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Developmental genetics of a lethal mutation, muscular dysgenesis (mdg), in the mouse
I)E\‘ELOPXIEiS’,‘AL
BIOLOGY
11,
93-109
( 1965 )
Developmental Genetics of a Lethal Mutation, Muscular Dysgenesis (mdg), in the Mouse II. Developmental
Analysis’
ANSA C. PA? Depurtment
of Genetics,
Albert Einstein College New York, New York
Accepted
Nocember
of
Medicine,
4, 1964
INTRODUCTION
Lethal mutations interfering with normal development in mice have contributed much to our present knowledge of mammalian development (Gluecksohn-Waelsch, 1963). Mutant genes which affect a specific cell type during embryogenesis are of special interest, since the analysis of their effects may yield clues to basic mechanisms underlying cell differentiation. A severe generalized deficiency of skeletal musculature in newborn mice homozygous for the recessive lethal mutation, muscular dysgenesis (mdg) has been described (Pai, 1965). Mutants died perinatally, presumably of asphyxiation due to inability to undergo respiratory movements. A developmental study of the muscular deficiency in mdg homozygotes was undertaken in order to investigate gene-controlled abnormal processes of muscle embryogenesis, which may elucidate some of the causal mechanisms of normal myogenesis and the interaction of skeletal musculature with other developing systems. MATERIAL
AND
METHODS
Embryos from 114 litters from matings of heterozygotes (+/mdg x +/mclg) were studied between the ages of 10% and 19% days after ’ 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 .%I6418 and lTl-GMllOA, and Research Grant HD-00193-39 from the National Institutes of Health, United States Public Health Service. 93
94
ANNA
C. PAI
fertilization; 22 control litters were dissected from matings of +/mdg X +/+ or +/+ X +/+ animals. The ages of the embryos were determined by the vaginal plug method. The gross morphology of embryos at different stages was described, and the embryos were fixed and sectioned serially for histological studies. For the examination of cytological details, mutant and normal embryos were fixed in Bouin’s fixative and stained with hematoxylin and eosin. The de Castro method of silver impregnation was used for the demonstration of striations in muscle cells. Motor end plates were studied with the method for cholinesterase visualization reported by Crevier and Belanger (1955) and modified by Mumenthaler and Engel (1962). In this investigation frozen sections at 40 p of 15-19 day embryos fixed in cold 10% formol-saline were incubated in medium containing thiolacetic acid as substrate, and silver ions to trap the released H,S. Control sections were incubated in medium without substrate, or with lo-” A4 eserine salicylate as described by Barrnett ( 1962). Spinal ganglion cells and anterior horn motor neurons in normal and mutant embryos were studied in hematoxylin and eosin preparations, and also in embryos of 14?4and 18?&days, sectioned and stained with Azure blue following fixation in Carnoy’s fluid. Silver-impregnated sections were used for visualizing peripheral nerves. RESULTS
Development
of Homozygous
(mdg/mdg)
Embryos
Table 1 summarizes the results of dissections of embryos from litters of matings of mice heterozygous for the mdg gene. Results of dissections of embryos from litters of control matings are shown in Table 2. Gross Morphology
of Embryos
13?&13jL days. As the data in Table 1 show, embryos homozygous for the mdg mutation can first be recognized at 13?i days; however, a deficiency of recognizable mdg/mdg embryos at this stage indicates that the effects of the gene might not become apparent in all homozygotes until a later stage. The earliest distinguishing characteristic of the mutant fetuses is severe and generalized edema, especially noticeable dorsally, causing the epidermis to be stretched away from under-
(:ENETIC
FAILURE
OF hIYOBLAST
ixumhcr of litters
Knmber of embryos mc/g/mr/g
s 14 5 10 11
Ii 12 9 13 9 6
+,‘+
or +/m,ig
OF EMBRYOS
~‘nclnssified
Resorbed I1 IO !J 16 IY 21 T 13 3 5 2
s ‘21 4:: 20 21 20 1s G
T.iBLli: S:IMM.IRY
95
DIFFEHENTIATION
__Total 51 I22 44 X3 OS lti2 57 53 11,s 8:) 30
2
FBOM LITTERS OF +/+ +/+ ?VI.\TINGS
X +/utdg
OR +/+
X
Xumher of embryos OXl3
I,itters
flmtlg x +/-I +/+
x
+/,rtcig
+/+
x +/+
1 9
12
Resorbed
Sorm:rl
0 i
7
Total 7
‘7
66 123
14
196
59 116
--
“”
1%
lying tissues. No other gross morphological differences between mutant and normal siblings can be seen at this stage. 14% &zys. Mutant fetuses are distinguishable by a dorsal curvature, in contrast to normal siblings, whose spine has begun to straighten (Figs. 1 and 2). The head of mutants is bent forward, and the limbs appear short owing to extension of the epidermis by edema (Figs. 1 and 2). A slight delay in fusion of sternal primordia in mutants as compared with normal sibs is noticeable. 15% dqs. Mandibles of mutant embryos appear shorter than normal. In the majority of mdg homozygotes, the tongue remains lodged in the cleft of the palate, whereas the palatine shelves of normal fetuses are
96
ANNA
C. PA1
fused at this stage. Ribs of mutants extend at right angles to the vertebral column, in contrast to normal sibs whose ribs are directed more posteriorly. Edema is still severe in the mutants. No movements of mdg homozygotes can be detected; however, slight spontaneous movement of head and limbs can be seen in normal fetuses. 16% days. Mutant embryos appear broader dorsoventrally than normal. Upon dissection, developing musculature of the mutants is found to be less firm than that of normal sibs. Neither motor response to tactile and electrical stimuli, nor spontaneous movement can be observed in mdg homozygotes, in contrast to normal fetuses. 17%18% &qs. Skeletal muscles of mutant fetuses are strikingly abnormal; the tissue is soft and easily separated from the bones. Edema, however, appears to be less severe. By 18% days only the extremities of the limbs are edematous, and the skin of mutant fetuses is no longer taut, but somewhat flabby, resembling the newborn. At this stage, homozygous fetuses have all the gross characteristics of the mutant newborn ( cf. Pai, 1965 ) . Results of Histological
