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Mesodermal tissue from the chick embryo at various stages of early differentiation was cultured in hydrated gels of type I collagen in the presence and absence of transferrin. Primary mesoderm explants from primitive-streak-stage embryos responded to the presence of avian transferrin by significantly improved outgrowth which appeared to be related to the ability of the cells to attach to, and migrate in, the collagen. No evidence was obtained which suggested that this observation was dependent on increased cell proliferation. This outgrowth enhancement was not duplicated by transferrin of human origin. The avian transferrin did not produce this effect on cells cultured on plastic substrata, suggesting that the species-specific effect involves modulation by the extracellular matrix. Mesoderm explants from somite stages of development showed no increase in outgrowth in the presence of either avian or human transferrin as judged by counting the number of outwandering cells. Ultrastructural immunocytochemistry indicated surface binding of transferrin by cells in the gels, and the presence of endogenous transferrin on the surfaces of mesoderm cells in situ and in their extracellular environment. It is suggested that by binding to cell surface receptors, transferrin may be able to influence the strength of cellular adhesion to collagen and hence the capacity for cell locomotion.

Chick embryos were treated in ovo and in vitro with L-phenylalanine from the intermediate streak stage (Hamburger & Hamilton stage 3, 12-13 h of incubation) to the 7-somite stage (H & H stage 9, 29-33 h of incubation). Treatment in ovo resulted in a large number of embryos developing somite blocks, i.e. imperfectly segmented somites. In embryos treated at an early developmental stage (12-21 h of incubation), the blocks of unsegmented somite mesoderm occurred mostly in the somite pairs 1-5, whereas treatment that began at a later stage (24-30 h of incubation) caused blocks in the somite pairs 5-10, i.e. the appearance of blocks of unsegmented somite mesoderm is correlated in time with the onset of the treatment. No difference regarding mitotic indices could be distinguished between normally segmented somites and blocks of unsegmented somite mesoderm. Autoradiography based on tritiated L-phenylalanine showed no regional differences in labelling of the chick embryo body. Electronmicroscopical observations indicate a slightly suppressed formation of microvilli in the cells of the unsegmented mesoderm blocks compared with cells in normally segmented somites. The observed disturbances are probably caused by a suppressed yolk granule decomposition in the developing somite cells. The experiments in vitro support the findings in the in ovo material; at the same time, they reveal an unexpectedly slow diffusion of L-phenylalanine through the vitelline membrane.

Tissue culture, time-lapse cinematographic and electron microscopic techniques have been used to study the properties of chick mesoderm at several stages of differentiation. Lateral plate, unsegmented mesoderm (segmental plate), and newly formed somites were dissected from stage-12 embryos, whilst dermo-myotomes and sclerotomes were dissected from stage-18 embryos. Each type of mesoderm was found to exhibit a characteristic pattern of behaviour. The explants from the unsegmented mesoderm from the newly formed somites and from the older embryos could be placed in a developmental sequence; with increasing differentiation they settled and spread on the substrate more readily, whether explanted as pieces of tissue or as individual cells, and it was concluded that this implied an increased adhesion to the substrate. Similarly, with increasing differentiation, the cells segmented at a faster rate. No significant differences could be discerned in the internal structure of the different types of cells, although differences in the general shape were apparent. The lateral plate mesoderm cells, which bear some resemblances to the unsegmented mesoderm cells in the embryo, also show some morphological resemblances to them in vitro. However, the lateral plate cells had a much greater success in attaching to glass or platic substrates. They were also found to have the highest speed of locomotion of all the tissues studied, whereas the unsegmented had the lowest. It is concluded therefore, that although cells may look similar to one another morphologically, their behaviour may differ greatly, probably because they are already partially determined.

