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Keywords:

  • craniofacial development;
  • skeletal muscle;
  • Tbx1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Vertebrate craniofacial and trunk myogenesis are regulated by distinct genetic programs. Tbx1, homologue of the del22q11.2 syndrome candidate gene TBX1, controls branchiomeric craniofacial muscle development. Here, we demonstrate using immunohistochemistry that myogenic regulatory factors are activated in Tbx1-positive cells within pharyngeal mesoderm. These cells are also Islet1 and Capsulin-positive and in the absence of Tbx1 persist in the core of the first arch. Sporadic hypoplastic mandibular muscles in Tbx1−/− embryos contain Pax7-positive myocytes with indistinguishable differentiation properties from wild-type muscles and have normal tendon attachments and fiber-type patterning. In contrast to TBX1 haploinsufficient del22q11.2 syndrome patients, no alteration in fiber-type distribution was detected in Tbx1+/− adult masseter and pharyngeal constrictor muscles. Furthermore, Tbx1-expressing limb muscles display normal patterning, differentiation, fiber-type growth, fiber-type distribution and fetal maturation in the absence of Tbx1. The critical requirement for Tbx1 during muscle development is thus in the robust onset of myogenic specification in pharyngeal mesoderm. Developmental Dynamics 237:3071–3078, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

The skeletal myogenic program is determined by the action of the myogenic regulatory factors (MRFs) of the MyoD family of basic helix–loop–helix (bHLH) transcription factors (Buckingham,2006). Despite this common nodal step in skeletal myogenesis the upstream regulators of myogenic progenitor cells differ at different sites of myogenesis in the developing embryo (Noden and Francis-West,2006). The three major groups of skeletal muscles in the vertebrate body are somitic, extraocular and branchiomeric muscles (Romer and Parsons,1977). Somitic muscles give rise to muscles of the trunk, limb muscles, ventral neck muscles, and the tongue musculature. Extraocular muscles are involved in eye movement and originate from prechordal and cranial mesoderm. Branchiomeric muscles originate in mesoderm of the branchial arches lateral to the pharynx and give rise to the muscles of mastication and facial expression, in addition to pharyngeal and laryngeal muscles.

While much work has revealed the intercellular signals and upstream transcription factors controlling MRF gene expression during development of trunk and appendicular muscles, the molecular control of skeletal muscle specification at sites of branchiomeric myogenesis is poorly understood (Noden and Francis-West,2006). Four transcription factors have been shown from mouse mutational analysis to affect the branchiomeric myogenic program (reviewed in Grifone and Kelly,2007; Shih et al.,2008). Mice doubly mutant for the bHLH repressor protein encoding genes Tcf21 (Capsulin) and Msc (MyoR) fail to activate Myf5 throughout the first arch and lack a subset of mandibular muscles (Lu et al.,2002); Pitx2 mutant mice also fail to normally activate Myf5 and cell death leads to mandibular muscle loss (Dong et al.,2006; Shih et al.,2007). Tbx1 mutant mice fail to activate MRF genes at sites of branchiomeric myogenesis (Kelly et al.,2004; Liao et al.,2004; Dastjerdi et al.,2007). Failure of outgrowth of the caudal pharynx suggests that this could in part be due to a requirement for Tbx1 in proliferation of caudal pharyngeal mesodermal progenitor cells (Jerome and Papaioannou,2001; Vitelli et al.,2002). However, in the mandibular arch progenitor cells are present but fail to activate MRF gene expression, suggesting that Tbx1 function is required at the myogenic specification step. Stochastic activation of MRF gene expression in the mesodermal core of the first arch leads to the development of sporadic, frequently unilateral, mandibular arch derived muscles in Tbx1 mutant embryos (Kelly et al.,2004).

