Investigation of ETV2/Etsrp Role in Lymphangiogenesis: A Potential Therapeutic Target in Lymphatic Disease

Abstract 614

Lymphangiogenesis, defined as novel growth of lymphatic vessels to constitute a functional vascular network, captures great clinical interest because of its essential role such pathologic settings as inflammation, vascular malformations, and tumor metastasis. Previous observational hurdles are now overcome by use of the optically clear and genetically modifiable zebrafish embryo, which provides a vertebrate model for understanding lymphangiogenesis and for revealing putative therapeutic targets for metastatic disease and vascular/lymphatic malformations that currently have no cure.

The zebrafish ets1-related protein, Etsrp, encodes a novel ETS domain transcription factor and is an early marker for vascular endothelial progenitors. Its function is necessary and sufficient to initiate vasculogenesis and is conserved during vertebrate development. Human and murine Er71/Etv2 proteins are functional orthologs of Etsrp. However, its role in lymphatic development is unknown. Our data show that both vascular and lymphatic vessels express etsrp driven GFP transgene. To analyze Etsrp role in lymphatic development, we performed functional Etsrp knockdown by photoactivatable morpholino oligonucleotide (MO) injection into fli1:GFP; flk1:mCherry transgenic reporter zebrafish embryos whose fluorescent background allowed distinction of lymphatic from vascular vessels.

Inhibition of etsrp function at early stages resulted in the absence of both lymphatic and vascular endothelial development. Inhibition of etsrp function at 1 day-post-fertilization, after the major axial vessels have already formed, specifically affected lymphangiogenesis without affecting vascular development. The major lymphatic vessel, the thoracic duct, was absent or discontinuous in Etsrp-depleted embryos, while the major axial and secondary intersegmental blood vessels were not affected. Further analysis demonstrated that the lymphatic progenitors, parachordal lymphangioblasts, failed to migrate from the posterior cardinal vein in the absence of Etsrp function. Control morpholino-injected but not photoactivated embryos did not show significant defects in the lymphatic development, demonstrating the specificity of the observed defects. Because expression of the major VegfC receptor, VegfR3 was strongly down regulated in Etsrp MO-injected embryos, our results suggest that Etsrp initiates lymphangiogenesis by regulating VegfR3 expression. In the absence of Etsrp function, lymphangioblasts would fail to respond to VegfC signaling.

We report for the first time the requirement of Etsrp for normal lymphangioblast migration and thoracic duct development in zebrafish. Additional studies are in process to determine the mechanism of Etsrp role in lymphangiogenesis. Our results suggest that Etsrp may represent a potential novel target for chemical modulation in multiple human lymphatic disorders.