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MyoD or Myf-5 is required for the formation of skeletal muscle
Cell, Vol. 75, 1351-1359,
December
31, 1993, Copyright
0 1993 by Cell Press
MyoD or Myf-5 Is Required for the Formation of Skeletal Muscle Michael A. Rudnicki, * Patrick N. J. Schnegelsberg,t Ronald H. Stead,* Thomas Braun,§ Hans-Henning Arnold,5 and Rudolf JaenischS *Institute for Molecular Biology and Biotechnology McMaster University Hamilton, Ontario Canada L8S 4Kl fwhitehead Institute and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02142 *Department of Pathology McMaster University Hamilton, Ontario Canada L8S 4Kl §lnstitut fur Biochemie und Biotechnologie Abteilung Zell und Molekularbiologie 38106 Braunschweig Federal Republic of Germany
Summary Mice carrying null mutations in the myogenic regulatory factors Myf-5 or YyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and Sl nuclease analyses indicated that Myf-5(-I-);MyoD(-I-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. lmmunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. Theseobservations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis. Introduction The myogenic basic-helix-loop-helix(bHLH)familyof transcription factors is believed to play an important regulatory role in the development of skeletal muscle. This group includes MyoD (reviewed by Weintraub et al., 1991) myogenin (Braun et al., 1989a; Edmondson and Olson, 1989; Wrightetal., 1989) Myfd(Braunetal., 1989b), and MRF4, also called Myf-6 or herculin (Rhodes and Konieczny, 1989; Braun et al., 1990; Miner and Wold, 1990). The myogenie bHLH genes are exclusively expressed in skeletal muscle, and forcing their expression in a wide range of cultured cells induces the skeletal muscle differentiation program. Thus, these transcription factors have been postulated to play a master regulatory role in determining the
identity of the skeletal muscle lineage (Olson, 1990; Weintraub et al., 1991; Buckingham, 1992). Vertebrate skeletal muscle is derived from cells in the somites and prechordal mesoderm that give rise to myoblasts that form the skeletal muscle of the head, trunk, and limbs (reviewed by Buckingham, 1992; Miller, 1992). In the mouse, primary myofibers develop first at 8.5 days of gestation, followed by secondary myofibers that start to develop around day 14. These two fiber types express distinctive isoforms of muscle proteins and have been postulated to be formed from distinct lineages of myoblasts (Miller, 1992). Cultured myoblast cell lines express Myf-5 mRNA, MyoD mRNA, or both before and after differentiation, whereas myogenin mRNA is expressed upon myotube fusion, and MRM mRNA several days after fusion (Braun et al., 1989b; Wright et al., 1989; Edmondson and Olson, 1989; Miner and Wold, 1990; Montarras et al., 1991). In mice, the myogenic bHLH genes are activated sequentially during the skeletal muscle developmental program. Mfl-5 mRNA is first detected in the 8 day somite and is markedly reduced after day 14 (Ott et al., 1991). myogenin mRNA appears on day 8.5 and is expressed throughout fetal development (Sassoon et al., 1989). MRF4 mRNA appears transientlyon days 10 and 11, and is re-expressed at day 16 to become the most abundant myogenic factor after birth (Bober et al., 1991). Finally, My00 mRNA appears around day 10.5 and is expressed thereafter throughout development (Sassoon et al., 1989). In the developing limb bud, a different pattern of expression is observed: Myf-5 mRNA is expressed transiently between day 10 and 12, myogenin and My00 mRNAs are coexpressed after day 11 (Ott, 1991; Sassoon et al., 1989), and MRM mRNA is expressed after day 16 (Bober et al., 1991). This pattern of activation of the four myogenic bHLH genes in vitro and in vivo suggests that they may have distinct functions in the developmental activation of the muscle differentiation program. Previously, we demonstrated that mice lacking a functional My00 gene are viable and fertile and exhibit no morphological or physiological abnormalities in skeletal muscle (Rudnicki et al., 1992). Mutant My00 mice did, however, exhibit a 3.5-fold increase in the amount of Myf-5 mRNA. Similarly, newborn mice lacking a functional My&5 gene display no obvious defects in skeletal muscle, but die perinatally, owing to severe rib abnormalities (Braun et al., 1992). The sustained induction of Myf-5 mRNA levels in mice lacking MyoD and the delay in the onset of musclespecific gene expression in mice lacking Myf-5 (Braun et al., 1992) raised the possibility that Myfd and MyoD can functionally substitute for one another in muscle development. To investigate further the role of Myf-5 and MyoD in myogenesis, we have generated mice lacking both myogenie factors by crossing Myf-5 and My00 mutant mice. Strikingly, mice lacking both Myfd and MyoD are completely devoid of skeletal myofibers and presumably de-
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Table 1. Offspring My&5 (+/-);MyoD(+/-)
of Mfi-5 and MyoD Mutant Mice Myki(+/-);MyoD(+/-)
x Myf-5 (+I-);MyoD(+/-)
x Mfl-S(+/+);MyoD(-/-)
Frequency Observed
Genotype Myf-5
MyoD
Predicted
+I+ +/+ +I+I-- l -- l -l+I-+I-+
+I+ +I+I+ -+I+I+ +I-- I -I-I-
6.25 12.5 12.5 25.0 6.25 12.5 6.25 12.5 6.25
Offspring of Myf-5 and MyoD mutant *The number of mice analyzed was b The number of mice analyzed was c The number of mice analyzed was
(o/o)
(%)
Frequency
Genotype
Newborn”
3 Weeksb
Myfd
MyoD
Predicted
4 12 9 19 10 6 7 16 15
17 24 20 36 0 0 0 0 3
1 2 3 4
+I+I-I-I-
25 25 25 25
mice were genotyped 137. 143. 139, at 3 weeks.
+I+ +Iii+I+
after weaning (3 weeks) or after cesarean
void of myoblasts. These experiments suggest that either Myfd or MyoD is required for the determination of skeletal myoblasts, their propagation, or both and indicate that Myf-5 and MyoD are, at least in part, functionally redundant in myogenesis. Results Interbreeding of Myf-5 and MyoD Mutant Mice To generate mice lacking both MyoD and Myfd, we first interbred homozygous Myoly” mice (derived from the ES line MyoD 177 and crossed into a BALBlc background [Rudnicki et al., 19921) and heterozygous MyW” mutant mice (derived from the ES line Myf-5 307 and crossed into a BALB/c background [Braun et al., 1992)). The offspring from this cross that were heterozygous for both Myf-5mi and MyoP’ mutations (i.e., MyRi(i-/-);MyoD (+/-)) were then intercrossed. As depicted in Table 1, nine different genotypes are expected to be distributed among the offspring of Myf-5(+/-);MyoD(+/-) mice, with Myf-5 (-/-);MyoD(-/-) mice having a frequency of 6.25%. The offspring of Myf-5(+/-);MyoD(+/-) mice were genotyped after weaning to assess the effect of the different genotypes on survival (Table 1). As expected, no viable Myf5(-/-J mice were found, presumably owing to the presence of truncated ribs (Braun et al., 1992). In addition, no viable Myf-5(+/F);MyoD(-/-) mice were found among the offspring of this cross, even though these mice should have normal ribs (expected frequency 12.5%; Table 1). Consistent with this result, Myf-5(+/-);MyoD(-I-) mice were also not found in viable offspring of Myf-5(+/k); MyoD(+/-) males crossed with Myf-5(+/+);MyoD(-I-) females(expectedfrequency25%; Table 1). Theseobservations support the hypothesis that MyoDdeficient mice are functionally rescued by Myf-5. Furthermore, these results suggest that postnatal survival in the absence of MyoD requires expression of two Myf-5 alleles. To determine the phenotype of My+5(-/-);MyoD(-I-) and Myf-5(+/F);MyoD(-I-) pups, offspring of Myf-5(+/-); MyoD(+/-) mice were delivered by cesarean section at
(%)
Observed
(%)
36 37 0 25
section (newborn).