The early development of the mesoblast in the intermediate and lateral regions of the chick embryo was examined with the scanning and transmission electron microscope. It was found that primary mesenchyme here becomes condenses into epithelial structures that emerge in a metameric pattern. Viewed in developmental sequence, the intermediate mesoblast condenses into a narrowing cord of axially oriented cells which divert medially at regular intervals into the intersegmental interfaces of somitomeres and somites. These cells give rise to the vascular channels of the posterior cardinal vein as well as to tubular elements of the pronephros. Intermediate mesenchyme cells become epithelial, forming zonular junctional complexes apically and depositing patchy basal lamina over their basal surfaces. The lateral plate mesenchyme organizes similarly into somatic and splanchnic epithelial sheets that utilize the body coelom as their lumenal surface. Cells of the lateral plate extend filopodia basally that interweave with adjacent cells, fibrillar extracellular matrix, as well as with interstitial bodies. The pattern in the lateral plate is subtly ribbed as bands of mesoblast undulate along the axis. The central region of each band is raised while ther are grooves created along lines of band abutment, corresponding to intersegmental clefts in the paraxial region and reflecting an underlying metameric pattern. These grooves are usually demarked medially by the protrusion of short segments of adjacent intermediate mesoblast. Most of the remaining primary mesenchyme develops into a non-metameric vascular epithelium, which forms a prominent anastamosing plexus between splanchnic mesoderm and endoderm. It is proposed that the emergence of primary mesenchyme into patterened epithelial anlage facilitates the distribution of neural crest cells introduced subsequently.

Transmission and scanning electron microscopy were used to study the epithelio-mesenchymal interface between the interacting mouse ureter-bud and the metanephric mesenchyme. The gap between the epithelial and mesenchymal cells varied in width. At the stalk of the ureter-bud the interspace was often about 1 mum, but in the inductively active areas at the tips of the branching ureter-bud epithelio-mesenchymal contacts were seen through discontinuities in the basal lamina. At these points the gap between the interacting cells was often less than 20 nm, in places less than 10 nm. The amount of electron-dense, ruthenium-red-positive material was greatest at the stalk of the ureter-bud, but only a small amount of extracellular material was found between the interacting cells at the tips. Whether epithelio-mesenchymal cell contacts play a role in kidney tubule induction is not yet known, but their existence in the inductively active areas and their absence in inactive zones suggests that they are morphogenetically significant. The finding also obviates the need to postulate long-range transmission of inductive signals to explain this example of embryonic induction.

Extraembryonic endoderm cells from gastrulating chick embryos undergo epiboly and change from a multilayered cell group to a single cell layer surrounding the yolk. Single cell suspensions from this cell layer can aggregate in vitro to form aggregates that cavitate. To study the stages of cavitation aggregates were harvested after different times in culture, and fixed and processed for light and electron microscopy. In aggregates harvested at 75 min of culture cell contact consisted of areas of parallel and close membrane apposition and interdigitation. Desmosomes were occasionally observed. Aggregates in the early stages of cavitation (24 h) contained numerous intercellular spaces bordered by irregularly shaped cells which appeared to be digesting their yolk and releasing material extracellularly. Long cytoplasmic projections were extended into these spaces. In addition to regions of parallel membrane apposition and interdigitation, desmosomes and adherens junctions were observed. Cells closer to the periphery of the aggregates displayed fewer cell projections and also showed signs of release of material extracellularly. After 48 h of culture, a single smooth-walled central cavity was present and cells still exhibited signs of extracellular release of material. These same cell shapes and intercellular junctions were also observed when area opaca tissue dissected from gastrulating embryos was examined. Aggregates of different sizes were created and cultured. The results suggest that a critical tissue mass may be important for cavitation.

A sequential morphological study of the initial cellular events in teratoma induction by displaced visceral yolk sac after foetectomy in rats was undertaken. This study led to the observation that apart from proliferation of cells displaying definite endodermal or mesodermal characteristics, a population of poorly differentiated cells appeared some days after the surgical procedure. It is very likely that these poorly differentiated cell are stem cells from which differentiated structures originate afterwards by a process of redifferentiation. The development of granulation tissue rich in capillaries seems to enhance this process. Similarities and differences with blastema formation are discussed.

