Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on September 22, 2004
Human Molecular Genetics 2004 13(22):2829-2840; doi:10.1093/hmg/ddh304

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Human Molecular Genetics, Vol. 13, No. 22 © Oxford University Press 2004; all rights reserved

The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis

Robert G. Kelly^*, Loydie A. Jerome-Majewska and Virginia E. Papaioannou

Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, NY 10032, USA

Received July 16, 2004; Accepted September 15, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Formation and remodeling of the pharyngeal arches play central roles in craniofacial development. TBX1, encoding a T-box-containing transcription factor, is the major candidate gene for del22q11.2 (DiGeorge or velo-cardio-facial) syndrome, characterized by craniofacial defects, thymic hypoplasia, cardiovascular anomalies, velopharyngeal insufficiency and skeletal muscle hypotonia. Tbx1 is expressed in pharyngeal mesoderm, which gives rise to branchiomeric skeletal muscles of the head and neck. Although the genetic control of craniofacial muscle development is known to involve pathways distinct from those operational in the trunk, the regulation of branchiomeric myogenesis has remained enigmatic. Here we show that branchiomeric muscle development is severely perturbed in Tbx1 mutant mice. In the absence of Tbx1, the myogenic determination genes Myf5 and MyoD fail to be normally activated in pharyngeal mesoderm. Unspecified precursor cells expressing genes encoding the transcriptional repressors Capsulin and MyoR are present in the mandibular arch of Tbx1 mutant embryos. Sporadic activation of Myf5 and MyoD in these precursor cells results in the random presence or absence of hypoplastic mandibular arch-derived muscles at later developmental stages. Tbx1 is also required for normal expression of Tlx1 and Fgf10 in pharyngeal mesoderm, in addition to correct neural crest cell patterning in the mandibular arch. Tbx1 therefore regulates the onset of branchiomeric myogenesis and controls normal mandibular arch development, including robust transcriptional activation of myogenic determination genes. While no abnormalities in branchiomeric myogenesis were detected in Tbx1^+/– mice, reduced TBX1 levels may contribute to pharyngeal hypotonia in del22q11.2 patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Formation and remodeling of the pharyngeal arches play central roles in development of the face and neck (1). Pharyngeal arches form in a rostro–caudal sequence and comprise ectoderm, endoderm and neural crest-derived mesenchyme surrounding a mesodermal core. Pharyngeal endoderm gives rise to glandular derivatives including the thymus and parathyroid glands, whereas neural crest-derived mesenchyme gives rise to cartilage, bone and connective tissue and contributes to septation of the cardiac outflow tract (2).

The mesodermal core of the pharyngeal arches is derived from cranial paraxial and anterior occipital mesoderm which migrates laterally into the pharyngeal region and gives rise to branchiomeric skeletal muscles of the head and neck (3–6). Branchiomeric muscles include muscles of mastication, derived from the first or mandibular arch; muscles of facial expression, derived from the second or hyoid arch; and muscles of the pharynx and larynx, derived from more caudal arches (2). Non-branchiomeric head muscles include extra-ocular muscles derived from the anterior-most paraxial and pre-chordal mesoderm, and tongue muscles, which are derived from the hypoglossal cord and originate in anterior somites (2,7). The genetic regulation of craniofacial myogenesis has remained enigmatic (6,8–10). Members of the MyoD family of basic helix–loop–helix myogenic regulatory factors (MRFs) act at a nodal point in the establishment of skeletal muscle lineages throughout the embryo and are first expressed in the pharyngeal arches of the mouse at embryonic day (E) 9.5 (reviewed in 11). However, distinct pathways regulate MRF expression in head versus trunk mesoderm (6,8–10). Mice lacking Myf5 and the paired homeodomain transcription factor Pax3 do not develop skeletal muscle in the trunk or limb, yet head muscles form normally (9). Furthermore, Wnt signals, which promote trunk myogenesis, have been recently shown to block head myogenesis (12). The transcription factors that activate Myf5 and MyoD expression in pharyngeal mesoderm are unknown, although mice lacking two bHLH repressor proteins, Capsulin and MyoR, fail to express Myf5 in the first arch and lack a subset of mandibular arch-derived muscles (13).

del22q11.2 (DiGeorge or velo-cardio-facial) syndrome is characterized by craniofacial defects, thymic hypoplasia, cardiovascular anomalies, velopharyngeal insufficiency and skeletal muscle hypotonia, associated in the majority of cases with a heterozygous multigene deletion of chromosome 22q11.2 (14). Through analysis of the homologous region of the murine genome, the gene encoding the T-box-containing transcription factor TBX1 has been identified as a major candidate gene for del22q11.2 syndrome (reviewed in 15). Point mutations in TBX1 have recently been identified in DiGeorge patients who do not carry a multigene deletion at 22q11.2 (16). Tbx1 is expressed in pharyngeal endoderm and mesoderm (17), and mice lacking Tbx1 display perinatal lethality and exhibit defects in the majority of structures perturbed in del22q11.2 patients, including failure of caudal pharyngeal morphogenesis and associated craniofacial and cardiovascular defects (18,19). Mice heterozygous for Tbx1 display abnormal development of the fourth pharyngeal arch artery at E10.5, a defect associated with the cardiovascular anomalies observed in del22q11.2 patients (18,20).

