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Developmental genetics of a lethal mutation, muscular dysgenesis (mdg), in the mouse
DEVELOPMENTAL
BIOLOGY
Developmental Muscular I. Genetic
11,
8%-92
(
1965)
Genetics
of a Lethal
Dysgenesis
(mdg),
Analysis
and Gross
Mutation,
in the Mouse Morphology’
ANNA C.PAI~ Department
of Genetics,
New Accepted
Albert Einstein College York, New York November
of Medicine,
4, 1964
INTRODUCTION
A spontaneous lethal mutation which produces a wide syndrome of abnormalities was recently discovered in our mouse colony. The most striking anomaly is a deficiency of all skeletal musculature in mutant newborn ( Gluecksohn-Waelsch, 1963). A study of the effects of this recessive mutation, designated muscular dysgenesis (mdg), shows that the hereditary myopathy of mdg homozygotes differs from known muscular dystrophies in man and animals. Further analysis of the mutational effects is expected to contribute to knowledge of developmental mechanisms and interactions occurring during normal myogenesis. This paper will present results of genetic studies of the mutation and will describe the gross morphology of the homozygous mutant newborn. The developmental analysis of this mutation will be presented separately ( Pai, 1965). GENETIC
ANALYSIS
The mutation muscular dysgenesis (mdg) arose in an inbred stock of tailless mice (T/t”). Matings of a tailless, otherwise normal male ’ Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine of Yeshiva University. *This investigation was supported by Training Grants 2M6418 and lTl-GMllOA, and Research Grant HD-00193-39 from the National Institutes of Health, United States Public Health Service.
82
GENETIC
FAILURE
OF
MYOBLAST
83
DIFFERENTIATION
with two tailless, otherwise normal sisters produced 20 tailless offspring, of which 13 were normal, two dead with peculiar abnormalities, and five partially eaten. When outcrossed to a normal Swiss female, the same male sired 7 offspring, 4 of which were normal-tailed and 3 short-tailed but otherwise normal; progeny tests proved 4 of the F, to carry a new mutant gene. Two such F, males, mated with 2 carrier sisters, produced 77 offspring, 10 of which were dead and showed a typical syndrome of abnormalities. The same 2 males, mated to 3 carrier daughters, produced 35 offspring, 9 of which were abnormal and dead. Altogether 44 males heterozygous for the mdg gene were mated with female heterozygotes; the results are summarized in Table 1. TABLE ~EIVBORN
OFFSPRING
Number of litters
Observed Expected
533
FROM
1 +/mdg
Number normal
2900 2837.2.5
X +/mdg
MATINGS
Number abnormal
Number eaten
709 945.75
IGS
Total
3783 3783
The data indicate segregation of mdg as a single autosomal recessive gene lethal in homozygous condition. The apparent deficiency in number of abnormal mice at birth disappears if the partially eaten animals are included as homozygotes. Justification for this comes from control matings where only one of 363 normal newborn in 38 litters was partially eaten. To investigate the mode of transmission of the mdg gene further, litters from +/mdg x +/mdg matings were dissected at different stages of gestation between 14% and 18% days, as summarized in Table 2. TABLE OFFSPRING
Values
Observed Expected
OBWINELI
2
FROM I~SSECTION OF LITTERS +,fmdg MATINGS Number mdg/mdg
104 101
FROM
Number resorbed
36
+/mdg
X
Total
440 404
84
ANNA
C. PA1
Since control litters showed the same percentage of resorbed sites as the experimental litters in Table 2, it is assumed that no preferential resorption of mdg/mdg fetuses occurs; the resorbed fetuses in Table 2 were therefore not included in the calculations of expected numbers. The results obtained in the different groups of fetuses closely correspond to the expected proportions of 3 normal to 1 abnormal. Segregation of the mdg gene was studied by testing normal offspring from +/mdg X +/mdg crosses in matings with known heterozygotes. Table 3 summarizes the results. TABLE 3 PROGENY TESTS OF ~VORMAL OFFSPRING FROM i-/r& Number +/+
Values Observrd Expeclcd
X -tlmdg
154 149.5
MUXWX Total
07 71.5
221 221
The numbers obtained are in close agreement with the proportions of 2/3heterozygotes and $5 normal homozygotes expected on the basis of segregation of a single autosomal recessive gene. Linkage tests of mdg with the T locus showed the new gene to segregate independently from the T locus on chromosome IX. MORPHOLOGY
Material
OF mdg HOMOZYGOUS
NEWBORN
and Methods
Altogether 264 newborn homozygotes were autopsied; 41 of these were used for detailed studies of bone and cartilage abnormalities, with normal littermates as controls. Twenty-four skeletons of mutant newborn were prepared for studies of bone with a modification of Dawson’s Alizarin Red S method. After dissection the animals were skinned and eviscerated, then fixed in 95% alcohol for 2 days and transferred to 1% KOH until the bones were clearly visible. They were placed in a solution of 0.01% Alizarin Red S in 1% KOH overnight, then in solutions of 2 parts KOH to one part glycerin, 1 part KOH to 1 part glycerin, and 1 part KOH to 2 parts glycerin for at least 1 day each, and stored in 100% glycerin. For studies of cartilage newborn mice were fixed in 10% formalin,
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
85
and stained with toluidine blue according to the method described by Sisken and Gluecksohn-Waelsch ( 1959). External Appearance of Newborn Homozygotes can be recognized immediately by their gross abnormalities and are usually found dead, The mutant newborn appears shorter and broader than its normal sib ( Fig. 1) . The skin is loose and smooth, and seems to lack normal adherence to underlying tissues. Head abnormalities include micrognathia in all mutants, and cleft palate in most. The heads of the mutants cannot be tilted back easily as is possible in the normal newborn. The limbs of mdg homozygotes seem to be short and are held in a typical clasping position. The extremities appear edematous. Internal
IllorplzoZogy
When the skin of mdg homozygotes, easily separable from underlying tissues, is removed, the animals as well as the extremities are found to be of normal length (Fig. 2). A general and severe deficiency of skeletal musculature is the most striking characteristic of homozygotes. The skeleton of thorax and limbs appears almost bare of muscle (Figs. 35), and whatever tissue is present is soft and abnormal. Most of the bones are easily separated during dissection. The tongue is smaller and softer than normal, and the diaphragm so thin that it is completely transparent. The severe degeneration of voluntary muscles suggested that mutants are incapable of movements, including those of respiration, and that they might have been alive at birth. That death occurs not long before birth is indicated by the normal size of the newborn and the absence of necrosis. Upon dissection the viscera are found to be well developed and in good condition, and the blood is often fresh iti color and of normal viscosity. Several homozygous newborn dissected immediately after birth showed strong and regular heartbeat, although no motor response was produced during the dissection. Death of mdg homozygotes therefore is assumed to occur perinatally due probably to poorly developed musculature of the thorax and diaphragm and inabihty to breathe, with resulting asphyxiation. Not a single homozygous newborn was found in which air had entered the lungs.
ANNA
C. PA1
FIG. 1. Mdg/mdg (left) and normal (right) newborn. Note short body, apx2. pendages, mandible, loose skin, and clasped limbs of mutant. Magnification: FIG. 2. Newborn skeletons. Alizarin Red S. Normal (left) and mdg/mdg (right). Deltoid tuberosity (arrow) of humerus is absent in mutant; ribs extend perpendicularly; micrognathia. Magnification: X 1.6. FIG. 3. Unfixed newborn forelimbs, with epidermis removed; normal (upper), mdg/mdg (lower). Note deficient muscle of mutant limb. Magnification: X4.4.
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
87
Dorsal adipose tissue in the region between the scapulae appears to be more extensive than in normal sibs. Examination of the viscera shows very little abnormality. In some mdg homozygotes the liver and kidneys appear slightly edematous, but usually there are no differences between mutant and normal siblings. Other abnormalities of the mdg homozygotes include the presence of a thoracic cavity wider and shorter than normal (Figs. 4 and 5); the sterna are short (Fig. 6) and ribs extend perpendicularly from the vertebral column (Fig. 2). Studies of Skeletal Preparations Consistent bone abnormalities. Studies of bone preparations of mdg homozygous newborn reveal certain consistent abnormalities. The head of the mutant is slightly shortened; however, individual skull bones are normal in shape, except for some minor abnormalities, for example, a slight enlargement of the interparietal and occipital bones. The mandible is most affected; it is reduced in length and has an abnormally pronounced curvature (Fig. 2). The proximal structures of the mandible, e.g., the coronoid and condyloid processes, are smaller than normal. The cervical vertebrae are abnormally broad and closely adjacent to each other (Fig. 7). P ersistence of kyphosis in the cervical region of the vertebral column, as well as the abnormality of the vertebrae themselves, probably contribute to the rigidity of the neck of the newborn. Decrease in size of the scapula is another abnormality consistently found. Also the clavicle is noticeably shorter than normal with a narrower sternal end. In addition to a reduction in size of the sternum the sternebrae are usually fused and/or duplicated (Fig. 6). The ossified portions of the ribs appear thinner than normal. The long bones of the limbs are normal in length, but thinner than usual, especially at the ends of the diaphysis. The deltoid tuberosity of the humerus is consistently absent in mdg homozygotes (Fig. 2). Consistent cartilage abnormalities. The sternum and ribs of mutants FIG. 4. Ventral view of thorax of normal newborn, unfixed. Magnification: x 5.7. FIG. 5. Ventral view of thorax of mutant newbo&. Note absence of muscles, short sternum, wide thoracic cavity. hlagnification: x Fj.7.
ANbi’A C. PA1
FIG. 6. Newborn sterna; mdg/mdg (left), normal (right). Alizarin Red S. Magnification: x 5. cervical vertebrae; ,mdg/mdg ( left), normal ( right ) . AliFIG. 7. Newborn zarin Red S. Mutant vertebrae are larger than normal. A, atlas. Magnification: x 6.7. FIG. 8. Ventral view of normal palate of mdg/mdg newborn. Alizarin Red S. PP, palatine process; ps: prespbenoid. Magnification: X 7.0. FIG. 9. Ventral view of cleft palate (C) of mdg/mdg newborn. Alizarin Red S. PS, presphenoid. Magnification: x 7.0.
show several abnormalities of their cartilaginous portions. Normally the sternochondral joints are fused; in the mutants the cartilage primordia, especially anteriorly, are smaller and not fused. The cartilagi-
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
89
nous portions of the ribs appear thinner in mutants; however, the 7th13th ribs are longer than normal. The epiphyseal cartilage at the ends of the limb bones appear to be normal, except for a slight reduction in size in the mutant newborn. Variable skeletal abnormalities. Approximately 77% of newborn mdg homozygotes studied in detail have a cleft palate (Figs. 8 and 9). In most of these the tongue remains lodged between the lateral palatine processes. Whenever the tongue is dislodged, the cleft appears narrower. Mdg homozygotes with cleft palate possess mandibles that appear to be slightly shorter than even those of mutant siblings with normal palates. Fusion of cervical and thoracic vertebrae was found in approximately 40% of mdg homozygotes studied in detail. There is no tendency for fusion of any particular cervical vertebrae; however, all fusions of thoracic vertebrae occur between the 4th and 7th vertebrae. DISCUSSION
The most striking aspect of the syndrome of abnormalities of newborn mice homozygous for the mutation “muscular dysgenesis” is the severe generalized deficiency of skeletal musculature. The specificity of effect of this single autosomal recessive gene on cells of one histotype is reflected in the apparent normality of cardiac and smooth muscle. The specificity of skeletal muscle abnormality resembles that reported in myopathies of man and experimental animals, such as in muscular dystrophy, or in degeneration of muscle as a consequence of denervation or other neuropathies (Adams et al., 1962). However, in other respects the skeletal muscle anomaly in mdg homozygotes differs strikingly from muscular dystrophies and other myopathies. Typical muscular dystrophies, for example, involve a progressive degeneration of fully differentiated muscle fibers; rarely is there a simultaneous effect on all muscles. Such a myopathy of adult skeletal muscles in the mouse, similar to muscular dystrophy in man (for review, cf. Harman et al., 1963), has been found to be transmitted as an autosomal recessive character (Michelson et al., 1955). In contrast, in the muscle deficiency of mdg homozygotes, abnormalities are apparently present in all muscle primordia prior to the mature stage; thus a progressive degeneration similar to that found in the dystrophies is excluded. In degeneration of skeletal muscle secondary to neuropathies, only
90
ANNA
C. PAI
those muscle cells are affected which normally are innervated by the missing or diseased nerves. If the profound deficiency of the entire skeletal musculature in mdg homozygotes were due to a neuropathy, a widespread abnormality of the nervous system would be expected to exist. Grossly, such an abnormality has not been detected in the newborn, although the histology of the nervous system in newborn animals and mutant embryos remains to be studied. It appears more likely at the present time that the mdg mutation exerts its effects on the muscle system not by way of a neuropathy, but directly, possibly by interfering with processes of differentiation of skeletal muscle cells. The possible consequences of muscular abnormalities for the morphogenesis of other systems are of interest. In view of the well known dependence of bone development on mechanical stress and strain ( Murray, 1936; Evans, 1957), several of the skeletal abnormalities observed in mdg homozygotes may be interpreted in terms of interaction of skeletal and muscle systems during embryogenesis. Skeletal anomalies of the mutants such as absence of the deltoid tuberosity of the humerus, decrease in size of the vertebral border of the scapula, abnormal shape of the clavicle, and decreased size of the mandible, involve structures that normally are the sites of muscle attachment; they probably are secondary effects of the absence of the respective muscles. Similar skeletal abnormalities correlated with myopathies have been observed in man. A further example of developmental interactions is demonstrated by the presence in the majority of mdg homozygous newborn, of cleft palate, with the tongue lodged in the cleft. Normal development of the palate has been shown to depend on growth of the mandible which is instrumental in lowering the tongue, thereby allowing the palatine shelves to fuse (Asling et al., 1960). Micrognathia in mdg homozygotes may have resulted in a retardation of the lowering of the tongue, thus impeding fusion of the palatine shelves in most mutants. Mandibles of mutant newborn with normal palates indeed appeared to be slightly longer than those of mutant sibs with cleft palates, possibly just long enough to have allowed normal lowering of the tongue during development. In man, an interesting syndrome of skeletal abnormalities has been reported by Banker et al. (1957) in two sibs afflicted with congenital myopathies. In a comparison of these cases with other similar ones reported in the literature, kyphoscoliosis, chest deformity, abnormality
