Fig. 7: NOTCH1/JAG1 cis interaction and HSC emergence is dependent on RFNG.

A IHC on E11.5 AGM sections for RFNG (yellow), GFI1 (red), CD31 (magenta) and DAPI (blue). Representative image from 4 different IHC with n = 4 embryos. Scale bar = 10 μm. B Flow cytometry plots for Sca1 and EPCR after gating for CD31 + /cKIT + /CD45 + . SCA-/EPCR- or SCA + /EPCR+ and further analysed for RFNG. RFNG gate is superimposed onto Sca1 and EPCR plot. C Quantification of the frequency of RFNG in either SCA-/EPCR- or SCA + /EPCR+ fraction of CD31 + /cKIT + /CD45 + . Vertical error bars indicate the mean and standard deviation values. Statistical significance was calculated with two-tailed t-tests. (3 independent experiments with n = 21 embryos). Source data are provided as a source data file. D CD31 + /cKIT + /CD45+Sca1+EPCR + T2-HSCs were gated for RFNG expression. RFNG- and RFNG + T-HSCs were further plotted for JAG1 and NOTCH1. E Quantification of the frequency of JAG1/NOTCH1 in either RFNG- or RFNG+ fraction of T2-HSCs. Statistical significance was calculated with two-tailed t-tests with data derived from (n = 3 independent with n = 21 embryos). Vertical error bars indicate the mean and standard deviation values. Source data are provided as a source data file. F Cartoon of experimental set up. E10.5 AGMs were cultured as explant with 10 μM control (scramble) FANA-ASO or 10 μM of RFNG FANA-ASO and processed for FACS analysis 48hrs later. Flow cytometry plots for Sca1 and EPCR after gating for CD31 + /cKIT + /CD45+ for scramble FANA-ASO or RFNG FANA-ASO. The T2-HSCs were then further analysed for JAG1 and NOTCH1 levels. G Quantification of the frequency of cKIT, cKIT+CD45+ and cKIT+CD45 + EPCR+Sca1 + T2-HSCs in either scramble or RFNG ASO. (n = 3 independent experiments, n = 24 embryos). Statistical significance was calculated with two-tailed t-tests. Vertical error bars indicate the mean and standard deviation values. Source data are provided as a source data file. H Scheme of proposed modulation of HSC specification through RFNG. GFI1 HE can be separated into HSC-HE that expresses higher levels of Jag1. Within the GFI1 + IAHC, some JAG1 positive cells co-express NOTCH1 on the surface, and this co-expression exists as a cis (inhibitory) conformation due to the presence of RFNG. RFNG expression is restricted to the T2-HSC population and this cis interaction is necessary to maintain stem cell identity by regulating cell cycle and differentiation-associated genes. (ASO: Antisense oligonucleotides).