o n of a 10 s o m i t e ICR/Bc embryo i n the p o s t e r i o r mesencephalon r e g i o n . The n e u r a l f o l d s a r e a wide "V" shape (xlOO) . F i g u r e 19d: P a r a f f i n s e c t i o n of a 10 s o m i t e SELH embryo i n the p o s t e r i o r mesencephalon r e g i o n . The n e u r a l f o l d s a r e l y i n g f l a t . Note th e r i p p l e d ectoderm and "squashed" mesenchyme (xlOO). F i g u r e 20a: P l a s t i c s e c t i o n of an 8 s o m i t e ICR/Bc embryo i n the p o s t e r i o r mesencephalon r e g i o n . The n e u r a l f o l d s a r e "V" shaped. Neuroectoderm c e l l s a r e b o t t l e shaped, mesenchyme c e l l s a r e g l o b u l a r w i t h e x t e n d i n g f i l o p o d l a ( x l O O ) . F i g u r e 20b: P l a s t i c s e c t i o n of an 8 s o mite SELH embryo i n the p o s t e r i o r mesencephalon r e g i o n . The n e u r a l f o l d s a r e f l a r e d wide. Neuroectoderm c e l l s however, s t i l l appear b o t t l e shaped, and mesenchyme c e l l s a r e g l o b u l a r w i t h e x t e n d i n g f i l o p o d l a (xlOO} F i g u r e 21a: P l a s t i c s e c t i o n of an 8 s o m i t e SELH embryo i n the a n t e r i o r mesencephalon r e g i o n ( x l O O ) . F i g u r e 21b: P l a s t i c s e c t i o n of the 8 s o m i t e SELH embryo i n f i g . 21a, l i p i d d r o p l e t s can be seen near the mesenchymal s i d e of the neuroectoderm. A p y k n o t i c c e l l can a l s o be seen i n the n euroectoderm ( 4 0 0 x ) . F i g u r e 22a: P l a s t i c s e c t i o n of a 6 s o mite ICR/Bc embryo i n t h e rhombencephalon r e g i o n . The n e u r a l f o l d s a r e r o u n d l y f l a r e d ( x l O O ) . F i g u r e 22b: P l a s t i c s e c t i o n of a 6 s o m i t e SELH embryo i n the rhombencephalon r e g i o n . N e u r a l f o l d s a r e f l a t . Gaps w i t h i n t h e n euroectoderm a r e v i s i b l e (the r i p p l e d ectoderm i s an a r t i f a c t ) (xlOO) . F i g u r e 23a: P l a s t i c s e c t i o n of a 6 s o m i t e ICR/Bc embryo i n t h e p r o s e n c e p h a l o n r e g i o n . The n e u r a l f o l d s a r e p a r a l l e l t o each o t h e r ( x l O O ) . F i g u r e 23b: P l a s t i c s e c t i o n of a 6 s o m i t e SELH embryo i n the p r o s e n c e p h a l o n r e g i o n . The n e u r a l f o l d s a r e w i d e r a p a r t t h a n t h o s e of f i g . 23a, and are more cup shaped t h a n p a r a l l e l ( x l O O ) . 97 100 IV. TEST OF THE HYPOTHESIS OF SEGREGATION OF AN EXENCEPHALY MUTATION IN THE SELH STOCK At the s t a r t of t h i s p r o j e c t , i t was not known whether the gene(s) t h a t cause e x e n c e p h a l y i n the SELH s t o c k were s e g r e g a t i n g , or whether the l i a b i l i t y t o e x e n c e p h a l y was g e n e t i c a l l y f i x e d i n the s t o c k . For t h i s r e a s o n , the embryos used f o r s t u d i e s of m o r p h o l o g i c a l development and h i s t o l o g y were from p a r e n t s t h a t had been proven t o t r a n s m i t the e x e n c e p h a l y t r a i t , as p r e v i o u s l y n o t e d . The q u e s t i o n of whether the genes c a u s i n g e x e n c e p h a l y were s e g r e g a t i n g i n the SELH s t o c k was approached d i r e c t l y i n an i n i t i a l s t u d y ((A) b e l o w ) , and was a l s o d e a l t w i t h i n d i r e c t l y i n 2 o t h e r s e t s of d a t a c o l l e c t e d d u r i n g the c o u r s e of the e x p e r i m e n t s , ((B) and (C) b e l o w ) . The s t u d i e s were: A. Day 14 mouse embryo s t u d y B. I d e n t i f i c a t i o n of e x e n c e p h a l y p r o d u c i n g mice C. C o n t r o l s from the g e n e t i c s t u d y of B C l males. A. Day 14 mouse embryo s t u d y , summer 1985 The aim of t h i s s t u d y was t o b e g i n t o d e t e r m i n e i f e x e n c e p h a l y was caused by a s i n g l e l e t h a l m u t a t i o n w i t h f u l l p e n e t r a n c e t h a t had a r i s e n i n the SELH s t o c k and was s e g r e g a t i n g . P r e v i o u s l y u n t e s t e d SELH males were mated t o n u l l i p a r o u s SELH females which were k i l l e d on day 14 of g e s t a t i o n and t h e i r l i t t e r s c o l l e c t e d and s c o r e d ( t a b l e XV). E x e n c e p h a l y was found i n 16.5% of f e t u s e s (65/393), w i t h a l l 8 males p r o d u c i n g e x e n c e p h a l y i n t h e i r o f f s p r i n g . Table XV. IDENTIFICATION OF EXENCEPHALY IN SELH FETUSES (observed on day 14 of g e s t a t i o n -1985) SELH male # t o t a l # # of # of # of pedigreed l i t t e r s implants moles f e t u s e s e x e n c e p h a l i c s 1059 5 56 2 54 3 -1060 5 47 3 44 8 -1061 4 50 2 48 19 -1062 5 54 3 51 7 •1063 5 63 1 62 4 .1064 3 34 2 32 4 1065 5 55 2 53 15 1066 5 51 2 49 5 TOTALS 36 410 17 393 65 * note: moles = r e s o r p t i o n s i t e s + :[= s i b l i n g s 102 B. I d e n t i f i c a t i o n of e x e n c e p h a l y - p r o d u c i n g mice As the g e n e t i c b a s i s of e x e n c e p h a l y i n SELH was not known, proven e x e n c e p h a l y - p r o d u c i n g mice were needed f o r use i n g e n e t i c and e m b r y o l o g i c a l s t u d i e s . P r e v i o u s l y u n t e s t e d male SELH mice were mated w i t h 1-5 SELH females each. Females ranged from b e i n g n u l l i p a r o u s t o h a v i n g had 3 p r e v i o u s l i t t e r s . Newborns (0-16 hours o l d ) or day 18 f e t u s e s were s c o r e d f o r e x e n c e p h a l y . Of 40 SELH males t e s t e d , a l l 40 produced e x e n c e p h a l y (Table X V I ) . N e i t h e r s p i n a b i f i d a , nor t a i l a n o m a l i e s were found. The o v e r a l l f r e q u e n c y of e x e n c e p h a l y (5.7%) i s lower i n t h i s s t u d y t h a n i n the s t u d i e s u s i n g o b s e r v a t i o n s b e f o r e b i r t h . T h i s i s not s u r p r i s i n g as female mice a r e known t o c a n n i b a l i z e malformed o f f s p r i n g a t b i r t h , and such c a n n i b a l i s m was o f t e n seen. F i f t e e n SELH l i t t e r s were c o l l e c t e d on day 18 of g e s t a t i o n from e l e v e n of t h e s e p r e v i o u s l y proven e x e n c e p h a l y - p r o d u c i n g SELH males mated t o n u l l i p a r o u s SELH f e m a l e s . The f r e q u e n c y of e x e n c e p h a l y i n the f e t u s e s was 14.1% (23/163) (Table X V I I ) . C. C o n t r o l s from the g e n e t i c s t u d y of B C l males, s p r i n g 1987 To p r o v i d e c o n t r o l s f o r B C l t e s t c r o s s males i n the g e n e t i c s t u d i e s , 5 p r e v i o u s l y u n t e s t e d n o n - l i t t e r m a t e SELH males were mated t o 8-9 n u l l i p a r o u s SELH females each, and t h e i r l i t t e r s o b s erved and r e c o r d e d on day 18 of g e s t a t i o n . The o v e r a l l f r e q u e n c y of e x e n c e p h a l y was 16.9% (70/413), w i t h a l l 5 males p r o d u c i n g e x e n c e p h a l y i n t h e i r l i t t e r s ( T a b le X V I I I ) . Table XVI. IDENTIFICATION OF EXENCEPHALY PRODUCING MICE IN SELH MALES # of SELH male # s c o r a b l e # of p e d i g r e e * l i t t e r s newborns e x e n c e p h a l i c s 1350 12 93 3 1348 3 28 1 1349 6 39 2 1351 4 28 2 1250 3 31 2 1251 2 21 1 1252 2 23 3 1253 3 23 3 1272 2 16 1 1270 5 43 5 1263 4 45 2 1271 5 48 4 1273 2 20 1 1260 3 28 1 1262 3 29 1 1261 5 39 2 1264 3 26 1 1377 6 54 1 1439 14 138 4 1437 11 96 11 1436 6 75 4 1438 8 61 2 1471 7 67 9 1467 7 65 8 1470 3 36 5 1521 12 136 2 1519 5 44 6 1520 6 55 5 1529 8 80 7 1528 5 52 1 1562 6 52 7 1563 6 69 3 1634 9 92 3 1635 13 128 2 1636 10 101 3 1702 10 99 6 1703 6 57 3 1704 9 72 4 1892 9 87 1 1962 6 68 2 40 MALES 249 2364 134 104 T a b l e X V I I . FREQUENCY OF EXENCEPHALY IN SELH FETUSES** (observed on day 18 of g e s t a t i o n -1986) # of SELH male pedigreetf # l i t t e r s t o t a l # i m p l a n t s # of moles* tt of f e t u s e s e x e n c e p h a l i c s (F:M) 1436 1 13 0 13 1:0 1437 2 26 0 26 4:1 1439 2 15 0 15 2:0 1471 1 9 1 8 1:0 1520 2 25 1 24 4:0 1521 1 14 2 12 0 1529 2 20 0 20 5:2 1562 1 7 0 7 2:0 1563. 1 12 0 12 0 1635 1 12 1 11 1:0 1636 1 15 0 15 0 TOTALS 15 168 5 163 (82:81) 20:3 * n o t e : moles = r e s o r p t i o n s i t e s **:These day 18 l i t t e r s a r e i n c l u d e d i n the d a t a shown i n t a b l e XVI. 105 Table X V I I I . SELH MALE CONTROLS (observed FOR THE GENETIC STUDY day 18 of g e s t a t i o n -OF B C l 1987) MALES SELH male p e d i g r e e # # of 1 i t t e r s # of i m p l a n t s # of f e t u s e s e x e n c e p h a l i c s (female:male) 2016 8 95 87 11 (5:6) 2031 8 80 77 11 (10:1) 2098 9 78 75 3 (2:1) 2173 8 97 91 36 (23:13) 2258 8 92 83 9 (6:3) TOTAL 5 MALES 41 442 413 (205F:208M) 70 (46:24) 106 The d a t a i n d i c a t e t h a t i t i s not l i k e l y t h a t a s i m p l e r e c e s s i v e , f u l l y p e n e t r a n t l e t h a l gene s e g r e g a t i n g i n the SELH s t o c k causes e x e n c e p h a l y . I f t h i s model were t r u e , one would e x p e c t 1/3 of the males t e s t e d t o be non-producers of e x e n c e p h a l y ( t h a t i s 1/3 of a l l s u r v i v i n g m a l e s ) . A l l of the 53 males t e s t e d produced e x e n c e p h a l y . T h i s l a c k of i d e n t i f i e d n on-producers of e x e n c e p h a l y i s s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the e x p e c t e d 1/3 p r o p o r t i o n (18/53) ( X 2 1 d f = 25.02; p<0.01) p r e d i c t e d by the s i m p l e s i n g l e gene r e c e s s i v e l e t h a l model. We can t h e r e f o r e r e j e c t t h e s i n g l e r e c e s s i v e s e g r e g a t i n g l e t h a l model as the l i k e l y g e n e t i c b a s i s f o r e x e n c e p h a l y i n the SELH s t o c k . A l t o g e t h e r , 53 SELH males were t e s t e d f o r p r o d u c t i o n of e x e n c e p h a l y i n t h e i r progeny and a l l 53 males produced e x e n c e p h a l y . The f r e q u e n c y ( f ) of "non-producer" males among SELH males can be e s t i m a t e d by P = ( l - f ) N , where N i s the number of males t e s t e d , and P i s the p r o b a b i l i t y of e r r o r . I f we s e t P a t 5% and s o l v e f o r f , the e s t i m a t e of the maximum f r e q u e n c y of nonproducers of e x e n c e p h a l y w i t h i n the SELH s t o c k i s 0.055. That i s , a t most, 5.5% of SELH males would not t r a n s m i t t h e gen e ( s ) c a u s i n g e x e n c e p h a l y . The r e s u l t s s u ggest t h a t a l l , or n e a r l y a l l SELH a n i m a l s a r e c a p a b l e of t r a n s m i t t i n g the genes t h a t cause e x e n c e p h a l y , and t h a t t h e g e n e t i c l i a b i l i t y t o e x e n c e p h a l y i s p r o b a b l y f i x e d i n the s t o c k . In o t h e r words, t h i s s u g g e s t s t h a t n e a r l y a l l SELH a n i m a l s may be homozygous f o r the e x e n c e p h a l y - c a u s i n g gene(s) which a r e e x p r e s s e d i n the 17% w i t h 107 e x e n c e p h a l y which d i e a t b i r t h . V. TEST OF HETEROGENEITY IN SELH MALES Having found t h a t most or a l l SELH males produce e x e n c e p h a l y i n t h e i r progeny, i t was of i n t e r e s t t o l e a r n whether t h e r e were d i f f e r e n c e s i n the f r e q u e n c y of the d e f e c t i n progeny from d i f f e r e n t s i r e s . There were two s e t s of d a t a a v a i l a b l e f o r t h i s c o m p a r i s o n : day 14 l i t t e r s c o l l e c t e d from 8 males i n 1985 ( t a b l e XV), and day 18 l i t t e r s c o l l e c t e d from 5 s i r e s i n 1987 ( t a b l e X V I I I ) . The f r e q u e n c y of e x e n c e p h a l y d i d not d i f f e r s i g n i f i c a n t l y between t h e s e two samples, ( X 2 1 d f = 0 . 0 0 4 ; p>0.90). The two samples a l s o d i d not d i f f e r ( t 1 1 d f = 0.02; p>0.2) when mean l i t t e r f r e q u e n c i e s t r a n s f o r m e d t o Freeman-Tukey a r c s l n e v a l u e s , were compared. The two samples were t h e r e f o r e p o o l e d ( t a b l e X I X ) . A one way a n a l y s i s of v a r i a n c e based on l i t t e r Freeman-Tukey a r c s i n e f r e q u e n c i e s of e x e n c e p h a l y i n d i c a t e d t h a t t h e r e i s s i g n i f i c a n t h e t e r o g e n e i t y among the SELH males i n the f r e q u e n c y of e x e n c e p h a l y produced ( F 1 2 6 4 = 4 . 