Fibronectin has been localized to basement membranes and cell surfaces with the light microscope by fluorescent staining of thick sections, and with the TEM by immunoperoxidase reaction. However, these methods are limited because it is difficult to appreciate the patterned distribution of fibronectin from sectioned material. We have developed a probe for fibronectin that facilitates its identification with the SEM. Our probe consists of two parts; the first component is a derivatized methacrylate microsphere 90 nm in diameter, linked to purified sheep anti-rabbit IgG. The second component is anti-fibronectin IgG raised in rabbits. Stage-3 to -12 chick embryos were fixed and the ectoderm covering the cranial mesoderm was removed. Embryos were treated with testicular hyaluronidase, exposed to rabbit anti-fibronectin IgG and finally to sheep anti-rabbit IgG conjugated microspheres. As expected, the basal lamina of surface and neural ectoderm as well as the remaining fibrous ECM were heavily decorated with microspheres, whereas control embryos treated with preimmune serum were beadless. Fibronectin was localized on the cell soma and processes of primary mesenchyme as early as stage 3. In addition, it was possible to decorate to various extents, populations of prosencephalic, mesencephalic, and rhombencephalic cranial neural crest cells. Our studies suggest that fibronectin is present in the cranium of chick embryos at earlier times than heretofore realized, and that fibronectin accumulates in a cranial to caudal gradient that reflects the sequential differentiation of the embryonic axis.

The development of the paraxial mesoderm in tadpoles of Xenopus, Bufo and Rana was observed with a scanning electron microscope. In addition to examination of the differentiation of the surface and the interior of the somites, some attention was also paid to the transformation of the material of the neural crests and to the innervation of the developing myotome.

The segmentation of somites in the chick embryo has been studied by transmission and scanning electron microscopy (stages 8-14). The segmental plate mesoderm consists of loosely arranged mesenchymal cells, whereas the newly formed somites are composed of elongated, spindle-shaped cells arranged radially around a lumen, the myocoele. The diamter of each somite is thus two cells plus the myocoele. Two major factors appear to be responsible for the change in cell shape at segmentation: (1) Each prospective somite cell becomes anchored at one end to the adjacent epithelia (i.e. the neural tube, the notochord, the ectoderm, the endoderm or the aorta) by means of collagen fibrils. These fibrils are already present in the segmental plate before the somites begin to form. (2) A change in cell-to-cell adhesiveness causes the free ends of these cells to adhere to one another. (Bellairs, Curtis & Sanders, 1978). This adhesion is then supplemented by the development of tight junctions proximally in the somite. Because it is anchored at both ends, each somite cell is under tension in much the same way as a fibroblast cell in tissue culture is under tension. Each somite cell therefore becomes elongated and the somite as a whole accommodates its general shape to that of the space available between the adjacent tissues. The arrangement of the cells in the more differentiated somites (stages 17-18) has also been examined and it has been found that the chick resembles Xenopus in that the myotome cells undergo rotation and become orientated in an anteroposterior direction.

Somitogenesis is described in two species of anuran amphibians, Xenopus laevis and Rana temporaria, in which the cellular mechanics of somite formation are distinctly different. Heat shocks are employed to demonstrate a wave of cellular change which precedes somite formation down the body axis. This prior wave is shown to be kinematic. It is not a propagated wave. It is a consequence of the temporal activities of the cells laid out in space, but there is no evidence that these activities depend upon an interpretation of their position. Heat shocks cause characteristic segmental abnormalities over a zone of somites which is formed several hours after the shock. Evidence from double heat shock experiments suggests that the pattern of abnormality is the result of (i) a disturbance of co-ordination between pre-somitic cells, and (ii) the time available to those cells for recovery before they are recruited into a segmental pre-pattern at the time of passage of the prior wave. It is a temporal co-ordination that is disturbed and subsequently recovered following a heat shock. This temporal co-ordination of pre-somitic cells does not depend upon position along the axis. The evidence for two physiologically independent temporal patterns of cellular processes, which interact to specify the segmental pattern of somites (their size, shape and number), gives experimental support for the theoretical account of somitogenesis proposed by Cooke & Zeeman (1976).

