ABSTRACT
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.
Subject(s)
Embryonic Stem Cells/drug effects , Lymphatic Vessels/drug effects , Tretinoin/pharmacology , Animals , Cell Differentiation/drug effects , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gestational Age , Glycoproteins/metabolism , Homeodomain Proteins/metabolism , Larva/drug effects , Larva/metabolism , Lymphatic Vessels/embryology , Lymphatic Vessels/metabolism , Membrane Transport Proteins , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/metabolism , Retinoic Acid Receptor alpha , Signal Transduction/drug effects , Tumor Suppressor Proteins/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Veins/drug effects , Veins/embryology , Veins/metabolism , Xenopus laevisABSTRACT
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.