2 2 ; p<0.05) ( t a b l e XX). T h i s h e t e r o g e n e i t y i s c l e a r l y e v i d e n t i n t h e f r e q u e n c i e s of e x e n c e p h a l y found. For example, 3 males c o n s i s t e n t l y produced h i g h f r e q u e n c i e s of e x e n c e p h a l y (28.3-39.6%) i n t h e i r progeny, w h i l e 5 males produced c o n s i s t e n t l y low f r e q u e n c i e s ( 4 . 5 - 1 0 . 2 % ) . The r e s t of the SELH male3 produced f r e q u e n c i e s of e x e n c e p h a l y 108 Table XIX ANALYSIS OF HETEROGENEITY OF EXENCEPHALY PRODUCTION AMONG SELH MALES day of SELH male g e s t a t i o n p e d i g r e e # observed I F e x e n s / t o t a l i n l i t t e r % x l i t t e r N exen a r c s i n e * 1059 1060 1061 1062 1063 1064 1065 1066 2016 2031 2098 2173 2258 14 1/16, 1/9, 1/4 0/12, 0/13 14 3/9, 0/11, 0/3 3/13, 2/8 14 4/12, 5/12, 4/11 6/13 14 1/14, 4/12, 2/14 0/7, 0/4 14 0/14, 0/10, 0/12 1/13, 3/13 14 0/13, 3/6, 1/13 14 2/6, 1/9, 1/13 4/14, 7/11 14 0/10, 1/14, 2/15 2/10 18 1/10, 1/13, 2/11 3/10, 0/11, 2/12 1/8, 1/12 18 0/6, 1/4, 1/9 0/11, 2/10, 3/13 2/10, 2/14 18 1/12, 1/9, 0/13 0/3, 0/2, 1/13 0/1, 0/11, 0/11 18 9/12, 5/7, 2/9 5/14, 5/11, 2/14 5/12, 3/12 18 0/8, 0/5, 2/11 0/5, 2/11,3/13 0/17,2/13 3/54 8/44 4/62 4/32 15/53 5/49 11/87 3/75 5.6 18.2 19/48 39.6 7/51 13.7 6.5 12.5 28.3 10.2 12.6 11/77 14.3 4.0 36/91 39.6 9/83 10.0 17.65 24.25 39.31 20.36 14.62 23.87 32.68 19. 62 22. 40 23.19 15.62 40.05 18. 56 * note t r a n s f o r m a t i o n = Freeman-Tukey a r c s i n e T a b l e XX. ANOVA TEST FOR HETEROGENEITY AMONG SELH MALES IN FREQUENCY OF EXENCEPHALY PRODUCED Source SS df MS * F Between males 5050.45 12 420.82 4.22* W i t h i n males 6378.64 64 99.67 TOTAL 11429.09 76 520.49 * F e r i t y . 0 5 ( 1 2 , 6 2 ) = 1 > 9 2 110 between 12.5 and 18.2%. The h e t e r o g e n e i t y s u g g e s t s t h a t w i t h i n the SELH s t o c k , g e n e t i c m o d i f i e r s t h a t a f f e c t the a b i l i t y of an embryo t o c l o s e i t s a n t e r i o r n e u r a l tube a r e s e g r e g a t i n g . However, i t s h o u l d be noted t h a t a l l 13 males produced e x e n c e p h a l y i n t h e i r progeny. The v a r i a t i o n among males was i n the p r o p o r t i o n of t h e i r o f f s p r i n g a f f e c t e d . V I . ESTIMATES OF THE FREQUENCY OF EXENCEPHALY IN THE SELH STOCK A. Frequency e s t i m a t e s There was no s i g n i f i c a n t d i f f e r e n c e among the f r e q u e n c i e s of ex e n c e p h a l y observed i n day 14 embryos from p r e v i o u s l y u n t e s t e d males i n 1985, day 18 f e t u s e s from p r e v i o u s l y proven males i n 1986, and day 18 f e t u s e s from p r e v i o u s l y u n t e s t e d males i n 1987 ( t a b l e s XV, X V I I , and X V I I I ) ; ( c o n t i n g e n c y X 2 2 d f=0.72; p>0.5) ( t a b l e X X I ) . No s p i n a b i f i d a or t a l l a n o m a l i e s were found i n the 1985, 1986, or 1987 SELH f e t u s e s . The f r e q u e n c y of e x e n c e p h a l y does not appear t o be c h a n g i n g over t i m e , nor does i t appear t o d i f f e r between progeny of proven c a r r i e r s and p r e v i o u s l y u n t e s t e d males. Table XXI. COMPARISON OF NUMBER OF EXENCEPHALIC FETUSES FOUND BETWEEN 3 SAMPLES (1985, 1986, AND 1987) Sample g e s t a t i o n a l age # Normal E x e n c e p h a l i c (%) 1985 - u n t e s t e d day 14 328 65 (16.5) 1987 - u n t e s t e d day 18 343 70 (16.9) 1986 - t e s t e d day 18 140 23 (14.1) C o n t i n g e n c y X 2 d f=0.72; p>0.5 Ta b l e X X I I . SEX RATIO IN DAY 18 SELH FETUSES- female s u s c e p t i b i l i t y * 1 ' # | it 1 I Normal (%)I E x e n c e p h a l i c (%) I N FEMALE I 221 (77) I 66 (23) 1 287 MALE 1 262 (91) 1 27 (9) I 289 *: same d a t a as t a b l e XVII and X V I I I 112 B. SEX RATIO The sex r a t i o w i t h i n SELH e x e n c e p h a l i c s i s not a 1:1 r a t i o . There i s a marked e x c e s s of a f f e c t e d females among e x e n c e p h a l i c f e t u s e s . There appeared t o be no r e l a t i o n s h i p between sex r a t i o among e x e n c e p h a l i c s and o v e r a l l f r e q u e n c y of e x e n c e p h a l y . For example, the sex r a t i o of e x e n c e p h a l i c s was 2F:1M, f o r progeny of male 2098 ( p r o d u c i n g 4.5% e x e n c e p h a l y ) , and 1.8F:1M f o r progeny of male 2173 ( p r o d u c i n g 39.6% e x e n c e p h a l y ) r e s p e c t i v e l y ( t a b l e X V I I I ) . The a v a i l a b l e d a t a on sex r a t i o In SELH i n c l u d e d the 1986 and 1987 SELH day 18 f e t u s s t u d i e s ( t a b l e s XVII and X V I I I ) . The p r o p o r t i o n of female and male e x e n c e p h a l i c s d i d not d i f f e r s i g n i f i c a n t l y between the two samples ( c o n t i n g e n c y X 2 3 c j f = 4.29; p>0.20), t h e r e f o r e the samples were p o o l e d . The combined number of f e t u s e s observed was 576 and the t o t a l sex r a t i o was 287 F :289 M (or 50.2% of a l l SELH f e t u s e s were m a l e ) . However, t h e t o t a l number of e x e n c e p h a l i c s o b s e r v e d was 93 (66F: 27 M), a sex r a t i o of 2.44 females a f f e c t e d f o r each male a f f e c t e d (or 29% of e x e n c e p h a l i c s were male) ( t a b l e X X I I ) . The e x c e s s of females among e x e n c e p h a l i c s i s s t a t i s t i c a l l y s i g n i f i c a n t ( c o n t i n g e n c y X 2 1 d f = 1 8 . 9 1 ; p<0.01). The o v e r a l l sex r a t i o does not s u g g e s t any d e f i c i e n c y of SELH male f e t u s e s ( t a b l e X X I I ) , and the low r a t e of p o s t i m p l a n t a t l o n l o s s ( t a b l e s XVII and X V I I I ) a l s o does not s u p p o r t the h y p o t h e s i s t h a t male e x e n c e p h a l i c s a r e l o s t as embryos. 5 and 29 r e s o r p t i o n s ("moles") were found i n the 1986 and 1987 s t u d i e s r e s p e c t i v e l y . The r e s o r p t i o n f r e q u e n c y d i d not d i f f e r s i g n i f i c a n t l y between the samples ( X 2 1 d f = 2 . 4 8 ; p>0 .10 ) , t h e r e f o r e the r e s o r p t i o n s were p o o l e d . In a t o t a l of 610 i m p l a n t a t i o n s , 34 moles were found f o r a f r e q u e n c y of 5.6%. I f a l l r e s o r p t i o n s were male e x e n c e p h a l i c s , the e x e n c e p h a l i c sex r a t i o would be 1 . 0 8 F:lM, and the t o t a l sex r a t i o would be . 89F : 1M compared t o the .99F:1M o b s e r v e d . T h i s l e v e l of p o s t -I m p l a n t a t i o n l o s s does not appear t o acc o u n t f o r the d i s p a r i t y i n the sex r a t i o of e x e n c e p h a l i c s . The a l t e r e d sex r a t i o of a f f e c t e d e x e n c e p h a l i c s appears t o i n d i c a t e a r e a l d i f f e r e n c e i n l i a b i l i t y t o e x e n c e p h a l y between males and f e m a l e s . A s e l e c t i v e p r e l m p l a n t a t l o n l o s s of male " f u t u r e e x e n c e p h a l i c s " cannot be r u l e d out by t h i s s t u d y , a l t h o u g h the t o t a l sex r a t i o of the 1986 and 1987 s t u d i e s (287F: 289 M) ( t a b l e X X I I ) does not I n d i c a t e t h a t t h e r e i s any d e f i c i e n c y of males. 114 V I I . GENETIC OUTCROSSES OF SELH AND ICR/BC O u t c r o s s e s of SELH mice t o the ICR/Be s t r a i n were used t o t e s t the g e n e t i c hypotheses of dominance, r e c e s s i v e n e s s , and sex l i n k e d i n h e r i t a n c e of the e x e n c e p h a l y t r a i t , and t o i d e n t i f y t h e number of genes d i f f e r i n g between SELH and ICR/Be t h a t cause the d e f e c t . A. F l G e n e r a t i o n Proven e x e n c e p h a l y - p r o d u c i n g SELH males and females were mated t o ICR/Be a n i m a l s and t h e i r l i t t e r s c o l l e c t e d on day 18 of g e s t a t i o n . 17 l i t t e r s were c o l l e c t e d from SELH females mated t o ICR/Be males (SELH.ICR F l ) . There were no e x e n c e p h a l i c s i n 121 F l f e t u s e s c o l l e c t e d ( t a b l e X X I I I ) . S i x SELH males were mated t o between 2-5 ICR/Be females each f o r a t o t a l of 21 l i t t e r s (ICR.SELH F l ) . I n 235 f e t u s e s c o l l e c t e d , t h e r e was one e x e n c e p h a l i c female ( t a b l e X X I I I ) . The i n c i d e n c e of e x e n c e p h a l y was 0% and 0.4% i n the SELH.ICR and ICR.SELH f e t u s e s r e s p e c t i v e l y . The f r e q u e n c y of e x e n c e p h a l y i n F l f e t u s e s was 0.3% o v e r a l l . The one e x e n c e p h a l i c found l i k e l y r e p r e s e n t s a background f r e q u e n c y of e x e n c e p h a l y i n the mice. I f e x e n c e p h a l y was due t o a d o m i n a n t l y i n h e r i t e d s i n g l e gene d e f e c t i n SELH w i t h p e n e t r a n c e of 16.9% ( t a b l e X X I I I ) , we would e x p e c t 60 e x e n c e p h a l i c s i n 356 f e t u s e s i n c o n t r a s t t o the observed 1. I f the d e f e c t was caused by a sex l i n k e d s i n g l e gene, we would e x p e c t SELH.ICR F l males t o be a f f e c t e d , whereas the r e c i p r o c a l c r o s s (ICR.SELH F l ) males would not be a f f e c t e d . 115 Female F l a n i m a l s would be e q u a l l y a f f e c t e d i n both c r o s s e s . E x e n c e p h a l y does not f i t e i t h e r a s i n g l e gene dominant (goodness of f i t X^ i d f ~ 68.83, p<.01), or a s i n g l e gene sex l i n k e d p a t t e r n of i n h e r i t a n c e . B. F2 G e n e r a t i o n F l females were mated t o one of two F l male s i b s ( I . S x I . S ; S . I x S . I ) . L i t t e r s were c o l l e c t e d on day 18 of g e s t a t i o n , the r e s u l t s of which a r e i n t a b l e s X X I I I and XXIV. I n t h e S.I c r o s s , 16 F l males were mated t o between 2 t o 8 F l female s i b s f o r a t o t a l of 68 l i t t e r s . 30 e x e n c e p h a l i c s (23 female:7 male) were found i n 829 f e t u s e s (394 female:435 male) f o r a f r e q u e n c y of 3.6% e x e n c e p h a l y (5.8% i n f e m a l e , 1.6% i n male) In the F2 ( t a b l e X XIV). The o b s e r v e d f r e q u e n c y produced by i n d i v i d u a l F l males ranged from 0-22.2% e x e n c e p h a l y , w i t h 7/16 F l males p r o d u c i n g no e x e n c e p h a l i c s . The h i g h e s t F l e x e n c e p h a l y p r o d u c e r , male 159 c o n s i s t e n t l y produced 2-3 e x e n c e p h a l i c f e t u s e s i n each of f o u r F2 l i t t e r s . There was s i g n i f i c a n t h e t e r o g e n e i t y observed between t h e s e S.I males i n t h e i r a b i l i t y t o produce e x e n c e p h a l y i n t o t a l f e t u s e s ( F 1 5 5 2 = 5.15; p<0.05), and i n female f e t u s e s ( F 1 5 5 2 = 4 . 8 3 ; p<0.05). There was no e v i d e n c e of h e t e r o g e n e i t y i n the f r e q u e n c y of e x e n c e p h a l y i n male f e t u s e s ( F 1 5 5 2 = 1 . 0 4 ; p>0.05). Twenty s i x r e s o r p t i o n s (moles) were found i n t o t a l from 68 l i t t e r s f o r a r e s o r p t i o n f r e q u e n c y of 3.0%. In the I.S F l r e c i p r o c a l c r o s s , 10 F l males were mated t o 116 Table X X I I I . FREQUENCIES OF EXENCEPHALY RECOVERED IN GENETIC STUDIES (TOTAL FETUSES) Genotype 1 # i m p l a n t s # m o l e s ( % ) # F e t u s e s # Exen+ % Exen ICR/BC I — — 420 0 — SELH** I 442 29 (6.6) 413 70 16.9 SELH.ICR F l I nd* nd 121 0 0 ICR.SELH F l I 243 8 (3.3) 235 1 0.4 SELH.ICR F2 1 855 26 (3.0) 829 30 3.6 ICR.SELH F2 1 nd nd 906 12 1.3 SELH X F l - B C l | 651 29 (4.5) 622 22 3.5 SELH X B C l - BC2 1 2348 109 (4.6) 2239 167 7.5 + Exen = e x e n c e p h a l y * nd= not done **: from t a b l e X V I I I Table XXIV. FREQUENCIES OF EXENCEPHALY RECOVERED IN THE F2 OF S.I x S.I F l MATINGS F l * # # # # EXEN % EXEN F % M % LITTERS IMPLANTS MOLES FETUSES ( F: M) * TOTAL EXEN EXEN 97 5 68 1 67 6(4:2) 9.0 11. 4 6.2 •105 6 83 0 83 6(3:3) 7.2 6.7 7.9 107 3 46 4 42 1(0.1) 2.4 0 4 . 