Studies
Histological study of serial cross sections of 17%-day fetuses confirmed the gross observation that abnormalities were present in every skeletal muscle of mdg homozygotes; cardiac musculature appeared normal as did smooth musculature. The study subsequently centered on the histogenesis of skeletal muscles. Because longitudinal sections of the supraspinatus muscle could be studied at the same level as cross sections of the occipitoscapularis muscle, these two muscles were chosen for detailed study. 12% days. No histological abnormalities were found in the muscles of a total of 26 embryos serially sectioned and stained with hematoxylin and eosin, or with the de Castro silver impregnation method. Presumptive skeletal muscle cells at this stage have rourd nuclei and are difficult to distinguish from embryonic mesenchymal cells. The muscles have not yet become discrete masses, although general outlines begin to appear. 13% days. Normal: At this stage, most skeletal muscle cells can be recognized as myoblasts. In longitudinal sections the nuclei appear slightly elongated and arranged parallel to each other. An occasional myotube is observed in cross sections. In silver preparations faint
GENETIC
FAILURE
OF MYOBLAST
DWFERENTIATION
97
striations are seen at the periphery of the myotubes, which are cylindrical multinucleated cells with central nuclei. Mutant: Muscle masses of mdg homozygotes appear to be less well organized than in the normal embryo, and there are fewer myotubes. Occasionally a cell is found with unusually acidophilic homogeneous cytoplasm in hematoxylin and eosin preparations, and homogeneous black regions in silver preparations. Most of the embryonic muscle cells, however, appear normal. 13% days. Normal: Myotubes have increased in number. Their nuclei are located centrally and are evenly distributed along the length of the cells. In silver -preparations peripheral cross striations can be seen in several cells. Mutant: Skeletal muscles of mutant embryos appear to be less well organized and compact than the normal, There are fewer myotubes and those that are present have less cross striation than in the normal. An increase in abnormal myotubes with swollen acidophilic cytoplasm in hematoxylin and eosin preparations, or homogeneous black areas in silver preparations, is apparent. In cross section abnormal cells are irregular in outline and they are filled with stained material. Most of the nuclei, however, appear normal, although some may be swollen. 14% days. Normal: Most of the muscle cells have reached the myotube stage. They are longer than in the previous stage and striations can be seen along the length of the cell (Figs. 4 and 6). Mutant: Skeletal muscles show increased abnormalities; only a few cells appear entirely normal. Swollen, homogeneously stained areas of cytoplasm, fragmentation of cells, abnormal swollen nuclei, or nuclei of irregular shape can be seen (Figs. 3 and 5a). Myotubes are reduced in number and have less striation than normal (Fig. 5). Cross sections show variations in the diameter of myotubes; some appear atrophic, others swollen. 15?i tlavs. Xormal: Normal myotubes have grown in length and width, and the amount of striation has increased. Muscle masses are compact with cells arranged parallel to each other, and there is a beginning of division into secondary groups by connective tissue. Mutunt: At this stage there appear to be fewer skeletal muscle cells than in the normal. Areas of amorphous, intensely acidophilic material resembling that found in floccular degeneration are observed, as well
98
ANNA
C. PA1
as chains and clumps of nuclei, mostly in atrophic cells. The muscle masses are generally diffuse, and there is no division into secondary groups. 16% days. Normal: Cross striations in muscle cells of normal fetuses are visible in hematoxylin and eosin preparations as well as in silverimpregnated material. All muscles show increased growth and organization (Figs. 7 and 9). Mutant: Some of the muscles in mutant fetuses, e.g., the occipitoscapularis muscle, are no longer distinct (Fig. 8). Atrophic cells, swollen homogeneous areas, increased floccular degeneration, fragmentation, chains and clumps of nuclei are characteristic of mutant muscle cells (Fig. 10). Peripheral striations in short regions of some muscle cells are observed only rarely. Although the number of cells appears to be reduced in the muscle masses, and these cells widely separated by connective tissue elements, the size of some muscles, e.g., the supraspinatus muscle, appears to be normal (Fig. 8). 17% days. Normal: Most skeletal muscle cells of normal fetuses are still in the myotube stage, although some have become fully striated, with nuclei situated closer to the periphery of the cells. Mutant: Further degeneration is evident in all skeletal muscles. At low magnification, the absence of some muscle masses, and the diffuse nature of the remaining ones, is striking. 1831days. Norm& The number of cells with complete striation and peripheral nuclei has increased. Myofibrils can be seen throughout the cells. FIG. 1. 14%day mdg/mdg embryo with severe subcutaneous edema (E, arrow). Magnification: X 5.3. FIG. 2. 14?kday normal embryo. Magnification: X5.3. FIG. 3. 14?&day mdg/mdg muscle, longit u d inal section, showing disorganization (A, left) and swollen homogeneous areas of some muscle cells (A, center). Hematoxylin and eosin. Magnification: X 265. FIG. 4. 14%day normal muscle, longitudinal section. Hematoxylin and eosin. Magnification: x 265. muscle, longitudinal section; only some myotubes FIG. 5. 14%day mdg/mdg showing limited peripheral cross striation (S). de Castro. Magnification: X 265. FIG. 5a. 14Gday mdg/mdg muscle, longitudinal section, with myotubes showing homogeneous areas of concentrated staining (A) de Castro. Magnification: x 265. FIG. 6. 14%day normal muscle, longitudinal section. Note many myotubes with peripheral cross striations. de Castro. Magnification: X265.
