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.
PLoS Biology   3(8): e254 (Journal)
Registered Authors
Ekker, Stephen C., Pickart, Michael
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
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.
Genes / Markers
Show all Figures
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes