Fine structural stud of cely l migration in the early mesoder omf … · Embryol. exp. Morph. Vol. 32, 3, pp. 723-738. 1974 723 Printed in Great Britain Fine structural stud of cely

/ . Embryol. exp. Morph. Vol. 32, 3, pp. 723-738. 1974 7 2 3

Printed in Great Britain

Fine structural study of cellmigration in the early mesoderm of normal and

mutant mouse embryos (T-locus: f/f)

By MARTHA SPIEGELMAN AND DOROTHEA BENNETT1

Department of Anatomy, Cornell University Medical College

SUMMARY

Mouse embryos homozygous for the f9-allele exhibit an enlarged primitive streak, scantymesoderm, and aberrant axial organization by the eighth day of development and die by thetenth day.

Light and electron microscopic observations of sections through mesodermal cells of thesemutant embryos reveal sheet-like arrangements of cells rather than the array of stellateindividual cells seen in mesoderm of normal embryos. The abnormal mesodermal cellsusually display lobate pseudopodia instead of the filiform pseudopodia typical of normalmesodermal cells. These lobopodia do not possess the subsurface microfLlaments characteristicof filopodia. Cellular junctions such as those regularly seen between adjacent filpopodia ofnormal mesodermal cells occur only very rarely between neighboring lobopodia of mutantmesodermal cells.

The deficiency of mesoderm may be due to the failure of cells to form the cellular associa-tions typical of gastrulation and to move normally from the primitive streak which con-sequently becomes enlarged due to the accumulation of apparently immobilized cells. Thosefew cells which do attain the proper location nevertheless do not appear well-differentiated.Their abnormalities of cell shape, cellular translocation, and cellular interactions may berelated to a lack of microfilament cytostructure and surface adhesiveness.

INTRODUCTION

Each of several mutant alleles at the complex T-locus in. the mouse whichresult in lethality in homozygous embryos has specific effects in space and timeon processes of development (Dunn & Bennett, 1964). The embryo homozygousfor the ?9-allele2 dies by the tenth day of gestation but can be distinguishedgrossly from its normal littermates at eight days by its retarded development ofaxial structures (Bennett & Dunn, 1960). Histologically, it is seen that the mutantembryo possesses a prominent primitive streak; primitive streak derivatives,however, are deficient or defective and neural ectoderm is grossly malformed.

We know surprisingly little about the role of various cell organelles and cellproducts during early stages of normal or abnormal cell differentiation. In the

1 Authors' address: Department of Anatomy, Cornell University Medical College, NewYork, New York 10021.

2 Our heterozygous stock carries the mutant allele t^19 as described by Bennett & Dunn,1960. The alleles ?wl8 and t9 have been shown to be identical genetically and embryo-logically (Van Valen, 1964).

724 M. SPIEGELMAN AND D. BENNETT

?9-mutant embryo, mesoderm fails to differentiate normally and to segregatefrom the primitive streak (Bennett & Dunn, 1960; Artzt & Bennett, 1972). It isknown (Gustafson & Wolpert, 1967; Trinkaus & Lentz, 1967; Trelstad, Hay &Revel, 1967; Johnson, 1972) that the first functional specialization evident innormal mesodermal cells is the ability to migrate, and further, that this abilityis correlated with stellate cell shape and contact zones between cells. This lightand electron microscopic investigation of the sequence of structural alterationsin cells of both the /9-mutant and normal mouse embryos at eight days of develop-ment was undertaken in order to describe in more detail the phenotype of themutant and to attempt to identify important morphogenetic structures in boththe mutant and its normal counterpart.

The mutant mesodermal cells show abnormalities of shape, microfilamentcontent and cellular contacts; presumably their inability to carry out thenormal movements of gastrulation and to complete differentiation is a reflexionof these abnormalities.

MATERIALS AND METHODS

Heterozygous mice (+t9/t9) and wild-type mice from stocks maintained inour own colony were mated inter se. Age of embryos was predicted by thevaginal plug method and was more precisely determined by counting somites ofnormal embryos at the time they were dissected and fixed. At eight days,mutant embryos {t*jf) are recognizable by their small size and retarded develop-ment; they rarely show normal axial organization, usually lacking completeneural folds, head folds, and somites.

