Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation

Abstract

Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.

Figure 1.

Analysis of EHT cell subsets. (A) Whole-mount image of a 34-sp Ly6aGFP embryo showing expression of CD31 (magenta), cKit (red), and GFP (green). The aorta, vitelline artery, and somatic vasculature are indicated. (B) Four types of aortic cells during EHT in a Ly6aGFP AGM section (36 sp) stained with anti-CD31 (magenta) and anti-cKit antibodies (red). ECs are CD31⁺cKit⁻GFP⁻, HECs are CD31⁺cKit⁻GFP⁺, HSCs are CD31⁺cKit⁺GFP⁺, and HCs are CD31⁺cKit⁺GFP⁻. (C) Transverse section through a 36-sp Ly6aGFP embryo showing expression of CD34 (red) and GFP (green). A hematopoietic cluster with some GFP⁺ cells is located ventrally. GFP⁺ ECs are scattered throughout the aorta. (D) Different GFP⁺ cell types (arrowheads) in an E10.5 Ly6aGFP aorta. (Endothelial) two flat GFP⁺ ECs; (bulging) rounding-up of a GFP⁺ EC; (juxtaposed) round HC closely adhering to an EC; (distal) round HC on the distal side of the cluster. The number of cells/aorta is listed below at the 32-, 34-, and 37-sp stages. Bars: (A) 100 µm; (B and D) 10 µm; (C) 50 µm. (E) Scatter plot showing the distribution and sorting gates for EHT subsets EC, HEC, HSC, and HC from E10.5/E11 Ly6aGFP AGMs. (F) Hematopoietic progenitor numbers (total CFU-C [CFU-culture]) per AGM. EHT subsets from E10.5 Ly6aGFP AGMs (34–39 sp) were plated in methylcellulose, and colonies were counted at day 12 (SD is shown; n = 4). (G) HSC long-term repopulating activity in E11 Ly6aGFP AGM EHT subsets (40–49 sp). Irradiated adult recipients (n = 4) were injected with 5–9 ee of ECs, 4–9 ee of HECs, 1–5 ee of HSCs, and 4–8 ee of HCs together with 2 × 10⁵ spleen cells (recipient type). Percentage of donor cell chimerism at 4 mo after injection is shown. Indicated above each bar is the number of repopulated recipients/number of recipients injected. (H) Normalized number of mapped fragments for genes encoding the markers used for sorting EHT fractions. FPKMs of CD31, cKit, and Ly6aGFP per fraction are shown (error bars are SD). (I) Heat map of FPKMs for genes encoding several relevant cell surface molecules: Cdh5, Tek, Esam, Kdr, and Eng in each of the sequenced cell fractions and the frequency of cells in each sorted fraction expressing the corresponding protein. Significant positive correlation is observed between FACS and RNAseq data (r² = 0.54; **, P = 0.01).

Figure 2.

RNAseq data analysis. (A) Distribution of raw counts per sample (left) and edgeR internal normalized counts (right). The normalized counts are used in all subsequent analyses. Datasets for three biological replicates are shown for ECs, HECs, HSCs, and HCs. Biological replicate 1 includes two 36-sp and two 37-sp embryos; replicate 2 includes four 34-sp, two 35-sp, three 36-sp, and five 37-sp embryos; replicate 3 includes two 35-sp, one 36-sp, two 38-sp, one 39-sp, one 40-sp, and one 41-sp embryos. (B) BCV in RNAseq samples from three biological replicates of relevant EHT cell fractions: EC, HEC, and HSC. (C) Venn diagram showing numbers of DEGs in comparisons of HECs versus ECs, HSCs versus HECs, and HSCs versus ECs. Total DEGs is 530 (see Table S2 for gene lists). (D) Heat maps showing all 530 DEGs and hierarchical clustering of the genes in each EHT cell fraction from the three biological replicates.

Figure 3.

