Vascular 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.
The authors declare no competing or financial interests.
M.S. designed and performed experiments for all aspects of this work and contributed to editing of the manuscript. I.M. and T.J.B. contributed to identification and maintenance of all zebrafish lines described in this study. J.A.V. generated and validated initial Fv2E-Flt4 constructs. F.O.K. generated the flt4um131 allele. L.J.Z. performed statistical analyses. N.D.L. designed experiments and wrote the manuscript.
This work was supported by grants from the National Heart, Lung, and Blood Institute (NHLBI) [R01HL122599 and R01HL079266 to N.D.L.]; by the Uehara Memorial Foundation (M.S.); and by the Japan Society for the Promotion of Science (JSPS). Deposited in PMC for release after 12 months.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.137901.supplemental
- Received March 21, 2016.
- Accepted August 25, 2016.