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Morphogenesis in micrasterias Lacalli, Thurston Castle 1973

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MORPHOGENESIS IN MICRASTERIAS by THURSTON CASTLE LACALLI B.Sc, University of Washington, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of 1 Zoology We accept t h i s thesis as conforming to the required standard. The University of B r i t i s h Columbia June, 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requ i rem*, i t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t he Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f Z O O L O G Y The U n i v e r s i t y o f B r i t i s h Co l umb i a V a n c o u v e r 8, Canada Date 23 A u g u s t , 1 9 7 3 1 Abstract The morphogenetic process responsible for elabora-t i o n of c e l l wall shape i n d i v i d i n g Micrasterias rotata c e l l s i s described and the associated u l t r a s t r u c t u r e discussed. The process i s dissected experimentally i n t o i t s contributing parts; l a s e r microbeam studies reveal the growth s i t e s re-sponsible f o r morphogenesis, and.autoradiography, the asso-ciated synthetic patterns. Tip growth s i m i l a r to that oc-curring i n fungal hypae, root hairs and pollen tubes makes a major contribution to c e l l shape. Tip growth, together with branching and broadening of the growths produced,accounts f o r the patterns of morphogenesis exhibited i n M. rotata and M. radlata, two species having d i s t i n c t i v e shape di f f e r e n c e s . The a b i l i t y of d i v i d i n g c e l l s to impose a b i r a d i a t e symmetry on the developing c e l l wall i s discussed and i s a t t r i -buted to a template. Evidence i s presented that f o r both the morphogenetic process and the formation of t h i s template, appropriate s p a t i a l information and organization i s embodied and maintained i n the c e l l cortex and i s not imposed by the cytoplasm or nucleus. Both nucleus and cytoplasm play only an i n d i r e c t , supportive r o l e ; cytoplasmic p o l a r i t y and s p e c i f i c structures such as microtubules are also not of primary impor-tance. Template formation and t i p growth are not reduced to t h e i r biochemical mechanisms, but are instead discussed i n terms of known c e l l wall microstructure and physical proper-ties'. A discussion of the nature of s c i e n t i f i c explanations i s included to demonstrate that the explanations employed i n 11 t h i s thesis are s c i e n t i f i c a l l y s a t i s f y i n g even though devoid of exact molecular mechanisms. ; i i i Table of Contents Introduction 1 Materials and Methods 11 Results and Discussion I. Observations on Sectioned Material 22 I I . Observations on L i v i n g Material 4 l I I I . Primary C e l l Wall Composition 54 IV. Laser Experiments 58 V. Autoradiography 70 VI. Sections through the Isthmus 78 Discussion I . Tip Growth i n Micrasterias 82 I I . The Template i n Micrasterias 91 I I I . Morphogenesis and Organization 9 5 IV. Morphogenesis and Explanation 101 Bibliography 107 i v L i s t of Tables Table I C e l l Cultures 12 II Treatments f o r C e l l Wall Preparations 17 I I I Chemicals Used ^ 19 IV Histochemical Analysis of Primary C e l l 55 Walls V S t a b i l i t y of D i f f e r e n t Symmetries i n 79 Haploid Micrasterias V L i s t of- Figures Figure 1 Interphase c e l l of M.rotata 8 2 Morphogenesis i n d i v i d i n g M.rotata 8 3 Normal and abnormal fac i e s of three Micrasterias species 9 4 Summary of published experiments concerning Micrasterias morphogenesis 10 5 Laser microbeam apparatus 20 6 Method of mounting c e l l s f o r i r r a d i a t i o n 20 7 Laser microbeam dimensions 21 8 Fine-structure of interphase c e l l s 32 9 Fine-structure of prophase c e l l s 33 10, 11 Fine-structure of telophase c e l l s 34, 35 12, 13 Fine-structure of a developing c e l l , stage 7 36, 37 14 Fine-structure of an early prophase c e l l 38 15 Reconstruction of the events of mitosis and cytokinesis 39 16 Comparison of microtubule positioning and the s i t e of primary wall i n i t i a t i o n 40 17 Scanning micrographs of c e l l isthmuses 40 18 Morphogenetic stages of M.jrotata 47 19 Rates of c e l l development i n M.rotata 47 20 E f f e c t of temperature on semicell development 48 21 C e l l shape i n d i f f e r e n t species 4$ 22 Morphogenetic stages of M.radlata 49 23 E f f e c t of osmotic conditions on semicell development 50 Figure 24 E f f e c t of enzymes on semicell development 5 l 25 Various experimental manipulations of developing c e l l s 52 26 Intermediate f a c i e s i n three species 53 27 Double c e l l s 53 28 Laser damage to polar lobes 64 29 Laser damage to wings 6.4 30 Laser damage to lobe t i p s 65 31 Laser damage to lobe notches 65 32 Summary of laser experiments 66 33 Idealized fate map of s i n g u l a r i t i e s 66 34, 35 Laser damage to young semicells 6-7 36, 37 Summary of laser experiments on young semicells 68 38 Laser fusion of membrane and wall 69 39 Glucose autoradiography of c e l l walls 7:4 40 Methionine autoradiography of c e l l walls 75 41 Tip l a b e l i n g i n autoradiograms 76 42 Idealized c e l l wall fate map 77 43 Summary diagram f o r autoradiography 7:7 44 Isthmus cross-sections 81 45 Robertson's model f o r hyphal t i p growth 90 46 Morphogenetic variations on t i p growth 96 47 Template regions at stage 2 94 48 Models of septum growth 94 49 Physical model of t i p growth dynamics 100 Acknowledgement I wish to thank the members of my thesis committee for t h e i r comments and Dr. Acton f o r his support and en-couragement over the past four years. I also thank Mrs. Chas. 0. Tolstad f o r typing the manuscript. 1 Introduction The desmids are a rather s p e c i a l i z e d group of fresh-water green algae d i s t i n c t i v e i n t h e i r methods of reproduction, both sexual (by conjugation) and vegetative (Fritsch, 1 9 3 5 ) . The placoderm desmids, of which Micrasterias i s an example, are characterized by a c e l l u l o s i c c e l l wall ( K l e i n and Cronquist, I967) comprising two d i s t i n c t halves connected by an isthmus ( f i g . 1 ) . Micrasterias appears deeply constricted at the isthmus as the two c e l l halves (semicells) are rather large. Each semicell i s divided i n t o lobes by a number of further c o n s t r i c t i o n s or notches. There are generally f i v e lobes; a central polar lobe and i n each of the l a t e r a l wings, two major lobes which branch repeatedly g i v i n g the wing perimeter a den-tated appearance. The c e l l nucleus l i e s at the very center of the c e l l , i n the isthmus. During vegetative c e l l d i v i s i o n , a daughter nucleus i s segregated to each semicell and a septum forms at the isthmus separating the cytoplasm of the two semicells ( f i g . 2 ) . The septum i s d i s t i n c t l y d i f f e r e n t from the semicell wall i n composition and structure, resembling more the primary walls of higher plants. Hence the septum and the wall i n t o which i t develops can be considered as the c e l l ' s primary wall ( M i x , I 9 6 I ) . Once formed, the septum bulges out i n t o the shape of a hemisphere ( f i g . 2 a ) : The hemisphere develops a pattern of lobes and notches as i t grows ( f i g . 2 b and c) so as to produce In several hours a new semicell exactly i n the image of 2 the old (Kiermayer and Jarosch, 1962; Lezenweger, 1966). When t h i s morphogenetic process i s complete, a thick layer of wall i s l a i d down immediately inside the primary wall and the primary wall i s cast o f f . The new semicell i s then l e f t enclosed by a secondary wall only, t h i s being the normal interphase condi-t i o n (Drawert and Mix, 1962b). The Micrasterias c e l l s so f a r figured are rather f l a t i n side view and are examples of c e l l types one would f i n d i n nature ( f i g . 3a, c and e ) . The c e l l s are haploid and show a normal or b i r a d i a t e symmetry. Other symmetry types called " f a c i e s " (by T e l l i n g ) have appeared spontaneously i n c e l l c u l -tures or have been induced to form (Heimans, 1942; T e l l i n g , 1950; Waris and K a l l i o , 1964; K a l l i o and H e i k k i l a , 1969). D i p l o i d c e l l s frequently become t r i r a d i a t e ( f i g . 3b) or quadriradiate, by producing one or two extra wings aligned i n d i f f e r e n t planes. Haploid c e l l s occasionally lose one or two wings to become uniradiate ( f i g . 3d and f ) or aradiate. The loss occurs i n a single daughter semicell, but the change may be h e r i t a b l e producing from t h i s one semicell a clone having a new symmetry. Symmetry type appears to be inherited through a non-genetic mechanism, at l e a s t i n the related genus Cosmarium (Starr, 1958; Tews, 1969). That c e l l s with increased nuclear ploidy tend to become t r i - or quadriradiate ( K a l l i o 1954, i960) can be ex-plained as a general rather than a s p e c i f i c e f f e c t of the nucleus, a r e s u l t of nuclear e f f e c t on c e l l turgor (Green, 1969). 3 The rather elegant process of primary c e l l wall mor-phogenesis has been the subject of my thesis work. I w i l l r e f e r to the process as morphogenesis or development and avoid c a l l i n g i t d i f f e r e n t i a t i o n . Various workers have studied the morphogenetic pro-cess i n Micrasterias and t r i e d to explain how the very s p e c i f i c pattern of lobes and notches a r i s e s . T e l l i n g (1950) referred to the a c t i v i t y of "meristematic cytoplasm," f e e l i n g that the peripheral cytoplasm i s r e g i o n a l l y d i f f e r e n t i a t e d i n t o areas which could support wall growth, hence producing lobes, and those which could not and would become notches. Kiermayer enlarged on these ideas suggesting f i r s t that the meristematic regions are distinguished by t h e i r a b i l i t y to deposit c e l l wall material (Kiermayer and Jarosch, 1962), and second that the meristematic areas are more loosely attached to the c e l l wall than the areas which become notches. The non-meristematic areas he c a l l e d f i x a t i o n zones, as they appeared to exhibit t i g h t f i b r i l l a r attachments between cytoplasm and c e l l wall (Kiermayer, 1964; see f i g . 4 a ) . Kiermayer has studied the u l t r a s t r u c t u r e of growing c e l l s (Kiermayer, 1968, 1970b) and suggested that a directed flow of v e s i c l e s containing wall precursors or a regional s p e c i a l i z a t i o n of the membrane re-ceiving these v e s i c l e s could also account f o r the d i f f e r e n t i a -t i o n of peripheral cytoplasm i n t o d i s t i n c t meristamatic regions. He has demonstrated that microtubules do not appear to be involved i n morphogenesis (Kiermayer, 1968a, 1968b). Kiermayer (1970b) was the f i r s t to t r y to break 4 Micrasterias morphogenesis down int o i t s contributing proces-ses. He distinguished f i v e major causal aspects of morpho-genesis; (1) production of c e l l wall precursors, (2) transport of precursors to the wall, (3) maintenance of a c o r t i c a l template of meristematic regions, (4) incorporation of precursors i n t o the wall, and (5) maintenance of a turgor threshold. Kiermayer considers these f i v e to be harmoniously operating parts of an organized whole. He comments that experimental manipulations w i l l damage the contributing processes d i r e c t l y aid not morphogenesis i t s e l f . Morphogenesis, l i k e a machine with a damaged part, would then be more l i k e l y to grind to a halt than to continue to function but i n an altered pattern. This may explain why i t i s d i f f i c u l t to experimentally i n t e r -fere with c e l l morphogenesis i n any but very t r i v i a l ways. Ce l l s generally grow eithe r normally or not at all} and i t i s p a r t i c u l a r l y d i f f i c u l t to experimentally a l t e r the symmetry of new semicells i n a predictable way (Waris and K a l l i o , 1964). Castle, i n his work on growth of Phycomyces sporangiophores, comments on morphogenesis as follows (Castle, 1953; pg. 369): "Growth i s very close to being l i f e i t s e l f , with everything included and implied thereby, so that multiple causation of any phenomenon of growth i s generally to be ant i c i p a t e d . The writer i s convinced, nevertheless, that problems of orient a t i o n are exceptional, and that the bulk of physical and chemical processes contributing to growth do not confer upon i t d i r e c t i o n a l properties. Ultimately these are undeniably 5 contributed by i n t e r a c t i o n between the given genetic make-up and the environment, but i n a more immediate sense d e f i n i t e facts of struc-ture - molecular or gross - must be involved." Kiermayer f e e l s he has pinpointed the d i r e c t i o n a l contribu-t i o n to. Micrasterias morphogenesis i n his t h i r d causal as-spect, the c o r t i c a l template. His template i s formed when the septum i s formed (Kiermayer, 1967). Its information, perhaps embodied i n the adjacent cytoplasm, d i r e c t s the f o r -mation of meristematic areas. The septum could contain i n -structions f o r b u i l d i n g the whole of the elaborate c e l l wall or f o r doing only the f i r s t few steps. In a long series of experiments, Waris and K a l l i o (1964) demonstrated that the nucleus i s not d i r e c t l y required f o r c e l l morphogenesis. C e l l s enucleated by centrifugation, UV i r r a d i a t i o n or drug treatment continue to grow and develop for several hours. No matter how early the enucleation i s done, d i v i d i n g c e l l s always produce at least three lobes, one corresponding to the polar lobe and one to each of the two l a t e r a l wings ( f i g . 4 b ) . Waris and K a l l i o , therefore, pro-posed that the morphogenetic template depends upon some aspect of the cytoplasm or of cytoplasmic structure. The idea that cytoplasm may have an oriented structure, that i t Is thixo-t r o p i c , i s a very a t t r a c t i v e one. For example, Picken ( i 9 6 0 ; pg. I63) has quoted Frey-Wyssling; "'...the structure of plasm must be of a wonderful co-ordination. The framework can-not represent an unordered p i l e , but must possess an organized and well-defined structure.'" 6 but on consideration of the experimental evidence (Moore, 1935; Crick and Hughes, 1950) Picken added; "We can agree whole-heartedly that the struc-ture of plasm must be of wonderful coordina-t i o n , i f the statement ref e r s simply to the f a c t that processes i n the cytoplasm are co-ordinated. But that there i s an organized and well-defined structure, i n the sense of a f i r m l y bonded framework, i s denied by the evidence i n those c e l l s that have so f a r proved accessible to examination." In Micrasterias the cytoplasm streams a c t i v e l y throughout morphogenesis (Kiermayer, 1964) and so i t i s p a r t i c u l a r l y d i f -f i c u l t to imagine that a f i r m l y bonded cytoplasmic structure i s present. Waddington (1962) suggested that a structure might s t i l l e xist but be dynamic i n nature. I t i s clear that Stentor, f o r example, shows dynamic organization. Minced c e l l s are able to reorganize t h e i r jumbled b i t s of c e l l cortex i n t o a normal whole (Tarter, i 9 6 0 ) . Whether dynamic or s t a t i c , cytoplasmic structure does o f f e r i t s e l f as a possible template f o r Micrasterias morphogenesis. Selman (1966) has shown that such a template does not depend d i r e c t l y on the old semicell as c e l l damage i s not h e r i t a b l e ( f i g . 4c) except i n cases of considerable general c e l l damage (Selman, 1966; f i g s . E to H, plate 1) . K a l l i o (1972b) has demonstrated h e r i t a b l e damage i n M.torreyi r e s u l t i n g from TJV i r r a d i a t i o n of the cytoplasm, but i n t e r p r e t a -t i o n of these experiments i s d i f f i c u l t as the damage produced i s very non-specific. M.torreyi i s , i n any case, a very mutable species ( K a l l i o , 1968). K a l l i o (1972b) has most recent-l y suggested that the template may depend on a p a r t i c u l a r spa-t i a l ordering of cytoplasmic organelles. This has also been 7 proposed as an explanation f o r zygospore morphogenesis i n Micrasterias (Kies, 1970), but no appropriate order has yet been found i n the vegetative c e l l (Kiermayer, 1970a). Waddington's ideas about dynamic structure are highly specu-l a t i v e . He i s at a loss when asked to give more concrete explanations of the template. " I t seems to me extremely d i f f i c u l t to en-visage a mechanism by which s p a t i a l arrange-ments of material on t h i s scale of magnitude could be brought about. The conventional deus ex machina to be invoked i s ' l o c a l s p e c i a l i z a t i o n of the c e l l surface'; but the pattern of the new h a l f - c e l l i s hardly af-fected by the pattern of the old surface." Hence morphogenesis i n Micrasterias remains an un-solved problem. I t i s a very s t r i k i n g process but s t i l l a mysterious one. I took a rather broad approach to the prob-lem, wanting to understand the growth process i t s e l f but also wanting to explore e x i s t i n g hypotheses, p a r t i c u l a r l y those of Waddington concerning dynamic structure; wanting also to f i n d out about the template, i t s p o s i t i o n and the nature of i t s substance. 