Fig. 4.

Novel ex vivo embryonic mandible culture system to assay SMG initiation. (A-D) E10.5 mandibular arch explants before (A) and after (B) 48 h of culture. (C,D) Mandibles before (C) and after (D) culture whole-mount immunostained for E-cadherin. Two dense spots of E-cadherin signal in cultured specimen correspond to sites of SMG initiation at the base of the tongue. (E,F) Surface rendering of E-cadherin immunofluorescence signal rotated to frontal view reveals SMG initial bud invaginated into underlying mesenchyme in cultured specimen (F). (G,H) Mandibles before (G) and after (H) culture whole-mount stained for SOX9. Two spots of SOX9 signals at the base of the tongue in cultured specimen correspond to initial SMG buds. (I,J) Merged whole-mount images with E-cadherin and SOX9. (J′) Single confocal optical slice shows regions of overlapping E-cadherin and SOX9 signals in the initiated SMG. (K,L) Frontal sections of mandibles co-stained for SOX9 and E-cadherin. (K) Optical frontal section of preculture mandible reveals no SOX9 staining in epithelium. (L) Frozen section of postculture mandible reveals two invaginated E-cadherin⁺ epithelial buds co-stained for SOX9. Yellow arrowheads indicate initiated SMG epithelial buds invaginated into underlying mesenchyme. mc, Meckel's cartilage; rc, Reichert's cartilage; s, submandibular gland initial bud. Scale bars: 500 µm in A,B; 100 µm in C-L.