Deletion of a conserved Gata2 enhancer impairs haemogenic endothelium programming and adult Zebrafish haematopoiesis

Abstract

Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2a Δi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2a Δi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.

Zebrafish Cancer Predisposition Models

“…Using CRISPR/Cas9, Dobrzycki and colleagues introduced a deletion to the conserved +9.5 enhancer within intron 4 of zebrafish gata2a ( gata2a Δ i4/ Δ i4 ) ( Dobrzycki et al, 2020 ). Unlike mouse Gata2 Δ+9.5 models ( Gao X. et al, 2013 ), gata2a Δ i4/ Δ i4 fish survive past embryogenesis, and had decreased expression of HSC markers, runx1 and cmyb , during primitive hematopoiesis.…”

“…Despite the recovery of runx1 and cmyb expression to wild-type levels by 48hpf, adult gata2a Δ i4/ Δ i4 fish had half the number of blood cells within their kidney marrow and a surplus of immature blasts was found in 10% mutants. Additionally, these fish had a high incidence of cardiac edema and infection within less than 6 months of age, suggestive of the immune deficiency and lymphatic defects seen in MonoMAC syndrome ( Dobrzycki et al, 2020 ).…”

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

“…GATA2 is required for HSC development and is known to act upstream of RUNX1 in the hemogenic endothelium 8,38,39 . Zebrafish contains two GATA2 paralogs, gata2a, which is expressed in the whole vasculature, included hemogenic endothelium 34 and gata2b, which is specifically expressed in hemogenic endothelium 33 . A previous publication showed that, at the onset of definitive hematopoiesis, gata2a Δ i4/Δi4 mutants present an initial delay in the induction of hemogenic endothelium but then can progress with a normal hematopoietic development 34 .…”

Table_1.docx

“…Using CRISPR/Cas9, Dobrzycki and colleagues introduced a deletion to the conserved +9.5 enhancer within intron 4 of zebrafish gata2a (gata2a i4/ i4 ) (Dobrzycki et al, 2020). Unlike mouse Gata2 +9.5 models (Gao X. et al, 2013), gata2a i4/ i4 fish survive past embryogenesis, and had decreased expression of HSC markers, runx1 and cmyb, during primitive hematopoiesis.…”

“…Despite the recovery of runx1 and cmyb expression to wildtype levels by 48hpf, adult gata2a i4/ i4 fish had half the number of blood cells within their kidney marrow and a surplus of immature blasts was found in 10% mutants. Additionally, these fish had a high incidence of cardiac edema and infection within less than 6 months of age, suggestive of the immune deficiency and lymphatic defects seen in MonoMAC syndrome (Dobrzycki et al, 2020).…”