Prospective Neural Areas and Their Morphogenetic Movements during Neural Plate Formation of Xenopus Embryos. I. Development of Vegetal Half Embryos and Chimera Embryos

Development of animal cap-less Xenopus gastrulae was examined. In vegetal halves from which the animal cap was removed 0.6 mm above the blastopore, an apparently normal array of craniocaudal structures developed. Histological examination showed differentiation of central nervous system (CNS) structures in the cap-less embryos, but differentiation of sensory organs, such as a lens and ear vesicle in only a few embryos. Only the dorsal midline of the embryos was covered with epidermis, and its lateral-ventral areas consisted of bare endoderm and mesoderm. The development of animal cap was also investigated by exchanging the animal cap of X. laevis embryos with that of X. borealis embryos, which can be distinguished by quinacrine fluorescence staining. The central nervous system of chimera embryos consisted mainly of X. laevis cells stained homogeneously with quinacrine but a small number of punctately-stained X. borealis cells was in the anterior tip of the forebrain. Cells of the lens and ear vesicle were punctately stained. More than two-thirds of the epidermal area consisted of punctately-stained cells and only the dorsal midline of the posterior head- and trunk-epidermis consisted of homogeneously-stained cells.
Areas of the prospective central nervous system and their movement during embryogenesis of Xenopus are discussed.     

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.     

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.     

Activin-treated ectoderm has complete organizing center activity in Cynops embryos

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops .     

Neural induction in embryos

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g ), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.     

Formation of the Neural Plate and the Mesoderm in Normally Developing Embryos of Xenopus laevis

Normally developing embryos of Xenopus were fixed at various stages between the blastula and early tail bud stage, and their serial sections were examined. The marginal belt of the blastula was characterized by abundance of cells with RNA-rich peripheral cytoplasm called mesoplasm. At the early gastrula stage, the marginal belt was folded into two layers giving rise to mesodermal material and marginal ectoderm. During gastrulation, the mesodermal material, which consisted of RNA-rich cells, spread to enclose the blastocoel and the endoderm, and a large part of it was shifted to the dorsal side of the embryo. It gradually established the mesodermal layer. The notochord was formed on the dorsal lip of the blastopore by involution, separately from preformed mesodermal material. The RNA-rich cells in the marginal ectoderm became columnar, forming a broad belt in the marginal zone. This belt was deformed and shifted to the dorsal side during gastrulation, eventually establishing the neural plate showing quantitative differentiation along the head-tail axis. Possible mechanisms involved in the formation of the neural plate and mesoderm were discussed with reference to the organizer and the mesoplasm.     

Inductive differentiation of two neural lineages reconstituted in a microculture system from Xenopus early gastrula cells.

Neural induction of ectoderm cells has been reconstituted and examined in a microculture system derived from dissociated early gastrula cells of Xenopus laevis. We have used monoclonal antibodies as specific markers to monitor cellular differentiation from three distinct ectoderm lineages in culture (N1 for CNS neurons from neural tube, Me1 for melanophores from neural crest and E3 for skin epidermal cells from epidermal lineages). CNS neurons and melanophores differentiate when deep layer cells of the ventral ectoderm (VE, prospective epidermis region; 150 cells/culture) and an appropriate region of the marginal zone (MZ, prospective mesoderm region; 5-150 cells/culture) are co-cultured, but not in cultures of either cell type on their own; VE cells cultured alone yield epidermal cells as we have previously reported. The extent of inductive neural differentiation in the co-culture system strongly depends on the origin and number of MZ cells initially added to culture wells. The potency to induce CNS neurons is highest for dorsal MZ cells and sharply decreases as more ventrally located cells are used. The same dorsoventral distribution of potency is seen in the ability of MZ cells to inhibit epidermal differentiation. In contrast, the ability of MZ cells to induce melanophores shows the reverse polarity, ventral to dorsal. These data indicate that separate developmental mechanisms are used for the induction of neural tube and neural crest lineages. Co-differentiation of CNS neurons or melanophores with epidermal cells can be obtained in a single well of co-cultures of VE cells (150) and a wide range of numbers of MZ cells (5 to 100). Further, reproducible differentiation of both neural lineages requires intimate association between cells from the two gastrula regions; virtually no differentiation is obtained when cells from the VE and MZ are separated in a culture well. These results indicate that the inducing signals from MZ cells for both neural tube and neural crest lineages affect only nearby ectoderm cells.     

Direct evidence of an essential role for extended involution in the specification of a dorsal marginal mesoderm during Cynops gastrulation

It has been indicated that specification of the dorsal marginal mesoderm of the Cynops gastrula is established by vertical interactions with other layers, which occur during its extended involution. In the present study, when the prospective notochordal area of the early gastrula was almost completely removed together with the dorsal mesoderm-inducing endoderm and most of the bottle cells, the D-less gastrulas still formed the dorsal axis with a well-differentiated notochord; in half of them, where the involution occurred bi-laterally, twin axes were observed. On the other hand, when the wound of a D-less gastrula was repaired by transplanting the ventral marginal zone and ectoderm, the formation of the dorsal axis was inhibited if the involution of the lateral marginal zone was prevented by the transplanted piece. The present study suggests that: (i) cells having dorsal mesoderm-forming potency distribute farther laterally than the fate map; and (ii) the extended involution plays an essential role in the specification of the dorsal marginal mesoderm, especially in notochordal differentiation in normal Cynops embryogenesis.     

