FUNCTIONAL CHARACTERIZATION OF SOX TRANSCRIPTION FACTORS IN ZEBRAFISH ANGIOGENESIS AND LYMPHANGIOGENESIS: KNOCKDOWN AND KNOCKOUT APPROACHES

My laboratory is interested in studying Sox (Sry-related HMG box) proteins, a family of transcription factors (TFs) present throughout the animal kingdom and important for the regulation of several fundamental processes during development (Pevny and Lovell-Badge, 1997; Wegner, 1999; Bowles et al., 2000). In particular, we focused on SOX18 that, together with SOX7 and SOX17, belongs to the SoxF subgroup (Bowles et al. 2000). In humans, mutations in SOX18 are associated with the Hypotrichosis-Lymphedema-Telangiectasia (HLT) syndrome, combining defects in hair, blood vessels and lymphatic development (Irrthum et al., 2003). In zebrafish, Sox18 and Sox7 have a redundant role in regulating arterio-venous differentiation (Cermenati et al. 2008; Herpers et al. 2008; Pendeville et al. 2008; Hermkens et al. 2015), while only Sox18 is involved in lymphatic development (Cermenati et al., 2013). However, a recent article questioned the relevance of Sox18 role in zebrafish lymphatic vessel development, because a sox18 mutant did not show a lymphatic phenotype (van Impel et al. 2014). To clarify Sox18 role in zebrafish lymphangiogenesis, we made use of a new, independent mutant allele: sox18sa12315. In vivo analyses show that sox18 mutant behaves as a null, indeed sox18 mutation, associated with sox7 perturbation, causes the absence of trunk tail circulation, the same phenotype observed in sox7/sox18 double partial morphants and in sox7/sox18 double mutants (Cermenati et al., 2008; Hermkens et al. 2015). Our data also point out that Sox18 has a conserved role in zebrafish lymphangiogenesis, with an interplay with VegfC in the formation of the thoracic duct (TD). The analyses of sox18sa12315 mutation in two transgenic backgrounds reveal that, even if the mutation does not cause strong alterations of lymphatic system development, TD defects are statistically significant in homozygotes. Perturbation of Vegfc signaling exacerbates TD defects in a genotype-dependent manner; TD defects are highly enhanced in homozygotes, but even heterozygotes show statistically significant alterations when vegfc is slightly perturbed. The ectopic expression of sox7 in the posterior cardinal vein (PCV), from which lymphatic precursors originate, can explain the only subtle lymphatic defects observed in sox18 mutants transgenic line used for TD analyses. However, sox7 ectopic expression is not seen in all the fish lines we have analyzed, suggesting that also in zebrafish, as in mouse, the fine regulation of soxF genes depends on the genetic background. On the other hand, we are interested in studying the complex interplay between Sox and Notch signaling. This year we published a collaborative article showing, with knockdown and knockout approaches, that SoxF transcription factors positively regulate notch1b and Notch1 vascular expression in zebrafish and mouse, respectively (Chiang et al., 2017). This work identifies SoxF responsive enhancers in Notch1 and notch1b loci in mouse and in zebrafish, respectively. SoxF binding to the zebrafish notch1b enhancer is functionally relevant: arterial ISV defects are indeed found in enhancer mutants under experimental conditions, which slightly perturb Notch signaling. My laboratory is also interested in studying the role of sox13 (subgroup D), a gene vaguely linked to angiogenesis in different models (Roose et al., 1998; McGary et al. 2010). We have the evidence that Sox13 is implicated in zebrafish angiogenesis and that SoxF positively regulate sox13 (subgroup D) endothelial expression. In particular, sox13 morphants show ISV defects and an upregulation of some genes implicating in the Notch pathway. In vivo SPIM time lapse was used to characterize in a dynamic way, through long-term time lapse analysis, the ISV defects observed in sox13 morphants. With a combination of knockdown approaches in zebrafish and overexpression studies in vitro on ECs, we gathered evidence of an involvement of Sox13 in promoting endothelial cell migration. Finally, we confirmed that SoxF positively regulate sox13 by performing ISH analyses both in morphants and mutants. Since SoxF positively regulate the Notch signaling and positively regulate the expression of sox13, a negative regulator of the Notch pathway, we can speculate the existence of a complex regulatory network between SoxF and Sox13 in fine-tuning vascular development.