TBX1 has been identified as a major gene underlying del22q11.2 (DiGeorge or Velocardiofacial) syndrome in man, characterized by craniofacial and cardiovascular defects (reviewed in Baldini,2005). In particular, del22q11.2 patients display pharyngeal muscle weakness, associated with velopharyngeal insufficiency and, in some patients, with general skeletal muscle hypotonia (Shprintzen et al.,1981; Gerdes et al.,1999; Scambler,2000; Zim et al.,2003). Del22q11.2 patients are heterozygous for a multigene deletion encompassing 30 genes, including TBX1 (Lindsay,2001). The etiology of skeletal muscle weakness in del22q11.2 patients is unknown. The finding that Tbx1 is required for robust bilateral branchiomeric myogenesis in homozygous mutant mice suggested that hapolinsufficiency for TBX1 may underlie the pharyngeal muscle weakness in human patients. We therefore investigated the properties of branchiomeric and somite-derived muscles in Tbx1 mutant mice. We show using immunohistochemistry that the myogenic program is activated in Tbx1 Isl1 Capsulin-positive cells and that sporadic mandibular arch derived muscles in Tbx1 homozygous mutant mice contain Pax7-positive myocytes and have normal tendon attachments and fiber-type distribution. Unlike the situation in human del22q11.2 syndrome patients we found no evidence for a shift in fiber-type in adult Tbx1 heterozygous branchiomeric muscles. We also investigated the role of Tbx1 in Tbx1-expressing nonbranchiomeric muscles and demonstrate normal muscle patterning, differentiation, maturation, and slow fiber-type distribution in Tbx1 mutant embryos. Together our results suggest that the predominant role of Tbx1 in skeletal muscle development is to ensure the robust bilateral onset of branchiomeric myogenesis.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Previous analysis of Tbx1−/− embryos revealed that Tbx1 is required for robust bilateral activation of the skeletal myogenic program in the mesodermal core of the mandibular arch (Kelly et al.,2004; Dasterdji et al.,2007). To study this process at the level of cellular resolution, we investigated the distribution of Tbx1 protein in the mesodermal core of the pharyngeal arches at the time of myogenic specification, embryonic day (E) 9.5. The mesodermal core is surrounded by neural crest-derived mesenchyme and pharyngeal endoderm and ectoderm. Tbx1 distribution was compared with that of cardiac neural crest-derived cells and two proteins expressed in pharyngeal mesoderm, the bHLH protein Capsulin (Lu et al.,2002) and the LIM-homeodomain protein Islet1 (Cai et al.,2003; Nathan et al.,2008), in addition to the myogenic regulatory factors (MRF) MyoD and Myf5. Consistent with Tbx1 transcript distribution, Tbx1 protein was observed in the mesodermal core of the branchial arches in addition to cranial mesoderm and pharyngeal endoderm (Fig. 1A). Intercalated Tbx1-negative cells revealed cellular heterogeneity within the core. Using Wnt1-Cre R26R embryos these cells were identified as neural crest-derived cells that have infiltrated core mesoderm (Fig. 1A). Tbx1-positive core cells are also positive for the LIM homeodomain protein Isl1 and the bHLH protein Capsulin, defining a Tbx1, Isl1, Capsulin-positive profile for core arch mesoderm (Fig. 1B). Accumulation of MyoD and β-galactosidase under control of Myf5 regulatory sequences (in yMyf5-nlacZ-96-16 transgenic embryos) was observed in these Tbx1 Isl1 Capsulin-positive cells (Fig. 1C). Initially observed in a subset of Tbx1-positive cells, almost all Tbx1-positive cells express the Myf5-nlacZ transgene by E10.5. In Tbx1−/− embryos Capsulin and Isl1, but not MyoD or Myf5, were observed in the mesodermal core of the mandibular arch, revealing failure of branchiomeric myogenic specification but persistence of the premyogenic mesodermal core (Fig. 1D). Tunel staining showed no evidence of elevated cell death in Capsulin-positive core mesoderm cells at E9.5 or E10.5 in the absence of Tbx1 (Fig. 1D). The branchiomeric myogenic program is thus activated in Tbx1 Isl1 Capsulin-positive cells, supporting a direct requirement for Tbx1 in MRF activation in core mesoderm. Conditional ablation experiments have suggested that both endodermal and mesodermal Tbx1 expression may be required for myogenic specification (Arnold et al.,2006; Dastjerdi et al.,2007). Furthermore, Tbx1, Isl1, and Capsulin provide a molecular signature for pharyngeal mesoderm myogenic progenitor cells that is distinct from that of premyogenic cells elsewhere in the embryo. This genetic program is shared by adjacent myocardial progenitor cells of the second heart field that contribute myocardium to the poles of the elongating heart tube, a process dependent on Isl1 and Tbx1 (Buckingham et al.,2005; Grifone and Kelly,2007; Nathan et al.,2008).