(XNETIC
FAILURE
OF
1\fYOBLAST
I)IFFEHENTIATION
91
of head posture, and fixed position of the limbs due to arthrogryposis multiplex are listed as abnormalities common to all. In addition, one of the sibs had a high palate and small mandible, and the skin over the extremities was loose and wrinkled. The similarity of this syndrome to that found in mdg homozygotes is striking and provides further illustration of developmental interaction between skeletal and muscular systems during mammalian embryogenesis. The existence of a wide syndrome of abnormalities apparently resulting from the specific effects of a mutation on a particular cell type illustrates and underscores the complexities of gene effects in higher organisms such as mammals. The abnormalities in mdg homozygotes are no doubt the end products of a complex series of intraand extracellular gene-controlled developmental interactions. How remote the effects observed are from direct results of mutant gene action, and just exactly where the primary effect occurs, are questions that remain to be clarified by further studies; obviously, in higher organisms, mutant gene action may interfere at any point in the complex sequence of events during morphogenesis. The basic error of the mdg mutation seems to lie in a genetically controlled specific abnormality of skeletal muscle cells. The normal appearance of cardiac and smooth muscles underscores the fundamental difference between these histotypes and skeletal musculature. Further studies of the mechanisms underlying the mutant effect in mdg homozygotes may be expected to contribute to an understanding of the unique nature of the skeletal muscle cell and its differentiation. SUXfRlARY
A new lethal mutation in the mouse designated muscular dysgenesis (mdg) was found to be transmitted as a single autosomal recessive gene. The most striking effect in newborn mice homozygous for the mdg mutation is a severe, general deficiency of skeletal musculature; cardiac and smooth muscles appear normal. The deficiency appears to be the result of a genetically determined abnormality of muscle cell differentiation. Various other abnormalities of the mdg syndrome are described which appear to be the result of skeletal muscle deficiency. The potential value of this mutation for studies of muscle cell differ-
92
ANNA
C. PA1
entiation and for the investigation of the interaction between skeletal musculature and other systems during development is discussed. The author would like to express her deep appreciation to Dr. Salome Gluecksohn-Waelsch, at whose suggestion this investigation was undertaken, and whose advice and guidance during the course of this study made its completion possible. I would like to thank Dr. Ernst Scharrer, Chairman of the Department of Anatomy, for his generous support and interest and Dr. Gertrude Moser and other colleagues for their help and encouragement in this investigation. I am also very grateful to Dr. Jane M. Oppenheimer for her suggestions in the preparation of this paper. REFERENCES ADAMS, R. D., DENNY-BHOWN, D., and PEARSON, C. (1962). “Diseases of Muscle, A Study in Pathology.” Harper & Row, New York. ASLING, C. W., NELSON, M. M., DOUGHERTY, H. L., WRIGHT, H. V., and EVANS, H. M. (1960). The development of cleft palate resulting from maternal pteroylglutamic (folic) acid deficiency during the latter half of gestation in rats. Surg. Gyneco2. Obstet. 111, 19-28. BANKER, B. Q., VICTOR, M., and ADA~IS, R. D. (1957). Arthrogryposis multiplex due to congenital muscular dystrophy. Brain 80, 319-334. Illinois. EVANS, R. G. ( 1957). “Stress and Strain in Bones.” Thomas, Springfield, GLUECKSOHN-WAELSCH, S. ( 1963). Lethal genes and analysis of differentiation. Science 142, 1269-1279. HARMAN, P. J., TASSONI, J. P., CURTIS, R. L., and HOLLINSHEAD, M. B. (1963). Dystrophy in Man and Muscular dystrophy in the mouse. In “Muscular Animals” (G. H. Bourne and M. N. Golarz, eds.), pp. 408-456. Hafner, New York. MICHELSON, A., RUSSELL, E. S., and HAHMAN, P. J. (1955). Dystrophia muscularis: a hereditary primary myopathy in the house mouse. Proc. Natl. Acad. Sci. U.S. 41, 1079-1084. MURHAY, P. E. F. ( 1936). “Bones.” Cambridge Univ. Press, London and New York. PAI, A. C. ( 1965). Developmental genetics of a lethal mutation, muscular dysgenesis (mdg), in the mouse. II. Developmental analysis. Develop. Biol. 11, 93-109. SISKEN, B. F., and GLUECKSOHN-WAELSCH, S. (1959). A developmental study of the mutation “phocomelia” in the mouse. J. Exptl. Zool. 142, 623-641.
