Fig. 2: Directed differentiation of NCC aggregates towards mdEM.

a, Schematic model illustrating the regional specification of pharyngeal arch ectomesenchyme along the proximal–distal axis in an embryo. mdEM, mandibular prominence ectomesenchyme. b, Differentiation strategy of mdEM from NCC aggregates. c, Bright-field images and immunofluorescence staining for SOX10 and TWIST1 in d5 and d5 treated with FEDB (combination of FGF8, EDN1 and BMP4) for 4 days (d5 + FEDB). Nuclei stained with DAPI (n = 6 from 3 biologically independent experiments). d, Phalloidin staining of d5 and d5 + FEDB (n = 6 from 3 biologically independent experiments). e, Triple-colour immunofluorescence staining for DLX2, DLX5 and HAND2 in d5 + FEDB. Bottom panels show magnified views of the white dashed boxes in the top panels (n = 15 from 8 biologically independent experiments). White dotted circle, DLX5-weak and HAND2-negative area; yellow dotted line, HAND2-negative area. f, Positive rates of DLX2, DLX5 and HAND2 to DAPI calculated using ImageJ (n = 6 from 3 biologically independent experiments). g, Schematic model illustrating the regional specification of embryonic PA1 and induced mdEM. h, Schematic model illustrating the strategy to examine the effect of EDN1 signal on the bifurcation of maxillary prominence ectomesenchyme (mxEM) and mdEM. i, Immunofluorescence staining for SOX10 and TWIST1 in d5 treated with FGF8 and BQ-123 for 7 days (d5 + FQ) (n = 3 from 3 biologically independent experiments). j,k, Gene expression analysis for mdEM marker (DLX5) (j) and mxEM markers (POU3F3 and CYP26A1) (k) in aggregates (n = 6 from 3 biologically independent experiments). l, Schematic model illustrating the strategy to examine the importance of initial A–P identity in mdEM differentiation. m, Immunofluorescence staining for SOX10 and TWIST1 in RA⁺ d5 treated with FEDB for 4 days (RA⁺ d5 + FEDB) (n = 3 from 3 biologically independent experiments). n, Gene expression analysis for mdEM marker (DLX5) and HOX genes (HOXB2 and HOXB4) in aggregates (n = 6 from 3 biologically independent experiments). In j, k and n, values are shown as mean ± s.d. P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. In c–e, i and m, representative data are shown. All scale bars, 100 μm.