8 Figure 1 Normal interphase c e l l of M.rotata (a) C e l l viewed face on to show ilfs shape. The c e l l i s packed with chloroplast and pyrenoids (arrow) except at the isthmus where a cle a r area indicates presence of the nucleus. (b) Diagram of the c e l l from (a) showing i t s parts. The c e l l consists of two semicells joined at an isthmus, each semicell having a central polar lobe with a wing on each side. Figure 2 Stages of morphogenesis i n M.rotata. The stages shown are separated from one another by about one hour. Figure 18 gives a more precise representation of stages. (a) Stage 3: Mitosis i s complete and the two daughter c e l l s have been separated by a septum of primary w a l l . The septum has begun to bulge out. (b) Stage 7: Five lobes have appeared on each new semi-c e l l . The chloroplast has advanced i n t o the new semicell and vacuoles populate the cytoplasm. (c) Stage 10: An almost complete complement of lobes i s present on each semicell. The chloroplast continues i t s invasion of the new semicells. F i g u r e 1 F i g u r e 2 9 Figure 3 Scanning and l i g h t micrographs of normal and abnormal f a c i e s of three Micrasterias species M.rotata b i r a d i a t e M.rotata t r i r a d i a t e M.torreyi b i r a d i a t e M.torreyi uniradiate M.thomasiana bira d i a t e FT.thomasiana uniradiate 10 Figure 4 A diagrammatic summary of published experiments concerning Micrasterias morphogenesis (a) Experiments by Kiermayer (1964) showing by plasmolysis that the protoplast i s most t i g h t l y at-tached to the c e l l wall at lobe notches. Under appropriate conditions, f i b r i l l a r connections between cytoplasm and c e l l wall can be demonstrated at these f i x a t i o n zones (FZ). (b) Experiments by Waris and K a l l i o (1964) i n which d i v i d i n g c e l l s were enucleated with UV r a d i a t i o n . C e l l s already developing can develop further i n the absence of a nucleus. C e l l s just preparing to divide also develop when enucleated and always produce at least three lobes representing the three major axes; polar lobe and two wings. (c) Experiments by Selman (1966) i n which developing semicell lobes were i r r a d i a t e d with a UV dose s u f f i -cient to arrest lobe growth. The abnormal c e l l thus produced showed normal semicell morphogenesis a f t e r i t s next d i v i s i o n and hence the UV induced abnormality i s shown not to be h e r i t a b l e . Figure 4 10a KIERMAYER b WARIS & K A L L I O C S E L M A N 11 Materials and Methods Certain of the species used i n t h i s work ( d e t a i l s i n Table I) were purchased from the Indiana Culture C o l l e c t i o n (Starr, 1964). Others were is o l a t e d l o c a l l y and unial g a l cultures established from single i n d i v i d u a l s with i d e n t i f i c a -t i o n made by reference to West and West (1905). On occasion abnormally shaped c e l l s were observed i n the cultures. These were i s o l a t e d and cultured and i n a few cases gave clones of an abnormal but stable c e l l type. C e l l s were grown i n a s a l t solution, the MS medium of Waris (1953). A simple l i g h t box served f o r routine work, supplying fluorescent l i g h t of 50 to 120 f t . candles on a cycle of 12 hrs. l i g h t , 12 hrs. dark. On t h i s l i g h t regime the c e l l s became roughly synchronized, about 20% d i v i d i n g each day. Those d i v i d i n g on any given day were synchronized to within about an hour of one another and began d i v i s i o n from one to two hours before the l i g h t period. Temperature could not be accurately controlled i n the l i g h t box but generally kept between 20 and 23° C. Stock cultures were kept i n an environment room at 20° C under s i m i l a r l i g h t conditions. About 30 c e l l s at a time were prepared f o r electron microscopy following the method of Kiermayer (1968) with some adjustments. I n i t i a l f i x a t i o n was i n 1% glutaraldehyde f o r 8 hrs. at 4 ° C followed by a 2 hr. wash and p o s t f i x a t i o n i n 2% OsO^ overnight. Dehydrating solutions were made up by mixing methanol with 0.25$ aqueous NaCI; dehydration was begun at 15$ 12 Table I C e l l Cultures Culture Source M. rotata Isolated February, 1970 Beaver Lake, Vancouver, B. C. M. radiata Isolated March, 1971 by Marion McCauley Loon Lake near Haney, B. C. M. denticulata Isolated February, 1970 Beaver Lake, Vancouver, B. C. M. thomasiana b i r a d i a t e Indiana C o l l e c t i o n LB 543 M. thomasiana uniradiate Indiana C o l l e c t i o n LB 548 M. t o r r e y i b i r a d i a t e Indiana C o l l e c t i o n LB 794 M. s o l Indiana C o l l e c t i o n LB 649 M. radiata pygmy K & S B i o l o g i c a l Supply Vancouver, B. C. M. t o r r e y i uniradiate Isolated from b i r a d i a t e cultures June and August 1972 M. rotata t r i r a d i a t e Isolated from b i r a d i a t e cultures June 1972 13 with changes of 10$ every 5 minutes. C e l l s were not centrifuged i n t o a p e l l e t but were processed i n d i v i d u a l l y through the solu-tions using a pip e t t e . C e l l s were taken through propylene oxide into Spurr's embedding medium (Spurr, 1969) and sectioned on an LKB microtome. Thick sections (1 yu) were cut f o r l i g h t microscopy, mounted on glass s l i d e s and stained with t o l u i d i n e blue. Thin sections were supported on grids coated with p a r a l o i d i n and carbon and stained with uranyl acetate and lead c i t r a t e or with uranyl acetate alone. Staining times of up to two hours were required; lead staining was done accord-ing to Reynolds (1963). S t a i n made from commercial lead c i t r a t e was not s a t i s f a c t o r y and l e f t a peppery residue. Micrographs were taken using the Hi t a s h l HS- 7S and AEI 801S microscopes. Electron microscopy was done only on M.rotata. Divid i n g c e l l s treated i n various v i t a l dyes were i r r a d i a t e d with a helium-neon gas la s e r microbeam, the la s e r (Metrologic Instruments) producing an 0.5 mWatt continuous beam i n the red region at 633 mp. The microbeam apparatus has been previously described ( L a c a l l i and Acton, 1972). The beam was focussed using the condenser ( L e i t z #77) of a L e i t z Ortholux microscope with the condenser front lens i n place ( f i g . 5 ) . Lasings were observed and photographed by means of standard microscope optics with a green interference f i l t e r interposed so as to reduce the beam i n t e n s i t y . Direct viewing of the 14 beam i s probably not dangerous under these conditions (Bloom, 1968). Photographic records of lasings were taken as double exposures, the beam being photographed with the con-denser front lens i n place and the object photographed with normal i l l u m i n a t i o n and the lens removed. A l l the l a s e r ex-periments reported here were done with M.rotata, though experiments with M.radiata gave s i m i l a r r e s u l t s . In a t y p i c a l laser experiment, 5 to 10 d i v i d i n g c e l l s were placed i n a dish containing a known concentration of a dye i n culture medium ( d e t a i l s i n Results, Part IV). After 30 minutes, a c e l l was removed and placed i n a drop of medium between a sandwich of coverslips ( f i g . 6 ) , and t h i s put on the microscope stage. The c e l l would be lased f o r from 1 to 60 seconds with spots of diameter 2 ju or greater, though t he c e l l i t s e l f r e a l l y experiences a cone of l a s e r l i g h t ( f i g . 7 ) . F i n a l l y , a photograph was taken to record the exact stage of the lased c e l l and the point of l a s i n g , and the c e l l removed to a spot plate depression f i l l e d with culture medium so that development and l a t e r c e l l d i v i s i o n s could be observed. In t h i s fashion, one d i v i d i n g c e l l could be lased about every 5 minutes. Afte r 15 minutes, the dye solution would be freshen-ed by adding s u f f i c i e n t dye from a stock solu t i o n to double the dye concentration, as many of the dyes tend to p r e c i p i t a t e from s o l u t i o n . Generally only 5 or 6 c e l l s were lased each day so that the dye solu t i o n was freshened only once. Patterns of c e l l wall synthesis were studied i n M.rotata by exposing d i v i d i n g c e l l s to t r i t i a t e d compounds, 15 i s o l a t i n g the c e l l walls as ghosts and preparing auto-radiograms from them. Usually 30 to 50 d i v i d i n g c e l l s were placed i n a small dish containing 0.3 to 0.4 ml of culture medium made up to a l a b e l concentration of 200 ^uC/ml (glucose, methionine or proline) or 50 mC/ml (water). C e l l s were l e f t i n the so l u t i o n f o r a measured length of time and then removed to a dish of d i s t i l l e d water, washed i n several changes of water, centrifuged to the bottom of a test tube containing d i s t i l l e d water to which grains of sand had been added and the tube immersed i n an ul t r a s o n i c cleaner f o r 20 to 30 seconds. The u l t r a s o n i c cleaner served to break c e l l s apart, releasing the protoplasm. Several standard commercial models were t r i e d and a l l worked. The time from f i r s t washing to rupture by ultrasonics was standardized at 5 minutes. Isolated walls were stored i n 5 to 10 ml d i s t i l l e d water to which a few drops of commercial formalin had been added. For mounting, a few drops of 0.1$ aqueous c r y s t a l v i o l e t were also added to the sol u t i o n . This colored the p r i -mary walls so that they could be picked out under a di s s e c t i n g microscope and dried onto albuminized s l i d e s , about 25 c e l l walls to a s l i d e . The sl i d e s were dipped i n acetone to re-move the dye and i n Carnoy's f i x a t i v e f o r 10 minutes to wash out small molecules. For autoradiography, s l i d e s were dipped once i n I l f o r d L -4 nuclear emulsion made up i n hot water as 2 gm/10 ml. Slides were l e f t i n l i g h t - t i g h t boxes with dessicant f o r one month at 4° C, developed f o r 4 minutes i n D-19, stained i n 0.1$ c r y s t a l v i o l e t and mounted. 16 A few simple staining tests were done on the p r i -mary wall to determine i t s chemical properties. C e l l wall ghosts were mounted as described above f o r autoradiograms. Various extraction methods were employed and these, with controls, were stained with PAS, a l c i a n blue or c r y s t a l v i o l e t (Table I I ) . In the case of enzyme extractions, controls were done with enzyme f i r s t dissolved i n a small volume of d i s -t i l l e d water and immersed i n a b o i l i n g water bath f o r an hour. Sets of s l i d e s which were to be d i r e c t l y compared were a l l stained together. Various chemicals and enzymes were tested i n d i v i d -ing c e l l s . These were dissolved i n culture medium, and several dozen d i v i d i n g c e l l s at various early stages were then introduced. Results were discarded i f they did not consistently point to one e f f e c t , as a chemical may produce diverse e f f e c t s , k i l l i n g some c e l l s while causing others to burst, swell or arrest t h e i r morphogenesis. E f f e c t i v e con-centration ranges were determined f o r each chemical thought to be of i n t e r e s t , though no attempt was made to determine to what extent the various chemicals could penetrate c e l l s . These experiments and those on c e l l wall analysis were done with M.rotata. The shapes of i n t e r e s t i n g c e l l s were recorded i n photographs, as the outline of c e l l s i s rather complex and a hand sketch was not thought precise enough. 17 Table I I Treatments f o r C e l l Wall Preparations Treatment Procedure Reference Wash f o r autoradiograms Carnoy's f i x a t i v e , 3 0 min. at 2 0 ° C Mil d a l k a l i e x t r a c t i o n 0 . 5 $ ammonium oxalate, 24 h r s . at 6 0 ° C Jensen ( 1 9 6 2 ) A l k a l i e x t r a c t i o n 4$ NaOH, 24 h r s . at 2 0 ° C Jensen ( 1 9 6 2 ) Strong a l k a l i e x t r a c t i o n 1 7 . 5 $ NaOH, 24 h r s . 2 0 ° C Jensen ( 1 9 6 2 ) M i l d methylation 2 $ S 0 C 1 ? i n abs. methanol, 6 h r s . at 2 0 ° C Pearse ( 1 9 6 8 ) Strong methylation 0 . 5 $ H C 1 i n abs. methanol, 1 2 h r s . at 5 0 ° C Pearse ( 1 9 6 8 ) S a p o n i f i c a t i o n 1 $ HaOH i n 8 0 $ ethanol, 1 h r s . at 2 0 ° C Pearse ( 1 9 6 8 ) Chromic a c i d 1 0 $ chromic a c i d , 4 h r s . at 2 0 ° C Mix ( 1 9 6 1 ) Pectinase 0 . 5 $ enzyme i n 0 . 2 M sodium acetate b u f f e r pH 4 . 0 , 24 h r s . at 2 7 ° C Kertesz ( 1 9 5 1 ) C e l l u l a s e 0 . 5 $ enzyme i n 0.2 M sodium acetate b u f f e r pH 4 . 5 , 24 hrs at 3 7 ° C Pigman ( 1 9 5 1 ) Hemicellulase 0 . 5 $ enzyme i n 0.2 M sodium acetate b u f f e r pH 5 . 5 , 24 h r s . at 3 7 ° C Pigman ( 1 9 5 1 ) T r y p s i n 0 . 1 $ enzyme i n 0 . 0 5 M T r i s with 0 . 0 2 M C a C l 2 pH 8 . 1 , 1 6 h r s . at 2 7 ° C Walsh ( 1 9 7 0 ) P e c t i n esterase 0 . 1 $ enzyme i n 0 . 1 M phosphate b u f f e r pH 7 , 24 h r s . at 2 7 ° C Kertesz ( 1 9 5 1 ) PAS 1 $ periodate f o r 2 0 min., S c h i f f reagent f o r 3 0 min., 2 f r e s h s u l f i t e washes Jensen ( 1 9 6 2 ) A l c i a n blue pH 2 . 5 0 . 3 $ a l c i a n blue i n 3 $ a c e t i c a c i d f o r 2 0 min., washed i n 3 $ a c e t i c a c i d Parker and D i b o l l ( 1 9 6 6 ) A l c i a n blue pH 0 . 5 0 . 3 $ a l c i a n blue ad-jus t e d t o pH 0 . 5 f o r 2 0 min., washed i n water adjusted t o pH 0 . 5 Parker and D i b o l l ( 1 9 6 6 ) C r y s t a l v i o l e t . 0 . 1 $ c r y s t a l v i o l e t , 1 0 min. at 2 0 ° C Drawert and Metzner-Kuster (1961) 18 C e l l s were prepared f o r scanning electron micro-scopy by freeze-drying c e l l s onto aluminum stubs and coating with gold. Stubs were observed using a Cambridge Stereoscan microscope. Sources f o r some of the chemicals used i n t h i s work are l i s t e d i n Table I I I . 19 Table I I I Chemicals Used Chemical Source L- methionine (Methyl-3H ) ICN D- glucose - 1 - 3R ICN Water - 3H ICN L- proline - 3H (generally labeled) ICN D-glucose-6 - 3H ICN Pectin methyl esterase (tomatoe) Sigma Cel l u l a s e , p r a c t i c a l type 1 (Aspergillus) Sigma Pectinase, p u r i f i e d (fungal) Sigma Trypsin Difco Hemicellulase, crude grade I I (Rhizopus) Sigma Pronase, B-grade Cal Biochem A l c i a n blue Gurr (ESBE) 20 Figure 5 Laser micro-beam apparatus u t i l i z i n g a standard microscope of which only the condenser, stage and objective are shown. A condenser front lens of high numerical aperture and an a d d i t i o n a l short f o c a l -length lens are shown introduced i n t o the l i g h t path to further reduce beam diameter. The mirrors shown are front-surfaced to reduce r e f l e c t i o n of multiple images. Figure 6 Method of mounting c e l l s f o r laser i r r a d i a t i o n . The chamber i s constructed of cover glasses and cover glass fragments and serves to sandwich a drop of culture medium containing the c e l l to be lased. 20a Figure 5 Figure 6 C ] 21 Figure 7 Laser microbeam dimensions. An experimental l a s i n g viewed through the microscope i s seen only as a spot of l i g h t (arrow). In side view the microbeam i s a double cone of l i g h t which may i r r a d i a t e a much larger area of wall than i s immediately apparent. 21g 22 Results and Discussion I. Observations on Sectioned Material Reports have been published on the f i n e structure of several Micrasterias species describing general character-i s t i c s of the c e l l s and t h e i r organelles; nucleus and nucleo-lus (Drawert and Mix, 196lb, 1961c), chloroplast and pyrenoids (Drawert and Mix, 196ld, 1962a), c e l l wall (Drawert and Mix, 196la; Kiermayer and Staehelin, 1972), g o l g i (Drawert and Mix, 196le; Staehelin and Kiermayer, 1970), mitochondria (Drawert and Mix, 196lf) and the microtubular system (Kiermayer, 1968b). Kiermayer (1970a) has studied the structure of d i v i d i n g c e l l s and Kies (1970) of the developing zygospore. My own EM studies have been b r i e f , but directed toward the solu t i o n of s p e c i f i c questions concerning morphogenesis. My survey of general structure i n M.rotata confirms the published reports, but I also looked at the u l t r a s t r u c t u r e of septum formation f o r which there i s no published d e s c r i p t i o n . Very early stages of septum formation cannot be distinguished by l i g h t microscopy. For t h i s study, about 30 c e l l s were selected from a synchronized culture i n which a number of c e l l s were beginning to d i v i d e . When sectioned, 15 of these could be seen to be i n interphase and the others i n various early d i v i s i o n stages; 4 i n post-telophase, 4 In telophase, 2 i n metaphase and 5 i n prophase. One c e l l was at a p a r t i c u l a r l y early stage of c e l l d i v i s i o n and showed only the barest suggestion of a forming primary w a l l . 23 Problems with f i x a t i o n and i n f i l t r a t i o n were en-countered. I solved the l a t t e r by using Spurr's embedding medium rather than Epon, as Spurr's i s much less viscous. The f i x a t i o n problem was not so e a s i l y solved. The cytoplasm of both d i v i d i n g and interphase c e l l s was found to be f i l l e d with large empty spaces, probably caused by the bursting of s l i m e - f i l l e d v e s i c l e s during f i x a t i o n . Kiermayer (1968b) found s i m i l a r damage i n the cytoplasm of M.dentlculata, but eliminated the spaces and maintained a population of large, membrane-bound vesicl e s by using glutaraldehyde very sparing-l y , f i x i n g i n 1$ glutaraldehyde f o r 10 minutes. I was unable to improve the appearance of M.rotata cytoplasm using Kiermayer's f i x a t i o n schedule and hence r e l i e d routinely on long g l u t a r a l -dehyde f i x a t i o n so as to minimize general shrinkage of the protoplast. This was important as I was interested i n the cytoplasm-cell wall i n t e r f a c e . Figure 8 summarizes the u l t r a s t r u c t u r e of a t y p i c a l interphase c e l l . Mitochondria are long and threadlike but generally are seen i n cross-section. A row of microtubules l i e s adjacent to the c e l l wall at the isthmus, forming rings around the nucleus. Dictyosomes show large and regularly stacked cisternae and l i n e the chloroplast membrane i n a l l parts of the c e l l . Small v e s i c l e s with dark centers appear throughout the cytoplasm but p a r t i c u l a r l y around the dictyo-somes. They correspond to Kiermayer's dark v e s i c l e s (DV) and appear to be associated with the dictyosome tubules. The d i c -tyosomes are c l e a r l y not producing v e s i c l e s from t h e i r cisternae. 24 Large vesic l e s appear throughout the cytoplasm; t h e i r con-tents are contracted to the center of each ve s i c u l a r space and resemble i n appearance the slime contained i n the pores of secondary w a l l . From thick sections the chloroplast can be seen to be a t h i n wafer, coated, as i s the c e l l wall, with a t h i n layer of cytoplasm. C e l l volume i s largely taken up with vacuole or packed with large v e s i c l e s . Figure 9 i l l u s t r a t e s c e l l s probably i n prophase. A g i r d l e of wall material encloses the isthmus and i s of d i f -ferent thickness i n d i f f e r e n t c e l l s . This g i r d l e appears to be primary wall and clo s e l y resembles i n structure the b i t s of primary wall l e f t from previous c e l l d i v i s i o n s . Dictyosomes throughout the c e l l appear a c t i v e . They are not so c l o s e l y associated with the chloroplast as i n interphase, and t h e i r outer cisternae are greatly enlarged. Stacks of ER are also present and l i e near the chloroplast. Microtubules do not appear to be as abundant at the isthmus as i n interphase c e l l s , but I have only a few micrographs on which to base t h i s conclusion. There i s nothing d i s t i n c t i v e about the nu-c l e i i n these c e l l s to indicate that the c e l l s are i n prophase of mitosis. Prophase as seen i n the l i g h t microscope may l a s t several hours and precedes septum formation (Waris, 1950a). These c e l l s appear to be at a stage of d i v i s i o n approaching septum formation and so I w i l l r e f e r to t h i s stage of wall development as a prophase stage. Figures 10 and 11 i l l u s t r a t e a telophase c e l l with the septum about h a l f formed. Nuclear envelope encloses the many small chromosomes, and spindle microtubules are s t i l l v i s i b l e . 