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/cytokines) 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.
Highlights
Cell-surface retention sequence (CRS)-binding protein-1 (CRSBP-1) was identified in cultured transformed cells
Like all other known vertebrate LYVE-1 congeners, including mammalian and salmon LYVE-1 (sLYVE-1), zebrafish LYVE-1 ortholog (zLyve-1) is a membrane glycoprotein possessing two N-glycosylation sites which are localized on both sides of the Link module
ZLyve-1 and sLYVE-1 exhibit amino acid sequence homology with other mammalian homologs but lack the acidic-amino-acid-rich (AAAR) domain which is believed to mediate its binding to growth factors/cytokines with CRS motifs [1,2,3,7,18]
Summary
Cell-surface retention sequence (CRS)-binding protein-1 (CRSBP-1) was identified in cultured transformed cells. The zebrafish offers several other advantages including small size, optical clarity (fluorescent blood and lymphatic vessel structures are easy to see in transgenic Tg(fli1:EGFP) zebrafish) and rapid development (days) of embryos [15] In this communication, we show the cloning of zlyve cDNA from zebrafish RNA at the 2 - 5 dpf stages by RT-PCR. We show that zLyve-1 expressed in cultured cells exhibits hyaluronic acid-binding activity but lacks the growth factor-binding activity seen in mammalian LYVE-1, as determined by cetylpyridinium chloride precipitation and 125I-labeled VEGF (vascular endothelial cell growth factor) peptide-affinity labeling [3], respectively. We show that zebrafish derived from both wild-type and fluorescent transgenic Tg(fli1:EGFP) embryos microinjected with a specific antisense morpholino oligonucleotide (MO2) exhibit defects in thoracic duct formation and impaired thoracic duct flow (as determined by intramuscular injection of FITC-dextran) These phenotypes can be reversed by co-injection with zlyve cDNA. Since PCV endothelial cells are known to be precursor cells for LECs forming the thoracic duct (TD), this suggests that zLyve-1 expressed in PCV endothelial cells may play an important role in lymphatic development in zebrafish
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