Tazumi S et al. (2010), Paraxial T-box genes, Tbx6 and Tbx1, are requir...

XB-ART-42286

Dev Biol 2010 Oct 15;3462:170-80. doi: 10.1016/j.ydbio.2010.07.028.

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Paraxial T-box genes, Tbx6 and Tbx1, are required for cranial chondrogenesis and myogenesis.

Tazumi S , Yabe S , Uchiyama H .

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We previously reported that Tbx6, a T-box transcription factor, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation. Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly similar to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. Tbx6 knockdown also reduced the expression of Tbx1, another T-box gene expressed in more anterior paraxial structures. Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9.

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Species referenced: Xenopus laevis
Genes referenced: fgf8 fzd7 gal.2 myf5 myod1 pcdh18 pcdh8l snai2 sox9 tbx1 tbx2 tbx6 wnt11 wnt11b wnt8a
GO keywords: neural crest cell development [+]
???displayArticle.antibodies??? Somite Ab1
???displayArticle.morpholinos??? tbx1 MO4 tbx6 MO1

???displayArticle.disOnts??? DiGeorge syndrome
???displayArticle.omims??? DIGEORGE SYNDROME; DGS

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	Fig. 2. Expression patterns of neural crest markers and neural crest inducers in Tbx6 knockdown embryos as revealed by whole-mount in situ hybridization. MO-injected sides (*) are shown by Magenta-GAL staining. Images of MO-injected sides are flipped horizontally to facilitate comparison. (A) Snail2 and Sox9 expressions at stage 16. Dorsal view, anterior to top. (B) Fgf8 and Wnt8 expressions at stage 14. Dorsal view, anterior to top. (C) Snail2, Sox9, and PCNS expressions in the head of stages 19, 25, and 27 embryos. Lateral view, anterior to the left. (D) Tbx6 expression at stage 47 (arrowhead). Lateral view, anterior to the left. (E) Sox9 expression in the head of stage 27 embryos injected into one cell at the 8-cell stage with 6 ng of Tbx6 exint-MO.
	Fig. 3. Cells from the neural crest transplants migrate to cartilage-forming areas in Tbx6 knockdown embryos. (A, B) Wnt11 and Fz7 were expressed in the cranial neural crest cells and adjacent ectoderm indicated by arrowheads in stage 17 embryos injected with Tbx6 exint-MO as revealed by whole-mount in situ hybridization. Dorsal view, anterior to top. MO-injected sides (*) are shown by Magenta-GAL staining. (C) Schematic diagram of the cranial neural crest-transplantation experiment. Distributions of fluorescent cranial neural crest cells in stage 27 (D) and stage 28 (E) host embryos. Both images are flipped horizontally.
	Fig. 4. Tbx6 knockdown causes defects in head muscles. MO-injected sides are marked with *. Skeletal muscle-specific 12/101 antibody staining in stage 47 larvae injected with Tbx6 5mis-MO (A–D) or Tbx6 exint-MO (E–H). Dorsal view (A, E), left lateral view (B, F), right lateral view (C, G), and ventral view (D, H). Some images are flipped horizontally to facilitate comparison (C, D, G, H). Lm, levator mandibulae; Eo, extraocular; Lab, levator arcuum branchialium; Im, intermandibular; QH, quadrato-hyoangularis; Oh, orbitohyoideus; Ih, interhyoid; Gh, geniohyoideus; Sr, subarcuales rectus; B, branchial; Vbw, ventral body wall.
	Fig. 5. Tbx6 regulates anterior mesodermal genes including Tbx1. MO-injected sides (*) are shown by Magenta-GAL staining. Myf-5 (A, B) and MyoD (C, D) expressions in stage 32 embryos injected with Tbx6 exint-MO. (E, F) Pcdh18 expression in stage 31 embryos injected with Tbx6 exint-MO. Tbx1 expression in stages 27 (G, H) and 14 (I) embryos injected with Tbx6 exint-MO. Images of MO-injected sides are flipped horizontally to facilitate comparison. Lateral view, anterior to the left (A–H), anterior view, dorsal to top (I). (J, K) Tbx6 (magenta) and Tbx1 (turquoise) expressions as revealed by double in situ hybridization. Stage 14, lateral view, anterior to the left (J), stage 16, dorso-anterior view (K). (L) PCNS (magenta) and Tbx1 (turquoise) expressions as revealed by double in situ hybridization. Stage 25, lateral view, anterior to the left. (M) A horizontal paraffin section (8 μm) at the position indicated in L.
	Fig. 6. Tbx1 knockdown causes defects in cranial cartilages and head muscle hypoplasia. MO-injected sides are marked with *. (A) RT-PCR analysis in Tbx1 exint-MO-injected embryos. (B, B′) Alcian blue-stained cartilages from a stage 47 larva injected with Tbx1 exint-MO before and after dissection, respectively. (C) Alcian blue-stained cartilage from a stage 47 larva injected with Tbx1/pCS2+ (10 pg) together with Tbx1 exint-MO. So, subocular; Et, ethmoid-trabecular; I, infrarostral; M, Meckel's cartilage; Q, quadrate; C, ceratohyal; Bh, basihyal; B, branchial. (D–G) Skeletal muscle-specific 12/101 antibody staining in a stage 47 larva injected with Tbx1 exint-MO. Dorsal view (D), left view (E), right view (F), and ventral view (G). Two images are flipped horizontally to facilitate comparison (F, G). Lm, levator mandibulae; Eo, extraocular; Lab, levator arcuum branchialium; Im, intermandibular; QH, quadrato-hyoangularis; Oh, orbitohyoideus; Ih, interhyoid; Gh, geniohyoideus; Sr, subarcuales rectus; B, branchial.
	Fig. 7. Expression patterns of neural crest marker and mesodermal genes in Tbx1 knockdown embryos. (A) Snail2, Sox9, and PCNS expressions in the head of stages 22 and 27 embryos as revealed by whole-mount in situ hybridization. Lateral view, anterior to the left. (B) Distribution of the fluorescent neural crest cells in the cranial neural crest-transplantation experiment. The image is flipped horizontally. (C, D) Myf-5, MyoD, and Tbx6 expressions as revealed by whole-mount in situ hybridization. (C) Stage 37, lateral view, anterior to the left. (D) Stage 16, dorsal view, anterior to top. MO-injected sides (*) are shown by Magenta-GAL staining. Images of MO-injected sides are flipped horizontally to facilitate comparison.
	Fig. 8. The phenotypes of Tbx6 morphants are rescued by Tbx1 plasmid injection. Alcian blue-stained cartilage from a stage 47 larva (A) or whole-mount in situ hybridization for Sox9 in a stage 27 embryo (B), injected with Tbx1/pCS2+ (10 pg) together with Tbx6 exint-MO. So, subocular; Et, ethmoid-trabecular; I, infrarostral; M, Meckel's cartilage; Q, quadrate; C, ceratohyal; Bh, basihyal; B, branchial. MO-injected sides (*) are shown by Magenta-GAL staining. Images of MO-injected sides are flipped horizontally to facilitate comparison. (C) RT-PCR analysis in stages 16, 22, and 28 embryos bilaterally injected with MOs alone (50 ng) or with Tbx6 or Tbx1/pCS2+ (20 pg).
	Suppl. Fig. 1. Expression patterns of neural crest markers, Snail2, Sox9, and PCNS in stage 22 embryos injected with Tbx6 5mis-MO, as revealed by whole-mount in situ hybridization. MO-injected sides (*) are shown by Magenta-GAL staining. Images of MO-injected sides are flipped horizontally to facilitate comparison.
	Suppl. Fig. 2. Sox9 expression in stage 27 embryos injected with Tbx6 exint-MO and Tbx6/pCS2+ (10 pg), and with Tbx1 exint-MO and Tbx1/pCS2+ (10 pg) as revealed by whole-mount in situ hybridization. MO-injected sides (*) are shown by Magenta-GAL staining. Images of MO-injected sides are flipped horizontally to facilitate comparison.
	Suppl. Fig. 3. Expression pattern of Xenopus Pcdh18, as revealed by whole-mount in situ hybridization. (A) Stage 12, blastoporal view. (B) Stage 14, dorsal view anterior to top. (C) Stage 14, anterior view, dorsal to top. (D) Stage 33, lateral view, anterior to the left. (E) A paraffin section (14 μm) at the position indicated in D.
	Suppl. Fig. 4. PCNS expression in stage 22 embryos injected with Tbx6 exint-MO and Tbx1 exint-MO as revealed by whole-mount in situ hybridization. MO-injected sides (*) are shown by Magenta-GAL staining. The left panel shows the loss of somitic PCNS expression in Tbx6 morphants. The right panel shows a great anterior-shift of somitic PCNS expression patterns in Tbx1 morphants. Both panels show the short distances of the cranial neural crest migration on the MO-injected side as compared to the control side.