term (day 16.5 of gestation). The pups were examined for abnormalities and subsequently genotyped by Southern blot analysis using restriction digests and probes described previously (Rudnicki et al., 1992; Braun et al., 1992). By means of this approach, all nine possible genotypes were observed at close to the predicted frequencies (Table 1). Newborn Myf-5(-/--);MyoD(-/-) mice, delivered by cesarean section, initially appeared alive and pink in color, but were completely immobile and quickly became cyanotic. These pups were runted and on average 75% the weight of Myf-5(-/-);MyoD(+/+) pups. Newborn Myf-5 (-/-);MyoD(-/-) mice displayed an arched spine and thin limbs through which the long bones could be discerned. Dissection of Myf-5(-/-);MyoD(-/-) pups revealed that the hearts were beating for a short time after delivery. Strikingly, we observed a complete absence of skeletal muscle in the trunk and limbs, with the spaces normally occupied by skeletal muscle appearing mostly devoid of tissue. As expected, these animals displayed the truncated rib phenotype typical of My65(-F) pups. In addition to truncated ribs, other abnormalities were observed in skeletons of Mfl-5(-/-);MyoD(-/-) mice. Insertion points where tendons make attachments to bone were absent or reduced in size (data not shown). For example, the deltoid tuberosity of the humerus was absent, and the olecranon process of the ulna and distal process of the calcaneus were greatly reduced in size. In addition, the sternum was shorter in length, and the anterior portion (manubrium) was split in Myf-5(-/-);MyoD(-I-) animals. Newborn Myf-5(+/-);MyoD(-/-) mutant mice, delivered by cesarean section, displayed the reflexive breathing movements typical of newborns. Initially, these animals breathed normally, but respiration became intermittent, and they gradually became cyanotic and eventually died. The pups appeared similar to their normal littermates, but did exhibit a slightly curved spine. Dissection revealed a normal ribcage and skeletal system. These results suggested that Myf-5(+/-);MyoD(-/-) mutant mice died owing to some deficiency in skeletal muscle function and imply
Skeletal Muscle Development 1353
Requires
MyoD or Myf-5
Probe
123456789 A
Skeletal-Actin
B
Cardiac-Actin
C
6-MHC
D
Embryonic-MHC
E
Fetal-MHC
F
MLC 183
G
Slow-MLCl
H
Troponin T
I
Fast-Troponin
J
Slow-Troponin
K
&AChR
L
PGK-I
I I
MyoD: t:t ti- tit +I- t/t +I- -I- -/- -IMyf-5: t:t t!t +‘- t/- -I- -I- -/- 4 t/t Figure 1. Skeletal Muscle-Specific ing Myf-5 and MyoD
mRNAs Are Absent in Mice Lack-
Total RNA isolated from newborn skeletal muscle was subjected to Northern blot analysis with probes specific for the following: (A), a-skeletal actin; (El), a-cardiac actin; (C), b (slow) myosin heavy chain (f3-MHC); (D), embryonic myosin heavy chain (embryonic MHC); (E), fetal MHC; (F), myosin light chain 1 and 3 (MLC 1 and 3); (G), slow myosin light chain 1 (slow MLCI); (H), troponin-T; (I), fast troponin I; (J), slow troponin I; (K), acetylcholine receptor 6 (SAChR); (L), phosphoglycerate kinase 1 (PGK-I).
that two functional copies of the Myf-5 gene are required to rescue MyoD(-/-) mice. Mice Lacking Myf-5 and MyoD Do Not Express Skeletal Muscle-Specific mRNAs To determine whether transcription of skeletal musclespecific genes was affected by null mutations in both Myf-5 and MyoD, we performed Northern blot analysis with a panel of cDNA and genomic probes using RNA isolated from animals of the different genotypes listed in Table 1. The expression of skeletal muscle-specific mRNAs appeared normal in all combinations of genotype except in Myf-5(-/-);MyoD(-/-) and Myi-5(+/-);MyoD(-/-) mice. Newborn mice lacking both Myf-5 and MyoD (Figure 1, lane 7) expressed no detectable skeletal muscle-specific mRNAs in the panel tested. This included a-cardiac and a-skeletal actins (Figures 1A and lB), embryonic, fetal, and 8 (slow) isoforms of myosin heavy chain (Figures lC, 1 D, and 1 E), myosin light chains 1 and 3 (Figure 1 F), slow myosin light chain 1 (Figure lG), troponin-T (TnT) (Figure
1 H), fast and slow troponin I (Figures 1 I and 1 J), and acetylcholine receptor 6 (GAChR) (Figure 1 K). The equal loading of RNA per lane was confirmed by hybridization with a probe for phosphoglycerate kinase 1 (PGK-1) (Figure 1 L). In addition, My+5(-/-);MyoD(-I-) mice did not express detectable ~43 mRNA (data not shown), which is normally expressed at high levels in myoblasts (Frail et al., 1989). These results indicate that virtually no skeletal muscles or myoblasts were present in My+5(-/-);MyoD(-/-) mutant mice. Although Myf-5(+/-);MyoD(-I-) mutant mice were capable of movement and respiration, they died shortly after birth (Table 1). As determined by densitometry and normalization to SK-7 mRNA levels, Northern blot analysis with skeletal muscle-specific probes indicated an overall reduction in the levels of muscle-specific mRNAs by about 60% (Figure 1, lane 8). This result suggests that in My&6 (+/-);MyoD(-/-,J mice, either the amount of skeletal muscle or the levels of muscle-specific mRNAs were reduced. In either case, this observation supports the notion that two functional alleles of Myf-5 are required for the development of a normal skeletal muscle system in the absence of MyoD (Rudnicki et al., 1992). In addition, this as well as the other genotypes exhibited no consistent alterations in the ratio between developmentally regulated isoforms of contractile protein-encoding mRNAs. For example, the ratio between a-cardiac and a-skeletal actin (Figures 1A and 1 B), between embryonic, fetal, and 6 (slow) isoforms of myosin heavy chain (Figures 1 C, 1 D, and 1 E), and between fast and slow troponin I (Figures 1 I and 1J) appeared normal. This suggests that a single copy of Myf-5 is sufficient for the normal progression of the skeletal muscle developmental program. To investigate whether expression of the myogenic transcription factors was altered in mutant mice, we employed Sl nuclease analysis to examine mRNA levels of MyoD, Myf-5, myogenin, and MRM (Figure 2). MyoD mRNA was reduced in MyoD(+/-) mice (Figure 2A, lanes 2, 4, and 6), and absent in MyoD(-/-) mice (Figure 2A, lanes 7, 8, and 9). Myf-5 mRNA was reduced in My-f-5(+/-J mice (Figure 28, lanes 3, 4, and 8), and absent in Mfl-5(-/-j mice (Figure 2B, lanes 5, 6, and 7). These mice also expressed achimericneo/Myf-5 mutant RNA most likelygenerated by transcription through the PGK-neo cassette in the Myf-5”’ allele (Figure 2B, lanes 3-8; see Braun et al., 1992). Myf-5 mRNA was elevated in MyoD(+/-) mice (Figure 2B, lane 2), and further elevated in MyoD(-/-) mice (Figure 28, lane 9) confirming our previous observations (Braun et al., 1992; Rudnicki et al., 1992). myogenin and MRF4 mRNA were expressed at varying but approximately normal levels in all but one genotype: Myf-5(-/-j; MyoD(-/-) mutant mice expressed no detectable myogenin or MRF4 mRNA (Figures 2C and 2D, lane 7). Myf5(+/-);MyoD(-/-) mutant mice appeared to express lower levels of myogenin and MRF4 mRNA than the other genotypes (Figures 2C and 2D, lane 8), but more quantitative studies are needed to assess a possible feedback regulation between the different members of the myogenic factor family. We conclude that mice lacking both Myf-5 and MyoD contain little or no skeletal muscle.
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MP123456789M -
Figure bHLH MRF4 Myf-5
819 nt MyoD Probe 541 nt Protected Fragment
-
550 nt Myf-5 Probe
-
289 nt Wild Type Fragment
-
180 nt Mutant Fragment
-
550 nt Myogenin Probe 365 nt Protected Fragment
-
567 nt MRF4 Probe
-
452 nt Protected Fragment
-
894 nt PGK-1 Probe
-
442 nt Protected Fragment
2. Nuclease Sl Analysis of Myogenic Factor mRNAs Indicates rnyogenin and Are Not Expressed in Mice Lacking Both and MyoD
Total RNAisolatedfrom newborn skeletal muscle was subjected to Sl nuclease analysis with end-labeled probes specific for the following: MyoD (A), Myf-5 (B), myogenin (C), MRF4 (D), and PGK-1 (E). M, end-labeled marker 6x174Haelll; P, probe with no RNA.
