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Control of Facial Muscle Development by MyoR and Capsulin

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Science  20 Dec 2002:
Vol. 298, Issue 5602, pp. 2378-2381
DOI: 10.1126/science.1078273

Abstract

Members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors control the formation of all skeletal muscles in vertebrates, but little is known of the molecules or mechanisms that confer unique identities to different types of skeletal muscles. MyoR and capsulin are related bHLH transcription factors expressed in specific facial muscle precursors. We show that specific facial muscles are missing in mice lacking both MyoR andcapsulin, reflecting the absence of MyoD family gene expression and ablation of the corresponding myogenic lineages. These findings identify MyoR and capsulin as unique transcription factors for the development of specific head muscles.

The myogenic bHLH transcription factors—MyoD, Myf5, myogenin, and MRF4—control vertebrate skeletal muscle development (1). MyoD andMyf5 act redundantly as myoblast specification genes (2), whereas myogenin is required for myoblast differentiation (3, 4). MyoDand MRF4 also play redundant roles in muscle differentiation (5). Although these myogenic genes control a developmental program shared by all skeletal muscles, there is evidence that muscles in the head and trunk differ with respect to the early steps in myogenic lineage specification (6). For example, inMyf5 −/− Pax-3 −/−double-mutant mice, the trunk musculature is eliminated, but head muscles are unaffected (7). The developmental control genes responsible for head muscle formation are unknown.

All skeletal muscle posterior to the head is derived from paraxial mesoderm that becomes segmented into somites (1,8). In contrast, head muscles are derived from multiple cell lineages, including prechordal mesoderm anterior to the first somite and paraxial mesodermal precursors that migrate into the branchial arches (9–11). MyoR/musculin and capsulin/Pod-1/epicardin are related bHLH proteins transiently expressed in migratory paraxial mesodermal cells in the branchial arches that appear to represent precursors of the muscles of mastication (12–18).

A null mutation in the mouse capsulin gene results in neonatal lethality due to pulmonary hypoplasia, but no overt skeletal muscle abnormalities (18, 19). We combined mutations in MyoR and capsulin to see if there were shared activities or effects not revealed with either single-gene deletion. The MyoR gene was targeted in embryonic stem (ES) cells by homologous recombination (fig. S1A). ES cell clones harboring the mutant allele were used to generate chimeric mice, which transmitted the mutation through the germ line (fig. S1B). Breeding of mice heterozygous for the mutant MyoR allele yielded homozygous mutants at predicted Mendelian ratios with no obvious abnormalities.

Mice homozygous for the MyoR and capsulin null mutations were obtained by breeding heterozygous mutant mice. Double mutants were born alive, but, like capsulin −/−mice (18, 19), they died within minutes after birth. Histological examination revealed a complete absence of the major muscles of mastication, including the masseter, medial and lateral pterygoids, and temporalis muscles in the majority of double-mutant embryos (Fig. 1, A to E). In their place was connective tissue. In a subset of double mutants, atrophic pterygoid myofibers persisted unilaterally. The missing muscles inMyoR −/− capsulin −/−double-mutant mice are derived from the first branchial arch and represent a distinct group of muscles that function in mastication (8). Other first arch–derived muscles, such as the anterior digastric and mylohyoid, which do not function in mastication, were present in the double mutants [Fig. 1, A to D and (20)]. Trunk muscles of double mutants were also indistinguishable from those of wild-type animals (20). Head muscle defects were not observed inMyoR+/ capsulin −/−or inMyoR −/− capsulin+/ embryos. These findings demonstrated that MyoR andcapsulin redundantly controlled the formation of a specific subset of first arch–derived facial skeletal muscles and that a single copy of either gene is sufficient to support normal muscle development.

Figure 1

Deficiency of head skeletal muscle and diaphragmatic hernia in MyoR−/−capsulin−/− neonates. Coronal sections fromMyoR −/− capsulin+/ (A and B) andMyoR −/− capsulin −/−(C and D); (B and D) show a higher magnification of (A and C), respectively. Note the cleft palate (p) in (C). gl, glands; m, mandible; ma, masseter muscle; p, palate; pt, pterygoid muscles; t, tongue; te, temporalis muscle. Asterisks in (C) denote missing muscles. In (B), the glands abut the masseter, whereas in (D), the masseter is missing and the glands abut the mandible. (E) Diagrams of muscle groups missing from the double mutant. (F) Sagittal section of aMyoR −/− capsulin −/−with diaphragmatic hernia. Arrowheads mark the boundaries of the diaphragmatic defect. Arrow marks the diaphragm. d, diaphragm; g, gut; l, liver; p, pancreas. Scale bars, 200 μm.

