The transcriptome of haemogenic endothelium in zebrafish embryos

Every day, billions of blood cells die in our body and need to be replaced. Haematopoietic (blood) stem cells (HSCs) located in the bone marrow maintain the blood system throughout life. They give rise to all of the blood cells in our body, and retain the ability to divide to generate more HSCs in a process called self-renewal. These characteristics also enable HSCs to reinitiate blood formation in patients who through disease, injury or therapeutic treatment have lost their own HSCs and are unable to produce blood cells themselves. HSC transplants are, however, not without risks. Transplant rejection and graft-versus-host disease are frequent complications that can lead to the death of the patient. Pluripotent stem cells (cells that have the potential to become any cell type in our body) are a potential source of patient-matched or patient-specific HSCs that would allow us to avoid these complications. While we are able to generate mature blood cells from pluripotent cells, we currently lack sufficient knowledge to guide the cells to become HSCs. All vertebrates first form HSCs during embryogenesis and they appear to do this in a very similar way. In all vertebrate embryos, HSCs arise from haemogenic (blood-forming) endothelium located in the wall of the dorsal aorta, the main trunk artery. Using a transgenic zebrafish line in which haemogenic endothelial cells are marked by the expression of a green fluorescent protein we want to isolate these cells and find out which genes are expressed (switched on) within them. This information will help us to unravel the genetic programme that steers cells in the embryo to become HSCs. This project on zebrafish embryos promises to make a valuable contribution to our knowledge about HSC formation in the embryo which will help us in the future to generate HSCs from pluripotent cells grown in a culture dish.

In the vertebrate embryo, haematopoietic stem cells arise in the ventral wall of the dorsal aorta via an endothelial intermediate, the haemogenic endothelium. Little is known about the molecular programme that drives this process. Here, we propose to analyse in detail the transcriptome of the haemogenic endothelium and to compare it to that of non-haemogenic endothelial cells. For this purpose, we make use of a novel unique transgenic zebrafish line that allows us to isolate haemogenic endothelial cells from the zebrafish embryo by fluorescence-activated cell sorting. Total RNA will be prepared, mRNA reverse transcribed and amplified. Amplified cDNAs are used to generate a library for SOLiD sequencing. Genes up- or down-regulated in haemogenic endothelium in comparison to non-haemogenic and arterial endothelial cells will be verified by quantitative RT-PCR and RNA in situ hybridisation. Genes verified to be differentially expressed will be examined further in loss- and gain-of-function experiments to study their role in the process of haematopoietic stem cell formation.

Haematopoietic stem cell (HSC) transplantations are the most common type of stem cell therapy performed in the clinic. However, allogeneic transplants bear the risk of transplant rejection or graft-versus-host disease, complications associated with significant morbidity and mortality. Pluripotent stem cells hold the promise to be an alternative source of transplantable HSCs that could overcome these problems. Unfortunately, efforts to generate HSCs from human embryonic stem cells have not been successful, mainly due to our limited understanding of the molecular program HSC precursors undergo during embryogenesis. In this project, we want to elucidate the transcriptome of the haemogenic endothelium, an endothelial intermediate that forms during HSC development. Thereby, we hope to provide insight into at least part of the molecular program that drives HSC formation in the vertebrate embryo. From this knowledge, new culture regimes may arise that will allow us to generate HSCs from pluripotent stem cells.