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Fig. 5. Molecular analysis of mouse perineal muscle development. (A-E) Whole mount in situ MyoD hybridisation on mouse embryos showing the development of perineal muscles from the ventral muscle mass of the leg. (A) E11.5 ventral view showing ventral muscle mass. (B) E12.5 caudal view showing extension in ventral direction. (C) E13.0 caudolateral view of the perineal anlage separating from the ventral leg muscle mass. (D) E13.5 caudolateral view of the anlage extending to the base of the genital tubercle. (E) E14.5 caudolateral view of perineal anlage forming individual muscles around the already septated cloaca, before they merge with the contralateral counterpart. Anal opening - arrow. (F,G) Muscle primordia in the pelvis of E15.0 wild type (F) and met^d/d mutant (G) embryos. Cryosections in the axis of rectum, stained for muscle (anti-MyHC) and counterstained by Haematoxylin and Alcian Blue. All perineal muscles are absent in met^d/d mutants mouse. Note the only muscles present in met^d/d mutants are PCL and medialis (not shown). E19.5 Lbx1 null mutant stained for MyHC show presence of the external anal sphincter (SA in Fig. H) and of bulbospongiosus and external urethral sphincter muscles (BS and SU respectively in Fig. I). (J) Meox2 null mutant E15.5 shows full complement of perineal muscles as detected by whole-mount in situ hybridisation using MyoD. abdo, abdomen; BS, m. bulbospongiosus; CFC, M. caudofemoralis (pars caudofemoralis of m. caudo-ilio-femoralis); ISC, m. ischiocavernosus; LA, m. levator ani; PCL, m. pelvicaudalis lateralis; SA, m. sphincter ani externus; SU, external urethral sphincter muscle.

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