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.     

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.     

A study of the effects of trypan blue injected into amphibian zygotes

Summary Trypan blue was injected (0.001–0.002 mg/egg) into zygotes ofRana. The trypan blue-injected series showed much greater mortality than the controls. A probable suppression of gastrulation was seen. 29.7% of the dye-injected and 50.3% of the control embryos developed beyond stage 14. Abnormalities seemed to appear only after neurulation began. Microcephaly, trunk and tail abnormalities occurred though in a small number. Sections of a few embryos revealed disorganized brain and degeneration of the neural tissue. There were no cases of mesodermalization of the notochord.The results are discussed from the view point that trypan blue alters the physical state of proteins in the cytoplasm of the egg.     

Studies on the Formation and State of Determination of the Trunk Organizer in the Newt, Cynops Pyrrhogaster III. Tangential Induction in the Dorsal Marginal Zone

Analysis of the course of differentiation of combinants between the presumptive prechordal plate (PcP) and presumptive ectoderm (PE) by time-lapse filming showed that the PcP of early gastrulae has the capacity to induce mesoderm (notochord, muscle cells and migrating cells) in the PE. The mesoderm-inducing capacity of the PcP decreases sharply during gastrulation. Following invagination in the mid-gastrula, the PcP completely loses its mesoderm-inducing capacity. This change also occurred when the PcP of the earliest gastrula was aged in vitro for 18 hr. This shows that the mesoderm-inducing capacity of the PcP decreases autonomously with aging.
PE transplanted into the presumptive trunk organizer region of the dorsal marginal zone of the earlist gastrula, became mesodermized within 12 hr. It is clear that this mesodermization of the transplanted PE is due to "tangential induction" from the PcP. The stepwise formation of the trunk organizer in Cynops pyrrhogaster is discussed in consideration of these results.     

A cyto-embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus , which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis , even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus . These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well-known species of sea urchins.     

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

Computer modelling of gastrulation and neurulation in amphibian embryos based on mechanical tension fields

The values of cell wall tensions were calculated with an assumption of mechanical equilibrium of every cell apex on schematic diagrams of histological sections of the common frog embryonic tissues. The maps of the main (the strongest) tensions were drawn for the early gastrula and early neurula. The further course of gastrulation and neurulation was simulated with an assumption that the cell apices are displaced due to active contractions of the most tensed cell walls (variant A of the model). In addition, a suggestion was studied that the capacity for contraction falls in the most extended cell walls (variant B). Up to seven steps of model morphogenesis were simulated and the tension field was recalculated at every step. The course of gastrulation and neurulation was reproduced during simulation with sufficient details, including regional peculiarities of neurulation in the trunk and head regions. Both variants gave roughly similar results for gastrulation, whereas variant B ensured a faster course of model morphogenesis for neurulation. A conclusion was drawn that the mechanical tension fields established by the onset of gastrulation and neurulation represent a sufficient informational base for their further course.     

Epithelial Cell Wedging and Neural Trough Formation Are Induced Planarly inXenopus,without Persistent Vertical Interactions with Mesoderm

In this study we investigate the induction of the cell behaviors underlying neurulation in the frog,Xenopus laevis.Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Kelleret al.,1992,Dev. Dyn.193, 218–234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a “giant sandwich” explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.     

Cell lineage analysis of neural induction: origins of cells forming the induced nervous system

Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.     

Stereotypical cell division orientation controls neural rod midline formation in zebrafish

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body-axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Here we show that SDO is required for formation of the neural rod midline during neurulation but dispensable for elongation of the body axis during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the noncanonical Wnt receptor Frizzled 7 (Fz7) and that interfering with cell division orientation leads to severe defects in neural rod midline formation but not body-axis elongation. These findings suggest a novel function for Fz7-controlled cell division orientation in neural rod midline formation during neurulation.     

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.     

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.     