BIOLOGY
Developmental Muscular I. Genetic
11,
8%-92
(
1965)
Genetics
of a Lethal
Dysgenesis
(mdg),
Analysis
and Gross
Mutation,
in the Mouse Morphology’
ANNA C.PAI~ Department
of Genetics,
New Accepted
Albert Einstein College York, New York November
of Medicine,
4, 1964
INTRODUCTION
A spontaneous lethal mutation which produces a wide syndrome of abnormalities was recently discovered in our mouse colony. The most striking anomaly is a deficiency of all skeletal musculature in mutant newborn ( Gluecksohn-Waelsch, 1963). A study of the effects of this recessive mutation, designated muscular dysgenesis (mdg), shows that the hereditary myopathy of mdg homozygotes differs from known muscular dystrophies in man and animals. Further analysis of the mutational effects is expected to contribute to knowledge of developmental mechanisms and interactions occurring during normal myogenesis. This paper will present results of genetic studies of the mutation and will describe the gross morphology of the homozygous mutant newborn. The developmental analysis of this mutation will be presented separately ( Pai, 1965). GENETIC
ANALYSIS
The mutation muscular dysgenesis (mdg) arose in an inbred stock of tailless mice (T/t”). Matings of a tailless, otherwise normal male ’ Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine of Yeshiva University. *This investigation was supported by Training Grants 2M6418 and lTl-GMllOA, and Research Grant HD-00193-39 from the National Institutes of Health, United States Public Health Service.
82
GENETIC
FAILURE
OF
MYOBLAST
83
DIFFERENTIATION
with two tailless, otherwise normal sisters produced 20 tailless offspring, of which 13 were normal, two dead with peculiar abnormalities, and five partially eaten. When outcrossed to a normal Swiss female, the same male sired 7 offspring, 4 of which were normal-tailed and 3 short-tailed but otherwise normal; progeny tests proved 4 of the F, to carry a new mutant gene. Two such F, males, mated with 2 carrier sisters, produced 77 offspring, 10 of which were dead and showed a typical syndrome of abnormalities. The same 2 males, mated to 3 carrier daughters, produced 35 offspring, 9 of which were abnormal and dead. Altogether 44 males heterozygous for the mdg gene were mated with female heterozygotes; the results are summarized in Table 1. TABLE ~EIVBORN
OFFSPRING
Number of litters
Observed Expected
533
FROM
1 +/mdg
Number normal
2900 2837.2.5
X +/mdg
MATINGS
Number abnormal
Number eaten
709 945.75
IGS
Total
3783 3783
The data indicate segregation of mdg as a single autosomal recessive gene lethal in homozygous condition. The apparent deficiency in number of abnormal mice at birth disappears if the partially eaten animals are included as homozygotes. Justification for this comes from control matings where only one of 363 normal newborn in 38 litters was partially eaten. To investigate the mode of transmission of the mdg gene further, litters from +/mdg x +/mdg matings were dissected at different stages of gestation between 14% and 18% days, as summarized in Table 2. TABLE OFFSPRING
Values
Observed Expected
OBWINELI
2
FROM I~SSECTION OF LITTERS +,fmdg MATINGS Number mdg/mdg
104 101
FROM
Number resorbed
36
+/mdg
X
Total
440 404
84
ANNA
C. PA1
Since control litters showed the same percentage of resorbed sites as the experimental litters in Table 2, it is assumed that no preferential resorption of mdg/mdg fetuses occurs; the resorbed fetuses in Table 2 were therefore not included in the calculations of expected numbers. The results obtained in the different groups of fetuses closely correspond to the expected proportions of 3 normal to 1 abnormal. Segregation of the mdg gene was studied by testing normal offspring from +/mdg X +/mdg crosses in matings with known heterozygotes. Table 3 summarizes the results. TABLE 3 PROGENY TESTS OF ~VORMAL OFFSPRING FROM i-/r& Number +/+
Values Observrd Expeclcd
X -tlmdg
154 149.5
MUXWX Total
07 71.5
221 221
The numbers obtained are in close agreement with the proportions of 2/3heterozygotes and $5 normal homozygotes expected on the basis of segregation of a single autosomal recessive gene. Linkage tests of mdg with the T locus showed the new gene to segregate independently from the T locus on chromosome IX. MORPHOLOGY
Material
OF mdg HOMOZYGOUS
NEWBORN
and Methods
Altogether 264 newborn homozygotes were autopsied; 41 of these were used for detailed studies of bone and cartilage abnormalities, with normal littermates as controls. Twenty-four skeletons of mutant newborn were prepared for studies of bone with a modification of Dawson’s Alizarin Red S method. After dissection the animals were skinned and eviscerated, then fixed in 95% alcohol for 2 days and transferred to 1% KOH until the bones were clearly visible. They were placed in a solution of 0.01% Alizarin Red S in 1% KOH overnight, then in solutions of 2 parts KOH to one part glycerin, 1 part KOH to 1 part glycerin, and 1 part KOH to 2 parts glycerin for at least 1 day each, and stored in 100% glycerin. For studies of cartilage newborn mice were fixed in 10% formalin,