Here we demonstrate that Tbx1 regulates the onset of branchiomeric myogenesis in the mouse. Loss of Tbx1 results in sporadic activation of Myf5 and MyoD in the mandibular arch and the random presence or absence of hypoplastic first arch-derived muscles at later developmental stages. Tbx1 is therefore required for robust transcriptional activation of myogenic determination genes in the mandibular arch. We demonstrate that Tbx1 also regulates the expression of Tlx1 and Fgf10 in the mesodermal core of the mandibular arch and the patterning of surrounding neural crest cells. Our results have important implications for the study of craniofacial development and suggest that reduced TBX1 levels may underlie pharyngeal hypotonia in del22q11.2 syndrome patients.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tbx1 is required for normal activation of Myf5 and MyoD in pharyngeal mesoderm
Tbx1 is expressed in the mesodermal core of the first and second pharyngeal arches during E9 (Fig. 1A and B). Transcripts encoding the MRFs Myf5 and MyoD are activated in the mesodermal core of the first and second arches at E9.5 (Fig. 1C, D and I). Tbx1 mutant mice have been previously generated and found to model multiple aspects of del22q11.2 syndrome (18). We investigated MRF gene expression in Tbx1 heterozygous and homozygous mutant embryos by in situ hybridization. Myf5 and MyoD are activated normally in the pharyngeal arches of Tbx1^+/– embryos (Fig. 1D, E, I and J). In contrast, in Tbx1^–/– embryos, no Myf5 transcripts were detected in the pharyngeal region at E9.5 (Fig. 1F), whereas Myf5 expression in the somites is indistinguishable in Tbx1^–/– and Tbx1^+/– embryos (Fig. 1E and F). Transcriptional activation of Myf5 fails not only in the second pharyngeal arch, which is severely hypoplastic in Tbx1 mutant embryos (18,19), but also in the mandibular arch. A transgene containing a nuclear-localized ß-galactosidase reporter gene controlled by 96 kb of DNA upstream from the Myf5 gene (y96–Myf5–nlacZ), which reproduces the endogenous Myf5 expression pattern (21), also fails to be activated throughout the mesodermal core of the first and second arches of Tbx1^–/– embryos (Fig. 1G and H). However, the cellular resolution provided by the reporter gene revealed sporadic patches of ß-galactosidase-positive cells in the mandibular arch of Tbx1^–/– embryos (Fig. 1H and H'). Similarly, sporadic patches of MyoD-expressing cells were observed in the mandibular arch of Tbx1^–/– embryos (Fig. 1K and K').

View larger version (88K): [in this window] [in a new window]   Figure 1. Tbx1 is required for robust expression of myogenic regulatory factors in the mandibular arch at E9.5. (A) Whole mount in situ hybridization showing Tbx1 expression in the core of the first (1) and second (2) pharyngeal arches and caudal pharynx (arrowhead). (B) Transverse section showing Tbx1 expression in core mesoderm. (C) Expression of a Myf5–nlacZ transgene is also restricted to core mesoderm. (D, E, F) Myf5 transcripts are observed in the first and second arches of Tbx1+/+ (D) and Tbx1+/– (E) embryos, but not in pharyngeal mesoderm of Tbx1–/– embryos (F). (G, G') X-gal stained Tbx1+/– embryo showing Myf5–nlacZ transgene expression in the branchial arches (1, 2), developing somites (m) and brain (asterisk). (H, H') A Tbx1–/– Myf5–nlacZ embryo with a unilateral patch of ß-galactosidase-positive cells in the left mandibular arch (arrowhead). (I, J, K) MyoD is expressed in the mandibular arch of Tbx1+/+ (I, I') and Tbx1+/– (J, J') embryos; a Tbx1–/– embryo showing abnormal restriction of MyoD expression to a unilateral patch in the mandibular arch (K, K'). cm, Cranial mesoderm; ov, otic vesicle; s, sclerotome; aa, aortic arch artery; mc, mesodermal core; m, myotome/dermomyotome. Scale bar: 25 µm (B, C).

 
Tbx1 is not required for migration of cranial paraxial mesoderm into the mandibular arch
The mesodermal core of the pharyngeal arches is derived from cranial paraxial mesoderm which migrates laterally into the pharyngeal region (3–6). Patches of MRF-expressing cells in the mandibular arch could result from sporadic transcriptional activation in a subset of cells within the mesodermal core, or from normal activation in a severely hypocellular core. Capsulin, encoding a bHLH transcriptional repressor, is expressed in the mesodermal core prior to Myf5 expression (Fig. 2A; 13). Capsulin and the related bHLH transcriptional repressor gene MyoR are required for Myf5 activation in the mandibular arch; Capsulin MyoR double mutant embryos subsequently fail to form a subset of skeletal muscles derived from the first arch (13). We analyzed Capsulin expression in Tbx1^–/– embryos in order to investigate whether normal lateral migration of cranial paraxial mesoderm to the pharyngeal arches takes place in the absence of Tbx1. Capsulin transcripts are observed in the mesodermal core of the mandibular arch of Tbx1^–/– embryos in a profile indistinguishable from that of Tbx1^+/– embryos (Fig. 2A–F); in contrast, no Capsulin transcripts accumulate in the region of the second arch of Tbx1^–/– embryos (Fig. 2B and D). MyoR is also expressed in the mandibular arch of Tbx1 mutant embryos at E9.5 (Figs 2H and 3B). This result demonstrates that migration of cranial paraxial mesoderm into the mandibular arch takes place in the absence of Tbx1. Pre-myogenic cells are therefore present in the mandibular arch of Tbx1^–/– embryos, although the majority of these cells fail to activate MRF transcription.

View larger version (76K): [in this window] [in a new window]   Figure 2. Pre-myogenic cells are present in the mandibular arch of Tbx1–/– embryos at E9.5. (A, C) Capsulin is expressed in the mesodermal core of the first (1) and second (2) arches of Tbx1+/– embryos on early (A) and late (C) embryonic day 9. Capsulin is also expressed in the caudal pharynx (arrowhead) and septum transversum (arrow). (B, D) In Tbx1–/– embryos Capsulin is expressed normally in the mandibular arch, but not in the region of the second arch. (E, F) Transverse sections showing Capsulin transcripts in the core of the mandibular arch in Tbx1+/– and Tbx1–/– embryos. (G) MyoR is expressed in the mesodermal core of the first and second arch of Tbx1+/– embryos and in the first arch of a Tbx1–/– embryo (H). nt, Neural tube. Scale bar: 100 µm (E, F).