M yo D: +I+ +I- +I+ +I- +I+ +I- -I- -I- -IM yf-5: +I+ +I+ +I- +I- -I- -I- -I- +I- +I+
Complete Absence of Skeletal Muscle in Mice Lacking Myf-5 and MyoD To assess further any possible alterations in skeletal muscle, we prepared Harris’ hematoxylin and eosin-stained tissue sections of mutant newborn animals (Figure 3). Stained sections were examined from the head, trunk, and limbs of newborn mice (day 18.5 of gestation) of the following genotypes: My&S(+/+);MyoD(+/-)(+/-!; Myf-5(-k); MyoDp-); Myf-5(i/-);MyoD(-/-); and Myf-5(-/F);MyoD (4-J Remarkably, mice lacking both Myf-5 and MyoD were completely devoid of skeletal muscle. The spaces normally occupied by skeletal muscle contained either amorphous loose connective tissues or expanded areas of adipose tissue (Figures 3A and 3E). Other organs, for example, the heart, lungs, liver, and bowel, appeared completely normal (data not shown). In newborn MyRi(+/-);MyoD(-/-) mice, the number of fibers was substantially reduced in all muscle groups (Figure 3 and Table 2). However, these fibers appeared normal in that they contained both peripherally and centrally lo-
Table 2. The Number of Muscle Fibers Is Dependent
cated nuclei and were of the normal size. Enumeration of the number of fibers in several muscle groups (e.g., diaphragm, intercostal, and biceps and triceps brachii; see Table 2) indicated a 350/b-55% reduction in the number of fibers. This decrease in muscle mass was consistent with the reduced expression of skeletal muscle-specific mRNAs observed by Northern blot and Sl nuclease analyses (see Figures 1 and 2). In contrast, My+5(-/-);MyoD (+/-) mice displayed no obvious morphological abnormalities in size, grouping, or form of muscles (Figures 3C and 3G). The developing fibers contained both peripherally and centrally located nuclei and were distributed normally with abundant mononucleated cells between the fibers. Therefore, one allele of MyoDappears capable of rescuing skeletal myogenesis in mice lacking Myfd, whereas one allele of Myf-5 can only partially rescue skeletal myogenesis in mice lacking MyoD. To determine whether a small number of skeletal muscle fibers might persist in Myf-5(-/-);MyoD(-I-) mutant mice that would not be readily apparent by light microscopic
on Genotype
Genotype
Diaphragm
Intercostal
Biceps Brachii
Triceps Brachii
MyRi(-/-);MyOD(-I-) My+5(+/-);Myoq-I-) Myf-q-/-);Myoq+/-) MyM(+/+),Myoq+/-)
0 4.0 f 8.0 f 0.0 f
0 2.4 f 0.2 6.8 f 0.6 6.6 f 0.2
0 16.6 f 30.0 f 29.4 f
0 11.0 -c 1.2 17.6 IT 3.6 20.0 f 7.2
1.0 0.4 1.4
2.2 4.4 1.0
The number of skeletal muscle fibers across the largest diameter of a muscle group was counted in Harris’ hematoxylin- and eosin-stained of newborn mice. Shown above is the mean and the standard deviation of this number from several muscle groupings.
sections
Figure 3. The Number of Muscle Fibers Is Dependent
on Genotype
Hematoxylin- and eosin-stained sections through the diaphragm (A to D) and intercostal muscles of the chest (E to H) suggest that the number of myofibers is dependent on the genotype of the animal (see Table 2). A.fyf-5(-/-J%fyoo(-/-,J mice are devoid of all skeletal muscle (A and E). !4fl-5(+/-);AfyoD( mice exhibit a reduction of about 35%-55% in the number of muscle fibers (6 and F). Myf-5(-/-);MyoD(+/-) (C and G) have numbers of muscle fibers similar to viable MyM(+/+);MyoD(+/-) mice (D and H). In (A) through (D), the lung and liver are located above and below the diaphragm (arrow), respectively. Note the expanded area of adipose tissue (arrow) in Mfl-5(-/-);MyoD(-/-) mice in (E). Representative intercostal muscles are indicated with arrows in (F). (G). and (H).
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Figure 4. Mice Lacking Both Myf-5 and MyoD Lack Skeletal Myocytes
and Myoblasts
lmmunohistochemical detection of skeletal muscle with a mouse monoclonal antibody reactive with smooth and striated muscle a-actin (A and B) and skeletal myoblasts with a mouse monoclonal antibody reactive with desmin (C and D). In My+5(+/-);MyoD(+/-) mice (A and C), anti-actin antibodies (A) stained skeletal muscle fibers (as well as smooth muscles), whereas anti-desmin antibodies (C) stained both skeletal muscle fibers (thick arrows) and myoblast-like cells (thin arrows). Sections from Myf-C(-/-);MyoD(-/-) mice (B and D) contained no detectable actin- or desmin-staining skeletal myocytes or desmin-staining skeletal myoblast-like cells. Note: the stained circular structures are smooth muscle cells in the walls of blood vessels.
Skeletal Muscle Development 1357
Requires MyoD or Myf-5
examination of hematoxylin- and eosin-stained sections, we performed immunohistochemistry on serial transverse sections through the trunk with a monoclonal antibody reactive to smooth and striated muscle a-actin (HHF35). Myf-5(+/-);MyoD(+/--J mice contained the normal amounts of skeletal muscle fibers reactive with ant a-actin antibodies (Figure 4A). However, using this procedure we did not detect any a-actin-staining skeletal myocytes in Myf-5 (-/-);MyoD(-/-j mice (Figure 48). lmmunohistochemistry was also performed with monoclonal antibodies reactive with desmin (DE-R-l l), an intermediate filament protein found in skeletal myoblasts and myofibers (George-Weinstein et al., 1993, and references therein). Whereas Myf5(+/-);MyoD(+/-) mutant mice contained extensive areas of anti-desmin-staining mononucleated myoblasts and skeletal myofibers (Figure 4C), Mfl-5(-I-);MyoD( mutant mice contained neither(Figure4D). These resultsconfirm that mice lacking MyoD and Myfd are indeed devoid of skeletal myocytes and myoblasts. Discussion In this study, we show that newborn mice deficient for the two myogenic bHLH factors Myfd and MyoD are totally devoid of all skeletal muscle. Mutant pups were born alive, but were completely immobile and died within minutes. Northern blot and Sl nuclease analyses demonstrated the absence of markers for skeletal muscle (a-actins, b-myosin heavy chain [MHC], embryonic MHC, fetal MHC, MLCl and MLC3, slow MLC, fast Tnl, slow Tnl, myogenin, MRF4, TnT, and GAChR). Histological examination revealed that these animals lacked all skeletal muscle, and the spaces normally occupied by muscle were filled with amorphous loose connective and adipose tissues. Immunohistochemistry indicated that desmin-containing myoblast cells were not detectable in Myf-5(-/F); MyoD(-/-) mice. Furthermore, except for truncated ribs and the lack of muscle tendon insertion points, the skeleton appeared normal. Our results have important implications for the understanding of the molecular control of skeletal muscle development by the myogenic bHLH factors. In previous studies we characterized myogenesis in mice carrying null mutations in either Myf-5 or MyoD genes. In animals lacking Myf-5, skeletal muscle development is delayed possibly until MyoD is expressed, resulting in apparently normal skeletal muscle at birth (Braun et al., 1992). Similarly, mice lacking MyoD are viable and have seemingly normal skeletal muscle. Importantly, Myf-5, which is normally down-regulated after day 14 of gestation, is expressed at 3.5-fold higher levels in newborn mutant My00 animals (Rudnicki et al., 1992; see Figure 2). These observations led to the hypothesis that Myf5 and MyoD have largely functionally redundant roles in myogenesis (Rudnicki et al., 1992). The complete absence of skeletal muscle in animals lacking both Myfd and MyoD strongly supports this hypothesis. Mice lacking Myf-5 die soon after birth not owing to deficient muscle function but rather as a consequence of a severe rib defect that prevents normal respiration (Braun
et al., 1992). The presence of one functional Myf-5 gene in homozygous MyoD mutant mice (My+5(+/-);MyoD (-/-)) results in the formation of normal ribs as well as skeletal muscle. Nevertheless, Myf-5(+/F);MyoD(-/-) were not viable and died soon after birth. Close inspection of this class of mutants revealed a reduced but normal expression pattern of developmentally regulated isoforms of muscle-specific mRNAs and demonstrated significantly reduced numbers of muscle fibers. This suggested that perinatal death was a consequence of an impairment in muscle function caused by a reduction in muscle mass. In contrast, one functional My00 gene in homozygous Myf-5 mutant mice (My+5(-/-);MyoD(+/-)) resulted in apparently normal skeletal muscle development. One interpretation of these results is that expression of these myogenic factors is important in determining the number of premyoblast cells in the somite that are recruited into the skeletal muscle lineage. Furthermore, these observations, while confirming that Myf-5 can functionally substitute for MyoD in myogenesis, suggest that either MyoD is inherently more active than Myf-5 in this role or that MyoD is present at higher levels in somite stage embryos. Alternatively, Myfd and MyoD may act to recruit different populations of cells into the myoblast lineage. Examination of myogenic factor expression during early somitogenesis will resolve this question. It is of interest to compare the roles played by the myogenie bHLH transcription factors in skeletal myogenesis as revealed by the phenotypes of mice carrying targeted mutations. Mice lacking a functional myogenin gene are immobile and die perinatally, owing to deficits in skeletal muscle differentiation (Hasty et al., 1993; Nabeshima et al., 1993). In contrast with Myf-5(-/-);MyoD(-/-) pups, myogenin mutant mice appear to form normal numbers of myoblasts, which are organized in groups similar to normal skeletal muscle, and express SAChR and TnT mRNA (Hasty et al., 1993). Myogenin-deficient myoblasts, however, are unable to undergo efficient fusion to form functional muscle fibers in vivo. This result suggests that myogenin plays an essential role in the differentiation of myoblasts into myotubes (Hasty et al., 1993; Nabeshima et al., 1993). The apparent lack of myoblasts in mice lacking both Myfd and MyoD, therefore, places these two fac-
Figure 5. Roles of Myogenic genesis
bHLH
Factors
During
Skeletal
Myo-
Targeted inactivation of myogenic bHLH genes suggests a model for the regulation of the skeletal muscle developmental program. The presence of either Myf-5 or MyoD is required for an essential role in the determination of myoblasts early in the develomsntal program. After myoblasts have been determined, myogenin has an essential role in the differentiation of myoblasts into multinucleated myotubes (Hasty et al., 1993; Nabeshima et al., 1993). MRF4 may act late in the developmental program, as suggested by its expression pattern (Sober et al., 1991).
