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First published online January 14, 2005
doi: 10.1242/10.1242/dev.01545

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A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells

¹ Veterinary Basic Sciences, Royal Veterinary College, London NW1 0TU, UK
² Anatomy Institute, First Medical Faculty, Charles University, Prague, Czech Republic
³ Brighton and Sussex Medical School, University of Sussex, Falmer, Brighton BN1 9PX, UK
⁴ INSERM UMR 623, Developmental Biology Institute of Marseille, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France

View larger version (68K): [in a new window]   Fig. 1. Anatomy of cloacal and perineal muscles. (A) Experimental procedures using newly formed quail somites for heterotopic single somite grafting into the chick recipient. Viral injection into interlimb somite was carried out for future exact determination of the operated segmental level. (B) Result of the single somite marking by retroviral alkaline phosphatase transfection. The second intercostal muscle is a derivative of somite 21 (Evans, 2003). Operated somite was 10 somites further caudally, therefore somite 31 was transplanted. (C) Lateral view of a chick HH36 with cloacal muscles (red), two leg muscles (green) and the tail region. Lines `a' and `b' show the approximate level of sections in Fig. 2A and 2B respectively. Orientation is marked: V, ventral; D, dorsal; Ca, caudal. The m. sphincter cloacae has 2 parts: m. sphincter circular (SCC) and m. sphincter sling-shaped (SCS). The sling region originates in the supracloacal septum and on the apex of pubis and forms a slightly dorsally positioned sling around the cloacal tubercle. These layers merge in the dorsocaudal aspect. The m. transversus cloacae (TCL) in the adult is a very prominent superficial muscle and with its almost transverse course it forms the boundary between the abdomen and the tail. The m. levator cloacae (m. retractor phalli caudalis) (LC) is a long narrow muscle arising from the underside of feather bulge of the lateral rectrices, inserting near the midline of the cloacal tubercle - phallus. Sexual dimorphism was not evident at the stages examined. Because of close topographical relationship to the cloacal muscles, we also focus on two hindlimb muscles. CFC begins from the proximal half of the femur and inserts on the ventral side of pygostyl on the fascia of m. depressor caudae in the vicinity of the rectrices. FCLP is the caudal-most muscle of the leg (C,D). (D) Lateral view of a skinned HH36 chick after in situ hybridisation with MyoD. (E) Caudolateral view of a mouse E15.5 with perineal muscles (red), leg muscles (green) and the tail region. (F) Caudolateral view of a skinned mouse E15.5 after in situ hybridisation with MyoD. g.t., genital tubercle; BS, m. bulbospongiosus; CFC, M. caudofemoralis (pars caudofemoralis of m. caudo-ilio-femoralis); FCLP, M. flexor cruris lateralis - pars pelvica; ISC, m. ischiocavernosus; LA, m. levator ani; LC, levator cloacae; PCE, m. pubocaudalis externus; PCI, m. pubocaudalis internus; SA, m. sphincter ani externus; SCC, m. sphincter circular; SCS, m. sphincter sling-shaped; TCL, m. transversus cloacae.

View larger version (81K): [in a new window]   Fig. 2. Fate mapping of the cloacal muscles using chick/quail chimeras and retroviral labelling. Identification of the contribution of the 31st somite to the cloacal musculature using chick/quail chimeras. Transverse parallel sections (see approximate levels in Fig. 1C) stained for quail cells (QCPN antibody) and counterstained with Eosin (A, B) and Alcian Blue (C). (A) Section through the level of the cloaca, showing all four cloacal muscles to be populated with quail cells. (B) Section through the level of the thigh and shank, showing quail derived cells from the somite 31 in some leg muscles. (C) Contribution of somite 31 to the hemivertebra and the adjacent epaxial muscle (arrow). (D) Detail of cloacal region of a chick embryo after marking somite 30 (at HH18) with lacZ-encoding retrovirus and X-gal whole-mount staining at HH36. X-Gal staining was detected in cloacal and leg muscles, see Table 1. Orientation is marked: V, ventral; D, dorsal. CFC, M. caudofemoralis (pars caudofemoralis of m. caudo-ilio-femoralis); CFP, m. caudofemoralis - pars pelvica; FCLP, M. flexor cruris lateralis - pars pelvica; FL, fibularis luingus; IF, iliofibularis; LC, levator cloacae; PCE, m. pubocaudalis externus; PCI, m. pubocaudalis internus; PIL, m. iliotibialis lateralis pre-acta bularis; SCC, m. sphincter circular; SCS, m. sphincter sling-shaped; TCL, m. transversus cloacae.

