Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors

Abstract

There was an error published in Development 143, 3785-3795.In the Materials and Methods section we provided an incorrect serine residue number for the map2k2b mutation. The corrected section of text is as follows:An activated myc-tagged form of MEK was generated from zebrafish map2k2b by mutating serines 221 and 225 to aspartic acid (Schramek et al., 1997) by PCR, followed by BP cloning to generate pME-myc-act-map2k2b.In addition, we have since verified that the pME-myc-act-map2k2b plasmid used in the original studies and submitted to Addgene contains the correct sequence. This plasmid contains several nucleotide changes in comparison to the GenBank reference sequence (accession number: NM_001128281). However, all of these changes have been noted in a previous annotation of single nucleotide polymorphisms in the zebrafish and are naturally occurring variants (Butler et al., 2015).We apologize for any confusion that this may have caused and we thank the researchers who discovered the error for bringing it to our attention. ABSTRACTVascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.

Vegfa signals through ERK to promote angiogenesis, but not artery differentiation

“…Thus, Vegfa signaling through ERK is acting to induce dynamically genes, such as flt4, that play a pro-angiogenic role in sprouting, and dll4, which is important to reduce angiogenic behaviors in neighboring cells. , kdrl um19 , flt4 um131 , plcg1 y13 and mib ta52b lines have been described elsewhere (Anderson et al, 2008;Asakawa and Kawakami, 2008;Bussmann et al, 2011;Covassin et al, 2009Covassin et al, , 2006Itoh et al, 2003;Lawson et al, 2003;Shin et al, 2016). Additional transgenic lines are described below.…”

“…For generation of UAS lines, pMEmyc-nlstdTomato-2a was cloned into pDest insUAS R1R2 CC using LR clonase II (Thermo Fisher Scientific), to give pinsUAS:nlstdTomato, whereas pME-myc-nlstdTomato-2a and p3E-dusp6dm were cloned into pDest insUAS R1R3 CC, resulting in pinsUAS:nlstdTomato-2a-dusp6dm-CC. Protocols for construction and application of Tol1 actMek2b plasmids can be found elsewhere (Shin et al, 2016). Plasmids are available at Addgene.…”

“…mcherry, vegf 121 and vegfc-2a-mcherry mRNAs were prepared and injected at the indicated amount as previously described . Tol1 constructs and mRNA were prepared and injected as described elsewhere (Shin et al, 2016). dll4 MO2 (15 ng per embryo) was injected as previously described (Siekmann and Lawson, 2007).…”

Vegfd modulates both angiogenesis and lymphangiogenesis during zebrafish embryonic development

“…Of relevance to human lymphatic disease, we show that vegfd gene dose broadly modifies Vegfc/Flt4 pathway phenotypes in a variety of mutant models and settings. Zebrafish with mutations in genes involved in this pathway are disease models of heritable lymphedema (Alders et al, 2009;Connell et al, 2010Connell et al, , 2009Crawford et al, 2016;Evans et al, 2003;Gordon et al, 2013;Hogan et al, 2009a,b;Karkkainen et al, 2000;Shin et al, 2016b;Villefranc et al, 2013). Given that ∼30% of cases of primary (inherited) lymphoedema are caused by mutations in the Ccbe1/Vegfc/Vegfr3 (Flt4) pathway (Mendola et al, 2013), it may be that Vegfd variants can modify patient outcomes.…”

“…Vegfd has been shown by MO knockdown to compensate for loss of Vegfc during facial lymphatic development but not elsewhere in the embryo . Growth factor-induced signaling downstream of Flt4 occurs through the activation of Erk, which is necessary and sufficient in the trunk to induce the normal sprouting of LECs and expression of the LEC marker Prox1 (Koltowska et al, 2015;Shin et al, 2016b). During facial lymphangiogenesis from the CCV, Erk signaling controls LEC sprouting but not Prox1 expression, suggesting spatially distinct mechanisms that remain to be fully understood (Shin et al, 2016b).…”