Title: Functional analysis of human hematopoietic stem cell gene expression using zebrafish
Authors: Eckfeldt, Craig E ×
Mendenhall, Eric M
Flynn, Catherine M
Wang, Tzu-Fei
Pickart, Michael A
Grindle, Suzanne M
Ekker, Stephen C
Verfaillie, Catherine #
Issue Date: Aug-2005
Publisher: Public Library of Science
Series Title: PLoS Biology vol:3 issue:8 pages:1449-1458
Abstract: 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.
ISSN: 1545-7885
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Interdepartemental Stem Cell Institute (-)
× corresponding author
# (joint) last author

Files in This Item:
File Description Status SizeFormat
Eckfelt et al 2005 Plos Biology.pdf Published 381KbAdobe PDFView/Open
Eckfeldt - Plos Biology - 2005.pdf Published 381KbAdobe PDFView/Open


All items in Lirias are protected by copyright, with all rights reserved.

© Web of science