Autoradiographic Analysis of Pleurodeles waltlii embryos Injected with Labelled Embryo Donor Cells

In Pleurodeles waltlii isolated embryonic cells, labelled with tritiated thymidine, were injected into a recipient embryo at the blastula stage. Three types of combinations were studied. Transplanted cells belonging to the presumptive ectoderm, endoderm and mesoderm were taken when the donor embryos were at the young gastrula stage. In each experimental series, localization and distribution of labelled donor cells in the host were analyzed by autoradiographic techniques.     

Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

The spatio-temporal distribution of single-stranded breaks in nuclear DNA in sections of clawed toad embryos during gastrulation and neurulation

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.     

An immunohistochemical study of early embryogenesis in the clawed toad Xenopus laevis by using monoclonal antibodies to intermediate filament proteins

Distribution of cytokeratin epitopes was studied in X. laevis embryos at stages 10-25 using 5 monoclonal antibodies against proteins of the human and rat keratin filaments. Specific staining was observed in chorda, outer layers of ectoderm and presumptive epidermis (late gastrula), and inner layer of presumptive epidermis. The cells of the stained zone (presumptive epidermis) were compressed while the cells of unstained zone (presumptive neuroectoderm) were extended tangentially.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.     

Expression of Na+, K+-ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na⁺, K⁺-ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na⁺, K⁺-ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na⁺, K⁺-ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na⁺, K⁺-ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8–16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li⁺ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na⁺, K⁺-ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.     

Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti-XHE (Xenopus hatching enzyme) as a marker. The anterio-dorsal ectoderm excised from stage 12-13 (mid-late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio-, but not the anterio-dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA-1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.     

Modulation of neural commitment by changes in target cell contacts in Pleurodeles waltl

In amphibian development, neural structures arise from the presumptive ectoderm at the gastrula stage by an inductive interaction with the chordamesoderm. It has been previously reported that early gastrula presumptive ectoderm can be neuralized when it is dissociated into single cells. A similar result is reported here with regard to Pleurodeles waltl presumptive ectoderm. Using this experimental model system we demonstrate: first, that neuronal and glial lineages can be specified from the presumptive ectoderm without any intervention of the natural inducing tissue; and second, that whereas rupture of cell-cell contacts evoked neural induction, dissociation immediately followed by reaggregation reduces the neuralizing response, pointing toward an active role played by cell-cell contacts of presumptive ectodermal cells in the modulation of neural commitment.     

Cell surface proteins in the early embryogenesis of Pleurodeles waltlii

Surface proteins in the first embryonic stages (8–32 cells, morula, blastula, early and late gastrula) of Pleurodeles waltlii were selectively labelled by ¹²⁵I using lactoperoxidase and glucose/glucose oxidase. Iodination was effected either on non-dissociated embryos or after their dissociation with EDTA. On the outer surface of non-dissociated embryos the two-dimensional electrophoresis revealed only three groups of ¹²⁵I-labelled proteins which did not change during all studied stages. Quite different results were obtained with the cells of dissociated embryos. In addition to the iodinated proteins of the embryonic outer surface seven major iodinated proteins were identified. These proteins originate from the regions of cell-cell contacts in intact embryo. Their two-dimensional pattern in dissociated cells changes between stages 8–32 cells and morula. The next important difference was observed during gastrulation, which corresponds in Pleurodeles waltlii to the first morphogenetic movements. Therefore the outside and inside cell surfaces of embryo are different already at stage 8–32 cells (and probably earlier), before the first step of morphogenesis. The changes of cell surface proteins at early embryonal development take place inside the embryo, in the regions of cell-cell interactions.     

Refinement of gene expression patterns in the early Xenopus embryo

During blastula and gastrula stages of Xenopus development, cells become progressively and asynchronously committed to a particular germ layer. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and single-cell RT-PCR. We show that at early gastrula stages, individual cells in the same region may express markers of two or more germ layers, and 'rogue' cells that express a marker outside its canonical domain are also observed at these stages. However, by the late gastrula stage, individual cells express markers that are more characteristic of their position in the embryo, and 'rogue' cells are seen less frequently. These observations exemplify at the gene expression level the observation that cells of the early gastrula are less committed to one germ layer than are cells of the late gastrula embryo. Ectodermal cells induced to form mesendoderm by the addition of Activin respond by activating expression of different mesodermal and endodermal markers in the same cell, recapitulating the response of marginal zone cells in the embryo.     

Spatial and temporal localization of FGF receptors in Xenopus laevis

Summary Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGF superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11⁺). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.     

STAGING OF NEWT BLASTULA EMBRYOS AND FIRST APPEARANCE OF PRIMARY MESODERMAL COMPETENCE

Using the swimbladder of the crusian carp ( Carrasius auratus ) as an inductor, the first appearance of mesodermal competence in the presumptive ectoderm of the newt ( Triturus pyrrhogaster ) blastula was investigated. The time course of embryonic development before the gastrula stage was determined by counting the number of surface cells on a 0.25 mm line at the animal pole. Pregastrula embryos with 2–3, 4–5, 6–7 and 7–8 cells roughly correspond to those at 14, 14–12, 8–6 and 4–0 hr before the beginning of gastrulation. Using presumptive ectoderm of the early gastrula stage, 15 min was found to be the minimum time of contact necessary for the realization of induction. The reactivity of the presumptive ectoderm from pregastrula embryos was tested by 30 min contact. Presumptive ectoderm up to the 4–5 cell stage did not react to the inductor. It may become competent within the next 4–8 hr, since the ectoderm from embryos in the 6–7 cell stage was reactive.     