25 Microtubules appear to be e n t i r e l y absent from the septum and isthmus area. The cytoplasm i s p a r t i c u l a r l y dense around the inner edge of the septum, perhaps because large v e s i c l e s ap-pear to be excluded from t h i s area. A few membrane-bound vesicles l i e along the primary wall external to the plasma membrane. Figures 12 and 13 i l l u s t r a t e a c e l l of about stage 7 of c e l l morphogenesis. Chloroplast lobes enter the new semi-c e l l on either side of the nucleus. Dictyosomes c l u s t e r about the ends of the chloroplast i n an area which i n l i v e c e l l s does not stream as a c t i v e l y as cytoplasm i n the semicell lobes i s observed to do (Jarosch, 1962). Lobes themselves are f i l l e d with vesicles and ER. By stage 7, microtubules should again be present at the surface of secondary wall at the Isthmus (Kiermayer, 1968b), but I have not observed them. Microtubules are present adjacent to the nucleus. Vesicles are occasionally seen between the plasma membrane and c e l l w a l l . Peculiar lamel l a r structures also occur i n these spaces and i n the cytoplasm, but t h e i r d i s t r i b u t i o n i n the c e l l follows no conspicuous pattern. The primary wall appears to be of s i m i l a r structure throughout and i s not noticeably d i f f e r e n t i n structure from the primary wall present at e a r l i e r stages. Figure 14 i l l u s t r a t e s sections of a single c e l l which appears to be at a very early stage of primary wall formation. I w i l l c a l l t h i s stage an early prophase stage, though again, EM does not properly reveal the state of nuclear organization. In the early prophase c e l l , a t h i n , uniform layer of primary w a l l - l i k e material g i r d l e s the isthmus. Deposits of s i m i l a r 26 material occur also i n interphase c e l l s and are found most often along the inside of secondary wall at notches i n old semicells. The early prophase c e l l represents the only case i n which such a deposit was found at the isthmus. In addition, dictyosomes appeared more active i n t h i s c e l l than i n i n t e r -phase c e l l s . They appear i n the cytoplasm at some distance from the chloroplast and have outer cisternae which are s l i g h t l y enlarged. Microtubules are present at the isthmus as i s a band of vesicles l y i n g external to the plasma membrane and e n c i r c l i n g the isthmus. Figure 15 shows semidiagrammatically the course of septum development as suggested by the EM evidence presented here. Of p a r t i c u l a r i n t e r e s t i s the movement of microtubules. Bands of wall microtubules si m i l a r to the isthmus band are seen i n other desmids, i n Closterium (Pickett-Heaps and Fowke, 1970a) and i n Penium (Gerrath, 1968). The isthmus band i s reminiscent of the preprophase band of microtubules found to enc i r c l e the nucleus i n some higher plants. These bands may supply microtubules to the mitotic spindle either i n subunit form or as whole tubules able to migrate from wall to spindle during prophase (Pickett-Heaps and Northcote, 1966). I t i s not surprising then that microtubules are absent from the isthmus wall during mitosis i n M.rotata. Microtubules also l i e along side the post-telophase nucleus. Kiermayer (1968a) suggests that these may anchor the nucleus and return i t to the isthmus following chloroplast migration i n a fashion s i m i l a r to that observed i n Closterium. When Micrasterias Is treated with c o l c h i c i n e (Kiermayer, 1968a), both nucleus 27 and chloroplast take up abnormal positions i n the new semi-c e l l . Post-telophase microtubules may act through t h e i r a b i l i t y to s t a b i l i z e cytoplasmic gels (Porter, 1966), strength-ening the cytoplasm around the invading chloroplast and nucleus. There i s considerable argument as to whether micro-tubules act to d i r e c t deposition of c e l l wall materials. There i s good evidence that they do d i r e c t secondary wall deposition (Pickett-Heaps, 1967), but f a r less as regards primary walls (Newcomb, 1969). Only with N l t e l l a has colchicine been shown to a l t e r synthetic patterns (Green, 1962) and here the observa-tions were rather cursory. Microtubules are present along many growing primary walls but absent from many others, p a r t i c u l a r l y from the growing regions of c e l l s showing t i p growth (Newcomb, I969). Only a very few microtubules are found along the grow-ing primary wall of M.denticulata, and col c h i c i n e does not a l t e r morphogenesis of t h i s species (Kiermayer, 1968a, 1968b) or of M.rotata. Microtubules may orient septa or d i r e c t the i n i t i a t i o n of cross-walls i n some c e l l s . Microtubules are present i n some c e l l s which divide by furrowing (Johnson and Porter, 1968) and are important i n the construction of c e l l plates, i n which they may l i e l o n g i t u d i n a l l y as i n phragomo-plasts or transversely as i n phycoplasts (Pickett-Heaps, 1972). The argument that preprophase bands of microtubules act to orient c e l l d i v i s i o n or c e l l plate formation has been d i s -credited (Pickett-Heaps, 1969a). Colchicine w i l l i n t e r f e r e with c e l l plate formation (Whaley et a l . , 1966) but does not affect septum formation i n Micrasterias (Kiermayer, 1968a). 28 It i s Interesting to speculate as to why the septum i n Micrasterias i n i t i a t e s at the isthmus and not somewhere else. Double c e l l s exist having two isthmuses but only one nucleus. These c e l l s can form a complete septum at both isthmuses ( K a l l i o , 1963) and hence the po s i t i o n of the nucleus i s not an important factor, rather there i s something peculiar about the isthmus i t s e l f . Note from figure 15 that the g i r d l e i n i t i a l l y appears at the isthmus roughly adjacent to the re-gion of wall microtubules. Kiermayer (1968b) supports the idea that these microtubules are an important positioning factor i n septum formation. Note the vesicles collected about the g i r d l e i n figure 14 ( f i g . l4c and f ) ; these could be taking part i n g i r d l e thickening or septum i n i t i a t i o n . And micro-tubules thus associated with vesic l e s at a time of g i r d l e thickening could be acting as guide elements i n g i r d l e deposi-t i o n and septum i n i t i a t i o n much as they are thought to do i n secondary wall thickening. Microtubule bands are c h a r a c t e r i s t i c Isthmus struc-tures, but the isthmus also lacks the pores found throughout the rest of the secondary wall ( f i g . 17). Slime contained i n these pores coats both the inside and outside of the wall, hence the isthmus may be the only part of the c e l l at which completely naked secondary wall i s exposed to the adjacent cytoplasm. Figure 16 shows the primary wall i n i t i a l stopping abruptly at the f i r s t pore body. Microtubules i n t h i s region t h i n out only gradually and l i e over wall which i s not accumulating primary wall material. The s p a t i a l correspondence between i n i t i a l 2 9 g i r d l e deposition and wall nakedness i s , therefore, better than that between deposition and microtubule p o s i t i o n . The wall could be o f f e r i n g , at the isthmus, a substratum f o r wall deposition found nowhere else i n the c e l l . And the argument could be made that microtubules are only one of several c o n t r i -buting f a c t o r s , that the substratum upon which deposition occurs i s also very important, being i n t h i s case the l i m i t i n g f a c t o r . Within the usual l i m i t a t i o n s of micrograph interpre-t a t i o n , g o l g i v e s i c l e s are generally thought of as a means of packaging and transporting c e l l wall substances i n plants (O'Brien, 1972). It i s not s u r p r i s i n g then, that i n Micrasterias the dictyosome cisternae. become very active just as primary c e l l wall i s f i r s t being l a i d down. The product of the dictyosome cisternae, large vesicles with rather f r a g i l e - l o o k i n g membranes, appear very l i t t l e d i f f e r e n t from the large v e s i c l e s present throughout interphase. These two sorts probably cor-respond to the single population of L v e s i c l e s found by Kiermayer (1970a) i n M.denticulata. I have not seen cl e a r e v i -dence of incorporation of large v e s i c l e s i n t o primary wall and neither has Kiermayer. He f e e l s that the L v e s i c l e s may supply slime to pores i n the secondary wall rather than being pre-cursors of primary wall. This conclusion i s strengthened by my observation ( f i g . 10b) that the inner edge of the growing septum, most l i k e l y i t s growing edge, i s surrounded by cytoplasm densely packed with dark v e s i c l e s but from which large v e s i c l e s are excluded. Kies (1970) has shown that the zygote of M. p a p i l l i f e r a forms a primary exospore very l i k e the vegetative primary wall i n structure. Small, dark vesi c l e s are present In 30 large number and appear to be associated with the dictyosomes; but no large slime vesi c l e s are present, and the dictyosome cisternae are i n a c t i v e . Related freshwater algae; Closterlum (Pickett-Heaps and Fowke, 1970a), Triploceras (Gerrath, 1968), Spirogyra (Fowke and Pickett-Heaps, 1969) and Oedogonlum (Pickett-Heaps and Fowke, 1970b), a l l have large v e s i c l e s i n both interphase and d i v i s i o n stages s i m i l a r to those I found i n M.rotata. Fowke and Pickett-Heaps (1969) c a l l these "fuzzy" vesic l e s and suggest that they contain slime which i s c o n t r i -buted to the c e l l cross-wall when i t forms. Kiermayer i s the only microscopist who has managed to f i x these vesi c l e s so as to maintain t h e i r membranes. In Closterlum and Spirogyra the vesic l e s show fusion p r o f i l e s with growing primary wall, and i n both cases these can be attributed to f i x a t i o n a r t i f a c t . In both Closterium and Spirogyra there i s a large population of smaller vesi c l e s associated with wall growth. In Spirogyra t h i s association i s p a r t i c u l a r l y clear as i t i s small vesi c l e s rather than large ones which form the phragmoplast or c e l l p l a te. From the evidence above, one might conclude that large ves i c l e s are not as important i n t h e i r contribution to primary wall as are other gol g i products despite the consider-able production of large ves i c l e s occurring at each c e l l d i v i s i o n . But large ves i c l e s are consistently associated with primary wall, p a r t i c u l a r l y at stages of g i r d l e thickening ( f i g . 14c and f ) , and so i t i s d i f f i c u l t to imagine these ve s i c l e s having no role at a l l . Dictyosomes i n other plant c e l l s have been shown to produce two sorts of vesicles 31 simultaneously, both associated with polysaccharide synthesis and transport (Pickett-Heaps, 1968); the tubular system sur-rounding distyocome cisternae may produce i t s own product d i s t i n c t from that of the cisternae (Mollenhauer and Morre, 1966). In M.rotata the role of the several sorts of g o l g i v e s i c l e s In production of primary c e l l wall has yet to be pre-c i s e l y determined. In conclusion, electron microscopy reveals some of the cytoplasmic machinery responsible f o r c e l l wall synthesis, but provides no f a c i l e explanation f o r the various patterns of wall morphogenesis. As a possible exception to t h i s , during g i r d l e i n i t i a t i o n , primary wall deposition correlates s p a t i a l l y with both microtubules and with regions of wall lacking pores. Both may p a r t i c i p a t e i n d i r e c t i n g wall deposi-t i o n at t h i s p a r t i c u l a r stage of development. 32 Figure 8 Fine-structure of interphase c e l l s (a) Thick section of an entir e c e l l . The nucleus with i t s n u c l e o l i i s at the center. The very dark areas are chloro-plast and contain occasional pyrenoids. Most of the l i g h t areas are either vacuole or are packed with large vesicles and contain very l i t t l e cytoplasm. Stained with t o l u i d i n e blue. (b) A pore i n the secondary wall having a canal through which pass threads of slime. These exit the canal on both outside and in s i d e , on the in s i d e forming a pore bulb. Stained with uranyl acetate and lead, x 52,200 (c) Part of the c e l l isthmus. The two semicells come together here, and t h e i r secondary walls can be seen to hook into one another (arrow). A row of microtubules are seen i n cross-section along the wall; they en-c i r c l e the isthmus. Stained with uranyl acetate only, x 20,800 (d) Mitochondrion i n long i t u d i n a l section. Mitochondria are seen as threads i n l i v i n g c e l l s but are most frequent-ly cut across i n sections. Stained with uranyl acetate and lead, x 16,000 (e,f) Interphase dictyosomes showing ti g h t stacks of flattened cisternae. Dictyosomes are clos e l y associated with chloroplast and l i e within the t h i n layer of cyto-plasm adjacent to the chloroplast. Elements of ER, both cisternae and tubules, are found immediately adjacent to the chloroplast membrane l y i n g between chloroplast and dictyosomes. Vesicles of various sorts including large ve s i c l e s and dark v e s i c l e s are present throughout the cytoplasm. Smaller vesicles and tubular elements are p a r t i c u l a r l y abundant at the margins of dictyosome cisternae. Stained with uranyl acetate and lead, x 18,700 x 16,400 Abbreviations: Nucleus (N), mitochondria (M), secondary wall (SW), chloroplast (Ch), large v e s i c l e s (LV), dark v e s i c l e s (DV), slime threads (Th), pore canal (C), pore bulb (B)# microtubules (Mt) 33 Figure 9 Fine-structure of prophase c e l l s (a) Thick section of an entire c e l l very l i t t l e changed from the interphase condition. The nucleus i s perhaps s l i g h t l y larger and the chloroplasts have withdrawn to some extent from the isthmus area. Stained with t o l u i d i n e blue. (b) C e l l isthmus showing the semicell walls no longer as t i g h t l y hooked as i n interphase. A t h i n g i r d l e of primary wall material l i n e s the inside of the secondary wall. A t u f t of s i m i l a r material i s also present between the two semicell walls and i s probably primary wall re-maining from a previous c e l l d i v i s i o n . Possible wall microtubules are v i s i b l e . Stained with uranyl acetate only, x 19,300 (c) A l a t e r prophase isthmus showing the thickened g i r d l e of primary wall material. Microtubules are not evident. Stained with uranyl acetate and lead, x 26,700 (d) Dictyosomes l y i n g loose i n the cytoplasm. The cisternae are active; that i s , they are i n f l a t e d to form large v e s i c u l a r spaces. Numerous dark v e s i c l e s are present (large arrow) and frequently one dictyosome cisterna stains more darkly than the others (small arrow). Stained with uranyl acetate and lead, x 22,000 Abbreviations: Nucleus (N), microtubules (Mt), primary wall (PW), secondary wall (SW) 3 3 . F i g u r e 9 34 Figure 10 Fine-structure of telophase c e l l s (a) Thick section of a whole c e l l . The septum i s complete except f o r a small gap at the center; some bulging of the septum has occurred. Both daughter nuclei are v i s i b l e . Stained with t o l u i d i n e blue (b) Growing primary wall septum with one end anchored to the secondary wall and the other l y i n g free i n the cytoplasm (arrow). This leading edge of the septum i s surrounded by a region of dense cytoplasm (double arrows) r i c h i n small v e s i c l e s and from which large vesicles are excluded, stained with uranyl acetate and lead, x 19,400 (c) Central region of the isthmus. Note the leading edge of the growing septum (arrow) and the surrounding area of dense cytoplasm. No c e l l plate structures are seen associated with septum formation and no microtubules are seen i n the cytoplasm adjacent to the septum. Stained with uranyl acetate and lead, x 14,200 (d) Telophase nucleus with condensed chromatin and an incompletely reformed nuclear envelope (arrows). Stained with uranyl acetate and lead, x 12,600 Abbreviations: Nucleus (N), large v e s i c l e s (LV), dark v e s i c l e s (DV), secondary wall (SW), primary wall (PW) 343 F i g u r e 10 35 Figure 11 Fine-structure of telophase c e l l s . Sections stained with uranyl acetate and lead. (a) Endoplasmic reticulum showing the large stacks of cisternae present i n d i v i d i n g c e l l s . These usually appear f i r s t i n prophase and telophase i n association with the chloroplast. x 24,800 (b) Dictyosomes with very active cisternae. x 23,000 Abbreviations: Chloroplast (Ch), starch grains (St) 36 Figure 12 Fine-structure of a developing semicell, stage 7 . Sections stained with uranyl acetate and lead. Note the central nucleus and invading chloroplast with surround-ing cytoplasm r i c h i n dictyosomes. Dark ve s i c l e s and the spaces i n d i c a t i n g large ves i c l e s are present through-out the cytoplasm and take up a major portion of the t i p s of lobes. Growing lobes are also r i c h i n ER and mito-chondria, x 3300 Inset: Primary wall and adjacent cytoplasm of a developing s e m i c e l l . x 9 2 , 0 0 0 Abbreviations: Nucleus (N), vacude (vac), regions r i c h i n dictyosomes (g), regions r i c h i n ER (er) F i g u r e 12 37 Figure 13 Fine-structure of a developing semicell, stage 7. Sections stained with uranyl acetate and lead. (a) Tangential section through the nucleus. Nuclear pores are evident; note microtubules flanking the nucleus on either side (arrows), x 18,400 (b) Cytoplasm near the nucleus containing dictyosomes with very active cisternae. Note the abundance of dark v e s i c l e s and t h e i r association with the outer tubular margin of dictyosome cisternae (double arrows). Occa-si o n a l microbodies are present, x 18,200 (c) Lamellar structure found i n the cytoplasm. Osmophillic patches are not due to s t a i n contamination but are consistent from section to section, x 16,000 (d) Growing t i p of a semicell lobe. A lamellar structure (arrow) and v e s i c l e appear between the membrane and c e l l w a ll, x 14,000 Abbreviations: Nucleus (N), chloroplast (Ch), microbody (Mb), v e s i c l e (V) 37? F i g u r e 13 38 Figure 14 Fine-structure of an early prophase c e l l (a) C e l l isthmus showing secondary wall and associated ve s i c l e s and microtubules. A t h i n layer of primary-w a l l - l i k e material l i e s along the secondary wall farrow). Stained with uranyl acetate and lead, x 2 9 , 0 0 0 (b) C e l l isthmus s i m i l a r to that i n (a), but stained with uranyl acetate only, x 3 4 , 0 0 0 (c) C e l l isthmus showing the extent of the primary-w a l l - l i k e material. The layer of material i s uniform i n thickness and stops abruptly (arrows), i n one case (upper arrow) abutting onto the bulb area of a pore