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Figure 1. Tbx1 expression in branchiomeric muscle progenitor cells. A:Tbx1 transcripts revealed by in situ hybridization and Tbx1 protein detected by immunofluoresecence accumulate in cells of the mesodermal core of the mandibular arch at embryonic day (E) 9.5. Tbx1-negative cells within the core are β-galactosidase positive in Wnt1-Cre R26R embryos (arrowheads). B: Tbx1-positive core mesodermal nuclei are also positive for the transcription factors Islet1 and Capsulin at E9.5. C: Immunofluorescence showing that the myogenic regulatory factor MyoD and β-galactosidase under control of Myf5 regulatory sequences accumulate in Tbx1-positive nuclei within the mesodermal core at E9.5. D: Islet1 and Capsulin accumulate in the mesodermal core of the mandibular arch of Tbx1−/− embryos at E9.5. No increase in Tunel staining is observed in the mesodermal core of Tbx1−/− embryos; increased staining is observed in adjacent endoderm (arrowhead). Scale bars = 20 μm in A–D.

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Sporadic Tbx1-independent MRF activation results in the generation of hypoplastic, frequently unilateral, first arch-derived muscles in Tbx1−/− embryos (Kelly et al.,2004). Somite-derived muscle development requires the continued differentiation of myogenic progenitor cells positive for the Paired homeodomain proteins Pax3 and Pax7 (Relaix et al.,2005: Gros et al.,2005; Kassar-Duchossoy et al.,2005). Pax7, but not Pax3, is expressed in developing branchiomeric muscles (Hacker and Guthrie,1998; Mootoosamy and Dietrich,2002; Horst et al.,2006). To investigate whether Pax7-positive cells also stem from the stochastic event leading to hypoplastic muscle development in Tbx1−/− embryos, we investigated Pax7 protein distribution in sporadic mandibular muscles at E17.5. Pax7 was present in hypoplastic Tbx1−/− muscles in an indistinguishable distribution from that of wild-type muscles (Fig. 2A). Primary myogenic cultures were prepared from Tbx1−/− and control masseter muscles to evaluate whether loss of Tbx1 impacted on the myogenic potential or differentiation capacities of fetal myoblasts. To identify sporadic muscles for dissection and primary culture in Tbx1−/− embryos, we crossed a Connexin40-eGFP allele expressed in skeletal muscle onto the Tbx1 differentiated myofibers onto the Tbx1 mutant background (Miquerol et al.,2004). Hypoplastic muscles were identified and dissected from Tbx1−/− heads at E17.5 based on activity of the green fluorescent protein reporter (Fig. 2B). Primary cultures prepared from such muscles were scored for MyoD-positive foci after 24 hr of culture and myosin heavy chain expression after 4 days (Fig. 2C). Despite the role of Tbx1 at the onset of branchiomeric muscle specification no differences were observed between the number of MyoD-positive foci or in the number or size of multinuclear myotubes between Tbx1−/− and control cultures seeded at equivalent densities (Fig. 2C). We also investigated primary myogenic cultures prepared at earlier time points: similar results to those above were obtained in E14.5 primary cultures (data not shown). At E12.5, primary cultures were prepared using microdissected tissue from the site of first and second arch myogenesis of embryos carrying the Mlc3f-nlacZ-2E transgene. Although observed at a lower frequency, myotubes in cultures prepared from Tbx1−/− embryos were qualitatively indistinguishable from control myotubes (Fig. 2C). Together these results suggest that Pax7-positive fetal myogenic progenitor cells arise downstream or independently of the stochastic event leading to sporadic muscle development, consistent with the observation that hypoplastic Tbx1−/− muscles contain both primary and secondary muscle fibers (Kelly et al.,2004).

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Figure 2. Pax7 expression and fetal myoblast potential in Tbx1 mutant embryos. A: Transverse sections showing Pax7 distribution detected by immunofluorescence in Tbx1+/− and hypoplastic Tbx1−/− masseter muscles at embryonic day (E) 17.5; for orientation, see Figure 3B. Note the absence of second arch–derived muscles (arrowhead) in the Tbx1−/− section. B: Fluorescence images showing Connexin40-eGFP expression in craniofacial muscles and arteries in left and right views of E17.5 heads. Note in the Tbx1−/− embryo that the left masseter is absent and the right masseter hypoplastic (arrowhead). C: Primary cultures prepared from dissected control and Tbx1−/− masseter muscles at E17.5 (left three panels) showing Hoechst staining and MyoD detection after 24 hr and myosin heavy chain (MHC) detection after 4 days. Right: X-gal–stained primary cultures prepared from the site of first and second arch myogenesis in control and Tbx1−/− embryos carrying the Mlc3f-nlacZ-2E transgene at E12.5, 72 hr after plating. Scale bars = 100 μm in A.

