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In amphibian embryos, fibronectin (FN) assembles as a fibrillar network on the roof of the blastocoel cavity, preceding mesodermal cell migration. Local inversion of the ectoderm to produce a site where no FN is available prevents mesodermal cell migration. Microinjection of monovalent antibodies to FN arrests gastrulation. A complete inhibition of mesodermal cell migration is obtained after microinjection of a synthetic peptide containing the cell binding site sequence of FN. Prevention of interactions between receptors and FN appears to be the primary cause for blockage of gastrulation.

Recent studies on temporal control of early amphibian development are reviewed. It is becoming clear that the development of an embryo is not timed by a single clock set in motion at fertilization, instead each developmental event seems to be timed by its own clock-like mechanism. The timing of developmental events is rigidly determined within embryonic cells, and usually can not be altered experimentally. One exception, however, is the timing of midblastula transition in amphibian embryos; recent studies have shown that its timing is regulated by the nucleocytoplasmic ratio. Several developmental events, particularly those associated with transcriptional activities, require DNA replication prior to their occurrence, suggesting an intimate relationship between DNA replication cycles and their onset. On the other hand, there are many other developmental events where timing is not controlled by the number of cell divisions, DNA replication cycles, or the nucleocytoplasmic ratio. Cytoplasmic machinery with autonomous oscillatory properties is thought to be involved in the timing of these events.

Different stages of thymus morphogenesis and thymocyte differentiation have been studied at the ultrastructural level in the urodele amphibian Pleurodeles waltlii Michah. From stage 38 to 42 the undifferentiated lymphoid stem-cells colonize the epithelial thymic buds. From stage 43 to 45 the lymphoid stem-cells differentiate into lymphoblasts and then transform into typical lymphocytes. A clasmatosis phenomenon seems to be involved in this transformation. From stage 46 to 52, a phase of intense proliferation occurs and relations between dense reticular epithelial cells and lymphocytes are described. At stage 53, numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. At the same time, lymphocytes undergo migrations through the intra- and peri-thymic blood vessels. These lymphocytes should populate the peripheral lymphoid organs, according to the previous finding that stage 52 was the last developmental step for an efficient abrogation of cell-mediated immunity by thymectomy in Pleurodeles.

In order to determine the origin of blood cells we performed embryonic grafts of different portions of mesoderm from various locations between diploid and tetraploid embryos at the tail-bud stage. The tetraploid animals were the hosts. The size differences between tetraploid and diploid cells made identification possible by direct microscopic examination of blood smears. In a previous report we showed the important role of truncal anterior mesoderm in the genesis of blood cells. We now establish that this effect is brought about by the inductive capacity of the hepatic endoderm or by the fact that the environmental conditions are more appropriate for blood stem cell development, whereas in the absence of the hepatic endoderm the blood stem cells fail to appear. Grafting of hepatic anlage containing endoderm and mesoderm gives rise to numerous graft-derived blood cells which last throughout the life span of the hosts. The same result is obtained by grafting truncal posterior ventral or lateral mesoderm onto hepatic endoderm. Heterotopic grafting of truncal anterior mesoderm isolated from its underlying hepatic endoderm leads to the formation of only a few blood cells which last only during larval life. This demonstrates that the whole lateral and ventral truncal mesoderm is able to differentiate into blood cells when associated with hepatic endoderm.

Neuronal differentiation has been studied in dissociated cell cultures from early neurulae of Pleurodeles waltl and Ambystoma mexicanum. Cocultures were prepared from the neural primordium and underlying chordamesoderm. NP and NF cultures were prepared from isolated neural plate and neural folds, respectively. Neuronal precursors in NP and NF cultures had distinctive aggregation properties already evident after 1-2 days in culture. After 10-15 days, mature neurones and synapses were observed by electron microscopy in the three culture types. The expression of neurofilament polypeptides and tetanus-toxin-binding sites was also present in these cultures. A small percentage of neurones contained cytochemically detectable catecholamine. Many neurones took up tritiated dopamine with a high affinity. Quantitative measurement of [3H]acetylcholine synthesis and storage from [3H]choline were negative at the early neurula stage and in 5 to 15-day-old NF cultures, and remained low in 5 to 15-day-old NP cultures. Acetylcholine production in cocultures increased linearly with time and was always much higher than in NP cultures. These results suggest that, at the early neurula stage, some neuronal precursors have acquired the capacity to express a high degree of morphological and biochemical differentiation even in the absence of further chordamesoderm influence. However, the chordamesodermal cells in the cultures increased acetylcholine synthesis.