 

View larger version (50K): [in this window] [in a new window]   Figure 3. Abnormal gene expression in the mesodermal core of the mandibular arch of Tbx1–/– embryos. (A) Left lateral view of the anterior pharyngeal region at E9.5 showing MyoR expression in the first (1) and second (2) arches of Tbx1+/– embryos and the first arch of Tbx1–/– embryos (B). (C, C') Left lateral and frontal views showing Tlx1 transcripts in the core of the first and second arches of Tbx1+/– embryos, in addition to surface ectoderm (arrowhead). (D, D') Tlx1 transcript levels are specifically reduced in the mesodermal core of Tbx1–/– embryos (arrows). (E) Fgf10 transcripts accumulate in the mesodermal core of arches 1 and 2 and surface ectoderm of the mandibular arch (arrowhead) in Tbx1+/– embryos. (F) Fgf10 expression in the mesodermal core is lost in Tbx1–/– embryos. (G, G') The Fgf10 enhancer-trap transgene Mlc1v–nlacZ–24 is expressed throughout the mesodermal core of arches 1 and 2, surface ectoderm (arrowhead) and heart (h), revealed by X-gal staining. (H, H') In Tbx1–/– embryos transgene expression is lost in the proximal mesodermal core but maintained in the distal core (arrows in H and H') and surface ectoderm. (I) Expression of a Myf5–nlacZ transgene in an Fgf10+/– embryo at E10.5, showing expression in the developing branchiomeric muscle masses of arches 1–6, in addition to extra-ocular (eo), forelimb (fl) and myotomal (m) muscle masses. (J) Normal transgene expression is observed in the branchiomeric region of an Fgf10–/– embryo; note the lack of forelimb outgrowth in the absence of Fgf10. as, Aortic sac.

 
In order to investigate whether Tbx1 directly activates Myf5 transcription in branchial arch mesoderm, we searched for T-box-binding sites in a previously identified 1.1 kb branchiomeric enhancer of the Myf5 gene (22). We identified a T-box motif (GTGTGAA) at nucleotide 636 which is conserved in six mammalian species (cow, dog, man, mouse, pig and rat). The in vivo role of this element was evaluated by site directed mutagenesis followed by transgenesis. Four out of six embryos carrying the wild-type enhancer upstream of a minimal TK promoter expressed an nlacZ reporter gene in myogenic cells of the branchial arches at E11.5. Four out of five embryos carrying the mutated enhancer expressed the reporter gene in a similar distribution in branchial arch muscle masses. This result demonstrates that Myf5 transcription in pharyngeal mesoderm is not dependent on this T-box site. The role of less conserved T-box sites present in the 1.1 kb enhancer, and in other branchiomeric enhancers at the Myf5 locus (23), remains to be evaluated.

Tbx1 regulates Tlx1 and Fgf10 expression in pharyngeal mesoderm
We investigated whether other genes normally expressed in the mandibular arch show aberrant expression patterns in Tbx1^–/– embryos. Tlx1, encoding a homeobox-containing transcription factor, is expressed in the mesodermal core of the first and second arches at E9.5 (Fig. 3C and C') (24). In the absence of Tbx1, Tlx1 transcript levels are severely reduced, but still detectable, in the mesodermal core, whereas Tlx1 expression in surface ectoderm of the mandibular arch is unaffected (Fig. 3D and D'). Mice lacking Tlx1 are asplenic but viable and fertile, and pharyngeal- and first arch-derived muscles are present and anatomically normal (25), demonstrating that Tlx1, although downregulated in the absence of Tbx1, is not required for branchiomeric myogenesis.

Fgf10, encoding a fibroblast growth factor, is also expressed in the mesodermal core of the pharyngeal arches at E9.5 (Fig. 3E) (26). Fgf10 transcripts do not accumulate in the mesodermal core of Tbx1^–/– mandibular arches, whereas expression in first arch ectoderm is unaffected (Fig. 3F) (27). To pursue this observation, we analyzed the expression profile of an Fgf10 enhancer-trap nlacZ transgene (Mlc1v–nlacZ–24) (26) in Tbx1 mutant embryos. Consistent with the loss of endogenous Fgf10 transcripts in the mesodermal core of the mandibular arch, the Fgf10 enhancer-trap transgene fails to be expressed in the proximal core region of the mandibular arch of Tbx1^–/– embryos (Fig. 3G and H). Transgene expression, however, was maintained in the distal region of the mesodermal core, in proximity to the aortic sac (arrow in Fig. 3H and H'), where Myf5 and MyoD are not expressed.

Fgf10 null mice die at birth, lacking lungs, limbs and displaying aplasia and hypoplasia of multiple other organs (28,29). Since Fgf10 is expressed in pharyngeal mesodermal prior to E9.5 (26), we investigated whether Fgf10 is required for MRF gene activation by crossing the y96–Myf5–nlacZ transgene into an Fgf10 mutant background (28). Normal activation of the transgene was observed in branchiomeric muscle masses in the absence of Fgf10 (Fig. 3I and J), indicating that Fgf10, although downstream of Tbx1, is not required for the activation of Myf5.

Tbx1 is required to pattern neural crest-derived mesenchyme in the mandibular arch
In addition to investigating gene expression in the mesodermal core, we examined gene expression in the surrounding mandibular mesenchyme of Tbx1^–/– embryos. Crabp1 in situ hybridization revealed that neural crest cells colonize the Tbx1^–/– mandibular arch (Fig. 4A and B), confirming the observations of Vitelli et al. (19). However, although neural crest cells are present in the mandibular arch of Tbx1^–/– embryos, mandibular mesenchyme is abnormally patterned. We observed altered distributions of transcripts encoding the regionally expressed transcription factors Tbx2, Tbx3, Dlx7, Msx2 and Prx2 in the mandibular arch of Tbx1^–/– embryos (Fig. 4C–L). The predominant defect was an expanded expression domain of these genes in the proximal arch region (arrows in Fig. 4D, F, H, J and L). Expansion of the Tbx2 expression domain was observed in three out of four Tbx1^–/– mandibular arches scored; in one arch Tbx2 transcripts were observed throughout the mandibular mesenchyme (Fig. 4D). In addition, a reduction of Dlx7 transcripts in the anterior region of the mandibular arch was noted (arrowhead in Fig. 4H and H'). These results suggest that, in addition to the defect in myogenic specification of the Tbx1^–/– mesodermal core, patterning defects in surrounding neural crest cells may contribute to the mutant phenotype.