Cell 1358
tom upstream of myogenin and suggests that they are involved in the determination of myoblasts, their propagation, or both (Figure 5). The fourth myogenic bHLH factor, MRF4, may function late in the myogenic pathway, as has been implied by its expression pattern in vivo and in cultured cells (Bober et al., 1991). We have previously suggested two alternative hypotheses to explain the defective rib cage formation in mice lacking Myf-5 (Braun et al., 1992). First, Myfd may act autonomously in some sclerotomal cells, although this seems unlikely, because MyfQ expression has not been detected in cells other than those in the dermamyotome of the somite (Ott et al., 1991). Second, dermamyotomederived myocytes may provide a permissive environment for sustaining continued proliferation of the rib rudiments. The presence of only relatively minor rib defects in myogenin-deficient mice suggests that myoblasts provide at least part of this signal. One possibility was that other skeletal elements were unaffected because the subsequent generation of skeletal muscle differentiation under the control of MyoD might provide these signals. Clearly, this study indicates that the formation of the rest of the skeletal system occurs independently of skeletal myogenesis. The generation of a normal rib cage, therefore, is unique, as it appears to require early skeletal myogenesis in the dermamyotomal compartment of the somite. MyoD and Myfd have been suggested to be functionally inactive in myoblasts for a variety of reasons, including the presence of Id proteins and phosphorylation of the DNA-binding domain (Benezra et al., 1990; Li et al., 1992). Yet, our data indicates a requirement for the expression of either Myfd or MyoD for the determination of skeletal myoblasts, their propagation, or both. Therefore, Myfd and MyoD must have a functional activity in myoblasts distinct from the transactivation of genes expressed after myotube fusion. This activity may function at the level either of myoblast commitment or of maintenance of myoblast propagation. Clearly, to understand how Myfd and MyoD specify the identity of myoblasts, it will be important to identify the target genes of these factors in myoblasts. Experimental
Procedures
Histological Analysis Cartilage and bone were stained using the method of McLeod (1980). Preparation, fixation, sectioning, and staining of tissue samples for light microscopy of histological preparations were performed using standard techniques (Bancroft and Stevens, 1990). Briefly, term embryos were delivered by cesarean section on day 18.5 of gestation, photographed, fixed in 4% paraformaldehyde in phosphate-buffered saline, dehydrated in steps to 70% ethanol, and stained with Harris’ hematoxylin and eosin. lmmunohistochemistry, as described elsewhere (Stead et al., 1989) was performed on paraformaldehyde-fixed sections with mouse monoclonal antibody HHF35 reactive with muscle a-actin (Enzo Diagnostics) and with a mouse monoclonal antibody (DE-R-l 1) reactive with desmin (Dakopatts). RNA Analysis RNA was isolated using the method of Auffray and Rougeon (1980) as described previously (Rudnicki et al., 1992; Braun et al., 1992). Northern blot analysis was carried out using standard techniques (Maniatis et al., 1982). The fast and slow troponin I (Koppe et al., 1989) were full-length rat cDNAs. The SK-7 probe (Adra et al., 1988) and the acetyl choline receptor 8 probe (LaPolla et al., 1984) were full-length
mouse cDNAs. The other probes used in the Northern blot analysis have been described previously (Rudnicki et al., 1992; Braun et al., 1992). Nuclease Si analysis was performed as previously described (Rudnicki et al., 1992) and was used to quantitate the myogenic bHLH and fgk-7 mRNAs. MyoD mRNA was detected with an 819 nt PvullStul probe that included vector sequences and the 5’ end of the fulllength mouse cDNA and was 5’ end-labeled. Myf-5 mRNA was detected with a 5’ end-labeled 550 nt Pvull-Hincll probe that included vector sequences and the Vend of the mouse cDNA. myogenin mRNA was detected with a 3’end-labeled 550 nt Smal probe containing exon 1 of the myogenin gene. MRF4 mRNA was detected with a 5’ endlabeled 567 nt Pvull-Accl probe that included vector sequences and the 5’end of the full-length mouse cDNA. Pgk-7 mRNA was detected with a 3’end-labeled 894 nt Xbal-Sphl probe that included the 3’end of the mouse cDNA and vector sequences. Signals from Northern blot and Si analyses were quantitated using an Apple Scanner and Image 1.4 software. Acknowledgments Address correspondence to M. A. R. We thank John Hassell and William Muller for critical comments on this manuscript, Ken Hastings for providing slow and fast Tnl probes, Steven Burden for providing the SAChR probe, and Chuyan Ying and Beth Colley for expert technical assistance. This work was supported by a grant from the Medical Research Council of Canada and by Public Health Service grant 5R35 CA44339 from the National Institutes of Health. M. A. R. is a Research Scientist of the National Cancer Institute of Canada. P. N. J. S. is supported by the Markey Biomedical Scientist Training Program. Received August 19, 1993; revised October 20, 1993. References Adra, C. N., Ellis, N. A., and McBurney, M. W. (1988). The family of mouse phosphoglycerate kinase gene and pseudogenes. Somat. Cell Mol. Genet. 74, 69-81. A&ray, C., and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 707, 303-314. Bancroft, J. D., and Stevens, A. (1990). Theory and Practice of Histological Techniques (Edinburgh: Churchill Livingston). Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 67, 49-59. Bober, E., Lyons, G. E., Braun, T., Cossu, G., Buckingham, M., and Arnold, H.-H. (1991). The muscle regulatory gene Myf-6 has a biphasic pattern of expression during early mouse development. J. Cell Biol. 773, 1255-1265. Braun, T. Grzeschik, of myogenic tion by the
G., Bober, E., Buschhausen-Denker, G., Kohtz, S., K. H., and Arnold, H.-H. (1989a). Differential expression determination genes in muscle cells: possible autoactivaMyf gene products. EMBO J. 8, 3617-3625.
Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E., and Arnold, H.-H. (1969b). A novel human muscle factor related to but distinct from MyoDl induces myogenic conversion in iOTl12 fibroblasts. EMBO J. 8, 701-709. Braun, T. G., Bober, E., Winter, B., Rosenthal, N., and Arnold, H.-H. (1990). My%6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12. EMBO J. 9, 821-831. Braun, T., Rudnicki, M. A., Arnold, H.-H., and Jaenisch, R. (1992). Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death. Cell 77, 369-382. Buckingham, 8, 144-148.
M. (1992). Making muscle in mammals.
Trends Genet.
Edmondson, D. G., and Olson, E. N. (1989). A gene with homology to the myc similarity region of MyoDl is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 3, 628-640.
s;$tal
Muscle Development
Requires MyoD or Myf-5
Frail, D. E., Musk, L. S., Buonanno, A., and Merlie. J. P. (1989). Expression of RAPsyn (43K protein) and nicotinic acetylcholine receptor genes is not coordinately regulated in mouse muscle. Neuron 2,10771086. George-Weinstein, M., Foster, R. F., Gerhart, J. V., and Kaufman, S. J. (1993). In vitro and in vivo expression of a7 integrin and desmin define primary and secondary myogenic lineages. Dev. Biol. 156,209229. Hasty, P., Bradley, A., Morris, J. H., Edmondson, J. M., Venuti, J. M., Olson, E. N.. and Klein, W. H. (1993). Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene. Nature 364, 501-506. Koppe, Ft. I., Hallauer, P. L., Karpati, G., and Hastings, K. E. M. (1989). cDNA clone and expression analysis of rodent fast and slow troponin I mRNAs. J. Biol. Chem. 264, 14327-14333. LaPolla, R. J., Mayne, K. M., and Davidson, N. (1984). Isolation and characterization of a cDNA clone for the complete protein coding region of the 8 subunit of the mouse acetylcholine receptor. Proc. Natl. Acad. Sci. USA 87, 7970-7974. Li, L., Zhou, J., James, G., Heller-Harrison, R., Czech, M. P., and Olson, E. N. (1992). FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 77, 1181-l 194. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1962). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press). McLeod, M. J. (1980). Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22, 299-301. Miller, J. 8. (1992). Myoblast diversity much and to what end? Cell 69, l-3.
in skeletal
myogenesis:
how
Miner, J. H., and Wold, 8. (1990). Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc. Natl. Acad. Sci. USA 87, 1089-1093. Montarras, D., Chelly, J., Bober, E., Arnols, H., Ott, M.-O., Gros, F., and Pinset, C. (1991). Developmental patterns in the expression of Myf-5, MyoD, myogenin, and MRF4 during myogenesis. New Biol. 3, 592-600. Nabeshima,Y., Hanaoka,K.,Hayasaka, M., Esumi,E., Li,S., Nonaka, I., and Nabeshima, Y. (1993). Myogenin gene disruption results in perinatal lethality because of a severe muscle defect. Nature 364, 532-535. Olson, E. N. (1990). MyoD family: a paradigm for development? Dev. 4, 1454-1461.
