Radar/Gdf6a function during zebrafish (Danio rerio)embryogenesis

Abstract

In the early vertebrate embryo, the vascular system is one of the first organ systems to form. Recently it has become evident that the development of mature, functional vessels requires not only signals derived from the endothelium itself, but a set of additional molecules that are not necessarily endothelium-specific. In zebrafish and Xenopus embryos two such tissues that are believed to secrete modulators of blood vessel assembly are the hypochord and primitive gut endoderm (PGE). These tissues intimately border the major axial vasculature. Radar/Growth/differentiation factor 6a (Gdf6a) represents a signalling molecule belonging to the GDF5, 6, 7 subgroup of the transforming growth factor-beta (TGF-β) superfamily. In the zebrafish, transcripts for radar are located in the hypochord, PGE and ventral tail mesenchyme (VTM), all tissues that border the developing axial blood vessels. This prompted an investigation into a potential role for this signalling bone morphogenetic protein (BMP) during the specification and assembly of the closely related vascular and haematopoietic systems in the zebrafish. Transient forced expression experiments confirmed an early ventralising activity for the Radar signal that resulted in the expansion of the haematopoietic/vascular compartment, known as the intermediate cell mass (ICM). However, a loss-of-function zebrafish model generated using morpholino technology demonstrated a critical requirement for this BMP signal in establishing the integrity of the axial blood vessels. Furthermore, this requirement was independent of the initial establishment of vascular patterning. Zebrafish embryos depleted of the Radar signal initiate a normal primitive circulation. However, soon after this commencement of normal flow, blood cells were observed to extravasate from the axial vasculature. Microangiography confirmed this leakage phenotype. Such an angiogenic/maturation role for Radar during vascular development was supported by a transgenic zebrafish line carrying an inducible copy of the radar gene. Homozygous transgenic embryos established a typical early circulation that became progressively restricted until no blood travelled throughout the entire embryonic tissue. In summary, the work presented in this thesis strongly suggests that Radar is involved in a signalling pathway required for establishing the integrity of the axial vessels during zebrafish development.