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
85
and stained with toluidine blue according to the method described by Sisken and Gluecksohn-Waelsch ( 1959). External Appearance of Newborn Homozygotes can be recognized immediately by their gross abnormalities and are usually found dead, The mutant newborn appears shorter and broader than its normal sib ( Fig. 1) . The skin is loose and smooth, and seems to lack normal adherence to underlying tissues. Head abnormalities include micrognathia in all mutants, and cleft palate in most. The heads of the mutants cannot be tilted back easily as is possible in the normal newborn. The limbs of mdg homozygotes seem to be short and are held in a typical clasping position. The extremities appear edematous. Internal
IllorplzoZogy
When the skin of mdg homozygotes, easily separable from underlying tissues, is removed, the animals as well as the extremities are found to be of normal length (Fig. 2). A general and severe deficiency of skeletal musculature is the most striking characteristic of homozygotes. The skeleton of thorax and limbs appears almost bare of muscle (Figs. 35), and whatever tissue is present is soft and abnormal. Most of the bones are easily separated during dissection. The tongue is smaller and softer than normal, and the diaphragm so thin that it is completely transparent. The severe degeneration of voluntary muscles suggested that mutants are incapable of movements, including those of respiration, and that they might have been alive at birth. That death occurs not long before birth is indicated by the normal size of the newborn and the absence of necrosis. Upon dissection the viscera are found to be well developed and in good condition, and the blood is often fresh iti color and of normal viscosity. Several homozygous newborn dissected immediately after birth showed strong and regular heartbeat, although no motor response was produced during the dissection. Death of mdg homozygotes therefore is assumed to occur perinatally due probably to poorly developed musculature of the thorax and diaphragm and inabihty to breathe, with resulting asphyxiation. Not a single homozygous newborn was found in which air had entered the lungs.
ANNA
C. PA1
FIG. 1. Mdg/mdg (left) and normal (right) newborn. Note short body, apx2. pendages, mandible, loose skin, and clasped limbs of mutant. Magnification: FIG. 2. Newborn skeletons. Alizarin Red S. Normal (left) and mdg/mdg (right). Deltoid tuberosity (arrow) of humerus is absent in mutant; ribs extend perpendicularly; micrognathia. Magnification: X 1.6. FIG. 3. Unfixed newborn forelimbs, with epidermis removed; normal (upper), mdg/mdg (lower). Note deficient muscle of mutant limb. Magnification: X4.4.
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
87
Dorsal adipose tissue in the region between the scapulae appears to be more extensive than in normal sibs. Examination of the viscera shows very little abnormality. In some mdg homozygotes the liver and kidneys appear slightly edematous, but usually there are no differences between mutant and normal siblings. Other abnormalities of the mdg homozygotes include the presence of a thoracic cavity wider and shorter than normal (Figs. 4 and 5); the sterna are short (Fig. 6) and ribs extend perpendicularly from the vertebral column (Fig. 2). Studies of Skeletal Preparations Consistent bone abnormalities. Studies of bone preparations of mdg homozygous newborn reveal certain consistent abnormalities. The head of the mutant is slightly shortened; however, individual skull bones are normal in shape, except for some minor abnormalities, for example, a slight enlargement of the interparietal and occipital bones. The mandible is most affected; it is reduced in length and has an abnormally pronounced curvature (Fig. 2). The proximal structures of the mandible, e.g., the coronoid and condyloid processes, are smaller than normal. The cervical vertebrae are abnormally broad and closely adjacent to each other (Fig. 7). P ersistence of kyphosis in the cervical region of the vertebral column, as well as the abnormality of the vertebrae themselves, probably contribute to the rigidity of the neck of the newborn. Decrease in size of the scapula is another abnormality consistently found. Also the clavicle is noticeably shorter than normal with a narrower sternal end. In addition to a reduction in size of the sternum the sternebrae are usually fused and/or duplicated (Fig. 6). The ossified portions of the ribs appear thinner than normal. The long bones of the limbs are normal in length, but thinner than usual, especially at the ends of the diaphysis. The deltoid tuberosity of the humerus is consistently absent in mdg homozygotes (Fig. 2). Consistent cartilage abnormalities. The sternum and ribs of mutants FIG. 4. Ventral view of thorax of normal newborn, unfixed. Magnification: x 5.7. FIG. 5. Ventral view of thorax of mutant newbo&. Note absence of muscles, short sternum, wide thoracic cavity. hlagnification: x Fj.7.