Genes
Ott, M.-O., Bober, E., Lyons, G., Arnold, H.-H., and Buckingham, M. (1991). Early expression of the myogenic regulatory gene, &fy+5, in precursor cells of skeletal muscle in the mouse embryo. Development 771.1097-1107. Rhodes, S. J.. and Konieczny, S. F. (1989). Identification of MRF4: a new member of the muscle regulatory factor gene family, Genes Dev. 3, 2050-2061. Rudnicki, M. A., Braun, T., Hinuma, S., and Jaenisch, R. (1992). inactiOf My00 in mice leads to up-regulation of the myogenic HLH gene r%ffi-5 and results in apparently normal muscle development. Cell 77, 383-390.
vation
Sassoon, D., Lyons, G.. Wright, W. E., Lin, V., Lassar, A., Weintraub, H., and Buckingham, M. (1989). Expression of two myogenic regutatory factors, myogenin and myoD1, during mouse embryogenesis. Nature 344, 303-307. Stead, R. H., Dixon, M. F., Bramwell, N. H., Riddell, R. H., and Bienensto nerves in the human tack, J. (1989). Mast cells are closelyapposed gaStrOinteSthal mucosa. Gastroenterology 97, 575-585. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, B., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., and Lasser, A. (1991). The myoD gene family: nodal point during specification of the mUSCle cell lineage. Science 251, 761-766. Wright. W. E., Sassoon, D. A., and Lin, V. K. (1989). Myooenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56, 607-617.
December
31, 1993, Copyright
0 1993 by Cell Press
MyoD or Myf-5 Is Required for the Formation of Skeletal Muscle Michael A. Rudnicki, * Patrick N. J. Schnegelsberg,t Ronald H. Stead,* Thomas Braun,§ Hans-Henning Arnold,5 and Rudolf JaenischS *Institute for Molecular Biology and Biotechnology McMaster University Hamilton, Ontario Canada L8S 4Kl fwhitehead Institute and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02142 *Department of Pathology McMaster University Hamilton, Ontario Canada L8S 4Kl §lnstitut fur Biochemie und Biotechnologie Abteilung Zell und Molekularbiologie 38106 Braunschweig Federal Republic of Germany
Summary Mice carrying null mutations in the myogenic regulatory factors Myf-5 or YyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and Sl nuclease analyses indicated that Myf-5(-I-);MyoD(-I-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. lmmunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. Theseobservations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis. Introduction The myogenic basic-helix-loop-helix(bHLH)familyof transcription factors is believed to play an important regulatory role in the development of skeletal muscle. This group includes MyoD (reviewed by Weintraub et al., 1991) myogenin (Braun et al., 1989a; Edmondson and Olson, 1989; Wrightetal., 1989) Myfd(Braunetal., 1989b), and MRF4, also called Myf-6 or herculin (Rhodes and Konieczny, 1989; Braun et al., 1990; Miner and Wold, 1990). The myogenie bHLH genes are exclusively expressed in skeletal muscle, and forcing their expression in a wide range of cultured cells induces the skeletal muscle differentiation program. Thus, these transcription factors have been postulated to play a master regulatory role in determining the
identity of the skeletal muscle lineage (Olson, 1990; Weintraub et al., 1991; Buckingham, 1992). Vertebrate skeletal muscle is derived from cells in the somites and prechordal mesoderm that give rise to myoblasts that form the skeletal muscle of the head, trunk, and limbs (reviewed by Buckingham, 1992; Miller, 1992). In the mouse, primary myofibers develop first at 8.5 days of gestation, followed by secondary myofibers that start to develop around day 14. These two fiber types express distinctive isoforms of muscle proteins and have been postulated to be formed from distinct lineages of myoblasts (Miller, 1992). Cultured myoblast cell lines express Myf-5 mRNA, MyoD mRNA, or both before and after differentiation, whereas myogenin mRNA is expressed upon myotube fusion, and MRM mRNA several days after fusion (Braun et al., 1989b; Wright et al., 1989; Edmondson and Olson, 1989; Miner and Wold, 1990; Montarras et al., 1991). In mice, the myogenic bHLH genes are activated sequentially during the skeletal muscle developmental program. Mfl-5 mRNA is first detected in the 8 day somite and is markedly reduced after day 14 (Ott et al., 1991). myogenin mRNA appears on day 8.5 and is expressed throughout fetal development (Sassoon et al., 1989). MRF4 mRNA appears transientlyon days 10 and 11, and is re-expressed at day 16 to become the most abundant myogenic factor after birth (Bober et al., 1991). Finally, My00 mRNA appears around day 10.5 and is expressed thereafter throughout development (Sassoon et al., 1989). In the developing limb bud, a different pattern of expression is observed: Myf-5 mRNA is expressed transiently between day 10 and 12, myogenin and My00 mRNAs are coexpressed after day 11 (Ott, 1991; Sassoon et al., 1989), and MRM mRNA is expressed after day 16 (Bober et al., 1991). This pattern of activation of the four myogenic bHLH genes in vitro and in vivo suggests that they may have distinct functions in the developmental activation of the muscle differentiation program. Previously, we demonstrated that mice lacking a functional My00 gene are viable and fertile and exhibit no morphological or physiological abnormalities in skeletal muscle (Rudnicki et al., 1992). Mutant My00 mice did, however, exhibit a 3.5-fold increase in the amount of Myf-5 mRNA. Similarly, newborn mice lacking a functional My&5 gene display no obvious defects in skeletal muscle, but die perinatally, owing to severe rib abnormalities (Braun et al., 1992). The sustained induction of Myf-5 mRNA levels in mice lacking MyoD and the delay in the onset of musclespecific gene expression in mice lacking Myf-5 (Braun et al., 1992) raised the possibility that Myfd and MyoD can functionally substitute for one another in muscle development. To investigate further the role of Myf-5 and MyoD in myogenesis, we have generated mice lacking both myogenie factors by crossing Myf-5 and My00 mutant mice. Strikingly, mice lacking both Myfd and MyoD are completely devoid of skeletal myofibers and presumably de-
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Table 1. Offspring My&5 (+/-);MyoD(+/-)
of Mfi-5 and MyoD Mutant Mice Myki(+/-);MyoD(+/-)
x Myf-5 (+I-);MyoD(+/-)
x Mfl-S(+/+);MyoD(-/-)
Frequency Observed
Genotype Myf-5
MyoD
Predicted
+I+ +/+ +I+I-- l -- l -l+I-+I-+
+I+ +I+I+ -+I+I+ +I-- I -I-I-
6.25 12.5 12.5 25.0 6.25 12.5 6.25 12.5 6.25
Offspring of Myf-5 and MyoD mutant *The number of mice analyzed was b The number of mice analyzed was c The number of mice analyzed was
(o/o)
(%)
Frequency
Genotype
Newborn”
3 Weeksb
Myfd
MyoD
Predicted
4 12 9 19 10 6 7 16 15
17 24 20 36 0 0 0 0 3
1 2 3 4
+I+I-I-I-
25 25 25 25
mice were genotyped 137. 143. 139, at 3 weeks.
