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.     

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.     

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.     

Developmental analysis of fs(1)gastrulation defective,a dorsal-group gene of Drosophila melanogaster

Summary The gastrulation defective (gd) locus is a maternally expressed gene in Drosophila required for normal differentiation of structures along the embryonic dorso-ventral axis. Cuticular defects of the offspring from females with different combinations of gd alleles comprised a phenotypic continuum. Complementation among several alleles produced normal offspring while progressively more severe mutations produced a graded loss of structures from ventral, and then lateral, blastoderm cells. The most severely affected embryos consisted entirely of structures derived from dorsal blastoderm cells. Histological examination of staged siblings from selected allelic combinations showed that internal tissues were similarly affected. The tissues observed in amorphic embryos support new, more dorsal, assignments of fate map positions for blastoderm precursors of the cephalopharyngeal apparatus, hindgut and ventral nerve cord. The loss of ventral and lateral structures did not occur through cell death and appeared to involve a change in blastoderm cell fate. A direct effect of the mutations on blastoderm cell determination, however, was insufficient to explain the development of the dorsalized embryos. Intermediate phenotypes suggested that cell interactions or movements associated with morphogenesis are required for the determination of some cell fates in the dorsoventral axis. Thus, the developmental fate of all blastoderm cells may not be fixed at the time of blastoderm formation.     

Development of Loach Eggs after Experimental Increase of Cell Mass in the Dorsal and Ventral Areas of Blastoderm

The fertilized loach eggs were injected, before the beginning of cleavage, with the nuclear dye Hoechst 33258 and left to develop until the late blastula stage. Some cells of the dorsal area of stained blastoderm were transplanted in the analogous area of intact embryos of the same age, which led to an earlier and more pronounced development of head and trunk structures in recipients. A relationship was established between specific features of the development of recipients and localization of descendants of the transplanted cells. Transplantation of cells of the dorsal area of stained blastoderm in the ventral area of embryos of the same age led to the formation of two axial complexes, both at the same level of development, but behind the control, and stained cells were located predominantly in one of twin embryos.     

Development of loach eggs after experimental increase of cell mass in the dorsal and ventral areas of blastoderm

The fertilized loach eggs were injected, before the beginning of cleavage, with the nuclear dye Hoechst 33258 and left to develop until the late blastula stage. Some cells of the dorsal area of stained blastoderm were transplanted in the analogous area of intact embryos of the same age, which led to an earlier and more pronounced development of head and trunk structures in recipients. A relationship was established between specific features of the development of recipients and localization of descendants of the transplanted cells. Transplantation of cells of the dorsal area of stained blastoderm in the ventral area of embryos of the same age led to the formation of two axial complexes, both at the same level of development, nut behind the control, and stained cells were located predominantly in one of twin embryos.     

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.     

Dorsal specification in blastoderm at the blastula stage in the goldfish, Carassius auratus

The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180°, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed: the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore, the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.     

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.     

Origin and organization of the zebrafish fate map

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.     

Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus

Dorsal or ventral blastomeres of the 16- and 32-cell stage animal hemisphere were labeled with a lineage dye and transplanted into the position of a ventral, vegetal midline blastomere. The donor blastomeres normally give rise to substantial amounts of head structures and central nervous system, whereas the blastomere which they replaced normally gives rise to trunk mesoderm and endoderm. The clones derived from the transplanted ventral blastomeres were found in tissues appropriate for their new position, whereas those derived from the transplanted dorsal blastomeres were found in tissues appropriate for their original position. The transplanted dorsal clones usually migrated into the host's primary axis (D1.1, 92%; D1.1.1, 69%; D1.1.2, 100%), and in many cases they also induced and populated a secondary axis (D1.1, 43%; D1.1.1, 67%; D1.1.2, 63%). Bilateral deletion of the dorsal blastomeres resulted in partial deficits of dorsal axial structures in the majority of cases, whereas deletions of ventral midline blastomeres did not. When the dorsal blastomeres were cultured as explants they elongated. Notochord and cement glands frequently differentiated in these explants. These studies show that the progeny of the dorsal, midline, animal blastomeres: (1) follow their normal lineage program to populate dorsal axial structures after the blastomere is transplanted to the opposite pole of the embryo; (2) induce and contribute to a secondary axis from their transplanted position in many embryos; (3) are important for the normal formation of the entire length of the dorsal axis; and (4) autonomously differentiate in the absence of exogenous growth factor signals. These data indicate that by the 16-cell stage, these blastomeres have received instructions regarding their fate, and they are intrinsically capable of carrying out some of their developmental program.     

