Paraxial T-Box Genes, Tbx6 and Tbx1, Are Required for Cranial Chondrogenesis and Myogenesis

Developmental Biology 346 (2010) 170–180

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Developmental Biology

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

Shunsuke Tazumi, Shigeharu Yabe 1, Hideho Uchiyama ⁎

International Graduate School of Arts and Sciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan article info abstract

Article history: We previously reported that Tbx6, a T-box transcription factor, is required for the differentiation of ventral Received for publication 19 February 2010 body wall muscle and for segment formation and somitic muscle differentiation. Here, we show that Tbx6 is Revised 12 June 2010 also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle Accepted 6 July 2010 development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the Available online 5 August 2010 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 Keywords: Neural crest neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. Sox9 Tbx6 knockdown also reduced the expression of Tbx1, another T-box gene expressed in more anterior Cartilage paraxial structures. Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, Head muscle including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, Xenopus laevis 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. © 2010 Elsevier Inc. All rights reserved.

Introduction et al., 2008; Carmona-Fontaine et al., 2008). Neural crest differentiation relies on intrinsic programs and extrinsic cues. Sry-related transcription In vertebrates, craniofacial structures are formed by the orches- factor Sox9 is required for cartilaginous, testicular, neural, and cardiac trated integration of multiple tissue interactions. The cranial bones, development (Akiyama et al., 2002; Stolt et al., 2003; Akiyama et al., cartilages, and attached muscles play important roles in determining 2004; Chaboissier et al., 2004). Sox9 is expressed in the neural crest and the shape and motor functions of the head. is required for cranial chondrogenesis in mice (Wright et al., 1995; Zhao The cranial neural crest is the source of nerves such as the cranial et al., 1997; Ng et al., 1997)andXenopus (Spokony et al., 2002). ganglia and sympathetic neurons, a lot of the cranial bones and The contribution of head mesoderm to muscle development has cartilages, and some of the musculature in mice, chicks, and fish (Noden, been well studied in mice and chicks (Trainor et al., 1994; Noden and 1991; Le Douarin and Kalcheim, 1999). During embryogenesis, the Francis-West, 2006). All muscle genes reflecting early steps of the neural crest cells develop through a course of three key events: myogenic program including Capsulin, Tbx1, MyoR, Myf-5, MyoD, and induction, migration, and differentiation. Neural crest induction desmin are expressed in the branchial arches following neural crest requires partial attenuation of BMP signaling by BMP antagonists ablation in chick embryos. However, in the absence of neural crest (Marchant et al., 1998; Nguyen et al., 1998; Tribulo et al., 2003), cells, jaw muscles were severely reduced, indicating that the neural canonical Wnt signaling (Wu et al., 2003; Heeg-Truesdell and LaBonne, crest cells are required for normal muscle differentiation after the 2006; Steventon et al., 2009), and Fgf signaling (Kengaku and Okamoto, onset of muscle specification (Tzahor et al., 2003; von Scheven et al., 1993; Mayor et al., 1995, 1997; Monsoro-Burq et al., 2003; Hong et al., 2006; Rinon et al., 2007). Head muscle differentiation in Xenopus has 2008). In Xenopus, the cranial neural crest cells migrate from the not been studied well, but the morphogenesis and histological hindbrain in three streams and populate the pharyngeal arches differentiation of the cranial muscles have been documented (Sadaghiani and Thiebaud, 1987). It has been suggested that neural (Ziermann and Olsson, 2007). crest migration requires noncanonical Wnt signaling and is controlled The T-box transcription factor family plays a crucial role in by contact inhibition of locomotion (De Calisto et al., 2005; Matthews embryonic development (Papaioannou and Silver, 1998; Showell et al., 2004). A member of this family, Tbx1, has been indicated by genetic analysis to be the major gene responsible for 22q11 deletion/ ⁎ Corresponding author. Fax: +81 45 787 2413. DiGeorge syndrome, in human and mouse (Merscher et al., 2001; E-mail address: [email protected] (H. Uchiyama). 1 Present address: Research Institute National Center for Global Health and Medicine, Meechan et al., 2009; Ryckebüsch et al., 2010). Tbx1 null mutant 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan. mice have craniofacial and cardiovascular defects associated with

