Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development

Bone morphogenetic protein (Bmp) signaling is crucial for the formation and patterning of zebrafish ventral and posterior mesoderm. Mutants defective in the Bmp pathway have expanded trunk muscle, abnormal tails and severely impaired development of ventral mesodermal derivatives such as vasculature, blood and pronephros. As Bmps continue to be expressed in the ventral and posterior mesoderm after gastrulation, it is likely that Bmp signaling continues to play an important developmental role during outgrowth of the posterior body. However, because Bmp signaling plays an essential role during the gastrula stages, it has not been possible with mutants or standard disruption techniques to determine the later functions of the Bmp pathway. To study the role of Bmp signaling in the ventral and posterior mesoderm during trunk and tail outgrowth, we generated a transgenic zebrafish line containing a heatshock-inducible dominant-negative Bmp receptor-GFP fusion. Our data show that Bmps are important for tail organizer formation and for patterning the ventral mesoderm during early gastrulation. However, from mid-gastrulation to the early somitogenesis stages, Bmp signaling is important for ventral tail fin development and for preventing secondary tail formation. We conclude that the role of Bmp signaling in the ventral and posterior mesoderm changes as gastrulation proceeds.     

eFGF regulates Xbra expression during Xenopus gastrulation.

We show that, in addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. We show that during blastula stages eFGF and Xbra are able to activate the expression of each other, suggesting that they are components of an autocatalytic regulatory loop. Moreover, we show that Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

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

Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis

During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.     

The bases for and timing of regional specification during larval development in Phoronis

A fate map has been constructed for Phoronis vancouverensis. The animal pole of the egg gives rise to the apical plate in the hood of the actinotroch larva. The vegetal pole of the egg marks the site of gastrulation. During the initiation of gastrulation the cells of the animal pole of the embryo are directly opposite those at the vegetal pole of the embryo. The plane of the first cleavage always goes through the animal-vegetal pole of the egg. In about 70% of the cases the plane of the first cleavage is perpendicular to the future anterior-posterior axis of the actinotroch larva; in the remaining cases the plane of the first cleavage is either oblique with reference to, or occurs along, the future anterior-posterior axis of the larva. Following gastrulation catecholamine-containing cells first make their appearance in the apical plate and gut cells first produce esterase. The timing of regional specification in these embryos has been examined by isolating animal or vegetal, anterior or posterior, or lateral regions at different time periods between the initiation of cleavage and gastrulation and examining their ability to differentiate. Animal halves isolated from early cleavage through late blastula stages do not gastrulate and do not form catecholamine-containing cells. When animal halves are isolated with endoderm during gastrulation, they differentiate catecholamine-containing cells. Vegetal halves isolated at the 8- to 16-cell stage gastrulate and form normal actinotroch larvae with esterase-positive gut and catecholamine-containing apical plate cells. When this same region is isolated at blastula stages it does not gastrulate and does not differentiate these cell types. Vegetal halves isolated during gastrulation subsequently form esterase-positive gut cells, but they do not form catecholamine-containing apical plate cells. When presumptive anterior, posterior, or lateral halves are isolated from early cleavage through blastula stages, each half forms a normal actinotroch larva. Lateral halves isolated during gastrulation also form normal larvae. Anterior halves isolated during late gastrulation differentiate only the anterior end of the actinotroch larva. These isolates have a hood with catecholamine-containing apical plate cells and the first part of an esterase-positive gut but lack the anlagen of the intestine and protonephridia. Posterior halves isolated during late gastrulation differentiate only the posterior end of the actinotroch which lacks a hood with catecholamine-containing cells but has an esterase-positive gut, protonephridia, and the anlagen of the intestine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Domains of movement in the zebrafish gastrula

The mechanisms controlling cell movements during vertebrate gastrulation are not known. Studies using the zebrafish embryo show promise at identifying these mechanisms, combining an embryo that is accessible and optically clear with mutations that affect early development. In this article we describe the movements of cells during the midblastula, early epiboly and gastrulation stages of the zebrafish, correlating 'domains of movement' with embryonic morphology. We suggest that these domains of movement may parallel the 'zones of movement' of Xenopus.     

The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.     