View larger version (67K): [in this window] [in a new window]   Figure 4. The distribution of neural crest cell markers in the mandibular arch of Tbx1–/– embryos at E9.5. (A) In situ hybridization showing Crabp1 transcripts in the first (1) and second (2) arches of a Tbx1+/– embryo, shown in a left lateral view. (B) Crabp1 transcripts are observed in the mandibular arch of Tbx1–/– embryos. (C, D) Tbx2 expression is observed in the anterior region of the first arch mesenchyme of Tbx1+/– embryos (C) and is expanded in the posterior proximal region of the mandibular arch of Tbx1–/– embryos (D, arrow). (E, F) Tbx3 is also expressed in anterior mandibular mesenchyme (E) and shows a broader expression domain in Tbx1–/– embryos (F). (G, G') Dlx7 is expressed in a restricted domain in the first and second pharyngeal arches of Tbx1+/– embryos. (H, H') The domain of Dlx7 expression is altered in the mandibular arch of Tbx1–/– embryos, showing an expansion in the posterior proximal region (arrow) and a reduction in the anterior region (arrowhead). (I, I') Msx2 is expressed in distal mesenchyme of the first and second pharyngeal arches. (J, J') In a Tbx1–/– embryo Msx2 transcripts are observed in an expanded domain in the posterior proximal region of the arch (arrow). (K) Prx2 is expressed in the distal mesenchyme of the first and second arches. (L) In Tbx1–/– embryos the domain of Prx2 transcription in the mandibular arch extends proximally (arrow). (M, N) Frzb1 transcripts are observed in mesenchyme of the first arch in Tbx1+/– and Tbx1–/– embryos. An expanded proximal domain of Frzb1 expression is observed in the Tbx1–/– arch (arrow). (O, P) A low level of Gremlin expression is observed in the mandibular arch of Tbx1+/– and Tbx1–/– embryos (white arrowhead). Gremlin expression is observed in the second arch of Tbx1+/– embryos, but not in the region of the second arch in Tbx1–/– embryos (black arrowhead in O).

 
BMP and Wnt inhibitors in mandibular mesenchyme have been shown to promote branchiomeric myogenesis by antagonizing anti-myogenic signals from pharyngeal epithelia, thus regulating the activation of MRF genes in the mesodermal core on neural crest influx (12). Tzahor et al. (12) demonstrated in the chick that Frzb and Gremlin are expressed in neural crest cells adjacent to the mesodermal core of the first and second pharyngeal arches at the time of myogenic induction. We investigated the distribution of Frzb1 and Gremlin transcripts in Tbx1^–/– embryos at E9.5 (Fig. 4M–P). Frzb1 expression was observed in the mandibular arch of control and Tbx1^–/– embryos (Fig. 4M and N). An expanded domain of Frzb1 expression was observed in the proximal mandibular arch in the absence of Tbx1 (Fig. 4N), similar to that observed for Tbx2 and Tbx3 (Fig. 4). Gremlin transcripts were only detectable in the mandibular arch at a low level, in both control and Tbx1^–/– embryos (Fig. 4O and P). In contrast, Gremlin expression in the second arch was absent from the hypoplastic Tbx1^–/– second arch region (Fig. 4O and P). These results suggest that Tbx1-dependent MRF gene activation in the mandibular arch is not mediated by Frzb1 or Gremlin transcription.

Sporadic mandibular myogenesis and failure of caudal branchiomeric myogenesis in the absence of Tbx1
At mid-gestation, Myf5 and MyoD expression is severely reduced in the pharyngeal region of Tbx1^–/– compared with Tbx1^+/+ and Tbx1^+/– embryos (Fig. 5A–C and H–J). Muscle masses derived from the first and second arches, in addition to muscle masses that normally form in the caudal pharyngeal region, are missing from Tbx1^–/– embryos (Fig. 5C and J). Small unilateral patches of MRF-expressing cells within the normal domain of mandibular arch-derived muscle mass development are present in almost all Tbx1^–/– embryos analyzed at E10.5 (Fig. 5D, E, G, K–L'). In addition to single unilateral patches of MRF-positive cells within the mandibular arch, we observed asymmetric bilateral patches and spatially separate unilateral patches of MRF expression (Fig. 5D and L). Rare spots of MRF expression were observed at the site of second arch muscle formation (arrow in Fig. 5E). These results are consistent with sporadic activation of MRF transcription in the absence of Tbx1. MRF expression at sites of non-branchiomeric myogenesis, including somites, limb-bud muscle masses, extra-ocular muscles and the hypoglossal cord, is normal in Tbx1^–/– embryos, although the length of the hypoglossal cord is reduced owing to perturbed caudal pharyngeal morphology (Fig. 5I–L).

View larger version (55K): [in this window] [in a new window]   Figure 5. Sporadic myogenic regulatory factor gene expression in the mandibular arch of Tbx1–/– embryos at E10.5. (A, B) Myf5–nlacZ transgene expression in branchiomeric muscle masses (1–6), myotome (m), extra-ocular (eo) and forelimb muscle masses (fl) of Tbx1+/+ (A) and Tbx1+/– (B) embryos. (C) A Tbx1–/– embryo showing no expression at sites of branchiomeric muscle formation but normal expression elsewhere. (D) A Tbx1–/– embryo with two patches of expression (arrowheads) in the left mandibular arch. (E) Patch of positive cells (arrowhead) in the right mandibular arch of the embryo in (C). A small patch of ß-galactosidase-positive cells is observed in the region of the second arch (arrow). (F, G) Transverse sections showing transgene expression in the mandibular arch of a Tbx1+/– embryo and a small patch of positive cells in a Tbx1–/– embryo. (H, I) MyoD is expressed in branchiomeric muscle masses (1–6), hypoglossal cord (hc), myotome and muscle masses of the forelimb of Tbx1+/+ (H) and Tbx1+/– (I) embryos. (J) A Tbx1–/– embryo with no MyoD expression at sites of branchiomeric muscle formation. (K) A Tbx1–/– embryo with a patch of MyoD expression (arrowhead) in the left mandibular arch. (L) A Tbx1–/– embryo with two patches of MyoD-positive cells (arrowheads) in the right mandibular arch. (K', L') Detailed views of the mandibular arches in (K) and (L). Scale bar: 100 µm (F, G).

