To investigate the effects of RA on tongue muscle development, we examined the expression of Myf5 and MyoD, early and late myogenic determination markers, respectively, in the developing tongue. We also analyzed tongue muscle differentiation, using an anti-myosin heavy chain fast antibody. There was no difference in the gross morphology of the tongue at E12.5 between control and RA-treated fetuses. RA treatment decreased both Myf5 and MyoD in the tongue, as compared with controls (Fig. 4A, D, G, J, M, P, S, V). Immunostaining using an anti-myosin antibody showed that tongue muscles begin to differentiate at E12.5 in control fetuses (Fig. 4B, H, N, T). Myosin was distributed both in extrinsic and intrinsic tongue muscle primordia, and Myf5- and MyoD-positive cells were observed not only in tongue primordial muscles but also in the tongue mesenchyme in both control and RA-treated fetuses (Fig. 4C, F, I, L, O, R, U, X). However, myosin-positive cells were significantly decreased in the RA-treated tongue (Fig. 4E, K, Q, W). Such a difference in myosin distribution between control and RA-treated fetuses was confirmed by observing the genioglossus muscle at higher magnifications, and some primordial myotubes were found to have no lumen and were collapsed in the RA-treated group (see Fig. 6E, F). At E14.0, numerous cells positive for Myf5 and MyoD were observed in both control and RA-treated fetuses (Fig. 5). In RA-treated fetuses, however, myosin-positive cells were not observed in the core of the primordial genioglossus muscle, which suggests that the number of primordial myotubes was decreased (Fig. 5B, E, H, K, N, Q, T, W). The abnormal attachment of the genioglossus and intrinsic muscles in RA-treated fetuses as observed by lacZ staining (Fig. 3H) was confirmed by immunostaining with an anti-myosin antibody (Fig. 5K), which was never found in control fetuses (Fig. 5I).
Figure 4. Immunohistochemical staining for myogenesis-associated markers at E12.5. A, D, G, J, M, P, S, V: Myf5 (an early muscle maker) and MyoD (a late muscle marker) are expressed in the developing tongue at E12.5, and appear to be decreased both in the anterior and posterior parts of RA-treated tongues, as compared with the controls. B, E, H, K, N, Q, T, W: Myosin-positive cells are distributed in intrinsic muscle primordia of RA-treated tongues less than control tongues. Primordial myotubes in not only the genioglossus muscle but also the geniohyoid and mylohyoid muscles of RA-treated tongues are not well organized (arrowheads). C, F, I, L, O, R, U, X: Myf5 or MyoD, myosin, and DAPI are merged in the tongue of control and RA-treated fetuses. Ant, anterior region of the tongue; Post, posterior region of the tongue; gg, genioglossus muscle primordium; gh, geniohyoid muscle primordium; mh, mylohyoid muscle primordium. Scale bar = 100 μm.
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Figure 5. Immunohistochemical staining for myogenesis-associated markers at E14.0. A, D, G, J, M, P, S, V: Myf5- and MyoD-positive cells are observed in the entire tongue in both control and RA-treated fetuses. B, E, H, K, N, Q, T, W: Myotubes are formed in control and RA-treated tongues, but myotubes in the core region of the genioglossus muscle primordium exhibit poor growth in RA-treated tongues. Aberrant attachment of the genioglossus muscle and intrinsic tongue muscle primordium is also observed in RA-treated tongues, compared with controls (arrowheads). C, F, I, L, O, R, U, X: Myf5 or MyoD, Myosin, and DAPI are merged in the tongue of control and RA-treated fetuses. Ant, anterior region of the tongue; Post, posterior region of the tongue; gg, genioglossus muscle primordium; gh, geniohyoid muscle primordium; mh, mylohyoid muscle primordium. Scale bar = 100 μm.
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Next, we counted the number of primordial myotubes in a fixed area within the genioglossus muscle at E12.5 and E14.0, to evaluate the effects of RA on tongue muscle development. At E12.5 and E14.0, there was no significant difference in the total cell number between the control and RA-treated fetuses (Fig. 6A). The ratio of Myf5-positive cells to the total cell number significantly decreased in the genioglossus muscle of RA-treated fetuses at E12.5 (P < 0.01), but the difference was not significant at E14.0 (Fig. 6B). The ratio of MyoD-positive cells significantly decreased in RA-treated fetuses both at E12.5 (P < 0.01) and at E14.0 (P < 0.05). The ratio of primordial myotubes significantly decreased after RA treatment (P < 0.01), but there was no difference in the size of primordial myotubes between the control and RA-treated fetuses (Fig. 6C, D). A high magnification of primordial myotubes in thegenioglossus muscle revealed that the myotubes were formed at E14.0 in both groups (Fig. 6G, H). In RA-treated fetuses, the lumina in primordial myotubes were round, but their size was variable.