The segmental plate mesoderm of snapping turtle embryos (Chelydra serpentina) was examined with stereoscanning electron microscopy imaging. A metameric pattern was detected along the entire length of the segmental plates. This pattern consisted of a tandem sequence of mesodermal units, called somitomeres. Each somitomere was oval to cubic in shape and the processes of the constituent mesodermal cells tended to be arranged in concentric rings about the centre of the somitomere. Several experiments from a previous study (Packard, 1980b) of snapping turtle segmental plates were repeated, but, instead of culturing the explants and observing the numbers of somites that formed, the explants were fixed immediately for scanning electron microscopy and the number of somitomeres was counted. The segmental plates were found to contain an average of 6.5 +/- 0.7 somitomeres, which is almost identical to the average number of somites formed by such segmental plates when cultured (6.6 +/- 1.2). Furthermore, the number of somitomeres was identical in right and left explants removed from the same embryo, and the number of somitomeres was consistent regardless of the length of the segmental plate. Both of these observations are identical to those made previously for somite formation in culture. This association between numbers of somitomeres and somites strongly suggests that one gives rise to the other. Finally, it was demonstrated that for each somite formed by a segmental plate in culture, the segmental plate contained one less somitomere. This showed in a direct manner that turtle somitomeres become somites. It was concluded that the segmental plate mesoderm of snapping turtle embryos is already segmented, and that the 'segmentation' seen under a dissecting microscope is actually the final stage of somitomere differentiation into an epithelial somite.

The axolotl pronephric duct rudiment is readily accessible to both SEM observation and surgical manipulation. The rudiment segregates from the dorsal part of the lateral mesoderm and then extends caudally along the ventrolateral border of the segmenting comites, eventually contacting the cloacal wall. The marked thinning of the rudiment which accompanies this migration is paralleled by a corresponding reduction in cell number across the duct's diameter and by caudad translocation and elongation of vital dye marks applied to the duct mesoderm. Duct extension thus involves appreciable cell rearrangement. The morphology of duct mesoderm and its substratum (somite and lateral mesoderm) suggests that active locomotion of cells near its tip marshals the duct's caudad elongation. Filopodia and small focal areas of intercellular contact may mediate the adhesions between the cells which must be broken and reformed as the cells rearrange.

It has been suggested that one of the major functions of the apical ectodermal ridge (AER) of the embryonic chick limb-bud is to maintain mesenchymal cells directly subjacent to it (i.e. cells extending 0.4-- 0.5 mm from the AER) in a labile, undifferentiated condition. We have attempted to directly test this hypothesis by subjecting the undiffertiated subridge mesoderm of stage-25 embryonic chick wing-buds to organ culture in the presence and absence of the AER and the ectoderm that normally surrounds the mesoderm dorsally and ventrally. During the period of culture, control explants comprised of the subridge mesoderm capped by the AER and surrounded by the dorsal/ventral ectoderm undergo progressive morphogenesis characterized by polarized proximal to distal outgrowth and changes in the contour of the developing explant, and ultimately form a structure grossly resembling a normal distal wing-bud tip. In contrast, explants from which the AER and dorsal/ventral ectoderm have been removed (minus ectoderm explants) or from which just the AER has been removed (minus AER explants) form compact, rounded masses exhibiting no signs of morphogenesis. During the polarized proximal to distal outgrowth control explants undergo during the first 3 days of culture, as cells of the explant become located greater than 0.4--0.5 mm from the AER, they concomitantly undergo a sequence of changes indicative of their differentiation into cartilage. However, those cells which remain 0.4--0.5 mm from the AER during this period retain the characteristics of non-specialized mesenchymal cells. In marked contrast to control explants, virtually all of the cells of minus ectoderm explants initiate chondrogenic differentiation during the first day of culture. Cells comprising the central core of minus AER explants also initiate chondrogenic differentiation during the first day of culture, but in contrast to minus ecotderm explants, non-chondrogenic tissue types form along the periphery of the explants subjacent to the dorsal/ventral ectoderm. These results indicate that the AER maintains cells directly subjacent to it in a labile, undifferentiated condition, and that when mesenchymal cells are freed from the AER's influence either artificially or as a result of normal polarized outgrowth, they are freed to commence cytodifferentiation. The results further suggest that the dorsal/ventral ectoderm may have an influence on the differentiation of the mesenchymal cells directly subjacent to it, once the cells have been removed from the influence of the AER.