5 127 3 39 2 37 0 0 0 0 •116 6 71 1 70 0 0 0 0 117 5 65 1 64 1(1.0) 1.6 2.8 0 130 4 57 1 56 0 0 0 0 131 4 53 2 51 1(1.0) 2.0 3.8 0 135 2 24 1 23 0 0 0 0 136 2 20 1' 19 0 0 0 0 158 4 46 0 46 1(1.0) 2.2 4 . 2 0 159 4 50 5 45 10(9:1) 22. 2 40.9 4.3 165 8 86 2 84 2(2:0) 2.4 5.0 0 166 7 91 3 88 0 0 0 0 182 3 33 1 32 0 0 0 0 183 2 23 1 22 2(2:0) 9.1 13.3 0 TOTAL 68 855 26 829 30(23:7) 3.6 5.8 1.6 (392F:435M) *: F= female, M= male +: [= s i b l i n g s 118 T a b l e xxv. FREQUENCIES OF EXENCEPHALY I.S x I.S F l RECOVERED MATINGS IN THE F2 OF F l ff # LITTERS If IMPLANTS If MOLES # ff EXEN FETUSES (F:M)* % EXEN TOTAL F % EXEN M% EXEN 12 8 nd + nd 100 2(1:1) 2.0 2.5 1.7 15 5 nd nd 67 0 0 0 0 22 7 nd nd 85 2(1:1) 2 . 4 3.3 1.8 23 7 nd nd 86 1(1:0) 1.2 2.1 0 36 7 nd nd 83 1(1:0) 1.2 2.8 0 45 8 nd nd 93 4(4:0) 4 . 3 9 . 3 0 62 8 99 4 95 0 0 0 0 63 8 103 1 102 1(1:0) 1.0 2.4 0 79 7 92 0 92 0 0 0 0 80 8 106 3 103 1(1:0) 1.0 2.0 0 TOTAL 73 *: F= female, M= male + nd= not done + +: [= s i b l i n g s 906 12(10:2) 1.3 (415F:491M) 2.4 0.4 119 between 5-8 female s i b s f o r a t o t a l of 73 l i t t e r s ( t a b l e XXV). In 906 f e t u s e s , 12 e x e n c e p h a l i c s (10 female:2 male) were found f o r a f r e q u e n c y of 1.3%. The f r e q u e n c y of e x e n c e p h a l y ranged from 0-4.3% w i t h 3/10 males p r o d u c i n g no e x e n c e p h a l y . There was no e v i d e n c e of h e t e r o g e n e i t y between I.S F l males f o r t h e f r e q u e n c y of e x e n c e p h a l y produced ( t o t a l f e t u s e s : F n _ = 1.21; y ,bo p>0.05, female f e t u s e s : F g 6 1 =1.68; p>0.05, male f e t u s e s : F g 6 3 = 0 . 5 9 ; p>0.05). R e s o r p t i o n s were not r e c o r d e d f o r a l l I.S F2 l i t t e r s , a l t h o u g h r e s o r p t i o n numbers from t h o s e F l males w i t h mole r e c o r d s a r e s i m i l a r t o the r e s o r p t i o n f r e q u e n c y i d e n t i f i e d i n S.I F2 l i t t e r s . The e x e n c e p h a l y f r e q u e n c y r e c o v e r e d i n I.S F2 f e t u s e s (1.3%) i s l e s s t h a n h a l f t h a t r e c o v e r e d i n S.I F2 f e t u s e s ( 3 . 6 % ) . T h i s d i f f e r e n c e c o u l d be e x p l a i n e d by the h e t e r o g e n e i t y of SELH i n the f r e q u e n c y of e x e n c e p h a l y t h e y produce. D i f f e r e n t s e t s of F l ' s a r e d i f f e r e n t samples of the SELH m o d i f i e r genes, and t h u s h e t e r o g e n e i t y i n the F l p a r e n t s c o u l d be e x p e c t e d . I t i s n o t a b l e t h a t i f the d a t a f o r F l male 159 i s d e l e t e d ( t a b l e X X I V ) , the r e m a i n i n g d i f f e r e n c e between F2 e x e n c e p h a l y f r e q u e n c i e s ( 2 . 6 % compared w i t h 1.3%) i s not s t a t i s t i c a l l y s i g n i f i c a n t ( X 2 1 d f = 3 . 4 0 ; p>0.05). I f t h e r e was an " X " - l i n k e d f a c t o r a f f e c t i n g e x e n c e p h a l y f r e q u e n c y , we would e x p e c t the f r e q u e n c y of e x e n c e p h a l y t o be h i g h e r i n S.I F2 females t h a n i n I.S F2 f e m a l e s . Such a d i f f e r e n c e i s o b s e r v e d (5.8% i n S.I F2 females v e r s u s 2.4% i n 120 I.S F2 f e m a l e s ) . However, we would e x p e c t no d i f f e r e n c e between male F2 f e t u s e s , and y e t t h e r e i s a d i f f e r e n c e (S.I F2- 1.6% v e r s u s I.S F2- 0.04%). I f the SELH "Y" c o n f e r r e d a h i g h e r l i a b i l i t y t o the t r a i t , we would e x p e c t the I.S F2 t o have a h i g h e r male e x e n c e p h a l y f r e q u e n c y , i n s t e a d , the r e v e r s e i s t r u e . A l t h o u g h the r e c i p r o c a l F2's d i f f e r i n e x e n c e p h a l y f r e q u e n c y , s e x - l i n k a g e does not appear t o be the cause. The sex r a t i o of a f f e c t e d e x e n c e p h a l i c s i s 3.3:1 (female:male) f o r S . I , and 5.0:1 (female:male) f o r I.S F2 f e t u s e s ( t a b l e XXVI) . A female e x c e s s i s a l s o observed i n SELH e x e n c e p h a l i c f e t u s e s (page 112). Because of t h i s sex d i f f e r e n c e , male and female f r e q u e n c i e s have been e v a l u a t e d s e p a r a t e l y . The f r e q u e n c y of female e x e n c e p h a l i c s i n the F2 was 5.8% i n S.I f e t u s e s , and 2.4% i n I.S f e t u s e s . In the SELH c o n t r o l s f o r the g e n e t i c s t u d y ( t a b l e X X V I I ) , 22.4% (46/205) of SELH female f e t u s e s were e x e n c e p h a l i c . I f the t r a i t was c o n t r o l l e d by a s i n g l e s i m p l e r e c e s s i v e l o c u s w i t h reduced p e n e t r a n c e , we would e x p e c t the F2 f r e q u e n c y t o be 1/4.of t h a t of SELH f e m a l e s , or 5.6%. The S.I F2 female f r e q u e n c y (5.8%) f i t s w e l l w i t h t h i s e x p e c t a t i o n , however the I.S F2 f r e q u e n c y (2.4%) does not ( t a b l e XXVII) . I n males, n e i t h e r the I.S, nor the S.I f r e q u e n c y (0.4%, and 1.6% r e s p e c t i v e l y ) agree w i t h the 2.9% f r e q u e n c y e x p e c t e d f o r the s i m p l e 1 l o c u s model ( t a b l e X X V I I I ) . However, the r e l a t i v e l y h i g h f r e q u e n c y of e x e n c e p h a l y r e c o v e r e d i n both F2 121 Table x x v i . SEX RATIOS AMONG EXENCEPHALIC AND NORMAL FETUSES r a t i o 1 T o t a l # Sex r a t i o i n exens T o t a l # Sex r a t i o i n normal O v e r a l l sex Genotype 1 exens (F:M)* normal (F:M)* ( F l M ) * SELH+ 1 70 1.92:1 343 0.86:1 0.99:1 ICR.SELH F l 1 1 1:0 234 0.97:1 0.97:1 SELH.ICR F l 1 0 0 121 0.86:1 0.86:1 I.S F2 1 12 5.00:1 894 0.83:1 0.85:1 S.I F2 1 30 3.29:1 799 0.87:1 0.91:1 B C l 1 20 4.00:1 601 1.00:1 1.03:1 BC2 1 167 2.80:1 2072 0.84:1 0.91:1 * n o t e : F:M= female:male r a t i o +: from t a b l e X V I I I 122 T a b l e XXVII. FREQUENCIES OF EXENCEPHALY RECOVERED IN GENETIC STUDIES COMPARED TO EXPECTATIONS BASED ON A 1 LOCUS SIMPLE RECESSIVE MODEL WITH 22.4% PENETRANCE FEMALE FETUSES % EXPECTED I F GENOTYPE N % EXENCEPHALY 1 LOCUS ICR/Bc 210 0 -SELH+ 205 22.4 -F l 172 0.6 0 I.S F2 415 2.4 5.6 S.I F2 394 5.8 5.6 B C l 316 5.1 11.2 BC2 1068 11. 5 16.8 T a b l e X X V I I I . FREQUENCIES OF EXENCEPHALY RECOVERED IN GENETIC STUDIES COMPARED TO EXPECTATIONS BASED ON A 1 LOCUS SIMPLE RECESSIVE MODEL WITH 11.5% PENETRANCE MALE FETUSES % EXPECTED I F GENOTYPE N % EXENCEPHALY 1 LOCUS ICR/Bc 210 0 -SELH+ 208 11.5 -F l 184 0 0 I.S F2 491 0.4 2.9 S.I F2 435 1.6 2.9 B C l 305 1.3 5.8 BC2 1171 3.8 8.6 +:from t a b l e X V I I I 123 c r o s s e s I n d i c a t e s t h a t t he number of genes i n v o l v e d i s not l a r g e . C. B C l G e n e r a t i o n A f i r s t b a c k c r o s s g e n e r a t i o n ( B C l ) t o SELH was made u s i n g 10 I.S F l males mated t o between 3-7 SELH females each. I n a t o t a l of 52 l i t t e r s and 622 f e t u s e s , 21 e x e n c e p h a l i c s were o b s e r v e d (16 female:4 male, + 1 dead e x e n c e p h a l i c f e t u s ) ( t a b l e X X I X ) . The t o t a l f r e q u e n c y of e x e n c e p h a l y was 3.5%. Female e x e n c e p h a l i c s outnumbered a f f e c t e d males 4:1 ( t a b l e X X V I ) . 29 moles were found i n a t o t a l of 651 i m p l a n t a t i o n s f o r a r e s o r p t i o n f r e q u e n c y of 4.5%. The f r e q u e n c y of e x e n c e p h a l y i n the B C l f e t u s e s ranged from 0 -11.9% among F l mal e s , w i t h 2/10 males p r o d u c i n g no e x e n c e p h a l y . The I.S F l males used t o ge n e r a t e the B C l were t h e same males used t o g e n e r a t e the I.S F2 ( t a b l e X X V ) . The f r e q u e n c y of ex e n c e p h a l y i n t h e F2 and B C l progeny from t h e s e males was c o r r e l a t e d u s i n g a Spearman's rank c o r r e l a t i o n c o e f f i c i e n t . L i t t e r f r e q u e n c i e s of e x e n c e p h a l y were t r a n s f o r m e d i n t o Freeman-Tukey a r c s i n e v a l u e s and mean l i t t e r a r c s i n e s f o r each s i r e c a l c u l a t e d . A p o s i t i v e , s i g n i f i c a n t (p<0.05) c o r r e l a t i o n was f o u n d ' f o r t o t a l f e t u s d a t a ( r s = 0 . 6 8 ) , and f o r female f e t u s d a t a ( r s = 0 . 5 8 ) , however no s i g n i f i c a n t c o r r e l a t i o n was found i n t h e f r e q u e n c i e s i n male f e t u s e s ( r s = 0 . 1 3 ) . These r e s u l t s i n d i c a t e t h a t t he h e t e r o g e n e i t y among F l s i r e s i n p r o d u c t i o n of ex e n c e p h a l y i s g e n e t i c . T a b l e XXIX. FREQUENCIES OF EXENCEPHALY RECOVERED IN THE B C l PROGENY OF I.S F l MALES X SELH FEMALES F l # # # # # EXEN % EXEN F % M % ft LITTERS IMPLANTS MOLES FETUSES (F:M)+ TOTAL EXEN EXEN 12 6 71 3 68 3(3:0) 4.4 8.6 0 15 5 66 3 63 1(1:0) 1.6 3.3 0 22 5 61 2 59 1(0:1) 1.7 0 3.8 23 5 68 1 67 1(1:0) 1.5 2.9 0 36 5 62 3 59 3 ( 2 : 0 ) * 5.1 7.7 0 45 6 68 1 67 8(8:0) 11.9 22.2 0 62 3 39 4 35 0 0 0 0 63 5 59 6 53 3(1:2) 5.7 3.7 7.7 79 5 64 1 63 0 0 0 0 80 7 93 5 88 1(0:1) 1.1 0 2.4 TOTAL : 52 651 29 622 21(16:4) * 3.5 5.1 1.3 ( l ? s e x ) (+1? sex) * n o t e : 1 dead e x e n c e p h a l i c f e t u s ( a p p r o x i m a t e l y day 16 of g e s t a t i o n ) t h a t c o u l d not be s c o r e d f o r sex i s i n c l u d e d i n the d a t a . +:F= f e m a l e , M= male 125 For a s i m p l e s i n g l e r e c e s s i v e t r a i t , we would e x p e c t a r e c o v e r y of 1/2 the f r e q u e n c y of e x e n c e p h a l y i n SELH i n B C l a n i m a l s , t h a t i s , 11.2% i n female and 5.8% i n male f e t u s e s . The o b s e r v e d f r e q u e n c y of 5.1% i n female and 1.3% i n male a r e t o o low ( X 2 1 d f = 19.9; p<0.01) t o f i t the s i m p l e 1 l o c u s model ( t a b l e s X X V I I , X X V I I I ) . D. BC2 G e n e r a t i o n One I.S F l male (#12) was mated t o 5 SELH females t o r a i s e a d u l t B C l males, which were i n t u r n mated t o 7-9 SELH f e m a l e s . 210 l i t t e r s (2239 f e t u s e s ) were c o l l e c t e d from 26 B C l males, i n which 167 (7.5%) e x e n c e p h a l i c s (123 female:44 male) were found ( t a b l e XXX). A l l males produced e x e n c e p h a l y . The f r e q u e n c y of e x e n c e p h a l y was 11.5% i n f e m a l e s , and 3.9% i n males. The f r e q u e n c y ranged from 2.0- 31.2% i n females and 0-9.4% i n males (4/26 B C l males had no male e x e n c e p h a l i c p r o g e n y ) . The sex r a t i o of a f f e c t e d a n i m a l s was 2.8 f e m a l e r l male ( t a b l e X X V I ) . There was an e x c e s s of a f f e c t e d females i n the progeny of a l l of the B C l s i r e s . No c o r r e l a t i o n between th e degree of female e x c e s s among e x e n c e p h a l i c s and the t o t a l f r e q u e n c y of e x e n c e p h a l y was found ( r s = 0 . 0 4 ) . The o v e r a l l sex r a t i o i n d i c a t e s a s l i g h t male e x c e s s ( t a b l e X X V I ) . 109 moles were found i n a t o t a l of 2348 i m p l a n t a t i o n s f o r a r e s o r p t i o n f r e q u e n c y of 4.6%. The r e s o r p t i o n f r e q u e n c i e s r e c o v e r e d i n t h e o u t c r o s s e s ( I . S F l , S.I F2, B C l , BC2) compared 126 Table XXX. FREQUENCIES OF EXENCEPHALY RECOVERED IN THE BC2 PROGENY OF BCl MALES X SELH FEMALES BCl # # # # EXEN < %> EXEN F % M % « LITTERS IMPLANTS MOLES FETUSES (F: M) + TOTAL EXEN EXEN [-110 8 94 0 94 6(5:1) 6.4 12.2 2.9 L l l l 8 91 4 87 4(3:1) 4.6 7.3 2.2 r-112 8 92 3 89 4(3:1) 4.5 7.5 2.0 1-113 8 96 4 92 10(7:3) 10.9 15.2 6.5 r l l 4 8 84 8 76 15(12:3) 19.7 27.3 9 . 4 L115 8 93 4 89 4(4:0) 4.5 9.1 0 rl23 8 97 8 89 7(5:2) 7.9 12.2 4.2 124 8 94 6 88 3(1:2) 3.4 2.4 4.3 141 8 96 2 94 8(7:1) 8.5 17.1 1.9 142 8 79 6 73 2(1:1) 2.7 2.9 2.6 1-143 8 90 4 86 8(5:3) 9 . 3 11.6 7.0 147 8 88 6 82 10(8:2) 12.2 17.8 5.4 rl50 8 76 2 74 6(4:2) 8.1 12.9 4.7 167 10 91 6 85 3(3:0) 3.5 8.3 0 168 7 80 1 79 5(4:1) 6.3 10 . 8 2.4 172 9 92 4 88 3(3:0) . 3.4 7.9 0 Ll73 8 81 3 78 12(10:2 15. 4 31.2 4.3 rl84 8 86 3 83 1(1:0) 1.2 2.0 0 185 8 73 1 72 4(3:1) 5 . 6 8 . 6 2.7 186 8 90 8 82 13(9:4) 15.9 26.5 8.3 193 8 86 8 78 2(1:1) 2.6 2 . 