Spatial-temporal dynamics of morphogenetic blastoderm potencies in early embryogenesis of the loach

The degree of differentiation of axial structures (notochord, neuroectoderm, and somites) in 24-hour explants (a total of 380) of the loach embryonic blastoderm was determined on histological sections according to a developed scale of estimates. Before the beginning of epiboly, axial structures were formed only from fragments of the dorsal sector of the blastoderm marginal zone. Its other sectors acquired the capacity of forming axial structure only with the beginning of epiboly, as the germ ring was formed in the marginal zone, unlike the cells outside the germ ring. The degree of differentiation of axial structures in the dorsal sector of marginal zone increased reliably with the appearance of embryonic shield, i.e. area of the convergence of cell flows. Here, statistically significant regional differences in morphogenetic potencies of the marginal zone first appeared, which corresponded to the differences in prospective significance of its materials; notochord and neuroectoderm better differentiate from the dorsal sector material, while somites better differentiate from the ventral sector material. Thus, distribution of morphogenetic potencies reflects precisely the spatial-temporal dynamics of collective movement of the blastoderm cells during the normal course of morphogenesis.     

Dorsoventral Differences of Morphogenetic Potencies of the Loach Blastoderm in Experiments with Alteration of Mass of Explanted Fragments

We studied the influence of doubling the mass of explanted fragments of the dorsal and ventral loach blastoderm at the early gastrula stage on their capacity for differentiation of axial structures. The dorsoventral differences are as follows: the differentiation of somites correlates, according to the results of factor analysis, with the shape complication only in double dorsal explants, while the notochord is more differentiated in the ventral fragments, if it is present, than in the dorsal ones. Doubling of the mass of dorsal fragments of the blastoderm enhances their morphogenetic potencies and shifts differentiation towards the formation of trunk axial structures. The increased mass of ventral fragments does not affect their differentiation and morphogenesis, but disturbs the correlation of these processes.     

Dorsoventral differences of morphogenetic potencies of the loach blastoderm in experiments with alteration of the mass of explanted fragments

We studied the influence of doubling the mass of explanted fragments of the dorsal and ventral loach blastoderm at the early gastrula stage on their capacity for differentiation of axial structures. The dorsoventral differences are as follows: the differentiation of somites correlates, according to the results of factor analysis, with the shape complication only in double dorsal explants, while the notochord is more differentiated in the ventral fragments, if it is present, than in the dorsal ones. Doubling of the mass of dorsal fragments of the blastoderm enhances their morphogenetic potencies and shifts differentiation towards the formation of trunk axial structures. The increased mass of ventral fragments does not affect their differentiation and morphogenesis, but disturbs the correlation of these processes.     

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

Variability of quantitative morphogenetic parameters during early morphogenesis of the loach, <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L.

Analysis of normal variation in quantitative morphological characters during the early embryonic development of the loach, based on observations on individual developmental trajectories of living embryos, shows that the dorsoventral differentiation of the blastoderm proceeds in two stages. Initially, at the onset of epiboly, the sagittal (short) and transverse (long) blastoderm meridians are marked off, and only then, upon germ ring (GR) formation, differentiation between the opposite poles of the sagittal meridian takes place. The embryonic shield (ES) usually appears in the segment of the blastoderm where the radius of its external curvature reaches a maximum and, therefore, the active surface tension at the blastoderm boundary with the YSL periblast) and yolk is the highest. In this case, the convergence of inner cells toward the future dorsal segment (leading to ES formation) is a mechanical consequence of surface tension anisotropy. The normal course of epiboly is associated with periodic changes in the curvature of the blastoderm external surface, with new structures (the dorsal segment, GR, and ES) are marked off only when the surface curvature becomes maximally uniform. Although the ES in most embryos appears within the initial dorsal segment, individual developmental trajectories have been traced where the GR starts to form at the dorsal pole of the blastoderm but the ES develops on its opposite site, at the point of GR closure. In both cases, GR formation is initiated at the point of convergence of centrifugal cell migration flows that arise in the marginal zone of the blastoderm upon GR initiation or closure.     

Suramin changes the fate of Spemann's organizer and prevents neural induction in Xenopus laevis.

Suramin, a polyanionic compound, which has previously shown to dissociate platelet derived growth factor (PDGF) from its receptor, prevents the differentiation of neural (brain) structures of recombinants of dorsal blastopore lip (Spemann's organizer) and competent neuroectoderm. Furthermore, the suramin treatment changes the prospective differentiation pattern of isolated blastopore lip. While untreated dorsal blastopore lip will differentiate into dorsal mesodermal structures (notochord and somites), suramin treated dorsal blastopore lip will form ventral mesoderm structures, especially heart structures. The results are discussed in the context of the current opinion about the mode of action of different growth factor superfamilies.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation

We identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial mesendoderm such as prechordal plate and notochord. dkk1 expression in the dorso-anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm. dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish.     