structure. Stained with uranyl acetate and lead. x 18,400 (d,e) Dictyosomes from the early prophase c e l l showing an i n d i c a t i o n of c i s t e r n a l a c t i v i t y (arrows). Stained with uranyl acetate and lead. (d) x 2 6 , 0 0 0 (e) x 4 0 , 8 0 0 ( f ) Tangential section of c e l l isthmus. Vesicles and microtubules can be seen to e n c i r c l e the isthmus i n a continuous band. Stained with uranyl acetate and lead, x 2 2 , 8 0 0 Abbreviations: Secondary wall (SW), microtubules (Mt), pore structure (p) F i g u r e 14 39 Figure 15 Semidiagrammatic reconstruction of the events of mitosis and cytokinesis compiled from EM information. Sections of the isthmus are shown. (a) Interphase isthmus with nucleus, n u c l e o l i and chloroplast (hatched) with associated dictyosomes. Mitochondria and large ves i c l e s are shown as i s the isthmus band of wall microtubules. (b) Early prophase isthmus showing the f i r s t appearance of primary wall as a t h i n uniform g i r d l e along the secondary w a l l . Dictyosomes become ac t i v e . (c) Prophase isthmus showing thickening of the primary wall g i r d l e . (d) Telophase isthmus i n which the septum has i n i t i a t e d and grown inward. Its inner leading edge i s surrounded by an area of dense cytoplasm. No c e l l plate or s i m i l a r structure appears to be present and microtubules are seen i n lon g i t u d i n a l array associated with the spindle and not with the isthmus wall. (e) Late telophase isthmus i n which the septum has completed i t s separation of the two daughter c e l l s . Abbreviations: Microtubules (Mt), primary wall (Pw) 393. F i g u r e 15 40 Figure 16 Microtubule p o s i t i o n i n g compared with the s i t e of primary wall i n i t i a t i o n . Diagrams are composites, each traced from several micrographs. (a) Part of the isthmus of an interphase c e l l . Note that microtubules t h i n out gradually and extend into the area of wall containing pores, x 1 4 , 9 0 0 (b) Part of the isthmus of an early prophase c e l l . The primary wall g i r d l e stops abruptly (arrows), p a r t i c u l a r l y where pores are present, and does not extend into a l l areas which i n interphase have associated microtubules. x 1 4 , 8 0 0 Abbreviations: Secondary wall (SW), primary wall (PW), microtubules (Mt), v e s i c l e s (V), pore structure (p) Figure 17 Scanning micrographs of c e l l isthmuses. Note that there i s a sharp boundary (arrows) between those areas of the wall containing pores and the isthmus area which does not. A pore i s indicated by each of the small blebs, the blebs probably being t u f t s of slime threads. (a) Isthmus of M.rotata showing a p a r t i c u l a r l y sharp boundary. x 750 (b) Isthmus of M.torreyi. x 1 , 5 0 0 40,T F i g u r e 16 F i g u r e 17 41 Results and Discussion I I . Observations on L i v i n g Material Figure 18 shows the progress of normal morphogenesis i n a c e l l of M.rotata. A series of stages are shown and numbered 1 to 14 f o r future reference. The pattern of stages i s constant as long as c e l l s are s u f f i c i e n t l y healthy to achieve a f u l l y dentated p r o f i l e . C e l l s subjected to stress may deviate from t h i s pattern, but usually only very s l i g h t l y and only i f morphogenesis i s greatly retarded. The normal pattern I observed i s i d e n t i c a l to that recorded by Kiermayer ( 1 9 6 4 ) , but the time scale i s s l i g h t l y d i f f e r e n t , probably due to temperature e f f e c t s . Figure 18 plots development at 2 0 ° C with i n t e r v a l s of 20 minutes between stages, the whole morphogenetic process taking about 4 hours. Most of my ex-periments were done at temperatures close to 2 0 ° and so when stages are mentioned, both development time and c e l l shape are being referred to. Figure 19 compares the rate of development at 30° with that at 2 0 ° . Development i s more rapid at higher temperatures and morphogenesis i s normal at 30° and 3 5 ° . At 3 8 ° a temperature l i m i t i s reached and no develop-ment occurs, while at 3 7 ° to 3 7 . 5 ° there i s growth but i n abnormal patterns ( f i g . 20 and 2 5 a-c). The lobes produced are frequently narrow and unbranched. M.radiata has a shape somewhat d i s t i n c t from that of M.rotata, though the two c l e a r l y represent variations on a singl e pattern. M.radiata i s smaller with slender lobes and wide notches, whereas M.rotata has rather broad lobes and 42 very narrow notches. M.torreyi represents an intermediate s i t u a t i o n . In healthy, a c t i v e l y d i v i d i n g cultures, c e l l s of M.torreyi resemble M.rotata and have broad lobes. In aging cultures, c e l l s frequently have narrowed lobes and broad notches and the round p r o f i l e of the c e l l i s much les s ob-vious ( f i g . 2 1 ) . S l i g h t modulations i n growth pattern are responsible f o r the shape differences between Micrasterias species. This becomes obvious i f one compares the growth stages of M.radiata ( f i g . 2 2 ) with those of M.rotata ( f i g . 1 8 ) . The culture medium contains about 0 . 0 0 3 M s a l t s . M.rotata c e l l s develop normally over a f a i r l y wide range of osmotic conditions. Development i s normal i n d i s t i l l e d water and i n solutions to which sucrose i s added up to a concentra-t i o n to about 0 . 0 6 M. In sucrose concentrations between 0 . 0 6 and 0 . 1 0 M, development i s abnormal; some lobes develop quite normally while others on the same c e l l arrest at early stages ( f i g . 2 3 a ) . At sucrose concentrations between 0 . 1 0 and 0 . 2 0 M, growth i s completely prevented and c e l l wall thickenings may develop on the Inside surface of the wall at lobe t i p s . Kiermayer and Jarosch ( 1 9 6 2 ) have described t h i s phenomenon and commented on the pattern of these wall thicken-ings which i s s i m i l a r to that sometimes produced when c e l l s are allowed to develop i n solutions of pectin methyl esterase ( f i g . 25 1 ) . In solutions of 0 . 2 M sucrose, c e l l s show v i s i b l e plasmolysis, the protoplast p u l l i n g away from the c e l l wall and the wall i t s e l f showing some shrinkage. C e l l s behave i n s a l t solutions much as they do In sucrose solutions and w i l l develop normally i n deionized water to which NaCI, L i C l or KC1 have been added up to concentrations of about 0 . 0 5 M. C e l l s w i l l not develop i n deionized water alone. The culture medium has a pH of 6 , but c e l l s w i l l develop normally within a pH range of 4 to 9 . In acid solu-tions (pH 2 - 3) c e l l s i n v a r i a b l y rupture within 1/2 hour. Rupture generally occurs at the t i p of a lobe, p a r t i c u l a r l y at the polar lobe t i p i n early stages. I f the rupture Is small, the c e l l can be rescued, transferred to culture medium at pH6 and growth resumes. A l l lobes show normal growth except the lobe s u f f e r i n g rupture which does not grow ( f i g . 25 d - f ) . The rupturing was f i r s t observed when c e l l s were placed i n 0 . 0 0 1 M glucuronic acid, but c e l l s be-haved s i m i l a r l y i n solutions of galacturonic, c i t r i c and a c e t i c acids. Plasmolysis inva r i a b l y upsets morphogenesis, but the subsequent patterns of growth are not c l e a r l y of any single type. E f f e c t s are frequently but not always s i m i l a r to those of turgor reduction. In a t y p i c a l experiment, c e l l s were taken from culture medium by steps to 0 . 2 M sucrose, the process taking several minutes. C e l l s were l e f t f o r up to one hour with t h e i r protoplasts v i s i b l y plasmolysed, then re-turned to culture medium by steps and allowed to develop. In a sampling of 60 c e l l s , h a l f died without developing further; and of the other h a l f , 10 showed no further growth and 2 0 produced short lobes of reduced diameter at various points over t h e i r surface ( f i g . 2 3 b ) . Various enzymes and chemicals have been tested f o r t h e i r e f f e c t s on Micrasterias morphogenesis. RNAase, puromycin, mitomycin and other compounds which i n h i b i t tran-s c r i p t i o n and t r a n s l a t i o n have effects s i m i l a r to those of enucleation ( K a l l i o , 1 9 6 3 ) . Indoleacetic acid has no speci-f i c e f f e c t s not found with other acids (Kiermayer and Jarosch, 1 9 6 2 ) . I t r i e d to produce s p e c i f i c pattern e f f e c t s i n M.rotata using enzymes, p a r t i c u l a r l y those enzymes af-f e c t i n g c e l l wall metabolism. Cellulase has no ef f e c t on growing c e l l s even though i t w i l l s o l u b i l i z e primary wall preparations. Pectinase has effects s i m i l a r to those of turgor reduction but less marked, c e l l s seldom develop com-p l e t e l y and lobes r e t a i n rounded t i p s ( f i g . 24a and 25 g, h). Occasionally one lobe may be much less developed than i t s neighbors. Hemicellulase has more s t r i k i n g e f f e c t s ; many lobes develop f u l l y but s p e c i f i c ones are consistently i n h i b -it e d ( f i g . 24b and 2 5 1 ) . The r e s u l t i n g pattern resembles that seen In unhealthy c e l l s of M.torreyi ( f i g . 21), i n -h i b i t e d lobes are frequently on the upper wing lobe and may face each other across a notch. Treatment with t r y p s i n causes c e l l s to arrest at early stages ( f i g . 24d), and small r e f r a c -t i l e areas may appear at the lobe t i p s . Pectin methyl esterase i s p a r t i c u l a r l y l i k e l y to cause c e l l s to rupture within several hours; t h i s i s not the case with the other enzymes mentioned. In pectin methyl esterase, lobe3 are rounded much as i n pectinase ( f i g . 24c), but may also become d i s t i n c t l y bulbous or show patterns pf c e l l wall thickening ( f i g . 25J - m ) . 45 I have watched f o r the appearance of new symmetry types In my c u l t u r e s . I Isolated one t r i r a d i a t e c e l l from a culture of M.rotata and established a clone. C e l l s of t h i s clone occasionally reverted to the b i r a d i a t e type by f i r s t forming a semicell which was only p a r t i a l l y t r i r a d i a t e ( f i g . 2 6 b ) . A p a r t i a l l y t r i r a d i a t e semicell might then pro-duce a b i r a d i a t e daughter semicell on subsequent d i v i s i o n , though i t could also produce a p a r t i a l or f u l l t r i r a d i a t e s . Cultures of b i r a d i a t e M.thomasiana produced no abnormal symmetry types, but uniradiate c e l l s reverted routinely to the b i r a d i a t e form, usually through a p a r t i a l l y uniradiate intermediate ( f i g . 2 6 a ) . P a r t i a l wings may appear at various points on these c e l l s and frequently l i e out of the normal plane of symmetry ( f i g . 2 6 c , d). Cultures of M.torreyi gave r i s e to uniradiate c e l l s on at least a dozen separate occa-sions. Clones were established from these c e l l s and some tended to revert more than others. Uniradiate and aradiate c e l l s were found i n cultures of the pygmy s t r a i n of M.radiata, but only i n very old cultures In which c e l l s were small and t h e i r shapes much s i m p l i f i e d . When placed i n fresh medium, these c e l l s reverted to b i r a d i a t e forms. I observed l i v e growing c e l l s under the l i g h t micro-scope with p a r t i c u l a r a t t e n t i o n to the a c t i v e l y streaming cyto-plasm. I noted nothing that does not already appear i n published reports. In general, the study of c e l l s i n culture revealed the natural and experimentally Induced modulations of c e l l shape which the morphogenetic machinery of Micrasterias i s capable of producing. Any good explanation of morphogenesis w i l l have be able to account f o r these i n addition to dealing with normal morphogenesis. 4 7 Figure 18 Morphogenetic stages of M.rotata showing the change i n semicell perimeter with" time. Fourteen stages are distinguished following one another at 2 0 minute i n t e r v a l s . Stage 1 i s placed just p r i o r to septum completion. Figure 19 Rate of c e l l development showing the number of developmental stages that various c e l l s were able to advance i n a 2 hour period. Data i s shown f o r de-velopment at 2 0 ° C ( 1 1 ) and 3 0 ° C ( tmm&llfflm ) i n normal culture medium and at 2 0 ° C i n medium dil u t e d with a lOx volume of d i s t i l l e d water ( 1/ H / / / / 7771 ). An arrow indicates the developmental rate reported f o r M.rotata by Kiermayer and Jarosch ( 1 9 6 2 ) f o r which the The temperature i s not reported. 47a 48 Figure 20 Semicell shape i n c e l l s placed at d i f f e r e n t tempera-tures at stages 4-5 and allowed to develop. Compare these with the normal semicell ( i n s e t ) . (a) Development at 35° C (b) Development at 37° C. Note long, unbranched lobes (arrow). (c) Development at 37.5° C. Note long, unbranched lobes (arrows). 48d Figure 20 £ % _ 3 7 - 5 -49 Figure 21 C e l l shape i n several species of Micrasterias, a l l to the same scale. One c e l l each i s shown of M. rotata (a) and M. radiata (b). The remaining c e l l s are a l T examples taken Trom a single aging culture of M. t o r r e y i and show consider-able v a r i a t i o n . Figure 22 Morphogenetic stages of M. radiata shown i n three parts for greater c l a r i t y . Stages are numbered 1 to 9 and are separated by 20 minute i n t e r v a l s . (a) Stages 1 - 7 . Note that notches remain fixed i n posi-t i o n once formed while t i p s change. (b) Stages 7 - 9 . Note the more complex changes i n notch p o s i t i o n . Inset: A comparison of one wing at stage 7 with the same wing at stage 9. Note that the notches change p o s i t i o n and that the width of each lobe (arrows) increases with l a t e r stages. * 50 Figure 23 Semicell shape i n c e l l s grown from stages 6 - 7 under d i f f e r e n t osmotic conditions. Compare these with the normal ( i n s e t ) . (a) Development i n 0 . 0 6 to 0.1 M sucrose solutions (b) Development i n culture medium a f t e r 40 min. to 1 hr. plasmolysis i n 0 .2 M sucrose 50a Figure 23 51 Figure 24 Semicell shape i n c e l l s allowed to develop from stages 3-4 i n solutions of enzymes. Compare these with the normal ( i n s e t ) . (a) Development i n 0.2 to 2 mg/ml pectinase (b) Development i n 2 to 4 mg/ml hemicellulase. Note c h a r a c t e r i s t i c abnormalities of lobes, p a r t i c u l a r l y of upper wing lobes (arrows). (c) Development i n 0.02 to 2 mg/ml pectin esterase. Bulbous lobes are evident i n a few c e l l s (arrow). (d) Development i n 0.02 mg/ml try p s i n 51a Figure 24 52 Figure 25 Experimental manipulations of developing c e l l s (a-c) C e l l development at d i f f e r e n t temperatures. Note long, unbranched lobes (arrows). (a) C e l l placed at 3 5 ° C at stage 3 , photo taken a f t e r 8 hrs. (b) C e l l placed at 3 7 ° C at stage 3 f o r 8 hrs., photo taken a f t e r 24 hrs. (c) C e l l placed at 3 7 . 5 ° C at stage 4 f o r 8 hrs., photo taken a f t e r 24 hrs. (d,f) C e l l s ruptured i n acid solutions (pH 2 . 5 ) (d,f) C e l l s placed i n 0.001 M glucuronic acid f o r 1/2 hr. and then returned to culture medium. The c e l l s each show rupture at one t i p . (e) C e l l from (d) a f t e r 24 hr. Ruptured t i p has not developed further, (g-m) C e l l s allowed to develop i n the presence of various enzymes (g) C e l l placed i n 1 mg/ml pectinase at stage 5 (hj C e l l placed i n 0.2 mg/ml pectinase at stage 5 ( i ) C e l l placed i n 3 mg/ml hemicellulase at stage 4. Note the c h a r a c t e r i s t i c abnormality of the upper wing lobe farrows) ( j ) C e l l (M.radiata) placed i n 0.2 mg/ml pectin esterase at stage 2 (k) C e l l (M.radiata) placed i n 1 mg/ml pectin esterase at stage 2 ( l ) C e l l placed i n 1 mg/ml pectin esterase at stage 5 . The c e l l i s ruptured, but shows a pattern of c e l l wall thickenings s i m i l a r to that caused by turgor stress farrows), (m) C e l l placed i n 2 mg/ml pectin esterase at stage 6. This c e l l also i s ruptured, but shows the development of d i s t i n c t l y bulbous lobes (arrows). 52.2 53 Figure 26 Scanning and l i g h t micrographs of intermediate faci e s i n three species. In each case an arrow i n d i -cates the p a r t i a l wing. (a) Uniradiate M.thomasiana with a p a r t i a l wing (b) Biradiate MTrotata with a p a r t i a l wing. The c e l l has probably reversed from a f u l l t r i r a d i a t e i n the follow-ing way: The p a r t i a l semicell arose from d i v i s i o n of a t r i r a d i a t e c e l l and at a l a t e r d i v i s i o n , t h i s semicell produced a b i r a d i a t e s e m i c e l l . (c) Uniradiate M.torreyi showing a l a t e r a l d u p l i c a t i o n of one wing. In some c e l l s two such wings may take up positions completely perpendicular to one another. (d) Uniradiate M.torreyi with a p a r t i a l wing Figure 27 Scanning micrographs of double c e l l s found i n old cultures (a) A double c e l l of M.rotata (b) A t r i p l e c e l l of which two isthmuses are shown (arrows). 53 a F i g u r e 26 F i g u r e 27 54 Results and Discussion I I I . Primary C e l l Wall Composition C e l l wall ghosts from M.rotata were mounted on s l i d e s , extracted i n various ways and stained f o r poly-saccharide (PAS), acid f r a c t i o n s ( a l c i a n blue) and slime ( c r y s t a l v i o l e t ) . P a r a l l e l extractions were done on radio-labeled walls and autoradiograms made. Table IV presents a summary of the re s u l t s of these experiments. The primary wall shows three d i s t i n c t f r a c t i o n s . There i s a "loosely bound" component which i s removed by a l l extractions with a l k a l i and which stains with c r y s t a l v i o l e t . The staining i s s i m i l a r to that of pores i n the secondary wall suggesting that t h i s component i s a slime, either simi-l a r to or i d e n t i c a l with the pore slime, though the s t a i n i s r e l a t i v e l y non-specific. The second component stains with a l c i a n blue at pH 2.5 and i s not removed by a l k a l i s . There i s no staining at pH 0 .5 , i n d i c a t i n g that t h i s a c i d i c f r a c -t i o n i s r i c h i n uronic and not s u l f o n i c acids. The acid groups can be methylated with loss of st a i n i n g and the s t a i n -ing returned by s a p o n i f i c a t i o n . Hot acid alcohol and chromic acid w i l l extract the a l c i a n blue p o s i t i v e f r a c t i o n leaving behind v i s i b l e walls which s t a i n with PAS, suggesting that a t h i r d and more stable component e x i s t s . Mix ( I 9 6 I ) found that chromic acid removed a l l matrix substances from the primary wall of a closely related desmid leaving a mat of randomly oriented m i c r o f i b r i l s . Des-mids are known to possess m i c r o f i b r i l s which are c e l l u l o s e -l i k e but contain several sugars i n addition to glucose 55 T a b l e IV H l s t o c h e m i c a l A n a l y s i s o f P r i m a r y C e l l W a l l s * Treatment R e s u l t s C o n t r o l A l c i a n b l u e + C r y s t a l v i o l e t + PAS + G l u c o s e + M e t h i o n i n e m e t h y l - J H + M i l d a l k a l i e x t r a c -t i o n + - + + - • A l k a l i e x t r a c t i o n + - + + -S t r o n g a l k a l i ex-t r a c t i o n + - + + Chromic a c i d - - + + -S t r o n g m e t h y l a t i o n - - + + • -S t r o n g m e t h y l a t i o n p l u s s a p o n i f i c a t i o n - - + + -M i l d m e t h y l a t i o n - + + + + M i l d m e t h y l a t i o n p l u s s a p o n i f i c a t i o n + + + + -P e c t i n e s t e r a s e -P e c t i n e s t e r a s e C o n t r o l + T r y p s i n + Pronase + C e l l u l a s e - - - - -C e l l u l a s e c o n t r o l + + + + + P e c t i n a s e - - - - + P e c t i n a s e c o n t r o l + + + + + H e m i c e l l u l a s e + + + + + H e m l c e l l u l a s e c o n t r o l + + + + + * S t a i n i n g o r a u t o r a d i o g r a p h i c l a b e l i n g i s e i t h e r p r e s e n t (+) absent (-) o r reduced (+) f o l l o w i n g t h e v a r i o u s t r e a t m e n t s . 