Functional analysis of human hematopoietic stem cell gene expression using zebrafish.
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.
Eckfeldt
Craig E
CE
Division of Hematology, Oncology, and Transplantation, Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.
Mendenhall
Eric M
EM
Flynn
Catherine M
CM
Wang
Tzu-Fei
TF
Pickart
Michael A
MA
Grindle
Suzanne M
SM
Ekker
Stephen C
SC
Verfaillie
Catherine M
CM
D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D005810:Multigene Family / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D020112:Rhodamine 123 / Q000032:analysis; D063268:Sialic Acid Binding Ig-like Lectin 3; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics / Q000502:physiology
D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C486055:SPRY1 protein, human; D063268:Sialic Acid Binding Ig-like Lectin 3; D029961:Zebrafish Proteins; C434248:spry4 protein, zebrafish; D020112:Rhodamine 123; D051997:ADP-ribosyl Cyclase 1