Regional expression, pattern and timing of convergence and extension during gastrulation of Xenopus laevis

We show with time-lapse micrography that narrowing in the circumblastoporal dimension (convergence) and lengthening in the animal-vegetal dimension (extension) of the involuting marginal zone (IMZ) and the noninvoluting marginal zone (NIMZ) are the major tissue movements driving blastopore closure and involution of the IMZ during gastrulation in the South African clawed frog, Xenopus laevis. Analysis of blastopore closure shows that the degree of convergence is uniform from dorsal to ventral sides, whereas the degree of extension is greater on the dorsal side of the gastrula. Explants of the gastrula show simultaneous convergence and extension in the dorsal IMZ and NIMZ. In both regions, convergence and extension are most pronounced at their common boundary, and decrease in both animal and vegetal directions. Convergent extension is autonomous to the IMZ and begins at stage 10.5, after the IMZ has involuted. In contrast, expression of convergent extension in the NIMZ appears to be dependent on basal contact with chordamesoderm or with itself. The degree of extension decreases progressively in lateral and ventral sectors. Isolated ventral sectors show convergence without a corresponding degree of extension, perhaps reflecting the transient convergence and thickening that occurs in this region of the intact embryo. We present a detailed mechanism of how these processes are integrated with others to produce gastrulation. The significance of the regional expression of convergence and extension in Xenopus is discussed and compared to gastrulation in other amphibians.     

The embryo-forming potency of the posterior marginal zone in stages X through XII of the chick

At stage X a small posterior marginal zone (PM) fragment, when transplanted into similar-size hole in the lateral marginal zone, can initiate the development of an ectopic axis. The laterally transplanted PM, inhibits the regeneration of an axis at the original posterior side from the lateral section of the marginal zone (LM) inserted to replace it. At stage XI both the axis-forming and inhibitory capacities of the PM fragment become weaker and an axis-forming capacity starts to build up anterior to the PM, resulting in the formation of two primitive streaks at 90 degrees to each other. At stage XII the change of potencies exhibited at stage XI is more pronounced, the ability of the transplanted PM to promote axis formation at the new site is lost, and an axis is formed from the original posterior side of the blastoderm.     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

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.     

Ambystoma maculatum gastrulae have an oriented, fibronectin-containing extracellular matrix.

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin-containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastula (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip-animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN-containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN-coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge.     

The anterior extent of dorsal development of the Xenopus embryonic axis depends on the quantity of organizer in the late blastula

In amphibian gastrulae, the cell population of the organizer region of the marginal zone (MZ) establishes morphogenesis and patterning within itself and within surrounding regions of the MZ, presumptive neurectoderm, and archenteron roof. We have tested the effects on pattern of reducing the amount of organizer region by recombining halves of Xenopus laevis late blastulae cut at different angles from the bilateral plane. When regions within 30 degrees of the dorsal midline are excluded from recombinants, ventralized embryos develop lacking the entire anterior-posterior sequence of dorsal structures, suggesting that the organizer is only 60 degrees wide (centered on the dorsal midline) at the late blastula stage. As more and more dorsal MZ (organizer) is included in the recombinant, progressively more anterior dorsal structures are formed. In all cases, when any dorsal structures are missing they are deleted serially from the anterior end. Thus, we suggest that the amount (lateral width) of the organizer in the MZ determines the anterior extent of dorsal development.     

Differential regulation of the chick dorsal thoracic dermal progenitors from the medial dermomyotome

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.     

Competence as the Main Factor Determining the Size of the Neural Plate

A piece of ectoderm was taken from the neural plate area of an early neurula ( Ambystoma mexicanum ) and replaced by competent gastrula ectoderm. Neural tissue was induced in these transplants, and the amount of neural tissue was used to estimate the spreading of the neural inductive stimulus. Dependence was tested of the amount of neural tissue formed in the transplants on the following factors: distance of the transplant from the dorsal midline of the host, age of the host, age of the transplant. The first two factors had no influence on the results but age of the transplant turned out to be decisive. The distance of the transplant from the midline of the host did not influence the amount of neural tissue found in the transplant, even in transplants out side the normal neural plate area neural differentiation was induced.
It is concluded that the spreading of neural induction is not controlled by a gradient but by the loss of neural competence of the ectoderm. A model for the spreading of neural induction is suggested using competence and homoiogentic induction as its main factors.     

Planar and vertical signals in the induction and patterning of the Xenopus nervous system.

The cellular mechanisms responsible for the formation of the Xenopus nervous system have been examined in total exogastrula embryos in which the axial mesoderm appears to remain segregated from prospective neural ectoderm and in recombinates of ectoderm and mesoderm. Posterior neural tissue displaying anteroposterior pattern develops in exogastrula ectoderm. This effect may be mediated by planar signals that occur in the absence of underlying mesoderm. The formation of a posterior neural tube may depend on the notoplate, a midline ectodermal cell group which extends along the anteroposterior axis. The induction of neural structures characteristic of the forebrain and of cell types normally found in the ventral region of the posterior neural tube requires additional vertical signals from underlying axial mesoderm. Thus, the formation of the embryonic Xenopus nervous system appears to involve the cooperation of distinct planar and vertical signals derived from midline cell groups.     

Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early beta-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator beta-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula beta-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.