Ventral cell rearrangements contribute to anterior-posterior axis lengthening between neurula and tailbud stages in Xenopus laevis

Studies of morphogenesis in early Xenopus embryos have focused primarily on gastrulation and neurulation. Immediately following these stages is another period of intense morphogenetic activity, the neurula-to-tailbud transition. During this period the embryo is transformed from the spherical shape of the early stages into the long, thin shape of the tailbud stages. While gastrulation and neurulation depend largely on active cell rearrangement and cell shape changes in dorsal tissues, we find that the neurula-to-tailbud transition depends in part on activities of ventral cells. Ventral explants of neurula lengthen autonomously as much as the ventral sides of intact embryos, while dorsal explants lengthen less than the dorsal sides of intact embryos. Analyses of cell division, cell shapes, and cell rearrangement by transplantation of labeled cells and by time lapse recordings in live intact embryos concur that cell rearrangements in ventral mesoderm and ectoderm contribute to the autonomous anterior-posterior axis lengthening of ventral explants between neurula and tailbud stages.     

Time-lapse cinemicrographic analysis of superficial cell behavior during and prior to gastrulation in Xenopus laevis

Time-lapse cinemicrography was used to show what changes in the number, size, shape, arrangement and what movements of apices of superficial cells occur during epiboly, extension, convergence and blastopore formation in the blastula or gastrula of Xenopus laevis. Epiboly of the animal region occurs by apical expansion of superficial cells at a nearly constant rate from the midblastula to the midgastrula stage. Egression of deep cells into the superficial layer does not occur. Extension of the dorsal marginal zone begins in the late blastula stage with the rapid spreading of the apices of cells in this region and this continues until the onset of neurulation when rapid shrinkage begins. Extension and convergence of the dorsal marginal zone occurs by a rearrangement in which individual cells exchange neighbors and by a change in the shape of the cell apices. Regional differences in apical expansion are accompanied by differences in rate of anticlinal division of superficial cells such that cells in all sectors of the animal region and the marginal zone show similar patterns of decrease in apparent apical area. Shrinkage of the apices of bottle cells during blastopore formation is described. From this and other studies, a model of the cellular behavior of epiboly, extension and convergence is constructed and several hypotheses as to how these activities might generate the mechanical forces of the gastrulation movements are presented.     

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. I. Independence in undisturbed embryos

We examined the spatial relationships between the meridian of sperm entry the plane of first cleavage, and the embryonic axis (defined by the neural groove) in eggs of Xenopus laevis. Direct measurement of the angular separations between these embryonic structures in gelatin-embedded eggs confirmed the classical conclusion that the sperm entry point and neural groove tend to form on opposite sides of the egg, and also revealed that the first cleavage plane has a nearly random orientation with respect to the neural groove. We next examined the distortion of the first cleavage plane that results from the normal processes of convergence and extension during gastrulation and neurulation. We permanently marked the first cleavage plane by injecting one blastomere of the two-cell embryo with a fluorescent lineage marker. At the start of gastrulation, the interface between the labeled and unlabeled regions was almost randomly oriented relative to the dorsal blastopore lip, confirming our first set of observations. In embryos with the interface less than 60 degrees to the plane passing through the midline of the dorsal lip, convergent movements of cells produced a confrontation of labeled and unlabeled cells along much of the dorsal midline. Thus, although the first cleavage plane and the bilateral plane were frequently not congruent, the morphogenetic movements of gastrulation and neurulation brought about an apparent congruence in many half-labeled embryos.     

Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements of the superficial layer.

The disposition of prospective areas and the course of morphogenetic movements during gastrulation and neurulation were investigated by vital staining. The prospective lining of the archenteron, the prospective neural area, and the prospective epidermal area are represented on the surface of the early gastrula. The prospective lining of the archenteron occupies the area within 65–70° of the vegetal pole and is divided into prospective archenteron roof and prospective archenteron floor by the blastopore pigment line which functions as the locus of invagination. A crescent-shaped neural area lies immediately above the prospective archenteron roof, rising from it at 125° lateral to the dorsal midline to a point 130° above the vegetal pole in the dorsal midline. In the early gastrula, most, if not all, mesoderm is deep to the surface layer and is mapped by the insertion of dyed agar spikes. Results thus far indicate that the prospective notochord lies in the dorsal deep marginal zone, followed laterally by the medial region of the somites, the lateral region of the somites, and the lateral plate.The morphogenetic significance of the comparative disposition of the anlagen in Xenopus is discussed.     

