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First published online 25 July 2007
doi: 10.1242/dev.002501

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Cranial neural crest cells regulate head muscle patterning and differentiation during vertebrate embryogenesis

¹ Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.
² Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
³ Swiss Federal Institute of Technology, ETH-Hoenggerberg HPM E38, CH-8093 Zürich, Switzerland.
⁴ Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

View larger version (121K): [in this window] [in a new window]   Fig. 1. CNC cells are not necessary for early myogenesis in Hoxa1/Hoxb1-3'RARE double-mutant embryos. (A,A') Whole-mount in situ hybridization (ISH) for Hoxa2 and the corresponding coronal section (inset) of control and double mutant, n=1. (B,B') Capsulin ISH in control and mutant embryos, n=3. (C,C') Tbx1 ISH in control and the mutant embryos, n=3. Arrowheads point to the branchial arches (ba1 and ba2); ht, heart. Scale bars: in A, 0.36 mm for A'-C'.

View larger version (128K): [in this window] [in a new window]   Fig. 2. Defects in cranial muscle patterning in Twist mutant embryos. (A-F') A comparative expression analysis for cranial mesoderm and neural crest markers in mouse embryos. Whole-mount and section in situ hybridization for Twist (A,A',C,C',E,E'), capsulin (B,B'D,D') and Dlx5 (F,F'). Broken lines indicate plane of section. (G,G') Whole-mount in situ hybridization (ISH) for capsulin in control and Twist mutants, n=6. Arrowheads point to the muscle anlagen in the branchial arches (ba1 and ba2). (H,H') Tbx1 ISH, n=7. (I,I') Myf5 ISH, n=7. (J,J') MyoD ISH, n=7. (K-M') Dlx5, cadherin 6 and cadherin 11 expression patterns in control and Twist mutants, n=5. Arrowheads point to the CNC cells in the branchial arches (ba1 and ba2) and open arrowheads indicate their absence. hf, head fold; hm, head mesenchyme; ht, heart; nt, neural tube; ph, pharynx. Scale bars: in A, 0.41 mm for B; in A', 0.25 mm for B'; in C and G, 0.3 mm for D,G',H-H',I',J',K',L',M'; in C' and E, 0.45 mm for D',E',F-F',I,J,K,L,M.

View larger version (102K): [in this window] [in a new window]   Fig. 3. Muscle patterning and differentiation defects in CA-ß-catenin/Wnt1-Cre mutant embryos. (A,A') Whole-mount in situ hybridization (ISH) for Dlx5 in control and CA-ß-catenin/Wnt1-Cre mutants, n=3. Arrowheads point to the CNC cells in the branchial arches (ba1 and ba2). (B,B') Capsulin ISH in control and mutants, n=6. Arrowheads point to the muscle anlagen in the branchial arches. (C,C') Tbx1 ISH, n=6. (D,D') Myf5 ISH, n=5. (E,E') MyoD ISH, n=4. (F,F') Myogenin ISH, n=4. Open arrowheads indicate on the loss of muscle anlagen. fl, forelimb; ht, heart; nt, neural tube. Scale bars: in A, 0.5 mm for A',D-D',E-E',F-F'; in B, 0.7 mm for B'-C'.

View larger version (102K): [in this window] [in a new window]   Fig. 4. Ablation of the CNC in chick embryos affects cranial muscle patterning. Images of control (A) or CNC-ablated (A') chick embryos at stage 8 (ablation boundaries are marked by white broken lines in A', dorsal view). (B-E') Whole-mount in situ hybridization in control and CNC-ablated embryos (lateral views) and the coronal/transverse sections (white broken line) of the corresponding embryos on the left: Tbx1 (B-B''', n=7/7), capsulin (C-C''', n=5/6), Myf5 (D-D''', n=8/9) and MyoD (E-E''', n=5/5). Arrowheads point to the muscle anlagen in the branchial arches (ba1 and ba2) and open arrowhead indicates their absence. ht, heart; nt, neural tube; ov, otic vesicle. Scale bars: in A, 0.4 mm for A'; in B, 0.5 mm for B'-E'.

