Fig. 6. Gut tube formation events impact neuronal patterning.

a–b Dual SMAD small molecule inhibitors LDN 193189 (LDN) and SB 431542 (SB) added before the EMLO elongation phase opposes mesenchymal and endodermal elongation a, and was quantified in b in representative ED-iPSC lines F3.5.2 (****p < 0.0001, t = 4.518, df = 61), H3.3.1 (****p < 0.0001, t = 7.293, df = 61), A2.1.1 (****p < 0.0001, t = 6.723, df = 61) by unpaired two-tailed t test. n = number of EMLOs counted (day 22). N = 3 biological replicates performed with similar results. Eight to 45 fields were analyzed per condition and data were quantified per field. Violin plot statistics are as follows: F3.5.2 (DMSO: max = 40, min = 0, median = 15.48, q1 = 0, q3 = 25.89; LDN + SB: max = 100, min = 0, median = 15.48, q1 = 25, q3 = 66.25); H3.3.1 (DMSO: max = 50, min = 0, median = 0, q1 = 0, q3 = 11.90; LDN + SB: max = 100, min = 0, median = 33.33, q1 = 18.33, q3 = 58.57); A.2.1.1 (DMSO: max = 48.86, min = 0, median = 13.39, q1 = 0, q3 = 20; LDN + SB: max = 100, min = 0, median = 60, q1 = 32.05, q3 = 73.21). c Identification of competing neuronal versus endodermal transcriptional programs in cluster 8 in day 16 H3.3.1 EMLOs by scRNAseq. Canonical endoderm markers FOXA2 and EPCAM were co-expressed with TUBB3 (243 cells). A subset of FOXA2/EPCAM/TUBB3 triple-positive cells (blue) also expressed MAP2 (128 cells, orange) and then further expressed SOX17 (16 cells, green). Cluster 8 is expanded. This phenomenon was previously described in vivo for enteric nervous system development²⁴. d IF of FOXA2 and TUJ1 in day 40 H3.3.1 EMLOs. High magnification Z-slices depict the emergence of FOXA2 + neurons in support of the dual embryonic origin model of the mammalian ENS. FOXA2 and TUJ1 staining on day 40 H3.3.1 EMLOs was performed in N = 3 biological replicate experiments with similar results. Individual scale bars provided. Data reported as (mean ± s.e.m.).