ANbi’A C. PA1
FIG. 6. Newborn sterna; mdg/mdg (left), normal (right). Alizarin Red S. Magnification: x 5. cervical vertebrae; ,mdg/mdg ( left), normal ( right ) . AliFIG. 7. Newborn zarin Red S. Mutant vertebrae are larger than normal. A, atlas. Magnification: x 6.7. FIG. 8. Ventral view of normal palate of mdg/mdg newborn. Alizarin Red S. PP, palatine process; ps: prespbenoid. Magnification: X 7.0. FIG. 9. Ventral view of cleft palate (C) of mdg/mdg newborn. Alizarin Red S. PS, presphenoid. Magnification: x 7.0.
show several abnormalities of their cartilaginous portions. Normally the sternochondral joints are fused; in the mutants the cartilage primordia, especially anteriorly, are smaller and not fused. The cartilagi-
GENETIC
FAILURE
OF
MYOBLAST
DIFFERENTIATION
89
nous portions of the ribs appear thinner in mutants; however, the 7th13th ribs are longer than normal. The epiphyseal cartilage at the ends of the limb bones appear to be normal, except for a slight reduction in size in the mutant newborn. Variable skeletal abnormalities. Approximately 77% of newborn mdg homozygotes studied in detail have a cleft palate (Figs. 8 and 9). In most of these the tongue remains lodged between the lateral palatine processes. Whenever the tongue is dislodged, the cleft appears narrower. Mdg homozygotes with cleft palate possess mandibles that appear to be slightly shorter than even those of mutant siblings with normal palates. Fusion of cervical and thoracic vertebrae was found in approximately 40% of mdg homozygotes studied in detail. There is no tendency for fusion of any particular cervical vertebrae; however, all fusions of thoracic vertebrae occur between the 4th and 7th vertebrae. DISCUSSION
The most striking aspect of the syndrome of abnormalities of newborn mice homozygous for the mutation “muscular dysgenesis” is the severe generalized deficiency of skeletal musculature. The specificity of effect of this single autosomal recessive gene on cells of one histotype is reflected in the apparent normality of cardiac and smooth muscle. The specificity of skeletal muscle abnormality resembles that reported in myopathies of man and experimental animals, such as in muscular dystrophy, or in degeneration of muscle as a consequence of denervation or other neuropathies (Adams et al., 1962). However, in other respects the skeletal muscle anomaly in mdg homozygotes differs strikingly from muscular dystrophies and other myopathies. Typical muscular dystrophies, for example, involve a progressive degeneration of fully differentiated muscle fibers; rarely is there a simultaneous effect on all muscles. Such a myopathy of adult skeletal muscles in the mouse, similar to muscular dystrophy in man (for review, cf. Harman et al., 1963), has been found to be transmitted as an autosomal recessive character (Michelson et al., 1955). In contrast, in the muscle deficiency of mdg homozygotes, abnormalities are apparently present in all muscle primordia prior to the mature stage; thus a progressive degeneration similar to that found in the dystrophies is excluded. In degeneration of skeletal muscle secondary to neuropathies, only
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those muscle cells are affected which normally are innervated by the missing or diseased nerves. If the profound deficiency of the entire skeletal musculature in mdg homozygotes were due to a neuropathy, a widespread abnormality of the nervous system would be expected to exist. Grossly, such an abnormality has not been detected in the newborn, although the histology of the nervous system in newborn animals and mutant embryos remains to be studied. It appears more likely at the present time that the mdg mutation exerts its effects on the muscle system not by way of a neuropathy, but directly, possibly by interfering with processes of differentiation of skeletal muscle cells. The possible consequences of muscular abnormalities for the morphogenesis of other systems are of interest. In view of the well known dependence of bone development on mechanical stress and strain ( Murray, 1936; Evans, 1957), several of the skeletal abnormalities observed in mdg homozygotes may be interpreted in terms of interaction of skeletal and muscle systems during embryogenesis. Skeletal anomalies of the mutants such as absence of the deltoid tuberosity of the humerus, decrease in size of the vertebral border of the scapula, abnormal shape of the clavicle, and decreased size of the mandible, involve structures that normally are the sites of muscle attachment; they probably are secondary effects of the absence of the respective muscles. Similar skeletal abnormalities correlated with myopathies have been observed in man. A further example of developmental interactions is demonstrated by the presence in the majority of mdg homozygous newborn, of cleft palate, with the tongue lodged in the cleft. Normal development of the palate has been shown to depend on growth of the mandible which is instrumental in lowering the tongue, thereby allowing the palatine shelves to fuse (Asling et al., 1960). Micrognathia in mdg homozygotes may have resulted in a retardation of the lowering of the tongue, thus impeding fusion of the palatine shelves in most mutants. Mandibles of mutant newborn with normal palates indeed appeared to be slightly longer than those of mutant sibs with cleft palates, possibly just long enough to have allowed normal lowering of the tongue during development. In man, an interesting syndrome of skeletal abnormalities has been reported by Banker et al. (1957) in two sibs afflicted with congenital myopathies. In a comparison of these cases with other similar ones reported in the literature, kyphoscoliosis, chest deformity, abnormality
