Ferrari Kaitlin, Gurung Suman, Loges Luiza N, Batta Surya Prakash Rao, Hammond Myles A, Griciunaite Martyna, DeMoya Ricardo, Restrepo Nicole K, Sumanas Saulius
University of South Florida, Department of Pathology and Cell Biology, USF Health Heart Institute, Tampa, FL, 33602, USA.
University of South Florida, Department of Pathology and Cell Biology, USF Health Heart Institute, Tampa, FL, 33602, USA.
Dev Biol. 2025 Sep;525:1-12. doi: 10.1016/j.ydbio.2025.05.018. Epub 2025 May 20.
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.
调节血管发育和造血的分子机制仍未完全明确。KLHL（kelch样）蛋白家族作为衔接蛋白，将靶蛋白靶向泛素化。然而，它们在血管发育中的作用此前尚未得到分析。在此，我们鉴定了斑马鱼中一种新型的血管发育调节因子——kelch样家族成员4（klhl4）。我们发现斑马鱼klhl4在早期血管内皮和造血祖细胞中表达，而在后期发育阶段其表达仅限于血管内皮细胞。为了确定klhl4的功能作用，我们使用CRISPR/Cas9基因组编辑技术构建了功能缺失的斑马鱼突变体。klhl4突变体胚胎能够存活，但它们表现出节间血管（ISV）出芽延迟，这与血管内皮和红细胞特异性分子标记物表达降低相关。延时成像显示，在klhl4突变体中，血管内皮和造血祖细胞向中线迁移延迟，凋亡增加且增殖减少。内皮和造血发育的两个主要调节因子npas4l和etv2/etsrp在klhl4突变体中的表达降低，这表明一些血管缺陷可能是由npas4l和etv2表达减少所致。然而，npas4l或etv2的过表达未能挽救klhl4突变体中的ISV出芽缺陷，这表明klhl4可能通过其他机制促进血管生成。总之，我们的研究结果证明了斑马鱼klhl4在胚胎发育过程中调节血管内皮和造血发育方面具有新的作用。由于Klhl4蛋白序列在不同脊椎动物之间高度保守，它很可能在其他生物体中发挥类似作用。