56 ( K l e i n and Cronquist, 1 9 6 7 ) . The t h i r d wall component i s therefore most l i k e l y a network of c e l l u l o s e - l i k e m i c r o f i b r i l s which may be oriented randomly or aligned i n some fashion. The a l c i a n blue and c r y s t a l v i o l e t p o s i t i v e f r a c t i o n s repre-sent two matrix substances. The a l c i a n blue f r a c t i o n could be c a l l e d hemicellulose-like because i t i s a l k a l i stable. I t i s probably the s t r u c t u r a l l y more Important of the two. Radiolabeled, externally-supplied glucose i s c l e a r l y Incorporated i n t o the m i c r o f i b r i l l a r f r a c t i o n and does not con-t r i b u t e greatly to the matrix, as considerable r a d i o a c t i v i t y remains a f t e r chromic acid treatment. Methyl-labeled methionine a c t i v i t y i s removed by a l k a l i , by pectin methyl esterase and to some extent by boiled esterase. The l a b e l i s not removed by tr y p s i n or pronase. This indicates, but does not prove, that l a b e l i s present i n e s t e r i f i e d methyl groups bound probably to the uronic acid rather than m i c r o f i b r i l l a r f r a c t i o n , and that i t i s not present i n loosely bound protein as methionine. The synthetic a c t i v i t i e s associated with incorpora-t i o n of glucose and methionine probably occur at the c e l l wall and not i n cytoplasmic structures such a s ( J o l g i . Golgl v e s i c l e s probably transport acid polysaccharides but are not thought to be involved i n ce l l u l o s e synthesis or transport except i n very sp e c i a l cases (Brown et a l . , 1 9 7 0 ) . Cellulose synthesis probably occurs r i g h t at the c e l l surface (Roelofsen, 1965; Northcote and Pickett-Heaps, I 9 6 6 ) , and par-t i c l e s perhaps responsible f o r t h i s synthesis have been found on the plasma membrane (Robertson and Preston, 1 9 7 2 ) . The glucose l a b e l Is contained i n a m i c r o f i b r i l l a r component 57 which i s probably synthesized i n t h i s fashion als o . Methy-l a t i o n of polysaccharides i s thought to p a r a l l e l methylation of other b i o l o g i c a l macromolecules. Rather than b u i l d i n g from methylated precursors, the assembled macromolecule i s methylated as a f i n a l step (Lamport, 1970) . Label appears i n the c e l l wall within about 10 minutes. This i s not so rapid that i t could not have come through the (golgi (Northcote and Pickett-Heaps, 1966), but the timing Is s i m i l a r f o r glucose and methionine and there are Independent reasons i n both cases f o r supposing that i n -corporation occurs at the wall i t s e l f . I therefore conclude that none of the l a b e l arrives via the g o l g i . Golgi c o n t r i -butions to the wall, probably a matrix precursor contained i n small dark v e s i c l e s and a c e r t a i n amount of slime contained i n large vesicles,are probably not labeled i n these experiments. This i s consistent with t h e i r presence i n separate cytoplasmic compartments which turn .over very slowly or into which l a b e l does not penetrate (Oaks and Bidwell, 1970) . In conclusion, primary wall comprises three d i s t i n c t f r a c t i o n s ; a slime matrix substance, an acid matrix substance and m i c r o f i b r i l s . The m i c r o f i b r i l f r a c t i o n incorporates l a b e l from t r i t i a t e d glucose; one of the f r a c t i o n s incorporates l a b e l from methyl-labeled methionine, probably as methyl esters. Both incorporations are rapid and probably are not associated d i r e c t l y with gol g i transport. 58 Results and Discussion IV. Laser Experiments Some means of damaging selected parts of the Micrasterias c e l l was required so as to determine t h e i r c o n t r i -bution to morphogenesis of the whole. A laser microbeam was chosen as i t can d e l i v e r large amounts of energy to a very small area. I mounted d i v i d i n g c e l l s as described and subjected them to laser i r r a d i a t i o n . The clear cytoplasm was found to be quite i n s e n s i t i v e to laser r a d i a t i o n , though c e l l s responded to lasings of t h e i r chloroplast by turning brown and dying. I then tested a number of blue and green dyes to see i f they would be taken up by c e l l s without k i l l i n g and whether they would promote the absorption of laser energy. Of about 20 dyes tested, 5 were c l e a r l y taken up by the c e l l s (malachite green, a l c i a n blue, janus green, n i l e blue and methylene blue) and of these 3, a l c i a n blue promoted the most s p e c i f i c and i n t e r e s t i n g sorts of lase r damage. Routinely, c e l l s were colored i n a 0.001$ s o l u t i o n of a l c i a n blue and lased with a 2 to 5 P- spot f o r no more than 5 seconds. At the concentrations used, a l c i a n blue was not toxic and did not a f f e c t the pH of the culture medium. C e l l s de-veloped normally i f l e f t i n a 0.001$ solution of a l c i a n blue and could survive there f o r several days. At 0 . 0 1 0 $ , c e l l s did begin to show contracted chloroplasts and other signs of t o x i c i t y . Lasing at the point between two developing semicells prevented formation of a polar lobe while the wings developed normally ( f i g . 2 8 a , b ) . Lasing t h i s same point at an e a r l i e r stage produced a s i m i l a r l o s s , but occasionally caused two polar 59 lobes to develop rather than one ( f i g . 2 8 c , d). As f o r wing development, the ef f e c t s were quite s t r i k i n g when the t i p s of lobes were lased ( f i g . 3 0 ) , but less so when other parts of the c e l l wall were lased. A lobe could be damaged so as to only retard i t s growth (by l a s i n g a notch as i n figu r e 31 f o r example) and the lobe would continue to grow and branch. These simple experiments demonstrated c l e a r l y that lobes can grow and develop independently of one another and suggested that l a s e r damage of t h i s sort i s quite l o c a l i z e d . The experiments also demonstrated that c e r t a i n points on the c e l l wall play a f a r more c r u c i a l r o l e i n lobe growth than others. The p a r t i c i p a t i o n of these points i s required i n some way f o r morphogenesis. In a series of l a s e r experiments involving about 300 c e l l s , I mapped out the p o s i t i o n on the c e l l wall of these se n s i t i v e points or " s i n g u l a r i t i e s " as I s h a l l c a l l them. The word " s i n g u l a r i t y " I have borrowed from Tokunaga and Stern ( I 9 6 5 ) . They use the term i n a broad sense to i n d i c a t e only that something i n t e r e s t i n g occurs at one point of an otherwise undistinguished surface and not elsewhere (Stern, 1972) . I w i l l use the word i n a s i m i l a r fashion so as to indicate p o s i t i o n while implying nothing about structure. Fate-mapping experiments are summarized i n figure 3 2 . Lasings i n which the lased lobe f a i l e d to develop are separated from those in. which i t recovered s u f f i c i e n t l y to produce a normal pattern of dentation. In each l a s i n g , one daughter was the experimental c e l l , and the other served as a contr o l . A l l c e l l s were kept and followed through one further d i v i s i o n , and the most malformed c e l l s were followed through several. In no 6o case were laser-induced shape abnormalities i n h e r i t e d . C e l l s lacking both polar lobe and one wing produced normal b i r a d i a t e daughter semicells at each d i v i s i o n . An i d e a l i z e d plot can be constructed showing the posi t i o n of s i n g u l a r i t i e s on the c e l l wall at d i f f e r e n t develop-mental stages. Such a plot, with the points connected by a l i n e , i s shown i n figur e 3 3 . S i n g u l a r i t i e s are generally located at lobe t i p s , but some lobes may show two i n associa-t i o n with branch formation. The implication, of course, i s that each s i n g u l a r i t y i s responsible for the growth of a lobe; and when s i n g u l a r i t i e s duplicate, lobes become branched. I s h a l l present further evidence before arguing t h i s point. Having surveyed stages 3 to 8 i n thi s fashion, I was p a r t i c u l a r l y interested i n tr a c i n g the p o s i t i o n of l a s e r s i n g u l a r i t i e s i n very early stages. I wanted to f i n d t h e i r points of o r i g i n ; presumably three existed, one each associated with the polar lobe and two wings. In f a c t , early stages re-cover f a r more re a d i l y from lase r i r r a d i a t i o n , l a s i n g may slow development of stage 1 to 3 c e l l s but i n a l l cases a normal semicell i s produced. Irreparable damage may occur i f much larger areas of c e l l wall are i r r a d i a t e d at these early stages or more concentrated solutions of a l c i a n blue used to color the c e l l s . I chose to use a 0 . 0 0 5 $ dye solution and r e t a i n the 2 to 5 ju beam diameter f o r studies of stages 1 to 3. C e l l s were again lased for 5 seconds or s l i g h t l y longer and frequently showed retarded development before recovering. Some of the c e l l s developed a mark on the c e l l wall corresponding to the point of l a s i n g . In c e l l s which recovered completely, t h i s 61 mark served to delimit the area from which lobes could have aris e n . Of 65 c e l l s lased at stages 1 to 3 , about 25 show suf-f i c i e n t marking that I could determine the point of o r i g i n of lobes r e l a t i v e to the l a s i n g . Two of the more clear-cut examples are shown i n figures 34 and 3 5 . The following important conclusion emerged from t h i s work; at an early stage the side lobe can a r i s e from any of several d i f f e r e n t places, i t s o r i g i n i s not t i e d to a single point. Note i n figure 36 that stage 3 c e l l s are well-behaved. Lasing small spots within an area, one can pick out s i n g u l a r i -t i e s f o r upper and lower wing lobes or h i t between the two. Lasing the large area including these prevents formation of one whole wing (as i n f i g . 2 9 ) though frequently the unlased wing appears to compensate f o r the missing wing opposite by hyper-trophy. At stage 1 and 2 l a s e r experiments become ambiguous ( f i g . 3 7 ) . Lasing at A ( f i g . 37b) leaves a mark, and the developing lobes must be assumed to have arisen from B (as i n f i g . 3 5 ) . Lasing at B ( f i g . 3 7 c ) leaves a mark and suggests that lobes arose at A or beyond (as i n f i g . 3 4 ) . Lasing at A and B both ( f i g . 37<3) leaves a mark and again a normal lobe which must have arisen from an area separated from B by at least the distance across A. Lasing at A and B together may also cause the lobes to grow out oblique to t h e i r normal plane, implying an o r i g i n l a t e r a l to the l a s i n g rather than above or below i t . So a rather large area of the stage 2 c e l l i s compe-tent to produce a normal wing, c e r t a i n l y the competent area i s larger than the area of a s i n g u l a r i t y as defined by l a s i n g l a t e r stages. The morphogenetic machinery of M.rotata, whatever i t s 62 material basis, i s therefore able to regulate to a considerable extent at early stages, but loses t h i s a b i l i t y as development proceeds. A note i s required here concerning the mechanism of laser e f f e c t s . Power output of my helium-neon laser i s on the order of 10 ^ times less than that of a pulsed ruby l a s e r . This output i s s t i l l s u f f i c i e n t to produce c e l l damage i f the energy can be absorbed. Absorption i s of a normal photochemical sort so that blue, green or black substances are required i f the laser produces red l i g h t . Energy absorbed by the dye may then be transferred to associated b i o l o g i c a l molecules, and proteins may be good candidates f o r t h i s . A number of dyes bind loosely to proteins i n sol u t i o n , and model studies show thermochemical and free r a d i c a l e f f e c t s when solutions of lip a s e and methylene blue are lased (Saks, 1 9 7 1 ) . In c e l l s then, one would expect very rapid l o c a l heat-ing and either protein denaturation or the formation of free r a d i c a l s and molecular c r o s s - l i n k s . In M.rotata, a l c i a n blue probably serves to color the c e l l wall * The wall appears s l i g h t -l y t i n t e d , and a l c i a n blue Is known to bind very t i g h t l y to c e l l walls (Benes, 1968) and i s used as a histochemical s t a i n f o r acid polysaccharides (Stadelmann and K i n z e l , 1 9 7 2 ) . I f a lased c e l l i s immediately plasmolysed, the c e l l membrane and wall s t i c k together i n the area cf l a s i n g . When the protoplast breaks away, a small b i t of membrane i s l e f t behind fused to the c e l l wall ( f i g . 3 8 ) . Lased portions of c e l l wall do not p a r t i -cipate f u l l y i n c e l l morphogenesis. Note i n fi g u r e 3 0 c that the 63 lased portion of wall retains i t s p o s i t i o n i n r i g i d i n d i f f e r -ence to the changes going on about i t . The wall Is not marked immediately by l a s i n g . Marks appear over a 10 to 15 minute period as surrounding wall continues to develop. Marks appear to be due to o p t i c a l r e f r a c t i o n , the r i g i d lased area having d i f f e r e n t r e f r a c t i l e properties from the surrounding, more p l a s t i c w a l l . Laser experiments do not d i s t i n g u i s h the morpho-genetic r o l e of c e l l wall from that of c e l l membrane as both are affected by l a s i n g . The l a s e r may act s o l e l y on wall, making parts of the wall p h y s i c a l l y too r i g i d to p a r t i c i p a t e i n morphogenesis f o r example. I t may act on membrane alone or perhaps i n t e r f e r e with the association between wall and mem-brane. I t i s appropriate then to say that the l a s e r has i t s e f f e c t on the c e l l cortex; meaning by cortex the c e l l wall, the membrane and the f i r s t micron or so of cytoplasm with whatever structures t h i s may contain. In conclusion, lase r experiments demonstrate p a r t i -cular regions of the c e l l cortex ( s i n g u l a r i t i e s ) which are e s p e c i a l l y important for maintaining morphogenesis. These re-gions f i r s t become fix e d and well-defined at about stage 2 and subsequently are associated with the t i p s of developing lobes. 64 Figure 28 Laser damage to polar lobes (a,b) C e l l 4 9 8 . An 8 sec. l a s i n g of the region shown res u l t s i n the absence of the polar lobe i n both daughter c e l l s , fa} l a s i n g at stage 4 (b) One daughter on the following day. Both daughter c e l l s were s i m i l a r . (c,d) C e l l 3 8 7 . An 8 sec. l a s i n g of the region shown resu l t s i n a du p l i c a t i o n of the polar lobe i n both daughter c e l l s . (c) Lasing at stage 3. (d) One daughter on the following day. Both daughter c e l l s were s i m i l a r . Figure 29 Laser damage to wings, c e l l 5 0 6 . A 10 sec. la s i n g of the region shown with a somewhat larger spot r e s u l t s i n the absence of one wing i n each daughter. At an intermediate stage of development (b), a re-f r a c t i l e area can be seen i n d i c a t i n g the region of las e r damage (arrow). (a) Lasing at stage 3 lb) Development 2 hrs. a f t e r l a s i n g (c) and (d) Daughter c e l l s on the following day 64.1 F i g u r e 28 F i g u r e 29 65 Figure 30 Laser damage to lobe t i p s , c e l l 5 9 0 . A 2 sec. l a s i n g of the lobe t i p r e s u l t s i n complete arrest of that lobe's subsequent development while other lobes remain unaffected. Compare the control c e l l (d) with the lased c e l l (c) (a) Lasing at stage 7 (b) Develpment 1 hr. 30 min. a f t e r l a s i n g (c) and (d) Daughter c e l l s on the following day Figure 31 Laser damage to lobe notches, c e l l 5 9 5 - A 2 sec. l a s i n g of the side of the lobe does not prevent formation of a complete pattern of lobe dentation, but does leave a s l i g h t abnormality (arrow i n c ) . (a) Lasing at stage 7 (b) Development 1 hr. 40 min. a f t e r l a s i n g (c) and (d) Daughter c e l l s on the following day F i g u r e 30 F i g u r e 31 66 Figure 32 Composite of la s e r experiments i n which regions of developing c e l l wall were i r r a d i a t e d . Representative growth stages from 2 to 6 are shown. Each c i r c l e repre-sents the p o s i t i o n of a l a s i n g of the c e l l wall i n one experiment; lasings are f o r 5 sec. with a beam diameter of 2-5 p. (a) Positions of lasings which i n each case f a i l e d to produce shape abnormalities. A l l lobes were present i n the f u l l y developed s e m i c e l l . (b) Positions of lasings which i n each case resulted i n l o s t morphogenetic capacity made evident by the absence of the associated lobe or lobes. Figure 33 Idealized fate map of the morphogenetically important regions of c e l l wall ( s i n g u l a r i t i e s ) as shown by l a s e r experiments. Stages 4 to 8 are represented. NO LOSS L O S S Figure 32 Figure 33 67 Figure 34 Laser damage to young semicells, C e l l 556. An 8 sec. l a s i n g leaves i t s mark on the semicells (arrows); but does not prevent a complete complement of lobes and notches from being produced, though the "recovered" wing i s abnormally small and the opposite wing somewhat en-larged. Note that the recovered wing must have originated from the c e l l wall at a point above the l a s i n g . fa) Lasing at stage 1 fb) Development 45 min. a f t e r l a s i n g fc) Development 1 hr. 15 min. a f t e r l a s i n g id) Development 1 hr. 45 min. afte r l a s i n g (e) One daughter on the following day. Both daughter c e l l s were s i m i l a r . Figure 35 Laser damage to young semicells, C e l l 554. An 8 sec. l a s i n g leaves i t s mark, but a p a r t i a l lobe i s able to develop from below the point of l a s i n g . The lobe i s small but shows branching and dentation of a f a i r l y normal s o r t . fa) Lasing at stage 2 fb) Development 20 min. a f t e r l a s i n g (c) Development 1 hr. a f t e r l a s i n g (d) and (e) Daughter c e l l s on the following day F i g u r e 3 5 68 Figure 36 Diagrammatic summary of laser experiments on young semicells. The semicell represented i s stage 2 or 3- Lasing with a large spot can prevent one entire wing from forming (as i n f i g . 