The course of gastrulation in embryos of a urodele, Cynops pyrrhogaster, was studied with 1 mum Epon sections and transmission and scanning electron microscopy. During the initial period of gastrulation, the bottle cells and a groove are formed in the dorsal part. The outer ends of the bottle cells have many microvilli, an electron-dense layer and many small vesicles. Microtubules are present parallel to the long axis of the bottle cells, and a yolk-platelet-free region is observed at the inner end. Thereafter, the archenteric roof makes contact with the inner surface of the blastocoelic wall. Cells of the archenteric roof form lobopodia, filopodia and lamellipodia. These cells make many focal contact, having gaps of less tha 20 nm, with the blastocoelic wall. Invaginating mesodermal cells of the lateral and ventral parts also form pseudopodia, and are in contact with the blastocoelic wall. Some of these cells appear to flatten against the wall. These observations suggest that, after the bottle cells and the blastoporal groove are formed, the invaginating cells actively migrate along the inner surface of the blastocoelic wall, and that these locomotive forces have an important role in the morphogenetic movements during gastrulation.

Vital dye staining and cell lineage tracers were used to mark superficial cells of early Ambystoma mexicanum gastrulae. Superficial marks placed between the equator and the blastopore, on the dorsal midline, stained notochord, whereas marks or injections made at similar animal-vegetal levels but 90 degrees to either side of the dorsal midline were later found in somitic mesoderm. Notochord marks remained on the dorsal surface of the archenteron throughout gastrulation, though they became elongate and narrow by the morphogenetic movements of extension and convergence. Marked somitic mesoderm disappeared from the superficial epithelial layer soon after passing over the blastoporal lip and could not be found on the archenteron surface. A possible mechanism for this de-epithelialization is proposed on the basis of correlated SEM. The significance of a method of gastrulation so distinctly different from that of certain other amphibians is discussed in terms of amphibian phylogeny.

The developmental capabilities of the iris rudiment in the chicken embryo, as well as the role of tissue interactions in the differentiation of the iris, were investigated in vitro. Sectors of the intact iris from 7 1/2- through 9-day embryos (stages 32 through 35) lost their morphological organization in vitro, but were capable of normal histodifferentiation. The pigmentation of the epithelium increased, and muscle differentiation occurred. Developing muscle was identified using immunocytochemistry with antiserum against chicken muscle myosin; this procedure permitted positive identification of myoblasts, myotubes, and muscle fibres in cultures in which histological features alone were equivocal. The proportion of irideal explants which developed muscle increased with the age of the embryo, and correlated with the incidence of epithelial buds and epithelial cells in the stroma. Irideal mesenchyme from stage-32 through stage-35 embryos was already populated with stromal epithelial cells when isolated, but growth and muscle differentiation in these cultures compared poorly with that in the intact iris in vitro. Isolated irideal epithelium (stages 32 through 37) demonstrated even more limited muscle differentiation in vitro, suggesting reciprocal interaction between irideal epithelium and mesenchyme during development. Irideal epithelium was also cultured in direct association with non-irideal mesenchyme from various embryonic organ rudiments, but muscle differentiation was not enhanced.