CESETIC
FAILURE
OF MYOBLAST
DIFFEREKTIATIOX
100
ANNA
C. PAI
Mutant: Sickle-shaped and nail-shaped pycnotic nuclei are found in some swollen, degenerating cells. Occasionally such nuclei appear to be half inside and half outside the cells as though no cell membrane existed (Fig. 11). The presence of phagocytosis is suggested for the first time by an apparent increase in the numbers of nuclei and mononuclear cells surrounding skeletal muscle cells; however, the origin of these is unclear: they may have been derived from phagocytes, or fibroblasts, or fragmented muscle cells. 19% days. Normal: All skeletal muscle cells of the normal fetuses are fully striated, organized muscle fibers with peripheral nuclei (Fig. 12).’ Mutant: Only large skeletal muscles such as the supraspinatus can still be recognized, and these are characterized by degenerating, fragmented pieces of muscle cells separated by connective tissue (Fig. 13). Nuclei are still centrally located. Tongue muscuZature. The striated muscle of the tongue differentiates later than the thoracic or limb musculature. In normal fetuses cross striation appears first at 16% days, and at term, muscle fibers are fully differentiated. Some disorganization is apparent in tongues of the mdg homozygotes at 15% days; by 16?&days, one or two cells with peripheral striations are found, but acidophilic staining and swelling of other FIG. 7. 16%day normal fetus, cross section. Note compact muscle masses. OSM, occipitoscapularis muscle; SSM, supraspinatus muscle; BF, brown fat. Hematoxylin and eosin. Magnification: X 9.6. FIG. 8. 16?4-day nrdg/mdg fetus, cross section. Note disorganized, diffuse muscle masses. Occipitoscapularis muscle is no longer distinct; brown fat appears larger and denser than normal. SP, space in edematous subcutaneous tissue; SSM: supraspinatus muscle; BF, brown fat. Hematoxylin and eosin. Magnification: x 9.6. FIG. 9. 16%day normal muscle, longitudinal section. Hematoxylin and eosin. Magnification: X 123. FIG. 10. 16?kday mdg/mdg muscle, longitudinal section. Note areas of fragmenting cells, and floccular degeneration (A). Hematoxylin and eosin. Magnification: X 123. muscle cells with pycnotic nuclei, longitudinal FIG. 11. 18%day mdg/mdg section. Note nail-shaped nucleus partly outside the cell (arrow). Hematoxylin and eosin. Magnification: X 370. section. de Castro. MagnificaFIG. 12. Newborn normal muscle, longitudinal tion: X 370. section. Fragmented deFIG. 13. Newborn mdg/mdg muscle, longitudinal generating cells; no cross striations. de Castro. Magnification: x370.
