Fine‐tune Regulation of Cpn1 (Carboxypeptidase N1) to Control Vascular Patterning during Zebrafish Development

Abstract

Vascular development is important for vertebrates and regulated by regulators via complicated signaling pathways. In our previous study, transcription factor islet2 regulated vein and ISV formation mediated by notch signaling. Genome‐wide transcriptional analysis showed that cpn1 is regulated by islet2/coupTFIb, suggested its function in vasculature. However, no direct evidence shown the role of Cpn1 in vascular function so far. Thus, we hypothesize that cpn1 encode enzymatic active subunits to perform CPN activity and function in vascular developmentIn‐situ hybridization showed cpn1 is expressed in developing vessels. Overexpression of cpn1 impairs the growth of ISV (intersegmental vessel) and CVP (cardinal vein plexus), which is similar to the phenotype of isl2 morphant. Knockdown of cpn1 by morpholino injection also causes vascular defects, suggesting the role of cpn1 in controlling ISV and CVP growth. We further showed the cpn1 MO knockdown works efficiently by probing cpn1 expression products and knockdown specificity by using 2nd MO. We found that a loss of cpn1 results in a decreased expression of vascular markers flk, stabilin, ephrinb2. This indicates the regulatory role of cpn1 in controlling vascular development. Promoter analysis showed cpn1 is likely regulated by isl2/coupTFIb. To examine the interaction between cpn1 and isl2/coupTFIb, we performed luciferase assay and showed cpn1 is regulated by isl2, which is consistent with our microarray results. We also revealed cpn1 is regulated by notch and VEGF signalings. Together, we showed cpn1 plays an important role for vascular development in zebrafish. We also uncovered a fine‐tune regulation of cpn1 that controls vascular patterning mediated by isl2‐Notch signaling.