GO terms/processes enriched in EHT subsets. (A) DEG patterns that are EC specific are shown (left): high-intermediate-low (HIL), HHL, HLI, and HLL. GO enrichment analysis was performed using WebGestalt, and enriched GO terms are summarized by REVIGO (right). (B) DEG patterns that are HEC specific are shown (left): LHL, IHL, IHI, and LHI. GO enrichment analysis and GO terms are summarized by REVIGO (right). (C) DEG patterns that are HSC specific are shown (left): LHH, LIH, IIH, LLH, and ILH. GO enrichment and GO terms are summarized by REVIGO. Rectangle size represents the number of DEGs in the accompanying GO term. See Table S3 for enriched ontology terms.

Figure 4.

Changing processes and cell surface molecules during EHT. (A) GSEA for VEGF, Notch, Hypoxia up-regulated genes, genes specifically expressed in HSCs, stem cell function–related gene sets like “telomere lengthening” and “DNA repair,” cell cycle–related gene sets, and early hematopoietic progenitor–specific genes. (B) Receptor-related genes with significant expression changes in EC to HEC and HEC to HSC transitions. Blue, increased expression; red, decreased expression; gray font, genes with low overall expression levels as defined by edgeR-calculated logCPM of <3 (and higher probability of being false positive); asterisks, genes differentially expressed during both transitions; logFC, log fold change.

Figure 5.

Gpr56 is a heptad target in mouse and human blood progenitors. (A) Mean FPKM values of heptad factors in EC, HEC, and HSC fractions. (B) A Venn diagram showing the overlap between sites with combinatorial binding of Scl, Gata2, Runx1, Erg, Fli1, Lyl1, and Lmo2 in HPC7 cells (Heptad targets) and 530 DEGs during EHT. (C) Heat map of top 10 heptad target DEGs based on highest expression in HSCs and with respective mean FPKM values inside heat map. (D) qPCR for TF enrichment at Gpr56-37 as compared with IgG and control in HPC7 mouse myeloid progenitor cells (n = 4). (E) Transfection assays in 416B and HPC7 mouse progenitors show enhancer activity of Gpr56-37 (n = 3). (F) Transactivation assays in Cos7 cells showing synergistic responsiveness of the Gpr56-37 element to Runx1, Gata2, and Fli1 (n = 4). (D–F) Error bars show SD. (G) TF binding at HsGPR56-48 (corresponding region to MmGpr56-37) in primary human CD34 HSCs.

Figure 6.

In silico and in vivo analysis of Gpr56. (A) ISH of WT mouse E10.5 AGM sections shows specific expression of Gpr56 in some HCs, a few cells lining the aorta (Ao), and the notochord (NT). The top images show low magnification of AGM cross-section, and the bottom images show high magnification of the boxed areas. (B) ISH of E10.5 Ly6aGFP AGM shows coexpression of GFP and Gpr56 in some HCs. (C) Homology relationships of the Gpr56 coding sequence of different vertebrate species. (D–F) Analysis of WT and gpr56 MO zebrafish for the presence of HSCs. (D) ISH with the HSC marker cmyb at 30 hpf. (E) Fluorescent analysis of WT and MO-injected CD41:GFP transgenic embryos at 48 hpf. Numbers in the panels indicate the number of embryos with the depicted phenotype. Arrowheads (left) indicate CD41-expressing HCs in the aorta. The dashed lines (right) indicate the outline of the morphant zebrafish embryo for orientation purposes. (F) ISH with arterial cell marker grl. No vascular or developmental abnormalities can be observed in gpr56 morphant embryos. (G) HSC rescue of gpr56 morphant zebrafish with gpr56 RNA (zebrafish and mouse) as shown by ISH for cmyb. Ectopic cmyb expression in the posterior cardinal vein is clearly visible. No vascular abnormalities can be observed by grl ISH. (D and G) Insets show boxed areas at higher magnification. Bars: (A) 30 µm; (B) 10 µm; (D–G) 100 µm. (H) Effect of human GPR56 activity in neutrophil differentiation of the 32D-CSF3R unipotent stem cell line. 32D-CSF3R cells cultured in medium containing CSF3 efficiently differentiated into neutrophils. Only constitutive active mutant GPR56 (MUT) could block differentiation. Diff ct, differential count; Cntl, empty vector control; WT, WT human GPR56 vector; mut, constitutively active human GPR56 mutant vector; Bl, blast morphology; In, intermediate morphology; Ne, neutrophil morphology.