Emergence of haematopoietic stem cells and cancer (K. Kissa)

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Summary

The origin of haematopoietic stem cells (HSCs) has been highly debated for a century. Indeed, while some authors described the existence of an aortic endothelium with ‘hemogenic’ properties because of the presence of clusters of hematopoietic cells in the ventral wall of the aorta (Minot, C. S. 1912), others reported the emergence of HSCs in the sub-aortic mesenchyme (Sabin, F. 1920; Murray, P.D. 1932). This question has remained unsolved for many years.

Because of its transparency, the zebrafish appears as a relevant model to address this question. We investigated whether these HSCs might arise directly from the aortic endothelium. For that we took advantage of the KDR:GFP transgenics in which the GFP highlights the whole vasculature, to address the sought issue of HSC. We observed that endothelial cells forming the floor of the aorta, have bent before leaving the endothelium to become free individual HSC. We concluded that HSCs emerge from the aorta endothelium by a new process we called the endothelial-hematopoietic transition or EHT (Kissa and Herbomel, Nature 2010).

Starting 2012, I initiated my PI project in the UMR5235 entitled ‘Emergence of hematopoietic stem cells: mechanism and fate’. We are addressing 2 questions:
(i) what is the mechanism allowing EHT process,
(ii) what is the fate of each HSC when they emerge in the AGM
(iii) what are the pathways governing the EHT process.
Research Projects
The zebrafish embryo, thanks to its transparency and the recent development of molecular tools and transgenic fluorescent reporter lines, is a relevant in vivo model to study the different steps lead-ing to definitive haematopoiesis both in terms of regulatory pathways and in term of cellular journey in the different compartments of the embryo.

Imaging of live zebrafish embryos during the first week of development showed the presence of in-creasing numbers of round cells displaying the morphology of haematopoietic precursors in the ven-tral part of the tail, around the caudal vein (CV) plexus. This haematopoietic population expanded dur-ing the development of this CV plexus. By 48 hpf, this tissue had extended largely beyond the ventral limit of the somitic muscles, and became entirely covered by them on either side within the next 24 hrs. Small cell clusters then started to develop at 3 dpf, and formed larger groups by 5–7 dpf.

We have called this CV plexus the Caudal Haematopoïetic Tissue (CHT) and we have shown that it is the functional homologue of the mammal foetal liver (Murayama et al, Immunity, 2006).

In mammals, the CD41 surface protein that is strongly expressed at the surface of platelets is also expressed, but to a lesser extent, by embryonic HSCs. Using the CD41:GFP transgenic zebrafish line, it has been shown that the CD41 surface protein is also strongly expressed in thrombocytes (homologous of mouse megakaryocytes). Our studies have shown that the first CD41:GFP+ cells appear in the fish AGM from 30 hpf. Then, they migrate to the CHT and later to the thymus and kidney. We then conducted a 4D confocal imaging experiment to investigate in vivo, for the first time, and in a vertebrate, the dynamics of multipotent haematopoietic precursors from the AGM to the developing thymus (Movie 1, Kissa et al, Blood, 2008).



Movie 1. Routes of the first hematopoietic immigrants to the nascent thymus. Kissa et al, Blood, 2008.

Finally, in order to image the emergence of HSCs from the aorta, we undertook a 4D confocal micros-copy analysis of the developing aorta in the trunk region between 18 and 72 hpf. We used the KDRl:GFP+ zebrafish transgenic line whose aortic endothelial cells express GFP under the control of the KDRl promoter (ortholog of Flk1/VEGF receptor 2). A strong GFP expression was detected at the surface of the arterial endothelium in this transgenic line. We systematically tracked each aortic cell in live embryos. Our results showed that multipotent precursors emerged directly from the aorta floor through a stereotyped process that involves a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. We named this process the endothelial haematopoietic transition (EHT, Kissa & Herbomel, 2010).

Thus, we have described that HSCs emerge from the ventral wall of the aorta, but the molecular and cellular events driving this process still have to be addressed. Moreover, the characterization and the long term fate of HSCs emerging from the ventral part of the aorta remain to be elucidated.
 

Research project:


Our research project is based on this recently published work and is split into three parts.

Aim 1: Characterising the identity of aorta-emerging HSCs

We developed different tools allowing the long-term fate of cells: live imaging, cell transplantation, cell tracing and parabiosis (see movie 2).


Movie 2. Cross-colonisation by primitive myeloid and erythroid cells in a gata1:dsred (right) // pu1:gfp (left) parabiote pair from 18.5–52 h.p.f. Demy et al, Nature Methods, 2014.

Aim 2: Regulatory pathways that govern the EHT process

The second part focuses on deciphering the regulatory pathways that govern the EHT process.

Aim 3: Aortic mechanical deformation and the EHT process

In collaboration with Andrea Parmeggiani (Biological Physics and Systems Biology, DIMNP - UMR 5235 CNRS/UM2/UM1 and Complex Systems & Nonlinear Phenomena, Laboratoire Charles Coulomb, UMR5221 CNRS/UM2, Montpellier II University), we are studying the relationship between aortic mechanical deformation and the EHT process (see movie 3).


Movie 3. Time-lapse confocal imaging of a zebrafish embryo from 1 to 4 days post fertilization, showing the correlation between aorta radial expansion then constriction and the period of endothelial to hematopoietic transition (EHT) of aorta floor cells. Kissa & Herbomel, Nature, 2010.



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Head of the team


Karima KISSA
Research Associate (CR) INSERM
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