Pregnant females were sacrificed by cervical dislocation and their uteri wereremoved immediately and placed in fixative. The embryos in fixative were freedof uterus, deciduae, and yolk sacs under the dissecting microscope. Fixationwas from 15 to 60 min, at room temperature, in 2 % glutaraldehyde in 0-05 Mphosphate buffer, with 0-00014 M calcium chloride, at pH 7-4; followed by45 min, at room temperature, in 1-5 % osmium tetroxide in 0-05 M phosphate

FIGURES 1-3

Light micrographs of 1 /^m-thick sagittal sections stained with toluidine blue.Fig. 1. Normal 8-day-old embryo.Fig. 2. Normal 7-day-old embryo.Fig. 3. Mutant 8-day-old embryo.x 250. The thickened epiblast (E), narrow hypoblast (H), and small number ofmesodermal cells (M) in the normal 7-day-old and mutant 8-day-old embryos arecomparable whereas all three primary germ layers show extensive development in the8-day-old normal embryo with head folds (HF) evident and primitive streak (P) inregression. Not only is development retarded in the 8-day-old mutant embryo, butalso the primitive streak is clearly enlarged and, with the overlying epiblast, bulgesinto the proamniotic cavity.

EM of mesoderm in mutant mice (i9) 725

2 V 3,

EM of mesoderm in mutant mice (f) 727buffer, with 0-035 M sodium chloride and 0-00014 M calcium chloride, at pH 7-4.(These fixative compositions were suggested by Dr Irwin Spiegelman and areapproximately 300 milli-osmolar.) Tissue was dehydrated in ethanol andpropylene oxide and embedded in Epon (Luft, 1961).

Sections were cut by glass or diamond knives on Sorvall ultramicrotomemodels MT-1 and MT-2. Thin sections were mounted on parlodion- and carbon-coated copper grids, stained for lOmin in uranyl acetate and in lead citrate(Venable & Coggeshall, 1965), and viewed in a Philips 200 electron microscope.

Epon sections 1 /im thick were mounted on glass slides and stained on a hotplate at approximately 60 °C for about 1 min with alkaline toluidine blue(Pease, 1964). Sections were examined in a Zeiss photomicroscope.

Observations were recorded on a total of 20 mutant embryos from 10 littersand 35 normal embryos from 15 litters.

RESULTS

The morphology of normal embryos at approximately seven and eight days,and the /9-mutant embryo at approximately eight days is compared at the lightmicroscopic level in sagittal sections through entire embryos (Figs. 1-3). Thenormal 8-day-old embryo is much larger than the 8-day-old mutant whichinstead, in size and organization of germ layers, more closely resembles the7-day-old normal. The normal 8-day-old embryos have head-folds, notochord,two to eight pairs of somites, and are undergoing neural tube closure andprimitive streak regression. In contrast, the 8-day-old mutant possesses none ofthese features, appearing, with the notable exception of its primitive streak,similar to the normal 7-day-old embryo. Both have a flat layer of hypoblastand a thickened layer of epiblast with the primitive streak along the mid-linein the posterior half. It is obvious, however, that the primitive streak of themutant is much larger, with an aggregation of cells causing the epiblast to bulgeinto the pro-amniotic cavity.

The primitive streak of the mutant was originally described as an 'overgrowth'(Bennett & Dunn, 1960), a term which implies that more cell proliferationhas occurred at this site than has occurred in the normal during the 24 h period

FIGURES 4-7

Fig. 4, 5. Primitive streak of the normal 8-day-old embryo. Fig. 4, x 650; Fig. 5,x1600.Fig. 6, 7. Primitive streak of the mutant 8-day-old embryo. Fig. 6, x650; Fig. 7,x1600.Cells of the primitive streak (P) and epiblast (E) in the normal embryo areslightly larger with cellular and nuclear contours more regularly rounded than arethe counterpart cells in the mutant embryo. Mutant cells show more vacuolization andgreater cytoplasmic density than do normal cells. Arrow, boundary betweenprimitive streak and epiblast.