 
At subsequent developmental stages, the expression of MRF genes in the second and caudal pharyngeal arches is sever-ely perturbed or absent, suggesting that the majority of branchiomeric muscles do not form in Tbx1^–/– embryos (Fig. 6), including muscles of the pharynx, larynx and part of the developing trapezius muscle (arrow in Fig. 6A, D and E). However, unilateral (or asymmetric bilateral) patches of MRF expression were observed in Tbx1^–/– embryos in the region where mandibular arch-derived muscle masses normally develop (arrowheads in Fig. 6C and H). Extremely hypoplastic laryngeal muscles were present in a subset of Tbx1^–/– embryos (data not shown). Expression of Myf5 or MyoD is essential for skeletal muscle determination, and mice lacking both genes develop neither differentiated skeletal muscle fibres nor skeletal myoblasts (30). Failure of branchiomeric myogenesis was confirmed by analysis of Myogenin transcripts, encoding a member of the MRF family essential for differentiation (Fig. 6I and J), and two differentiation markers, a myosin light chain transgene (Mlc3f–nlacZ–2E; Fig. 6K–M) and embryonic myosin light chain transcripts (Mlc1a/emb; data not shown). No differences in MRF or differentiation marker expression were observed between Tbx1^+/– and Tbx1^+/+ littermates (Fig. 6K and L). Normally developing hypobranchial and tongue muscle development was apparent in the absence of branchiomeric muscles, consistent with the presence of the hypoglossal cord at earlier developmental stages (Fig. 6N and O).

View larger version (82K): [in this window] [in a new window]   Figure 6. Failure of branchiomeric myogenesis in Tbx1–/– embryos. (A) At E12.5 MyoD is expressed in muscle masses of the first (1) and second (2) arches, the caudal branchiomeric region (arrow), the trunk and forelimb (fl). (B) No MyoD transcripts are observed at sites of branchiomeric muscle formation in this left view of a Tbx1–/– embryo. (C) In a right view of the same embryo a patch of MyoD expression is observed in the region where first arch muscles normally develop (arrowhead). (D) Dorsal view showing MyoD expression in axial and caudal branchiomeric muscle masses (developing trapezius muscle, arrow); (E) no caudal branchiomeric expression is observed in Tbx1–/– embryos. (F) Myf5–nlacZ transgene expression in developing first and second arch-derived muscles in addition to extra-ocular, trunk and forelimb muscle masses. (G) A Tbx1–/– embryo showing normal extra-ocular, forelimb and trunk expression but no expression in muscle masses derived from the first and second arch. (H) A Tbx1–/– embryo showing a unilateral patch of transgene expression at the site of normal first arch-derived muscle development (arrowhead). (I) Myogenin transcripts are detected in developing first and second arch-derived muscles in addition to trunk and forelimb muscle masses. (J) No Myogenin transcripts are observed in branchiomeric muscles in this left view of a Tbx1–/– embryo. (K, L) At E13.5 the Mlc3f–nlacZ–2E transgene is expressed in all differentiated skeletal muscles including developing muscles of the first and second arch of Tbx1+/+ (K) and Tbx1+/– (L) embryos. (M) The Mlc3f–nlacZ–2E transgene is not expressed in branchiomeric muscles of a Tbx1–/– embryo (M), with the exception of a patch of expression where the left temporalis muscle normally develops (arrowhead). (N) Inferior view of a dissected Tbx1+/– head at E13.5 showing Myf5–nlacZ expression in first (1) and second (2) arch-derived muscles and muscles of the pharynx (p). (O) No transgene expression is detected at sites of branchial arch-derived muscle formation in Tbx1–/– embryos, revealing underlying tongue muscles (t). ma, Mandible.

 
A subset of the normal complement of mandibular arch-derived muscles was observed in all Tbx1^–/– embryos scored at late fetal stages (n=11; Fig. 7; Table 1). Tbx1^–/– mandibular arch-derived muscles are frequently hypoplastic and asymmetric or unilateral. Each mandibular arch-derived muscle was absent in at least one embryo. When present, Tbx1^–/– first arch-derived muscles contain both primary and secondary muscle fibers, indicating that the normal sequence of myofiber development can take place in the absence of Tbx1 (Fig. 7I and J). Individual muscles were either unilaterally present on the right or left side (Fig. 7B–D), smaller on one side than the other (Fig. 7C), bilaterally symmetric (Fig. 7E) or bilaterally absent (Fig. 7F; Table 1). Muscle loss is significantly more severe on the left than right side (P<0.01). Although almost all second arch-derived muscles were absent (Fig. 7G and H), rare hypoplastic fibers were observed at sites of normal second arch muscle formation (arrowhead in Fig. 7B). Our data suggest that the formation of first arch-derived muscles in Tbx1^–/– embryos is a direct consequence of sporadic activation of MRF transcription in the mandibular arch, and that the precise sub-regions of the mesodermal core in which MRF activation occurs define the resulting complement of first arch-derived muscles. Furthermore, although sporadic Tbx1-independent MRF activation can occur in either the left or right mandibular arch, the probability of activation occurring in the right arch is greater than that in the left arch.