0012-1606/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2010.07.028 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 171 pharyngeal hypoplasia (Baldini, 2005). In Xenopus, an injection of a AL, USA). Tbx1, full-length cDNA clone, was obtained from a Lambda repressor form of Tbx1 caused cranial cartilage disorganization, ZAP II cDNA library derived from embryos at stages 22–25 with a PCR reduction or absence (Ataliotis et al., 2005). Another member of this product as a probe, according to the screening procedures as family, Tbx6, is expressed in the primitive streak, paraxial mesoderm, previously described (Uchiyama et al., 2001). To generate Wnt8/ muscle lineage blastomeres, and tailbuds in various species (Chapman pBluescript II, PCR products were amplified from Xwnt-8/pSP64T et al., 1996; Yasuo et al., 1996; Uchiyama et al., 2001). Tbx6 is required (Christian et al., 1991) and ligated into the T-vector made from for somite segmentation and differentiation and for ventral body wall pBluescript II (Uchiyama et al., 2001). To produce Fgf8b/pBluescript II, muscle differentiation (Chapman and Papaioannou, 1998; Tazumi Snail2/pBluescript II, and Sox9/pBluescript II, PCR fragments were et al., 2008). amplified from whole embryonic cDNA and ligated into the T-vector. Here, we show that Tbx6 is also involved in differentiation of more Forward (F) and reverse (R) PCR primers for Tbx1, Wnt8, Fgf8b, Snail2, anterior structures such as cranial cartilages and head muscles by and Sox9 were as follows: Tbx1 (F: 5′-GTGTCAATCATCTGTGTA- regulating more anterior genes, including Tbx1 and Sox9. GAGCTGCG-3′;R:5′-CAACACTGGAGGAACAGCATTAATGC-3′), Wnt8 (F: 5′-CCTTCATCATGCAAAACACC-3′;R:5′-TCTGGAATGCCGTCATCTCC-3′), Materials and methods Fgf8b (F: 5′-CTGAGCAACATGAACTACATCACC-3′;R:5′-CCTACCGA- GAACTTGAATATCGAG-3′), Snail2 (F: 5′-GACCGTTACTTGTGCGTCC-3′;R: Plasmid constructs 5′-GCAGCTACACACTGCTTCTCC-3′), and Sox9 (F: 5′-CGCATGAATCTCTTG- GATCC-3′;R:5′-CTAGGGTCTTGTGAGCTGTGTGTAC-3′). Tbx6/pCS2+ and β-gal/pBluescript II were described previously A partial protocadherin-18 (Pcdh18) sequence was identified from (Tazumi et al., 2008). cDNA clones including the full open reading two nonoverlapping EST clones of Xenopus laevis (BX846839 and frame of PCNS, Wnt11,andFz7 (GenBank accession numbers: BQ732394). Using primers derived from these clones (F: 5′-GGAGCA- BG020773, BC042228, and BC084745; each inserted in pCMV- GATGGTCGGTACCG-3′;R:5′-CCAAGAGCACAGCACATATGGC-3′), we SPORT6 vector) were obtained from Open Biosystems Inc. (Huntsville, obtained a PCR fragment of 2030 bp that encodes 676 amino acids of

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. 172 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 the putative X. laevis Pcdh18 (DDBJ accession number: AB573177 3′;R:5′-CTAGGGTCTTGTGAGCTGTGTGTAC-3′)andTbx1 (F: 5′-CACCGTG- currently in preparation for submission). It was ligated into the T-vector CAGCTGGAGATGAAAGC-3′;R:5′-ATCGGCCATGGGATCCATCCC-3′). to generate Pcdh18/pBluescript II. The obtained clone had 91% nucleotide and 96% amino acid identities with X. tropicalis Pcdh18. Magenta-GAL staining, whole-mount in situ hybridization, and immunohistochemistry