2 9 ) . Smaller lasings within t h i s large area can s e l e c t i v e l y cause the ab-sence of the upper or lower wing lobe or can h i t between the two. Figure 37 Diagrammatic summary of lase r experiments on very young semicells (a) Diagram of a stage 1-2 isthmus d i s t i n g u i s h i n g two general regions, A_ and B. (b) Results of l a s i n g a¥ A. A lobe develops below the lased spot (as i n f i g . 35T a n d hence must have developed from region B. (c) Result of l a s i n g a^ B. A lobe develops above the lased spot (as i n f i g . 3^) and hence must have developed from region A. (d) Results of l a s i n g a large area including both A_ and B. A lobe w i l l s t i l l develop i n many cases from an area which i s neither B nor A. A lobe may also develop not above or below the lased" spot but l a t e r a l to i t on either side, producing a wing projecting somewhat out of the normal plane of symmetry. These experiments rough-l y define the area from which lobes can be Induced to form (shown i n f i g . 4 7 ) . Figure 36 69 Figure 38 Laser fusion of c e l l membrane to c e l l w a ll. Fusion i s shown by plasmolysing c e l l s i n 0.2 M sucrose immediately a f t e r l a s i n g of the wall. Protoplast s t i c k s to the wall at the point of l a s i n g and eventually i s torn away leaving a remnant of membrane behind. (a-d) Small laser spotj 3 sec. l a s i n g followed by plasmo-l y s i s (e-h) Large laser spot, 3 sec. l a s i n g followed by plasmolysis 70 Results and Discussion V. Autoradiography Autoradiography was used i n order to reveal patterns of c e l l wall synthesis i n M.rotata. C e l l s at d i f f e r e n t de-velopmental stages were incubated f o r fix e d times with radio-active compounds and preparations made from the primary walls. In a l l cases, autoradiograms are of c e l l wall ghosts only. The protoplast has been removed by rupturing the c e l l s i n d i s t i l l e d water and the preparations washed to remove small molecules (see Methods). T r i t i a t e d water, proline, glucose and methyl-labeled methionine were tested as possible wall pre-cursors. The former two did not show any incorporation i n t o walls whereas methionine and glucose produced heavily labeled preparations and revealed a d i s t i n c t and consistent pattern of la b e l i n g . With both methionine and glucose, incorporated label was extractable under conditions consistent with i t s presence i n c e l l wall polysaccharides and not i n cytoplasmic or membrane residue or as small molecules (see Table IV and asso-ciated discussion); glucose incorporated i n t o m i c r o f i b r i l s , and methionine probably incorporated i n t o matrix substances i n the form of methyl esters. Glucose showed very l i t t l e incorporation i n t o c e l l s stage 1 to 6, but did lab e l the t i p s of lobes i n l a t e r stages. In stages 11 to 14,tips were heavily labeled; but i n addition, label appeared i n a pattern over the c e l l wall resembling some-what the veining i n leaves ( f i g . 3 9 a-i). In very l a t e stages, la b e l i n g was frequently uniform over the whole semicell and considerably denser ( f i g . 39J-1). This I concluded to 71 represent incorporation i n t o secondary wall l a i d down "by apposition i n c e l l s which had completed morphogenesis. Most of the completely dentated primary wall ghosts labeled i n t h i s fashion, and frequently labeled portions appear to have been torn away from the primary wall. A l l three patterns of l a b e l were seen i n c e l l s treated for as l i t t l e as 10 minutes ( i n c l u d -ing the 5 minute washing procedure). Label was denser and more extensive i n c e l l s exposed f o r longer times, but the pat-terns were the same. Methionine-treated c e l l s showed si m i l a r t i p and vein-l i k e label patterns. The t i p pattern was noticeable i n stages as early as 4, and i n general a l l l a b e l i n g was much denser and the pattern more d i s t i n c t ( f i g . 40a-k). Of 30 f u l l y developed primary walls i n t h i s experiment, none showed the uniform pat-tern c h a r a c t e r i s t i c of glucose l a b e l i n g ( f i g . 4 o 1). C e l l s treated 10 minutes with methionine showed a pattern of l a b e l somewhat d i f f e r e n t from that seen i n c e l l s treated with glucose for a s i m i l a r length of time ( f i g . 4la,b). I t i s clear that the methionine l a b e l i s more l o c a l i z e d , and i n p a r t i c u l a r , l o c a l i z e d to the very t i p area of lobes. When glucose l a b e l f i r s t appears, i t occupies a much larger area around and includ ing the t i p s of lobes. It i s c h a r a c t e r i s t i c of these experi-ments that density of l a b e l may vary greatly from wall to wall, even though the autoradiographic technique was standardized. For a given length of exposure to l a b e l , however, the area of labeling for any given stage i s always very constant ( f i g . 4lc, d). Hence i t was easy to construct the diagrams i n figure 43. For these I drew the upper side lobes of about 30 stage 10 72 c e l l s , using a camera l u c i d a , and outlined the area of radio-l a b e l i n g . As I selected c e l l s labeled f o r longer lengths of time, the area showing incorporation increased progressively i n si z e as shown. For glucose and methionine the patterns were d i f f e r e n t . Note now figure 4 2 , representing a model of c e l l wall fate constructed on the assumption that a l l increase i n wall perimeter from stage to stage originates as new wall formed at the t i p s . The l i n e s mark off portions of the wall which, by the assumption, must be less than 10 minutes old, less than 20 min-utes old and so on. The time scale here i s appropriate to de-velopment at 20° C as the model i s constructed from measure-ments done on f i g u r e 18. Compare figure 42 with f i g u r e 33 showing the fate of c e l l wall s i n g u l a r i t i e s as demonstrated i n l a s e r experiments. I f growth originates at the t i p s of lobes only, these alterna-t i v e forms of fate mapping become complementary. Figure 33 i s derived from experimental work and figure 42 shows patterns i d e n t i c a l to those observed f o r methionine incorporation i n t o c e l l walls ( f i g . 43b). I take t h i s as good evidence that lobes i n M.rotata increase t h e i r s i z e by growth o r i g i n a t i n g at the t i p s of lobes ( t i p growth), and that methionine l a b e l acts as a good i n d i c a t o r of the areas i n t o which new wall material i s contributed. In conclusion, autoradiograms reveal s p e c i f i c t i p and v e i n - l i k e patterns of incorporation i n t o primary wall i n d i c a t i n g l o c a l i z e d synthesis of m i c r o f i b r i l s ( i n the case of glucose l a b e l ) and probably of matrix ( i n the case of methionine). 73 Methionine incorporation i s p a r t i c u l a r l y l o c a l i z e d and follows the pattern predicted f o r t i p growth. 74 Figure 39 Glucose autoradiography of c e l l walls. Times l i s t e d include the time c e l l s were l e f t i n l a b e l plus 5 min. f o r the standard washing procedure. (a-c, j ) Walls of c e l l s labeled f o r 10 minutes j d - f , kj Walls of c e l l s labeled f o r 30 minutes ( g - i , 1) Walls of c e l l s labeled f o r 1 hour Note the t i p l a b e l pattern i n a - i and the veined pattern i n f, i and h. Walls j-1 show la b e l patterns probably asso-ciated with secondary wall synthesis. 75 Figure 40 Methionine autoradiography of c e l l walls. Times l i s t e d include the time c e l l s were l e f t i n l a b e l plus 5 min. f o r the standard washing procedure, (a) Wall of a c e l l labeled f o r 10 minutes ib,c) Walls of c e l l s labeled f o r 15 minutes (d,f1 Walls of c e l l s labeled for 20 minutes (g-1) Walls of c e l l s labeled f o r 35 minutes Note the t i p la b e l pattern i n a-k and the veining i n j and k. C e l l wall 1 i s t y p i c a l of a number of walls probably i n stages of secondary wall synthesis. 75 F i g u r e 40 76 Figure 4 l Tip l a b e l i n g as shown by autoradiography (a,b) Two stage 11 c e l l walls both labeled f o r 10 minutes and showing patterns of t i p l a b e l i n g . The area of uni-form t i p l a b e l i n g with glucose (a) i s much larger than that with methionine (b). (c,d) Two stage 9 c e l l walls labeled f o r 10 minutes with glucose and showing t i p l a b e l i n g . The amount of la b e l i n g i s somewhat d i f f e r e n t , but the area labeled i s roughly the same. 76 77 Figure 42 Idealized map of c e l l wall fate f o r the upper wing lobe (arrow) of a stage 10 semicell. The model as-sumes that a l l new wall s t a r t s at the t i p s of lobes and i s pushed back: by subsequent t i p addition of wall as rapidly as wall perimeter i s added to ( t h i s rate measured from f i g . 18). Contours d i v i d e the wall i n t o age categories; a l l wall within the f i r s t contour i s less than 10 minutes old, a l l within the second contour i s less than 20 minutes old and so on. Figure 43 A composite diagram of l a b e l i n g experiments, the tracings made from the upper wing lobes of about 30 stage 10 semicells using a camera l u c i d a . The area of uniform and dense t i p l a b e l i s shown f o r c e l l s labeled f o r 10 minutes, 20 minutes and so on. fa) Patterns of t i p l a b e l i n g with glucose (b) Patterns of t i p l a b e l i n g with methionine. Note the close correspondence between th i s methionine pattern and the model pattern ( f i g . 42). 77a. Figure 42 Figure 43 G L U C O S E METHIONINE 78 Results and Discussion VI. Sections through the Isthmus I have commented already on the occurrence of d i f -ferent symmetry types i n M i c r a s t e r i a s . I have also observed that some species have a marked tendency to produce c e l l s of abnormal symmetry types whereas others do not. K a l l i o has noted differences i n both frequencies of spontaneous symmetry mutation and the ease with which these changes can be induced. Table V summarizes my observations and the information a v a i l -able from the l i t e r a t u r e . It i s clear that some species and clones are notoriously mutable (M.torreyi and uniradiate M.thomasiana) and others extremely stable (M.rotata and M. d e n t l c u l a t a ) . I reasoned as follows: I f there i s a structure i n the parent semicell responsible f o r continuity of symmetry i n daughter semicells, i n mutable species or clones the struc-ture must be ei t h e r more l a b i l e or i t s d i s p o s i t i o n i s i n some way r a d i a l l y more uniform than i n very stable species. I began looking at cross-sections through the c e l l isthmus hoping that i n mutable species or p a r t i c u l a r l y mutable clones the cross-section would be more nearly round than i n stable species. In a preliminary study of 3 species (including 6 symmetry types) t h i s proved to be the case. About 70 c e l l s were sectioned, and f o r 4 to 5 c e l l s i n each of the six groups I got appropriate sections i n good condi-t i o n ( f i g . 4 4 ) . My p r e d i c t i o n was borne out as follows: Biradiate M.torreyi, uniradiate M.thomasiana, p a r t i a l l y Table V S t a b i l i t y of Di f f e r e n t - Symmetries ( f a c i e s ) i n Haploid M i c r a s t e r i a s Species and pacies Personal Observations Published Reports Comments Reference M. r o t a t a b i r a d i a t e u n i r a d i a t e t r i r a d i a t e Stable S i n g l e occurrence, un-s t a b l e , perhaps d i p l o i d Stable S i n g l e occurrence i n pygmy clone, very unstable K a l l i o (1972) K a l l i o (1954) M. d e n t i c u l a t a b i r a d i a t e Stable Very s t a b l e K a l l i o (1972) M. r a d i a t a b i r a d i a t e u n i r a d i a t e Stable Occurs only i n un-, healthy pygmy s t r a i n Very s t a b l e K a l l i o (1972) M. s o l b i r a d i a t e u n i r a d i a t e a r a d i a t e Stable Stable ) E a s i l y induced by UV ) E a s i l y induced, ) somewhat non-viable ) K a l l i o (1968) M. thomasiana b i r a d i a t e u n i r a d i a t e Stable Unstable Stable Occurs only r a r e l y , i s unstable K a l l i o (1951) K a l l i o ( I 9 6 0 ) Waris (1950b) M. americana b i r a d i a t e u n i r a d i a t e a r a d i a t e Stable ) Occurs only r a r e l y , ) i s unstable ) Stable ) K a l l i o ( i 9 6 0 ) K a l l i o and H e i k k i l a (1969) M_. f i m b r i a t a b i r a d i a t e u n i r a d i a t e j K a l l i o (1972) M. t o r r e y i b i r a d i a t e u n i r a d i a t e a r a d i a t e Unstable Occurs f r e q u e n t l y , i s unstable Unstable, e a s i l y induced E a s i l y induced by UV, 1 i s unstable ) Induced by UV, ) i s unstable ) K a l l i o (1957) K a l l i o and H e i k k i l a (1969) 80 uniradiate and revertant b i r a d i a t e M.thomasiana a l l have isthmus cross-sections which appeared to be round. The cross-sections f o r b i r a d i a t e and t r i r a d i a t e M.rotata were c l e a r l y not round. As a r u l e , i n M.rotata the Isthmus i s flattened s l i g h t -ly at points from which wings a r i s e . In b i r a d i a t e c e l l s t h i s gives the isthmus the cross-sectional shape of a s l i g h t l y squashed c i r c l e , a shape asymmetry which could be related to preformed template required by theory to impose or transmit symmetry information to daughter semicells. Other s t r u c t u r a l components of the parent semicell do not show appropriate asymmetries, do not maintain a fixed asymmetric po s i t i o n or have an asymmetry which has proven to be i r r e l e v a n t . In the l a t t e r category are included semi-c e l l s lacking one or more wings or the chloroplast contained therein. Wings are not necessary f o r normal morphogenesis (see f i g . 29) , and c e l l s also develop normally though lacking large pieces of chloroplast, a l l the chloroplast of one wing being absent f o r example. In conclusion, parent and daughter semicells i n M.rotata share a b i r a d i a l symmetry with the isthmus cross-section. M.torreyi and M.thomasiana are more mutable and hence may have a less determinate template. These species lack the isthmus asymmetry thus suggesting a possible l i n k between isthmus cross-section and the morphogenetic template. 81 Figure 44 Isthmus cross-sections. Sample sections are taken from Epon-embedded c e l l s sectioned as shown i n (a) so that secondary and primary wall are both included i n the section. Lines are drawn i n to show the orien t a t i o n i n which l a t e r a l wings would l i e i f deeper sections were taken. A l l photos are X 6 5 0 . (b) M. t o r r e y i b i r a d i a t e (c) M. thomasiana uniradiate (d) M. thomasiana b i r a d i a t e reverted from a uniradiate culture (e,f) M. rotata b i r a d i a t e , two d i f f e r e n t c e l l s (g,h) M. rotata t r i r a d i a t e , two d i f f e r e n t c e l l s 813 82 Discussion I. Tip Growth i n Micrasterias Area growth i n c e l l walls generally occurs i n walls of a p a r t i c u l a r sort; they are t h i n and constructed of loosely packed m i c r o f i b r i l s with matrix f i l l i n g the spaces. Such walls are call e d primary walls and grow i n substance by the intus-susception of material i n t o e x i s t i n g wall. Various physical, chemical and biochemical processes contribute to primary wall growth. Wall stretching, wall loosening and wall deposition are a l l important aspects of growth, and one would l i k e to know i f any one of these i s more important than the others. For example, growth might occur because the f a b r i c of the wall i s loosened, f o r t h i s would allow the wall to be stretched by turgor and new material would f i l l up the spaces. This i s a d i f f i c u l t hypothesis to prove (Cleland, 1971) f o r the processes c o n t r i -buting to wall growth are as d i f f i c u l t to separate from one another as are Kiermayer's f i v e causal aspects of morpho-genesis i n M i c r a s t e r i a s . In general i t does appear that turgor stretching alone cannot drive the growth process (Roelofsen, 1965), but may act to a l i g n growth i f the wall i s not i s o t r o p i c . I tend to accept the argument that c e l l wall deposition i s rather important, p a r t i c u l a r l y the deposition of matrix substances ( S e t t e r f i e l d and Bayley, I96I; Frey-Wyssling, i n Preston, 1964). S e t t e r f i e l d and Bayley (1961, pg. 56) view growth as follows: 83 "Dependence of wall deposition on extension might be v i s u a l i z e d as some process by which stretching of the wall frees active surface areas required f o r addition of new materials ... The a l t e r n a t i v e r e l a t i o n , dependence of wall elongation on deposition, might be explained on the basis that a given amount of wall material i s only cap-able of a certain amount of stretching by turgor, and f o r continued expansion to occur, new material capable of being stretched must be added." Obviously we are faced with multiple causation (Castle, 1953) and want to discover how d i r e c t i o n a l information i s c o n t r i -buted . Two categories of primary wall growth may be contrasted; extension and t i p growth. Extension growth i s the means by which c y l i n d r i c a l c e l l s elongate uniformly along t h e i r length. The multi-net hypothesis (Roelofsen and Houwink, 1953) explains changes observed i n m i c r o f i b r i l orien-t a t i o n during extension growth, but does not explain the mechanism of growth I t s e l f . S e t t e r f i e l d and Bayley, as quoted above, come closer to that. Tip growth i s demonstrated when marking experiments or radiolabeling show that wall extension and deposition are l o c a l i z e d at a small area of the w a l l . This area may be hemispherical and act so as to produce a cylinder of non-growing wall behind as i n the extensively studied case of hyphal growth i n fungi (Robertson, 1959, 1968; Grove et a l . , 1970). Tip growth also occurs i n a l g a l rhizoids and moss protonema (Sievers, 1967) and i n higher plants; i n root hairs (Sievers, 1963; Bonnett and Newcomb, 1966), p o l l e n tubes 84 (Rosen et a l . , 1964; Franke et a l . , 1972) and f i b e r s and s c l e r i d s (Esau, 1967). I t i s to be expected that i n some cases t i p and extension growth could both occur as the mechanisms of each may be s i m i l a r . Mix (1961) looked at m i c r o f i b r i l o r i e n t a t i o n i n the primary walls of three desmid species. M i c r o f i b r i l s were unoriented i n walls of Cosmarium but l o n g i t u d i n a l l y oriented i n parts of the elongated wall of Pleurotaenium. Longitudinal m i c r o f i b r i l o r i e n t a t i o n i s usual-ly associated with extension growth while i n a l l cases so f a r studied, growing t i p s show randomly oriented m i c r o f i b r i l s (Houwink and Roelofsen, 1954; Sassen, 1964; Green and King, 1966; Bartnicki-Garcia, 1972). The apical c e l l i n N l t e l l a has randomly oriented m i c r o f i b r i l s at i t s t i p , but these become transversely rather than l o n g i t u d i n a l l y aligned at the base of the growth hemisphere. Microtubules are Impli-cated i n primary wall extension growth but have not yet been found i n the cytoplasm of growing t i p s (Newcomb, 1969). Cytoplasmic p o l a r i t y i s a s t r i k i n g u l t r a s t r u c t u r a l feature of c e l l s showing t i p growth. Tip cytoplasm Is usual-ly packed with vesi c l e s to the exclusion of a l l other organ-e l l e s . Golgi and ER l i e at some distance from the t i p and larger structures, such as n u c l e i , are even more di s t a n t . With phase microscopy, vesi c l e s can be seen to wander from the golgi region to the v e s i c l e - f i l l e d t i p and to remain there. As the c e l l wall i s d i f f e r e n t i a t e d i n t o only two areas, a r a p i d l y growing one (the t i p ) and a non-growing one, i t i s frequently argued that the observed cytoplasmic p o l a r i t y i s s u f f i c i e n t to explain t i p growth of the w a l l . 