3 2.9 1-19 4 8 100 4 96 5(5:0) 5.2 10.6 0 r202 9 98 7 91 10(6:4) 11.0 14 . 3 8.2' 203 8 101 1 100 9(6:3) 9 . 0 12 . 5 5.8 204 8 101 4 97 5(2:3) 5.2 4 . 8 5.5 L205 8 99 2 97 8(5:3) 8.2 10 .0 6.4 TOTAL :210 2348 109 2239 167 7.5 11. 5 3.8 (123:44) +:F= f e m a l e , M= m a l e [ : = s i b l i n g s 127 t o the SELH r e s o r p t i o n f r e q u e n c y do not d i f f e r s i g n i f i c a n t l y ( t a b l e X X I I I ) . I f t h e t r a i t were due t o a s i m p l e s i n g l e r e c e s s i v e l o c u s t h a t d i f f e r e d between ICR/Be and SELH, 1/2 of B C l males would be e x p e c t e d t o produce e x e n c e p h a l y a t the same f r e q u e n c y as SELH, and 1/2 of the B C l males would be e x p e c t e d t o produce the d e f e c t a t t h e same f r e q u e n c y as i n B C l f e t u s e s . C o n v e r s e l y , i f m u l t i p l e l o c i were r e s p o n s i b l e f o r the d e f e c t , the d i s t r i b u t i o n of B C l b r e e d i n g v a l u e s would be e x p e c t e d t o be t i g h t , u n i m o d a l , and c e n t e r e d h a l f way between SELH and F l b r e e d i n g v a l u e s . A n a l y s i s of the f r e q u e n c i e s produced i n t h e BC2 f e t u s e s ( t o t a l , f e m a l e , and male f e t u s e s ) by each male do not i n d i c a t e any c l e a r 1:1 bimodal d i s t r i b u t i o n of B C l males ( f i g u r e 24a-c) about the two e x p e c t e d v a l u e s , nor do the f r e q u e n c i e s i n d i c a t e a smooth unimodal d i s t r i b u t i o n . The f r e q u e n c i e s of e x e n c e p h a l y i n female progeny suggest t h a t a t l e a s t 3 of the 26 B C l s i r e s produced e x e n c e p h a l y a t f r e q u e n c i e s a t l e a s t as h i g h as SELH. The f r e q u e n c y of e x e n c e p h a l y e x p e c t e d f o r the 1 l o c u s model i n BC2 a n i m a l s i s 16.8% i n females and 8.6% i n males (3/4 of the f r e q u e n c y i n SELH) ( t a b l e s XXVII and X X V I I I ) . A g a i n , the s i n g l e l o c u s model does not f i t the observed f r e q u e n c y . I t seems c l e a r t h e n , t h a t the h i g h f r e q u e n c y of e x e n c e p h a l y i n SELH i s not due t o one s i m p l e r e c e s s i v e l o c u s . C o n s i d e r i n g the s e g r e g a t i o n of 1, 2, 3, 4, or 5 l o c i , the e x p e c t e d p r o p o r t i o n of homozygotes l i k e the p a r e n t a l s t r a i n i n a B C l g e n e r a t i o n are 1/2, 1/4, 1/8, F I G U R E 2 4 ( « - c ) I Of s i res D I S T R I B U T I O N OF BC MALES IN FREQUENCY OF E X E N C E P H A L Y PRODUCED ( BC ) 2 TOTAL FETUSES 0 2 4 6 10 12 14 16 18 20 22 24 26 28 30 32 34 36 I E x e n c e p h a l y ( T o t a l F e t u s e s ) F E H A L E FETUSES of s i res 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34. 36 I E x e n c e p h a l y ( F e m a l e F e t u s e s ) HALE F E T U S E S of s i res 0 2 4 6 B 10 12 14 16 18 20 22 24 26 28 30 32 34 36 BC mean 2 o b s e r v e d SELH mean o b s e r v e d 8 C , mean 129 1/16, and 1/32 r e s p e c t i v e l y . At l e a s t 3/26 or 1/9, of the B C l s i r e s were c l e a r l y l i k e the p a r e n t a l s t r a i n , SELH, f o r f r e q u e n c y of e x e n c e p h a l y i n female o f f s p r i n g or t o t a l o f f s p r i n g . The d a t a s u g g e s t , t h e r e f o r e , t h a t 2-4 l o c i may cause the h i g h f r e q u e n c y of e x e n c e p h a l y i n SELH mice. The observed h e t e r o g e n e i t y w i t h i n SELH f o r the f r e q u e n c y of e x e n c e p h a l y o b s c u r e s f u r t h e r c o n c l u s i o n s . I t may not be p o s s i b l e t o come t o a f i n a l c o n c l u s i o n about the number of genes c a u s i n g e x e n c e p h a l y i n SELH ( i n comparison t o ICR/Be) u n t i l the genes i n f l u e n c i n g f r e q u e n c y of e x e n c e p h a l y are f i x e d i n the SELH a n i m a l s . 130 V I I I . ENZYME LINKAGE TESTING 25 B C l males were typed f o r t h e i r genotype a t the 4 p o l y m o r p h i c l o c i known t o d i f f e r between SELH and the ICR/Be s t r a i n ( G p i - 1 , Mod-1, Gpt-1, Pgk-2) (Table X X X I ) . Each l o c u s was t e s t e d f o r a s s o c i a t i o n w i t h e x e n c e p h a l y f r e q u e n c y produced by the B C l s i r e s , however, no a s s o c i a t i o n was found. That i s , none of the t e s t s were s i g n i f i c a n t ( a l l p>0.05; Mann-Whitney U t e s t ) . The gene(s) d i f f e r i n g between ICR/Be and SELH t h a t cause or i n f l u e n c e r i s k f o r e x e n c e p h a l y do not appear t o be l i n k e d t o Gpi- 1 on chromosome 7, Mod-1 on chromosome 9, Gpt-1 on chromosome 15, nor Pgk-2 on chromosome 17. IX. MISCELLANEOUS STUDIES- Newborn a d r e n a l g l a n d s Anencephaly i n humans i s a s s o c i a t e d w i t h the l a c k of a normal f u n c t i o n i n g hypothalamus and a h y p o p l a s t i c or absent p i t u i t a r y , accompanied by h y p o p l a s t i c a d r e n a l g l a n d s (Emery and Rimo i n 1983). The a d r e n a l g l a n d s of 16 normal SELH, and 11 e x e n c e p h a l i c SELH newborns were o b s e r v e d . No d i f f e r e n c e i n a d r e n a l shape was found between normal and e x e n c e p h a l i c SELH newborns. The a d r e n a l g l a n d s of SELH e x e n c e p h a l i c s t h e r e f o r e do not appear t o be h y p o p l a s t i c . Table XXXI. TYPING OF MARKER LOCI GPI-1, MOD-1, PGK-2, AND GPT-1 IN B C l MALES P e d i g r e e #/ s t r a i n of male c o n t r o l s : SELH 1702 ICR/Bc 1547 F l ICR/SELH 79 B C l males: GPI-1 MOD-1 a b ab a b ab PGK-2 b a ab GPT-1 b a ab 110 1 a a b b 111 1 a ab ab b 112 1 ab ab b ab 113 1 a ab ab ab 114 1 a ab b b 115 1 a a ab b 123 1 ab ab ab b 124 1 ab ab b ab 141 1 ab a ab ab 142 1 ab a ab b 143 1 a a ab ab 147 i a a ab b 150 1 a a b b 167 1 ab ab b b 172 1 ab a b ab 173 1 a a b ab 184 1 a ab b b 185 1 a a ab b 186 1 ab a b ' b 193 1 ab ab ab b 194 1 a ab b b 202 1 a ab ab ab 203 1 ab a ab ab 204 1 ab a b b 205 1 ab a b ab 132 DISCUSSION A. The embryopathogenesis of e x e n c e p h a l y i n SELH mice The most i m p o r t a n t and i n t r i g u i n g f i n d i n g was t h a t p r a c t i c a l l y a l l SELH embryos f a i l e d t o make the normal i n i t i a l c o n t a c t of n e u r a l f o l d s i n the p o s t e r i o r prosencephalon-mesencephalon r e g i o n ( c l o s u r e 2 ) , u n l i k e both ICR/Bc and SWV/Bc embryos. T h i s i n d i c a t e d t h a t SELH embryos were not s i m p l y s l o w In c l o s i n g t h e c r a n i a l n e u r a l f o l d s , but t h a t t h e y were m i s s i n g a s t e p i n n e u r a l tube c l o s u r e , and the whole s t o c k was a p p a r e n t l y abnormal i n c r a n i a l f o l d c l o s u r e . D e s p i t e t h i s , over 80% of SELH embryos s t i l l manage t o complete a n t e r i o r n e u r a l tube c l o s u r e . The c r a n i a l f o l d s of SELH embryos manage t o make c o n t a c t t h r o u g h an a l t e r n a t e , p a s s i v e mechanism of f u s i o n i n i t i a t e d a t the r o s t r a l base of the p r o s e n c e p h a l o n which extends c a u d a l l y t o the rhombencephalon. T h i s f u s i o n a t the r o s t r a l base of the p r o s e n c e p h a l o n o c c u r s i n normal s t r a i n SWV/Bc and ICR/Bc embryos a t the same st a g e i n development as i n SELH embryos. However, n o r m a l l y t h i s f u s i o n ( c l o s u r e 3) t a k e s p l a c e a f t e r the i n i t i a l c o n t a c t i n the p o s t e r i o r p r o s e n c e p h a l o n r e g i o n , and a c t s as a minor c l o s u r e , f u s i n g o n l y about the r o s t r a l 1/3 of the p r o s e n c e p h a l o n . Delayed c o m p l e t i o n of a n t e r i o r n e u r a l tube c l o s u r e i n SELH o c c u r s because c l o s u r e s t a r t s from t h i s u n i d i r e c t i o n a l t h i r d f u s i o n , and has t o proceed over the e n t i r e f o r e and m i d b r a i n r e g i o n s . 133 SELH embryos d i d not d i f f e r from ICR/Bc and SWV/Bc normal s t r a i n embryos i n the r a t e of g e n e r a l development. The p a t t e r n s of som i t e development over time d i d not suggest any major d e v i a t i o n s of SELH from normal. SELH embryos were d e l a y e d i n t h e i r c r a n i a l n e u r a l tube c l o s u r e r e l a t i v e t o somite c o u n t , compared t o both ICR/Bc and SWV/Bc embryos. The c l o s u r e d e l a y was e v i d e n t as e a r l y as 8-10 s o m i t e s i n development when SELH embryos lagged behind normal embryos i n c u r l i n g i n the a n t e r i o r n e u r a l f o l d s i n t o the m i d l i n e . The a n t e r i o r n e u r a l f o l d s of SELH embryos a r e more w i d e l y open and f l a r e d wide i n s t e a d of e l e v a t i n g and a p p r o x i m a t i n g each o t h e r as i n normal s t r a i n embryos d u r i n g c l o s u r e . Without a f u s i o n a t the p r o s e n c e p h a l o n base a p p a r e n t l y p u l l i n g t he f o l d s t o g e t h e r , the c r a n i a l n e u r a l f o l d s would l i k e l y not make c o n t a c t . SELH n e u r a l t i s s u e does not seem t o have any d i f f i c u l t y f u s i n g once the f o l d s a r e c l o s e l y a l i g n e d . I t i s i n t e r e s t i n g t o note t h a t the c a p a c i t y f o r f u s i o n of the f o l d s appears t o be f l e x i b l e enough t o a l l o w more th a n 80% of SELH embryos time t o fu s e the c r a n i a l f o l d s w e l l a f t e r normal s t r a i n embryos have completed f u s i o n . The a b i l i t y of SELH embryos t o fuse by t h i s a l t e r n a t e method a c c o u n t s f o r the s u r v i v a l of t h i s s t o c k i n the absence of normal c r a n i a l c l o s u r e . I t i s s i g n i f i c a n t t h a t a l t h o u g h SELH embryos a r e m i s s i n g a s t e p i n n e u r a l tube c l o s u r e , SELH mice appear t o be normal. B r a i n and head development appears f o r the most p a r t , t o be u n a f f e c t e d by the l a c k of normal c l o s u r e ( n o t e : a low f r e q u e n c y (about 4%) of a t a x i a which does not s e g r e g a t e as a m endelian r e c e s s i v e has been observed i n the SELH s t o c k . These a t a x i c mice are m i s s i n g p o r t i o n s of the c e r e b e l l u m which may be the consequence of abnormal c l o s u r e i n some a n i m a l s or may be an independent phenomenon). E x e n c e p h a l i c SELH embryos f a i l t o e l e v a t e and f u s e t h e i r c r a n i a l n e u r a l f o l d s . E x e n c e p h a l y i n SELH i s the r e s u l t of a f a i l u r e t o f u s e i n t h e mesencephalon r e g i o n of the n e u r a l f o l d s . The a l t e r n a t i v e c l o s u r e method must f a i l t o compensate abnormal c l o s u r e i n a p p r o x i m a t e l y 17% of SELH f e t u s e s . Some SELH embryos of 20-22 s o m i t e s were found w i t h f u s i o n of a l l or o n l y p a r t of the p r o s e n c e p h a l o n , w h i l e the mesencephalon remained open, and t h e y were thought t o be i n the e a r l y s t a g e s of development of e x e n c e p h a l y . These p a r t i a l l y f u s e d embryos i n d i c a t e d t h a t a n t e r i o r n e u r a l tube c l o s u r e may be a t h r e s h o l d t r a i t . W ith the absence of c l o s u r e ( 2 ) , SELH embryos a r e a l l d e l a y e d i n c r a n i a l n e u r a l tube c l o s u r e t o v a r y i n g degrees because f u s i o n b e g i n s from a l a t e r - o c c u r r i n g s i t e ( c l o s u r e 3) and has t o p r o g r e s s a l o n g e r d i s t a n c e than u s u a l from t h a t s i t e . Because of t h i s , SELH may l i e c l o s e t o the t h r e s h o l d f o r f a i l u r e of a n t e r i o r n e u r a l tube c l o s u r e . Those embryos w i t h f o l d s f l a r e d w i d e r than most so t h a t t h e y cannot be " p u l l e d " i n , and those w i t h s l o w e r f u s i o n of the n e u r a l f o l d s t h a t "run out of t i m e " , may f a i l t o complete c l o s u r e . The v a r i a t i o n i n " f l a r e " of