Embryo manipulation, microinjection, antisense morpholino oligos, and For lineage tracing, mRNA of β-gal with the nuclear localization RT-PCR signal of SV40 middle T antigen at the N-terminus (Tazumi et al., 2008) was injected together with MOs at a dose of 200 pg per one-half X. laevis embryos were prepared according to Uchiyama et al. (2001) of the embryo. The procedures followed were in accordance with Yabe and embryonic stages were determined according to Nieuwkoop and et al. (2006), except that Magenta-GAL (INALCO, Milano, Italy) was Faber (1956). Microinjection was performed according to Tazumi et al. used as a chromogen. Whole-mount in situ hybridization, immuno- (2008). Antisense morpholino oligos (MOs) were obtained from Gene histochemistry using the skeletal muscle-speciﬁc antibody 12/101 Tools LLC (Philomath, OR, USA). Tbx6 exint-MO and Tbx6 5mis-MO were (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and described previously (Tazumi et al., 2008). For Tbx1 knockdown, exon1– cRNA probes for Tbx6, Myf-5, and MyoD were described previously exon2 of the Tbx1 gene was PCR-ampliﬁed from tadpole genomic DNA (Tazumi et al., 2008). Tbx1, Fgf8b, Wnt8, Wnt11, Fz7, Snail2, Sox9, PCNS, and sequenced, and exint-MO (5′-CAAAAGATGGCCCTTACCTGCCAGC-3′) and Pcdh18 were transcribed with T3 or T7 RNA polymerase from the was designed at the exon1–intron1 junction. All MOs were injected at a following linearized templates: Tbx1/pCS2+, Fgf8b/pBluescript II, dose of 25 ng per one-half of the embryo. RT-PCR primers for ornithine Wnt8/pBluescript II, Wnt11/pCMV-SPORT6, Fz7/pCMV-SPORT6, decarboxylase (ODC) were described previously (Tazumi et al., 2008), and Snail2/pBluescript II, Sox9/pBluescript II, PCNS/pCMV-SPORT6, and for Sox9 and Tbx1 were as follows: Sox9 (F: 5′-ACTCAGCAAGACGCTGGG- Pcdh18/pBluescript II.

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. S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 173

Alcian blue staining markers Snail2/Slug, Sox9,andPCNS in the MO-injected embryos. In stage 16 mid-neurulae, no remarkable changes in Snail2 and Sox9 Stage 47 larvae were fixed in neutral-buffered 4% formaldehyde expressions were detected (85% of embryos, n =13; 92% of embryos, for 90 min at room temperature (RT) and dehydrated in 100% n =13, respectively; Fig. 2A), suggesting that neural crest induction methanol. Fixed embryos were preincubated in acidic alcohol (80% was accomplished in Tbx6 knockdown embryos. Consistent with this ethanol, 20% acetic acid) for at least 10 min and were stained for 6 h at finding, the neural crest inducers Fgf8 and Wnt8 appeared to be RT in 0.05% Alcian blue (A3157; Sigma, St. Louis, MO, USA) in acidic expressed normally in stage 14 early-neurulae (93% of embryos, alcohol. The embryos were destained in acidic alcohol overnight at RT, n =42; 100% of embryos, n =44, respectively; Fig. 2B). At stage 19, rehydrated using 50% and 25% ethanol mixed with phosphate- the neural crest migration had started, but migration paths on the buffered saline (PBS;137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, exint-MO-injected side were shorter than that on the uninjected side 1.47 mM KH2PO4; pH 7.4), and bleached in 2–3% hydrogen peroxide, as revealed by Snail2 and PCNS in situ hybridization (100% of 5% formamide, and 0.5× SSC. After dehydration and rehydration to embryos, n = 19; 94% of embryos, n =18, respectively; Fig. 2C). In remove bleach solution, the embryos were transferred into 1% stage 25 tailbud embryos, Snail2 expression had declined, showing potassium hydroxide for 2 h at RT. Then the tissue was rinsed in only residual expression in the cells that had already migrated to saturated sodium tetraborate, and cleared with 0.05% trypsin (207- lateral positions on the uninjected side, while Snail2 expression on 09891; Wako Chemicals, Osaka, Japan) in this borate solution for 2 h the MO-injected side was confined to the dorsal side (100% of at RT. After being washed with PBS, the skin was removed, the embryos, n =23). Sox9 expression in the neural crest was slightly junction between the ethmoid-trabecular cartilage and quadrate shortened in the dorso-ventral direction (100% of embryos, n =25). cartilage on the MO-injected side was cut open, and the specimens The most posterior stream of migrating neural crest cells expressing were flat-mounted. PCNS on the uninjected side had already begun to branch off, whereas this stream on the MO-injected side was still single (100% of Transplantation experiments and sandwich assay embryos, n =21). These results suggest that Tbx6 knockdown caused a slight delay in neural crest migration. In 5mis-MO-injected control At the 4-cell stage, 25 ng of Tbx6 exint-, Tbx6 5mis-, or Tbx1 exint- embryos, even milder or no delay in neural crest migration was MO dissolved in sterile water with or without 5 ng of Oregon Green 488 dextran amine (ODA, D7173; Invitrogen, Paisley, UK) was injected into the marginal zone on the right side of the embryos. The cranial neural crest region was isolated from these embryos at stages 14–16, and inserted into the same region of the recipients according to the procedures described (Borchers et al., 2000). At the 2-cell stage, 50 pg of Tbx6 or Tbx1 mRNA or 5 ng of ODA was injected into both blastomeres in the animal pole region. Injected and uninjected animal caps were excised at stages 8 and 9, conjugated by pressing the outer surfaces with the inner surfaces facing together, and cultured at 20 °C in Steinberg's solution on an agar-coated dish. At stages 14–16, the ODA-labeled cranial neural crest region was isolated and sandwiched between the conjugated animal caps, which were partially cut reopen. The resulting sandwich explants and whole embryos were cultured until stage 47, fixed in neutral-buffered 4% formaldehyde overnight at RT, embedded in paraffin, and sectioned at 6 μm. The sections were stained with Alcian blue solution (pH 2.5, 015-13805; Wako Chemicals) and counterstained with hematoxylin for light microscopy.

