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First published online 15 November 2006
doi: 10.1242/dev.02693

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Pitx2 promotes development of splanchnic mesoderm-derived branchiomeric muscle

¹ Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA.
² Department of Craniofacial Development, King's College, London Guy's Tower, London SE1 9RT, UK.
³ Program in Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

View larger version (116K): [in this window] [in a new window]   Fig. 1. Pitx2 is required for survival of branchiomeric muscle precursors. (A,B) Right and left views of 9.5 dpc mouse embryos after whole-mount in situ hybridization with a Pitx2 probe (arrows denote hybridization signal). (C-H) Expression in chick embryos of Pitx2 (C,E,F,H) and MyoD (D,G) analysed by whole-mount in situ hybridization (C,F) and radioactive in situ hybridization to tissue sections (D,E,G,H) of stage 10 (C,F), 24 (D,E) and 26 (G,H) chick embryos. C is a dorsal view and D,E,G,H are frontal sections through the facial primordial. F is a transverse vibratome section through the head of the embryo in C. Arrows in D,G indicate developing muscles, in E the ectodermal and mesodermal expression of Pitx2, in H the ectomesenchymal Pitx2 expression. do, dorsal oblique muscle; e, eye; f, frontonasal mass; hy, hyoid arch; md, mandibular primordia.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Defective specification of first branchial arch undifferentiated core mesoderm in Pitx2 mutants. (A-C) Myogenin and (D,E) MyoD fail to be expressed normally in Pitx2 mutant embryos. (F-I). MyoR, a marker of undifferentiated core mesoderm, fails to be expressed in the Pitx2null mutant embryos while another marker of undifferentiated progenitors. (J,K) Pitx2 expression in control and Tbx1null mutant embryos. Arrows denote first branchial arch hybridization signal or absence of signal. Arrowheads in F,G denote signal in the second branchial arch.

View larger version (109K): [in this window] [in a new window]   Fig. 3. Specified, Myf5-positive myoblasts are drastically reduced in the Pitx2null mutant branchial arch. (A,B) Lineage tracing with the Myf5cre allele indicates that the Myf5 lineage is drastically reduced in the first branchial arch of 9.5 dpc Pitx2null mutant embryos (arrows). (C,D) By 10.5 dpc, the Myf5 lineage has expanded in the wild-type first branchial arch (arrow in C) but is still greatly reduced in the Pitx2null branchial arch. LacZ-positive cells are detectable dorsal to the branchial arch in both control and Pitx2null embryos (arrow in D). Also the Myf5 lineage that contributes to extraocular muscle is mislocalized in Pitx2null embryos (arrowhead in D). (E,F) In the 11.5 dpc embryo, a small number of Myf5cre-labeled cells can be detected, indicating that a few specified myoblasts are present in the Pitx2null mutant branchial arch (arrow in E,F). Abnormal localization of the EOM precursors is still observed in the Pitx2null embryo (arrowhead in F). e, eye; fn, frontonasal mass; md, mandible; mx, maxilla; ov, otic vesicle.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Fate mapping Pitx2 descendants in Pitx2 mutant embryos. (A,B) Lineage tracing with the Wnt1cre transgenic line that outlines core mesoderm of the first branchial arch. (C,D) Lineage tracing with the Pitx2cre allele showing Pitx2 lineage contributes to the core mesoderm in both control (arrow in C) and Pitx2null embryos (arrow in D). (E,F) Lineage tracing with the Pitx2cre allele in control (E) and Pitx2 hypomorphic embryos (F) at 16.5 doc. Pitx2 descendents contribute to branchiomeric muscle in the control, but in the mutant branchiomeric muscle is absent (arrow). (G,H) Lineage tracing and TUNEL double labeling in control and Pitx2null mutant embryos. The LacZ-positive cells are Pitx2 descendents that show upregulated TUNEL-positive cells in the mutant (arrow). mo, molar tooth; to, tongue.

View larger version (79K): [in this window] [in a new window]   Fig. 5. Splanchnic mesoderm-derived Mef2cAHFcre-marked muscle precursors are reduced in the Pitx2null mutant branchial arch. (A,B) Lineage tracing with the Mef2c AHF cre transgenic driver and the R26R allele indicated that the Mef2c-marked splanchnic mesoderm lineage contributes to first branchial arch branchiomeric muscle (denoted by arrows). (C-H) Lineage tracing with the Mef2c AHF cre in the control and Pitx2null mutant embryos indicated a deficiency in the splanchnic mesoderm component of the first branchial arch muscle precursors (denoted by arrows) in Pitx2null mutant embryos at 9.5 dpc (C,D), 10.5 dpc (E,F) and 11.5 dpc (E,F). It is notable that in the 11.5 dpc Pitx2 mutant embryo, Mef2c AHF cre-marked cells are found diffusely scattered in the caudal branchial arches (arrowhead). e, eye; md, mandibular process; mx, maxillary process.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Elevated and reduced Pitx2 activity in chick primary myoblasts. (A) Fluorescent images of mandibular micromass cultures, which have been infected with a control, Pitx2a or Pitx2a RCASBP retrovirus showing myocytes that have been visualized with the pan MyHc antibody A4.1025. (B) Bar chart quantifying these effects and also showing the effect on slow (red bar) and fast (yellow bar) myocyte development. *P<0.05.

View larger version (123K): [in this window] [in a new window]   Fig. 7. Conditional ablation of Pitx2 in mesoderm-derived cells reveals an autonomous role for Pitx2 in branchiomeric muscle development. Pitx2 deletion in mesoderm using the Mesp1cre and Pitx2flox alleles. (A,B) Lineage tracing indicates a reduction in the number of LacZ-marked Pitx2 mutant descendents in the masseter muscle (outlined, arrow). (C,D) Myogenin expression (arrow) is greatly reduced in the Pitx2 mutant compared with control. Sections through the masseter muscle of control (E,F) and Mesp1cre; Pitx2 n/f mutant (G,H) indicate a muscle deficiency in the Pitx2 conditional mutant (arrows). (I-L) Myosin immunostaining with the MF20 antibody in 14.5 control (I,J) and Pitx2 conditional null mutant (K,L) to mark branchiomeric muscle in control and show deficiency in mutant (arrows). Note that in the mutant (L) there is residual subcutaneous muscle that shows myosin reactivity (arrowhead). e, eye; mc, Meckel's cartilage; md, mandible; mx, maxilla.

View larger version (47K): [in this window] [in a new window]   Fig. 8. Ablation of Pitx2 in splanchnic mesoderm with Mef2cAHFcre- reveals an autonomous role for Pitx2 in branchiomeric muscle development. (A-F) Lineage tracing with the Mef2c AHF cre transgenic driver (arrows) indicated that the Mef2c-marked splanchnic mesoderm lineage is reduced in the first branchial arch of Mef2c AHF cre; Pitx2 n/f conditional mutant embryos at 9.5 dpc (A,B), 11.5 dpc (C,D) and 12.0 dpc (E,F). e, eye; md, mandibular process; mx, maxillary process.

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