Divergence of zebrafish and mouse lymphatic cell fate specification pathways

Abstract

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

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“…KD of prox1a using ATG antisense morpholino oligonucleotides (MOs) results in reduced numbers of PACs and a defective thoracic duct (Yaniv et al, 2006). By contrast, prox1a homozygous mutants display rather mild lymphatic defects (Koltowska et al, 2015a;van Impel et al, 2014). This discrepancy between phenotypes has recently been shown to result from the presence of maternally contributed transcripts in prox1a −/− embryos, as maternal zygotic mutants carrying a mutation in the same gene were shown to display a severe lymphatic phenotype, including reduced numbers of PACs and absence of the thoracic duct (Koltowska et al, 2015a).…”

“…The expression pattern, as well as the function, of prox1b remains controversial, with one study reporting expression of prox1b mRNA in the PCV and venous sprouts at 48 hpf, but not in PACs (Del Giacco et al, 2010), and another showing expression of a prox1b transgenic reporter in lymphatic progenitors at comparable developmental stages (Tao et al, 2011). Moreover, whereas the MO-based KD of prox1b results in defective lymphangiogenesis (Del Giacco et al, 2010), two different prox1b mutant alleles display no apparent lymphatic phenotypes (Tao et al, 2011;van Impel et al, 2014). Additional experiments analyzing the potential maternal contribution of prox1b transcripts might be required in order to ascertain the exact role of this gene during zebrafish lymphatic development.…”

“…In zebrafish, sox18 is expressed throughout the entire CV and is also detected in the dorsal aorta (DA) (Cermenati et al, 2008(Cermenati et al, , 2013. As for prox1b, various differences in MO-induced versus mutant phenotypes have been reported for sox18 (Cermenati et al, 2013;van Impel et al, 2014) ( Table 1), and again the analysis of mutants lacking maternal sox18 might be required in order to resolve these discrepancies.…”

“…No lymphatic defects were detected in coup-TFII homozygous zebrafish mutants , whereas MO-based KD experiments have suggested that coup-TFII is indispensable for lymphatic development in zebrafish and Xenopus (Aranguren et al, 2011). Here too, therefore, discrepancies between KD (Aranguren et al, 2011) and KO (Kok et al, 2015;van Impel et al, 2014) phenotypes have presented difficulties in understanding the exact role of this transcription factor during lymphangiogenesis in these animal models (Table 1).…”

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“…35 Nonetheless, people have used different lymphatic reporter fish lines to study the functionality of the lymphatic system in zebrafish, and the key features are congruent with their mammal counterparts. For example, lymphatics in zebrafish are important for removal of subcutaneous fluids, and defects in lymphatics result in edema.…”