Neuronal induction and regional identity by co-culture of adherent human embryonic stem cells with chicken notochords and somites

The role of somites and notochords in neuroectoderm differentiation from the embryonic ectoderm and its subsequent patterning into regional compartments along rostro-caudal and dorso-ventral axes, especially in humans, remains elusive. Here, we demonstrate the co-culture effect of somites and notochords isolated from chicken embryos on the neuronal differentiation and regional identity of an adherent culture of human embryonic stem cells (hESCs). Notochord increased the efficiency and speed of neuronal induction, whereas somites had a weak neuronal inducing effect on hESCs. However, a synergistic effect was not observed when notochords and somites were used together. Moreover, in somite and notochord co-culture groups, hESCs-derived neuronal cells expressed HOXB4, OTX2, IRX3 and PAX6, indicative of dorsal hindbrain and ventral anterior identities, respectively. Our results reveal the influence of embryonic notochord and somite co-culture in providing neuronal induction as well as rostro-caudal and dorso-ventral regional identity of hESCs-derived neuronal cells. This study provides a model through which in vivo neuronal induction events may be imitated.     

Embryogenesis in mouth-breeding cichlids (Osteichthyes,Teleostei) structure and fate of the enveloping layer

The eggs of African mouth-brooders are of unusual size and shape. Studying their development may help to more clearly understand epiboly, gastrulation, and the relation between enveloping layer (periderm) and epidermis. When epiboly has progressed over just one fifth of the yolk mass, the germ ring and embryonic shield are already well established. Behind the germ ring very few deep cells are present at this early stage of epiboly, except in the embryonic shield. When the blastodisc covers the animal half of the yolk mass, the future body is already well established with notochord, somites and developing neural keel. Apart from these structures, no deep cells can be detected between enveloping layer and yolk surface; not even a germ ring remains behind the advancing edge of the enveloping layer. Epiboly over the greater part of the yolk is achieved only by the enveloping layer and the yolk syncytial layer. As the margin of the enveloping layer begins to reduce its circumference when closing around the vegetal pole, groups of cells in the advancing edge become spindle-shaped, with a single cell in between of each of these groups broadening along the edge. The enveloping layer (called periderm after epiboly) remains intact until after hatching, when, together with the underlying ectoderm, it forms the double-layered skin of the larval fish. Thereafter, cells deriving from the subperipheral ectoderm gradually replace the decaying periderm cells to form the final epidermis. Thus, in the cichlids studied, the enveloping layer alone forms the yolk sac to begin with, and it covers the larval body until some days after hatching.     

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

Experimental approaches to the study of the formation of axial structures during early development of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The effect of mechanical extension on the differentiation of axial mesoderm in double explants (sandwiches) of Xenopus laevis embryonic tissues isolated during the early gastrula–late neurula developmen-tal period is studied. In explants at the early gastrula stage, artificial extension orients and stimulates isolated differentiation of the notochord and somites as well as their joint formation. Moreover, extension facilitated the formation of the normal anatomical structure of the notochord and affected expression of Chordin gene. At the late gastrula stage, the effect of artificial extension on joint somite–notochord differentiation was weaker. At the stage of late neurula, somites were sometimes formed in explants lacking a notochord anlage. Thus, at earlier stages, the formation of somites was stimulated by contacts with the notochord and joint development of both structures was mechanical dependent, while at the later stages, somites developed inde-pendently of the notochord. Thus, the role of tissue extension is primarily the establishment of normal mor-phology and expression of Chordin was located in the direction of extension.     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in Xenopus laevis embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90 degrees rotation, (5) pi-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90° rotation, (5) Π-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

The influence of axial structures on chick somite formation

The influence of the axial structures on somite formation was investigated by culturing, on a nutritive agar substrate, segmental plates from chick embryos having 8 to 20 pairs of somites. In the first set of experiments, segmental plate was explanted together with adjacent notochord and approximately the lateral halves of the neural tube and node region. These explants formed 18 to 20 somites within 30 hr. In a second series of experiments, the notochord and neural tube were included as before, but further regression movements in the explants were prevented by removing the node region. These explants formed only 11.9 ± 1.1 somites. Finally, explants of segmental plate that included no neural tube, notochord, or node region were made. These explants had formed 10.7 ± 1.1 somites 14 to 17 hr later. When such explants were cultured for periods longer than 17 hr, there was a marked tendency for the more posterior somites to disperse and for all of the somites to develop a peculiar “hollow” morphology. It was concluded from these results that during the period of development when chick embryos possess 8 to 20 pairs of somites, the segmental plate mesoderm (1) represents about 12 prospective somites, (2) may segment into its full complement of somites without further contact with the axial structures, but (3) requires continued intimate contact with the axial structures for normal somite morphologic differentiation and stability.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis .
Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells.
These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.