Comparative Approach to the Hematopoietic Stem Cell





A comparison of hematopoiesis in the zebrafish and mouse species.  Note the theme of multiple waves of hematopoiesis, but the differences in the location between sequences.

Hematopoiesis involves the differentiation and proliferation of stem cells, which are specified during embryogenesis.  The vasculature and hematopoietic cells appear closely associated in embryonic sites and are thought to derive from a common cell known as the hemangioblast. Transcription factors such as SCL and LMO2 are critical for both vasculogenesis and hematopoiesis. To further our understanding of these molecular programs, we formed a consortium of investigators with expertise in stem cell biology and genomics.  Our aim is to create cDNA libraries from stem cell enriched populations from the mouse and zebrafish.  The stem cell sources include blast colonies from differentiated murine ES cells and marrow cells from transgenic zebrafish expressing green fluorescent protein (GFP) under the LMO2 promoter.  Global gene expression patterns will be evaluated by microarray analysis of wild type and mutant mice and zebrafish.  In situ hybridization studies performed on 6598 cDNA clones from zebrafish libraries have uncovered 128 zebrafish genes expressed in hematopoietic and vascular tissues during embryogenesis.  This includes genes such as draculin, a novel zinc finger transcription factor, which is the earliest marker of ventral mesoderm fated to become blood.  We plan to screen this zebrafish stem cell library by in situ hybridization to obtain new markers of stem cells and hemangioblasts.  Mammalian orthologs of these zebrafish genes will be characterized.  The genes will be positioned on the zebrafish genome map and correlated to known zebrafish mutations. Several factors will be studied by gene knockdown and overexpression in zebrafish, and by gene targeting experiments in murine embryonic stem cells.  Transgenic mice and zebrafish will be used to evaluate gene regulatory elements predicted by a novel computer algorithm.  The information developed in this proposal will be distributed through a WWWeb site. These studies should produce a better molecular characterization of hematopoietic and vasculogenic stem cells and could provide therapies for patients with anemia or leukemia.

Research Objectives:


This proposal utilizes a comparative approach to understanding hematopoietic stem cells.   By evaluating the gene expression program in similar populations of mouse and zebrafish cells, themes of conservation  (and divergence) will be elucidated.  Our experiments should also provide a view of the gene regulatory elements utilized by the vertebrate stem cell.  These studies will have impact on the functional characterization of the hematopoietic stem cell, and given the apparent plasticity of stem cell populations, may lead to new treatments for a variety of hematopoietic and non-hematopoietic diseases.


Aim1.     Characterization and Definition of Cell Populations:  Define genes expressed in hematopoietic stem cells (HSCs) and hemangioblasts (HAs).


Aim 2.      mRNA Expression Profile: Examine gene expression of the HSCs and HAs.


Aim 3.      Histology and Functional Genomics:  Examine regulatory elements and function of newly obtained genes.


Aim 4.       Establish Databases, Research Tools, and Create Outreach Programs.