The wing mesoderm of the chick embryo cultured in vitro without ectoderm is able to differentiate into cartilage from stage 17 (Hamburger & Hamilton, 1951). But before this stage the presence of ectoderm is necessary. In transfilter cultures of wing-bud ectoderm and mesoderm, the mesodermal response as measured by chondrogenesis was directly related to the pore size (0.2--1 micrometer) of the filter. Filters of 0.2 micrometer pore size and 10 micrometer thickness gave no increase in chondrogenesis over that of mesoderm cultures alone. The lower face of filters on the upper face of which mesoderm or ectoderm has been cultured was observed by scanning electron microscopy. With ectoderm, no cell processes crossed the filter. In contrast, with mesoderm, cell processes crossed the filter and this was also related to pore size. A good correlation was observed between the mass and density of processes crossing the filter and the mesodermal response. It is concluded that induction of cartilage in limb mesoderm cannot be classified as a 'long-range transmission' system. It requires ectoderm and mesoderm to be separated by a very narrow gap and this condition can be brought about in vitro by extension of mesodermal processes through the filter close to the ectoderm. The results are discussed in relation to a possible role of the basement membrane and associated extracellular matrix in limb cartilage induction.

A scanning electron microscopic, comparative survey of notochord and somite formation including some details of change in cell morphology and arrangement, was made of selected stages of two species of anuran amphibians (Xenopus laevis and Rana pipiens) and two species of urodeles (Ambystoma mexicanum and Pleurodeles waltlii). The ectoderm or neural plate was removed from fixed embryos and the dorsal aspect of the developing notochord and somite mesoderm was photographed. Micrographs of comparable stages of all species were arranged together to form an atlas of notochord and somite formation. Similar morphogenetic events occur in the same sequence in the four species. Notochordal cells become distinguishable from paraxial mesodermal cells by shape, closeness of packing, and arrangement. Notochordal elongation is accompanied by a decrease in cross-sectional area and by cell rearrangement. Somitic mesoderm becomes distinguished from lateral mesoderm by a change in cell shape and orientation, followed by segmentation of somites. The schedule of somite formation was compared and related to the staging series for each species. The urodeles differ from the anurans in that the notochordal region in the early neurula stages in triangular, with the broadest part in the posterior region of the embryo. In anurans it is uniform in width. This difference may reflect differences in gastrulation and in the mechanism of elongation of the posterior part of the embryo in the neurula.

Limb ectoderm induces cartilage differentiation in mesoderm from chick embryo limb buds, Transfilter cultures have shown that this interactions requires 'contact' conditions and cannot take place at a distance. In vivo, a basement membrane is always present between ectoderm and mesoderm. The present paper demonstrates that the relationship between ectoderm and mesoderm is similar in vivo and in transfilter cultures. In culture conditions, the filter appears to be infiltrated by mesodermal cell outgrowths which form a continuous mesodermal cover on the filter. A basement membrane is always present between the mat of mesodermal cell processes and the ectoderm. Mesodermal cell processes are able to cross the Nuclepore filters (pore size 0.6-0.8 micrometer) within 15 min. After 2 h in culture, the surface of the filter opposite to the mesodermal explant is completely covered with mesodermal outgrowths. The extracellular material accumulating at the ectoderm-mesoderm interface appears to be mainly of mesodermal origin.

Sub-ridge, core, anterior and posterior borders of mesoderm were dissected from stages 22-24 chick wing buds to investigate whether structures for intercellular coupling develop between mesenchymal cells. Fine structure was examined using techniques of transmission electron microscopy, freeze-fracture and scanning electron microscopy. Gap (communicating) junctions which were observed between mesenchymal cells of all limb bud regions were distributed between apposed cell bodies, points of contact between cell processes and other cell bodies, and between contacting tips of slender cell projections. In addition particularly in the the subridge region, filopodia were observed to extend through the intercellular matrix to contact other cells several micrometers distant. The observations reported in this paper show that mesodermal cells throughout the limb have the structural capability for electrotonic and metabolic coupling during a critical period of morphogenesis in the avian limb. Whether intercellular signals which are thought to be transmitted through gap junctions are active in normal limb development remains to be investigated.