GSE114464
Mesoderm-derived PDGFRA+ cells regulate emergence of hematopoietic stem cells in the dorsal aorta
During embryonic development, the first hematopoietic stem cells (HSCs) arise from a transient population of endothelial cells lining the ventral surface of the dorsal aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic day (E) 10.5. This region contains resident PDGFRA+ stromal cells (PSCs), but their identity and role in HSC generation in the AGM are not well understood. Using a library of compound transgenic mice, we identified a population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with PSC activity in the E10.5 and E11.5 mouse AGM. Freshly isolated PSCs were adept at forming blood vessels with CD31+ luminal endothelium enveloped by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of PSCs respectively, with severe loss of endothelial and pericyte-like cells, and concomitant loss of blood formation in the AGM. Lineage tracing studies using tamoxifen induction in PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothelial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) cells in the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice, we showed that MesP1 (mesoderm) derived PDGFRA+ cells dominated the sub-endothelial and deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1 (neural crest) derived cells at E13.5. Re-aggregation of E11.5 Mesp1 derived PSC cells with E13.5 aortic or adult cardiac non-hemogenic endothelial cells resulted in the generation of endothelial cell derived LT-HSCs. RNA-sequencing analysis of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes, WNT, BMP and Notch cell signalling pathways when re-aggregated with E11.5 Mesp1 derived PSCs. LT-HSC generation from these re-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA signalling. Taken together, we report that PSC populations in the AGM are temporally dynamic, and that MesP1-derived PSCs regulate hemogenic potential of the endothelium and that this cooperativity is dependent on PDGF-AA/PDGFRA signalling. This submission represents the RNAseq component of the study. Overall design: E11.5- and E13.5 AGM PSCs were isolated from Mesp1DsRed and Wnt1DsRed embryos and cultured as previously described. Dissected AGMs were transferred into collagenase Type II (263 U/ml; Worthington Biosciences, Lakewood, NJ) and placed on a shaker at 37°C for 15 min. The supernatant was passed through a 40 µm filter into a fresh tube and inactivated with 100% FCS. Cells were washed twice in 2% FCS in PBS and plated in alpha-MEM (Invitrogen, Carlsbad, CA) with 20% FCS, and penicillin/ streptomycin/ glutamine (P/S/G, Invitrogen)2 and cultured in the incubator at 37°C, 5% CO2 for 72 h. At the end of 72 hrs, cells were washed in PBS to remove non-adherent cells and cultures were continued in fresh medium. Cells were passaged on reaching 80% confluence. After passaging, cells were placed back in tissue culture flasks with alpha-MEM + 20% FCS + P/S/G for bulk passaging. Cells were routinely cryopreserved in 10% DMSO and 90% culture medium.   Re-aggregates were made by reconstituting FAC sorted cells from one AGM equivalent or 50,000 adult cardiac endothelial cells with 200,000 AGM PSCs. Dissociated cells were resuspended in 10µl of IMDM+ (IMDM Invitrogen) containing 20% fetal calf serum, 4mM L-glutamine, 50units/ml penicillin/streptomycin, 0.1mM mercaptoethanol, 100ng/ml IL, 100ng/ml SCF and 100ng/ml Flt3L (Peprotech). Re-aggregates were made by centrifugation in a yellow tip occluded by parafilm at 300 x g for 5 min and cultured on top of a 0.65µm Durapore filter (Millipore, Cat. No. DVPP02500) at the gas-liquid interface as described. Tissues were maintained in 5% CO2 at 370C in a humidified incubator.