Fig. 8: Formation of the 1st BA (first branchial arch)-like aggregates containing both proximal and distal-type cells.

a Schema of the axial organization of the 1st BA. The proximal (dorsal) side is POU3F3⁺ MX (maxillary arch), and the distal (ventral) side is HAND2⁺ distal MN (mandibular arch). b Schematic of experiment. The duration of treatment with EDN1 (endothelin 1) and BMP4 (bone morphogenetic protein 4) differed among conditions 1, 2, and 3. The aggregates were analyzed using real-time PCR and immunohistochemistry on day 21. bFGF: basic fibroblast growth factor. c Immunostaining of the day-21 aggregates cultured in each condition with antibodies for POU3F3 (green), HAND2 (red), and DLX2 (blue). Scale bars, 100 µm (the left one) and 200 µm (the others). Three independent cultures were used for experiment and representative images are shown. Cond. 1–3 condition 1–3. d Real-time PCR analysis of the region-specific genes. Data are presented as mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey–Kramer test was used. Source data are provided as a Source Data file. e Scheme for differentiation of first branchial arch-like aggregates. When cultured with bFGF alone, the aggregates differentiated into MX-like states, whereas they differentiated into MN-like states in the presence of EDN1 and BMP4. Temporary treatment with EDN1 and BMP4 induced the formation of single aggregates containing both POU3F3⁺ Mx-like and HAND2⁺ Mn-like regions.