Results

Tbx6 knockdown caused severe defects in cranial cartilages

In our previous study, MOs targeting the exon1–intron1 junction of the Tbx6 gene (Tbx6 exint-MO) and a control 5 bases-mismatch (Tbx6 5mis-MO) were effective in knockdown (Tazumi et al., 2008). When these knockdown embryos were cultured until later stages, Tbx6 exint-MO-injected tadpole (stage 47), unexpectedly, showed apparent morphological abnormalities in the head (Figs. 1AandB). Alcian blue staining of these larvae revealed no effect of 5mis-MO (100% of larvae, n=29; Figs. 1CandC′) but a severe loss of the mandibular, hyoid, and branchial arch derivatives on the exint-MO-injected side (100% of larvae, n=51; Figs. 1DandD′). The loss of cartilages was rescued overall by co-injecting the closed circular plasmid DNA containing the full coding sequence (CDS) of Tbx6 (Tbx6/pCS2+) Fig. 3. Cells from the neural crest transplants migrate to cartilage-forming areas in Tbx6 together with exint-MO (85% of larvae, n=26; Fig. 1E). 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 knockdown caused a delay in cranial neural crest migration 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 Because a lot of cranial cartilages are derived from the neural fluorescent cranial neural crest cells in stage 27 (D) and stage 28 (E) host embryos. crest, we examined the expression patterns of the neural crest Both images are flipped horizontally. 174 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 observed (Supplementary Fig. 1). Remarkably, at stage 27, Sox9 Tbx6 knockdown caused severe defects of head muscles expression was severely reduced (100% of embryos, n = 12) despite continuous expression of PCNS in the migrating neural crest cells of For further analysis of the morphological abnormalities in the head the exint-MO-injected side (88% of embryos, n =16). This reduction of Tbx6 knockdown stage 47 larvae, we performed skeletal muscle- of Sox9 expression was rescued by co-injecting Tbx6/pCS2+ together specific 12/101 antibody staining. It was revealed that 5mis-MO had with the MO (70% of embryos, n=20; Supplementary Fig. 2). no effect (100% of larvae, n=21; Fig. 4 A–D), while most of the Whole-mount in situ hybridization has revealed that Tbx6 is skeletal muscles in the larval head were severely fragmentary or lost exclusively expressed in the tailbud (Uchiyama et al., 2001; Tazumi on the exint-MO-injected side (100% of larvae, n=43; Figs. 4E–H). et al., 2008), and at the tip of the larval tail (Fig. 2D). To examine Whole-mount in situ hybridization revealed that expressions of Myf-5 whether Tbx6 acts on the neural crest development in a cell- and MyoD, the early markers of the muscle progenitors, were reduced autonomous or non-cell-autonomous manner, targeted injections of in the head of embryos injected with exint-MO (97% of embryos, exint-MO were carried out at the 8-cell stage, into a dorso-animal n=30; 90% of embryos, n=31, respectively; Figs. 5A–D). To analyze (DA; neural crest lineage) or a ventro-vegetal (VV; mesodermal more properties of the pharyngeal structures in exint-MO-injected lineage) blastomeres. As shown in Fig. 2E, DA injection had no embryos, partial cDNA of Pcdh18, which has been reported to be significant effect on Sox9 expression in the neural crest cells of 93% clearly expressed in the pharyngeal arches of zebrafish (Kubota et al., embryos (n =14), while attenuation of Sox9 expression occurred in 2008; Aamar and Dawid, 2008), was newly identified in X. laevis. VV injection (77% of embryos, n =13). Similar to zebrafish Pcdh18, Xenopus Pcdh18 expression was observed To monitor noncanonical Wnt signaling, which is essential for in the pharyngeal arches and neuroectoderm (Supplementary Fig. 3), neural crest migration, the expressions of Wnt11 and Fz7 were and was reduced in the pharyngeal arches by Tbx6 knockdown (100% analyzed; no significant changes in the expressions of either gene in of embryos, n=26; Figs. 5E and F). the cranial neural crest and adjacent ectoderm were detected in Tbx6 knockdown embryos (85% of embryos, n=13; 100% of embryos, Tbx6 regulates Tbx1 expression n =11, respectively; Figs. 3A and B). We next performed a transplantation experiment for the direct assessment of the migration In Xenopus, Tbx6 is expressed specifically in nascent paraxial and capacity in Tbx6-knockdown neural crest cells. At the 4-cell stage, ventral mesoderm (Uchiyama et al., 2001). In order to clarify the reasons exint- or 5mis-MO was injected alone or with ODA. At the early why Tbx6 knockdown affects the development of head and pharyngeal neurula stage, the prospective neural crest region was transplanted muscles and cartilages, we noticed on Tbx1 as a candidate to mediate (Fig. 3C). Whether the exint-MO-loaded neural crest region was Tbx6 function. Although Tbx1 and Tbx6 are expressed in the paraxial transplanted to 5mis-MO-loaded recipient, or vice versa, a large part of mesoderm, Tbx1-expressing areas are located anteriorly (Ataliotis et al., the grafted cells showed migration along three streamlines around 2005). At the early neurula and tailbud stages, Tbx1 expression was the pharyngeal arches (76% of host embryos, n=41; Figs. 3D and E). severely reduced in Tbx6 knockdown embryos (at stage 27, 94% of These results suggest that the cranial neural crest cells migrated and embryos, n=17; at stage 14, 91% of embryos, n=45;Figs. 5G–I). Double finally reached the prospective cartilage-forming area in Tbx6 in situ hybridization of normal embryos revealed that the Tbx1-and knockdown embryos. Tbx6-expressing areas did not overlap, and there was a gap in between