Xoom is maternally stored and functions as a transmembrane protein for gastrulation movement in Xenopus embryos

Xoom has been identified as a novel gene that plays an important role in gastrulation of Xenopus laevis embryo. Although Xoom is actively transcribed during oogenesis, distribution and function of its translation product have not yet been clarified. In the present study, the polyclonal antibody raised against Xoom was generated to investigate a behavior of Xoom protein. Anti-Xoom antibodies revealed that there are two forms of Xoom protein in Xenopus embryos: (i) a 45 kDa soluble cytoplasmic form; and (ii) a 44 kDa membrane-associated form. Two forms of Xoom protein were ubiquitously detected from unfertilized egg to tadpole stage, with a qualitative peak during blastula and gastrula stages. Immunohistochemical examination showed that Xoom protein is maternally stored in the animal subcortical layer and divided into presumptive ectodermal cells during cleavage stages. Enzymatic digestion of membrane protein and immunologic detection of Xoom showed that Xoom exists as a membrane-associated protein. To examine a function of Xoom protein, anti-Xoom antibodies were injected into blastocoele of stage 7 blastula embryo. Anti-Xoom antibodies caused gastrulation defect in a dose- dependent manner. These results suggest that maternally prepared Xoom protein is involved in gastrulation movement on ectodermal cells.     

Histones and histone acetylation during the embryonic development of Drosophila hydei

Histones and histone acetylation have been investigated during three stages of Drosophila hydei embryogenesis--early gastrula, late gastrula and organogenesis. No essential changes in the electrophoretic pattern of the histones have been revealed during the stages examined. However, we established an enhanced level of [14C]acetate incorporation at the time of extensive gene activation during gastrulation as well as some quantitative differences in the pattern of acetylation during gastrula and organogenesis. We consider most of them to be related to chromatin assembly during the stage of gastrulation and suggest that the correlation between histone acetylation and gene activity during Drosophila embryogenesis concerns histone H3 acetylation. The involvement of both acetylation and deacetylation in the steady-state acetylation level has been examined as well. We have found that the higher acetyltransferase activity is responsible for the enhanced level of acetate incorporation during gastrulation.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.     

Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.     

INTER AND INTRATISSUE COMMUNICATION DURING AMPHIBIAN DEVELOPMENT*

The changes of electrical communication between various tissues of the newt (Cynops pyrrhogaster) embryo during development have been investigated by measuring electrotonic potentials at various interelectrode distances. In general, cells of the same tissue are electrically coupled from gastrulation up to closure of the neural tube. Notochordal cells, however, are an exception in that cell coupling decreases during stages 22–23 in comparison to earlier stages. Neuroectoderm cells are coupled to adjacent chorda-mesoderm cells during the initial stages of gastrulation (st. 12c). Subsequently coupling of these tissues diminishes (st. 15–16) and finally disappears (st. 22–23). The similar decrease of coupling was observed in inter-tissues of the chorda-mesoderm cells and the somitic mesoderm cells during the mesodermal differentiation. In contrast, coupling values of less than 0.1 recorded between somite cells and cells of the neural tube or epidermis still remain at st. 22–23. The neural plate cells remain coupled to the lateral ectoderm cells at st. 18 and then become insulated from the epidermis by st. 22–23, even though a coupling ratio of 0.1 remains between these tissues. These developmental patterns of coupling are discussed with respect to cellular movements of neuroectoderm and mesoderm during gastrulation, and with special reference to neural competence.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. © 1995 Wiley-Liss, Inc.     

When does the anterior endomesderm meet the anterior-most neuroectoderm during Xenopus gastrulation?

During amphibian gastrulation, the anterior endomesoderm is thought to move forward along the inner surface of the blastocoel roof toward the animal pole where it comes into physical contact with the anterior-most portion of the prospective head neuroectoderm (PHN), and it is also believed that this physical interaction occurs during the mid-gastrula stage. However, using Xenopus embryos we found that the interaction between the anterior endomesoderm and the PHN occurs as early as stage 10.25 and the blastocoel roof ectoderm at this stage contributed only to the epidermal tissue. We also found that once the interaction was established, these tissues continued to associate in register and ultimately became the head structures. From these findings, we propose a new model of Xenopus gastrulation. The anterior endomesoderm migrates only a short distance on the inner surface of the blastocoel roof during very early stages of gastrulation (by stage 10.25). Then, axial mesoderm formation occurs, beginning dorsally (anterior) and progressing ventrally (posterior) to complete gastrulation. This new view of Xenopus gastrulation makes it possible to directly compare vertebrate gastrulation movements.