the f o l d s and i n speed of c l o s u r e may be due i n p a r t t o g e n e t i c 135 m o d i f i e r s demonstrated t o be s e g r e g a t i n g i n the SELH s t o c k , and a l s o t o minor and u n i d e n t i f i e d e n v i r o n m e n t a l v a r i a b l e s . Some of the v a r i a t i o n may a l s o be p u r e l y s t o c h a s t i c i n o r i g i n ( K u r n i t , L a y t o n , and M a t t h y s s e 1987). The SELH s t o c k demonstrates a new model f o r the mechanism of development of e x e n c e p h a l y and o t h e r c l o s u r e f a i l u r e s (a missed s i t e ( c l o s u r e 2) i n the m u l t i f o c a l i n i t i a t i o n s i t e s i n the sequence of c l o s u r e ) , t o be added t o the c u r r e n t hypotheses of absence of f u s i o n , and d e l a y e d f u s i o n . I t i s not known whether o t h e r e x e n c e p h a l y mutants a l s o omit a c l o s u r e s i t e . Many s t u d i e s of abnormal n e u r a l tube c l o s u r e i n mice i n c l u d e s i m u l t a n e o u s use of a m u t a t i o n and a t e r a t o g e n so t h a t when one o b s e r v e s a b n o r m a l i t i e s i n the embryo and i t s c e l l s , i t i s not c l e a r whether t h e s e a b n o r m a l i t i e s a r e the r e s u l t of the m u t a t i o n , the t e r a t o g e n , or t h e i r i n t e r a c t i o n . I f the t e r a t o g e n were the cause, i t would not be known whether the a b n o r m a l i t y c o n t r i b u t e d t o the f a i l u r e of c l o s u r e or whether i t was a g e n e r a l i z e d t o x i c e f f e c t . One might p r e d i c t t h a t SELH embryos would be s e n s i t i v e t o a l m o s t any t e r a t o g e n i n t e r f e r i n g w i t h n e u r a l tube c l o s u r e . T h i s i n i t s e l f would not be s u r p r i s i n g because SELH as a s t o c k w i t h abnormal c l o s u r e , c o u l d be seen t o be c l o s e t o the t h r e s h o l d f o r n e u r a l tube c l o s u r e d e f e c t s . A l a c k of response t o a t e r a t o g e n however, c o u l d i n d i c a t e t h a t the c h e m i c a l a f f e c t e d the same system a l r e a d y i n c a p a c i t a t e d by the mutant g e n e ( s ) , ( f o r example: c l o s u r e (2) i n SELH) so t h a t no f u r t h e r damage c o u l d be i n f l i c t e d . One would have t o t e s t the a l t e r n a t i v e p o s s i b i l t y t h a t a l a c k of r e s p o n s i v e n e s s were due s i m p l y t o the i n f l u e n c e of the the background g e n e t i c makeup of the s t o c k or dosage t o l e r a n c e . A s m a l l v a r i a t i o n was observed i n a n t e r i o r n e u r a l tube c l o s u r e between the two normal s t r a i n s , ICR/Bc and SWV/Bc. A d i f f e r e n c e i n the l o c a t i o n of i n i t i a l c o n t a c t of the a n t e r i o r f o l d s i n " c l o s u r e 2" was o b s e r v e d . SWV/Bc f o l d s tended t o c o n t a c t i n the m i d d l e of the p r o s e n c e p h a l o n , whereas ICR/Bc f o l d s c o n t a c t e d i n the p o s t e r i o r p r o s e n c e p h a l o n . Comparison of o t h e r s t u d i e s of n e u r a l tube c l o s u r e a l s o s u ggest some v a r i a t i o n i n the t i m i n g and p o s i t i o n of c l o s u r e s i n the c r a n i a l n e u r a l f o l d s (Geelan and Langman 1977, Golden and C h e r n o f f 1983). Normal v a r i a t i o n s i n development, such as the one observed h e r e , may a l s o e x i s t i n o t h e r d e v e l o p m e n t a l p r o c e s s e s and would h e l p e x p l a i n d i f f e r e n c e s among normal s t r a i n s i n s u s c e p t i b i l i t y t o m a l f o r m a t i o n s . H i s t o l o g i c a l s t u d i e s d i d not r e v e a l the cause of t h e n e u r a l tube c l o s u r e a b n o r m a l i t i e s and e x e n c e p h a l y i n SELH a t the c e l l u l a r l e v e l . The n e u r o e p i t h e l i u m of SELH c r a n i a l f o l d s a r e f l a r e d w i d e r t h a n those of ICR/Bc embryos as e a r l y as 8 s o m i t e s i n development, p r i o r t o any c r a n i a l n e u r a l f o l d c l o s u r e and the u n d e r l y i n g l a t e r a l mesenchyme appears c o l l a p s e d . I n c r e a s e s i n the number of s m a l l u n s t a i n e d i n c l u s i o n s ( r e f e r r e d t o as l i p i d d r o p l e t s ) , and i n the number of p y k n o t i c c e l l s were seen i n SELH c r a n i a l f o l d s compared t o ICR/Bc. The c e l l u l a r d i f f e r e n c e s may however r e p r e s e n t u n r e l a t e d v a r i a t i o n among s t r a i n s , and no c e r t a i n p a t h o l o g i c a l cause f o r the c l o s u r e d e f e c t s was 137 i d e n t i f i e d . SELH mesenchyme, n e u r o e p i t h l e l i u m and s u r f a c e e p i t h e l i u m a l l appeared normal i n morphology, c e l l d e n s i t y , and t i s s u e volume d u r i n g the i n i t i a l s t a g e s of c r a n i a l n e u r a l tube c l o s u r e . The normal c e l l shape of neuroectoderm c e l l s s u g g e s t s t h a t m i c r o f i l a m e n t s and m i c r o t u b u l e s a r e f u n c t i o n i n g w i t h i n c e l l s . SELH embryos a l l manage the i n i t i a l e l e v a t i o n of the c r a n i a l f o l d s which o c c u r s between 1-6 s o m i t e s i n development. The p r o c e s s t h a t i s d e f i c i e n t i s t h a t of c o n t i n u e d e l e v a t i o n and a p p r o x i m a t i o n of the n e u r a l f o l d s . I t i s p o s s i b l e t h a t the c o n c e n t r a t i o n s of m i c r o f i l a m e n t s and m i c r o t u b u l e s a re low, however the observed normal c e l l shapes of neuroectoderm c e l l s do not s u p p o r t t h i s . There may be a d e f i c i e n c y of h y a l u r o n i c a c i d and/or o t h e r components of the e x t r a c e l l u l a r m a t r i x which c o u l d p r e v e n t the movement of the n e u r a l ectoderm towards the m i d l i n e by f a i l i n g t o f o r c e the f o l d s t o g e t h e r . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y of neuroectoderm c e l l s , as w e l l as immuno-f l u o r e s c e n c e and h i s t o c h e m i c a l s t u d i e s of a c t i n , c a l c i u m , and e x t r a c e l l u l a r m a t r i x components of SELH embryos d u r i n g n e u r a l tube c l o s u r e may h e l p i d e n t i f y the cause of the c l o s u r e f a i l u r e . The presence of m o r p h o l o g i c a l l y normal a d r e n a l g l a n d s found i n SELH e x e n c e p h a l i c newborns i s c o n s i s t e n t w i t h the f a i r l y i n t a c t c o n d i t i o n of the exposed b r a i n i n the s e newborns. The g e s t a t i o n p e r i o d of a mouse i s 19 days, 1/12 the g e s t a t i o n p e r i o d of a human f e t u s , and the l e n g t h of time of exposure of the open 138 b r a i n t o the a m n i o t i c f l u i d i s about 10 days i n the mouse and about 8 months i n humans. The p i t u i t a r y i s p r o b a b l y s t i l l i n t a c t and r e g u l a t i n g the proper f u n c t i o n i n g of the a d r e n a l c o r t e x i n the mouse newborn, whereas the p i t u i t a r y of a human a n e n c e p h a l i c newborn i s o f t e n d e generated and not s t i m u l a t i n g the a d r e n a l g l a n d s . B. Sex r a t i o About 17% of SELH f e t u s e s a r e e x e n c e p h a l i c . S p i n a b i f i d a and or t a i l a n o m a l i e s a r e not found. The observed f r e q u e n c y of e x e n c e p h a l y i n female SELH f e t u s e s i s about 22%, and i n males about 12% (about 1/2 of t h a t of females) i n the SELH (1987) c o n t r o l s t u d y . T h i s sex d i f f e r e n c e , which was a l s o o b s e r v e d i n e x e n c e p h a l i c F2, B C l , and BC2 f e t u s e s , i s not e a s i l y e x p l a i n e d by p o s t - i m p l a n t a t i o n l o s s of p o t e n t i a l l y e x e n c e p h a l i c males, as the number of r e s o r p t i o n s and f e t a l d e aths i s low i n SELH, and i n the F2, B C l , and BC2 l i t t e r s . N o t a b l y , the I.S F l r e s o r p t i o n f r e q u e n c y was observed t o be 3.3%, w i t h 0% e x e n c e p h a l y . I f a l l the r e s o r p t i o n s were males w i t h e x e n c e p h a l y , the sex r a t i o of e x e n c e p h a l i c s would be c l o s e t o 1:1 i n SELH, but would show a male exc e s s i n the o u t c r o s s e s ( t h a t i s , .70F:1M i n the S.I F2, •48F:1M i n the B C l , and .80F:1M i n the BC2 [ d e r i v e d from t a b l e X X I I I ] ) . The sex r a t i o among a l l f e t u s e s s u g g e s t s t h a t t h e r e i s not a s u b s t a n t i a l embryonic l o s s of males, as i n a l l c r o s s e s the sex r a t i o of t o t a l f e t u s e s ranged from 1.03 F : l M t o 0.85 F : l M ( t a b l e X XVI). I t appears t h a t t h e r e may be a d i f f e r e n c e i n r i s k of n e u r a l tube maldevelopment, r a t h e r than s u r v i v a l , i n males. 139 A d i f f e r e n c e i n the c l o s u r e p a t t e r n between male and female SELH embryos does not appear t o be the b a s i s f o r the e x c e s s of female e x e n c e p h a l i c s . Almost a l l SELH embryos l a c k c l o s u r e 2 and fuse the c r a n i a l n e u r a l f o l d s from the p r o s e n c e p h a l o n base ( c l o s u r e 3 ) . T h e r e f o r e , the cause of the sex d i f f e r e n c e i s not a d i f f e r e n c e i n the sequence of n e u r a l tube c l o s u r e between males and f e m a l e s . S i m i l a r d i f f e r e n c e s i n the sex r a t i o of e x e n c e p h a l i c s have been observed f o r the m u t a t i o n s " c r a n i o r a c h i s c h i s i s " ( K a l t e r 1985), " e x e n c e p h a l y " ( W a l l a c e , K n i g h t and Anderson 1978), and " c u r l y - t a i l " (Embury, S e l l e r , A d i n o l f i e t a l 1979). A marked female e x c e s s has been found i n a l l of t h e s e s t u d i e s , w i t h sex r a t i o s v a r y i n g from 2F:1M t o 4F:1M i n e x e n c e p h a l i c s . These v a l u e s a r e a l s o c o n s i s t e n t w i t h the sex r a t i o of a n e n c e p h a l i c s i n humans which has been observed t o range from 3-7F:lM (Shulman 1979) . At t h e time of n e u r a l tube c l o s u r e (days 8-9 of g e s t a t i o n ) , gonad d i f f e r e n t i a t i o n has not y e t o c c u r r e d . X chromosome i n a c t i v a t i o n o c c u r s on about day 4. I t may be t h a t some a s p e c t of normal e a r l y development i n females i n c r e a s e s t h e i r r i s k of d e v e l o p i n g a n t e r i o r n e u r a l tube d e f e c t s . One p o s s i b l e d i f f e r e n c e between male and female e a r l y embryos c o u l d be t h a t male embryos c l o s e t h e i r n e u r a l tubes e a r l i e r t han females and t h e r e f o r e have a d e v e l o p m e n t a l advantage i n c o m p l e t i n g n e u r a l 140 tube c l o s u r e . In s u p p o r t of t h i s S e l l e r and P e r k i n s - C o l e (1987) found t h a t male c u r l y t a i l embryos were g e n e r a l l y more de v e l o p e d and had l a r g e r s o mite c o u n t s on day 9 ( p l u g day = day 1) t h a n f e m a l e s . The c r i t i c a l q u e s t i o n however, of whether n e u r a l tube c l o s u r e i s advanced i n males r e l a t i v e t o somite count compared w i t h females i s not y e t answered. A s t u d y of n e u r a l tube c l o s u r e and g e n e r a l development i n day 8 and 9 male and f e m a l e SELH embryos would answer t h i s q u e s t i o n f o r e x e n c e p h a l y i n the SELH s t o c k . Another p o s s i b l e d i f f e r e n c e between male and female e a r l y embryos i s a s s o c i a t e d w i t h X chromosome i n a c t i v a t i o n i n f e m a l e s . I f d e l a y e d X - i n a c t i v a t i o n o c c u r r e d i n females i t c o u l d make a mammalian embryo e f f e c t i v e l y a n e u p l o i d f o r a p e r i o d i n development, d i s r u p t i n g n e u r a l tube c l o s u r e ( H a l l 1987). T h i s h y p o t h e s i s however, does not e x p l a i n the o c c u r r e n c e of e x e n c e p h a l y w e l l above any background f r e q u e n c y i n males i n mice or human p e d i g r e e s g e n e t i c a l l y a t r i s k f o r n e u r a l tube d e f e c t s . C. G e n e t i c s of e x e n c e p h a l y i n SELH and c r o s s e s L i a b i l i t y t o e x e n c e p h a l y appears t o be f i x e d i n the SELH s t o c k . A l l SELH a n i m a l s appear t o be c a p a b l e of p r o d u c i n g e x e n c e p h a l y i n t h e i r o f f s p r i n g . 