Embryos of the ac/ac maternal-effect mutant in Pleurodeles waltl show disturbed epibolic movement during gastrulation. At the early gastrula stage, ectoderm cells begin to sink in at random sites in the animal half of the embryo. At the advanced gastrula stage the ectodermal pits develop into grooves. Electron microscopical analysis shows that many cells in the bottom of the pits and grooves have narrowed apices and bear many microvilli, while the cortical cytoplasm is dense, filamentous and underlain by a stratum of vesicles. These findings are interpreted as indicating that ectoderm cells contract rather than expand leading to disturbed epibolic movement.

In Pleurodeles waltl, the early neuronal differentiation of precursor cells from late gastrula stage has been studied by culture in vitro from either isolated neural plate (NP) or isolated neural fold (NF). The aim of this study was to delineate the information acquired by ectodermal target cells during neural induction. By culturing these cells in vitro either with or without the underlying chordamesoderm, we showed that in the absence of chordamesodermal influence such NP or NF cells exhibited a high degree of biochemical and morphological differentiation as revealed by the synthesis and the storage of neurotransmitters, the activity of specific enzymes, as well as by the expression of neuronal markers: specific changes in cell surface carbohydrates, tetanus toxin binding sites and neurofilament polypeptides. Remarkable changes in the cell adhesive properties were the first events observed in the different central (NP) and peripheral (NF) types. In cocultures the chordamesodermal cells exert a beneficial influence on this differentiation, specially increasing acetylcholine synthesis. There are some differences between central (NP) or peripheral (NF) neuroblast response to this further notochord or mesodermal influence.

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.

The competent ectoderm of Pleurodeles waltl comprises two cell layers with characteristic differences in their morphology, their composition and the molecular arrangement of the various constituents. The use of labelled lectin probes for observations of ectoderm tissue in vitro with u.v. microscopy (epi-illumination) and the quantification of the results show the following: Differences in labelling according to the nature of the lectins (SBA, PSA, LCA and Con A). These differences provide information on the nature of the carbohydrates which are present at this stage and on the number of receptors. Differences in fluorescence intensity of the surfaces studied. The internal surface of the ectoderm is labelled more densely than the external surface. Rearrangement of the lectin receptors with a new molecular configuration, stressing the fluidity of the membrane (by the mobility of the receptors throughout the membrane) and its importance for the occurrence of neural induction. Existence of membrane glycoconjugate turnover. A difference in behavioural characteristics between the internal and the external surfaces with respect to the lectins and the formation of an extracellular matrix on the internal surface alone. The extracellular matrix seems to have a role in morphogenetic movements.

Detailed SEM observations of the changes in cellular morphology, arrangements, and contacts that occur during the process of somite formation were made in two species of urodele amphibians, Ambystoma mexicanum and Pleurodeles waltlii, and one species of anuran amphibian, Rana sphenocephala. After fixation, embryos were fractured transversely, horizontally, and parasagittally, and the intrasomitic cellular arrangement pattern was examined with the SEM. It was found that Ambystoma and Pleurodeles embryos followed exactly the same development sequence in rosette formation and myoblast fusion. Rana somites did not, however, appear to form rosettes. Those myotomal cells underwent fusion immediately after a few segmentations occurred. Patterns of cellular rearrangement were also described during urodele rosette formation at the time of somite segmentation and during myoblast fusion. Extensive changes in cell shape and orientation appeared to occur during those processes. When cells changed their orientation, they often exhibited a triangular configuration. Probable roles of these triangular-shaped cells in rosette formation and myoblast fusion are discussed. During the initial period of myoblast or myotomal cell fusion, cells first send out specialized cell processes and then establish their cell-cell contacts. The establishment of such contacts eventually leads to tight membrane appositions and fusion. Since myoblast fusion appeared to occur between two cells which were tandemly arranged in a rosette, the origin of multi-nuclearity in the fused cells is discussed. Finally, comparative analyses of the pattern of somite formation and subsequent muscle development were made between different species of amphibians. The possibility is discussed that patterns of somitogenesis may provide useful indicators for determining how different families of amphibians evolved.

The possible involvement of target membrane specific receptor(s) in the transmission of the neural signal leading to activation of the intracellular machinery involved in the process of neural determination, has been examined using lectin probes (Con A, succinylated-ConA, LcA, PsA and SB A). Not only Con A binding sites but many different glycoconjugated molecules (α-D-galactose, N-acetyl-D-galactosamine, α-D-fucose, N-acetyl-D-glucosamine, etc.) would have to be involved, if neural receptor(s) are invoked to explain initiation of neural induction. We show here that the close involvement of such receptor molecules in neural induction is so far hypothetical and remains to be demonstrated. Moreover we are inclined to the view of Barth and others who suggested that ionic fluxes and physicochemical and electrophysiological properties of the target membrane could play a crucial role in neural induction.

Journal Article, info:eu-repo/semantics/published