Fig. 7: Mandibular arch (MN)-like differentiation of the aggregates.

a Schema of the expression of EDN1 (endothelin 1) and DLX5/6 in first branchial arch. MX: maxillary arch. b Feature plot of EDNRA in scRNA-seq data. c Scheme of long-term culture of aggregates. Aggregates were cultured with or without EDN1 and BMP4 (bone morphogenetic protein 4) in the presence of bFGF (basic fibroblast growth factor) from day 4 and analyzed by real-time PCR and immunohistochemistry at day 14. NCC: neural crest cell. d Real-time PCR analysis of POU3F3, and DLX5, which are expressed in MX and MN, respectively. Data are presented as mean ± SEM (n = 4 independent experiments). Two-way ANOVA with Tukey–Kramer test was used. Source data are provided as a Source Data file. e Schema of the spatial organization of domains in the first branchial arch. f Real-time PCR analysis of the region-specific genes. Data are presented as mean ± SEM (n = 4 independent experiments). Two-way ANOVA with Tukey–Kramer test was used. Source data are provided as a Source Data file. g Hypothetical representation of the effect of EDN1 and BMP4 on regional identity of the aggregates. The aggregates switched their identity from the MX-like to the MN-like, when treated with EDN1 and BMP4 during the long-term culture. h Immunostaining of non-treated aggregates and EDN1/BMP4-treated aggregates on day 14 with antibodies for DLX5 (green), GSC (red), and DLX2 (blue). Scale bar, 100 µm. i Immunostaining of non-treated aggregates and EDN1/BMP4-treated aggregates on day 14 with antibodies for POU3F3 (green), HAND2 (red), and DLX2 (blue). Scale bar, 100 µm. Three independent cultures were used for experiment and representative images are shown.