85 In the various mechanisms proposed for t i p growth, cytoplasm i s responsible d i r e c t l y f o r supporting areas of rapid c e l l wall synthesis or i n d i r e c t l y through cytoplasmic p o l a r i t y , allowing a s p a t i a l d i f f e r e n t i a t i o n of c e l l wall functional a c t i v i t y to e x i s t . Mechanisms appropriate to the former argument include; (1) d i r e c t i o n a l transport of v e s i c l e s to c e r t a i n regions of the c e l l wall by microtubules or oriented cytoplasmic stream-ing, (2) regional s p e c i a l i z a t i o n of the membrane to a t t r a c t v e s i c l e s , or (3) the elaboration of a special organelle responsible f o r t i p growth. Some of these organelles can a c t u a l l y be seen, such as the spitzenkorper found i n some hyphae (McClure e_t a l . , 1968; Grove and Bracker, 1970); others are purely hypo-t h e t i c a l (Bartnicki-Garcia and Lippman, 1969). The growth of lobes i n M.rotata shows many p a r a l l e l s with t i p growth i n these other plant and fungal systems. Lobe cytoplasm shows a s i m i l a r p o l a r i t y i n d i s t r i -bution of organelles with v e s i c l e s only at the t i p , then ER and g o l g i . I f growth i s arrested, lobes deposit a thick layer of c e l l wall at t h e i r t i p s as i s c h a r a c t e r i s t i c of root hairs (Schroter and Sievers, 1971). The l a s e r serves to mark M.rotata wall i n the same way that dyes and various p a r t i c l e s have been used to mark other c e l l s f o r wall fate studies (Rosen et a l . , 1964; Green and King, 1966), and autoradio-graphic studies s i m i l a r to mine are common i n the l i t e r a t u r e on t i p growth (Bartnicki-Garcia and Lippman, 1 9 6 9 ; Galun, 1 9 7 2 ) . Hyphae w i l l rupture at t h e i r t i p s much as M.rotata lobes do i n acid solutions. Hyphal t i p s w i l l begin to harden over i f growth i s arrested, f o r i f growth i s then a l -lowed to resume, t i p s produce hyphae of a much reduced diameter (Robertson, 1 9 5 9 ) . M.rotata shows s i m i l a r narrowing of t i p s a f t e r plasmolysis ( f i g . 2 3 b ) , but the time course of t i p hardening could not be determined as i t has been i n hyphae. Pollen tube growth i s enhanced by the presence of pectinase and c e l l u l a s e (Roggen and Stanley, 1 9 6 9 ) ; M.rotata lobes do not show t h i s e f f e c t . In short, I have f a i r l y good evidence that Micrasterias lobes o f f e r an example of t i p growth comparable with the growth of fungal hyphae, root hairs and perhaps pollen tubes. And as well as being just another example, Micrasterias offers p o t e n t i a l l y i n t e r e s t i n g information not available f o r other t i p growth systems. In p a r t i c u l a r , M.rotata c e l l wall preparations are very t h i n and auto-radiograph can be done close to the t h e o r e t i c a l l i m i t of res o l u t i o n (Schultze, 1 9 6 9 ) . Figure 45 represents the model f o r t i p growth developed by Robertson ( 1 9 5 9 ) to explain his observations on hyphae. In general, t i p growth i s a steady state s i t u a t i o n . The hemispherical growth zone must maintain i t s e l f ; new wall material must be supplied to the growth hemisphere as rapid-l y as completed stable wall passes to the cylinder behind. Wall i n the growth zone i s therefore p l a s t i c as i t i s both gaining new substance and enlarging i n area. Wall passing from the growth zone cannot be p l a s t i c and i s o t r o p i c both while s t i l l contributing to a cylinder of fixed radius. Robertson proposes two fundamental parts to t i p growth. Wall area must be increased and new wall incorporated, and these w i l l occur at a maximum rate at the very t i p (A i n f i g . 45). Over the rest of the growth zone wall area may increase, but hardening of p l a s t i c wall must also occur (B i n f i g . 45). The two processes, incorporation and harden-ing, could occur by any of a number of mechanisms. P l a s t i c wall may be added at A, expand and become s t r a i n hardened at B. P l a s t i c wall may be biochemically altered at B, enzymes added at A may be degraded, or chemical bonds altered to a f f e c t wall r i g i d i t y . Or a second wall component may be added at B strengthening and hardening the p l a s t i c component. This l a t t e r p o s s i b i l i t y was envisaged by Robertson f o r hyphae and appears to be the case i n M.rotata, as two d i s t i n c t pat-terns of wall incorporation are revealed by autoradiography. The pattern of methionine incorporation f i t s well with i t s being associated with synthesis of p l a s t i c wall at A. The hardening process at B i s then associated with the incorpora-t i o n of glucose and hence with synthesis of m i c r o f i b r i l s . This c o r r e l a t i o n of two d i s t i n c t synthetic a c t i v i t i e s with Robertson's two model processes i s an important r e s u l t . Note that the two processes must be exactly balanced f o r t i p growth to produce a cylinder uniform radius. If the hardening reaction begins to overtake incorporation of p l a s t i c wall, the cylinder produced w i l l begin to narrow. This appears to be the case i n normal lobe development i n M.rotata for lobes do become progressively narrower and eventually come to a point. Narrowing i s not so marked i n c e l l s grown at 37° C and can be completely reversed, as i n the presence of pectin methyl esterase. One would expect that chemicals a l t e r i n g methionine metabolism would be l i k e l y to af f e c t narrowing or broadening of t i p s . I have so fa r discussed lobe growth i n M.rotata as a t i p growth system. I wish now to propose that Micrasterias wings are produced by a combination of processes Including t i p growth and branching. I t may s t r i k e the read-er that M.rotata wings do not much resemble a branched hypha. The pattern i s f a r more obvious i n M.radiata, i n the c e l l ' s shape and i t s developmental stages. Note figur e 22, stages 4 to 7. Here the four wing lobes lengthen while the notches remain fi x e d ; c l e a r l y a case of pure t i p growth. Note stages 7 to 9 of the same figure; here both t i p s and notches change i n r e l a t i v e p o s i t i o n and t i p s branch. In M.radiata t i p growth i s only part of normal morphogenesis; a second sort of growth which acts to broaden lobes also contributes ( f i g . 22 i n s e t ) . In M.rotata, t i p growth and broadening growth are less separable. Throughout de-velopment both lobe t i p s and notches change r e l a t i v e p o s i -t i o n , though i n late developmental stages (stages 11 to 14 i n f i g . 18) c e l l size continues to increase a f t e r lobe denta-t i o n i s complete. I t i s during these stages that the 8 9 v e i n - l i k e pattern of c e l l wall l a b e l i n g appears i n auto-radiograms. The conclusion i s inescapable that broadening growth i s associated with a synthetic process s i m i l a r to t i p growth, but synthesis occurs i n various patterns over the surface of the c e l l wall rather than exclusively at lobe t i p s ( f i g . 46). Note that branching and lobe broadening can be separated experimentally from pure t i p growth. Growth of M.rotata lobes at 37° i s an example where, as i n normal de-velopment of M.radiata, branching and lobe broadening are both absent. To t h i s extent branching and broadening appear to be associated with one another. This discussion has neglected almost e n t i r e l y the morphogenesis of the polar lobe. Polar lobe growth does not f i t n i c e l y i n t o a model depending on l o c a l i z e d synthesis. The lobe does not appear to grow at i t s t i p but rather by some more d i f f u s e mechanism, perhaps by extension growth. An EM study of m i c r o f i b r i l o rientation might c l a r i f y t h i s prob-lem . In conclusion then, the growth patterns observed i n Micrasterias wings are associated with c h a r a c t e r i s t i c pat-terns of wall synthesis. This implies that Micrasterias shape should be explained i n terms of the patterns of c e l l wall synthesis and the ways that these a r i s e and are maintained. 90 Figure 45 A model for hyphal t i p growth according to Robertson (1959). New wall i s i n i t i a t e d at the t i p and grows i n area to pass eventually i n t o the cylinder behind, Two processes are associated with t h i s : (1) Incorporation of large amounts of p l a s t i c wall sub-stance i n region A and (2) Hardening of t h i s wall over the surface of the hemi-sphere (region B). Figure 46 Morphogenetic variations on t i p growth 'a) Tip growth showing some degree of narrowing [bi Tip growth with branching c; Tip growth with branching and broadening (arrows) as occurs i n stages 7 to 9 of M.radiata development (fig.22) Inset: The patterns of labeT which would be associated with the three variations on t i p growth i f wall synthesis and l o c a l i z e d wall expansion always occurred together. Note that (c) resembles the veined pattern seen i n la t e stages of M.rotata growth. 90a Figure 45 91 Discussion I I . The Template i n Micrasterias There is some agreement that at least part of Micrasterias morphogenesis depends on the actions of a template ( K a l l i o , 1972b). The template may embody s p e c i f i c information concerning the ultimate p o s i t i o n of each semicell lobe and notch (Kiermayer, 1970b) or i t may provide only general i n -structions concerning symmetry. The l a t t e r s i t u a t i o n i s more l i k e l y i f , as I w i l l argue i n part I I I , the growth of each lobe i s independent and s e l f - o r g a n i z i n g . The template then need be responsible only f o r I n i t i a t i o n of the wings and polar lobe, presumably by establishing the i n i t i a l growth s i n g u l a r i t y f o r each. As a f a i r l y large area of the stage 2 septum i s com-petent to give r i s e to wing s i n g u l a r i t i e s ( f i g . 47), any template associated with the septum at t h i s stage probably contains only general information s u f f i c i e n t to i n i t i a t e s i n g u l a r i t i e s i n roughly the appropriate symmetry r e l a t i o n s h i p . An understanding of septum structure and growth may be c r u c i a l to any explanation of the Micrasterias template. The mechanism of wall growth i n the septum i s probably s i m i l a r to or i d e n t i c a l with that occurring at l a t e r stages of morpho-genesis. Certainly the wall appears to have a s i m i l a r structure throughout morphogenesis. I f we consider the mechanics of septum growth, we must allow that a number of processes are involved. The septum wall must grow i n substance through addition of matrix and the lengthening of e x i s t i n g m i c r o f i b r i l s , probably also by the 92 addition of new m i c r o f i b r i l s . The c e l l membrane must also grow and may do so by the fusion of ve s i c l e s or intussusception of membrane subunits. Any of these processes might be d i s -posed i n a p a r t i c u l a r pattern so as to contribute a s t r u c t u r a l or f u n c t i o n a l asymmetry to the septum thereby causing i t to act as a template. As we have seen ( f i g . 15), the septum i n i t i a t e s at the Isthmus g i r d l e and grows inward. I presume that synthetic a c t i v i t y i s concentrated around the inner edge of the advancing septum and that t h i s edge, with i t s associated membrane, forms the substratum upon which new wall and membrane must be deposited. I f the Inner septum edge Is d i f f e r e n t i a t e d i n t o d i f f e r e n t regions about i t s perimeter, synthetic a c t i v i t i e s could be correspondingly s p a t i a l l y d i f f e r e n t i a t e d . I f the isthmus cross-section i s perf e c t l y c i r c u l a r , t h i s d i f f e r e n t i a -t i o n cannot occur as a l l parts of the r e s u l t i n g septum edge are i d e n t i c a l ( f i g . 48a). C e l l s with an asymmetric isthmus cross-section, however, have the opportunity to produce an asymmetric septum. I f template formation were related to cross-sectional asymmetry, we would expect c e l l s with the most asymmetric cross-sections to have the least mutable templates, and t h i s appears to be the case. At an isthmus s i m i l a r i n cross-section to that found i n M.rotata ( f i g . 48b), c e r t a i n regions of the septum edge (A In f i g . 48b) w i l l always have a greater curvature than others. I f the e f f i c i e n c y or or i e n t a t i o n of membrane addition or wall synthesis depended upon curvature of the substratum, then the septum formed at such an isthmus would have to be 93 b i r a d i a l l y symmetric i n structure. This deviation from r a d i a l symmetry offers i t s e l f as a r e l a t i v e l y plausible candidate for a template. Consider a hypothetical case i n which the synthesis of wall m i c r o f i b r i l s i s proportional to the density of micro-f i b r i l s i n the e x i s t i n g inner septum edge. The more r a d i a l l y disposed m i c r o f i b r i l s would converge and become r e l a t i v e l y dense at points of greatest curvature (A i n f i g . 48c). The r e s u l t i n g septum would have a b i r a d i a l l y symmetric gradient of m i c r o f i b r i l density with points of least density f a l l i n g rough-ly where, i n M.rotata, the i n i t i a t i o n of s i n g u l a r i t i e s may be demonstrated. This example i s presented to demonstrate that wall structure has, i t s e l f , considerable potential as a bearer of morphogenetic information. A case could s i m i l a r l y be made for s p e c i f i c s t r u c t u r a l organization i n the c e l l membrane or associated cytoplasm. In conclusion, the septum produced i n M.rotata could contain information concerning the symmetry of the parent semi-c e l l , possibly as asymmetries b u i l t i n t o the structure of the septum during i t s formation. The septum could f u l f i l l the minimum required of a template for Micrasterias morphogenesis i f t h i s information were translatable i n t o instructions for the i n i t i a t i o n of three s i n g u l a r i t i e s and could thus represent the template sought by other authors. 94 Figure 47 One daughter of a d i v i d i n g pair at stage 2 of c e l l development. Regions of the new semicell competent to produce wing lobes are cross-hatched and labeled (R). Figure 48 Three models of septum growth. In each case the cross-sectional isthmus space i s to be f i l l e d i n by uniform inward growth of c e l l wall material. (a) A p e r f e c t l y round cross-section such as that found i n M.torreyi and M.thomasiana. Note that a l l points of the inner septum edge (arrows) w i l l be i d e n t i c a l regard-less of the mechanism by which the septum i s constructed. (b) A cross-section s i m i l a r to that found i n M.rotata showing the regions of greatest (A) and least (B) curvature at the inner septum edge. (c) A cross-section i d e n t i c a l to that i n (b) demonstrating that points spaced uniformly at septum i n i t i a t i o n w i l l converge non-unlformly as the septum grows inward. I f the points represented some r e a l structure, the r e s u l t -ing septum would demonstrate a b i r a d i a l l y symmetric gradient i n the density of the structure. 94c? Figure 47 Figure 48 95 Discussion III. Morphogenesis and Organization I t i s natural to hope f o r some unity underlying the di f f e r e n t morphogenetic contributions to f i n a l c e l l wall shape. For one, the raw material of morphogenesis, the primary c e l l wall, appears to have the same physical structure throughout c e l l development, hence the physical and biochemical mechanisms of growth may be the same. Perhaps also the organization re-sponsible f o r morphogenesis i s i n some way the same regardless of the pattern of growth, even i n two such d i s t i n c t growth processes as template formation and t i p growth. As mentioned, wall growth i s a coordinated a c t i v i t y demanding a number of preconditions, and most of the contrib-uting processes do not also contribute d i r e c t i o n a l information. With t i p growth, though i t also i s a complex a c t i v i t y , we expect to be able to point to some part responsible for d i r e c t i o n a l information; to f i n d , somewhere, organization d i r e c t l y relevant to morphogenesis rather than j u s t to growth. It i s usual to look either to the cytoplasm or to the c e l l wall i t s e l f f o r hints concerning t h i s organization. "Theories of the mechanism of s p i r a l growth ( i n Phycomyces sporangiophores) f a l l i n t o two categories: (1) those that a t t r i b u t e the o r i g i n primarily to protoplasmic ac-t i v i t y of some sort, not always well de-fined; and (2) those that look to e x i s t i n g oriented wall structure, on the whole or i n part, f o r i t s immediate source." (Castle, 1953; pg. 365) Castle's comments are as appropriate to t i p growth as to s p i r a l growth; i t i s fashions i n research which d i c t a t e where one i s most l i k e l y to look f o r organization. 