102
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C. PA1
cells is observed. Tongue musculature in the mutant newborn shows many of the abnormalities characteristic of other mutant skeletal musculature, e.g., fragmentation, chains of nuclei, floccular degeneration. Motor end plate studies. Normal and mutant embryos were studied at 15-19 days for acetylcholinesterase activity at the motor end plates in skeletal muscle. Whereas enzyme activity at the myoneural junction is present in normal muscles at all stages studied, no activity which might indicate the presence of normal end plates can be seen in mutant skeletal muscle at any stage (Figs. 14 and 15). Control sections of normal and mutant muscle incubated in medium without substrate are completely unstained; sections incubated in medium with lo-” M eserine salicylate show light background staining in tissues such as cartilage, but not the black concentration of staining at the motor end plates. Peripheral nervous system. Peripheral nerves of mdg homozygotes appear morphologically normal at the time of onset of skeletal muscle abnormalities. No differences have been observed between cranial nerves of normal and mutant embryos at 14X days when traced in serial cross sections from their origins to their terminations. Nerve endings are visible even on extremely abnormal skeletal muscle cells of 17%day mutant embryos. Some of the nerve endings appear swollen (Fig. 16), others are found on loose cells which may be connective tissue or fragmented muscle cells (Fig. 17). Central nervous system. The brain and spinal cord of mutant fetuses, studied in serial cross sections at different stages, appear to be FIG. 14. 18-day normal muscles with motor end plates (MEP) in the middle of the muscle fibers. Thiolacetic acid method for cholinesterase. Magnification: x 22.5. muscle showing the absence of staining at motor FIG. 15. 18-day mclg/m& end plates. N, nerve fibers. Thiolacetic acid method for cholinesterase. Magnification: X 110. FIG. 16. 17%day mdg/mdg muscle with swollen nerve ending (arrow). de Castro. Magnification: X 480. FIG. 17. 17%day mdg/mdg muscle with nerve endings between muscle cells (arrow). de Castro. Magnification: x 810. 17%day normal joint cavity (arrow) between humerus (H) and FIG. 18. scapula ( S ) . Hematoxyhn and eosin. Magnification: X 143. FIG. 19. 17%day mdg/mdg humerus (H) and scapula (S). Note absence of joint cavity (arrow). Hematoxylin and eosin. Magnification: x 110.
MYOBLAST
DIFFERENTIATIOK
I
104
ANNA
C. PAI
morphologically normal. There is some indication of occasional degeneration of anterior horn motor and spinal ganglia neurons at late stages in gestation; however, most of the neurons appear normal. Edema. The subcutaneous edema grossly distinguishing mdg homozygotes from normal sibs at 13% days appears to have developed very rapidly, since no signs of edema are observed grossly or histologically at 12% days. As the edema progresses, large spaces resembling expanded lymphatic vessels are seen in the subcutaneous connective tissues (Fig. 8). When traced serially, however, the spaces soon disappear in the connective tissue. At 18% days, when the edema subsides, these spaces are no longer seen. No morphological abnormalities were found in the major blood vessels of the head and neck of the mutants at 14% days. At no time during development did the viscera appear edematous and swollen. Joint cavity development. The development of joint cavities between limb and girdle primordia is abnormal in mutant fetuses. Although the process of cavitation begins in the mesenchyme between cartilage primordia in both normal and mutant embryos at 14% days of gestation, it is not completed in the mutants. At 16% days no joint cavities are found in mdg homozygotes in contrast to normal sibs (Figs. 18 and 19). Brown fat. Brown adipose tissue appears first at 15% days in the dorsal mesenchyme of normal and mutant embryos. By 16% days this tissue has become hyperplastic in mutants. The fat bodies do not show as much lobulation as the normal, and appear larger and denser (Figs. 7 and 8). DISCUSSION
The specificity of effect of the mutation muscular dysgenesis ( mdg ) , indicated by studies of newborn mutants, was confirmed by studies of muscle histogenesis in homozygous embryos: abnormalities were confined to the differentiating skeletal musculature; in contrast, cardiac and smooth muscles were normal. It could be shown that the deficiency of muscles in newborn mutant mice resulted from abnormality of differentiation and subsequent degeneration of embryonic skeletal muscle cells. Abnormalities in mutants make their appearance at a stage of histogenesis when myoblasts differentiate into myotubes with cross stria-
CENETIC
FAILURE
OF MYOBLAST
DIFFERENTIATIOS
105
Cons, regardless of the gestational age at which this occurs in the various muscles. Tongue musculature, for example, normally differentiates 3 days later than thoracic muscle, and shows the first signs of degeneration in mutant fetuses correspondingly later, indicating an abnormality of the very process of muscle differentiation in the mutants. Further support for the assumption of an inherent abnormality of muscle cell differentiation may be seen in the almost complete absence of fully differentiated skeletal muscle cells in homozygous newborn animals. Even a few exceptional cells found to be striated were not normal, and had centrally located nuclei in contrast to normal muscle fibers. The failure of any mutant embryonic skeletal muscles to develop normally, and the subsequent degeneration of the undifferentiated cells distinguish the myopathy caused by the mdg mutation from all known hereditary muscular dystrophies, which show degeneration of fully differentiated muscle fibers only (Adams et al,, 1962). The normal appearance of the embryonic nervous system at the time of onset of muscle abnormalities, supports the previous interpretation ( Pai, 1965)) which excluded a neuropathy as the cause of degeneration of