Generation of a c-myb reporter line for functional testing of haematopoietic stem cells isolated from zebrafish embryos
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biology
Abstract
We are dissecting the molecular mechanisms that control formation and maintenance of blood/haematopoietic stem cells (HSCs) in zebrafish embryos.
In vertebrates, HSCs form during embryogenesis. In adult humans, they reside in the bone marrow from where they replace short-lived mature blood cells throughout life. Thus, they are essential for our survival. They are the active component of bone marrow transplants that are used to re-establish blood formation (haematopoiesis) in patients that have lost their blood system due to blood disorders, accidents or medical treatments.
To develop new therapies, we need to be able to generate HSCs in vitro and to maintain and expand their numbers. To date, we know little about the molecular mechanisms that control these processes.
We study zebrafish embryos with the aim to reveal these mechanisms. While we can identify the earliest potential HSCs by gene expression, we currently lack an assay to test them. Therefore, we are generating a transgenic fish line in which these cells express a green fluorescent protein. Fluorescent cells will be transplanted from a transgenic to a wild-type embryo to see whether they participate in long-term haematopoiesis. This assay will be an invaluable tool for dissecting the molecular mechanisms that control HSC biology.
In vertebrates, HSCs form during embryogenesis. In adult humans, they reside in the bone marrow from where they replace short-lived mature blood cells throughout life. Thus, they are essential for our survival. They are the active component of bone marrow transplants that are used to re-establish blood formation (haematopoiesis) in patients that have lost their blood system due to blood disorders, accidents or medical treatments.
To develop new therapies, we need to be able to generate HSCs in vitro and to maintain and expand their numbers. To date, we know little about the molecular mechanisms that control these processes.
We study zebrafish embryos with the aim to reveal these mechanisms. While we can identify the earliest potential HSCs by gene expression, we currently lack an assay to test them. Therefore, we are generating a transgenic fish line in which these cells express a green fluorescent protein. Fluorescent cells will be transplanted from a transgenic to a wild-type embryo to see whether they participate in long-term haematopoiesis. This assay will be an invaluable tool for dissecting the molecular mechanisms that control HSC biology.
Technical Summary
Haematopoietic stem cells (HSCs) can self-renew and give rise to mature blood cells of all lineages. Thus, HSCs are essential for the homeostasis of the blood system and the long-term survival of the individual. HSCs first form during vertebrate embryogenesis in the second or definitive wave of haematopoiesis. The first or primitive wave merely gives rise to primitive red blood cells that are required for immediate gas exchange and die off quickly. The definitive wave generates the HSCs and occurs in close association with the ventral wall of the dorsal aorta (DA).
Little is known about the molecular and cellular steps that lead to the formation of HSCs and that distinguish HSCs from the primitive blood cells. This is due to the late appearance of HSCs during ontogeny and the fact that primitive and definitive haematopoiesis share a number of key regulators as well as a common origin with the endothelium. Thus, loss of primitive blood or loss of blood circulation causes lethality prior to HSC emergence in mammalian embryos. By contrast, zebrafish embryos survive even in the absence of primitive blood circulation as they are small and develop externally, and gas exchange through passive diffusion through the skin is sufficient. This makes zebrafish an ideal vertebrate model to study HSC formation in. In zebrafish embryos, putative HSCs are found at 24 hours post fertilisation. They are defined by (a) the expression of HSC genes, like those for the transcription factors Runx1, c-Myb and Ikaros, and (b) the location of the cells in association with the ventral wall of the DA (Gering and Patient, 2005). A functional test of these cells is yet to be done. Here, I propose to functionally test the putative HSCs.
To tag the putative HSCs, an RFP reporter gene shall be expressed in HSCs under the control of the c-myb regulatory elements in transgenic zebrafish. Red fluorescent cells will be isolated by flow cytometry and injected into host embryos. The cells will then be followed in the host to examine whether they can generate blood cells of all lineages for several months and, therefore, constitute HSCs or their precursors. Furthermore, these experiments will provide a functional HSC promoter that can be used for misexpression of transgenes in HSCs, and will allow the isolation of HSCs for transcriptome analysis in the future.
Little is known about the molecular and cellular steps that lead to the formation of HSCs and that distinguish HSCs from the primitive blood cells. This is due to the late appearance of HSCs during ontogeny and the fact that primitive and definitive haematopoiesis share a number of key regulators as well as a common origin with the endothelium. Thus, loss of primitive blood or loss of blood circulation causes lethality prior to HSC emergence in mammalian embryos. By contrast, zebrafish embryos survive even in the absence of primitive blood circulation as they are small and develop externally, and gas exchange through passive diffusion through the skin is sufficient. This makes zebrafish an ideal vertebrate model to study HSC formation in. In zebrafish embryos, putative HSCs are found at 24 hours post fertilisation. They are defined by (a) the expression of HSC genes, like those for the transcription factors Runx1, c-Myb and Ikaros, and (b) the location of the cells in association with the ventral wall of the DA (Gering and Patient, 2005). A functional test of these cells is yet to be done. Here, I propose to functionally test the putative HSCs.
To tag the putative HSCs, an RFP reporter gene shall be expressed in HSCs under the control of the c-myb regulatory elements in transgenic zebrafish. Red fluorescent cells will be isolated by flow cytometry and injected into host embryos. The cells will then be followed in the host to examine whether they can generate blood cells of all lineages for several months and, therefore, constitute HSCs or their precursors. Furthermore, these experiments will provide a functional HSC promoter that can be used for misexpression of transgenes in HSCs, and will allow the isolation of HSCs for transcriptome analysis in the future.