96 With t i p growth i n Micrasterias, the choice of c e l l cortex ( c e l l wall plus plasma membrane) as the seat of organiza t i o n i s f a i r l y e a s i l y made. We can f i r s t eliminate the nucleus as a d i r e c t o r of wall growth. Certainly the nucleus provides something to the growing c e l l , f o r the enucleate c e l l runs down af t e r several hours. But a certa i n amount of morphogenesis does take place i n the absence of a nucleus (Selman, 1966), and the various i n t e r e s t i n g effects of nuclear ploidy on growth patterns can a l l be interpreted as due to the increased turgor available to polyploid c e l l s (Green, 1969). The general cytoplasm i s also a poor candidate f o r organization. Oriented structures such as microtubules are absent from areas of active c e l l wall growth (Kiermayer, 1968b), and the cytoplasm shows very active streaming (Kiermayer, 1964). I t Is d i f f i c u l t to imagine any cytoplasmic organization or structure maintain-ing I t s e l f i n such a s i t u a t i o n though Waddington (1962, 1966) has t r i e d to get around the problem of cytoplasmic a c t i v i t y by proposing various dynamic structures, a l l of which remain hypothetical. The c e l l cortex i s a much better candidate f o r the embodiment of organization. F i r s t , the cortex (the wall por-t i o n , at least) i s a fixed structure, and i t i s easiest to imagine organization i n something r e l a t i v e l y f i x e d . Second, morphogenesis i s altered i n very s p e c i f i c ways when the c e l l cortex i s damaged. Neither of these arguments i s e n t i r e l y compelling, but the t h i r d i s more so; the c e l l cortex exhibits a s p a t i a l d i f f e r e n t i a t i o n of a c t i v i t i e s at a f a r f i n e r scale 97 than any seen i n the cytoplasm. That i s , the c e l l cortex shows a s i n g u l a r i t y with p a r t i c u l a r growth and synthetic c a p a b i l i t i e s which i s of the order of microns i n size whereas cytoplasmic p o l a r i t y i s expressed over tens of microns. Hence the organiza-t i o n most relevant to t i p growth must reside i n the c e l l cortex, and the seat of organization i s roughly i d e n t i c a l with the s i n g u l a r i t y as described from l a s e r experiments. Note that Robertson's model for t i p growth has s i m i l a r implications. His region of incorporation (A i n f i g . 45) i s self-perpetuating as long as the hardening process (B i n f i g . 45) does not catch up; the t i p growth zone tends to maintain i t s e l f once established. Hence i n hyphae or i n Micrasterias lobes, t i p growth i s a self-organizing behavior of the cortex. Tip growth i s an organized system, the organization associated with s i n g u l a r i t i e s . By using t h i s vague word " s i n g u l a r i t y , " I stress that we know nothing of the structure of these s p e c i f i c s i t e s or even whether they should be thought of i n s t r u c t u r a l terms at a l l . We can ask i n what part of the c e l l cortex the s i n g u l a r i t y resides, whether i n membrane, i n c e l l wall or whether In both. I have no experimental evidence to support a p a r t i c u l a r a l t e r n a t i v e , but I favor c e l l wall as the most l i k e l y place f o r reasons analogous to those given by Waddington f o r r e j e c t i n g cytoplasmic structure as a possible template. That i s , the wall i s probably the most fixed and stable component of the c e l l cortex whereas, I f animal c e l l s are any i n d i c a t i o n , c e l l membrane i s probably much more l a b i l e (Chapman-Andresen, 1972). In any case, I f e e l i t a worthwhile 98 exercise to show that c e l l wall could i n theory have structure and organization s u f f i c i e n t to explain morphogenesis, without the imposition of s p e c i f i c s t r u c t u r a l order from cytoplasm or membrane. A s i n g u l a r i t y i n Micrasterias could be thought of as a point of i n s t a b i l i t y on the c e l l w a l l . I f stable wall com-prised a feedback-controlled balance of m i c r o f i b r i l s and matrix, then the s i n g u l a r i t y would be a point at which the balance was upset, the i n i t i a l upset occurring when turgor stretched the wall enough to allow a b i t of extra matrix to be incorporated. F i b r i l s would begin to be synthesized i n the area of new matrix incorporation to return the m a t r i x - t o - m i c r o f i b r i l balance, but would always be just a few steps behind. Matrix and micro-f i b r i l synthesis would race one another; matrix synthesis could not get too f a r ahead as a wall of matrix alone becomes un-stable. So we have a s i n g u l a r i t y established, one which perpetuates i t s e l f . Figure 49 shows by graphical example the sorts of interactions one would expect i n such a system. The si n g u l a r i t y i s an i n s t a b i l i t y i n the sense that wall precursors flow through i t on t h e i r way to becoming mature wall (Prigogine and N i c o l i s , 1971). The s i n g u l a r i t y remains, not as a r e a l structure but as a dynamic one. One can now imagine how s i n g u l a r i t i e s might a r i s e i n the Micrasterias septum. When the septum Is being formed, there i s equal turgor on both sides and no net stress, so areas of d i f f e r e n t m i c r o f i b r i l density can be formed . Only when the septum bulges out and i s subjected to i n t e r n a l turgor do these 99 areas expose themselves as weak points i n t o which extra b i t s of matrix may be able to sneak. Badly coordinated osmotic events could account f o r the production of double c e l l s ( f i g . 27), f o r regardless of whether the septum were complete when septum bulging occurred, the template would be expressed. The model presented i n figure 49 has a number of biases. I t focusses on c e l l wall as a s i t e of organization ignoring other p o s s i b i l i t i e s . I t exploits dynamics at the ex-pense of r e a l mlcrostructure f o r no s p e c i f i c s t r u c t u r a l order i s required; there are no ordered f i b r i l l a r networks or pave-ments of matrix to which subunits must be added i n s p e c i f i c configuration. Events are ordered rather than structures. The model r e l i e s heavily on physical rather than biochemical p r i n c i p l e s . I t assumes that synthetic enzymes and precursor substances are available i n abundance as part of the preconditions f o r growth, and so biochemical regulation does not enter i n t o the arguments. Explanations i n terms of i n s t a b i l i t i e s and e q u i l i b r i a are perhaps too simple to endure, but they are appropriate to the l i m i t e d information a v a i l a b l e f o r M i crasterias. In conclusion, the organization relevant to morpho-genesis resides i n the c e l l cortex, perhaps i n the c e l l w a ll. Organization may be thought of i n s t r i c t l y s t r u c t u r a l terms, as embodied i n a s p e c i f i c a l l y ordered microstructure f o r example; but plausible a l t e r n a t i v e s to t h i s view can be offered. 100 Figure 49 A physical model of t i p growth dynamics. The wall i s considered to have two major components as shown i n the two upper insets; a f i b r i l l a r network and a globular matrix. Appropriate physical variables include the following: (1) R, the r a t i o of matrix to f i b r i l s (a r a t i o of mass per unit area of wall f o r each); (2) M and F, the synthesis (incorporation)rates f o r matrix and f i b r i l s respectively (measured as mass per unit time added to a unit area of wall) and (3) D, t o t a l wall density (the sum of mass per unit area of wall of matrix and of f i b r i l s ) . (a) The graph shows that M responds, to changes i n both R arid D. For a given R there i s a D at which the wall i s stable and no matrix i s being added to the wall. I f D i s lowered, matrix begins being added so as to r e - e s t a b l i s h a stable D. The response curve s h i f t s as R changes. (b) The graph shows that F responds to changes i n R but not to changes i n D. I f matrix i s added to the wall, thereby increasing R, F w i l l act to r e - e s t a b l i s h the o r i g i n a l R by incorporation of f i b r i l s . (c) The graph shows that matrix added to walls with d i f -ferent R values contributes d i f f e r e n t l y to increase i n wall area. In walls of low matrix content, additional matrix f i r s t f i l l s up e x i s t i n g spaces between the f i b r i l s . In walls r i c h i n matrix, a d d i t i o n a l matrix gets i n only by pushing aside e x i s t i n g wall to create space. Note now the Interesting behaviors of the system defined by these graphs. Take a stable wall of density Do and r a t i o R 0 and subject i t to increased turgor. I f parts of the wall can be stretched s u f f i c i e n t l y to cause l o c a l s h i f t s i n wall density - a s h i f t from D G to Di (graph a) -then matrix w i l l be added at these points to f i l l up spaces. This has the e f f e c t of increasing both D and R together. As R increases (to Ri or R2) f i b r i l synthesis i s brought i n t o play (graph b) and acts to increase D while decreas-ing R. Thus R can be balanced by c o n f l i c t i n g a c t i v i t i e s while D s t e a d i l y returns to i t s i n i t i a l value. We are re-turned to ,R0 and D 0 but an increase i n wall area has occurred. We could imagine a case, however, i n which f i b r i l synthesis was unable to dominate matrix synthesis i n t h i s way (a low F m a x ) . Local areas could exist at which matrix addition outpaced the e f f e c t of F to lower D (and hence to lower M), but would be surrounded by areas In which f i b r i l synthesis was catching up by t h i s same mechanism. The regions of maximum matrix incorporation would have high R values and so substantial l o c a l i n -creases i n wall area would accompany matrix addition there (graph c ) . In effect we would have at those regions, a l l the c h a r a c t e r i s t i c s required of a t i p growth s i n g u l a r i t y . Figure 49 100a a b MATRIX/FIBRIL RATIO c % OF MATRIX SYNTHESIS CONTRIBUTING TO INCREASE IN WALL AREA 101 Discussion IV. Morphogenesis and Explanation I have devoted considerable space to a discussion of morphogenesis i n Micrasterias and wish now to examine whether our understanding of the process has been thereby im-proved. On the one hand, I have t r i e d to undermine c e r t a i n of the published arguments concerning morphogenesis. In p a r t i c u -l a r , I have shown the c e l l cortex to be f a r more active i n organizing i t s own morphogenesis than K a l l i o or Kiermayer appear w i l l i n g to admit. Waddington i s closer to my p o s i t i o n i n his recognition of the importance of regional s p e c i a l i z a -tion of c e l l surface, but he Is at a loss as to the mechanism responsible. I w i l l admit that I have not come to grips with the exact mechanisms, but I have suggested the form that an explanation might take, using by example a r e l a t i v e l y plausible argument stated i n terms of c e l l wall microstructure and syn-t h e t i c dynamics. I have broken morphgenesis of the whole c e l l i n t o manageable parts, analysed the parts and shown that they can be reassembled. And again, I have t r i e d to state my argu-ments as I f e e l an explanation of morphogenesis must be stated. I have avoided discussing "causes" of morphogenesis, for example; I have not asked whether turgor causes morphogenesis or organization causes t i p growth. I now want to examine whether my arguments are r e a l l y appropriate and e s p e c i a l l y whether we should expect that better explanations e x i s t . In preparing t h i s section I have consulted a number of works concerned with s c i e n t i f i c explanation p a r t i c u l a r l y 102 as regards developmental biology (Woodger, 1929; Waddington, 1934, 1962; Needham, 1934; Hempel and Oppenheim, 1948; Bonner, i960; Picken, i960; Blandino, 1971; Pattee, 1971; Steward, 1971; Rosen, 1970, 1972). Each author deals with the problem i n a d i f f e r e n t way, the most useful I found to be the machine analogy used by Needham (1934). I f appropriately developed, th i s one analogy can embrace a l l the other arguments I c o l l e c t e d . Therefore l e t us take a machine, a Swiss watch f o r example, and t r y to explain i t s functioning. I f l i v e and mechanical systems have the s i m i l a r i t i e s suggested by Descartes, or more recently by Loeb (Blandino, 1971), then a s a t i s f a c t o r y explanation of the watch w i l l suggest how we must best explain b i o l o g i c a l systems. We would probably want to f i r s t take the watch apart and examine Its parts, perhaps l i s t i n g a c e r t a i n number; springs, gears, shafts and so on, and diagramming the way they f i t together. Knowing the important parts and the manner of f i t t i n g together, we could describe a l l the mechanical actions which take place when the watch functions. This gives us an a n a l y t i c explanation, but the watch i s not yet explained as well as i t could I f we also knew something of the p r i n c i p l e s of mechanical motion. Knowing about momentum, moments of i n e r t i a or simple harmonic motion adds a dimension to the explanation which does not exist i n a simple descrip-t i o n of the parts and the s t r u c t u r a l r e l a t i o n s between them. What we add to the a n a l y t i c d e s c r i p t i o n i s a group of con-s t r a i n t s (Pattee,1971). In naming constraints, i n e r t i a and harmonic motion are examples, we are b u i l d i n g a nonanalytic 103 language i n which names r e f e r to concepts, though each concept may be associated with i t s own c o n s t e l l a t i o n of s p e c i f i c physical experiments. Without constraint language, a decent explanation of almost anything would become Impossibly cumber-some and generalization would be very d i f f i c u l t . Now an enzyme i s a chemical machine of sorts, and enzyme chemists are close to a complete a n a l y t i c d e s c r i p t i o n of t h e i r machine. Yet they are not s a t i s f i e d that enzyme action has been explained. A gap exists; concepts such as ac-t i v e s i t e , microscopic r e v e r s i b i l i t y , entropy, even organization, cannot yet be replaced by a n a l y t i c d e s c r i p t i o n . Some concepts may be discarded or modified when a complete analytic description emerges, because they w i l l then be unnecessary or misleading. But some of the concepts w i l l remain i n t h e i r abstract form. To deny th i s i s to suggest that, knowing how the Swiss watch works, one need no longer bother about i n e r t i a or harmonic motion. The need f o r constraint language does not r e f l e c t the r e a l i t y that we wish to explain so much as i t does our own i n t e l l e c t u a l demands. Man cannot systematize nor use his know-ledge and cannot learn without the use of abstractions, and abstractions are found only i n constraint language. Phy s i c i s t s and chemists cannot get along without such a language and we cannot a_ p r i o r i expect b i o l o g i s t s to be any d i f f e r e n t . A science has the job, not only of analysing i t s p a r t i c u l a r machines into t h e i r parts, but of s o r t i n g through and r e f i n i n g the c o l l e c t i o n of words that make up i t s own language, par t i c u -l a r l y as regards the more abstract parts of that language. To 104 have the Swiss watch explained only i n terms of constraints such as i n e r t i a and harmonic motion may be unsatisfying, but t h i s does not mean that the terms themselves are useless or old fashioned. "Things are what they are; and i t i s use-less to disguise the fact that 'what things are' i s often very d i f f i c u l t f o r our i n t e l l e c t s to follow." (Whitehead, 1920; pg. 119) With Mic r a s t e r i a s , a l l that I observed concerning morphogenesis could have been described using terms borrowed from embryology, a l l of them e s s e n t i a l l y constraint terms. Thus the septum has i t s morphogenetic p o t e n t i a l progressively delimited as regions within the semicell f i e l d are determined as presumptive polar or wing lobe. The o r i g i n a l f i e l d i s s e l f -organizing and can regulate as one would expect. Subsidiary f i e l d s are also self-organizing and may r e t a i n the character of the o r i g i n a l f i e l d (as i n the polar lobe) or may not (as i n the wings). This d e s c r i p t i o n i s quite appropriate, but f o r the purposes of t h i s thesis I agree with Waddington (1962): "As a l l operationally defined terms, they (embryological terms) are useful f o r describing the r e s u l t s of experiments, but are feeble guides, or perhaps even deceptive ones, to the nature of the underlying elements whose properties bring about the processes which the experiments discovered." I have found i t f a r more useful to Introduce terms such as wall organization and s i n g u l a r i t y , and to add as a c o r o l l a r y that they show f i e l d - l i k e behavior. In a s i m i l a r fashion I have t r i e d to avoid the term c o r t i c a l Information, though c l e a r l y the Micrasterias c e l l wall and plasma membrane make up a c e l l 105 cortex which I have shown to contain information. A biochemist, now, might object to s i n g u l a r i t y and wall organization as ab-stractions so fuzzy as to be meaningless, and claim that a biochemical explanation i s to be preferred. But biochemistry, even i n i t s study of r e l a t i o n s between molecular structures rather than just structures themselves i s a d i s c i p l i n e heavily committed to analysis and a n a l y t i c explanations. Analysis i n the absence of a c a r e f u l l y framed constraint language w i l l not by i t s e l f give the best explanation of Micrasterias morpho-genesis, just as i t does not do so f o r enzyme a c t i v i t y or Swiss watches. The problem of explanation i s more pr e c i s e l y stated i n the language of systems theory. I f we want to work out the dynamics of systems behavior, p a r t i c u l a r attention must be paid to f i n d i n g the state variables appropriate to interactions within the system (Rosen, 1970, 1972). Systems theory i s es-p e c i a l l y f o r c e f u l on t h i s point; the state variables which present themselves to us as most obvious and measurable may not be the ones necessary for describing system i n t e r a c t i o n s . Therefore, there are t h e o r e t i c a l reasons f o r doubting that a n a l y t i c a l l y measureable variables are important i n themselves. Rosen (1972) comments that the best state variables are frequent-l y constructed from combinations of the observable vari a b l e s . These combinations may be rather abstract i n appearance and re-semble c l o s e l y the constraints of Pattee's argument. It would be absurd to suggest that a n a l y t i c technique w i l l not eventually provide a f u l l d e s c r i p t i o n of t i p growth 106 and c e l l morphogenesis. 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