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First published online 21 July 2004
doi: 10.1242/dev.01276

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Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401, USA

View larger version (57K): [in a new window]   Fig. 1. Transplantation design for this study. Medial or lateral halves of newly-formed somites or pieces of segmental plate mesoderm from quail embryos were grafted into pockets cut into chick head mesoderm. Also shown are the sites of origin within head paraxial mesoderm of extraocular and jaw muscles discussed in this paper. These foci have been identified by orthotopic transplantations (Noden, 1986; Couly et al., 1992) and retroviral injections (Noden, 2002), and were confirmed in this study. The lateral rectus and pyramidalis primordia arise medial and ventral to the first branchial arch progenitors.

View larger version (97K): [in a new window]   Fig. 2. The distribution of grafted quail mesoderm cells 1.5 to 2.5-days after surgery (stages 19-22) is revealed by the application of anti-quail (QCPN) antibody. (A-D) Whole embryos. Asterisks indicate ectopic somite-like condensations; double arrowheads indicate a mesenchymal condensation adjacent to the isthmus. DO (dorsal oblique) and LR (lateral rectus) are sites where eye muscle primordia condense. (E,F) Sections from segmental plate grafted hosts fixed at stage 22. These show an abundance of graft-derived cells within and scattered around an ectopic somite-like condensation (arrow, E), and a mesenchymal aggregate adjacent to the isthmus (Isth). (G,H) Segmental plate hosts fixed at stages 22 (G) and 26 (H). These reveal the presence of mesenchymal bridges (arrows) extending to the LR muscle during and after the translocation of the LR to its definitive position. Note the presence of quail cells circumscribing and within the trigeminal ganglion (T). Arrowheads indicate dense cords of graft-derived cells that extend ventrally from the isthmus region.

View larger version (50K): [in a new window]   Fig. 3. (A,B) Adjacent sections from a stage 19 segmental plate recipient embryo stained with QCPN (A), to reveal the locations of all quail cells, and QH1 (B), to identify quail endothelial cells. These show that most of the cells that migrated from transplanted trunk mesoderm are angioblasts. (C) A QH1-stained section from a stage 28 lateral somite recipient, showing the substantial contributions of trunk-derived angioblasts to endothelial cells within and surrounding the brain.

View larger version (80K): [in a new window]   Fig. 4. Pattern of gene expression following head-into-head or trunk-into-head mesoderm transplantation. Column headings indicate the donor tissue; row headings are the gene products assayed. Sites of ectopic expression (black arrows) are evident in all heterotopic categories. myf5/myod and paraxis are expressed in aggregates, and also in cells dispersed around the site of implantation. lbx1 is evident in ectopic aggregates but not in dispersed cells. Neither paraxis nor lbx1 mRNA was detected in the first branchial arch (BA1). DR, dorsal rectus; VO, ventral oblique.

View larger version (58K): [in a new window]   Fig. 5. (A,C) Segmental plate recipients fixed 24 hours after surgery and processed for myf5 expression. (B,D) Vibratome sections from the embryos shown in A and C at the planes indicated. Myf5 is activated on a normal trunk timetable, which is earlier than is appropriate for head muscles. (E,F) Adjacent sections from a stage 20 segmental plate recipient. Graft-derived cells within a somite-like condensation synthesize MyHC, nearly a day earlier than it would be found in developing head muscle cells.

View larger version (83K): [in a new window]   Fig. 6. Reconstructions of stage 29-30 (6-6.5 day) embryos showing the distribution and differentiation of cells derived from mesoderm transplants. A shows an enlargement of the LR and adjacent structures. All grafts contribute to the LR and the pyramidalis muscles, and usually to proximal first arch muscles, especially the palpebral depressor primordium. Many embryos also exhibited labeling in the DO. In contrast to control grafts (A), all trunk-into-head grafts (B-E) produce a large number of ectopic muscles. These are not randomly distributed, but form in large irregular aggregates beside the isthmus, and as small clusters and single myotobes dispersed in a supra-orbital band deep to and paralleling the dorsal margin of the eye.

View larger version (124K): [in a new window]   Fig. 7. Control (A,B, orthotopic) and trunk-into-head (C,E, lateral half somite; D, medial half somite) transplantations. Embryos were fixed at stage 28-30 and double stained with F59, to show myosin heavy chain (purple), and QCPN, to show quail nuclei (brown). (A) The labels are on the unoperated side. (B) Graft-derived cells contributing to the lateral rectus (box in A). (C) A lateral rectus muscle in which nearly all cells are derived from the transplant. Blazes of unlabeled intramuscular cells are connective tissues derived from neural crest cells. (D) Dorsal oblique muscle, and (E) lateral rectus and proximal first branchial arch (BA1) muscles, formed by grafted trunk mesoderm. Proximal BA1 muscles normally initiate MyHC synthesis at a later stage, and graft-derived myocytes follow this head timetable.

View larger version (133K): [in a new window]   Fig. 8. Low (A) and high (B) magnification images of a stage 29 embryo, showing the ability of grafted medial somitic mesoderm to form the lateral rectus muscle. Note that grafted cells do not contribute to the adjacent dorsal rectus muscle. Purple, myosin heavy chain; brown, quail nuclear marker. Arrows indicate ectopic muscles.

View larger version (139K): [in a new window]   Fig. 9. Ectopic muscles formed by grafted trunk mesoderm. (A) A large mesenchymal aggregate dorsal to the isthmus is shown; note that myosin is synthesized primarily in abaxial cells (stage 29 embryo, segmental plate graft). This ectopic condensation has not disrupted the emergence of trochlear nerve fibers (n. IV) from the roof of the isthmus. (B) Multiple finger-like projections of ectopic muscle extend ventrally from the isthmus (stage 29 embryo, medial half somite graft). (C,D) Multiple small, ectopic muscle clusters are dispersed along a band deep to and paralleling the dorsal margin of the eye; the edge of the pigmented retina is visible. (E) An ectopic muscle adjacent to the dorsal rectus muscle; there is no mixing between them. Labels: MyHC is purple in A-C and red in D,E; quail nuclei are brown in A-C and black in D,E.

View larger version (145K): [in a new window]   Fig. 10. Transplanted trunk mesoderm forms scattered myocytes and myotubes, as shown in sections from stage 28-29 embryos. (A) An ectopic aggregate (bottom of figure) containing non-aligned myotubes, identified by their shape and synthesis of MyHC (F59-positive, blue), with individual MyHC-positive cells dispersed deep to the dorsal quadrant of the eye. Some MyHC-positive cells are adjacent to the pigmented epithelium of the eye (right side), which normally is populated by connective tissues (e.g. sclera) formed by periocular neural crest cells. (B,C) Scattered myoytubes in the supra-orbital band; both cases shown are from segmental plate grafts. (D) A multinucleated myotube (MyHC, magenta) adjacent to the eye; the pigmented epithelium is visible on the right. (E) A triple-stained section from a lateral half somite recipient, showing graft-derived endothelial cells (QH1, brown), myocytes (MyHC, magenta) and quail nuclei (QCPN, black). Although invasive movements of grafted angioblasts have been reported, comparable behavior by myoblasts is novel. No double-labeled myoendothelial cells were found.

View larger version (120K): [in a new window]   Fig. 11. MyHC-positive quail cells (arrows) are present in the root of the trigeminal ganglion. Epineural connective tissues and endothelial cells within the ganglion and brainstem were found in all transplant categories, but only segmental plate grafts generated myotubes within the ganglion. The other quail cells evident within the trigeminal root are endothelial cells. An ectopic muscle is visible to the left. A stage 29 embryo is shown.

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