Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

Movements of cellular material of the dorsal wall in clawed-toad embryos during gastrulation and neurulation

The pieces of dorsal ectoderm of the Rana temporaria embryos at the early and midgastrula stages were transplated onto the dorsal surface of the X. laevis embryos of the same age and the movements and changes in the form and area of the transplants were followed from early gastrula to neurula. During the first period (early--midgastrula) all movements of the transplants were directed towards the blastopore and related ma- In the beginning of the second period the transplants moved toward the blastopore only in the most caudal region, whereas in all other regions the material was markedly displaced craniad. Until the early neurula stage these movements were related to the longitudinal expansion of the material in the dorsal area and later, during neurulation, to its transverse compression. The head region material was first markedly expanded in the transverse direction and then also contracted. Alternation of active contractions and expansion of the suprablastopore material has been revealed and mediocaudal (gastrulation) vs. craniopetal (neurulation) cell movements were distinctly shown.     

Expression of Brachyury-like T-box transcription factor, Xbra3 in Xenopus embryo

The Xenopus Brachyury-like Xbra3 gene is a novel T-box gene that is closely associated with Xenopus Brachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation Xbra3 is expressed in the marginal zone, with a gradient of increasing expression from ventral to dorsal. In the early neurula stage Xbra3 is expressed in the notochord and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.     

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.     

A quantitative study of regional and stage specific reaction of African clawed frog embryonic tissues on mechanical stress

Residual deformation of fragments of the embryonic tissues preserved after relaxation of the stretching force serve as a criterion of active redistribution of their cells caused by this stretching. We measured residual deformations of the Xenopus laevis ventral and dorsal ectoderm at the early gastrula and lateral ectoderm at the late gastrula-early neurula after stretching of varying time and force. While the samples responded to moderate (up to 40%) short-term stretching as elastic bodies (residual deformations were absent), residual deformation appeared in the early gastrula tissues after 30-60-min stretching, which were more pronounced in the ventral tissues than in the dorsal ones. On the contrary, a contractile reaction developed in the late gastrula-early neurula tissues in response to 60-min stretching, which almost relaxed residual deformation within 20 min after unloading. A conclusion was drawn that gastrulation and neurulation proceed under the conditions of relaxing and nonrelaxing mechanical tensions, respectively. Mechanical bases and morphogenetic role of the described reactions is discussed.     

A Quantitative Study of Regional and Stage Specific Reaction of Xenopus laevisEmbryonic Tissues on Mechanical Load

Residual deformation of fragments of the embryonic tissues preserved after relaxation of the stretching force serve as a criterion of active redistribution of their cells caused by this stretching. We measured residual deformations of the Xenopus laevis ventral and dorsal ectoderm at the early gastrula and lateral ectoderm at the late gastrula-early neurula after stretching of varying time and force. While the samples responded to moderate (up to 40%) short-term stretching as elastic bodies (residual deformations were absent), residual deformation appeared in the early gastrula tissues after 30–60-min stretching, which were more pronounced in the ventral tissues than in the dorsal ones. On the contrary, a contractile reaction developed in the late gastrula-early neurula tissues in response to 60-min stretching, which almost relaxed residual deformation within 20 min after unloading. A conclusion was drawn that gastrulation and neurulation proceed under the conditions of relaxing and nonrelaxing mechanical tensions, respectively. Mechanical bases and morphogenetic role of the described reactions is discussed.     

ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC-rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.     

A quantitive evaluation of gap junctions and their morphological alteration during differentiation of amphibian notochord cells

Summary The early development of notochord cells may be divided into three phases according to the quantitative evaluation of gap junctions: late gastrula, neurula and from tailbud to tadpole. In late gastrula, the percentage of the area of gap junctions to total membrane is 0.054% and most of the gap junctions are small in size. During the stages of neurulation, the ratios of gap junctions to total membrane area increase and remain high (0.106–0.181 %), and the majority of the gap junctions are of medium and large size. The high ratios of gap junctions to membrane area during neurulation suggests that intercellular communication via gap junctions is important during this period. In the stages from tailbud to tadpole the ratios decrease and drop drastically to 0.001 % and most of the gap junctions found are small in size. It is in the last phase that gap junctions of altered configuration appear.     

Alteration of cell adhesion system in amphibian ectoderm cells during primary embryonic induction: changes in reaggregation pattern of induced neurectoderm cells and ultrastructural features of the reaggregate

Summary Cell adhesion was studied during primary embryonic induction. The disaggregation rate and reaggregation patterns were analysed in the ectoderm cells of various developing Cynopus gastrulae and neurulae. The neurectoderm cells disaggregated more slowly with gastrulation, and the neural plate cells of early neurula showed a lesser capacity for disaggregation. Although no differences in reaggregation were found between dorsal and ventral ectoderm at the early gastrula stage, there were significant differences between the induced neurectoderm and the non-induced ventral epidermal cells at the late gastrula stage. Neural plate cells of the early neurula stage were seen to form a chain-like reaggregate, but the ventral epidermal cells of the same embryo formed a cluster-like spherical reaggregate. Scanning electron microscope observations of reaggregates also showed significant differences in adhesive properties between induced neurectoderm and non-induced epidermal cells. The adhesion field of the induced neurectoderm cells was smooth, differing from the distinct ridges of the non-induced epidermal cells. These results suggest that changes in the cell adhesion system, resulting in the formation of a columnar cell shape, may occur immediately after a neural-inducing action.     

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.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

A Relevant Method for Estimating Cell Cycle of Developing Cell: The Conspicuous Lengthening of Ts during the Early Neural Development of Cynops Embryo

A new method for estimating cell cycle was proposed and the cell cycles of the presumptive neural cells of Cynops embryo from the gastrula to neurula stages were estimated. Up to the onset of gastrulation, Tg₂ and Tg₁ became recognizable and Ts lengthened more than 10 times of that in the morula stage. The respective phases of cell cycle, espetially Ts became prominently longer as gastrulation and neurulation proceeded. However, the Ts retained a correlation with Tgc as expressed in the following regression equation, Ts=0.795Tgc–0.090, through the early development of presumptive neural cells.