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The cardiovascular system (CVS) consists of heart and blood vessels, forming a close circulatory loop. All tissues depend on the nutrients and molecular oxygen (O2) delivered by the blood. Therefore, it is not surprising that the CVS is one of the first working systems and the heart is the first functional organ in the forming embryo (Baldwin 1996). The building blocks of blood vessels are endothelial cells (ECs), which form the endothelium, a specialized epithelium that defines the luminal surface of the vessels (Pugsley and Tabrizchi 2000). The process of blood vessel development comprises several steps. The first events occurring are the formation of new vessels de novo to constitute the primary vascular loop known as vasculogenesis. During vasculogenesis the vascular precursors, known as angioblasts, migrate and coalesce to form the axial vessels. Subsequently, the main vessels undergo a specification step where they acquire either arterial or venous identity. As the embryo increases in size, the main vascular loop needs to increase in complexity. In order to reach all the different parts of the developing organs, new blood vessels are formed from pre-existing ones, a phenomenon known as angiogenesis (Gore et al. 2012).
Mature blood cells have a short lifespan. Therefore, hematopoietic stem cells (HSCs) are required throughout lifetime to constantly form new blood cells in a process called hematopoiesis. Interestingly, endothelial and immune cells development have been shown to converge at different points during their development, one of which is developmental hematopoiesis. During embryogenesis, definitive hematopoiesis occurs in a tissue called hemogenic endothelium (HE), a specialized subset of ECs at the ventral wall of the dorsal aorta (DA). HE acquires hematopoietic potentials and gives rise to HSCs, through a process known as endothelial-to-hematopoietic transition (EHT). During EHT, these specialized ECs extrude from DA and colonize the so-called aorta-gonadmesonephros (AGM) region, forming the native HSCs (Paik and Zon 2010).
As vascular development requires different steps, the molecular pathways involved are many. The Notch signaling pathway has been demonstrated to be one of the main players in vascular development. Among other functions, Notch signaling has been shown to be important during EHT. In the murine model, Runx1, a master regulator of HSC formation, has been shown to be transcriptionally regulated by NOTCH1 through GATA2 activation. This observation was later corroborated by knockdown studies for notch1a and notch1b in zebrafish (Butko, Pouget, and Traver 2016). Another essential pathway for vascular development is the HIF pathway. Hif-1α, Hif-1β and Hif-2α mouse mutants show severe vascular defects that result in early embryonic lethality (Simon and Keith 2008), which hinders a deep analysis of the phenotypes incurring in the mutant embryos. In addition, deletion of Hif-1α specifically in myeloid cells showed abnormalities in the motility, invasiveness, and adhesion of macrophages (Cramer et al. 2003). Intriguingly, Hif-1α deletion in vascular endothelial cadherin-expressing cells led to a significant but partial reduction of HSC number, suggesting that other players may be involved in this pathway (Imanirad et al. 2014).
Zebrafish embryos have been shown to be tolerant to hypoxia at very early stages of development (Padilla and Roth 2001). Also, zebrafish embryos develop externally and this allows to finely manipulate the environment where they grow (Lieschke and Currie 2007). These features make zebrafish an ideal model to investigate how hypoxia and Hif transcription factors affect vertebrate vascular development. In this study, I will examine the impact of hypoxia on zebrafish vascular development. Specifically, I will dissect the role of hif-1α in macrophage-EC interactions during vascular development and repair. Moreover, I show redundant functions for hif-1α and hif-2α in HSC development upstream of Notch signaling.