View larger version (130K): [in this window] [in a new window]   Figure 7. Sporadic mandibular muscle development in Tbx1–/– embryos. (A) Frontal section of a Tbx1+/– embryo after X-gal and eosin staining showing Myf5–nlacZ transgene expression in craniofacial muscles at E16.5, including mandibular arch-derived muscles (m, masseter; my, mylohyoid; ad, anterior digastric), second arch-derived muscles (arrowhead), tongue muscles (t) and extra-ocular muscles (eo). (B, C) A Tbx1–/– littermate with unilateral right masseter, pterygoid (pt) and temporalis (te) muscles. The site of the absent left masseter is indicated (arrow). A unilateral patch of second arch-derived fibers is observed (arrowhead in B). The left anterior digastric muscle is hypoplastic (arrowhead in C). (D) A Tbx1–/– embryo with absence of all left-sided mandibular arch-derived muscles including the masseter and mylohyoid (arrows). (E) A Tbx1–/– embryo with bilateral masseter muscles and hypoplastic mylohyoid and anterior digastric muscles. (F) A Tbx1–/– embryo with bilateral absence of masseter muscles (arrows). (G, H) Anti-myosin antibody staining showing second arch-derived muscle fibers (arrowheads) adjacent to vibrissae (v) in a Tbx1+/– but not Tbx1–/– embryo. (I, J) Detailed view of myofibers in the masseter of Tbx1+/– and Tbx1–/– embryos showing primary fibers with centrally located nuclei (arrowhead) flanked by small secondary fibers (arrow). Green, MF20-positive myofibers; red, propidium iodide-stained nuclei. ma, Mandible; tb, tooth bud. Scale bars: 400 µm (A–F); 200 µm (G, H); 20 µm (I, J).

 

View this table: [in this window] [in a new window]   Table 1. The status of different mandibular arch-derived muscles in 11 late fetal Tbx1 mutant embryos

 
Branchiomeric muscles form normally in Tbx1^+/– mice
Patients with del22q11.2 syndrome are haploinsufficient for TBX1 and, in most cases, about 30 linked genes on chromosome 22q11.2 (14). Although we observed no differences in MRF or myogenic differentiation-specific gene expression between Tbx1^+/+ and Tbx1^+/– littermates during in utero development (Figs 5 and 6), we examined branchiomeric muscles in Tbx1^+/+ and Tbx1^+/– mice at 5 weeks of age (Fig. 8). The use of a fast fiber myosin light chain nlacZ transgene (Mlc3f–nlacZ–2E) permitted detailed comparison of heterozygous and wild-type pharyngeal and laryngeal muscles by whole-mount and histological analysis. All Tbx1^+/– branchiomeric muscles scored were present and appeared anatomically normal (n=6), including mandibular arch-derived muscles (Fig. 8A and B), pharyngeal constrictor muscles (Fig. 8C–F and M–R) and intrinsic laryngeal muscles (Fig. 8G–L and O–R) derived from the caudal branchiomeric region. Furthermore, expression of the fast fiber muscle transgene was comparable in Tbx1^+/+ and Tbx1^+/– muscles (Fig. 8).

View larger version (81K): [in this window] [in a new window]   Figure 8. Comparison of branchiomeric muscles in Tbx1+/+ and Tbx1+/– mice. (A–L) Whole-mount partially dissected X-gal-stained branchiomeric muscles from 5-week-old Mlc3f–nlacZ–2E mice. No differences in anatomy or transgene expression were observed between wild-type and Tbx1+/– littermates for first arch-derived muscles (A, B, ventral views), pharyngeal muscles (C, D, lateral views; E, F, dissected in the sagittal plane) or intrinsic laryngeal muscles (G, H, dorsal views; I, J, superior views; K,L, after bisection in the sagittal plane). (M–R) Sagittal sections of Tbx1+/+ (M, O, Q) and Tbx1+/– (N, P, R) pharynxes after X-gal and eosin staining to reveal pharyngeal and laryngeal muscle histology. ad, Anterior digastric; m, masseter; sth, sternohyoid; my, mylohyoid; spc, superior pharyngeal constrictor; mpc, middle pharyngeal constrictor; stp, stylopharyngeus; pp, palatopharyngeus; a, arytenoid; ta, thyroarytenoid; ct, cricothyroid; pca, posterior cricoarytenoid; ae, aryepiglottis. Scale bars: 100 µm (M, N); 200 µm (O, P); 300 µm (Q, R).

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In this paper we have demonstrated that Tbx1 regulates the onset of branchiomeric myogenesis in the mouse. The failure of branchiomeric myogenesis in Tbx1^–/– mice uncovers an evolutionarially conserved role for Tbx1 in pharyngeal muscle development. The zebrafish van gogh (vgo) mutation shows loss and reduction of certain pharyngeal muscles (31). Vgo alleles have recently been shown to result from point mutations in the zebrafish tbx1 gene (32). Our results in the mouse significantly extend these observations and reveal a low-level of sporadic myogenesis in the mandibular arch which is Tbx1-independent. We demonstrate that Tbx1 is not required for myogenic differentiation but rather functions during the initiation of branchiomeric myogenesis to ensure robust bilateral activation of MRF genes in pharyngeal mesoderm. Our findings have direct implications for the understanding of skeletal muscle heterogeneity, craniofacial development and the pathogenesis of del22q11.2 syndrome.

Here we discuss two possible mechanisms by which Tbx1 could regulate MRF activation in the mandibular arch. Myf5 and MyoD could be direct transcriptional targets of Tbx1 (Fig. 9, pathway A); alternatively, or in addition, Tbx1 could indirectly regulate MRF gene activation through modulation of pro-myogenic signals in surrounding neural crest cells (pathway B).

View larger version (27K): [in this window] [in a new window]   Figure 9. Myogenic specification in the mandibular arch at E9.5. Tbx1, expressed in the mesodermal core of the mandibular arch, is required for normal activation of the myogenic regulatory factor (MRF) genes Myf5 and MyoD which control branchiomeric myogenesis. Tbx1 is proposed to regulate MRF activation by one or both of two pathways: direct activation of MRF transcription (pathway A) or indirect modulation of signaling molecule activity (X) in surrounding neural crest cells (pathway B). Candidate signaling molecules include Wnt and BMP inhibitors which promote branchiomeric myogenesis by antagonizing anti-myogenic signals originating from pharyngeal epithelia (dark gray) (12). Capsulin and MyoR encode transcription factors expressed in the mesodermal core which are required for Myf5 activation in a subset of mandibular muscle precursor cells (13). The expression of Capsulin and MyoR is Tbx1 independent and may contribute to low level sporadic MRF activation in the absence of Tbx1. In addition, Tbx1 is required for high level Tlx1 expression and activation of Fgf10 in proximal core mesoderm.

