The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses, and promotes cancer metastasis. To address the role microRNAs (miRNAs) play in the development and function of the lymphatic vascular system, we defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) and identified four BVEC signature and two LEC signature miRNAs. Their vascular lineage-specific expression patterns were confirmed in vivo by quantitative real-time PCR and in situ hybridization. Functional characterization of the BVEC signature miRNA miR-31 identified a novel BVEC-specific posttranscriptional regulatory mechanism that inhibits the expression of lymphatic lineage-specific transcripts in vitro. We demonstrate that suppression of lymphatic differentiation is partially mediated via direct repression of PROX1, a transcription factor that functions as a master regulator of lymphatic lineage-specific differentiation. Finally, in vivo studies of Xenopus and zebrafish demonstrated that gain of miR-31 function impaired venous sprouting and lymphatic vascular development, thus highlighting the importance of miR-31 as a negative regulator of lymphatic development. Collectively, our findings identify miR-31 is a potent regulator of vascular lineage-specific differentiation and development in vertebrates.Vertebrates have developed two parallel but structurally and functionally distinct vascular systems: the blood and lymphatic vascular systems (1, 7). The lymphatic vascular system controls tissue fluid homeostasis, absorbs lipids and fat-soluble vitamins from the intestine, and mediates afferent immune responses by transporting lymphocytes and antigen-presenting cells to regional lymph nodes (1, 7). In addition, malignant cancers can induce lymphatic vessel activation and growth (lymphangiogenesis) within primary tumors and draining lymph nodes, which enhances cancer metastasis to draining lymph nodes and beyond (1, 22). These findings have fueled a surge in studies aimed at defining the molecular characteristics and functional activities of lymphatic vessels and identifying molecules that regulate lymphangiogenesis.Genomic and proteomic studies have identified novel molecular markers and growth factors for lymphatic vessels (2,23,48,52). Mouse genetic models have characterized the transcription factors PROX1 and SOX18 as master regulators of lymphatic vascular development and differentiation in vivo (12,56,65). These studies indicate that SOX18 expression in a subset of cardinal vein endothelial cells initiates lymphatic vascular development by inducing PROX1 expression (12). The resulting lymphatic vascular progenitor cells bud off and migrate away from the cardinal vein and form primitive lymph sacs, which subsequently develop into functional lymphatics (12, 56). PROX1 and SOX18 expression in cultured blood vascular endothelial cells (BVECs) triggers these cells to adopt lymphatic lineage-specific molecular and phen...

The molecular mechanisms that regulate the earliest steps of lymphatic vascular system development are unknown. To identify regulators of lymphatic competence and commitment, we used an in vitro vascular assay with mouse embryonic stem cell-derived embryoid bodies (EBs). We found that incubation with retinoic acid (RA) and, more potently, with RA in combination with cAMP, induced the expression of the lymphatic competence marker LYVE-1 in the vascular structures of the EBs. This effect was dependent on RA receptor (RAR)-α and protein kinase A signaling. RA-cAMP incubation also promoted the development of CD31+/LYVE-1+/Prox1+ cell clusters. In situ studies revealed that RAR-α is expressed by endothelial cells of the cardinal vein in ED 9.5–11.5 mouse embryos. Timed exposure of mouse and Xenopus embryos to excess of RA upregulated LYVE-1 and VEGFR-3 on embryonic veins and increased formation of Prox1-positive lymphatic progenitors. These findings indicate that RA signaling mediates the earliest steps of lymphatic vasculature development.

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