View larger version (105K): [in a new window]   Fig. 4. Cloacal muscle precursors reside in the hindlimb. (A) Quail leg bud transplanted onto chick hindlimb stump at HH23 results in cloacal muscles consisting of quail cells at HH36. Proximal skeletal elements are of only chick origin (asterisk). (B) Injection of retrovirus into the proximoventral region of the leg bud at stage HH25 gives rise to cloacal muscles (TCL and SCS) labelled with lacZ. There is concomitant signal in the leg muscles. GC, gastrocnemius muscle; LC, levator cloacae; PCE, m. pubocaudalis externus; PCI, m. pubocaudalis internus; SCC, m. sphincter circular; SCS, m. sphincter sling-shaped; TCL, m. transversus cloacae.

View larger version (84K): [in a new window]   Fig. 3. Molecular analysis of chick cloacal muscle development. (A-F) Ventrolateral views of whole-mount in situ for MyoD on chick embryos at stages HH25-35 show the development of the cloacal muscles from the ventral muscle mass of the leg. (A) Ventral muscle mass (arrow) of the hindlimb at HH25. (B) Elongation of ventral muscle (arrow) mass ventrocaudally towards the cloaca at HH27. (C) Further elongation of cloacal anlage (arrow) at HH29; the cleavage of leg muscle mass has started. (D) Cloacal anlage arrives to the vicinity of cloacal tubercle (arrow) at HH31, note the discontinuity with the anlage of FCLP and CFC muscles of the leg (arrowhead). (E) Cloacal anlage extends around the tubercle at HH33 (arrow). (F) Single muscles of the cloacal complex are discernible at HH35. (G) Pax7 expression at HH27 (arrow). (H) Details of the Pax7 expression of the proximal limb anlage at HH28, limb has been removed. Cloacal anlage (arrow) extends from the ventral muscle mass (cross). Dorsal muscle mass (red arrowhead) began to split into single muscles. LC, levator cloacae; SCC, m. sphincter circular; SCS, m. sphincter sling-shaped; TCL, m. transversus cloacae.

View larger version (89K): [in a new window]   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 metd/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 metd/d mutants mouse. Note the only muscles present in metd/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.

View larger version (65K): [in a new window]   Fig. 6. Experimental evidence for cloacal muscle dependency on leg muscle development. (A) Whole-mount in situ hybridisation with MyoD probe on chick embryo at stage HH34 after surgical leg bud ablation at HH18. Ventral view of the cloacal region shows the unilateral absence of all cloacal muscles on the operated side (arrow). (B) Left operated side, lateral view; (C) right control side, lateral view (mirror image). Unilateral absence of all cloacal muscles on the operated side (asterisk in B) and normal pubocaudal and tail muscles are shown. Orientation is marked: V, ventral; D, dorsal. PCE, m. pubocaudalis externus; PCI, m. pubocaudalis internus.

View larger version (52K): [in a new window]   Fig. 7. Genetic evidence for the development of cloacal/perineal muscles depending on leg field. (A) limbless chick mutant HH36 before skinning for hybridisation. (B) Ventral view of the cloaca of limbless chick mutant after MyoD hybridisation reveals complete bilateral (arrows) absence of all cloacal muscles. Pubocaudal (PCI and PCE) and tail muscles are again normal. (C) p63-null E16.5 mutant mouse shows the presence of all perineal muscles. BS, m. bulbospongiosus; ISC, m. ischiocavernosus; LA, m. levator ani; PCE, m. pubocaudalis externus; PCI, m. pubocaudalis internus; PCL, m. pelvicaudalis lateralis; SA, m. sphincter ani externus.