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. S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 175

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. them (Figs. 5J and K). In addition, the comparison between Tbx1 and phase (at 30 cycles). Although the sizes of the other amplicons were PCNS expression patterns at the tailbud stage (stage 25) showed that not similar to those of the fragments obtained from genomic PCR, Tbx1 was expressed exclusively in the pharyngeal mesoderm and splicing perturbations and the resultant translational frameshift in the endoderm adjacent to the PCNS-expressing neural crest cells (Figs. 5L Tbx1 mRNA were confirmed by sequence analysis. and M). Tbx1 exint-MO-injected larvae showed a high rate of cranial cartilage hypoplasia including severe loss of subocular, Meckel's, and Tbx1 knockdown also caused cranial cartilage and head muscle quadrate cartilages, partial loss of ethmoid-trabecular and ceratohyal hypoplasia cartilages, and malformations of branchial cartilages (96% of larvae, n=23; Figs. 6B and B′). This hypoplasia was rescued by co-injecting For Tbx1 knockdown, an MO targeting the exon1–intron1 junction Tbx1/pCS2+ closed circular plasmid DNA containing the full CDS of of Tbx1 gene (Tbx1 exint-MO) was designed. To examine the Tbx1, together with exint-MO (47% of larvae, n=19; Fig. 6C). Tbx1 performance of Tbx1 exint-MO, MO was injected into the marginal knockdown also caused head muscle hypoplasia including severe loss zone at the 4-cell stage and the injected embryos were cultured until of interhyoid, quadrato-hyoangularis, orbitohyoideus, and branchial stage 16 or 26. In RT-PCR analysis, several amplicons were detected in muscles, partial loss of extraocular muscles, and reductions of other the MO-injected embryos (Fig. 6A). The length of the shortest head muscles (100% of larvae, n=23; Figs. 6D–G). These phenotypes amplicon was indistinguishable in size from that of the normal of Tbx1 knockdown larvae were morphologically milder than those of amplicon, but it showed little amplification before the PCR plateau Tbx6 knockdown larvae. 176 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180

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.