EM ofmesoderm in mutant mice (fi) 729between seven and eight days of development. The series of sections shownhere suggests that this is not the case. Inspection of the primitive streak andmesoderm reveals approximately equivalent proportions of division figuresper microscopic field in both mutant and normal embryos. There are, however,fewer cells in the mutant than in the normal embryo. The total cellular pro-geny, therefore, is less in the mutant than in the normal embryo between sevenand eight days of development and, moreover, the cells deriving from theprimitive streak in the mutant are not in appropriate locations (Figs. 1-3). Theenlarged primitive streak of the mutant may result from the slow pile-up ofcells which seem to be immobilized. This 'paralysis' probably began at aboutseven days of development, the onset of gastrulation, and is more obvious byeight days because of the retarded development of the mutant embryo and itsdeficiency of mesoderm and protuberant form of its primitive streak.

Comparing the primitive streaks of normal and mutant embryos at thelight microscopic level, the cells of the mutant appear somewhat smaller andtheir nuclei have more irregular profiles than those of the normal (Figs. 4-7).Cells of the mutant exhibit more vacuolization and greater cytoplasmic densitythan do cells of the normal embryos. In both, the boundary between presump-tive neural ectoderm and primitive streak is usually distinct.

Striking contrasts in cell form, cell structure, and cellular associationsbetween the normal and mutant mesoderms are readily apparent at the lightmicroscopic level (Figs. 8-11). Whereas the normal mesoderm is arrayed as anetwork of stellate cells, the mutant mesoderm has a sheet-like configuration.These cells appear as irregularly rounded profiles with large areas of cellsurfaces in apposition to their neighbors. In normal mesoderm, each cell alsoappears to be in contact with its neighbors, but principally by means of slendercytoplasmic processes, orfilopodia. The mutant mesodermal cells rarely exhibitfilopodia; instead, they have numerous rounded, broad projections which wecall lobopodia. These lobopodia are often palely stained and appear to containfew cell organelles; most organelles seem to have a juxtanuclear location. Inaddition, a number of the mutant mesodermal cells contain dense cytoplasmicinclusions, many of which are Feulgen-positive and may be residual bodies.The nuclear profiles of these cells are often irregular.

FIGURES 8-11

Fig. 8, 9. Mesoderm of the normal 8-day-old embryo. Fig. 8, x 650; Fig. 9, x 1600.Fig. 10,11. 'Mesoderm' of the mutant 8-day-old embryo. Fig. 10, x 650; Fig. 11,x 1600. Neural ectoderm (E) is at the left in all micrographs. Stellate cells of normalmesoderm (M) constitute a continuous tissue by means of adjoining cellular pro-cesses having relatively small contact zones in section. In contrast, mutant'mesoderm' forms a continuous sheet in section with extensive areas of cell surfacesin close apposition. Instead of the narrow cytoplasmic processes {arrow) of normalcells, the mutant cells exhibit broad, lobose cytoplasmic projections {arrow). Densecytoplasmic inclusions are probably residual bodies (R).

EM of mesoderm in mutant mice (f9) 731

There has apparently been a failure in the mutant embryo to make thetransition from primitive streak to mesoderm. The stellate cell shape, typical ofnormal migrating mesoderm, is not attained in the mutant embryo. Thisdefect in differentiation may arise at an earlier time in cells of the primitivestreak.

The initial impressions obtained by light microscopic images of the meso-dermal cell structure in normal and mutant embryos are confirmed by electronmicroscopy (Figs. 12, 13). The nuclear outlines in cells of the mutant are oftenirregular and even appear lobulated at times while the nuclear profiles innormal cells are usually smoothly oval. Polysomes and individual ribosomesseem to be more abundant in cells of the mutant than in cells of the normal. Inthe mutant, most cellular organelles, except ribosomes and polysomes, areconcentrated near the nucleus while organelles are more evenly distributedthrough the cytoplasm of normal cells.