+I+ +Iii+I+
after weaning (3 weeks) or after cesarean
void of myoblasts. These experiments suggest that either Myfd or MyoD is required for the determination of skeletal myoblasts, their propagation, or both and indicate that Myf-5 and MyoD are, at least in part, functionally redundant in myogenesis. Results Interbreeding of Myf-5 and MyoD Mutant Mice To generate mice lacking both MyoD and Myfd, we first interbred homozygous Myoly” mice (derived from the ES line MyoD 177 and crossed into a BALBlc background [Rudnicki et al., 19921) and heterozygous MyW” mutant mice (derived from the ES line Myf-5 307 and crossed into a BALB/c background [Braun et al., 1992)). The offspring from this cross that were heterozygous for both Myf-5mi and MyoP’ mutations (i.e., MyRi(i-/-);MyoD (+/-)) were then intercrossed. As depicted in Table 1, nine different genotypes are expected to be distributed among the offspring of Myf-5(+/-);MyoD(+/-) mice, with Myf-5 (-/-);MyoD(-/-) mice having a frequency of 6.25%. The offspring of Myf-5(+/-);MyoD(+/-) mice were genotyped after weaning to assess the effect of the different genotypes on survival (Table 1). As expected, no viable Myf5(-/-J mice were found, presumably owing to the presence of truncated ribs (Braun et al., 1992). In addition, no viable Myf-5(+/F);MyoD(-/-) mice were found among the offspring of this cross, even though these mice should have normal ribs (expected frequency 12.5%; Table 1). Consistent with this result, Myf-5(+/-);MyoD(-I-) mice were also not found in viable offspring of Myf-5(+/k); MyoD(+/-) males crossed with Myf-5(+/+);MyoD(-I-) females(expectedfrequency25%; Table 1). Theseobservations support the hypothesis that MyoDdeficient mice are functionally rescued by Myf-5. Furthermore, these results suggest that postnatal survival in the absence of MyoD requires expression of two Myf-5 alleles. To determine the phenotype of My+5(-/-);MyoD(-I-) and Myf-5(+/F);MyoD(-I-) pups, offspring of Myf-5(+/-); MyoD(+/-) mice were delivered by cesarean section at
(%)
Observed
(%)
36 37 0 25
section (newborn).
term (day 16.5 of gestation). The pups were examined for abnormalities and subsequently genotyped by Southern blot analysis using restriction digests and probes described previously (Rudnicki et al., 1992; Braun et al., 1992). By means of this approach, all nine possible genotypes were observed at close to the predicted frequencies (Table 1). Newborn Myf-5(-/--);MyoD(-/-) mice, delivered by cesarean section, initially appeared alive and pink in color, but were completely immobile and quickly became cyanotic. These pups were runted and on average 75% the weight of Myf-5(-/-);MyoD(+/+) pups. Newborn Myf-5 (-/-);MyoD(-/-) mice displayed an arched spine and thin limbs through which the long bones could be discerned. Dissection of Myf-5(-/-);MyoD(-/-) pups revealed that the hearts were beating for a short time after delivery. Strikingly, we observed a complete absence of skeletal muscle in the trunk and limbs, with the spaces normally occupied by skeletal muscle appearing mostly devoid of tissue. As expected, these animals displayed the truncated rib phenotype typical of My65(-F) pups. In addition to truncated ribs, other abnormalities were observed in skeletons of Mfl-5(-/-);MyoD(-/-) mice. Insertion points where tendons make attachments to bone were absent or reduced in size (data not shown). For example, the deltoid tuberosity of the humerus was absent, and the olecranon process of the ulna and distal process of the calcaneus were greatly reduced in size. In addition, the sternum was shorter in length, and the anterior portion (manubrium) was split in Myf-5(-/-);MyoD(-I-) animals. Newborn Myf-5(+/-);MyoD(-/-) mutant mice, delivered by cesarean section, displayed the reflexive breathing movements typical of newborns. Initially, these animals breathed normally, but respiration became intermittent, and they gradually became cyanotic and eventually died. The pups appeared similar to their normal littermates, but did exhibit a slightly curved spine. Dissection revealed a normal ribcage and skeletal system. These results suggested that Myf-5(+/-);MyoD(-/-) mutant mice died owing to some deficiency in skeletal muscle function and imply
Skeletal Muscle Development 1353
Requires
MyoD or Myf-5
Probe
123456789 A
Skeletal-Actin
B
Cardiac-Actin
C
6-MHC
D
Embryonic-MHC
E
Fetal-MHC
F
MLC 183
G
Slow-MLCl
H
Troponin T
I
Fast-Troponin
J
Slow-Troponin
K
&AChR
L
PGK-I
I I
MyoD: t:t ti- tit +I- t/t +I- -I- -/- -IMyf-5: t:t t!t +‘- t/- -I- -I- -/- 4 t/t Figure 1. Skeletal Muscle-Specific ing Myf-5 and MyoD
mRNAs Are Absent in Mice Lack-
Total RNA isolated from newborn skeletal muscle was subjected to Northern blot analysis with probes specific for the following: (A), a-skeletal actin; (El), a-cardiac actin; (C), b (slow) myosin heavy chain (f3-MHC); (D), embryonic myosin heavy chain (embryonic MHC); (E), fetal MHC; (F), myosin light chain 1 and 3 (MLC 1 and 3); (G), slow myosin light chain 1 (slow MLCI); (H), troponin-T; (I), fast troponin I; (J), slow troponin I; (K), acetylcholine receptor 6 (SAChR); (L), phosphoglycerate kinase 1 (PGK-I).
that two functional copies of the Myf-5 gene are required to rescue MyoD(-/-) mice. Mice Lacking Myf-5 and MyoD Do Not Express Skeletal Muscle-Specific mRNAs To determine whether transcription of skeletal musclespecific genes was affected by null mutations in both Myf-5 and MyoD, we performed Northern blot analysis with a panel of cDNA and genomic probes using RNA isolated from animals of the different genotypes listed in Table 1. The expression of skeletal muscle-specific mRNAs appeared normal in all combinations of genotype except in Myf-5(-/-);MyoD(-/-) and Myi-5(+/-);MyoD(-/-) mice. Newborn mice lacking both Myf-5 and MyoD (Figure 1, lane 7) expressed no detectable skeletal muscle-specific mRNAs in the panel tested. This included a-cardiac and a-skeletal actins (Figures 1A and lB), embryonic, fetal, and 8 (slow) isoforms of myosin heavy chain (Figures lC, 1 D, and 1 E), myosin light chains 1 and 3 (Figure 1 F), slow myosin light chain 1 (Figure lG), troponin-T (TnT) (Figure
1 H), fast and slow troponin I (Figures 1 I and 1 J), and acetylcholine receptor 6 (GAChR) (Figure 1 K). The equal loading of RNA per lane was confirmed by hybridization with a probe for phosphoglycerate kinase 1 (PGK-1) (Figure 1 L). In addition, My+5(-/-);MyoD(-I-) mice did not express detectable ~43 mRNA (data not shown), which is normally expressed at high levels in myoblasts (Frail et al., 1989). These results indicate that virtually no skeletal muscles or myoblasts were present in My+5(-/-);MyoD(-/-) mutant mice. Although Myf-5(+/-);MyoD(-I-) mutant mice were capable of movement and respiration, they died shortly after birth (Table 1). As determined by densitometry and normalization to SK-7 mRNA levels, Northern blot analysis with skeletal muscle-specific probes indicated an overall reduction in the levels of muscle-specific mRNAs by about 60% (Figure 1, lane 8). This result suggests that in My&6 (+/-);MyoD(-/-,J mice, either the amount of skeletal muscle or the levels of muscle-specific mRNAs were reduced. In either case, this observation supports the notion that two functional alleles of Myf-5 are required for the development of a normal skeletal muscle system in the absence of MyoD (Rudnicki et al., 1992). In addition, this as well as the other genotypes exhibited no consistent alterations in the ratio between developmentally regulated isoforms of contractile protein-encoding mRNAs. For example, the ratio between a-cardiac and a-skeletal actin (Figures 1A and 1 B), between embryonic, fetal, and 6 (slow) isoforms of myosin heavy chain (Figures 1 C, 1 D, and 1 E), and between fast and slow troponin I (Figures 1 I and 1J) appeared normal. This suggests that a single copy of Myf-5 is sufficient for the normal progression of the skeletal muscle developmental program. To investigate whether expression of the myogenic transcription factors was altered in mutant mice, we employed Sl nuclease analysis to examine mRNA levels of MyoD, Myf-5, myogenin, and MRM (Figure 2). MyoD mRNA was reduced in MyoD(+/-) mice (Figure 2A, lanes 2, 4, and 6), and absent in MyoD(-/-) mice (Figure 2A, lanes 7, 8, and 9). Myf-5 mRNA was reduced in My-f-5(+/-J mice (Figure 28, lanes 3, 4, and 8), and absent in Mfl-5(-/-j mice (Figure 2B, lanes 5, 6, and 7). These mice also expressed achimericneo/Myf-5 mutant RNA most likelygenerated by transcription through the PGK-neo cassette in the Myf-5”’ allele (Figure 2B, lanes 3-8; see Braun et al., 1992). Myf-5 mRNA was elevated in MyoD(+/-) mice (Figure 2B, lane 2), and further elevated in MyoD(-/-) mice (Figure 28, lane 9) confirming our previous observations (Braun et al., 1992; Rudnicki et al., 1992). myogenin and MRF4 mRNA were expressed at varying but approximately normal levels in all but one genotype: Myf-5(-/-j; MyoD(-/-) mutant mice expressed no detectable myogenin or MRF4 mRNA (Figures 2C and 2D, lane 7). Myf5(+/-);MyoD(-/-) mutant mice appeared to express lower levels of myogenin and MRF4 mRNA than the other genotypes (Figures 2C and 2D, lane 8), but more quantitative studies are needed to assess a possible feedback regulation between the different members of the myogenic factor family. We conclude that mice lacking both Myf-5 and MyoD contain little or no skeletal muscle.