Constitutive and stress‐inducible expression of the endoplasmic reticulum heat shock protein 70 gene family member,immunoglobulin‐binding protein (BiP), during Xenopus laevis early development

We have characterized the constitutive and stress‐inducible pattern of immunoglobulin‐binding protein (BiP) gene expression during Xenopus early development. Whole mount in situ hybridization analysis revealed that BiP mRNA was detected in unfertilized eggs, cleavage and blastula stage embryos. In gastrulae, BiP mRNA was present across the surface of the embryo, while in neurulae BiP mRNA was enriched in the neural plate, neural fold, and around the blastopore. In early and late tailbud embryos, BiP mRNA was found primarily in the dorsal region. Tunicamycin and A23187, the calcium ionophore, enhanced BiP mRNA accumulation first at the neurula stage, while heat shock induced BiP mRNA accumulation first at the gastrula stage. Compared to control, A23187‐ and heat shock‐treated neurulae displayed relatively high levels of BiP mRNA in selected tissues, including the neural plate, neural folds, around the blastopore, and ectoderm. At the early tailbud stage, A23187 and heat shock enhanced BiP mRNA accumulation primarily in the head, somites, tail, and along the spinal cord. A similar situation was found with A23187‐ and heat shock‐treated late tailbud embryos, except that heat‐shocked embryos also displayed enhanced BiP mRNA accumulation in the epidermis. These studies demonstrate a preferential accumulation of BiP mRNA in selected tissues during development and in response to stress. Dev. Genet. 25:31–39, 1999. © 1999 Wiley‐Liss, Inc.     

Accumulation and decay of DG42 gene products follow a gradient pattern during Xenopus embryogenesis

The DG42 gene is expressed during a short window during embryogenesis of Xenopus laevis. The mRNA for this gene can be first detected just after midblastula, peaks at late gastrula, and decays by the end of neurulation. The sequence of the DG42 cDNA and genomic DNA predicts a 70,000-Da protein that is not related to any other known protein. Antibodies prepared against portions of the DG42 open reading frame that had been expressed in bacteria detected a 70,000-Da protein in the embryo with a temporal course of appearance and decay that follows that of the RNA by several hours. Localization of the mRNA in dissected embryos and immunohistochemical detection of the protein showed that DG42 expression moves as a wave or gradient through the embryo. The RNA is first detected in the animal region of the blastula, and by early gastrula is found everywhere except in the outer layer of the dorsal blastopore lip. By midgastrula DG42 protein is present in the inner ectodermal layer and the endoderm; it disappears from dorsal ectoderm as the neural plate is induced and later decays in a dorsoventral direction. The last remnants of DG42 protein are seen in ventral regions of the gut at the tailbud stage.     

Experimental reversal of the normal dorsal-ventral timing of blastopore formation does not reverse axis polarity in Xenopus laevis embryos

During gastrulation in Xenopus laevis, the dorsal lip of the blastopore normally appears before the ventral lip. Metabolic gradient models propose that the dorsal lip develops from the region of highest metabolic activity and somehow dominates other regions to prevent them from becoming dorsal. To test these ideas, I applied a temperature gradient of 12 degrees C across the embryo. Localized heating of the prospective ventral vegetal region from early in the first cleavage period until gastrulation causes the blastopore lip to form first by 2 hr at the prospective ventral meridian rather than at the prospective dorsal meridian. Despite this reversal of the timing of blastopore formation, gastrulation is completed, and the neural plate forms at its usual position on the prospective dorsal meridian. This demonstrates that the earliest gastrulating regions of the blastopore do not necessarily become dorsal, nor do they inhibit dorsal development by other regions. It is unlikely that axis polarity is based on regional differences in energy metabolism.     

