VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 14-29.     

Wnt5 signaling in vertebrate pancreas development

Background  

Signaling by the Wnt family of secreted glycoproteins through their receptors, the frizzled (Fz) family of seven-pass transmembrane proteins, is critical for numerous cell fate and tissue polarity decisions during development.     

Frodo proteins: modulators of Wnt signaling in vertebrate development

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.     

A conserved role for retinoid signaling in vertebrate pancreas development

Retinoic acid (RA) signaling plays critical roles in the regionalization of the central nervous system and mesoderm of all vertebrates that have been examined. However, to date, a role for RA in pancreas and liver development has only been demonstrated for the teleost zebrafish. Here, we demonstrate that RA signaling is required for development of the pancreas but not the liver in the amphibian Xenopus laevis and the avian quail. We disrupted RA signaling in Xenopus tadpoles, using both a pharmacological and a dominant-negative strategy. RA-deficient quail embryos were obtained from hens with a dietary deficiency in vitamin A. In both species we found that pancreas development was dependent on RA signaling. Furthermore, treatment of Xenopus tadpoles with exogenous RA led to an expansion of the pancreatic field. By contrast, liver development was not perturbed by manipulation of RA signaling. Taken together with our previous finding that RA signaling is necessary and sufficient for zebrafish pancreas development, these data support the hypothesis that a critical role for RA signaling in pancreas development is a conserved feature of the vertebrates.     

Molecular signaling in zebrafish development and the vertebrate phylotypic period

SUMMARY During development vertebrate embryos pass through a stage where their morphology is most conserved between species, the phylotypic period (approximately the pharyngula). To explain the resistance to evolutionary changes of this period, one hypothesis suggests that it is characterized by a high level of interactions. Based on this hypothesis, we examined protein–protein interactions, signal transduction cascades and miRNAs over the course of zebrafish development, and the conservation of expression of these genes in mouse development. We also investigated the characteristics of genes highly expressed before or during the presumed phylotypic period. We show that while there is a high diversity of interactions during the phylotypic period (protein–DNA, RNA–RNA, cell–cell, and between tissues), which is well conserved with mouse, there is no clear difference with later, more morphologically divergent, stages. We propose that the phylotypic period may rather be the expression at the morphological level of strong conservation of molecular processes earlier in development.     

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.     

Wnts and wing: Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis

In the past twenty years, secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development from hydra to human. In the developing vertebrate limb, Wnt signaling is required for limb bud initiation, early limb patterning (which is governed by several well-characterized signaling centers), and, finally, late limb morphogenesis events. Wnt ligands are unique, in that they can activate several different receptor-mediated signal transduction pathways. The most extensively studied Wnt pathway is the canonical Wnt pathway, which controls gene expression by stabilizing beta-catenin in regulating a diverse array of biological processes. Recently, more attention has been given to the noncanonical Wnt pathway, which is beta-catenin-independent. The noncanonical Wnt pathway signals through activating Ca(2+) flux, JNK activation, and both small and heterotrimeric G proteins, to induce changes in gene expression, cell adhesion, migration, and polarity. Abnormal Wnt signaling leads to developmental defects and human diseases affecting either tissue development or homeostasis. Further understanding of the biological function and signaling mechanism of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. This review provides a critical perspective on how Wnt signaling regulates different developmental processes. As Wnt signaling in tumor formation has been reviewed extensively elsewhere, this part is not included in the review of the clinical significance of Wnt signaling.     

VEGF signaling has distinct spatiotemporal roles during heart valve development

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.     

FGF-FGFR signaling in vertebrate organogenesis.

Fibroblast growth factor and FGF receptor (FGFR) system play significant roles in many biological events including pattern formation in many tissues during vertebrate embryogenesis at early stages. The functions of each of FGFs and their receptors have recently been revealed by a variety of approaches among species. We have recently generated FGF10-deficient mice by gene targeting. This KO mice had complete truncation of the fore-and hindlimbs and with no lung. Analyses of the embryos and marker gene expression showed that FGF10 triggers sequential events, which are essential for formations of limb and lung. Focusing on FGF10 function, the FGF-FGFR system is discussed.     

MicroRNAs in vertebrate development

The vertebrate genome contains hundreds of small non-coding 'microRNAs' that have been implicated in controlling the expression of potentially thousands of target genes. Presently, only a handful of these targets have been characterized. Recent reports of microRNA 'sensors', microRNA microarrays and the creation of vertebrates that lack all microRNA activity will aid in determining the roles played by microRNAs, and the genes that they regulate, during vertebrate development.     

Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development

The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. Here we report that PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-beta-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. We conclude that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to beta-catenin, possibly via targets other than Dishevelled.