View larger version (113K): [in this window] [in a new window]   Fig. 5. CNC cells influence paraxial mesoderm migration and axial registration in chick embryos. (A) An image of a stage 8 embryo injected with DiI in the CPM (arrow indicates dye location, dorsal view). (A') A lateral view of the embryo in A after 48 hours; arrowhead points to the labeled cells migrating toward BA1. (B,B') DiI labeling of the CPM in CNC-ablated embryos. In some ablated embryos cell migration was arrested (n=8/13) whereas in others partial migration towards BA1 was observed (n=4/13) compared with normal migration of CPM cells in controls (n=13/14). (C-D') Embryos were labeled with both DiI and DiO simultaneously (DiO, green, arrowheads in C and D; DiI, red, arrows in C and D). CPM cell migration was monitored after 48 hours. A mixture of the DiO- and DiI-labeled cells streaming toward BA1 is seen in the ablated embryo (D', n=6/8) compared with the separate streams seen in controls (C', n=4/5). Ablation boundaries are marked by broken line. (E-H'') Quail-chick (Q-C) transplantation assay; E, a scheme of the experiment. Stage 8 quail CPM grafts labeled with DiI at the level of rhombomere 4 and then transplanted into stage-matched chick embryos. (F) A lateral view of the Q-C chimeric embryo after 24 hours. (F'-F'') Transverse sections through the BAs of the embryo on the left (F', ba1, F'', ba2) stained with the quail-specific antibody (QCPN, in green). Note the quail-derived cells exclusively in BA2 (F'', higher magnification in the inset, n=4/4). (G-H'') Similar images as shown in E-F'' except that the host chick embryo was CNC ablated. In the CNC-ablated embryo, quail-derived cells are seen in BA1 (arrows in H', inset, n=3/4) but not in BA2. Lateral views of embryos (A',B',C',D',F,H) are shown as an overlay of bright field and fluorescence images. ec, ectoderm; ht, heart; nt, neural tube; ov, otic vesicle; ph, pharynx. Scale bars: in A, 0.4 mm for B,C,D; in A', 0.5 mm for B',C',D'; in F, 0.36 mm for H; in F', 0.2 mm for H' and 0.1 mm for F'',H''.

View larger version (85K): [in this window] [in a new window]   Fig. 6. Increased cell proliferation and Fgf8 upregulation after CNC ablation in chick embryos. (A-C'') A combined in situ hybridization for Myf5 (purple) and BrdU staining (red) in transverse sections of stage 18 embryos in the trunk (A-A'') and BA2 region (B-B'', control) or CNC-ablated embryo (C-C''). Arrowheads point to Myf5 expression and BrdU staining in the muscle anlagen. (D) A quantification analysis of proliferating myoblasts in branchial arches of control and CNC-ablated embryos at 26 and 45 hours of incubation. Similar results were obtained in three independent experiments and in each experiment the bars represent counts from three adjacent sections. *P<0.05, **P<0.01. (E-G'''') A double immunostaining for Myf5 (red) and BrdU (green). (E-E'''') A transverse section at the trunk level. (F-F'''') A transverse section at the level of BA2 in control embryos. (G-G'''') A transverse section at the level of BA2 in CNC-ablated embryos. Merged BrdU/Myf5 images plus higher magnifications (inset) are shown on the right. In situ hybridization for Fgf8 in control (H,H') or CNC-ablated embryos (I,I'). (J) RT-PCR results of CPM explants from control or 100 ng/ml FGF8b-treated explants incubated for 3 days in culture, n=2/2. (K) RT-PCR results of CPM explants from control or CNC-ablated embryos. Whereas CPM explants underwent myogenesis in control cultures, a reduction in myogenesis was observed following CNC ablation (n=4/4). my, myotome; nt, neural tube; ov, otic vesicle; ph, pharynx. Scale bars: in A and E, 0.15 mm for B,C,F,G and 75 µm for A'-A'',B'-B'',C'-C'',E'-E'',F'-F'',G'-G''; in H, 0.5 mm for H'-I'.

View larger version (95K): [in this window] [in a new window]   Fig. 7. Myogenic differentiation, myofiber architecture and positioning are perturbed in the absence of the CNC in chick embryos. (A) An illustration of genes that regulate the transition from muscle progenitors to mature myofibers, adapted from Tajbakhsh (Tajbakhsh, 2005). (B-G'') Immunofluorescence stainings on transverse sections of E4.5-5 control (B,D,F) or CNC-ablated embryos (C,E,G) for the indicated muscle markers desmin and myosin heavy chain (MHC). The typical organization of BA1-derived jaw muscle fibers is clearly seen in the controls (B',D'; n=5/5), but is much less visible in the ablated embryos (C',E'; n=3/4). Note the absence of the mandibular adductor in the ablated embryo (compare G' with F'). (H-K''') Higher resolution images of BA1 myofibers: F-actin (phalloidin, red) filaments (H',I',J',K'); desmin, green (H'',I''); MHC, green (J'',K''). DAPI (blue) stains nuclei (H,I,J,K). (L) A model illustrating the multiple roles played by the CNC in the regulation of head skeletal muscles in vertebrate embryos. ad, mandibular adductor; im, intermandibular; nc, notochord; nt, neural tube; ov, otic vesicle. Scale bars: in B, 0.2 mm for C,D,E,F,G, 0.1 mm for B',C',D',E',F',G' and 50 µm for B'',C'',D'',E'',F'',G'' and 66 µm for H'-K'''.

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