ZFIN ID: ZDB-PUB-050818-2
Functional analysis of human hematopoietic stem cell gene expression using zebrafish
Eckfeldt, C.E., Mendenhall, E.M., Flynn, C.M., Wang, T.F., Pickart, M.A., Grindle, S.M., Ekker, S.C., and Verfaillie, C.M.
Date: 2005
Source: PLoS Biology   3(8): e254 (Journal)
Registered Authors: Ekker, Stephen C., Pickart, Michael
Keywords: Zebrafish, Embryos, Gene expression, Genetic screens, Hematopoiesis, Hematopoietic stem cells, Blood, Blood cells
MeSH Terms:
  • ADP-ribosyl Cyclase 1/analysis
  • Animals
  • Antigens, CD/analysis
  • Antigens, CD34/analysis
  • Antigens, Differentiation, Myelomonocytic/analysis
  • Cell Differentiation
  • Cell Lineage
  • Cell Proliferation
  • Fetal Blood/cytology
  • Flow Cytometry
  • Gene Expression Profiling/methods*
  • Genomics/methods*
  • Hematopoietic Stem Cells/cytology
  • Hematopoietic Stem Cells/metabolism*
  • Humans
  • Membrane Proteins/genetics
  • Membrane Proteins/physiology
  • Multigene Family/physiology
  • Nerve Tissue Proteins/genetics
  • Nerve Tissue Proteins/physiology
  • Oligonucleotide Array Sequence Analysis
  • Phosphoproteins/genetics
  • Phosphoproteins/physiology
  • RNA, Messenger/metabolism
  • Rhodamine 123/analysis
  • Sialic Acid Binding Ig-like Lectin 3
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/physiology
PubMed: 16089502 Full text @ PLoS Biol.
Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+)(CD33-)(CD38-)Rho(lo)(c-kit+) cells, enriched for hematopoietic stem/progenitor cells with (CD34+)(CD33-)(CD38-)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.