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

The molecular repertoire of the 'almighty' stem cell

Stem cells share the defining characteristics of self-renewal, which maintains or expands the stem-cell pool, and multi-lineage differentiation, which generates and regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem-cell niche, but the specific signals involved are poorly understood. Recently, several studies have sought to identify the genetic mechanisms that underlie the stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an understanding of the true potential of stem cells.status: publishe

The transcription factors, Scl and Lmo2, act together during development of the haemangioblast in zebrafish

The transcription factors, Scl and Lmo2, are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl(-/-) embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of haemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important haematopoietic regulators is lost, reduced or delayed, pan-endothelial gene expression is downregulated and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in haemangioblast development within a multi-protein complex, as shown for erythropoiesis. Consistent with this, we find that Scl morphants cannot be rescued by a non-Lmo2 binding form of Scl but can be rescued by non-DNA binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other haematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell extrinsic signals.status: publishe

Representative Hematopoietic Phenotypes Observed in MO-Targeted Zebrafish Fluorescence microscopic images of <i>gata1</i>:DsRed Tg zebrafish embryos display the hematopoietic phenotypes observed for six MO-targeted zebrafish embryos compared to an uninjected control

<p>Phenotypes shown are representative of greater than 70% of injected embryos at 48 hpf (greater than or equal to three experiments of <i>n</i> > 40 embryos). Hematopoietic defects were quantified by Q-RT-PCR for the expression of erythroid-specific <i>hbae1</i> and myeloid-specific <i>lcp1</i> transcripts in MO-targeted embryos relative to uninjected clutchmate controls (greater than or equal to three experiments of <i>n</i> = 5 embryos; * <i>p</i> < 0.05)</p

spry4 Is Required for Normal Hematopoietic Development in Zebrafish Embryos

<div><p>(A) The observed frequency of hematopoietic defects in <i>gata1:</i>DsRed Tg zebrafish embryos are indicated for two independent <i>spry4</i>-targeted MOs (MO1 and MO2; black bars), a four-base mismatched control MO (MM MO; no bar indicates a 0% frequency), a low-dose injection of MO1 and MO2 individually and in combination (gray bars), and MO2 coinjected with a human <i>SPRY1</i> DNA expression vector or a GFP control (white bars) (error bars = standard deviation of the mean, *<i>p</i> < 0.05, **<i>p</i> ≤ 0.01).</p> <p>(B) Representative pictures of the phenotypes seen with <i>spry4</i> MO1 at 48 h using <i>fli1:</i>EGFP/<i>gata1:</i>DsRed double Tg zebrafish embryos that have EGFP⁺ vascular endothelial cells and DsRed⁺ erythroid cells. The embryos display a more drastic reduction in DsRed⁺ blood cells with some blood pooling and pericardial edema at higher MO doses (++MO) without major vasculature defects. The <i>gata1:</i>DsRed and <i>fli1:</i>EGFP Tg images are representative of three experiments of <i>n</i> > 40 embryos each.</p> <p>(C) Q-RT-PCR analysis of <i>hbae1</i> and <i>lcp1</i> transcripts in <i>spry4</i> MO1, control mismatch MO and a <i>gata1</i> MO-injected zebrafish at 48 hpf (*<i>p</i> < 0.05).</p> <p>(D) Injection of 30 pg of human <i>SPRY1</i> DNA expression vector resulted in an expansion of DsRed⁺ hematopoietic cells in the posterior ICM in greater than 50% of successfully injected embryos at 32 hpf (three experiments of <i>n</i> = 30), and a representative bright-field image is pictured with the fluorescence micrograph overlaid (left). Q-RT-PCR analysis of <i>hbae1</i> and <i>lcp1</i> transcripts in <i>SPRY1</i> overexpressing embryos relative to uninjected clutchmate controls (three experiments of <i>n</i> = 5 embryos) (right).</p></div

spry4 Is Required for Early Hematopoietic Development, but Not Mesodermal Commitment, in Developing Zebrafish Embryos

<div><p>(A) Expression of the early hematopoietic marker <i>scl</i> was reduced at the four-somite stage (arrow) and virtually absent at the 20-somite stage in spry4^MO embryos compared to uninjected controls.</p> <p>(B) The more mature hematopoietic marker <i>gata1</i> was also reduced in spry4^MO compared to uninjected zebrafish embryos at 20 somites.</p> <p>(C and D) In contrast to hematopoietic genes, the mesodermal-specific <i>flk1</i> (vasculature) transcripts were expressed at similar levels to uninjected controls at 26 hpf, and <i>myod</i> (muscle) had a slight defect (arrowhead) at 10 hpf, while expression was similar to controls at 26 hpf. All images are representative of greater than or equal to two experiments of <i>n</i> ≥ 8 embryos.</p></div

Fluorescence-Activated Cell Sorting and Gene Expression Analysis

<p>UCB or adult BM CD34⁺CD33⁻CD38⁻Rho^loc-kit⁺ (Rho^lo; stem cell enriched) and CD34⁺CD33⁻CD38⁻Rho^hi (Rho^hi; stem cell depleted) cell populations were sorted for subsequent global gene expression profiling. Total RNA was isolated from Rho^lo and Rho^hi cell populations prior to linear amplification and labeling for hybridization to the Affymetrix HG-U133 GeneChip set (approximately 45,000 probe sets) and subsequent data analysis.</p