Cell 1354
MP123456789M -
Figure bHLH MRF4 Myf-5
819 nt MyoD Probe 541 nt Protected Fragment
-
550 nt Myf-5 Probe
-
289 nt Wild Type Fragment
-
180 nt Mutant Fragment
-
550 nt Myogenin Probe 365 nt Protected Fragment
-
567 nt MRF4 Probe
-
452 nt Protected Fragment
-
894 nt PGK-1 Probe
-
442 nt Protected Fragment
2. Nuclease Sl Analysis of Myogenic Factor mRNAs Indicates rnyogenin and Are Not Expressed in Mice Lacking Both and MyoD
Total RNAisolatedfrom newborn skeletal muscle was subjected to Sl nuclease analysis with end-labeled probes specific for the following: MyoD (A), Myf-5 (B), myogenin (C), MRF4 (D), and PGK-1 (E). M, end-labeled marker 6x174Haelll; P, probe with no RNA.
M yo D: +I+ +I- +I+ +I- +I+ +I- -I- -I- -IM yf-5: +I+ +I+ +I- +I- -I- -I- -I- +I- +I+
Complete Absence of Skeletal Muscle in Mice Lacking Myf-5 and MyoD To assess further any possible alterations in skeletal muscle, we prepared Harris’ hematoxylin and eosin-stained tissue sections of mutant newborn animals (Figure 3). Stained sections were examined from the head, trunk, and limbs of newborn mice (day 18.5 of gestation) of the following genotypes: My&S(+/+);MyoD(+/-)(+/-!; Myf-5(-k); MyoDp-); Myf-5(i/-);MyoD(-/-); and Myf-5(-/F);MyoD (4-J Remarkably, mice lacking both Myf-5 and MyoD were completely devoid of skeletal muscle. The spaces normally occupied by skeletal muscle contained either amorphous loose connective tissues or expanded areas of adipose tissue (Figures 3A and 3E). Other organs, for example, the heart, lungs, liver, and bowel, appeared completely normal (data not shown). In newborn MyRi(+/-);MyoD(-/-) mice, the number of fibers was substantially reduced in all muscle groups (Figure 3 and Table 2). However, these fibers appeared normal in that they contained both peripherally and centrally lo-
Table 2. The Number of Muscle Fibers Is Dependent
cated nuclei and were of the normal size. Enumeration of the number of fibers in several muscle groups (e.g., diaphragm, intercostal, and biceps and triceps brachii; see Table 2) indicated a 350/b-55% reduction in the number of fibers. This decrease in muscle mass was consistent with the reduced expression of skeletal muscle-specific mRNAs observed by Northern blot and Sl nuclease analyses (see Figures 1 and 2). In contrast, My+5(-/-);MyoD (+/-) mice displayed no obvious morphological abnormalities in size, grouping, or form of muscles (Figures 3C and 3G). The developing fibers contained both peripherally and centrally located nuclei and were distributed normally with abundant mononucleated cells between the fibers. Therefore, one allele of MyoDappears capable of rescuing skeletal myogenesis in mice lacking Myfd, whereas one allele of Myf-5 can only partially rescue skeletal myogenesis in mice lacking MyoD. To determine whether a small number of skeletal muscle fibers might persist in Myf-5(-/-);MyoD(-I-) mutant mice that would not be readily apparent by light microscopic
on Genotype
Genotype
Diaphragm
Intercostal
Biceps Brachii
Triceps Brachii
MyRi(-/-);MyOD(-I-) My+5(+/-);Myoq-I-) Myf-q-/-);Myoq+/-) MyM(+/+),Myoq+/-)
0 4.0 f 8.0 f 0.0 f
0 2.4 f 0.2 6.8 f 0.6 6.6 f 0.2
0 16.6 f 30.0 f 29.4 f
0 11.0 -c 1.2 17.6 IT 3.6 20.0 f 7.2
1.0 0.4 1.4
2.2 4.4 1.0
The number of skeletal muscle fibers across the largest diameter of a muscle group was counted in Harris’ hematoxylin- and eosin-stained of newborn mice. Shown above is the mean and the standard deviation of this number from several muscle groupings.
sections
Figure 3. The Number of Muscle Fibers Is Dependent
on Genotype
Hematoxylin- and eosin-stained sections through the diaphragm (A to D) and intercostal muscles of the chest (E to H) suggest that the number of myofibers is dependent on the genotype of the animal (see Table 2). A.fyf-5(-/-J%fyoo(-/-,J mice are devoid of all skeletal muscle (A and E). !4fl-5(+/-);AfyoD( mice exhibit a reduction of about 35%-55% in the number of muscle fibers (6 and F). Myf-5(-/-);MyoD(+/-) (C and G) have numbers of muscle fibers similar to viable MyM(+/+);MyoD(+/-) mice (D and H). In (A) through (D), the lung and liver are located above and below the diaphragm (arrow), respectively. Note the expanded area of adipose tissue (arrow) in Mfl-5(-/-);MyoD(-/-) mice in (E). Representative intercostal muscles are indicated with arrows in (F). (G). and (H).
Cell 1356
Figure 4. Mice Lacking Both Myf-5 and MyoD Lack Skeletal Myocytes
and Myoblasts
lmmunohistochemical detection of skeletal muscle with a mouse monoclonal antibody reactive with smooth and striated muscle a-actin (A and B) and skeletal myoblasts with a mouse monoclonal antibody reactive with desmin (C and D). In My+5(+/-);MyoD(+/-) mice (A and C), anti-actin antibodies (A) stained skeletal muscle fibers (as well as smooth muscles), whereas anti-desmin antibodies (C) stained both skeletal muscle fibers (thick arrows) and myoblast-like cells (thin arrows). Sections from Myf-C(-/-);MyoD(-/-) mice (B and D) contained no detectable actin- or desmin-staining skeletal myocytes or desmin-staining skeletal myoblast-like cells. Note: the stained circular structures are smooth muscle cells in the walls of blood vessels.