N-CAM alterations in splotch neural tube defect mouse embryos.

The splotch (Sp) mouse is a model for both neurulation defects and defects in neural crest cell (NCC) derivatives. Since neurulation and NCC emigration from the neural tube occur at similar times in development, we suggest that these two events share a mechanism that, if disrupted, leads to malformations in both developmental pathways. Previous studies have shown that the underlying defect in these mutants may involve a mechanism that alters cellular organization and communication. Cell adhesion molecules (CAMs) have been linked with such interactions and because some, including N-CAM, are involved in neural development, we were interested in their pattern of expression in the splotch mutant. Immunolocalization studies showed similar temporospatial distributions of N-CAM antibody in embryonic day 9 mutants and controls. However, mutant embryos had a much higher intensity of anti-N-CAM fluorescence compared to controls. Further characterization using immunoblot analysis revealed that Sp mutants have an altered N-CAM polypeptide profile. Two N-CAM isoforms (Mr 140K and 180K, K = 10(3] are normally present at this time of development. However, extracts from Sp embryos display a heavier N-CAM species (Mr 200K), as well as an altered 140K isoform. Heterozygotes also exhibit a different N-CAM profile, displaying a band between 180K and 200K in addition to the normal 180K and 140K species. Microheterogeneity was also observed in mutant and heterozygous embryos carrying Spd, an allele of Sp. However, these differences were less dramatic than that of Sp. The Sp locus may be involved in post-translational modification of N-CAM. An aberration in N-CAM processing could be the primary target of the mutation that leads to the development abnormalities observed in this mouse mutant.     

Expression of N-CAM-180 and N-cadherin during development in two South-American anuran species (Bufo arenarum and Hyla nana)

Cadherins and N-CAM are Ca++-dependent and Ca++-independent cell adhesion molecules respectively. These molecules play a key role in morphogenesis and histogenesis. We determined the spatiotemporal pattern of N-cadherin and N-CAM-180 kDa expression by immunohistochemistry during development in two South-American anuran species (Bufo arenarum, toad and Hyla nana, frog). Both N-cadherin and N-CAM were not detectable during early developmental stages. Expression of N-cadherin appeared between the inner and the outer ectoderm layers at stages 19-20. At stages 24-25, N-cadherin was expressed in the neural tube and the heart. In early tadpoles, N-cadherin expression increased along with the central nervous system (CNS) morphogenesis, and reached its maximum level at metamorphic climax stage. N-Cadherin expression was not uniformly distributed. At stage 42, olfactory placodes and retina expressed N-cadherin. Contrary to N-CAM, the strongly myelinated cranial nerves were not labeled. N-Cadherin was present in several mesoderm derivatives such as the notochord, heart and skeletal muscle. The non-neural ectoderm and the endoderm were always negative. Expression of N-CAM appeared first in the neural tube at stages 24-25 and the level of expression became uniform from pre-metamorphic to metamorphic climax tadpoles. At this latter stage, a clear N-CAM immunolabeling appeared in the nerve terminals of pharynx and heart. N-Cadherin and N-CAM were found mainly co-expressed in the CNS from early tadpole to metamorphic climax tadpole. Our results show that the expression of N-CAM and N-cadherin is evolutionary conserved. Their increased expression during late developmental stages suggests that N-CAM and N-cadherin are involved in cell contact stabilization during tissue formation.     

Behavior of pigment cells in gastrula-stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis

The behavior of pigment cells in sea urchin embryos, especially at the gastrula stage, is not well understood, due to the lack of an appropriate method to detect pigment cells. We found that pigment cells emanated autofluorescence when they were fixed with formalin and irradiated with ultraviolet or green light. In Hemicentrotus pulcherrimus, fluorescent pigment cells became visible at the archenteron tip at the mid-gastrula stage. The cells detached from the archenteron slightly before the initiation of secondary invagination and migrated toward the apical plate. Most pigment cells entered the apical plate. This entry site seemed to be restricted, because pigment cells could not enter the ectoderm and remained in the blastocoele at the vegetal pole side when elongation of archenteron was blocked. Pigment cells that had entered the apical plate soon began to migrate in the aboral ectoderm toward the vegetal pole. In contrast, pigment cells of Scaphechinus mirabilis embryos were first detected in the vegetal plate before the onset of gastrulation. Without entering the blastocoele, these cells began to migrate preferentially in the aboral ectoderm toward the animal pole. When the archenteron tip reached the apical plate, pigment cells had already distributed throughout the aboral ectoderm. Thus, the behavior of pigment cells was quite different between H. pulcherrimus and S. mirabilis.     

A comparative analysis of notochord formation in amphibian embryos]

We studied the origin, structure, and development of the notochord in Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura) embryos. The notochord rudiment is formed in both species at the early gastrula stage as a cluster of polarized chorda-mesoderm cells located along the sagittal plane of the embryo. In Pl. waltlii the notochord rudiment is separated from the gastrocoele roof as a result of contraction of apical cell surfaces. The contraction wave spreads forward and backward along craniocaudal axis, i.e., segregation of the notochord rudiment progresses in two directions simultaneously. Similar process takes place in X. laevis embryos; however, propagation of the contraction wave in the anterior part of the body somewhat differs from that in the posterior part. While the "anterior" contraction wave resembles that in Pl. waltlii embryos, progression of the wave in the posterior part of the body is distinguished by a closer association of the notochord rudiment with ectoderm and the presence of its delamination boundaries with the somite mesoderm.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.