An early developmental phase of pp60c-src expression in the neural ectoderm

The expression of the normal cellular src protein (pp60c-src) was investigated in the early chick embryo during gastrulation and neurulation by immunoperoxidase staining using antisera, raised against bacterially expressed pp60v-src, that recognizes pp60c-src specifically in normal cells. During gastrulation pp60c-src immunoreactivity appeared primarily in the neural ectoderm and was much less prominent in the mesoderm, endoderm, and nonneural ectoderm. During neurulation pp60c-src immunoreactivity began to disappear from the wall of the closing neural tube so that by the completion of neural tube closure no specific pp60c-src immunoreactivity appeared in any of the neuroepithelial cells composing the neural tube. These studies reveal a developmental phase of pp60c-src expression even earlier than reported previously, when neuroepithelial cells of later embryos undergo terminal neuronal differentiation. These findings raise the possibility that pp60c-src may mediate two different differentiation signals in the neuronal lineage.     

Avian sickle endoblast induces gastrulation or neurulation in the isolated area centralis or isolated anti-sickle region respectively

By the quail-chicken chimera technique, we studied, in culture, the inducing effect of sickle endoblast (derived from Rauber's sickle by centripetal and cranial migration) on the isolated Rauber's sickle-free central part of the area centralis or on the isolated Rauber's sickle-free anti-sickle region from unincubated chicken blastoderms. Just as Rauber's sickle, the flat one-cell-thick sickle endoblast (Stage 2-3, Hamburger & Hamilton, 1951) induces a primitive streak (PS) and a neural plate in the area centralis. If a vitelline membrane is interposed between the sickle endoblast and the area centralis, then a small primitive streak is still induced, suggesting the effect of a diffusible factor on PS formation. In the adjacent upper layer of an isolated anti-sickle region the apposed sickle endoblast induces only a (pre)neural plate. By contrast, this (pre)neural plate inducing effect is rapidly and totally suppressed after grafting on the anti-sickle region of whole unincubated blastoderms. This suggests dominating positional information phenomena emanating from Rauber's sickle over the whole blastoderm. After grafting sickle endoblast either on the isolated area centralis or on isolated anti-sickles, no junctional endoblast and no blood islands developed. This suggests that the differentiation of Rauber's sickle material into sickle endoblast is irreversible. Our results indicate that Rauber's sickle material under the form of sickle endoblast also influences early neurulation phenomena (at distance in space and time). The present study indicates the existence of a temporo-spatially bound cascade of gastrulation and neurulation phenomena and blood island formation in the avian blastoderm, starting from Rauber's sickle, the primary major organizer with inducing, inhibiting and dominating potencies. The latter not only plays a role by secretion of signalling molecules (positional information) but it also influences development by its cell lineages (junctional endoblast and sickle endoblast).     

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.     

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.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

TEMPORAL RELATIONS BETWEEN EXTENSION OF ARCHENTERON ROOF AND REALIZATION OF NEURAL INDUCTION DURING GASTRULATION OF NEWT EMBRYO

This investigation was performed in order to analyze the basic relationships between the archenteron roof and the overlying ectoderm in primary induction in the Cynopus (Triturus) pyrrhogaster embryo.
The part of the archenteron roof that is active in inducing capacity extends linearly after invagination at the speed of 0.15 mm per hr at 23°C until stage 13b. The period of contact at each position of the presumptive neuro-ectoderm with the active archenteron roof could be estimated by the formula described in the Discussion.
Pieces of the presumptive neuro-ectoderm were isolated from gastrulae at three developmental stages and cultured separately in Holtfreter solution after being divided caudo-cranially into 4 parts. The result showed that some of them were able to differentiate into neural tissues even in the mid-gastrula stage and that the presumptive neuro-ectoderm acquired the capacity to differentiate into neural tissue along a caudocranial axis from the part adjacent to the blastopore during gastrulation.
It could be estimated that 3 hr of contact with the active archenteron roof is sufficient for the presumptive neuro-ectoderm to differentiate into neural tissue.
The present study also showed that the neuralizing capacity of the whole prospective neuro-ectodermal area has already been determined before the end of stage 13, i.e., within less than 14 hr after first contact of the ectoderm with the active archenteron roof at 23°C.