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Introduction: There is increasing evidence that the endothelium is able to transduce circulatory force into signals which influence angiogenic behaviour. The Von Hippel-Lindau homozygous mutant zebrafish (vhl-/-) has increased HIF-1α signalling and aberrant angiogenesis. In this study I therefore set out to characterise the angiogenic phenotype of the vhl-/- mutant and examine the effect of manipulating blood flow. Both nitric oxide (NO) and Notch regulated intracellular signalling have been shown to influence endothelial angiogenic behaviour in development and disease, so I studied the role of these on development of the angiogenic phenotype in the vhl-/- mutant. Hypothesis: Does angiogenesis stimulated by up-regulated hypoxic signalling require blood flow? Methods: vhl mutant zebrafish were crossed with endothelial reporter transgenics to assess and quantify the angiogenic phenotype. Blood flow was manipulated using genetic and pharmacological manipulation in developing zebrafish embryos. To study the effect of flow on endothelial notch signalling I utilised a novel transgenic line Tg(CSL:venus), which expresses a fluorescent reporter upon activation of a Notch regulated transcription factor. The role of individual notch signalling components was investigated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for various ligands in developing zebrafish tissue during development in the absence of flow and in control embryos. Results: The vhl-/- mutant showed early vessel changes including enlargement and sprouting. These angiogenic vessel changes were dependent on the presence of circulatory flow and are lost in mutant embryos treated with troponin t2 morpholino or 2,3-Butanedione monoxime (BDM). Imaging of aortic endothelium showed a significant up regulation of notch signalling in the Tg(CSL:venus) line in the absence of flow, induced by troponin t2 knockdown or BDM treatment. This increase in endothelial notch signalling in the absence of flow was mediated by increased expression of the notch ligand dll4 in RT-qPCR and whole mount in-situ hybridisation experiments. Conclusion: The aberrant angiogenesis stimulated by increased HIF-1α signalling in the vhl-/- mutant is dependent on blood flow. My data suggests a novel flow dependent regulation of notch signalling in the developing vasculature mediated by the suppression of dll4 by blood flow.

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Haematopoietic stem cells (HSCs) are multipotent progenitors that can self-renew and give rise to cells of all blood lineages. In vertebrates, HSCs are specified during embryogenesis from haemogenic endothelial cells (HECs) in the ventral wall of the dorsal aorta (vDA). Previously, a transposon-based gene trap approach was used in zebrafish to identify a transgenic line called qmc551 that expresses a GFP reporter gene in primitive red blood cell (prRBC) progenitors and in HECs of the vDA. In this line, GFP expression is retained as the cells undergo endothelial to haematopoietic transition and seed sites of larval and adult haematopoiesis. In the adult kidney marrow, GFP expression is found in progenitors, lymphocytes and myelomonocytes. The transposon that encodes qmc551:GFP is located in intron 1 of gfilaa, one of three zebrafish gfil paralogs along with gfilab and gfilb. In this study, it was shown that qmc551 :GFP is expressed from a spliced transcript that carries the GFP reading frame downstream of exon 1 of gfilaa, demonstrating that GFP expression is under the control of the gfilaa promoter. Consequently, GFP recapitulates gfilaa expression in development. The presence of the transposon in intron 1 interferes with normal splicing of the gfilaa transcript leading to a loss of gfilaa mRNA in primitive and definitive haematopoietic cells as shown in WISH and qRT-PCR experiments. Gfilaa depletion is compatible with the normal development of prRBC which retain the expression of the paralogue, Gfilb. Morpholino-mediated depletion of Gfilb in homozygous qmc551 embryos blocked prRBC maturation, leading to a severe reduction in haemoglobinization. This phenotype is rescued by Gfilaa in a dose dependent manner as morphant embryos carrying one or two wt copies of gfilaa have increasingly normal levels of haemoglobin. This shows that Gfilaa and Gfilb play essential redundant roles in prRBC maturation. During the onset of definitive haematopoiesis, gfilaa expression is induced downstream of Vegf and Notch signalling and is parallel to Runxl in HECs of the vDA. Its depletion in qmc551 homozygous embryos does not interfere with HSC specification or maintenance which is likely due to the Runxl dependent upregulation of its paralog, gfilab, in the vDA. Interestingly, the loss of Gfilb in qmc551 homozygous embryos leads to the hyperproliferation of haematopoietic stem and progenitor cells (HSPCs) in the caudal haematopoietic tissue (CHT), suggesting that these genes, like their mammalian counterparts, are involved in the maintenance of HSPC quiescence. Together, results presented here show that, in contrast to previously published morphant data, zebrafish Gfil factors and their mammalian homologs play functionally conserved roles during primitive and definitive haematopoiesis.

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