Tbx1 knockdown caused a delay in the neural crest migration and a all, but rather the area expanded anteriorly (Fig. 7D). Similarly, reduction in the muscle progenitor gene expression PCNS expression in the somites was shifted anteriorly by Tbx1 knockdown (100% of embryos, n=16), while Tbx6 knockdown As observed in Tbx6 knockdown embryos, the expression patterns severely reduced somite expression of PCNS (100% of embryos, of Snail2, Sox9,andPCNS on the Tbx1 exint-MO-injected side n=10; Supplementary Fig. 4). These results suggest that Tbx1 is indicated a delay in the neural crest migration at stage 22 (94% of hypostatic to Tbx6 and is involved in the cranial neural crest and head embryos, n =16; 100% of embryos, n=15; 100% of embryos, n =16, muscle development. respectively; Fig. 7A). PCNS was still clearly expressed in the neural crest cells, migrating in a slightly delayed manner at stage 27 (94% of embryos, n =18) with a marked reduction in Sox9 expression (100% Tbx1 plasmid injection rescued phenotypes of Tbx6 morphants of embryos, n = 19). This reduction of Sox9 expression was rescued by co-injecting Tbx1/pCS2+ together with the MO (68% of embryos, To examine whether Tbx1 mediates Tbx6 functions, Tbx1/pCS2+ n =22; Supplementary Fig. 2). The effect of Tbx1 knockdown on the was co-injected together with Tbx6 exint-MO. At stage 47, injected neural crest migration was also veriﬁed by transplantation of Tbx1- larvae showed a partial rescue of cranial cartilages, relatively much knockdown ODA-loaded neural crest cells into Tbx1-knockdown higher levels in subocular, ethmoid-trabecular, Meckel's, and embryos in which the transplanted neural crest cells ﬁnally reached quadrate cartilages (70% of larvae, n =23; Fig. 8A). Furthermore, the prospective cartilage-forming area (Fig. 7B). the restoration of Sox9 expression was observed at stage 27 by Consistent with defects in the head muscles of Tbx1 knockdown whole-mount in situ hybridization (85% of embryos, n =26;Fig. 8B). larvae, the expressions of Myf-5 and MyoD in the head were reduced RT-PCR analysis revealed that the reduction of Sox9 expression in on the Tbx1 exint-MO-injected side (Fig. 7C). Importantly, Tbx6 Tbx6 morphants was recovered by Tbx1 as well as Tbx6 plasmid expression on the Tbx1 exint-MO-injected side had not reduced at injections at stage 28 (Fig. 8C). S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 177

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.

Tbx6-injected animal cap induces cartilage formation from the neural neurula stage. ODA-labeled neural crest regions were isolated from crest sibling neurulae and inserted into the conjugated explants; the sandwiches were then cultured until stage 47 (Figs. 9B–D). At this As mentioned above, Tbx6 and Tbx1 are suggested to be involved in stage, cartilages have been formed only in the larval head (Fig. 9E). In cartilage differentiation from the neural crest. To test this issue all uninjected sandwiches, melanophores were differentiated but no directly, we performed the following experiment using animal cap cartilaginous tissues were found (n =24; Fig. 9F). In contrast, explants (Fig. 9A). Tbx6 or Tbx1 mRNA-injected animal caps and cartilaginous tissues were differentiated in some of the sandwiches uninjected animal caps were conjugated and cultured until the injected with Tbx6 mRNA (16% of explants, n=25; Fig. 9G). In all

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). 178 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180

