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. 2021 May 21;12:3020. doi: 10.1038/s41467-021-23294-7

Fig. 3. Default dorsal-intermediate spinal cord identity in EMLOs can be ventralized.

Fig. 3

a IF of day 13 and day 16 H3.3.1 EMLOs depicts TFAP2α (green) and GATA4 (cyan) (top), and TFAP2α (green) and GATA6 (cyan) (bottom), counterstained with TUJ1 (red). Position of gut tube is shown to highlight TFAP2α cell migration. b N-cadherin is restricted to SOX2 domain and gut tube anterior pole (dotted circle). c Schematic representation of spinal cord domains along dorsal–ventral axis with ventral (Hh) and dorsal (BMP) morphogen signaling gradients. Somite (S) and notochord (ventral, blue circle) are shown. d Day 22 spinal cord interneuron domain subtypes PAX2 (dI4, dI6, V0, V1), LBX1 (dI4-dI6), LHX9 (dI1), TLX3/ISL1 (dI3), CHX10 (V2a) in neural SC compartment. e Nkx-6.1 expression at day 22 suggests ventralization of spinal cord phenotype by addition of 500 nM Hh-Ag1.5 on day 10. f Quantification of neuronal subtype biomarkers in H3.3.1 EMLOs under control (DMSO, gray) or ventralizing (Hh-Ag1.5, blue) conditions. LHX9 (****p < 0.0001, t = 11.66, df = 15), ISL1 (n.s. p = 0.36, t = 0.9480, df = 19), LBX1 (*p = 0.047, t = 2.133, df = 18), PAX2 (***p = 0.0002, t = 4.352, df = 23), CHX10 (***p = 0.0005, t = 4.518, df = 14), Nkx-6.1 (****p < 0.0001, t = 23.09, df = 17) by unpaired two-tailed t test. n = 6000 cells minimum counted per condition from N = 1 experiment (Source Data file). The box and whisker plot statistics are as follows: LHX9 (Hh-Ag1.5: max = 8.35, min = 1.79, median = 4.52, q1 = 2.51, q3 = 6.97; DMSO: max = 48.50, min = 24.25, median = 35.45, q1 = 37.80; q3 = 30.86); ISL1 (Hh-Ag1.5: max = 88.07, min = 52.89, median = 67.52, q1 = 63.34, q3 = 80.90; DMSO: max = 90.68, min = 21.66, median = 68.15, q1 = 49.29, q3 = 74.60); LBX1 (Hh-Ag1.5: max = 98.24, min = 81.02, median = 94.29, q1 = 90.48, q3 = 96.91; DMSO: max = 50.24, min = 21.43, median = 39.72, q1 = 30.74, q3 = 45.77); PAX2: (Hh-Ag1.5: max = 94.68, min = 67.22, median = 79.94, q1 = 75.94, q3 = 90.63); CHX10 (Hh-Ag1.5: max = 39.62, min = 10.03, median = 19.87, q1 = 13.29, q3 = 26.98; DMSO: max = 8.30, min = 2.87, median = 5.54, q1 = 3.67, q3 = 5.75); Nkx-6.1 (Hh-Ag1.5: max = 98.57, min = 81.78, median = 94.81, q1 = 90.99, q3 = 97.16; DMSO: max = 33.67, min = 10.03, median = 19.87, q1 = 13.29, q3 = 28.34). g Dorsal (dI) and ventral (V) interneuron subtypes in scRNAseq neuronal cluster 10. Color-coded biomarker combinations are listed. Histogram data reported as (mean ±s.e.m.). Individual scale bars provided. For scRNAseq, N = 1 biological sample was analyzed with n = 15,576 cells. For IF, CDH2, GATA4, GATA6, TUJ1, and TFAP2α immunostain was performed for each of the N = 11 H3.3.1 EMLO formation experiments with similar results a, b. The experiment in d–f was performed N = 1 time with a parallel analysis performed using the scRNAseq data.