The normal stellate mesodermal cells are rather widely separated and theirnumerous filopodia terminate in contacts with similar filopodia of neighboringcells (Fig. 12). In contrast, mesodermal cells of the mutant embryo usually lieside-by-side with adjacent plasma membranes conforming to each other(Fig. 13). Filopodia are occasionally observed but most cells have roundedcontours rather than a stellate form. Although the closely packed mesodermalcells of the mutant are apparently in contact, there is a deficit of filpodia coupledby contact zones, i.e. the stellate cell reticulum typical of early mesodermaltissue is lacking in the mutant embryo.

The occurrence of broad, blunt, cytoplasmic projections, or lobopodia, inthe mesodermal cells of the mutant is in sharp contrast to the appearance ofnormal mesoderm. These lobopodia contain abundant ribosomes and faintlyfibrillar ground plasm but few subsurface filaments are encountered (Figs. 14,15). In. grazing sections through filopodia of normal mesodermal cells, however,a lattice of fine filaments, each about 6 nm wide, is regularly seen in the sub-surface cytoplasm (Fig. 16).

Cellular junctions of several types usually occur between cytoplasmic pro-cesses of mesodermal cells of the normal embryo; some of these are small focaljunctions and some are more extensive (Figs. 17-19). None of the junctions isidentical to those depicted in mature, stable tissues. The apposing plasma

FIGURES 12 AND 13

Fig. 12. Electronmicrograph of mesoderm of the normal 8-day-old embryo. Inter-cellular spaces are extensive except at small contact zones (arrows) where neigh-boring cellular processes meet, x 5000.Fig. 13. 'Mesoderm' of the mutant 8-day-old embryo. Adjacent cells are in closeapposition over extensive areas with some contact zones. Broad cytoplasmic pro-cesses, or lobopodia, are prominent and a few small, dense processes are present,x 5000.


FIGURES 14-16

Fig. 14, 15. Lobopodia of 'mesoderm' of the mutant 8-day-old embryo containribosomes but no other organelles. A few filaments are present but a sub-surfacemicrofilament lattice is not evident, x 75000.Fig. 16. Sub-surface microfilament lattice (arrow) in tangential section throughfilopodium of normal mesodermal cell, x 75000.

EM of mesoderm in mutant mice (t9) 733

FIGURES 17-19

Fig. 17. Small focal junction (arrow) of neighboring cellular processes. Filopodia (F)containing filamentous cytoplasm extend from central junction. Intercellular spaceat junction is 5-8 nm and dense material appears in the cleft, x 75000.Fig. 18. Extensive junction (arrows) with densely filamentous material just insidethe plasmalemma. Dense material appears in the intercellular cleft which is 5-8 nmwide. Filopodia (F), containing filamentous cytoplasm, extend from the junctionzone, x 75000.Fig. 19. Extensive junction (arrowheads) that may be a gap junction, although theintercellular cleft is about 8 nm wide. Neighboring junctions (D) have somecharacteristics of desmosomes with associated plaques (arrow) and fibrils. Inter-cellular space is about 25 nm and fine filaments bridge the cleft. An intermediatedense line is not apparent. Filopodia (F), containing filamentous cytoplasm, extendfrom the junction zone, x 75000.

47 EMB 32


FIGURES 20-22

Fig. 20-22. Apposing cell surfaces of lobopodia in mutant mesodermal cells. A fewcytoplasmic filaments are present but a microfilament lattice is of rare occurrence.Apposing cell membranes usually remain 20 nm or more apart (Fig. 20). Rarely,adjacent surfaces approach to 10 nm and a few fine filaments (arrow) extend acrossthe intercellular cleft (Fig. 21) and, very rarely, densely amorphous material(arrow) appears in the intercellular space bstween adjacent lobopodia (Fig. 22). Noneof these zones resembles contact specializations observed in normal mesoderm;filopodia and sub-surface microfilaments are typically lacking, x 75000.