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). sandwiches injected with Tbx6 or Tbx1 mRNA, melanophores were Wnt8, allowing the activation of both placodal markers and neural differentiated. In addition, nerve-like structures were differentiated in crest specifiers (Callery et al., 2010), and Fgf8 and Wnt8 are immediate 52% of explants injected with Tbx6 (n=25) or 49% of explants early response genes of Tbx6 in animal cap assays (Li et al., 2006). injected with Tbx1 mRNA (n=51; Fig. 9H), whereas no nerve-like However, in vivo, the expression of Fgf8 and Wnt8 did not change in structures were found in uninjected sandwiches. the early neurulae after injection with Tbx6 exint-MO. In mice, it was reported that Tbx1 is involved in cardiac neural crest cell migration via Discussion the Slit/Robo signaling pathway (Calmont et al., 2009); similar mechanisms might exist in Xenopus. Alternatively, because Xenopus Tbx6 and Tbx1 are involved in cranial neural crest development cranial neural crest cell migration was promoted on the fibronectin- coated slide (Alfandari et al., 2003), Tbx6 or Tbx1 knockdown might In X. laevis, it has been proposed that the paraxial mesoderm is cause quantitative alteration of fibronectin between the neural crest involved in neural crest induction via FGF signals, mainly based on the and the underlying mesoderm. However, the results of PCNS in situ recombination assay (Monsoro-Burq et al., 2003). In the present hybridization and the neural crest-transplantation experiments study, a knockdown of Tbx6, a master gene for the paraxial mesoderm, clearly showed the presence of cranial neural crest cells and their inhibited the cartilage differentiation from the cranial neural crest. In migration on a near complete scale in Tbx6 morphants. Therefore, we Tbx6 knockdown embryos, neural crest induction appeared to be think that Tbx6 is mainly involved in the differentiation of the neural attained. A recent report suggests that Tbx6 can induce both Fgf8 and crest. In animal cap explants inserted with the neural crest, Tbx6 S. Tazumi et al. / Developmental Biology 346 (2010) 170–180 179 induced cartilaginous tissues as well as nerve-like tissues, although at MO-injected Xenopus larvae, suggesting that there are some func- a moderate rate, while Tbx1 induced only nerve-like tissues even with tional differences between Xenopus and mouse Tbx1 genes. a high dose of mRNA. These results suggest that Tbx1 is only a partial mediator of the functions of Tbx6. Indeed, cranial cartilage hypoplasia Conclusions observed in Tbx1 knockdown larvae was milder than that observed in Tbx6 knockdown larvae. The possibility of the incompleteness of Tbx1 Tbx6 knockdown produces faulty paraxial mesodermal tissues and knockdown could not be ruled out. Both Tbx6 and Tbx1 knockdowns affects the development of not only their derivatives but also their caused a reduction in Sox9 expression in the neural crest. Although adjacent cells such as neural crest. Tbx6 has thus been shown to be many studies have reported that Fgf, insulin-like growth factor I, more than a master gene of myogenesis along a wide range of human cartilage glycoprotein 39, transient receptor potential vanil- anterior–posterior axes. The contribution of Tbx6 in chondrogenesis loid 4, retinoic acid receptor agonists, and a Src inhibitor increased from the cranial neural crest cells has not been reported in other Sox9 expression (Murakami et al., 2000; Afonja et al., 2002; Shakibaei organisms; hence, Xenopus is a good animal model for investigating et al., 2006; Jacques et al., 2007; Bursell et al., 2007; Muramatsu et al., this issue. 2007), the mechanism underlying the regulation of Sox9 expression in Supplementary data to this article can be found online at chondrocytes is still unclear (Akiyama, 2008). Our results suggest that doi:10.1016/j.ydbio.2010.07.028. the regulator of Sox9 exists in downstream targets of Tbx6 or Tbx1.

Acknowledgment The vertical vs planar interaction in paraxial tissues We are grateful to Dr. R. T. Moon (University of Washington) for Tbx6 is expressed in the nascent paraxial and ventral mesoderm providing the Xwnt-8/pSP64T plasmid. during gastrulation; afterward, its expression persists in the most posterior mesoderm (Uchiyama et al., 2001). Because there was a gap between Tbx1- and Tbx6-expressing areas in neurula embryos, we References speculated that during normal development, the presumptive para- Aamar, E., Dawid, I.B., 2008. Protocadherin 18a has a role in cell adhesion, behavior and xial or ventral mesodermal cells involute and then express Tbx6 for a migration in zebrafish development. Dev. Biol. 318, 335–346. while, during which cells continue gastrulation movements anterior- Afonja, O., Raaka, B.M., Huang, A., Das, S., Zhao, X., Helmer, E., Juste, D., Samuels, H.H., ly. Next, at some point turning off Tbx6 expression (Hitachi et al., 2002. 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