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Figure 3. Tendon and muscle fiber-type distribution in Tbx1 mutant embryos A:Scleraxis expression in Tbx1 mutant embryos. Sagittal sections through Tbx1+/− and Tbx1−/− embryonic day (E) 15.5 heads showing MyoD and Scleraxis transcripts revealed by in situ hybridization. MyoD is expressed in all forming skeletal muscles and Scleraxis transcripts accumulate in the tendons of all muscles, including hypoplastic mandibular muscles in Tbx1−/− embryos. B: Immunofluorescence with slow myosin heavy chain type I (MHC I) antibodies on transverse sections through E16.5 Tbx1+/+, Tbx1+/− and Tbx1−/− heads (right three panels). The sections correspond to the boxed region in the X-gal stained section through the head of an E16.5 Mlc3f-nlacZ-2E embryo (left panel; m, masseter; t, tongue muscle). Slow MHC fibers are observed in the perimandibular region of the masseter (arrows). Note the loss of second arch derived muscles in the Tbx1−/− section (arrowheads). C: Transverse cryostat sections through the masseter of 4-month-old adult Tbx1+/+ and Tbx1+/− mice after immunohistochemistry to detect myosin heavy chain type IIA fibers. High magnification images of four different areas of the masseter are compared on the right. D: Histograms showing the average number (plus standard deviation) of MHC type IIA–positive fibers (left) and the maximum fiber diameter (right) for the different areas illustrated in panel C. Scale bars = 200 μm in A, 100 μm in B, 50 μm in C.

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We subsequently investigated tendon development and myosin heavy chain (MHC) fiber-type distribution in hypoplastic Tbx1−/− muscles. Scleraxis expression has been shown to label mouse head tendons (Pryce et al.,2007). At embryonic day (E) 15.5, we observed Scleraxis transcripts associated with sites of sporadic branchiomeric muscle development in Tbx1−/− embryos (Fig. 3A). Tendon attachment sites as defined by Scleraxis expression are, therefore, maintained in hypoplastic Tbx1 null muscles. The distribution of slow MHC type I fibers was scored in sporadic masseter, temporalis, and pterygoid muscles of E16.5 Tbx1−/− embryos and compared with that of wild-type and Tbx1+/− muscles (Fig. 3B). Within the masseter, type I fibers were observed adjacent to the mandible in wild-type and Tbx1+/− mice; in sporadic Tbx1−/− masseter muscles MHC I-positive fibers were also located in a perimandibular position. Together these data demonstrate that hypoplastic muscles forming in the absence of Tbx1 are correctly patterned and suggest that once the bottleneck of loss of Tbx1 is circumnavigated, sporadic muscles arising in Tbx1−/− embryos develop normally.

Del22q11.2 syndrome patients are haploinsufficient for a multigene deletion including TBX1 and display velopharyngeal insufficiency associated with pharyngeal muscle weakness and occasionally general hypotonia. An increase in slow type I MHC fiber numbers has been observed in muscle biopsies from pharyngeal constrictor muscles of del22q11.2 patients (Zim et al.,2003). We investigated whether a similar shift to a slow skeletal muscle phenotype occurs in adult Tbx1+/− mice. The distribution of slow type I and fast type IIA MHC-positive fibers was analyzed in the masseter and superior pharyngeal constrictor muscles of 4-month-old adult Tbx1+/− and wild-type littermates (Fig. 3C). No type I fibers were observed in the masseter or superior pharyngeal constrictor muscles of either wild-type or Tbx1+/− mice. The distribution and number of MHCIIA-positive fibers were scored in four different regions of the masseter and were found to be indistinguishable in wild-type (n = 4) and Tbx1+/− (n = 5) mice (Fig. 3C,D). No MHCIIA-positive fibers were observed in the superior pharyngeal constrictor muscles of wild-type (n = 4) or heterozygous (n = 2) mutant animals (Supp. Fig. S1, which is available online). Similar results were observed in mixed and C57Bl/6 genetic backgrounds. A shift in the distribution of type IIA fibers was observed in one subregion of the masseter of C57Bl/6 mice compared with mice of a mixed genetic background; a similar shift, however, was observed in both wild-type and Tbx1+/− mice (data not shown). Fiber diameter in the masseter and pharyngeal constrictor muscles was also scored and found to be equivalent in wild-type and Tbx1+/− mice (Fig. 3D, data not shown).