skeletal musculature in mdg homozygotes. Furthermore, since the differentiation of skeletal muscles has been shown by Harrison (1904) and Hamburger (1939) to be independent of innervation, the degeneration of embryonic muscle cells in mutant homozygotes cannot be ascribed to an abnormality of innervation and appears to be due to a primary deficiency of muscle differentiation. The inability of skeletal muscle cells of mdg homozygous embryos to differentiate is further reflected in the absence of various products of normal cell differentiation. During the course of normal myogenesis, fine structures such as cross striations of myofibrils are formed (Holtzer, 1961). Also, in the middle third of each cell the cell membrane forms the subneural folds of motor end plates in which the enzyme acetylcholinesterase, believed to be a product of the muscle cell itself (Koelle, 1963; Shen, 1958), is concentrated. Abnormal differentiation of cross striations in the muscle cells of mutant fetuses was apparent even at the earliest stages. Not only was the number of cells showing any striation decreased strikingly, but in any particular cell the areas of striation appear to be reduced. The other product of muscle cell differentiation, the motor end
106
ANNA
C. PA1
plate, may be visualized normally by staining its active enzyme, acetylcholinesterase. The failure to demonstrate motor end plates in mdg homozygotes at all stages studied is most probably due to the inability of mutant myotubes to produce active enzyme. The hypothesis of a myogenic source of acetylcholinesterase at myoneural junctions therefore derives support from the absence of the enzyme in mutant muscle cells unable to form normal differentiation products. The deficiency of differentiated skeletal musculature may be expected to affect other developmental systems of the mutant. An example of this is the syndrome of skeletal anomalies described in newborn homozygous mutant mice (Pai, 1965). In addition to these, histological studies revealed a failure of development of joint cavities in mutant fetuses indicating a possible causal role of muscle function in normal joint development. Joint abnormalities have also been reported in humans (Banker et al., 1957) and in sheep ( Hadlow, 1961) afflicted with congenital myopathies. Although the development of the nervous system is known to be affected by the peripheral field (Hamburger, 1958): the methods used did not reveal any obvious degeneration of motor neurons in homozygous mutants even at birth, in spite of the general abnormality of skeletal muscles and the loss of some muscle masses. The hyperplasia of brown fat bodies in mutant animals cannot at present be related directly to the muscle degeneration. A possible interpretation might refer to the lowered energy requirements of these mutant fetuses and decreased utilization of fat stores. The etiology of the edema in md,0 mutant fetuses is unclear, although the occurrence of this anomaly simultaneously with the first appearance of abnormalities in differentiating skeletal muscles suggests a relationship between these events. The loss of edema shortly before birth may be due to the activation of water-regulating mechanisms more efficient in the elimination of excess fluids than excretion via placental circulation alone. Fetal kidneys, in rats for example, begin to function 2 days before term (McCance, 1948 ) . It is not possible to ascertain from the present study whether the processes of muscle cell differentiation themselves are defective in mdg homozygotes, or whether the death of mutant myoblasts prevents them from undergoing differentiation. It seems significant, however,
GENETIC
FAILURE
OF MYOBLAS’I‘
DIFFEHENTIATIOK
107
that no evidence of cell death can be detected before the time that differentiation products normally become apparent. Furthermore, large muscles such as the supraspinatus muscle in mutant homozygotes appear to increase in size during development, indicating that some mutant muscle cells grow, a fact not reconcilable with cell death as the basic effect of the mutation. It therefore appears more likely that the mdg mutation interferes directly with differentiation of the muscle cell, by affecting, for example, the formation of striations. This may be due to a specific inherent defect of skeletal muscle protein synthesis, in which case the mdg gene would be considered a cell lethal with autonomous effects, as discussed by Hadorn (1961) and by Gluecksohn-Waelsch (1963). On the other hand, it is possible that the mutation is responsible for the presence of an extrinsic detrimental factor to which skeletal muscle cells are particularly susceptible and which interferes with the differentiation process. In this case, the effect would be nonautonomous, and removal of cells from the detrimental environment by explantation before they have become affected might permit normal differentiation. Studies with in &TO techniques of mutant myoblasts may be able to answer this question. Further investigation of the cellular nature of the effects of the mdg mutation may be expected to contribute to knowledge of mechanisms of normal myogenesis and their genetic control. SUMhlARY
The developmental analysis of the lethal mutation, muscular dysgenesis (mdg), reveals that the myopathy results from a genetically determined specific interference with skeletal muscle cell differentiation. A description of the morphogenesis of mutants is presented, with particular emphasis on the histogenesis of skeletal muscle. Abnormalities are first apparent in embryos homozygous for the mdg mutation at the time when myoblasts differentiate into striated myotubes, regardless of the embryonic age at which this occurs in the various muscles. Subsequently, all skeletal muscle cells degenerate. Other abnormalities of the rn,dg syndrome, such as the skeletal anomalies, appear to result from the muscle deficiency, demonstrating interaction between skeletal musculature and other systems during embryogenesis.