EM of mesoderm in mutant mice (79) 735

membranes are usually from 5 to 20 nm of each other and filamentousmaterial is located in the intercellular cleft at the junctions. Just inside the cellsurfaces at these regions there is usually a local accumulation of fibrillar material,sometimes a considerable amount. Many junctions are rather inconspicuousand are possibly incipient or transitory structures (Fig. 17) while some junctionsare more extensive, as much as 1 /tm long in section, and exhibit abundant sub-surface fibrils (Fig. 18). In some junctions, the apposing membranes followeach other's contours exactly, and little subsurface fibrillar material is present(Fig. 19). These may be gap junctions although the intercellular cleft is about8 nm wide. Contact specializations which may be incipient desmosomes arealso present with plaque formation and subsurface fibrillar material evident(Fig. 19). The intercellular cleft here, about 25 nm wide, is bridged by filaments.Filopodia border all of the junctions in the normal mesodermal cells.

On the contrary, in the mesoderm of the mutant these features are not ob-served. Instead of abundant filopodia, the cells are characterized by large,rounded cytoplasmic projections which rarely exhibit a subsurface network ofmicrofilaments. Plasma membranes of adjacent cells usually remain more than20 nm apart (Fig. 20). In rare instances, apposing plasma membranes of thelobopodia approach to within 10 nm and a few filaments appear to bridge theintercellular cleft (Fig. 21). Amorphous dense material has been occasionallyseen in the space between two apposing lobopodia but the surfaces remain20 nm or more apart (Fig. 22). In none of these regions of membrane appositionare local accumulations of subsurface fibrillar material found nor are typicalfilopodia observed nearby.

In conclusion, primitive streak cells of the ?9-mutant embryo do not completethe transition to mesoderm; cells of this germ layer fail to attain the stellateform, to make appropriate junctions with neighboring cells, to generate filopo-dia, to migrate, and, finally, to differentiate into typical mesodermal rudiments.The ultrastructural findings reported here strongly suggest some causal relation-ships among these abnormalities of morphogenesis, although of course thesequence of events remains at this time a matter for speculation.

DISCUSSION

It has been shown that the subsurface cytoplasm of the leading lamellae ofmoving cells in vitro contains a network of microfilaments (Ingram, 1969;Spooner, Yamada & Wessells, 1971; Ludueila & Wessells, 1973; Heaysman,1973). In the few studies at the fine structural level of mesoderm segregation insome embryos (Trinkaus & Lentz, 1967; Trelstad et al. 1967; Johnson, 1972) themigrating cells display contact zones.

The present work suggests that the normal processes of segregation and migra-tion of mesodermal cells in the mammalian embryo can be correlated withthe presence of filopodia containing a microfilament lattice and sharing cellular

47-2


junctions with one another. The deficiency of these elements of extension andadhesion in presumptive mesodermal cells of the mutant embryo appearsto be reflected in the impairment of migration.

The microfilament lattice, if it has insertions on the plasma membrane, mayconstitute the mechanism of extension and withdrawal of cytoplasmic processes(Abercrombie, Heaysman & Pegrum, 1971; Behnke, Kristensen & Nielsen,1971; Goldman et al. 1973; Luduena & Wessells, 1973; Spooner et al. 1973).Adhesion requires that some elements of the outer surface of the cell recognizeand attach to a substrate, which in the in vivo situation is likely to be neighboringcells or extracellular matrix. Following upon adhesion, nascent filopodia maybe generated adjacent to these sites and, with advancing cytoplasmic flow, newadhesions could form at suitable interfaces as the earlier ones decay. Thus,filopodia may spread along surfaces, such as adjacent cells in the embryo, andplay a role in translocation.

The observations reported here on genetically defective embryos whosemesoderm fails to migrate and to differentiate normally support the conten-tion that the ability of cells to move depends on their ability to form filopodiawhich contain microfilament lattices and to organize adhesive contact zones.That the defective mesodermal cells come to occupy the middle layer of themutant embryo at all is surprising and perhaps results from passive displace-ment from the proliferating primitive streak. These mesodermal cells rarely formtypical filopodia and typical cellular junctions between cytoplasmic processesare almost never seen. The paucity of filopodia and their accompanying cellularjunctions is probably an expression of a diminished capacity for cell-cellrecognition. Contact and interaction between cells is doubtless a prerequisitefor cell movement, as well as a likely condition for normal differentiation.

