Fig. 3. Severe and selective muscular hypoplasia of Six1^–/– fetuses. Histological sections of E18.5 Six1^–/– (A,C,E,F,H,J,L,N) and Six1^+/– (B,D,G,I,K,M,O) fetuses. (A,B) Haematoxylin-stained transverse sections at the distal forelimb level reveal a drastic reduction of the muscular masses in Six1^–/–, compared to normal littermates: most dorsal and ventral masses are missing in Six1^–/– embryos; R, radius; U, ulna. (C,D) Transverse sections of distal hindlimb stained with Haematoxylin and fast MHC (MY32) antibody reveal an important hypoplasia and absence of most ventral and intermediate muscle masses in Six1^–/– fetus (2, medial and lateral gastrocnemius; 3, soleus), while most dorsal muscles are present but much smaller (1, tibialis anterior); T,- tibia; F, fibula. (C2-C4 and D2-D4) Higher magnifications of C1 and D1, respectively, at the tibialis anterior level. While the size of tibialis anterior is reduced by approximately 33% in Six1^–/– fetuses, the density of the myogenic cells and their size are similar in Six1^–/– and Six1^+/– mice. As a result, the total number of myofibers is reduced by approximately 33%. (E) X-gal/Eosin-stained sagittal section at forelimb level of an E18.5 Six1^–/– fetus: a strong hypoplasia characterizes both triceps brachii (4) and biceps brachii (5), but tendons (arrows) of these muscles seem correctly developed and attached. (F-I) Immunochemistry and Haematoxylin coloration of triceps muscle sections showing slow (F,G) and fast (H,I) MHC. Whereas this muscle is reduced in size in Six1^–/– fetuses, slow and fast myofibers are present in equivalent relative proportions. (J-K) Haematoxylin/Eosin-stained sagittal sections at the thoracic level show that most superficial back muscles (11-13) are strikingly reduced and disorganized in Six1^–/– fetuses, whereas intercostal muscles (9-10) are less affected. 9, intercostal interni, 10, intercostal externi, 11, spinotrapezius; 12, latissimus dorsi; 13, serratus dorsalis. (L,M) X-gal/Eosin-stained transverse sections at the head level. The tongue (6) is significantly reduced; the genioglossus muscle (7) is absent, but the masseter (8) seems correctly developed. Note also the Meckel's cartilage hypoplasia (arrows). (N,O) Fast myosin immunochemistry of diaphragm sections. In the Six1^–/– embryo the diaphragm is reduced to a thin layer of connective tissue, without any detectable muscle fiber.