Abstract
A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.
Highlights
Recent studies have demonstrated the use of zebrafish to study vascular development (Chan et al, 2005; Kidd and Weinstein, 2003; Sumanas et al, 2005; Zhu et al, 2005)
A clone designated GA2692 was one of the roughly 50 clones – from a total of approximately 4000 cDNAs screened by wholemount in situ hybridization in zebrafish – that showed expression largely restricted to developing vessels
We found that negative control siRNA (NC)-transfected human umbilical vein endothelial cells (HUVECs) form well-connected tube-like structures, whereas si1 and si3 transfected HUVECs barely formed tube-like structures, resembling the effect seen with potent antiangiogenic molecules (Fig. 9), and being consistent with our zebrafish phenotype
Summary
Recent studies have demonstrated the use of zebrafish to study vascular development (Chan et al, 2005; Kidd and Weinstein, 2003; Sumanas et al, 2005; Zhu et al, 2005). We have sought to discover new genes of functional importance in early vessel formation by a two-step process: (A) the screening of 4000 transcripts from a zebrafish kidney marrow cDNA library (Galloway et al, 2005) to identify those whose expression was relatively restricted to developing vessels in the 24-72 hours post fertilization (hpf) period and, (B) the use of antisense morpholino-oligonucleotide-mediated knockdown in embryos to assess a vascular phenotype. Of the approximately 50 transcripts from the library whose expressions were localized mainly to vasculature during early development, we focused attention on one, designated as GA2692. This transcript, at the time these studies were initiated, had homology to the human cDNA FLJ32150, and nothing was known in terms of its expression or function. Mammalian Hsp70s associate with unfolded nascent precursor peptides to stabilize them prior to their folding into mature proteins and reaching their ultimate cellular
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