MyoR−/−capsulin−/−double mutants also displayed cleft palate (Fig. 1C). Other branchial arch–derived skeletal elements were normal in double mutants [Fig. 1and (20)]. In addition, the visceral organs ofMyoR−/−capsulin−/−double-mutant neonates were displaced into the chest through a defect in the posterior region of the diaphragm (Fig. 1F). This abnormality suggests that MyoR and capsulin are required to maintain integrity of the diaphragm muscle. The diaphragm develops from fusion of the septum transversum, the pleuroperitoneal membrane, and the dorsal esophageal mesentery and is populated by muscle precursor cells that migrate from the cervical somites (21).MyoR is expressed in diaphragmatic myoblasts (12,13), and capsulin is expressed in the septum transversum and the pleuroperitoneal membrane (14–18).

The head muscle deficiency in MyoR −/− capsulin −/− double mutants could arise from a defect in specification, migration, or proliferation of the affected myogenic lineages; a block in myoblast differentiation; or an increase in apoptosis. To determine the basis for this muscle deficit and to pinpoint the time of its onset, we compared the expression of alacZ reporter integrated into the targetedcapsulin allele in staged embryos of the different genotypes. From embryonic day 7.5 (E7.5) to E8.0, when mesodermal precursors of first arch muscle cells initially appear subjacent to the metencephalon (10), lacZ staining was localized to this muscle precursor population in embryos of the different genotypes (Fig. 2, A to C). At E9.5, these lacZ-positive cells could be seen migrating into the newly formed branchial arches of wild-type and double-mutant embryos (Fig. 2, D and G). By E10.5, a swathe of lacZ-positive cells extended into the first and second branchial arches. This myogenic precursor pool appeared to migrate properly, but there were noticeably fewer of these lacZ-positive cells in the first branchial arch of the double mutants (Fig. 2, E and H). In contrast, other embryonic sites of lacZ expression showed similar staining in embryos of the different genotypes. At E11.5, lacZ expression was observed in presumptive myoblasts within the first and second branchial arches of normal embryos (Fig. 2F). These lacZ-positive cells within the first branchial arch surrounded the fifth cranial nerve (Fig. 2J). There was a dramatic reduction of lacZ staining in the myogenic cores of the first branchial arches of double mutants at E11.5 (Fig. 2, I and K), suggesting that thecapsulin-expressing myogenic lineage of the first branchial arch was specifically affected in these mutant embryos.

Figure 2

Expression of capsulin-lacZallele. Expression of lacZ in capsulin −/− mice at E7.5 (A) and E8.0 (B and C), as indicated by arrowheads. (C) A coronal section through the embryo in (B). (D to F) LacZ staining is present within the first and second branchial arches inMyoR −/− capsulin+/ . (G to I) Staining gradually disappears from the first arch ofMyoR −/− capsulin −/−. (J and K) Sections of the first arch of embryos in (F) and (I), respectively. 1, branchial arch 1; 2, branchial arch 2. Arrowheads in (J) and (K) indicate the muscle developing around the fifth cranial nerve. LacZ-positive cells are missing from this region of the double mutant. Scale bars: (A, B, and C), 200 μm; (D to I), 500 μm; (J and K), 100 μm.

The striking lack of specific facial skeletal muscles inMyoR −/− capsulin −/−double mutants was reminiscent of the phenotype associated with the combined absence of Myf5 and MyoD, except that the latter phenotype affects all skeletal muscles (2). To define the temporal sequence of expression of these genes and to determine whether myogenic bHLH genes were expressed in the affected muscle lineage of the double mutant, we performed in situ hybridization with adjacent sections of staged embryos between E8.5 and 15.5.Capsulin transcripts were detected in mesodermal precursors of the first arch at E8.5 (Fig. 3A). At this stage, Myf5, MyoD, and MyoRexpression was undetectable. There has been some disagreement about the timing of expression of Myf5 and MyoD in the branchial arches, depending on the method of detection, but the earliest reported expression of these genes in this region is E9.25 and E9.5, respectively (22–24). By E9.5,Myf5 and capsulin were expressed in the same cell population within the first branchial arch, and by E10.5,Myf5, capsulin, and MyoR were coexpressed in these cells of wild-type embryos (Fig. 3A). In contrast,Myf5 was not expressed in first branchial arch precursors ofMyoR −/− capsulin −/−double mutants at E9.5 or E11.5 (Fig. 3B). There was also no evidence for expression of Myf5, MyoD, ormyogenin at E15.5 in the region of affected facial muscles (Fig. 3C), whereas these genes were expressed in other developing head and trunk muscles.

