Fig 2: Immunohistochemical analysis of myenteric plexus in the 3 HSCR subgroups (white bars) (HSCR patients with obstructive symptoms (OS), with HAEC or diarrhoea (HAEC/D) and without complications (w/oC)) and in ARM (grey bar) patients. (A) Immunohistochemical labeling of myenteric plexus with anti-HuC/D and anti-nNOS antibodies (Scale bar = 100 µm). (B) Number of HuC/D-IR myenteric neurons per ganglion (*p = 0.0011). (C) Number of nNOS-IR myenteric neurons per ganglion (*p = 0.0095; **p = 0.002; ***p = 0.006). (D) Number of non nNOS-IR myenteric neurons per ganglion (*p = 0.019; **p = 0.009; ***p = 0.002). (E) ENS area occupied by tissue surface (*p = 0.007; **p = 0.02; ***p = 0.001). Medians with ranges. ENS: enteric nervous system.

Fig 3: Single cell RNA-seq reveals the diversity of radial glia progeny in the adult forebrain. (A) Violin plots showing the expression of known radial glia markers (top), pan-neuronal markers (middle, elavl3 and map2), an early neurogenic fate marker (middle, insm1a) and markers of mature synaptically integrated neurons (bottom). (B) tSNE plot revealing five different clusters from a total of 264 RG, NBN and MN cells (top). The cell number per cluster is: RG, 76 cells; NBN.1, 80 cells; NBN.2, 54 cells; MN, 44 cells; and OPC, 10 cells. Smaller panels underneath show expression of characteristic marker genes that separate the different clusters. (C) Heat map with the top 15 cluster-specific genes for the five identified clusters.

Fig 4: Neuron specific activation of mHTT-?N17-exon1 causes motor behavioral phenotype. a elavl3:cre (n = 23), b gfap:cre (n = 24), c mylpfa:cre (n = 34), and d etv2:cre (n = 21) transgenic lines were incrossed to mHTT-?N17-exon1 to activate the transgene specifically in neurons, glia, muscle, and vascular cell lineages respectively. GFP fluorescence in living embryos demonstrates the spatially restricted expression of each line at 5 days post fertilization. e A disease free survival curve demonstrating that only elavl3:cre/mHTT-?N17-exon1 fish develop a behavioral phenotype similar to ubiquitous activation of mHTT-?N17-exon1, p < 0.01, Kaplan Meier survival analysis. f Disease progression of elavl3:cre/mHTT-?N17-exon1 fish is similar to ubiquitious mHTT-?N17-exon1 fish although temporally delayed, note the discontinuous x-axis (n = 23). Behavioral Stages are described in Fig. 2

Fig 5: Functional impacts of a HSCR-associated SNP (rs2435357) and the deletion of a novel S-HSCR enhancer on RET expression. (A) ATAC-seq and ChIP-seq profiles of hPSC and hNC in intron 1 of RET show that rs2435357 is residing in a hNC-specific ATAC-seq peak. (B) Location of rs2435357 in the RET gene locus and in the sgRNA used for CRISPR-Cas9-mediated HDR for editing the C allele to the HSCR-associated risk allele T. The electrographs of Sanger sequencing show the successful introduction of the risk allele at rs2435357 in the UE-rs2435357 hPSC line. (C) Differentiation strategy to generate human neural crest (hNC) and neuronal progenitor (hNP). HU is encoded by the ELAVL4 gene. Immunostaining of SOX10 and TUJ1 in hNC and hNP of the control and the mutant (UE-rs2435357) lines. Scale bars: (hNC) 100 µm; (hNP) 200 µm. RT-qPCR analysis showing the comparable ELAVL4 expression level in hNP in the control (n = 5) and the mutant (UE-rs2435357) (n = 3) lines. t-test, (ns) not significant. (D) RT-qPCR analysis showing RET expression in the hPSC and hNP stages of the control (n = 5) and the mutant (UE-rs2435357) (n = 3). t-test, (ns) not significant. (E) Hi-C data from neural progenitor cells show that the enhancer in intron 1 of RASGEF1A (marked in yellow on the right) has physical interaction with the promoter of RET (marked in yellow on the left) at 10-kbp bin size. (F) ATAC-seq and ChIP-seq data from hPSC and hNC at the RASGEF1A intron 1 locus. (G) The design of sgRNAs used for the CRISPR-Cas9 system for deleting the DNA fragment in RASGEF1A intron 1. Genotyping reveals the specific deletion of RASGEF1A intron 1 in the UE-RASGEF1A-int1-KO hPSC line. (WT) Wild type, (KO) knockout. (H) Immunostaining of SOX10, TUJ1, and HU in hNC and hNP of the control and the mutant (RASGEF1A-int1-KO) lines, respectively. Scale bars: (hNC) 100 µm; (hNP) 200 µm. (I) RT-qPCR reveals the expression level of RET in the hPSC and hNP stages of the control (n = 4–5) and the mutant (RASGEF1A-int1-KO) (n = 6–7). t-test, (ns) not significant.