108
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C. PAI
The developmental effects of the mdg mutation illustrate gene action on the cellular level by the specific interference with differentiation of cells of a particular histotype, the skeletal myoblasts. 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-BROWN, D., and PEARSON, C. ( 1962). “Diseases of Muscle, A Study in Pathology.” Harper & Row, New York. BANKER, B. Q., VICTOR, M., and ADAMS, R. D. ( 1957). Arthrogryposis multiplex due to congenital muscular dystrophy. Brain 80, 319-334. BAHRNETT, R. J. (1962). The fine structural localization of acetylcholinesterase at the myoneural junction. J. Cell Bid. 12, 247-262. CREVIER, M., and B~LANGER, L. F. (1955). Simple method for histochemical detection of esterase activity. Science 122, 556. GLUECKSOHN-WAELSCH, S. (1963). Lethal genes and analysis of differentiation. Science 142, 1269-1276. HADLOW, W. J. ( 1961). Genetic muscular diseases in animals. Z’roc. 2nd Intern. Congr. Human Genet., Rome, 1961, E81. Excerpta Medica Foundation, New York. HADORN, E. (1961). “Developmental Genetics and Lethal Factors.” Wiley, New York. HAMBURGER, V. ( 1939). The development and innervation of transplanted limb primordia of chick embryos. J. Exptl. 2001. SO, 347-389. HAMBURGER, V. ( 1958). Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anat. 102, 365-410. HARRISON, R. G. ( 1904). An experimental study of the relation of the nervous system to the developing musculature in the embryo of the frog. Am. .I. Anut. 3, 197-220. HOLTZER, H. ( 1961). Chondrogenesis and myogenesis. In “Synthesis of Molecular and Cellular Structure” (D. Rudnick, ed.), pp. 233-243. Ronald Press, New York. KOELLE, G. B. ( 1963). Cytological distributions and physiological functions of cholinesterase. In “Handbuch der Experimentellen Pharmakologie,” Vol. 15, pp. 189-298. MCCANCE, R. A. ( 1948). Renal function in early life. Physiol. Reo. 28, 331-348. MUMENTHALER, M., and ENGEL, W. K. ( 1961). Cytological localization of cholinesterase in developing chick embryo skeletal muscle. Actu Anut. 47, 274-299.
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FAILURE
OF MYOBLAST
DIFFERENTIATIOX
109
PAI, A. ( 1965). Developmental genetics of a lethal mutation, muscular dysgenesis (mdg) in the mouse. I. Genetic analysis and gross morphology. Develop. Biol. 11, 82-92. SHEN, S. C. (1958). Changes in enzymic patterns during development. In “The Chemical Basis of Development” ( W. D. McElroy and B. Glass, eds. ), pp. 416432. The Johns Hopkins Press, Baltimore.
BIOLOGY
11,
93-109
( 1965 )
Developmental Genetics of a Lethal Mutation, Muscular Dysgenesis (mdg), in the Mouse II. Developmental
Analysis’
ANSA C. PA? Depurtment
of Genetics,
Albert Einstein College New York, New York
Accepted
Nocember
of
Medicine,
4, 1964
INTRODUCTION
Lethal mutations interfering with normal development in mice have contributed much to our present knowledge of mammalian development (Gluecksohn-Waelsch, 1963). Mutant genes which affect a specific cell type during embryogenesis are of special interest, since the analysis of their effects may yield clues to basic mechanisms underlying cell differentiation. A severe generalized deficiency of skeletal musculature in newborn mice homozygous for the recessive lethal mutation, muscular dysgenesis (mdg) has been described (Pai, 1965). Mutants died perinatally, presumably of asphyxiation due to inability to undergo respiratory movements. A developmental study of the muscular deficiency in mdg homozygotes was undertaken in order to investigate gene-controlled abnormal processes of muscle embryogenesis, which may elucidate some of the causal mechanisms of normal myogenesis and the interaction of skeletal musculature with other developing systems. MATERIAL
AND
METHODS
Embryos from 114 litters from matings of heterozygotes (+/mdg x +/mclg) were studied between the ages of 10% and 19% days after ’ 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 .%I6418 and lTl-GMllOA, and Research Grant HD-00193-39 from the National Institutes of Health, United States Public Health Service. 93
94
ANNA
C. PAI
fertilization; 22 control litters were dissected from matings of +/mdg X +/+ or +/+ X +/+ animals. The ages of the embryos were determined by the vaginal plug method. The gross morphology of embryos at different stages was described, and the embryos were fixed and sectioned serially for histological studies. For the examination of cytological details, mutant and normal embryos were fixed in Bouin’s fixative and stained with hematoxylin and eosin. The de Castro method of silver impregnation was used for the demonstration of striations in muscle cells. Motor end plates were studied with the method for cholinesterase visualization reported by Crevier and Belanger (1955) and modified by Mumenthaler and Engel (1962). In this investigation frozen sections at 40 p of 15-19 day embryos fixed in cold 10% formol-saline were incubated in medium containing thiolacetic acid as substrate, and silver ions to trap the released H,S. Control sections were incubated in medium without substrate, or with lo-” A4 eserine salicylate as described by Barrnett ( 1962). Spinal ganglion cells and anterior horn motor neurons in normal and mutant embryos were studied in hematoxylin and eosin preparations, and also in embryos of 14?4and 18?&days, sectioned and stained with Azure blue following fixation in Carnoy’s fluid. Silver-impregnated sections were used for visualizing peripheral nerves. RESULTS
Development
of Homozygous
(mdg/mdg)
Embryos