 
According to pathway A, Tbx1 is a direct regulator of MRF genes and loss of Tbx1 would directly impair the robustness of branchiomeric enhancers of Myf5 and MyoD. Enhancer elements have been argued to act in a stochastic fashion to increase the probability that a regulated gene will be transcribed in each cell within the domain of enhancer action (33). Impaired branchiomeric enhancer function would result in reduced probability of transcriptional activation in pre-myogenic cells of the mandibular core, consistent with our observations of sporadic MRF activation in the absence of Tbx1. Such a hypothesis is in agreement with observations that a T-box-binding site mediates activation of Xenopus Myf5 in trunk muscle and that ascidan and sea-urchin T-box genes are implicated in activation of the skeletal myogenic program (34–36). Furthermore, a Caenorhabditis elegans Tbx1 subfamily member, MLS-1, acts as a fate switch during specification of non-striated muscle, a role similar to that of muscle founder identity genes in Drosophila (37). Myf5 expression in branchiomeric muscles is regulated by at least five elements in an extended region upstream of and within the Myf5 gene (8,21–23). We have identified T-box-binding sites in defined Myf5 pharyngeal arch enhancer elements, including a highly conserved site GTGTGAA in the 1.1 kb branchial arch enhancer defined by Summerbell et al. (22). However, site directed mutagenesis of this T-box target site in transgenic embryos did not impair enhancer activity in pharyngeal mesoderm. Future experiments will address whether additional T-box target sites in the 1.1 kb or other branchial arch enhancer elements mediate activation of Myf5 by Tbx1.

Tlx1 and Fgf10 are additional targets of Tbx1 in the mesodermal core of the mandibular arch. However, neither of these genes appears to act downstream of Tbx1 in the regulation of branchiomeric myogenesis, since first arch-derived muscles form normally in Tlx1 mutant mice (25), and a Myf5–nlacZ transgene is expressed normally in branchiomeric muscle masses of Fgf10^–/– embryos. Our observations concerning Tbx1-dependent core expression of Fgf10 are consistent with those of Vitelli et al. (27), and the recent demonstration that Tbx1 can activate an Fgf10 promoter in COS-7 cells through a conserved T-box-binding element (38). However, analysis of an Fgf10 enhancer-trap transgene in Tbx1 mutant embryos reveals that transgene expression in the distal region of the mesodermal core is Tbx1-independent. Although the extent of overlap between endogenous Fgf10 and Tbx1 expression in the distal mesodermal core remains to be determined, this result suggests that Tbx1 specifically regulates transcription in the proximal region of the mesodermal core where MRF gene activation takes place.

In contrast to Myf5, MyoD, Fgf10 and Tlx1, the genes encoding the transcriptional repressors Capsulin and MyoR are expressed normally in the mesodermal core of the Tbx1^–/– mandibular arch. The presence of Capsulin and MyoR transcripts demonstrates that Tbx1 does not regulate the lateral movement of cranial paraxial mesoderm into the mandibular arch. In contrast, MyoR and Capsulin transcripts are absent from the second and more caudal arches, which are severely hypoplastic in Tbx1^–/– embryos (18). Thus, unlike the situation in the mandibular arch, failure of branchiomeric myogenesis in the second and more caudal arches of Tbx1^–/– embryos is essentially a result of failure of caudal pharyngeal morphogenesis, possibly involving proliferation defects as documented for progenitor cells of the outflow tract of the heart (38). However, the expression pattern of Tbx1 in pharyngeal mesoderm and the observation of sporadic muscle fibers at the sites of second arch-derived muscle formation (Figs 5E and 7B) suggest that Tbx1 may play a series of roles, regulating pharyngeal outgrowth and subsequently ensuring robust bilateral MRF expression at sites of caudal branchiomeric myogenesis, analagous to our observations in the mandibular arch.

Capsulin and MyoR are together required for Myf5 activation in the mandibular arch, and for subsequent development of a subset of first arch-derived muscles (13). Capsulin and MyoR, however, are not sufficient to activate robust MRF expression in the absence of Tbx1, although they may contribute to the low level of sporadic MRF activation observed in Tbx1^–/– embryos. Since Capsulin, but not MyoR, is expressed prior to MRF activation, and since branchiomeric myogenesis is normal in Capsulin^–/– embryos (13), it appears unlikely that Capsulin/MyoR act upstream of Tbx1 in the mandibular arch. We conclude that Capsulin/MyoR and Tbx1 define independent parallel inputs into branchiomeric myogenesis, which converge at the level of MRF activation (Fig. 9).

Tbx1 could also control MRF expression indirectly, through the regulation of pro-myogenic signals in the mandibular arch (Fig. 9, pathway B). Candidate signals include secreted Wnt and BMP inhibitors expressed in mandibular mesenchyme at the time of MRF gene activation. Frzb and Gremlin, Wnt and BMP antagonists, respectively, have been proposed to isolate the mesodermal core from anti-myogenic signals originating from pharyngeal endoderm or ectoderm (12). Our results demonstrate that Frzb1 and low level Gremlin expression are maintained in the Tbx1^–/– mandibular arch. However, Tbx1 regulated signals from pharyngeal mesoderm to surrounding neural crest cells could interfere with signaling at the post-transcriptional level or regulate transcription of as yet unidentified BMP or Wnt antagonists. Indeed, transcripts encoding regionally expressed transcription factors in the mandibular neural crest mesenchyme are abnormally distributed in the absence of Tbx1. Aberrant neural crest migration has been documented in Tbx1 mutant mice (19) and may underlie the altered gene expression domains documented here. In addition to a defect in specification of the mesodermal core, loss of Tbx1 therefore results in abnormalities of the surrounding neural crest cells, which could in turn lead to further developmental defects in core mesoderm (Fig. 9). Our results demonstrate that Tbx1 plays a central role in mandibular development and suggest that Tbx1 may control MRF activation and subsequent branchiomeric myogenesis through both direct and indirect mechanisms. Since Tbx1 is expressed in pharyngeal endoderm in addition to core mesoderm, conditional mutagenesis will be required to define the relative importance of each of these expression domains in the regulation of branchiomeric myogenesis.