Previous experiments have shown that adult skeletal muscle is a major site of Tbx1 transcription (Chieffo et al.,1997). In addition to investigating branchiomeric muscle development, we examined Tbx1 expression and function in nonbranchiomeric myogenesis (Fig. 4). Tbx1 transcripts accumulate in certain appendicular muscle masses, in particular in dorsal limb muscle masses, from E12.5, consistent with a recent report by Dastjerdi et al. (2007). The expression profile of Tbx1 transcripts in developing limb muscles was highly comparable to that of β-galactosidase in a lacZ knock-in Tbx1 allele (Fig. 4A; Lindsay et al.,2001). Expression of Tbx1 in the developing tibialis anterior and extensor digitorum longus muscles was maintained throughout development (Fig. 4A). We investigated a potential role of Tbx1 in patterning and differentiation of these muscles. Using both MyoD whole-mount in situ hybridization and the Mlc3f-nlacZ-2E transgene, these muscles were observed to form normally in the absence of Tbx1 (Fig. 4B, data not shown). Our data support those of Dastjerdi et al. (2007) and show that Tbx1 does not play an early role in patterning these muscles. The role of Tbx1 in appendicular myogenesis must, therefore, be fundamentally different from that during branchiomeric muscle specification. This conclusion is consistent with the observation that MRF expression precedes Tbx1 expression in limb muscle masses as opposed to the prior expression of Tbx1 at sites of branchiomeric myogenesis (Kelly et al.,2004; Dasterdji et al.,2007). We also investigated whether Tbx1 affects myogenic potential or differentiation properties in primary cultures isolated from Tbx1 mutant hindlimbs at E17.5, E14.5 and E12.5. No differences in the number of MyoD-positive myogenic foci after 24 hr or in the size and number of differentiated myofibers after 4 days were observed between mutant and wild-type derived cultures (Fig. 4B, data not shown). In addition, the distribution of slow type I MHC-positive fibers in developing hindlimb muscle masses was indistinguishable in mutant and control hindlimbs (Supp. Fig. S1B). The tibialis anterior and extensor digitorum longus muscles are known to initiate the fetal myogenic program before ventral muscle groups in the hindlimb, including the activation of fetal-specific muscle genes such as Mlc3f and MCK (Ontell et al.,1993). The early regional expression profile of Tbx1 suggested that Tbx1 may play a role in establishing this specific program of gene expression. We investigated the distribution of a myosin light chain 3f transgene activated during the fetal period, Mlc3f-nlacZ-9, in Tbx1−/− embryos (Zammit et al.,2008). This transgene was activated at the same time point in the tibialis anterior and extensor digitorum longus of mutant and wild-type embryos, suggesting that Tbx1 is not required for the specific activation of late expressed genes in these muscles (Fig. 4B).

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Figure 4. Analysis of Tbx1 expression and function in limb muscle development. A: Expression analysis showing Tbx1 transcript accumulation detected by in situ hybridization in limb muscles (arrowheads) at embryonic day (E) 12.5 compared with β-galctosidase expression under control of a Tbx1-lacZ allele detected by X-gal staining. Note other expression sites including the cervical domain of the trapezius muscle and the vibrissae. Cryostat sections show Tbx1 transcript accumulation in a subset of αc-actin–positive hindlimb muscles at E18.5. B: Left two panels, E12.5: hindlimb muscle masses from control and Tbx1−/− embryos carrying the Mlc3f-nlacZ-2E transgene visualized in whole mount after X-gal staining. Myotubes in primary cultures prepared from hindlimbs of control and Tbx1−/− embryos carrying the Mlc3f-nlacZ-2E transgene, 72 hr after plating. Right three panels, E17.5: cryostat sections showing normal Mlc3f-nlacZ-9 transgene activation in the tibialis anterior and extensor digitorum longus muscles of control and Tbx1−/− embryos (arrowheads). t, tibia; f, fibula. No differences are observed in primary cultures of control and Tbx1−/− embryos after immunofluoresecence for MyoD after 24 hr and Myosin heavy chain after 4 days. Scale bars = 100 μm.

