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Distinct regulatory cascades for head and trunk myogenesis

King’s College London, Department of Craniofacial Development, Floor 27 Guy’s Tower, Guy’s Hospital, London Bridge, London SE1 9RT, UK

View larger version (109K): [in a new window]   Fig. 1. Expression of Lbx1 in the pre-otic head mesenchyme. (A-D) HH16-23 chick heads stained for Lbx1 expression; lateral views, anterior towards the top. (A) Lbx1 expression in the head mesenchyme rostral to the otic vesicle commences at HH16 (arrow). (B) At HH19, while Lbx1-expressing, somitic tongue muscle precursors (t) migrate towards the mandibular arch, Lbx1 cells in the cranial mesenchyme remain in residence (arrow). (C) Higher magnification of the Lbx1 domain at HH20. Using an anti neurofilament antibody to identify the cranial ganglia (brown), we located the Lbx1-positive cells (arrow) beneath the developing trigeminal (Vth) ganglion at the axial level of rhombomere 2 (r2). (D) The cranial Lbx1 spot is still in the same location by HH23, by which time it has been overgrown by the eye. m, midbrain; ma; mandibular arch; nt, neural tube; ov, otic vesicle; r2, rhombomere 2; t, tongue muscle precursors; V, trigeminal ganglion; VII, facial ganglion. Scale bars: 500 µm in A,B,D; 200 µm in C.

View larger version (96K): [in a new window]   Fig. 2. Mesodermal origin of the Lbx1-expressing pre-otic mesenchyme. (A) DiI labellings at HH8-8+, before cranial neural crest cell migration. The cranial paraxial mesoderm was labelled on the right side at the axial levels indicated. To record the position of the injection, a further injection was made in the neural plate. (B-D) Lateral views on the trigeminal region of chick heads, analysed for Lbx1 expression (blue) 36-48 hours after DiI injection (red). (B) Mesoderm labelled at the level of the posterior midbrain (arrowhead) resides anterior to the Lbx1 domain (arrow). (C) Mesoderm labelled at the level of rhombomeres 3/4 is seen posterior to the Lbx1 domain (arrow), migrating into the hyoid arch, (hy, arrowheads). (D) The fluorescent signal coincides with Lbx1 expression when mesoderm was labelled at the level of rhombomere 2 (arrowhead and arrow). (E) Vibratome cross-section through the embryo shown in D, confirming co-localisation of Lbx1 and DiI signals. hy, hyoid arch; np/nt, neural plate/neural tube; ma, mandibular arch; mes, mesoderm; ov, otic vesicle; r, rhombomere. Scale bar in B: 200 µm in B-D; 100 µm in E.

View larger version (100K): [in a new window]   Fig. 3. Comparison between Lbx1 and markers for the somitic dermomyotome, epaxial/hypaxial programmes and myogenic precursors at HH19/20. Lateral views of chick heads (A,C,E,G,I,K,M,O,Q) and cross sections at rhombomere 2 levels (B,D,F,H,J,L,N,P,R). To facilitate comparison, Lbx1 expression is shown in the centre of the figure (I,J). To provide anatomical landmarks, Isl1 was used to stain the cranial ganglia in red (except M,N). (A,B) Pax3, a master regulator of trunk myogenesis, is not expressed in the head mesoderm. (C,D) Pax7 and (E,F) Paraxis, co-expressed with Lbx1 during hypaxial muscle precursor migration in the trunk, coincide with Lbx1 in the pre-otic mesoderm (arrows). (G,H) The hypaxial programme marker Sim1 is expressed throughout the cranial mesenchyme (arrows). (H) Note that Sim1 expression is highest next to the neural tube, avoiding the territory beneath the Vth ganglion (arrow). (K,L) The epaxial programme marker Wnt11 is absent from the head mesoderm, instead labelling in the ectoderm around the eye (arrows). (M-R) Cranial expression patterns for the myogenic markers Dach2, Six1 and Pitx2. (M) Dach2, besides signals in the peri-optic mesenchyme (black arrows) appears to be expressed underneath the trigeminal ganglion (white arrow). Cross-sections show that staining resides at the interface between the trigeminal ganglion and the hindbrain (N, arrow). (O,P) Six1 shows widespread expression throughout the head mesenchyme (arrows). (Q,R) Pitx2, besides the peri-optic mesenchyme, shows prominent expression in the pre-otic mesoderm under the trigeminal (arrows). V, trigeminal ganglion. Scale bars: in A, 500 µm for A,C,E,G,I,K,M,O,Q; in B, 100 µm in B,D,F,H,J,L,N,P,R.