Skeletal Muscle Development 1357
Requires MyoD or Myf-5
examination of hematoxylin- and eosin-stained sections, we performed immunohistochemistry on serial transverse sections through the trunk with a monoclonal antibody reactive to smooth and striated muscle a-actin (HHF35). Myf-5(+/-);MyoD(+/--J mice contained the normal amounts of skeletal muscle fibers reactive with ant a-actin antibodies (Figure 4A). However, using this procedure we did not detect any a-actin-staining skeletal myocytes in Myf-5 (-/-);MyoD(-/-j mice (Figure 48). lmmunohistochemistry was also performed with monoclonal antibodies reactive with desmin (DE-R-l l), an intermediate filament protein found in skeletal myoblasts and myofibers (George-Weinstein et al., 1993, and references therein). Whereas Myf5(+/-);MyoD(+/-) mutant mice contained extensive areas of anti-desmin-staining mononucleated myoblasts and skeletal myofibers (Figure 4C), Mfl-5(-I-);MyoD( mutant mice contained neither(Figure4D). These resultsconfirm that mice lacking MyoD and Myfd are indeed devoid of skeletal myocytes and myoblasts. Discussion In this study, we show that newborn mice deficient for the two myogenic bHLH factors Myfd and MyoD are totally devoid of all skeletal muscle. Mutant pups were born alive, but were completely immobile and died within minutes. Northern blot and Sl nuclease analyses demonstrated the absence of markers for skeletal muscle (a-actins, b-myosin heavy chain [MHC], embryonic MHC, fetal MHC, MLCl and MLC3, slow MLC, fast Tnl, slow Tnl, myogenin, MRF4, TnT, and GAChR). Histological examination revealed that these animals lacked all skeletal muscle, and the spaces normally occupied by muscle were filled with amorphous loose connective and adipose tissues. Immunohistochemistry indicated that desmin-containing myoblast cells were not detectable in Myf-5(-/F); MyoD(-/-) mice. Furthermore, except for truncated ribs and the lack of muscle tendon insertion points, the skeleton appeared normal. Our results have important implications for the understanding of the molecular control of skeletal muscle development by the myogenic bHLH factors. In previous studies we characterized myogenesis in mice carrying null mutations in either Myf-5 or MyoD genes. In animals lacking Myf-5, skeletal muscle development is delayed possibly until MyoD is expressed, resulting in apparently normal skeletal muscle at birth (Braun et al., 1992). Similarly, mice lacking MyoD are viable and have seemingly normal skeletal muscle. Importantly, Myf-5, which is normally down-regulated after day 14 of gestation, is expressed at 3.5-fold higher levels in newborn mutant My00 animals (Rudnicki et al., 1992; see Figure 2). These observations led to the hypothesis that Myf5 and MyoD have largely functionally redundant roles in myogenesis (Rudnicki et al., 1992). The complete absence of skeletal muscle in animals lacking both Myfd and MyoD strongly supports this hypothesis. Mice lacking Myf-5 die soon after birth not owing to deficient muscle function but rather as a consequence of a severe rib defect that prevents normal respiration (Braun
et al., 1992). The presence of one functional Myf-5 gene in homozygous MyoD mutant mice (My+5(+/-);MyoD (-/-)) results in the formation of normal ribs as well as skeletal muscle. Nevertheless, Myf-5(+/F);MyoD(-/-) were not viable and died soon after birth. Close inspection of this class of mutants revealed a reduced but normal expression pattern of developmentally regulated isoforms of muscle-specific mRNAs and demonstrated significantly reduced numbers of muscle fibers. This suggested that perinatal death was a consequence of an impairment in muscle function caused by a reduction in muscle mass. In contrast, one functional My00 gene in homozygous Myf-5 mutant mice (My+5(-/-);MyoD(+/-)) resulted in apparently normal skeletal muscle development. One interpretation of these results is that expression of these myogenic factors is important in determining the number of premyoblast cells in the somite that are recruited into the skeletal muscle lineage. Furthermore, these observations, while confirming that Myf-5 can functionally substitute for MyoD in myogenesis, suggest that either MyoD is inherently more active than Myf-5 in this role or that MyoD is present at higher levels in somite stage embryos. Alternatively, Myfd and MyoD may act to recruit different populations of cells into the myoblast lineage. Examination of myogenic factor expression during early somitogenesis will resolve this question. It is of interest to compare the roles played by the myogenie bHLH transcription factors in skeletal myogenesis as revealed by the phenotypes of mice carrying targeted mutations. Mice lacking a functional myogenin gene are immobile and die perinatally, owing to deficits in skeletal muscle differentiation (Hasty et al., 1993; Nabeshima et al., 1993). In contrast with Myf-5(-/-);MyoD(-/-) pups, myogenin mutant mice appear to form normal numbers of myoblasts, which are organized in groups similar to normal skeletal muscle, and express SAChR and TnT mRNA (Hasty et al., 1993). Myogenin-deficient myoblasts, however, are unable to undergo efficient fusion to form functional muscle fibers in vivo. This result suggests that myogenin plays an essential role in the differentiation of myoblasts into myotubes (Hasty et al., 1993; Nabeshima et al., 1993). The apparent lack of myoblasts in mice lacking both Myfd and MyoD, therefore, places these two fac-
Figure 5. Roles of Myogenic genesis
bHLH
Factors
During
Skeletal
Myo-
Targeted inactivation of myogenic bHLH genes suggests a model for the regulation of the skeletal muscle developmental program. The presence of either Myf-5 or MyoD is required for an essential role in the determination of myoblasts early in the develomsntal program. After myoblasts have been determined, myogenin has an essential role in the differentiation of myoblasts into multinucleated myotubes (Hasty et al., 1993; Nabeshima et al., 1993). MRF4 may act late in the developmental program, as suggested by its expression pattern (Sober et al., 1991).
Cell 1358
tom upstream of myogenin and suggests that they are involved in the determination of myoblasts, their propagation, or both (Figure 5). The fourth myogenic bHLH factor, MRF4, may function late in the myogenic pathway, as has been implied by its expression pattern in vivo and in cultured cells (Bober et al., 1991). We have previously suggested two alternative hypotheses to explain the defective rib cage formation in mice lacking Myf-5 (Braun et al., 1992). First, Myfd may act autonomously in some sclerotomal cells, although this seems unlikely, because MyfQ expression has not been detected in cells other than those in the dermamyotome of the somite (Ott et al., 1991). Second, dermamyotomederived myocytes may provide a permissive environment for sustaining continued proliferation of the rib rudiments. The presence of only relatively minor rib defects in myogenin-deficient mice suggests that myoblasts provide at least part of this signal. One possibility was that other skeletal elements were unaffected because the subsequent generation of skeletal muscle differentiation under the control of MyoD might provide these signals. Clearly, this study indicates that the formation of the rest of the skeletal system occurs independently of skeletal myogenesis. The generation of a normal rib cage, therefore, is unique, as it appears to require early skeletal myogenesis in the dermamyotomal compartment of the somite. MyoD and Myfd have been suggested to be functionally inactive in myoblasts for a variety of reasons, including the presence of Id proteins and phosphorylation of the DNA-binding domain (Benezra et al., 1990; Li et al., 1992). Yet, our data indicates a requirement for the expression of either Myfd or MyoD for the determination of skeletal myoblasts, their propagation, or both. Therefore, Myfd and MyoD must have a functional activity in myoblasts distinct from the transactivation of genes expressed after myotube fusion. This activity may function at the level either of myoblast commitment or of maintenance of myoblast propagation. Clearly, to understand how Myfd and MyoD specify the identity of myoblasts, it will be important to identify the target genes of these factors in myoblasts. Experimental
Procedures
Histological Analysis Cartilage and bone were stained using the method of McLeod (1980). Preparation, fixation, sectioning, and staining of tissue samples for light microscopy of histological preparations were performed using standard techniques (Bancroft and Stevens, 1990). Briefly, term embryos were delivered by cesarean section on day 18.5 of gestation, photographed, fixed in 4% paraformaldehyde in phosphate-buffered saline, dehydrated in steps to 70% ethanol, and stained with Harris’ hematoxylin and eosin. lmmunohistochemistry, as described elsewhere (Stead et al., 1989) was performed on paraformaldehyde-fixed sections with mouse monoclonal antibody HHF35 reactive with muscle a-actin (Enzo Diagnostics) and with a mouse monoclonal antibody (DE-R-l 1) reactive with desmin (Dakopatts). RNA Analysis RNA was isolated using the method of Auffray and Rougeon (1980) as described previously (Rudnicki et al., 1992; Braun et al., 1992). Northern blot analysis was carried out using standard techniques (Maniatis et al., 1982). The fast and slow troponin I (Koppe et al., 1989) were full-length rat cDNAs. The SK-7 probe (Adra et al., 1988) and the acetyl choline receptor 8 probe (LaPolla et al., 1984) were full-length
mouse cDNAs. The other probes used in the Northern blot analysis have been described previously (Rudnicki et al., 1992; Braun et al., 1992). Nuclease Si analysis was performed as previously described (Rudnicki et al., 1992) and was used to quantitate the myogenic bHLH and fgk-7 mRNAs. MyoD mRNA was detected with an 819 nt PvullStul probe that included vector sequences and the 5’ end of the fulllength mouse cDNA and was 5’ end-labeled. Myf-5 mRNA was detected with a 5’ end-labeled 550 nt Pvull-Hincll probe that included vector sequences and the Vend of the mouse cDNA. myogenin mRNA was detected with a 3’end-labeled 550 nt Smal probe containing exon 1 of the myogenin gene. MRF4 mRNA was detected with a 5’ endlabeled 567 nt Pvull-Accl probe that included vector sequences and the 5’end of the full-length mouse cDNA. Pgk-7 mRNA was detected with a 3’end-labeled 894 nt Xbal-Sphl probe that included the 3’end of the mouse cDNA and vector sequences. Signals from Northern blot and Si analyses were quantitated using an Apple Scanner and Image 1.4 software. Acknowledgments Address correspondence to M. A. R. We thank John Hassell and William Muller for critical comments on this manuscript, Ken Hastings for providing slow and fast Tnl probes, Steven Burden for providing the SAChR probe, and Chuyan Ying and Beth Colley for expert technical assistance. This work was supported by a grant from the Medical Research Council of Canada and by Public Health Service grant 5R35 CA44339 from the National Institutes of Health. M. A. R. is a Research Scientist of the National Cancer Institute of Canada. P. N. J. S. is supported by the Markey Biomedical Scientist Training Program. Received August 19, 1993; revised October 20, 1993. References Adra, C. N., Ellis, N. A., and McBurney, M. W. (1988). The family of mouse phosphoglycerate kinase gene and pseudogenes. Somat. Cell Mol. Genet. 74, 69-81. A&ray, C., and Rougeon, F. (1980). Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 707, 303-314. Bancroft, J. D., and Stevens, A. (1990). Theory and Practice of Histological Techniques (Edinburgh: Churchill Livingston). Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990). The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 67, 49-59. Bober, E., Lyons, G. E., Braun, T., Cossu, G., Buckingham, M., and Arnold, H.-H. (1991). The muscle regulatory gene Myf-6 has a biphasic pattern of expression during early mouse development. J. Cell Biol. 773, 1255-1265. Braun, T. Grzeschik, of myogenic tion by the
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