53/53 SELH males s i r e d e x e n c e p h a l i c s when t e s t e d , i n d i c a t i n g t h a t t h e major gene(s) c a u s i n g e x e n c e p h a l y i n the s t o c k a r e l i k e l y f i x e d i n a t l e a s t 94.5% of a n i m a l s . T h i s r e s u l t f i t s w e l l w i t h the p r e v i o u s o b s e r v a t i o n t h a t a l m o s t a l l SELH embryos l a c k c l o s u r e 2 d u r i n g n e u r a l tube development, 141 s u g g e s t i n g t h a t a l l SELH are both g e n e t i c a l l y and d e v e l o p m e n t a l l y abnormal i n t h e i r n e u r a l tube c l o s u r e . A l t h o u g h the f r e q u e n c y of e x e n c e p h a l y i n SELH i s o n l y about 17%, the u n d e r l y i n g d e f e c t , t h a t of the l a c k of a c l o s u r e s i t e , i s p r e s e n t i n al m o s t a l l SELH embryos. The f i n d i n g t h a t l i a b i l i t y t o e x e n c e p h a l y i s t r a n s m i t t e d by a t l e a s t 94.5% i n d i c a t e s t h a t the g e n e t i c cause i s not a f u l l y p e n e t r a n t r e c e s s i v e l e t h a l , where one t h i r d of the a d u l t c o l o n y would be n o n - c a r r i e r s . The phenomenon of low f r e q u e n c i e s of a b i r t h d e f e c t i n a s t o c k " f i x e d " f o r the d e f e c t i s not unu s u a l i n mouse mutant s t r a i n s . For example, the A/WySn s t r a i n w i t h over 200 g e n e r a t i o n s of i n b r e e d i n g has a f r e q u e n c y of c l e f t l i p of o n l y about 30% ( J u r i l o f f and H a r r i s 1985). A t h r e s h o l d model i s commonly used t o e x p l a i n t h i s , whereby a n o r m a l l y d i s t r i b u t e d p o p u l a t i o n l i e s c l o s e t o the t h r e s h o l d f o r a d e f e c t , and s m a l l e n v i r o n m e n t a l f a c t o r s , or random chance, push embryos a t the t a i l of the d i s t r i b u t i o n over the t h r e s h o l d . SELH males were heterogeneous i n the f r e q u e n c y of e x e n c e p h a l y each produced. Some SELH s i r e s produced v e r y h i g h f r e q u e n c i e s of the d e f e c t ( 3 9 . 6 % ) , whereas o t h e r males produced much lower f r e q u e n c i e s ( 4 . 0 % ) . I t appears t h e n , t h a t t h e r e a r e genes s e g r e g a t i n g w i t h i n SELH which a f f e c t the a b i l i t y t o a c c o m p l i s h a n t e r i o r n e u r a l tube f u s i o n s t a r t i n g from the p r o s e n c e p h a l o n base. These genes may not be c a u s a l f o r e x e n c e p h a l y i n SELH, but may i n s t e a d enhance or l i m i t the e f f e c t i v e n e s s of the a l t e r n a t i v e c l o s u r e found i n the s t o c k . These f a c t o r s might be 142 a f f e c t i n g the speed of n e u r a l tube c l o s u r e , or the degree of f l a r e i n the n e u r a l f o l d s . ICR/Bc was s e l e c t e d as the normal s t r a i n t o c r o s s t o the SELH s t o c k f o r g e n e t i c a n a l y s e s i n o r d e r t o o u t c r o s s SELH t o the most c l o s e l y r e l a t e d normal s t r a i n p o s s i b l e . The purpose of t h i s "narrow c r o s s " g e n e t i c d e s i g n was t o i d e n t i f y the number of major genes t h a t cause e x e n c e p h a l y i n the SELH s t o c k r a t h e r than t o i d e n t i f y a l l the l o c i d i f f e r i n g between 2 s t r a i n s t h a t c o u l d m o d i f y the l i a b i l i t y t o n e u r a l tube d e f e c t s . The r e s u l t s of the s t u d y of SELH, and the F l ' s , F2's, B C l ' s , and BC2's i n d i c a t e t h a t e x e n c e p h a l y i n SELH mice i s not caused by a dominant, s e x - l i n k e d , or a f u l l y p e n e t r a n t s i m p l e r e c e s s i v e gene. The h e t e r o g e n e i t y w i t h i n the SELH s t o c k appears t o have been t r a n s m i t t e d t o the F l and B C l p a r e n t s . The c o r r e l a t i o n between the I.S F2 and B C l e x e n c e p h a l y f r e q u e n c i e s of F l males i n d i c a t e d t h a t the h e t e r o g e n e i t y i s g e n e t i c . The e f f e c t s of t h i s h e t e r o g e n e i t y would i n c l u d e h e t e r o g e n e i t y between F l s i r e s i n the f r e q u e n c y of e x e n c e p h a l y produced i n the F2, as w e l l as d i f f e r e n c e s between the f r e q u e n c y of e x e n c e p h a l y produced i n the F2 by the r e c i p r o c a l F l c r o s s e s ( t h a t i s , S.I F l compared t o I.S F l s i r e s ) . H e t e r o g e n e i t y would a l s o cause g r e a t e r v a r i a t i o n among BC l s i r e s than t h a t of s i m p l e s e g r e g a t i o n of h e t e r o z y g o u s l o c i from the F l p a r e n t ( t h e i r SELH mothers would a l s o 143 c o n t r i b u t e v a r i a t i o n ) , more v a r i a n c e among BC2 l i t t e r s t h a n t h a t due t o s i m p l e s a m p l i n g e r r o r because of m a t e r n a l (SELH) g e n e t i c d i f f e r e n c e s , and consequent l e s s a c c u r a t e e s t i m a t e s of B C l b r e e d i n g v a l u e s . The r e c o v e r e d f r e q u e n c i e s of e x e n c e p h a l y i n the F2, B C l , and BC2 f e t u s e s a r e too low t o be e x p l a i n e d by a s i n g l e r e c e s s i v e gene model. E i t h e r the c r o s s e s have reduced the gene p e n e t r a n c e , or t h e r e a r e more l o c i r e q u i r e d f o r the d e f e c t . Beyond t h i s , t h e h e t e r o g e n e i t y w i t h i n SELH makes i t d i f f i c u l t t o a n a l y s e the o u t c r o s s d a t a f o r the number of major genes i n SELH c a u s i n g e x e n c e p h a l y . The l a r g e f r e q u e n c i e s of e x e n c e p h a l y r e c o v e r e d i n the F2, B C l , and BC2 do however c l e a r l y suggest t h a t not many l o c i a r e r e s p o n s i b l e f o r the d e f e c t . For example, an e p i s t a t i c , r e c e s s i v e 2 l o c u s model w i t h 22.4% p e n e t r a n c e i n females (as i n SELH) would p r e d i c t 1.4%, 5.6%, and 12.6% r e c o v e r y of the d e f e c t i n the F2, B C l , and BC2 r e s p e c t i v e l y . The observed female f r e q u e n c i e s ( I . S F2=2.4%, BC1=5.1%, and BC2=11.5%) ar e c l o s e t o those p r e d i c t e d by t h i s 2 l o c u s model. There are two b a s i c hypotheses which can account f o r the observed g e n e t i c d a t a . The f i r s t h y p o t h e s i s i s t h a t of a major c a u s a l gene f o r the d e f e c t , which causes the o m i s s i o n of c l o s u r e 2, and has m o d i f i e r s w i t h i n SELH and between SELH and ICR/Be t h a t a r e s e g r e g a t i n g i n the o u t c r o s s e s , and a f f e c t the r a t e of e x p r e s s i o n of the major gene. The a l t e r n a t e h y p o t h e s i s i s t h a t t h e r e a r e a s m a l l number of genes (2-4) which i n 144 c o m b i n a t i o n and i n the absence o£ any 1 major gene, l e a d t o the f a i l u r e of c l o s u r e 2, and thus cause the d e f e c t . The s t u d y p r e s e n t e d cannot d i s t i n g u i s h between t h e s e two hy p o t h e s e s . To t e s t f o r the presence of a s i n g l e c a u s a t i v e m u t a t i o n , a l a r g e sample of B C l male b r e e d i n g v a l u e s would be needed ( g i v e n the amount of g e n e t i c v a r i a t i o n i n SELH and between the s t r a i n s ) t o a s s e s s whether t h e s e v a l u e s f i t a bimodal d i s t r i b u t i o n ( w i t h 1 mode l i k e SELH and 1 l i k e F l b r e e d e r s ) p r e d i c t e d by the major gene model. A l t e r n a t e l y , s e v e r a l l o c i would tend t o make a unimodal d i s t r i b u t i o n w i t h the mode i n t e r m e d i a t e between SELH and F l b r e e d e r s . U n t i l the g e n e t i c v a r i a t i o n s which i n f l u e n c e the l i k e l i h o o d of f a i l u r e of c l o s u r e i n SELH a r e f i x e d , i t w i l l be d i f f i c u l t t o detemine the number of major genes c a u s i n g e x e n c e p h a l y i n t h e s t o c k . Genes c a u s i n g l i a b i l i t y t o e x e n c e p h a l y i n SELH d i d not have d e t e c t a b l e a s s o c i a t i o n w i t h Gpi-1 on chromosome 7, Mod-1 on chromosome 9, Gpt-1 on chromosome 15, or Pgk-2 on chromosome 17. In summary, what has been l e a r n e d about e x e n c e p h a l y i n SELH mice i s t h a t a l t h o u g h the f r e q u e n c y of the d e f e c t i s about 17%, a l l SELH mice appear t o be l i a b l e f o r the t r a i t , i n t h a t a l l SELH a n i m a l s f a i l t o make c l o s u r e 2 i n the c r a n i a l n e u r a l f o l d s and a l l SELH males t e s t e d (53/53) produced e x e n c e p h a l y . As w e l l , p r a c t i c a l l y a l l SELH f o l l o w an abnormal p a t t e r n of c r a n i a l n e u r a l tube c l o s u r e i n i t i a t e d a t the r o s t r a l base of the p r o s e n c e p h a l o n , d e s p i t e normal g e n e r a l development (compared t o 145 ICR/Be and SWV/Bc s t r a i n s ) . E x e n c e p h a l y i n SELH i s due t o the f a i l u r e of c l o s u r e i n the mesencephalon r e g i o n and i s not a s s o c i a t e d w i t h s p i n a b i f i d a or t a i l a n o m a l i e s . C o l l a p s e d mesenchyme and a b n o r m a l l y f l a r e d neuroectoderm t i s s u e a r e found i n the h i s t o l o g i c a l s e c t i o n s of SELH embryos, a l o n g w i t h i n c r e a s e s i n the number of p y k n o t i c c e l l s i n SELH neuroectoderm and mesenchyme, and i n the number of l i p i d d r o p l e t s i n SELH neuroectoderm. There i s h e t e r o g e n e i t y w i t h i n the SELH s t o c k i n the f r e q u e n c y of e x e n c e p h a l y produced by males, i n d i c a t i n g t h a t t h e r e a r e genes s t i l l s e g r e g a t i n g which a f f e c t the embryos a b i l i t y t o complete a n t e r i o r n e u r a l tube c l o s u r e . The sex r a t i o w i t h i n e x e n c e p h a l i c s i n d i c a t e s a female e x c e s s i n a l l c r o s s e s , w i t h a sex r a t i o of 2.44 f e m a l e s : l male i n SELH. An i n c r e a s e i n l i a b i l i t y i n female embryos f o r e x e n c e p h a l y i s s u g g e s t e d . The g e n e t i c c r o s s e s have been a b l e t o e l i m i n a t e the hypotheses of dominance, s e x - l i n k a g e , and f u l l y p e n e t r a n t s i m p l e r e c e s s i v i t y as the cause of e x e n c e p h a l y i n SELH. Genes c a u s i n g l i a b i l i t y t o e x e n c e p h a l y i n SELH a r e not d e t e c t a b l y a s s o c i a t e d w i t h Gpi-1 on chromosome 7, Mod-1 on chromosome 9, Gpt-1 on chromosome 15, or Pgk-2 on chromosome 17. F i n a l l y , the F2, B C l , and BC2 f r e q u e n c i e s of e x e n c e p h a l y suggest t h a t the number of l o c i c a u s i n g the d e f e c t i s not l a r g e . REFERENCES Amuso S., Mankln H. (1967): H e r e d i t a r y s p o n d y l o l i s t h e s i s and s p i n a b i f i d a - r e p o r t of a f a m i l y i n which l e s i o n i s t r a n s m i t t e d as an autosomal dominant t h r o u g h 3 g e n e r a t i o n s . J o u r n a l of Bone and J o i n t S u r g e r y 49A:507-513. B a t c h e l o r A., P h i l l i p s R., S e a r l e A. (1966): A comparison of the mutagen e f f e c t i v e n e s s of c h r o n i c n e u t r o n - and If i r r a d i a t i o n of mouse spe r m a t o g o n i a . M u t a t i o n R e s e a r c h 3:218-129. B i d d l e F. (1975): T e r a t o g e n e s i s of A c e t a z o l a m i d e i n the CBA/J and SWV s t r a i n s of mice. T e r a t o l o g y 11:37-46. Bronson F., Dagg C , S n e l l G. (1966): " R e p r o d u c t i o n " i n B i o l o g y  of the L a b o r a t o r y Mouse, ed. E. L. Green, M c G r a w - H i l l , New York ppl94-195. Brun R., Garson J . (1983): N e u r u l a t i o n i n the Mexican salamander (Ambystoma mexicanum): a drug s t u d y and c e l l shape a n a l y s i s of the e p i d e r m i s and the n e u r a l p l a t e . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 74:275-295. Burman G., Smith A., Howard J . (1986): R i s k f a c t o r s i n the p r e v a l e n c e of anencephalus and s p i n a b i f i d a i n New Z e a l a n d . T e r a t o l o g y 33:221-230. Bush K., Marchander D., Nguyen H., Nagele R., Lee H. (1986): S t u d i e s on the mechanisms of n e u r u l a t i o n i n the c h i c k : L o c a l i z e d changes i n c e l l shape v s . bending of the n e u r o e p i t h e l i u m . New J e r s e y Academy of S c i e n c e , B u l l e t i n 31:8. Campbell L., Dayton D., S o h o l G. (1986): N e u r a l tube d e f e c t s : A r e v i e w of human and a n i m a l s t u d i e s on the e t i o l o g y of n e u r a l tube d e f e c t s . T e r a t o l o g y 34:171-187. C a r t e r C. (1974): Recurrence r i s k of common c o n g e n i t a l m a l f o r m a t i o n s . The P r a c t i t i o n e r 213:667-676. C a r t e r C. (1974b): C l u e s t o the a e t i o l o g y of n e u r a l tube m a l f o r m a t i o n s . Developmental M e d i c i n e and C h i l d N e u r o l o g y . , supp. 16:3-14. C a r t e r C , Evans K. (1973): S p i n a b i f i d a and anencephalus i n g r e a t e r London. J o u r n a l of M e d i c a l G e n e t i c s 10:209-220. C o f f e y V., J e s s o p W. (1957): A s t u d y of 137 cases of a n e n c e p h a l y . B r i t i s h J o u r n a l of P r e v e n t a t i v e S o c i a l M e d i c i n e 11:174-180. Co l e W., T r a s l e r D. (1980): Gene-teratogen i n t e r a c t i o n i n i n s u l i n - i n d u c e d mouse e x e n c e p h a l y . T e r a t o l o g y 22:125-139. 147 Collmann R., S t o l l e r A. (1962): E p i d e m i o l o g y o£ c o n g e n i t a l a n o m a l i e s of the c e n t r a l nervous system w i t h s p e c i a l r e f e r e n c e t o p a t t e r n s i n the s t a t e of V i c t o r i a , A u s t r a l i a . J o u r n a l of M e n t a l D e f i c i e n c i e s Research 6:22-37. Copp A., S e l l e r M., P o l a n i P. (1982): N e u r a l tube development i n mutant ( c u r l y t a i l ) and normal mouse embryos: the t i m i n g of p o s t e r i o r neuropore c l o s u r e i n v i v o and i n v i t r o . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 69:151-167. Curnow, R., S m i t h , C. (1975): M u l t i f a c t o r i a l models f o r f a m i l i a l d i s e a s e s i n man. J o u r n a l of the R o y a l S t a t i s t i c a l S o c i e t y A. 138:131-168. DeLorenzo R., Ruddle F. (1969): G e n e t i c c o n t r o l of two e l e c t r o -p h o r e t i c v a r i a n t s of Glucosephosphate Isomerase i n the mouse (Mus m u s c u l u s ) . Biochem Genet. 3:151-162. Dempsey E., T r a l s e r D. (1983): E a r l y m o r p h o l o g i c a l a b n o r m a l i t i e s i n S p l o t c h mouse embryos and p r e d i s p o s i t i o n t o gene and R e t i n o i c a c i d - i n d u c e d n e u r a l tube d e f e c t s . T e r a t o l o g y 28:461-472. D i c k i e M. (1964): New S p l o t c h a l l e l e s i n the mouse. J o u r n a l of H e r e d i t y 55:97-101. E i c h e r E., Womach J . (1977): Chromosomal l o c a t i o n of s o l u b l e G l u t a m i c - P y r u v i c Transaminase-1 (GPT-1) i n the mouse. Biochem Genet 15:1-8. E i c h e r E., C h e r r y M., F l a h e r t y L. (1978): Autosomal P h o s p h o g l y c e r a t e K i n a s e l i n k e d t o mouse major h i s t o c o m p a t i b i l i t y complex. Molec Genet 158:225-228. Embury S., S e l l e r M., A d i n o l f i M., P o l a n i P. (1979): N e u r a l tube d e f e c t s i n c u r l y - t a i l mice. I . I n c i d e n c e , e x p r e s s i o n , and s i m i l a r i t y t o the human c o n d i t i o n . P r o c e e d i n g s of the R o y a l S o c i e t y of London B 206:85-94. Elwood J . , Elwood J . (1980): E p i d e m i o l o g y of anencephalus and  s p i n a b i f i d a , O x f o r d U n i v e r s i t y P r e s s , T o r o n t o . Embury S., F e d r i c k J . (1976a): Anencephalus i n S c o t l a n d 1961-92. B r i t i s h J o u r n a l of P r e v e n t a t i v e S o c i a l m e d i c i n e 30:132-137. F e d r i c k J . (1976): Anencephalus i n the O x f o r d r e c o r d l i n k a g e s t u d y a r e a . Developmental M e d i c i n e and C h i l d N e u r o l o g y 18:643-656. F i n n e l l R., Moon S., A b b o t t L., Golden J . , C h e r n o f f G. (1986): S t r a i n d i f f e r e n c e s i n h e a t - i n d u c e d n e u r a l tube d e f e c t s i n mice. T e r a t o l o g y 33:247-252. F r a s e r F.C. (1976): The m u l t i f a c t o r i a l / t h r e s h o l d concept - uses and m i s u s e s . T e r a t o l o g y 14:267-280. 148 Freeman M., W i l l i a m s , D., schmike R., Temtamy S., V a c h i e r E,, German J . (1974): The R o b e r t s syndrome. C l i n i c a l G e n e t i c s 5:1-16. F r e z a l J . , K e l l e y J . , G u i l l e m o t M., Lamy M. (1964): Anencephaly i n F r a n c e . American J o u r n a l of Human G e n e t i c s 16:336-350. F r i e d K., L i b a n E., L u r i e M., Friedman S., R e i s n e r S. (1971): P o l y c y s t i c k i d n e y s a s s o c i a t e d w i t h m a l f o r m a t i o n s of the b r a i n , P o l y d a c t y l y and o t h e r b i r t h d e f e c t s i n newborn s i b s . J o u r n a l of M e d i c a l G e n e t i c s 8:285-290. Geelan J . , Langman J . (1977): C l o s u r e of the n e u r a l tube i n the c e p h a l i c r e g i o n of the mouse embryo. A n a t o m i c a l Record 189:625-640. G i r o u d A. (1960): "Causes and morphogenesis of a n e n c e p h a l y " . I n C i b a F o u n d a t i o n Symposium on C o n g e n i t a l M a l f o r m a t i o n s r London, 1960, ed. G. W o l l s t e n h o l m e , C. O'Connor. C h u r c h i l l , London, ppl99-240. Golden J . , C h e r n o f f G. (1983): A n t e r i o r n e u r a l tube c l o s u r e i n the mouse: f u e l f o r disagreement w i t h the c l a s s i c a l t h e o r y . C l i n i c a l R e s e a r c h 31:127A. Gordon R. (1985): A r e v i e w of the t h e o r i e s of v e r t e b r a t e n e u r u l a t i o n and t h e i r r e l a t i o n s h i p t o the mechanics of n e u r a l tube b i r t h d e f e c t s . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 89 (supp) 229-255. Green M. ed. (1981): G e n e t i c v a r i a n t s and s t r a i n s of the l a b o r a t o r y mouse, Gustav F i s c h e r V e r l a g New York pp38-270. Gropp A., Grohe A. (1981): S t r a i n background dependence of e x p r e s s i o n of chromosome t r i p l i c a t i o n i n the mouse embryo. H e r e d i t a s 94:7-8. Gruneberg H. (1954): G e n e t i c a l s t u d i e s on the s k e l e t o n of the mouse. V I I . C u r l y - t a i l . G e n e t i c s 52:52-67. H a l l J . (1987): L e c t u r e : G e n e t i c h e t e r o g e n e i t y i n n e u r a l tube d e f e c t s i n B r i t i s h Columbia. A p r i l 14, 1987. H a r r i s M., McLeod M. (1982): E y e l i d growth and f u s i o n i n f e t a l mice - a s c a n n i n g e l e c t r o n m i c r o s c o p y s t u d y . Anatomy and Embryology 164:207-220. Hol l o w a y A., Hornby J . , Muntz L. (1984): An SEM s t u d y of n e u r u l a t i o n i n normal and l e t h a l g r a y (Lg) s y r i a n hamsters M e s o c r i c e t u s a u r a t u s . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 89 (supp) 229-255. 149 Holmes, L., D r i s c o l l S., A t k i n s L. (1976): E t i o l o g i c h e t e r o g e n e i t y of n e u r a l - t u b e d e f e c t s . The New England J o u r n a l of M e d i c i n e 294:365-369. Jacobson A., O d e l l G., Os t e r G. (1985): The c o r t i c a l t r a c t o r model f o r e p i t h e l i a l f o l d i n g : A p p l i c a t o n t o the n e u r a l p l a t e . M o l e c u l a r D e t e r m i n a n t s of Animal Form, ppl43-166. Jacobson A., Tarn P. (1982): C e p h a l i c n e u r u l a t i o n i n the mouse embryo a n a l y z e d by SEM and morphometry. A n a t o m i c a l Record 203:375-396. J a n e r i c h D. (1973): Epidemic waves i n the p r e v e l a n c e of an e n c e p h a l y and s p i n a b i f i d a i n N.Y. S t a t e . T e r a t o l o g y 8:253-256. Johnson D. (1967): E x t r a t o e s : a new mutant gene c a u s i n g m u l t i p l e a b n o r m a l i t i e s i n the mouse. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 17:543-581. Johnson D. (1969): B r a c h y p h a l a n g y , an a l l e l e of e x t r a - t o e s i n the mouse. G e n e t i c Research 13:275-280. Johnson D. (1976): The i n t e r f r o n t a l bone and mutant genes i n the mouse. J o u r n a l of Anatomy 121:507-513. J u r i l o f f D. (1987): M a t e r n a l t r e a t m e n t w i t h c o r t i s o n e a c c e l e r a t e s e y e l i d c l o s u r e and o t h e r d e v e l o p m e n t a l f u s i o n p r o c e s s e s i n f e t a l mice. Development 100:611-618. J u r i l o f f D., H a r r i s M. (1985): T h y r o x i n e - i n d u c e d d i f f e r e n t i a l m o r t a l i t y of c l e f t l i p mouse embyros: Dose and time response s t u d i e s of the A/WySn s t r a i n . T e r a t o l o g y 31:319-329. J u r i l o f f D., H a r r i s M., F r o s t e r - I s k e n i u s U. (1987): H e m i f a c i a l d e f i c i e n c y i n d uced by a s h i f t i n dominance of the mouse m u t a t i o n f a r : A p o s s i b l e g e n e t i c model f o r h e m i f a c i a l m i c r o s o m i a . J o u r n a l of C r a n i o f a c i a l G e n e t i c s and Developmental B i o l o g y 7:27-44. J u r i l o f f D., S u l i k K., R o d e r i c k T., Hogan B. (1985): G e n e t i c and d e v e l o p m e n t a l s t u d i e s of a new mouse m u t a t i o n t h a t produces o t o c e p h a l y . J o u r n a l of C r a n i o f a c i a l G e n e t i c s and Developmental B i o l o g y 5:121-145. K a l t e r H. (1983): Noninbred mutant mouse s t o c k s d i f f e r i n t e r a t o l o g i c r e s p o n s i v e n e s s t o r e t i n o i c a c i d . T e r a t o l o g y 27:54A. K a l t e r H. (1985): Sex and t e m p o r a l v a r i a t i o n s i n h e r e d i t a r y mouse e x e n c e p h a l y . T e r a t o l o g y 31:69A. K a r f u n k e l P. (1974): The mechanisms of n e u r a l tube f o r m a t i o n . I n t e r n a t i o n a l Review of C y t o l o g y 38:245-272. 150 K a r f u n k e l P., Hoffman M., P h i l l i p s M., B l a c k J . (1978): Changes i n c e l l a d h e s i v e n e s s i n n e u r u l a t i o n and o p t i c cup f o r m a t i o n . Zoon 6:23-31. Kaufman M. (1979): C e p h a l i c n e u r u l a t i o n and o p t i c v e s s i c l e f o r m a t i o n i n the e a r l y mouse embryo. American J o u r n a l of Anatomy 155:425-444. Karp G., B e r r i l l N. (1981): Development Second e d i t i o n . McGraw- H i l l Book Co. Toronto p.374. Keeton W. (1980): B i o l o g i c a l Sciences,. W. W. N o r t o n and Company, New York p.906. Khoury M., E r i c k s o n J . , James L. (1982): E t i o l o g i c h e t e r o g e n e i t y of n e u r a l tube d e f e c t s : C l u e s from e p i d e m i o l o g y . American J o u r n a l of E p i d e m i o l o g y 115:538-548.. K u r n i t D., L a y t o n W., Mathysse S. (1987): G e n e t i c s , Chance, and Morphogenesis. American J o u r n a l of Human G e n e t i c s . 