Table 1 summarizes the results of dissections of embryos from litters of matings of mice heterozygous for the mdg gene. Results of dissections of embryos from litters of control matings are shown in Table 2. Gross Morphology
of Embryos
13?&13jL days. As the data in Table 1 show, embryos homozygous for the mdg mutation can first be recognized at 13?i days; however, a deficiency of recognizable mdg/mdg embryos at this stage indicates that the effects of the gene might not become apparent in all homozygotes until a later stage. The earliest distinguishing characteristic of the mutant fetuses is severe and generalized edema, especially noticeable dorsally, causing the epidermis to be stretched away from under-
(:ENETIC
FAILURE
OF hIYOBLAST
ixumhcr of litters
Knmber of embryos mc/g/mr/g
s 14 5 10 11
Ii 12 9 13 9 6
+,‘+
or +/m,ig
OF EMBRYOS
~‘nclnssified
Resorbed I1 IO !J 16 IY 21 T 13 3 5 2
s ‘21 4:: 20 21 20 1s G
T.iBLli: S:IMM.IRY
95
DIFFEHENTIATION
__Total 51 I22 44 X3 OS lti2 57 53 11,s 8:) 30
2
FBOM LITTERS OF +/+ +/+ ?VI.\TINGS
X +/utdg
OR +/+
X
Xumher of embryos OXl3
I,itters
flmtlg x +/-I +/+
x
+/,rtcig
+/+
x +/+
1 9
12
Resorbed
Sorm:rl
0 i
7
Total 7
‘7
66 123
14
196
59 116
--
“”
1%
lying tissues. No other gross morphological differences between mutant and normal siblings can be seen at this stage. 14% &zys. Mutant fetuses are distinguishable by a dorsal curvature, in contrast to normal siblings, whose spine has begun to straighten (Figs. 1 and 2). The head of mutants is bent forward, and the limbs appear short owing to extension of the epidermis by edema (Figs. 1 and 2). A slight delay in fusion of sternal primordia in mutants as compared with normal sibs is noticeable. 15% dqs. Mandibles of mutant embryos appear shorter than normal. In the majority of mdg homozygotes, the tongue remains lodged in the cleft of the palate, whereas the palatine shelves of normal fetuses are
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fused at this stage. Ribs of mutants extend at right angles to the vertebral column, in contrast to normal sibs whose ribs are directed more posteriorly. Edema is still severe in the mutants. No movements of mdg homozygotes can be detected; however, slight spontaneous movement of head and limbs can be seen in normal fetuses. 16% days. Mutant embryos appear broader dorsoventrally than normal. Upon dissection, developing musculature of the mutants is found to be less firm than that of normal sibs. Neither motor response to tactile and electrical stimuli, nor spontaneous movement can be observed in mdg homozygotes, in contrast to normal fetuses. 17%18% &qs. Skeletal muscles of mutant fetuses are strikingly abnormal; the tissue is soft and easily separated from the bones. Edema, however, appears to be less severe. By 18% days only the extremities of the limbs are edematous, and the skin of mutant fetuses is no longer taut, but somewhat flabby, resembling the newborn. At this stage, homozygous fetuses have all the gross characteristics of the mutant newborn ( cf. Pai, 1965 ) . Results of Histological
Studies
Histological study of serial cross sections of 17%-day fetuses confirmed the gross observation that abnormalities were present in every skeletal muscle of mdg homozygotes; cardiac musculature appeared normal as did smooth musculature. The study subsequently centered on the histogenesis of skeletal muscles. Because longitudinal sections of the supraspinatus muscle could be studied at the same level as cross sections of the occipitoscapularis muscle, these two muscles were chosen for detailed study. 12% days. No histological abnormalities were found in the muscles of a total of 26 embryos serially sectioned and stained with hematoxylin and eosin, or with the de Castro silver impregnation method. Presumptive skeletal muscle cells at this stage have rourd nuclei and are difficult to distinguish from embryonic mesenchymal cells. The muscles have not yet become discrete masses, although general outlines begin to appear. 13% days. Normal: At this stage, most skeletal muscle cells can be recognized as myoblasts. In longitudinal sections the nuclei appear slightly elongated and arranged parallel to each other. An occasional myotube is observed in cross sections. In silver preparations faint
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striations are seen at the periphery of the myotubes, which are cylindrical multinucleated cells with central nuclei. Mutant: Muscle masses of mdg homozygotes appear to be less well organized than in the normal embryo, and there are fewer myotubes. Occasionally a cell is found with unusually acidophilic homogeneous cytoplasm in hematoxylin and eosin preparations, and homogeneous black regions in silver preparations. Most of the embryonic muscle cells, however, appear normal. 13% days. Normal: Myotubes have increased in number. Their nuclei are located centrally and are evenly distributed along the length of the cells. In silver -preparations peripheral cross striations can be seen in several cells. Mutant: Skeletal muscles of mutant embryos appear to be less well organized and compact than the normal, There are fewer myotubes and those that are present have less cross striation than in the normal. An increase in abnormal myotubes with swollen acidophilic cytoplasm in hematoxylin and eosin preparations, or homogeneous black areas in silver preparations, is apparent. In cross section abnormal cells are irregular in outline and they are filled with stained material. Most of the nuclei, however, appear normal, although some may be swollen. 