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of head posture, and fixed position of the limbs due to arthrogryposis multiplex are listed as abnormalities common to all. In addition, one of the sibs had a high palate and small mandible, and the skin over the extremities was loose and wrinkled. The similarity of this syndrome to that found in mdg homozygotes is striking and provides further illustration of developmental interaction between skeletal and muscular systems during mammalian embryogenesis. The existence of a wide syndrome of abnormalities apparently resulting from the specific effects of a mutation on a particular cell type illustrates and underscores the complexities of gene effects in higher organisms such as mammals. The abnormalities in mdg homozygotes are no doubt the end products of a complex series of intraand extracellular gene-controlled developmental interactions. How remote the effects observed are from direct results of mutant gene action, and just exactly where the primary effect occurs, are questions that remain to be clarified by further studies; obviously, in higher organisms, mutant gene action may interfere at any point in the complex sequence of events during morphogenesis. The basic error of the mdg mutation seems to lie in a genetically controlled specific abnormality of skeletal muscle cells. The normal appearance of cardiac and smooth muscles underscores the fundamental difference between these histotypes and skeletal musculature. Further studies of the mechanisms underlying the mutant effect in mdg homozygotes may be expected to contribute to an understanding of the unique nature of the skeletal muscle cell and its differentiation. SUXfRlARY
A new lethal mutation in the mouse designated muscular dysgenesis (mdg) was found to be transmitted as a single autosomal recessive gene. The most striking effect in newborn mice homozygous for the mdg mutation is a severe, general deficiency of skeletal musculature; cardiac and smooth muscles appear normal. The deficiency appears to be the result of a genetically determined abnormality of muscle cell differentiation. Various other abnormalities of the mdg syndrome are described which appear to be the result of skeletal muscle deficiency. The potential value of this mutation for studies of muscle cell differ-
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entiation and for the investigation of the interaction between skeletal musculature and other systems during development is discussed. The author would like to express her deep appreciation to Dr. Salome Gluecksohn-Waelsch, at whose suggestion this investigation was undertaken, and whose advice and guidance during the course of this study made its completion possible. I would like to thank Dr. Ernst Scharrer, Chairman of the Department of Anatomy, for his generous support and interest and Dr. Gertrude Moser and other colleagues for their help and encouragement in this investigation. I am also very grateful to Dr. Jane M. Oppenheimer for her suggestions in the preparation of this paper. REFERENCES ADAMS, R. D., DENNY-BHOWN, D., and PEARSON, C. (1962). “Diseases of Muscle, A Study in Pathology.” Harper & Row, New York. ASLING, C. W., NELSON, M. M., DOUGHERTY, H. L., WRIGHT, H. V., and EVANS, H. M. (1960). The development of cleft palate resulting from maternal pteroylglutamic (folic) acid deficiency during the latter half of gestation in rats. Surg. Gyneco2. Obstet. 111, 19-28. BANKER, B. Q., VICTOR, M., and ADA~IS, R. D. (1957). Arthrogryposis multiplex due to congenital muscular dystrophy. Brain 80, 319-334. Illinois. EVANS, R. G. ( 1957). “Stress and Strain in Bones.” Thomas, Springfield, GLUECKSOHN-WAELSCH, S. ( 1963). Lethal genes and analysis of differentiation. Science 142, 1269-1279. HARMAN, P. J., TASSONI, J. P., CURTIS, R. L., and HOLLINSHEAD, M. B. (1963). Dystrophy in Man and Muscular dystrophy in the mouse. In “Muscular Animals” (G. H. Bourne and M. N. Golarz, eds.), pp. 408-456. Hafner, New York. MICHELSON, A., RUSSELL, E. S., and HAHMAN, P. J. (1955). Dystrophia muscularis: a hereditary primary myopathy in the house mouse. Proc. Natl. Acad. Sci. U.S. 41, 1079-1084. MURHAY, P. E. F. ( 1936). “Bones.” Cambridge Univ. Press, London and New York. PAI, A. C. ( 1965). Developmental genetics of a lethal mutation, muscular dysgenesis (mdg), in the mouse. II. Developmental analysis. Develop. Biol. 11, 93-109. SISKEN, B. F., and GLUECKSOHN-WAELSCH, S. (1959). A developmental study of the mutation “phocomelia” in the mouse. J. Exptl. Zool. 142, 623-641.