	Fig. 1. Tbx6 knockdown causes defects in cranial cartilages. The heads of stage 47 larvae injected with Tbx6 5mis-MO (A) or exint-MO (B) plus ODA and viewed from the ventral side (C–E). Dotted lines indicate the dorsal midline and rostral border of the larvae. MO-injected sides are marked with *. Alcian blue-stained cartilages from stage 47 larvae injected with Tbx6 5mis-MO or Tbx6 exint-MO before (C, D) and after (C′, D′) dissection. So, subocular; Et, ethmoid-trabecular; I, infrarostral; M, Meckel's cartilage; Q, quadrate; C, ceratohyal; Bh, basihyal; B, branchial. (E) Alcian blue-stained cartilage from stage 47 larvae injected with Tbx6/pCS2+ (10 pg) together with Tbx6 exint-MO.
	Fig. 9. Sandwich assay of Tbx6 or Tbx1 to access histogenesis from the cranial neural crest. (A) Schematic diagram of the production of sandwich explants composed of animal caps and cranial neural crest. Uninjected explants (B) or explants injected with Tbx6 mRNA (C) and injected with Tbx1 mRNA (D) at stage 47. Paraffin sections of the cranial cartilages of a stage 47 larva (E), an uninjected explant (F), or explants injected with Tbx6 mRNA (G) and Tbx1 mRNA (H).
	Expression pattern of Xenopus pcdh18, as revealed by whole-mount in situ hybridization.Stage 33, lateral view, anterior to the left.
	Expression pattern of Xenopus pcdh18, as revealed by whole-mount in situ hybridization. A paraffin section (14 Î¼m).