The Ortholog of LYVE-1 Is Required for Thoracic Duct Formation in Zebrafish*

Abstract

LYVE-1 (also termed CRSBP-1), a 120-kDa disulfide-linked dimeric type I membrane glycoprotein, is a specific marker for lymphatic endothelial cells (LECs) and exhibits multiple ligand (hyaluronic acid and growth factors/cyto- kines) binding activity in mammals. Recent studies indicate that LYVE-1/CRSBP-1 ligands (VEGF-A165, PDGF-BB, oligopeptides containing the cell-surface retention sequence (CRS) motifs of VEGF-A165 and PDGF-BB) induce opening of lymphatic intercellular junctions in vitro and in vivo. To determine the function of the ortholog of mammalian LYVE-1 in zebrafish, we cloned it (zLyve-1). The cloned cDNA (zLyve1) encodes a 328-amino-acid type I membrane glycoprotein. The protein and genomic structure evidence supports the notion that the cloned zLyve-1 is the ortholog of LYVE-1 in zebrafish. zLyve-1 expressed in cultured cells by transfection exhibits hyaluronic acid binding activity but lacks the growth factor binding activity seen in mammalian homologs. Knockdown of zLyve-1 levels by embryo microinjection with a specific antisense morpholino oligonucleotide (MO2) in wild-type and Tg(fli1:EGFP)-transgenic zebrafish causes defects in thoracic duct (TD) formation. Such zebrafish injected with MO2 also exhibit impaired TD flow (as determined by intramuscular injection of FITC-dextran). The phenotypes in these zebrafish injected with MO2 are reversed by co-injection with zLyve1cDNA. In situ hybridization reveals that zLyve-1 is expressed in the posterior cardinal vein (PCV). Expression of zLyve-1 at the highest level in the PCV occurs at 3 dpf which coincides with the time for TD formation in zebrafish development. These results suggest that zLyve-1 is required for TD formation. They also suggest that zLyve-1 is distinct from mammalian LYVE-1 in its role in lymphatic function.