Functional Analysis of the Differential Gene Expression Profile of Human HSC Using a Functional Genomics Screen in the Zebrafish.

Self-renewal and lineage differentiation of hematopoietic stem cells (HSC) is likely regulated by a combination of intrinsic and extrinsic signals. At present these signals are poorly understood. Recently, numerous groups have identified the expressed gene profile of HSC in an attempt to identify novel genes that regulate HSC fate decisions. Likewise, our group compared the expressed gene profiles of CD34⁺CD33⁻CD38⁻Rho^loc-kit⁺ (Rho^lo) cells, enriched in primitive progenitors and presumed human HSC, compared to CD34⁺CD33⁻CD38⁻Rho^hi (Rho^hi) cells, depleted of such cells, from umbilical cord blood (UCB) and bone marrow (BM) to identify conserved signaling pathways active in these ontogenically distinct populations using the Afftymetrix™ HG-U133 GeneChip® set. We identified a putative molecular signature for human HSC containing 286 genes, expressed more highly in Rho^lo or Rho^hi cells from both cell sources using a p<0.05 and fold change of 1.5 cutoff. To assess the role of this series of genes in a high-throughput fashion, we developed an in vivo functional genomics screen in the zebrafish that allows for the determination of hematopoietic function of the differentially expressed genes. Candidate gene expression was knocked down by injecting morpholino antisense oligonucleotides (MO) into 1–4 cell embryos from GATA1:DsRed transgenic zebrafish that have red fluorescent GATA1⁺ blood cells and blood production was scored by fluorescence microscopy at 30 and 48 hours post-fertilization. Of the 286 differentially expressed genes, 128 were deemed too universal or likely non-specific for hematopoiesis, and were not targeted. These included histones, HLA-antigens, hemoglobin genes, and genes encoding proteins involved in general cellular metabolism. Of the remaining 158, MO were designed for 70 (44%), no zebrafish ortholog could be identified for 72 (46%), inadequate sequence information was available to design morpholinos for 9 (6%) and multiple zebrafish orthologs were identified for 7 (4%). Fluorescence microscopy of GATA1:DsRed fish has revealed a reproducible reduction in GATA1⁺ blood cell number for 13 of 55 MO-targeted genes analyzed to date, giving a 24% frequency for hematopoietic phenotypes in the screen, that compares very favorably with the noted 0.5–1% frequency of hematopoietic genes identified in ENU mutagenesis screens that mutate genes in a near random fashion. The functionally-validated genes identified thus far include known genes that lack a known hematopoietic function such as SPRY1, CRTAP, IRAK3 and UCP2, as well as genes that currently lack a functional annotation such as C12orf2, DKFZp564D137, MGC15875 and FLJ21269. Hematopoietic phenotypes are being further characterized by whole-mount in situ hybridization for hematopoietic genes and rescue of hematopoietic phenotypes by co-injection of overexpression vectors in the zebrafish. In addition, we are confirming the role of the genes in mammalian hematopoiesis using overexpression and knockdown studies in murine and human repopulating HSC. This is the first description of a high-throughput functional genomics screen in the zebrafish to functionally validate differentially expressed genes, an essential step in obtaining meaningful functional data from global gene profiling studies.