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From these results we conclude that heterozygosity for Tbx1 does not lead to altered muscle morphogenesis or fiber-type composition in laboratory mice. Species-specific differences in Tbx1 dosage requirements or the involvement of other genes in the multigene deletion region may account for the association of pharyngeal muscle weakness and general hypotonia with del22q11.2 syndrome; species differences have been noted for other aspects of the del22q11.2 phenotypic spectrum (Zhang and Baldini,2008). A potential role of Tbx1 in nonbranchiomeric myogenesis remains obscure, although our data have demonstrated that Tbx1 is not required for skeletal muscle specification, differentiation, patterning or activation of the fetal myogenic program, in contrast to the pivotal role of Tbx1 in the initial stages of branchiomeric myogenesis. Future experiments will investigate whether Tbx1 function in nonbranchiomeric muscles overlaps with that of other T-box family members, several of which are known to be expressed in the developing limb (King et al.,2006). Together our data suggest that the critical requirement for Tbx1 in skeletal myogenesis is restricted to conferring robustness on the early specification step of branchiomeric muscle development.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Mice

Mice carrying the Tbx1 null allele Tbx1tm1Pa (here referred to as Tbx1) were kindly provided by Virginia Papaioannou (Columbia University, New York) and heterozygous and homozygous mutant mice were genotyped as described by Jerome and Papaioannou (2001). Tbx1+/− mice were maintained on mixed and C57Bl/6 genetic backgrounds. Mice carrying Tbx1lacZ and Connexin-40 eGFP alleles were kindly provided by Antonio Baldini and Lucile Miquerol, respectively (Lindsay et al.,2001; Miquerol et al.,2004). Wnt1-Cre R26R mice and mice carrying the Mlc3f-nlacZ-2E, Mlc3f-nlacZ-9, and yMyf5-nlacZ-96-16 transgenes have been described by Jiang et al. (2001), Kelly et al. (1997), and Hadchouel et al. (2000).

Histology and Immunochemistry

Embryos and fetuses were dissected and fixed in 4% paraformaldehyde for 20 min to 1 hr, washed in phosphate buffered saline (PBS), embedded in OCT and frozen on dry ice. Adult muscles were directly embedded in OCT and frozen on dry ice. Primary myogenic cultured cells were fixed for 5 min, before several washes in PBS. Twelve-micrometer frozen sections and cultured myoblasts were permeabilized with 0.5% Triton X-100/PBS for 5 min, and washed several times with PBS before adding the blocking agent (20% BSA, Sigma). Primary antibodies were applied overnight at 4°C, and sections and cultured cells were washed 3 times with PBS. Secondary antibodies were applied for 2 hr at room temperature. For Pax7 staining, antigen retrieval involved boiling for 20 min in Antigen Unmasking solution (Vector), cooling for 20 min at room temperature, washing in PBS and blocking 30 min. Apoptosis detection analysis on frozen section were performed using Apoptag Fluorescent in situ detection kit according to the protocol provided by the manufacturer (Q-Biogene).

Antibodies

Antibodies used were as follows: Tbx1 (polyclonal, 1/200, Zymed), MyoD (monoclonal, 1/100, Dako), Slow skeletal Myosin (monoclonal, 1:2,000, Sigma), Capsulin (1/1,000, clone E-13, Santa Cruz), Pax7 (monoclonal, 1/20), and Islet1 (1/100, clone 40.2D6) from Developmental Studies Hybridoma Bank antibodies, β-galactosidase (monoclonal, 1:2,000, Promega; polyclonal, 1/2000, Capel), MyHCIIA (1/20 SC-71 clone, German Collection of Microorganisms and Cell Cultures). Secondary antibodies used were AlexaFluor anti-mouse and rabbit 546 and 488 (1/200, Molecular Probes) and anti-goat Texas Red (1/200, Jackson).

X-gal Staining

To visualize β-galactosidase activity, embryos and frozen sections were incubated overnight at 37°C in X-gal solution (4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2, 400 μg/ml X-gal, and 0.02% NP40 in PBS), washed in PBS, post-fixed in 4% paraformaldehyde and observed under a Zeiss Lumar stereomicroscope.

