BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.     

Connexin43 expression during Xenopus development

The polypyrimidine tract binding protein (PTB) and its recently discovered homologue brain-enriched PTB (brPTB) are RNA binding proteins involved in the control of alternative splicing. We have characterized expression patterns of the PTB and brPTB in course of mouse brain development, using mRNA in situ hybridization. PTB is expressed in choroid plexi and ependyma at all the stages of development and temporarily in the mantle layer of migrating neuroblasts of fore-, mid- and hindbrain and in the external granular layer of cerebellum. In the neurons of adult mouse cerebrum and cerebellum expression of PTB is undetectable. In contrast to this, brPTB is expressed ubiquitously in neuroblasts of various parts of embryonic brain and in the differentiated neurons of postnatal cerebrum and cerebellum. brPTB mRNA is not observed in choroid plexi and ependymal layer. Thus, in the embryonic brain expression patterns of PTB and brPTB overlap, but in the course of brain development the patterns become complementary to each other.     

Xenopus brain factor-2 controls mesoderm, forebrain and neural crest development

The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurula forebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.     

The role of cell mixing in branchial arch development

Compartmental structures are the basis of a number of developing systems, including parts of the vertebrate head. One of the characteristics of a series of compartments is that mixing between cells in adjacent units is restricted. This is a consequence of differential chemoaffinity between neighbouring cells in adjacent compartments. We set out to determine whether mesenchymal cells in the branchial arches and their precursors show cell-mixing properties consistent with a compartmental organisation. In chimaeric avian embryos we found no evidence of preferential association or segregation of neural crest cells when surrounded by cells derived from a different axial level. In reassociation assays using mesenchymal cells isolated from chick branchial arches at stage 18, cells reformed into clusters without exhibiting a preferential affinity for cells derived from the same branchial arch. We find no evidence for differential chemoaffinity in vivo or in vitro between mesenchymal cells in different branchial arches. Our findings suggest that branchial arch mesenchyme is not organised into a series of compartments.     

The expression of the imprinted gene Ipl is restricted to extra-embryonic tissues and embryonic lateral mesoderm during early mouse development

Genes with restricted expression within the developing embryo represent valuable tools as they allow distinct tissue types to be distinguished and studied. In order to identify genes that are expressed within a particular germ layer, a differential screen was performed using germ layer-specific cDNA libraries derived from gastrulation stage mouse embryos. The gene expression profiles of the germ layers were compared following the hybridisation of some 20,000 cDNA clones with probes derived from germ layer-specific Ectoderm, Mesoderm and Endoderm libraries. A cDNA clone (50c15) was identified that hybridised with the Mesoderm-derived probe but not Ectoderm or Endoderm. 50c15 derives from Ipl/Tssc3/BWR1C, an imprinted gene which in human maps to chromosome 11p15.5. This region has been associated with Beckwith-Weidemann Syndrome, Wilms' tumour and ovarian, breast and lung cancer. In the gastrulating mouse embryo, wholemount RNA in situ hybridisation revealed that Ipl expression is restricted not only to the mesodermal germ layer, but specifically to lateral mesoderm and the most posterior extent of the primitive streak from which lateral and extra-embryonic mesoderm is derived. Moreover, Ipl is expressed in extra-embryonic tissues prior to gastrulation and afterwards in extra-embryonic mesoderm, ectoderm and endoderm. This expression profile indicates that Ipl is a good molecular marker for embryonic mesoderm and extra-embryonic tissues. In addition heterotopic grafting studies indicate that nascent mesoderm, which expresses Ipl, is restricted in its potential and therefore may be committed to its fate.     

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish

Background

Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

Methodology/Principal Findings

In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp ⁺ cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli ⁺ etsrp ⁺ hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp ⁺ cell numbers at the LPM.

Conclusions/Significance

This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.     

Spatiotemporal expression of Prdm genes during Xenopus development

Epigenetic regulation is known to be important in embryonic development, cell differentiation and regulation of cancer cells. Molecular mechanisms of epigenetic modification have DNA methylation and histone tail modification such as acetylation, phosphorylation and ubiquitination. Until now, many kinds of enzymes that modify histone tail with various functional groups have been reported and regulate the epigenetic state of genes. Among them, Prdm genes were identified as histone methyltransferase. Prdm genes are characterized by an N-terminal PR/SET domain and C-terminal some zinc finger domains and therefore they are considered to have both DNA-binding ability and methylation activity. Among vertebrate, fifteen members are estimated to belong to Prdm genes family. Even though Prdm genes are thought to play important roles for cell fate determination and cell differentiation, there is an incomplete understanding of their expression and functions in early development. Here, we report that Prdm genes exhibit dynamic expression pattern in Xenopus embryogenesis. By whole mount in situ hybridization analysis, we show that Prdm genes are expressed in spatially localized manners in embryo and all of Prdm genes are expressed in neural cells in developing central nervous systems. Our study suggests that Prdm genes may be new candidates to function in neural cell differentiation.     

Cyclic expression of esr9 gene in Xenopus presomitic mesoderm

During somitogenesis, the cycling expression of members of the Notch signalling cascade is involved in a segmentation clock that regulates the periodic budding of somites in chicken, mouse, and zebrafish. In frog, genes with cycling expression in the presomitic mesoderm have not been reported. Here, we describe the expression of Xenopus esr9 and esr10, two new members of the Hairy/Enhancer of split related family of bHLH proteins. We show that they are expressed in a highly dynamic fashion, with their mRNA levels oscillating periodically in the presomitic mesoderm during somitogenesis. This dynamic expression is independent of de novo protein synthesis. Thus, expression of esr9 and esr10 is an indicator of the segmentation clock in the amphibian embryo. This confirms the evolutionary conservation of a molecular pathway involved in vertebrate segmentation clock.     

Constitutive over-expression of VEGF results in reduced expression of Hand-1 during cardiac development in Xenopus

During heart development, various signaling cascades are tightly regulated in a stage- and region-dependent manner. Vascular endothelial growth factor (VEGF) is one of the important molecules required for both vascular development and cardiac morphogenesis. VEGF receptors are present in the embryonic heart, so we focused on heart formation in VEGF-over-expressing Xenopus embryos. Over-expression of VEGF(170) caused disorganized vessels, while the expression of an endothelial marker, Tie-2, was increased. The embryo's heart was distinctly larger than that of control, and showed abnormal morphology. Histological analysis of these embryos showed failure of heart looping. In situ hybridization with Hand-1, which controls intrinsic morphogenetic pathways, revealed that the expression level of Hand-1 was decreased in the heart region. These results suggest that increased VEGF(170) levels disturb Hand-1 expression in the region required for normal heart morphogenesis. VEGF expression level may be important in heart morphology during embryonic development.     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.