Zebrafish Kelch-like family member 4 is required for vasculogenesis and hematopoiesis

Molecular mechanisms regulating vascular development and hematopoiesis are still incompletely understood. The KLHL (Kelch-like) family of proteins function as adapters to target proteins for ubiquitination. However, their role in vascular development has not been previously analyzed. Here we have characterized a novel regulator of vascular development, kelch-like family member 4 (klhl4) in zebrafish. We show that zebrafish klhl4 is expressed in early vascular endothelial and hematopoietic progenitors, while its expression is restricted to vascular endothelial cells during later developmental stages. To determine the functional role of klhl4, we generated loss-of-function zebrafish mutants using CRISPR/Cas9 genome editing. klhl4 mutant embryos were viable, yet they exhibited delayed sprouting of intersegmental vessels (ISVs), which correlated with reduced expression of vascular endothelial and erythroid specific molecular markers. Time-lapse imaging showed that vascular endothelial and hematopoietic progenitor cells exhibit delayed migration towards the midline and undergo increased apoptosis and reduced proliferation in klhl4 mutants. Expression of npas4l and etv2/etsrp, two master regulators of endothelial and hematopoietic development, was reduced in klhl4 mutants, suggesting that some vascular defects could be caused by the reduction of npas4l and etv2 expression. However, npas4l or etv2 overexpression failed to rescue ISV sprouting defects in klhl4 mutants, suggesting that klhl4 may promote vasculogenesis by additional mechanisms. In summary, our findings demonstrate a novel role for zebrafish klhl4 in regulating vascular endothelial and hematopoietic development during embryogenesis. Because the Klhl4 protein sequence is highly conserved between different vertebrates, it is likely that it may play a similar role in other organisms.