Cellular interactions resulting in normal morphogenesis may be due to thepresence of appropriate differentiation antigens on cell surfaces of certain groupsof cells at particular stages of development (Bennett, Boyse & Old, 1971). Ithas been suggested by the same authors that the lethal alleles at the JT-IOCUSmay identify genes which are switched on sequentially but transiently to providecell surface recognition factors important at morphogenetic transition pointsin early development. A wild-type allele ( + *12) has recently been shown tospecify an antigenic cell surface component on cells of the normal morula(Artzt, Bennett & Jacob, 1974); the presence of this antigen appears to benecessary for subsequent normal development since t12/tn embryos are unableto differentiate beyond the morula stage (Smith, 1956). By analogy, we canspeculate that a specific antigen determined by the wild-type allele of t9 is a com-ponent of the normal mesodermal cell surface that is essential for gastrulation.The mutant allele t9, on the other hand, may determine cell surface componentsat this critical time which do not function adequately either as recognition sitesor as sites where elements of the apparatus for adhesion or motility mayassemble.

EM of mesoderm in mutant mice (t9) 737

Furthermore, the deficiency of microfilament-containing filopodia in themutant mesodermal cells may result from the same abnormal membrane com-ponents. Filopodia with their constituent microfilaments appear regularlycoupled with cellular junctions in the normal mesodermal cells; these coupledstructures are regularly lacking in the mutant mesodermal cells. Specifically,we suggest that the presence of a microfilament lattice may be related to thepresence of adjacent adhesive sites. The same defect in the plasma membranecomponents that may account for the failure of recognition and adhesionbetween cells may likewise furnish an inhospitable polymerization site formicrofilament sub-units and thus prevent the normal insertion and alignmentof microfilaments at the inner face of the plasma membrane. In short, theexternal and the cytoplasmic faces of the plasma membrane may share the samestructural defect.

It may also be speculated that other aspects of cell structure that appearabnormal in the mutant, including irregular nuclear conformations and thejuxta-nuclear location of most cellular organelles, may be due to forces similarto or related to those suggested for abnormalities of cell shape and cell adhesion,lntracellular architecture may be affected by cell shape or by interactions atapposing cell surfaces. In addition, the abundant ribosomal content of cellsof the mutant perhaps reflects the failure of differentiation of abnormal cells,in particular, of their migratory and adhesive apparatus.

The mesodermal cells of the mutant embryo, with their normal counterparts,provide suitable material for studying the role of cellular junctions and micro-filaments in cell migration without resort to drugs, such as cytochalasin B, thatmay produce a multitude of effects on cell components. In addition, the func-tional role of mesoderm in development can be explored by means of this'mesodermal mutant'. It has been shown that /9 mutant embryos transplantedto suitable hosts produce embryonal tumors that are almost exclusively ecto-dermal in nature, and many of which histologically resemble neuroepithelialmalignancies (Artzt & Bennett, 1972). The disturbance of mesodermal dif-ferentiation may not only prevent the normal differentiation of structuresdependent on mesenchymal interactions, but also lead to malignancy in theabsence of influences which normally would be present.

This work was presented in preliminary form at the 13th International Congress of Geneticsat Berkeley, California in August, 1973.

The authors are grateful to Miss Victoria Neufeld for expert technical assistance. We thankDr Roy C. Swan for reading the manuscript. This work was supported by National ScienceFoundation Grant GB 33804X.


REFERENCESABERCROMBIE, M., HEAYSMAN, J. E. M. & PEGRUM, S. M. (1971). The locomotion of fibro-

blasts in culture. IV. Electron microscopy of the leading lamella. Expl Cell Res. 67, 359—367.

ARTZT, K. & BENNETT, D. (1972). A genetically caused embryonal ectodermal tumor in themouse. / . natn. Cancer Inst. 48, 141-158.

ARTZT, K., BENNETT, D. & JACOB, F. (1974). Primitive teratocarcinoma cells express a dif-ferentiation antigen specified by a gene at the J-locus in the mouse. Proc. natn. Acad. Sci.U.S.A. 71, 811-814.

