Functional analysis of human hematopoietic stem cell gene expression using zebrafish

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.

Figure 1. Fluorescence-Activated Cell Sorting and Gene Expression Analysis

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.

Figure 2. Functional Genomics Screen in Zebrafish

The hematopoietic function of differentially expressed candidate genes was determined by injecting MOs into one- to two-cell embryos from gata1:DsRed Tg zebrafish to disrupt gene expression. Injected embryos were scored at 30–48 hpf for the presence of DsRed⁺ blood cells by fluorescence microscopy. Subsequently, MO-targeted embryos with gross hematopoietic defects were analyzed for the expression of the early hematopoietic markers gata1 and scl by whole-mount in situ hybridization, and the late hematopoietic markers hbae1 and lcp1 by Q-RT-PCR.

Figure 3. Representative Hematopoietic Phenotypes Observed in MO-Targeted Zebrafish Fluorescence microscopic images of gata1:DsRed Tg zebrafish embryos display the hematopoietic phenotypes observed for six MO-targeted zebrafish embryos compared to an uninjected control

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

Figure 4. spry4 Is Required for Normal Hematopoietic Development in Zebrafish Embryos

(A) The observed frequency of hematopoietic defects in gata1:DsRed Tg zebrafish embryos are indicated for two independent spry4-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 SPRY1 DNA expression vector or a GFP control (white bars) (error bars = standard deviation of the mean, *p < 0.05, **p ≤ 0.01). (B) Representative pictures of the phenotypes seen with spry4 MO1 at 48 h using fli1:EGFP/gata1: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 gata1:DsRed and fli1:EGFP Tg images are representative of three experiments of n > 40 embryos each. (C) Q-RT-PCR analysis of hbae1 and lcp1 transcripts in spry4 MO1, control mismatch MO and a gata1 MO-injected zebrafish at 48 hpf (*p < 0.05). (D) Injection of 30 pg of human SPRY1 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 n = 30), and a representative bright-field image is pictured with the fluorescence micrograph overlaid (left). Q-RT-PCR analysis of hbae1 and lcp1 transcripts in SPRY1 overexpressing embryos relative to uninjected clutchmate controls (three experiments of n = 5 embryos) (right).

Figure 5. spry4 Is Required for Early Hematopoietic Development, but Not Mesodermal Commitment, in Developing Zebrafish Embryos

(A) Expression of the early hematopoietic marker scl was reduced at the four-somite stage (arrow) and virtually absent at the 20-somite stage in spry4^MO embryos compared to uninjected controls. (B) The more mature hematopoietic marker gata1 was also reduced in spry4^MO compared to uninjected zebrafish embryos at 20 somites. (C and D) In contrast to hematopoietic genes, the mesodermal-specific flk1 (vasculature) transcripts were expressed at similar levels to uninjected controls at 26 hpf, and myod (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 n ≥ 8 embryos.