41:979-995. Laurence K.M., James N., M i l l e r M., Tennant G., Campbell H. (1981): Double b l i n d randomized c o n t r o l l e d t r i a l of f o l a t e t r e a t m e n t b e f o r e c o n c e p t i o n t o p r e v e n t r e c u r r e n c e of n e u r a l tube d e f e c t s . B r i t i s h M e d i c a l J o u r n a l 282:1509-1511. Leek I . , Rogers S. (1967): Changes i n the i n c i d e n c e of a n e n c e p h a l u s . B r i t i s h J o u r n a l of P r e v e n t a t i v e S o c i a l M e d i c i n e 21:177-180. Lee H., K o s c i u k M., Nagele R. (1983): S t u d i e s on the mechanisms of n e u r u l a t i o n i n the c h i c k : p o s s i b l e i n v o l v e m e n t of myosin i n e l e v a t i o n of n e u r a l f o l d s . J o u r n a l of E x p e r i m e n t a l Zoology 225:449-457. Lee H., Nagele R. (1985): S t u d i e s on the mechanism of n e u r u l a t i o n i n the c h i c k : I n t e r r e l a t i o n s h i p s of c o n t r a c t i l e p r o t e i n s , m i c r o f i l a m e n t s , and the shape of n e u r o e p i t h e l i a l c e l l s . J o u r n a l of E x p e r i m e n t a l Zoology 235:205-215. Lemire R., B e c k w i t h J . , Warkany J . (1978): Anencephaly. Raven P r e s s , New York. Lewis W. (1947): Mechanics of i n v a g i n a t i o n . A n a t o m i c a l Record 97:139-156. Lyon M. (1959): A new dominant T - a l l e l e i n the house mouse. J o u r n a l of H e r e d i t y 50:140-142. Lyon M., M o r r i s T., S e a r l e A., B u t l e r J . (1967): Occurrence and l i n k a g e r e l a t i o n s of the mutant <<extra-toes>> i n the mouse. G e n e t i c R e s e a r c h 9:383-385. 151 MacMahon B., Yen S. (1971): U n r e c o g n i z e d e p i d e m i c of anencephaly and s p i n a b i f i d a . L a n c e t i:31-33. M a r i n - P a d i l l a M. (1966): Mesodermal a l t e r a t i o n s induced by h y p e r v i t a m i n o s i s A. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 15:261-269. McBride M. (1979): S i b r i s k s of a n e n c e p h a l y and s p i n a b i f i d a i n B r i t i s h Columbia. American J o u r n a l of M e d i c a l G e n e t i c s 3:377-387. Moase C , T r a s l e r D. (1987): H i s t o p a t h o l o g y of s p i n a l g a n g l i o n i n i d e n t i f i e d S p l o t c h - d e l a y e d mouse n e u r a l tube d e f e c t s mutants. T e r a t o l o g y 35:26A. Morgan W. (1954): A new c r o o k e d t a i l m u t a t i o n i n v o l v i n g d i s t i n c t i v e p l e o t r o p i s m . J o u r n a l of G e n e t i c s 52:354-373. M o r r i s s - K a y G. (1981): Growth and development of p a t t e r n i n the c r a n i a l n e u r a l e p i t h e l i u m of r a t embryos d u r i n g n e u r u l a t i o n . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 65 (supp) 225-241. M o r r i s s G., S o l u r s h M. (1978): R e g i o n a l d i f f e r e n c e s i n mesenchymal c e l l morphology and g l y c o s a m i n o g l y c a n s i n e a r l y n e u r a l f o l d s t a g e r a t embryos. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 46:37-52. M o r r i s s - K a y G., P u t z B. (1986): Abnormal n e u r a l f o l d development i n mouse t r i s o m y 12 and t r i s o m y 14. I I LM and REM. B r a i n R e s e a r c h B u l l e t i n 16:825-832. Nagele R., P i e t r o l u n g o J . , Lee H. (1981): S t u d i e s on the mechanisms of n e u r u l a t i o n i n the c h i c k : The i n t r a c e l l u l a r d i s t r i b u t i o n of Ca++1. E x p e r e n t i a 37:304-306. O'Shea K., M e t c a l f W. (1980): Chromosome unbalance and mouse n e u r a l tube c l o s u r e : an SEM and TEM s t u d y . T e r a t o l o g y 21:60A. Penrose L. (1957): G e n e t i c s of anencephaly. J o u r n a l of M e n t a l D e f i c i e n c y R e s e a r c h 1:4-15. P u t z B., M o r r i s s - K a y G. (1981): Abnormal n e u r a l f o l d development i n t r i s o m y 12 and t r i s o m y 14 mouse embryos. I Scanning e l e c t r o n m i c r o s c o p y . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 66:141-158. R i c h a r d s I . (1973): F e t a l and i n f a n t m o r t a l i t y a s s o c i a t e d w i t h c o n g e n i t a l m a l f o r m a t i o n s . B r i t i s h J o u r n a l of P r e v e n t a t i v e S o c i a l M e d i c i n e 27:85-90. Ri m o i n D. ( 1 9 8 3 ) : " G e n e t i c d i s o r d e r s of the p i t u i t a r y g l a n d " i n P r i n c i p l e s and P r a c t i c e of M e d i c a l G e n e t i c s , ed. A. Emery, D. R i m o i n , C h u r c h h i l l L i v i n g s t o n e I n c . , New York, p755. 152 S a d l e r T., Greenberg D., C o u g h l i n P. (1982): A c t l n d i s t r i b u t i o n p a t t e r n s i n the mouse n e u r a l tube d u r i n g n e u r u l a t i o n . S c i e n c e 215:172-174. Schoenwolf G. (1982): On the morphogenesis of the e a r l y r u diments of the d e v e l o p i n g c e n t r a l nervous system. S c a n n i n g E l e c t r o n M i c r o s c o p y I 289-308. Schoenwolf G., F i s h e r M. (1983): A n a l y s i s of the e f f e c t s of Streptomyces h y a l u r o n i d a s e on f o r m a t i o n of the n e u r a l t u b e . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 73:1-15. S e a r l e A. (1966): C u r t a i l e d , a new dominant T- a l l e l e i n the house mouse. G e n e t i c Research 7:86-95. S e l l e r M. (1985): P e r i c o n c e p t i o n a l v i t a m i n s u p p l e m e n t a t i o n t o p r e v e n t r e c u r r e n c e of n e u r a l tube d e f e c t s . L a n c e t 1:1392. S e l l e r M., A d i n o l f i M. (1979): N e u r a l tube d e f e c t s i n c u r l y - t a i l mice . I n c i d e n c e , e x p r e s s i o n and s i m i l a r i t y t o the human c o n d i t i o n . P r o c e e d i n g s of the R o y a l S o c i e t y of London ( B i o l ) 206:85-94. S e l l e r M., Embury S., P o l a n i P., A d i n o l f i M. (1979): N e u r a l tube d e f e c t s i n c u r l y - t a i l mice. I I E f f e c t of m a t e r n a l a d m i n i s t r a t i o n of v i t a m i n A. P r o c e e d i n g s of the R o y a l S o c i e t y of London. B 206:95-107. S e l l e r M., P e r k i n s K. (1982): P r e v e n t i o n of n e u r a l tube d e f e c t s i n c u r l y - t a i l mice by m a t e r n a l a d m i n i s t r a t i o n of v i t a m i n A. P r e n a t a l D i a g n o s i s 2:297-300. S e l l e r M., P e r k i n s K., A d i n o l f i M. (1983): D i f f e r e n t i a l response of h e t e r o z y g o u s c u r l y - t a i l mouse embryos t o v i t a m i n A t e r a t o g e n e s i s depending on m a t e r n a l genotype. T e r a t o l o g y 28:123-129. S e l l e r M., P e r k i n s - C o l e K. (1987): Sex d i f f e r e n c e i n mouse embryonic development a t n e u r u l a t i o n . J o u r n a l of R e p r o d u c t i v e F e r t i l i t y 79:159-161. S e l l e r M., P e r k i n s - C o l e K. (1987): H y p e r t h e r m i a and n e u r a l tube d e f e c t s of the c u r l y t a i l mouse. J o u r n a l of C r a n i o f a c i a l G e n e t i c s and Developmental B i o l o g y . 7:321-330. Sheenan D. C , Hrapchk B.B. (1980): Theory and P r a c t i c e of  H i s t o t e c h n o l o q y second e d i t i o n . C V . Mosby Co., T o r o n t o , ppl39-142. Shows T., Ruddle F. (1968): M a l a t e Dehydrogenase: e v i d e n c e f o r t e t r a m e r i c s t r u c t u r e i n mus musculus. S c i e n c e 160:1356-1357. 153 Shulman K. (1979): "Anencephaly", i n B i r t h d e f e c t s compendium second e d i t i o n , ed. D. Bergsma. A l a n R. L i s s I n c , New York p.83. S i e g e l S. (1956): N o n p a r a m e t r i c S t a t i s t i c s f o r the B e h a v i o r a l S c i e n c e s , M c G r a w - H i l l , New York, p p l l 6 - 1 2 1 . Smedley M., S t a n i s t r e e t M. (1986): C a l c i u m and n e u r u l a t i o n i n mammalian embryos. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 93:167-178. Smith L., S t e i n K. (1962): A x i a l e l o n g a t i o n i n the mouse and i t s r e t a r d a t i o n i n homozygous l o o p t a i l mice. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 10:73-87. S m i t h e l l s R. (1968) : I n c i d e n c e of c o n g e n i t a l a b n o r m a l i t i e s i n L i v e r p o o l , 1960-1964. B r i t i s h J o u r n a l of P r e v e n t a t i v e S o c i a l M e d i c i n e 22:36-37. S m i t h e l l s R., Sheppard S., Schorah C , S e l l e r M., N e v i n N., H a r r i s R., Read A., F i e l d i n g D. (1981): Apparent p r e v e n t i o n of n e u r a l tube f e d e c t s by p e r i c o n c e p t i o n a l v i t a m i n s u p p l e m e n t a t i o n . A r c h i v e s of D i s e a s e s of C h i l d h o o d 56:911-918. Snedecor G., Cochran W. (1967): S t a t i s t i c a l Methods 6th  e d i t i o n Iowa S t a t e U n i v e r s i t y P r e s s , Iowa, pp432-436. S t a a t s J . (1985): S t a n d a r d i z e d nomenclature f o r i n b r e d s t r a i n s of mice. J o u r n a l of Cancer Research 45:965. S t e i n K., R u d i n I . (1953): Development of mice homozygous f o r the gene f o r l o o p - t a i l . J o u r n a l of H e r e d i t y 44:59-68. S t r o n g L., H o l l a n d e r W. (1949): H e r e d i t a r y l o o p - t a i l i n the house mouse. J o u r n a l of H e r e d i t y 40:329-334. S u l i k k., J o h n s t o n M. (1983): Sequence of d e v e l o m e n t a l a l t e r a t i o n s f o l l o w i n g a c u t e e t h a n o l exposure i n mice: C r a n i o f a c i a l f e a t u r e s of the f e t a l a l c o h o l syndrome. The American J o u r n a l of Anatomy 166:257-269. T h e i l e r K., Stevens L. (1960): The development of R i b F u s i o n s , a m u t a t i o n i n the house mouse. American J o u r n a l of Anatomy 106:171-183. Theo d o s i s D., F r a s e r F.C. (1978): E a r l y changes i n the mouse n e u r o e p i t h e l i u m p r e c e e d i n g e x e n c e p h a l y induced by h y p e r v i t a m i n o s i s A. T e r a t o l o g y 18:219-232. T r i m b l e B., B a i r d P. (1978): C o n g e n i t a l a n o m a l i e s of the c e n t r a l nervous s y s t e m : I n c i d e n c e i n B r i t i s h Columbia, 1952-1972. T e r a t o l o g y 17:43-49. 154 T r u s l o v e G. ( 1 9 5 2 ) : G e n e t i c a l s t u d i e s on the s k e l e t o n o£ the mouse V " i n t e r f r o n t a l " and " p a r t e d f r o n t a l s " . J o u r n a l of G e n e t i c s 51:115-122. T u c k e t t F., M o r r i s s - K a y G. (1985): The k i n e t i c b e h a v i o r of the c r a n i a l n e u r a l e p i t h e l i u m d u r i n g n e u r u l a t i o n i n the r a t . J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 85:111-119. Unger A., H a r r i s M., B e r n s t e i n S. (1983): H e m o l y t i c anemia i n the mouse- r e p o r t of a new m u t a t i o n and c l a r i f i c a t i o n of i t s g e n e t i c s . J o u r n a l of H e r e d i t y 74:88-92. W a l l a c e M., K n i g h t s P., Anderson J . (1978): I n h e r i t a n c e and morphology of e x e n c e p h a l y , a n e o n a t a l l e t h a l r e c e s s i v e w i t h p a r t i a l p e n e t r a n c e , i n the house mouse. G e n e t i c R e s e a r c h Cambridge 32:135-149. W a l l a c e M., K n i g h t s P., Dye A. (1976): P i l o t s t u d y of the m u t a g e n i c i t y of DDT i n mice. E n v i r o n m e n t a l P o l l u t i o n 11:217-222. Warkany J . (1971):"Anencephaly" i n C o n g e n i t a l M a l f o r m a t i o n s , notes and comments. Yearbook M e d i c a l p u b l i s h e r s I n c . Chicago ppl96-198. Warkany J . (1971): C o n g e n i t a l M a l f o r m a t i o n s n o t e s and comments Year Book M e d i c a l P u b l i s h e r s , C h i c a g o , PP189-200. W i l s o n D. (1985): H i s t o c h e m i c a l a n a l y s i s of the n e u r a l b a s a l l a m i n a i n the h i n d b r a i n r e g i o n of e x e n c e p h a l i c mutant mice. J o u r n a l of C r a n i o f a c i a l G e n e t i c s and Developmental B i o l o g y 5:363-371. W i l s o n D., Center E. (1974): The n e u r a l c e l l c y c l e i n the l o o p - t a i l (Lp) mutant mouse. J o u r n a l of Embryology and E x p e r i m e n t a l Morphology 32:697-705. W i l s o n D., F i n t a L. (1979): Gap j u n c t i o n a l v e s i c l e s i n the n e u r a l tube of the S p l o t c h (Sp) mutant mouse. T e r a t o l o g y 19:337-340. Wright S, ( 1 9 3 4 ) : The r e s u l t s of c r o s s e s between i n b r e d s t r a i n s of g u i n e a p i g s , d i f f e r i n g i n number of d i g i t s . G e n e t i c s 19 .-537-551. Yen S., MacMahon B. (1968): G e n e t i c s of a n e n c e p h a l y and s p i n a b i f i d a . L a n c e t i i : 6 2 3 - 6 2 6 . 

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:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0097709/manifest

Comment

Related Items