Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium

Florian Bonkhofer,Janina Schneider-Swales,Marella De Bruijn,Roger Patient,Rui Monteiro,Philip Pinheiro,Rossella Rispoli,Monika Krecsmarik,Ingrid Ho Ching Tsang,Tessa Peterkin

doi:10.1038/s41467-019-11423-2

Abstract

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1−/− mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

Highlights

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA)
HE maintains an arterial programme in runx1−/− mutants, but it is dependent on prior arterial specification, strongly suggesting that HE derives from aortic endothelium
A Citrine cassette followed by a polyA stop signal was recombineered downstream of the P2 ATG (Fig. 1d) to establish a stable TgBAC(runx1P2:Citrine) line, which expresses Citrine under the control of runx[1] regulatory elements

Summary

Introduction

Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). We present a transgenic zebrafish runx[1] reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding nonaortic endothelium Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Runx1−/− mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx[1] represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs. 1234567890():,; Definitive haematopoietic stem cells (HSCs) drive lifelong reconstitution of the blood system and are pivotal in the treatment of haematological disorders including leukaemia[1]. HE maintains an arterial programme in runx1−/− mutants, but it is dependent on prior arterial specification, strongly suggesting that HE derives from aortic endothelium

Methods

Results

Conclusion