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.     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.     

The gastrocoel roof plate in embryos of different frogs

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.     

Development of the dendrobatid frog Colostethus machalilla

To provide a developmental correlate with other frogs, we prepared a normal table of development for the dendrobatid, Colostethus machalilla and analyzed the morphology of its early development. This frog reproduces in captivity and deposits moderately sized eggs (1.6 mm in diameter) in terrestrial nests. The father guards the embryos until tadpole hatching. We divided development until hatching into 25 stages and implemented methods for in vitro culture of the embryos. The external and internal morphology of embryos were evaluated by observations in whole mount and in sections. Neural, notochord and somite specific antibodies were used to analyze gene expression patterns by immunostaining of embryos. Embryonic development of C. machalilla is slow and deviates from Xenopus laevis. In C. machalilla the elongation of the notochord, neural plate and somite formation occur after blastopore closure, possibly due to a delay in the dorsal convergence and extension movements. The gastrula of C. machalilla also deviates from X. laevis. The archenteron remains small until blastopore closure, where small cells accumulate at the blastopore lips. Simultaneously, the blastocoel roof thins until it becomes a monolayer of cells. Although C. machalilla does not form an embryonic disk, its thick blastopore lips resemble the embryonic disk of the marsupial frog Gastrotheca riobambae and represent an interesting deviation from the gastrulation pattern observed in X. laevis.     

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.     

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed <Emphasis Type="Italic">Xenopus</Emphasis> embryos

Influence of the relaxation of mechanical tensions upon collective cell movements, shape formation, and expression patterns of tissue-specific genes has been studied in Xenopus laevis embryos. We show that the local relaxation of tensile stresses within the suprablastoporal area (SBA) performed at the early-midgastrula stage leads to a complete arrest of normal convergent cell intercalation towards the dorsal midline. As a result, SBA either remains nondeformed or protrudes a strip of cells migrating ventralwards along one of the lateral lips of the opened blastopore. Already, few minutes later, the tissues in the ventral lip vicinity undergo abnormal transversal contraction/longitudinal extension resulting in the abnormal cell convergence toward ventral (rather than dorsal) embryo midline. Within a day, the dorsally relaxed embryos acquire pharyngula-like shapes and often possess tail-like protrusions. Their antero-posterior and dorso-ventral polarity, as well as expression patterns of pan-neural (Sox3), muscular cardiac actin, and forebrain (Otx2) genes substantially deviate from the normal ones. We suggest that normal gastrulation is permanently controlled by mechanical stresses within the blastopore circumference. The role of tissue tensions in regulating collective cell movements and creating pharyngula-like shapes are discussed.     

Expression patterns of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl: implications on the evolutionary relationships between the balancers and cement gland in amphibians

We report the characterization of an Otx2 and an Otx5 orthologue in the urodele Pleurodeles waltl. These two genes, termed PwOtx2 and PwOtx5, share highly conserved expression domains with their gnathostome counterparts at tailbud stages, like the developing forebrain ( PwOtx2), or the embryonic eye and epiphysis ( PwOtx5). As in Xenopus laevis, both are also transcribed in the dorsal lip of the blastopore during gastrulation and in anterior parts of the neural plate during neurulation. In addition, PwOtx5 displays a prominent expression in the developing balancers and the lateral non-neural ectoderm during neurulation, from which they derive. By contrast, PwOtx2 expression remains undetectable in the balancers and their presumptive territory. These data suggest that PwOtx5, but not PwOtx2, may be involved in the differentiation and early specification of balancers. Comparisons of Otx5 expression patterns in P. waltland X. laevis embryos suggest that, as previously shown for Otx2, changes in the regulatory mechanisms controlling Otx5 early expression in the non-neural ectoderm may occur frequently among amphibians. These changes may be related to the rise of cement glands in anurans and of balancers in urodeles. This hypothesis could account for some similarities between the two organs, but does not support a homology relationship between them.