Figure 3

Expression of myogenic bHLH genes in wild-type and MyoR −/− capsulin −/− embryos. (A)MyoR, capsulin, and Myf5 expression were detected in the branchial arches of wild-type embryos by in situ hybridization. Capsulin was detected at E8.5 in the first arch (ba1). Expression of Myf5 and MyoRoverlapped that of capsulin at later stages. (B)Myf5 expression was detected in the first branchial arches of MyoR+/+capsulin+/ , designated wild type, but not inMyoR −/− capsulin −/−embryos. The middle panels show higher magnification of the upper panels. ba1, first branchial arch; ba2, second branchial arch; hc, hypoglossal cord. m, Myotome. (C) Myf5,myogenin, and MyoD expression was detected in wild-type andMyoR −/− capsulin −/−embryos at E15.5. The masseter muscle (ma) is completely absent inMyoR −/− capsulin −/−mutants (designated ma*). Arrow indicates cleft palate. Scale bars: 200 μm for (A and B), 1 mm for (C).

To determine the fate of first arch muscle precursors that failed to activate expression of Myf5 and MyoD, we performed TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) on histological sections of double-mutant embryos at E10.5, when cells marked by expression of capsulin-lacZ were disappearing. As shown in Fig. 4, TUNEL- positive cells were observed among the lacZ-positive muscle precursors of double mutants, but not ofMyoR −/− capsulin+/ embryos. We conclude that these cells, which fail to initiate the normal program for muscle development in the double mutant, undergo apoptosis with resulting ablation of muscles of mastication. Similar observations have been made in muscle precursor cells in the limb buds of mice lacking MyoD and myf5 (25).

Figure 4

Apoptosis of muscle cells in the first branchial arch inMyoR −/− capsulin −/−embryos. First branchial arch of MyoR −/− capsulin+/ andMyoR −/− capsulin −/−embryos at E10.5 stained for lacZ expression and viewed by differential interference contrast microscopy. (A and B). The same sections were stained with propidium iodide, and apoptotic cells were identified by TUNEL labeling (C and D). At this stage, lacZ-positive cells are disappearing from the first branchial arch of the double mutant because of apoptosis, as revealed by TUNEL labeling (yellow) in the lacZ-positive cores. LacZ staining interferes with detection of propidium iodide. Arrowheads point to lacZ-positive cores in the first branchial arches. Scale bar, 100 μm,

The absence of specific head muscle cells, as well as markers of the corresponding myogenic lineages, inMyoR −/− capsulin −/−mutants resembles the effect ofMyoD −/− Myf5 −/− double mutations on all skeletal muscles (2) and is distinct from the phenotype of Myf5 −/− Pax3 −/− mutants, which exhibit a specific deficiency of trunk skeletal muscles (7). This phenotype also differs from that of myogenin mutant mice, in which myoblasts express myogenic bHLH genes, but are unable to differentiate (3, 4). These findings demonstrate that MyoR and capsulinredundantly regulate an initial step in the specification of a specific subset of facial skeletal muscle lineages and that, in the absence of these factors, myogenic bHLH genes are not switched on, and cells from these lineages undergo programmed cell death. There may also be a modest effect on migration of precursors, as is seen in Lbx1mutant mice (21). MyoR and capsulin act as transcriptional repressors in transfection assays (12,20). Whether they act to repress an inhibitor of myogenesis or have a transcriptional-activating function during development of facial muscle remains to be determined. The phenotype ofMyoR −/− capsulin −/−mutant mice reveals a previously unanticipated complexity in the development of head skeletal muscles, and these findings identify MyoR and capsulin as unique transcriptional regulators for the development of specific head muscles.

Supporting Online Material

www.sciencemag.org/cgi/content/full/298/5602/2378/DC1

Materials and Methods

Fig. S1

References

  • * To whom correspondence should be addressed. E-mail: eolson{at}hamon.swmed.edu

REFERENCES AND NOTES

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