The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

(A) Schematic illustration of the human EREG gene locus.The location of the guide RNAs for CRISPR/Cas9-mediated ablation of EPIREGULIN expression (gEREG KO1 + 2) is shown, as well as the location of primer binding sites (Fwd, forward; Rev, reverse) for the generation of DNA templates for in vitro gRNA efficiency testing.(B) Guide RNA efficiencies were tested in vitro.The effects of the gEREG KO1 + 2 RNAs to direct Cas9-mediated cutting of PCR templates was analyzed by agarose gel electrophoresis.Schemes of the sizes of PCR templates, guide RNA binding sites, and expected sizes of cut fragments are indicated below.(C) CRISPR/Cas9-mediated targeting of EREG was confirmed in the CRTDi004-A iPSC line by electroporation of Cas9/gRNA ribonucleoprotein complexes together with a GFP plasmid, followed by FACS of GFP-positive cells, PCR amplification of the target region and Sanger sequencing.The sequencing results are shown for gEREG KO1 + 2. (D) DAPI staining and immunofluorescence for GFP, KI67, and SOX2 of an electroporated human cortical organoid derived from the HPSI0114i-kolf_2 iPSC line.(E-H) Quantifications of KI67 and SOX2 in the VZ and SVZ/CP.(I) DAPI staining and immunofluorescence for GFP and CTIP2 of an electroporated human cortical organoid from the CRTDi004-A iPSC line.(J, K) Quantifications of CTIP2 in the VZ and SVZ/CP.Data information: Scale bar, 100 µm.Bar graphs represent mean values.Error bars represent SD of 12-15 organoids from three batches.**p < 0.01; Student's t-test.

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Figure EV3 .
Figure EV3.Gene expression analysis upon addition of EPIREGULIN to the mouse neocortex.(A)Schematic illustration of the experimental workflow.Mouse brain hemispheres (E14.5)from the Tubb3::GPF line(Attardo et al, 2008)  were isolated and cultured under rotation in the presence of 50 ng/mL of EPIREGULIN for 24 h, dissociated, stained for Sox2 and Tbr2, and cell populations isolated by immuno-FACS based on the indicated marker combinations.(B) Gating strategy of RG (top, left) based on high levels of Sox2 and low levels of Tbr2; IP (top, right) based on high levels of Tbr2, irrespective of other markers; and neurons (bottom) based on enrichment of GFP expressed from the Tubb3 promoter and low level of Tbr2, followed by exclusion of Sox2positive cells.(C) Confirmation of cell type identity by RT-qPCR expression analysis of marker genes characteristic of RG (Sox2, Prom1), IP (Eomes), and neurons (Dcx, Tubb3) for control and hemispheres treated with EPIREGULIN for 24 h relative to Gapdh.(D) Expression of Sox2, Eomes, Tubb3, and Ereg in RG, IP, and neurons analyzed by RNA-seq.(E) Volcano plots of log10 (p value) against log2 fold change representing the differences in gene expression in the indicated cell types analyzed by RNA-seq.Gray, non-significant; blue, downregulated.(F) Principal component analysis (PCA) based on the 500 most divergent genes.The percentage of variance covered by the first two components is indicated.Data information: Bar graphs represent mean values.Error bars represent SD of 3 mNcx samples from different litters.E, Wald test of DESeq2 was used and P Values were corrected for multiple testing with the Independent Hypothesis Weighting package (IHW 1.18.0).

Figure EV4 .
Figure EV4.NSC proliferation upon exposure to different growth factors.(A) Schematic illustration of the experimental workflow.Early (p0) and late (p9) passage mouse NSC cultures were treated with different growth factors for 3 days and their proliferation was assessed.(B) Images of mNSC cultures following treatment with EPIREGULIN in culture medium containing FGF and with or without EGF.The control mNSCs were cultured in a medium with EGF and FGF.(C) Quantification of cells on days 1, 2, and 3 following EPIREGULIN treatment, either with or without EGF, shown as fold change relative to 1 h.(D) Staining for DAPI and immunofluorescence for PH3 of mNcx slices treated with 10 µM of the receptor inhibitor Dacomitinib for 24 h.Note the reduced tissue integrity and the apoptotic nuclei in the inset (right).Data information: Scale bars, 100 µm.C, Data points are from two different mNSC lines.

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Figure EV5.Editing of histone methylation at the Ereg locus in mNSCs.(A) Epigenome editing (EE) employing the catalytic domain of KDM6B (JMJC_6B) fused to nuclease deficient Cas9 (dCas9) in mNSCs.Histone methylation and gene expression were analyzed 2 days post-nucleofection following FACS isolation of GFP-positive cells.(B) The location of the gRNAs and primer binding sites (PP, primer pair) for ChIP-qPCR is shown for the Ereg locus.Guide RNAs gEreg EE1 + 2 and gEreg EE3 + 4 were co-expressed from one plasmid, respectively.(C) Level of H3K27me3 at Hoxb, Eomes, Actb, and Ereg (PP1 to PP3) as determined by ChIP-qPCR in mNSCs.(D) Bright-field and GFP fluorescence images of mNSCs 2 days post nucleofection with a dCas9-JMJC_6B-T2A-EGFP-gLacZ plasmid.(E) ChIP-qPCR analysis of H3K27me3 around the TSS of Ereg and two unrelated genes (Hoxb5, Eomes) after epigenome editing at the Ereg locus.(F, G) Expression of Sox2 and Ereg as determined by RT-qPCR upon epigenome editing using gEreg EE1 + 2 and gEreg EE3 + 4. Expression normalized to Gapdh and relative to gLacZ EE.Data information: Scale bar, 100 µm.Bar graphs represent mean values.Error bars represent the SD of three replicates (from two to three independent experiments).One-way ANOVA with Dunnett post hoc test; no statistically significant changes were detected.