View larger version (83K): [in a new window]   Fig. 4. Myogenic differentiation markers co-localise with cranial Lbx1 expression. Lateral views and cross sections of HH19/20 chick heads as in Fig. 3. Cranial ganglia in A-D,F-I are highlighted with Isl1 (red). (A,B) Myf5 labels all cranial muscle precursors in the process of differentiation. Note that beneath the trigeminal ganglion, Myf5 stains the anlage of the lateral rectus muscle (arrow, lr). (C,D) MyoD, (F,G) Pitx3 and (H,I) R-Cadherin all resemble the expression pattern of Myf5, with expression evident in the cells beneath the trigeminal ganglion, and in the mandibular and hyoid arches. (E,J) Double labelling depicting Lbx1 in blue and MyoD (E) or R-Cadherin (J) in red. Note that transcripts for both myogenic markers and Lbx1 co-localise (arrows). do, dorsal oblique; dr, dorsal rectus; hy, hyoid arch; lr, lateral rectus; ma, mandibular arch; t, tongue muscle precursors; vr, ventral rectus. Scale bars: in A, 500 µm in A,C,F,H; in B, 100 µm in B,D,G,I; in E, 100 µm in E,J.

View larger version (97K): [in a new window]   Fig. 5. Identity of Lbx1-expressing head muscle precursors. Internal views of HH20 bisected chick heads, stained for Lbx1 (blue) and an anti-neurofilament antibody (brown). (A) The abducens nerve (cranial nerve VI) axons, with nerve rootlets in rhombomeres 5 and 6 (arrowheads), has innervated the Lbx1 domain (arrow). (B) Higher magnification of the same embryo demonstrates that the accessory branch of the abducens (small arrows) avoids the Lbx1 domain (large arrow). This indicates that at HH20, Lbx1 labels the lateral rectus extrinsic eye muscle, but not the pyramidalis and quadratus muscles. Rhombomeres denoted by r2, r5 and r6; VI, abducens nerve. Scale bars are: 200 µm in A; 50 µm in B.

View larger version (91K): [in a new window]   Fig. 6. Heterotopic grafting experiments reveal head-specific cues for lateral rectus development. (A-E) Dorsolateral view of chick embryos whose somites at forelimb levels were replaced (A) orthotopically with quail somites or (B-E) heterotopically with quail pre-otic mesoderm. (F-J) Lateral view of the trigeminal area of chick embryos whose head mesoderm at the level of rhombomere 2 was (F) replaced orthotopically with quail head mesoderm or (G-J) heterotopically with quail somites from forelimb levels. Quail tissues were detected in brown, using the QCPN antibody, in addition to blue staining for Lbx1 (A,B,F,G), Pax7 (C,H), Paraxis (D,I) and Myf5 (E,J). Note that orthotopic grafting results in normal marker gene expression (A,F, arrows). Heterotopic grafting of head mesoderm into the trunk prevents marker gene expression, indicating that the graft is deaf to signals that pattern the somite (B-E, arrowheads). Somites transplanted into the head express Pax7 (H), paraxis (I) and Myf5 (J) in a segmented fashion (arrows) with Lbx1 always absent (G, arrowheads). Thus, the ectopic somites show marker gene expression reminiscent of the epaxial half of the somite. hy, hyloid arch; ov, otic vesicle; ma, mandibular arch; sc, spinal cord; som, somites. Scale bar: 200 µm.