Tbx1 mutant mice display defects in the majority of structures perturbed in del22q11.2 patients, including mesenchymal derivatives of the first pharyngeal arch, such as the inner ear, tooth and mandible (18). Our finding that specification of skeletal myogenesis is abnormal in the mandibular arch of Tbx1^–/– mice suggests that many of these craniofacial defects may be secondary to abnormal specification of the mesodermal core. Skeletal muscle hypotonia has been noted in >50% of del22q11.2 patients (39–41). Furthermore, defects in pharyngeal muscles contribute to velopharyngeal insufficiency, which occurs in 90% of del22q11.2 patients (42). We observed no differences between Tbx1^+/+ and Tbx1^+/– branchiomeric muscles during in utero development or at 5 weeks of age. However, the cardiovascular and craniofacial phenotype of Tbx1^+/– mice is milder than that of haploinsufficient del22q11.2 patients, while almost all structures affected in the human syndrome are severely affected in Tbx1^–/– embryos (18). Our results suggest that this is also true of branchiomeric myogenesis, and that reduced TBX1 levels may lead to pharyngeal hypotonia in del22q11.2 patients.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mice carrying the null allele Tbx1^tm1Pa (here referred to as Tbx1^–) have been previously described (18), as have mice carrying the y96–Myf5–nlacZ, Mlc1v–nlacZ–24 and Mlc3f–nlacZ–2E transgenes (21,26,43). Fgf10^+/– mice were kindly provided by Drs N. Itoh and S. Kato (28). Mice were maintained on a mixed genetic background. Tbx1 genotypes were determined using PCR on DNA isolated from tail tips or yolk sacs using the primer combinations defined by Jerome and Papaioannou (18); Fgf10 genotypes were carried out as described by Sekine et al. (28).

X-gal revelation, whole mount in situ hybridization and histology were performed as previously described (18,43). MF20 antibody developed by D. Fishman, Cornell University Medical College, was obtained from the Developmental Studies Hybridoma Bank, NICHD, University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. Immunohistochemistry was carried out on 14 µm cryostat sections as described previously (43). The statistical significance of right–left differences in Tbx1^–/– mandibular arch-derived muscles was determined by the chi square test.

Riboprobes to detect transcripts of myogenic regulatory and structural genes were prepared as described elsewhere (9). Templates for MyoR (503 bp), Capsulin (460 bp) and Frzb1 (426 bp) were prepared by PCR from mouse strain 129 DNA using the following primers: MyoR 5'-TCTAGATCTACGTGGCCATTCGTGGTCTCTGGAC, MyoR 3'-CCGAAGAGCTCTAATGCCGGCCTTC; Capsulin 5'-TCTAGATCTCTCTAAACATGTCCACTGGCTCC, Capsulin 3'-CAGGTTGACTGGGTGAATGTAACCG; Frzb1 5'-TCTAGATCTTAAGGGGCACACTGGAATCAGTAGC, Frzb1 3'-GTGCAATGGCTTAAACCTACCCACC.

Products were subcloned in pGEM-TEasy prior to synthesis of antisense riboprobes using the restriction endonuclease BglII and T7 RNA polymerase (Capsulin) and BglII and SP6 RNA polymerase (MyoR and Frzb1). Tlx1 riboprobe was synthesized using T7 RNA polymerase and BamHI digested IMAGE clone 4164519. A plasmid generating a 1700 nucleotide Dlx7 riboprobe was kindly provided by T. Lufkin (Mount Sinai Medical Centre, NY, USA). Other probes have been described elsewhere: Tbx1, Tbx2 and Tbx3 (17), Prx2 (44), Gremlin (45), Crabp1 (46) and Msx2 (47).

A 1.1 kb NheI–BsaBI Myf5 branchial arch enhancer (22) was amplified from mouse DNA using primers M5A-F and M5A-R and subcloned in pGEM-TEasy. Mutagenesis of the T-box target site at nucleotide 636 was carried out using the QuikChange XL site directed mutagenesis kit (Stratagene) and primers M5A-mut1 and M5A-mut2. Mutant and wild-type enhancers were DNA sequence verified and subcloned into a NotI site upstream of the TK promoter and nlacZ reporter gene in pSKT–TK–nlacZ (48). Transgenic embryos were generated by pronuclear injection and analyzed at E11.5; 4 independent X-gal-positive embryos were obtained for both wild-type and mutant constructs. Primers: M5A-F CAGCTAGCAAGTGCAGTGATTGGC, M5A-R GATGGTCATCTCATGGGGATGACAG; M5A-mut1 GGCTTTTGTCACCAAAGTACTAGAAGCCACTCTTTTAAGAGGAG, M5A-mut2 CTCCTCTTAAAAGAGTGGCTTCTAGTACTTTGGTGACA AAAGCC.

	ACKNOWLEDGEMENTS

 
We thank Sharaghim Tajbakhsh for discussion and probes, Juliette Hadchouel and Margaret Buckingham for providing y96–Myf5–nlacZ mice, Marcia Ontell for providing Mlc3f–nlacZ–2E mice; Nobu Itoh and Shigeaki Kato for providing Fgf10^+/– mice and Thomas Lufkin and Richard Harland for probes. This work was supported by NIH grant HD33082 (V.E.P.). R.G.K. is an INSERM research fellow.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 2123054791; Fax: +1 2129232090; Email: rk2149{at}columbia.edu

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