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.     

The role of tensile fields and contact cell polarization in the morphogenesis of amphibian axial rudiments

Summary The role of stretching-generated tensile stresses upon the organization of axial rudiments have been studied. Pieces of the dorsal wall ofXenopus laevis andRana temporaria embryos at the late gastrula stage were rotated through 90°, transplanted into the field of neurulation tensions of another embryo and replaced by ventral tissues already insensitive to inductive influences. The axial rudiments which developed from rotated and transplanted dorsal tissues oped from rotated and transplanted dorsal tissues almost completely reorientated according to the tensile patterns in adjacent host tissues. Some of the donor cells changed their presumptive fates in accordance with their new positions in the host tensile field. Transplanted ventral tissues were involved in the morphogenetic movements specific for the dorsal regions and imitated some typical dorsal structures. In the regions without pronounced tensions the structure of transplanted axial rudiments was chaotic. It is suggested that the organization of the axial structures is established and maintained by tensile fields created by uniformly polarized cells. Cell polarization can be transmitted by contact from host to donor tissues. The specificity of this propagating process and its morphogenetical role is discussed.     

Determination During Early Embryogenesis in Drosophila melanogaster

Ligation of developing embryos of Drosophila melanogaster wasperformed at three different stages of nuclear multiplicationand at the cellular blastoderm stage. Egg fragments of variablesizes are able to continue development up to the hatching stage.Partial embryos differentiate larval structures, anterior fragmentsforming larval head and posterior fragments larval abdominalstructures. These fragments differentiate a variable numberof the twelve larval cuticular bands formed by intact embryos.We found that ligation at cellular blastoderm can lead to anteriorand posterior fragments which differentiate together all thetwelve bands, indicating that at this stage the embryo developsthese patterns in a mosaic fashion. Ligation of younger embryosprevents the differentiation of some intermediate larval cuticularbands, while the terminal ones are consistently differentiated.The number and position of the deleted bands is correlated withthe time and position of ligation. This indicates that the mosaicpattern present in the egg at blastoderm is not fully formedat earlier stages in development.     

A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis

In Xenopus laevis, dorsal cells that arise at the future dorsal side of an early cleaving embryo have already acquired the ability to cause axis formation. Since the distribution of cytoplasmic components is markedly heterogeneous in an egg and embryo, it has been supposed that the dorsal cells are endowed with the activity to form axial structures by inheriting a unique cytoplasmic component or components localized in the dorsal region of an egg or embryo. However, there has been no direct evidence for this. To examine the activity of the cytoplasm of dorsal cells, we injected cytoplasm (dorsal cytoplasm) from dorsal vegetal cells of a Xenopus 16-cell embryo into ventral vegetal cells of a simultaneous recipient. The cytoplasm caused secondary axis formation in 42% of recipients. Histological examination revealed that well-developed secondary axes included notochord, as well as a neural tube and somites. However, injection of cytoplasm of ventral vegetal cells never caused secondary axis and most recipients became normal tailbud embryos. Furthermore, about two-thirds of ventral isolated halves injected with dorsal cytoplasm formed axial structures. These results show that dorsal, but not ventral, cytoplasm contains the component or components responsible for axis formation. This can be the first step towards identifying the molecular basis of dorsal axis formation.     

Developmental effects of monensin on Drosophila melanogaster

Extracellular matrix and membrane proteins and their correct secretion probably are key elements in morphogenesis and differentiation in Drosophila. In this study, we have analysed the effects of monensin, a Na⁺-H⁺-ionophore which blocks normal secretion, applied during cellular blastoderm formation on further development. Normal cell morphology and intercellular contacts are lost and the extracellular matrix becomes disorganized. Gastrulation is blocked and abnormal foldings can be observed. Cuticle phenotypes showed different degrees of ventral, dorsal, head and posterior defects. The results are discussed in the context of what is known about membrane and extracellular matrix proteins in Drosophila.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

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.     

Cell proliferation and protein synthesis as initial factors in determination of axial polarity

The rate of cell proliferation relative to that of protein synthesis appears to have an initial role in establishment of axial polarities in developing animal embryos. An increase in this ratio leads to anterior or dorsal differentiation, while reduction allows posterior or ventral differentiation in a number of organisms. The role that various growth factors play in the regulation of proliferation and protein synthesis is examined.     

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.