BEHNKE, O., KRISTENSEN, B. I. & NIELSEN, L. E. (1971). Electron microscopical observationson actinoid and myosinoid filaments in blood platelets. / . Ultrastruct. Res. 37, 351-369.

BENNETT, D., BOYSE, E. A. & OLD, L. J. (1971). Cell surface immunogenetics in the study ofmorphogenesis, pp. 247-263. Proc. Ill Lepetit Colloqium {London, 1971). Amsterdam:North Holland Publ. Co.

BENNETT, D. & DUNN, L. C. (1960). A lethal mutant (/!018) in the house mouse showing partialduplications. / . exp. Zool. 143, 203-219.

DUNN, L. C. & BENNETT, D. (1964). Abnormalities associated with a chromosome region inthe mouse. Science, N. Y. 144, 260-267.

GOLDMAN, R. D., BERG, G., BUSHNELL, A., CHANG, C-M., DICKERMAN, L., HOPKINS, N.,MILLER, M. L., POLLACK, R. & WANG, E. (1973). Fibrillar systems in cell motility. InCIBA Foundation Symposium on Locomotion of Tissue Cells, pp. 83-107. (London, .1972)New York: Associated Scientific Publishers.

GUSTAFSON, T. & WOLPERT, L. (1967). Cellular movement and contact in sea urchin morpho-genesis. Biol. Rev. 42, 442-498.

HEAYSMAN, J. (1973). General discussion. I. In CIBA Foundation Symposium on Locomotionof Tissue Cells, pp. .187-194. New York: Associated Scientific Publishers.

INGRAM, V. M. (1969). A side view of moving fibroblasts. Nature, Lond. 222, 641-644.JOHNSON, K. E. (1972). The extent of cell contact and the relative frequency of small and large

gaps between presumptive mesodermal cells in normal gastrulae of Rana pipiens and thearrested gastrulae of Rana pipiens $ x Rana catesbiana $ hybrid. J. exp. Zool. 179, 227-238.

LUDUENA, M. A. & WESSELLS, N. K. (1973). Cell locomotion, nerve elongation, and micro-filaments. Devi Biol. 30, 427-440.

LUFT, J. H. (1961). Improvements in epoxy embedding methods. / . biophys. biochem. Cytol.9, 409-414.

PEASE, D. C. (1964). Histological Techniques for Electron Microscopy, 2nd ed., pp. 260. NewYork: Academic Press.

SMITH, L. J. (1956). A morphological and histochemical investigation of a pre-implantationlethal (f12) in the house mouse. J. exp. Zool. 132, 51-83.

SPOONER, B. S., ASH, J. F., WRENN, J. T., FRATER, R. B. & WESSELLS, N. K. (1973). Heavymeromyosin binding to microfilaments involved in cell and morphogenetic movements.Tissue and Cell 5, 37-46.

SPOONER, B. S., YAMADA, K. M. & WESSELLS, N. K. (1971). Microfilaments and cell locomo-tion./. Cell Biol. 49, 595-613.

TRELSTAD, R. L., HAY, E. D. & REVEL, J. P. (1967). Cell contact during early morphogenesisin the chick embryo. Devi Biol. 16, 78-106.

TRINKAUS, J. P. & LENTZ, T. L. (1967). Surface specializations of Fundulus cells and theirrelation to cell movements during gastrulation. / . Cell Biol. 32, 139-153.

VAN VALEN, P. (1964). Genetic Studies on /w18 and t9: lack of complementation in rwl8//9

embroys. Ph.D. Dissertation, New York, Columbia University.VENABLE, J. H. & COGGESHALL, R. E. (1965). A simplified lead citrate stain for use in electron

microscopy. / . Cell Biol. 25, 407^08.

{Received 1 April 1974)

Documents

Fine structural stud of cely l migration in the early mesoder omf … · Embryol. exp. Morph. Vol. 32, 3, pp. 723-738. 1974 723 Printed in Great Britain Fine structural stud of cely