Figure 10. Tongue frenum in Ofd1 mutant mice.

(A, B) Frontal sections showing in situ hybridization of Myf5 (A) and LacZ staining (B) in wild-type (A) and WntCre;R26R^LacZ (B). Arrows indicating tongue frenum region. (C–E) Oral view of whole-mount in situ hybridization of Lgr5 (C, E) and Tbx22 (D) in wild-type at embryonic day (E) 11.5 (C, D) and E10.5 (E). Red and blue arrows indicating anterior expression domain and midline region, respectively. (F, H) Schematic diagram showing oral view of mandible with AP (light red) and TE region (light green), and midline (orange line). (G, G’, I, I’) Nuclear fluorescent image showing oral view of wild-type mandible before (G, I) and after (G’, I’) culture. Red and green arrows indicating DiD and DiO, respectively. Yellow arrowheads indicating midline. (G’, I’) Tongue-like structure was partially removed and presumptive tongue-like structure are outlined by white dots. (J) Schematic diagram showing oral view of mandible with movement of cells in AP (light red), LT (light yellow), and TE region (light green line). (K, K’) Schematic diagram showing oral view of mandible with incision (black line) and midline (orange line). (L, M) Frontal sections showing histological images of cultured jaw without incision (L) and with incision (M). Arrows indicating the tongue frenum region. Tongue frenum formation with, n=1/6 and without incision, n=8/10. (N, O) Frontal sections showing histological images in Ofd1^fl/WT;Wnt1Cre(HET) mice obtained from same mouse (O; more posterior site than N). (P, Q) Frontal sections showing immunohistochemistry of p-c-Jun in wild-type at E12 (P) and E11 (Q). Arrows indicating positive cells in the tongue frenum (P) and AP region (Q). (R, S) Frontal sections showing histological images of cultured jaw with (S) and without U73122 (R). Arrows indicating the tongue frenum region. Tongue frenum formation with U73122, n=0/4. (T) Oral view of mandible in Ofd1^fl/WT;Wnt1Cre(HET);Hprt^fl mice. Arrows indicating Gfp-negative domains around AP region before culture. (U) Frontal sections showing histological images of T after culture. Arrow indicating tongue frenum region. (V) Schematic diagram showing oral view of mandible with replacement (A, yellow circle=adipose, blue circle=cranial neural crest-derived cell [CNCC]). (W, X) Frontal sections showing histological images of cultured jaw without replacement (W) and with replacement (X). Arrows indicating tongue frenum region. Tongue frenum formation with replacement, n=1/5. Scale bars: 200 µm (A, B, L, M, N, O, R, S, U, W, X), 500 µm (C-E­)­­.

Figure 10—figure supplement 1. Tongue frenum formation in wild-type mice.

(A, B) Frontal section showing histological image (A) and in situ hybridization of Lgr5 (B) of tongue frenum (A) and mandible (B) in wild-type mice at embryonic day (E) 10.5 (B) and E12.5 (A). Arrow indicating mandible region. (C) Schematic diagram showing oral view of mandible with LT (yellow ceircle) and AP (right red circle) region, and midline (orange line). (D, E) Frontal sections showing in situ hybridization of Tbx22 (D) and Lgr5 (E) at E11.5. Left panels, dark field; right panels, bright fields. (F) Frontal image showing mandibular process with DID injection before culture. Arrow indicating DID. (G) Sagittal section showing mandible with DID after culture. Tongue was outlined by white dots. (H–H’’) Oral view of wild-type mandible with DiO injection (H, 0 hr, H’, 12 hr, H’’, 36 hr after injection). Blue arrowheads and red arrows indicating DiO at LT and AP region, respectively. Yellow arrowhead indicating midline. (I) Frontal sections showing in situ hybridization of Ptch1 at E12.5. (J) Frontal view of mandible in LacZ stained Axin2^LacZ mice at E12.5. Arrows and arrowhead indicating tongue frenum region (E, F).