In Situ Hybridization

The following riboprobes were used: Tbx1 (Chapman et al.,1996), MyoD (Kelly et al.,2004), αc-actin (Sassoon et al.,1988), and Scleraxis (Schweitzer et al.,2001). Antisense digoxigenin (DIG)-labeled riboprobes were generated using a Boehringer transcription kit, following the manufacturer's instructions. For whole-mount Tbx1 in situ hybridization (ISH), embryos were fixed and processed as described in Kelly et al. (2004). For ISH on sections, fixed embryos were placed overnight in PBS with 20% sucrose, embedded in OCT and frozen on dry ice. Twelve-micrometer cryostat sections were washed briefly with PBS, rinsed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris, pH 8.0), post-fixed in 4% paraformaldehyde for 15 min at room temperature, and washed with PBS. After a 30 min incubation with 0.5% H2O2 in methanol followed by PBS washes the slides were transferred to 100 mM triethanolamine, pH 8.0, acetylated by adding dropwise acetic anhydride (0.25% final concentration) for 15 min at room temperature while being rocked, and washed in PBS-T (PBS, 0.05% Tween). The slides were prehybridized briefly with 500 μl of hybridization solution (50% formamide, 5× SSC, 5× Denhardt's, 500 μg/ml herring sperm DNA, 250 μg/ml yeast RNA) and hybridized overnight at 70°C with the same solution in the presence of the heat-denatured DIG-labeled RNA probes. The following day, slides were washed twice in 0.2× standard saline citrate (SSC) for 60 min at 70°C and finally in 0.2× SSC at room temperature for 5 min. Immunological detection of DIG-labeling is performed as for whole-mount ISH and described.

Primary Culture

E17.5 and E14.5 Tbx1+/+, Tbx1+/−, and Tbx1−/− mice were killed by cervical dislocation and hindlimb muscles removed. Hypoplastic Tbx1−/− head muscles were identified by enhanced green fluorescent protein (eGFP) staining under a Zeiss Lumar stereomicroscope. Dissected muscles were finely chopped with small scissors in a drop of Dulbecco's Modified Eagles Medium (DMEM) and digested in a solution of 0.1% collagenase D and 0.25% trypsin (Roche) in DMEM at 37°C for 20 min under agitation. The supernatant was then added to inhibition medium (DMEM supplemented with 20% fetal calf serum [FCS] and filtered through a 37-μm nylon filter to remove cell clumps. The suspension was centrifuged at 350 × g for 10 min and resuspended in 20% FCS before cell counting and plating at a seeding density of 5 × 105 cells per 3.5-cm dish in 10% FCS in DMEM. For E12.5 cultures, the site of first and second arch myogenesis was microdissected (avoiding sites of extraocular and tongue myogenesis), together with hindlimbs, from Mlc3f-nlacZ-2E transgenic embryos. Dissected tissue was treated with 0.25% tryrpsin for 5 min before disaggregation using a micropipette and plating in 10% FCS in DMEM. Plates were harvested at 24 and 72 hr and analyzed by X-gal staining.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank Virginia Papaioannou for mice carrying the Tbx1tm1Pa allele, Antonio Baldini and Peter Scambler for mice carrying the Tbx1lacZ allele, Lucile Miquerol for Connexin40eGFP mice, and Nigel Brown for Wnt1-Cre R26R embryos. This work was supported by the Association Française contre les Myopathies and the Inserm Avenir Program. RG was supported by postdoctoral fellowships from the AFM and Fondation pour la Recherche Mèdicale.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY21718SuppFigS1.tif12762KSupp. Fig. S1. Fiber type analysis in <I>Tbx1</I> mutant embryos. <B>A:</B> Myosin heavy chain type IIA and type I distribution after immunohistochemistry on sagittal cryosections through the pharyngeal region of 4-month-old control and <I>Tbx1</I><SUP>+/−</SUP> mice including 5× magnification of the boxed regions. No myosin heavy chain (MHC) type I or type IIA fibers are detected in the pharyngeal constrictor (pc) muscles of control or <I>Tbx1</I><SUP>+/−</SUP> embryos. <B>B:</B> MHC I distribution in transverse cryosections through hindlimbs of E17.5 <I>Tbx1</I><SUP>+/+</SUP>, <I>Tbx1</I><SUP>+/−</SUP> and <I>Tbx1</I><SUP>−/−</SUP> embryos. t, tibia; f, fibula. Scale bars = 200 μm.

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