Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification

Abstract

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

A Zebrafish Model for a Human Myopathy Associated with Mutation of the Unconventional Myosin MYO18B

“…The locus is within the final 1.5 Mb at the end of the chromosome and the myo18b gene sequence is distributed across several fragments in GRCz10, with one fragment placed further toward the telomere (LOC100331349) and two large fragments from the 59 (LOC100537963) and 39 (LOC10033206) ends of the gene in the correct location ( Figure 2B). This fragmentation of the gene is likely to be due to errors in the sequence assembly in the subtelomeric region, something that has been observed for other chromosomes, for example in the course of cloning the cloche gene (Reischauer et al 2016). The myo18b locus is also fragmented in the genome assemblies of several other fish species, with many incomplete transcripts aligning within this genomic region.…”

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

“…These mutants include, among many others: cloche/npas4 2223, alk8 24, med12 25, prpf8 26, plcg1 27 and runx1 28.…”

Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish

“…7B,C), suggesting that steatosis and HSC proliferation can be suppressed by VEGF inhibition without impairment of angiogenesis. In a second approach, we performed acute ethanol treatment on cloche mutants that carry a null mutation in the cloche gene encoding a bHLH-PAS transcription factor (Reischauer et al, 2016). These animals lack most hematopoietic and endothelial cells but form normal numbers of HSCs during liver development (Stainier et al, 1995; Yin et al, 2012).…”

“…HSCs and endothelial cells, but not hepatic parenchymal cells, exhibit robust changes in the expression of VEGF receptor genes upon acute ethanol exposure and are likely the direct targets of VEGFR inhibition. By conducting ethanol treatment experiments on cloche (also known as npas4l ) mutants lacking hepatic endothelial cells (Reischauer et al, 2016), we revealed that the effect of VEGFR inhibition on hepatic steatosis and fibrogenesis could be uncoupled from angiogenesis.…”