14% days. Normal: Most of the muscle cells have reached the myotube stage. They are longer than in the previous stage and striations can be seen along the length of the cell (Figs. 4 and 6). Mutant: Skeletal muscles show increased abnormalities; only a few cells appear entirely normal. Swollen, homogeneously stained areas of cytoplasm, fragmentation of cells, abnormal swollen nuclei, or nuclei of irregular shape can be seen (Figs. 3 and 5a). Myotubes are reduced in number and have less striation than normal (Fig. 5). Cross sections show variations in the diameter of myotubes; some appear atrophic, others swollen. 15?i tlavs. Xormal: Normal myotubes have grown in length and width, and the amount of striation has increased. Muscle masses are compact with cells arranged parallel to each other, and there is a beginning of division into secondary groups by connective tissue. Mutunt: At this stage there appear to be fewer skeletal muscle cells than in the normal. Areas of amorphous, intensely acidophilic material resembling that found in floccular degeneration are observed, as well
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as chains and clumps of nuclei, mostly in atrophic cells. The muscle masses are generally diffuse, and there is no division into secondary groups. 16% days. Normal: Cross striations in muscle cells of normal fetuses are visible in hematoxylin and eosin preparations as well as in silverimpregnated material. All muscles show increased growth and organization (Figs. 7 and 9). Mutant: Some of the muscles in mutant fetuses, e.g., the occipitoscapularis muscle, are no longer distinct (Fig. 8). Atrophic cells, swollen homogeneous areas, increased floccular degeneration, fragmentation, chains and clumps of nuclei are characteristic of mutant muscle cells (Fig. 10). Peripheral striations in short regions of some muscle cells are observed only rarely. Although the number of cells appears to be reduced in the muscle masses, and these cells widely separated by connective tissue elements, the size of some muscles, e.g., the supraspinatus muscle, appears to be normal (Fig. 8). 17% days. Normal: Most skeletal muscle cells of normal fetuses are still in the myotube stage, although some have become fully striated, with nuclei situated closer to the periphery of the cells. Mutant: Further degeneration is evident in all skeletal muscles. At low magnification, the absence of some muscle masses, and the diffuse nature of the remaining ones, is striking. 1831days. Norm& The number of cells with complete striation and peripheral nuclei has increased. Myofibrils can be seen throughout the cells. FIG. 1. 14%day mdg/mdg embryo with severe subcutaneous edema (E, arrow). Magnification: X 5.3. FIG. 2. 14?kday normal embryo. Magnification: X5.3. FIG. 3. 14?&day mdg/mdg muscle, longit u d inal section, showing disorganization (A, left) and swollen homogeneous areas of some muscle cells (A, center). Hematoxylin and eosin. Magnification: X 265. FIG. 4. 14%day normal muscle, longitudinal section. Hematoxylin and eosin. Magnification: x 265. muscle, longitudinal section; only some myotubes FIG. 5. 14%day mdg/mdg showing limited peripheral cross striation (S). de Castro. Magnification: X 265. FIG. 5a. 14Gday mdg/mdg muscle, longitudinal section, with myotubes showing homogeneous areas of concentrated staining (A) de Castro. Magnification: x 265. FIG. 6. 14%day normal muscle, longitudinal section. Note many myotubes with peripheral cross striations. de Castro. Magnification: X265.
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Mutant: Sickle-shaped and nail-shaped pycnotic nuclei are found in some swollen, degenerating cells. Occasionally such nuclei appear to be half inside and half outside the cells as though no cell membrane existed (Fig. 11). The presence of phagocytosis is suggested for the first time by an apparent increase in the numbers of nuclei and mononuclear cells surrounding skeletal muscle cells; however, the origin of these is unclear: they may have been derived from phagocytes, or fibroblasts, or fragmented muscle cells. 19% days. Normal: All skeletal muscle cells of the normal fetuses are fully striated, organized muscle fibers with peripheral nuclei (Fig. 12).’ Mutant: Only large skeletal muscles such as the supraspinatus can still be recognized, and these are characterized by degenerating, fragmented pieces of muscle cells separated by connective tissue (Fig. 13). Nuclei are still centrally located. Tongue muscuZature. The striated muscle of the tongue differentiates later than the thoracic or limb musculature. In normal fetuses cross striation appears first at 16% days, and at term, muscle fibers are fully differentiated. Some disorganization is apparent in tongues of the mdg homozygotes at 15% days; by 16?&days, one or two cells with peripheral striations are found, but acidophilic staining and swelling of other FIG. 7. 16%day normal fetus, cross section. Note compact muscle masses. OSM, occipitoscapularis muscle; SSM, supraspinatus muscle; BF, brown fat. Hematoxylin and eosin. Magnification: X 9.6. FIG. 8. 16?4-day nrdg/mdg fetus, cross section. Note disorganized, diffuse muscle masses. Occipitoscapularis muscle is no longer distinct; brown fat appears larger and denser than normal. SP, space in edematous subcutaneous tissue; SSM: supraspinatus muscle; BF, brown fat. Hematoxylin and eosin. Magnification: x 9.6. FIG. 9. 16%day normal muscle, longitudinal section. Hematoxylin and eosin. Magnification: X 123. FIG. 10. 16?kday mdg/mdg muscle, longitudinal section. Note areas of fragmenting cells, and floccular degeneration (A). Hematoxylin and eosin. Magnification: X 123. muscle cells with pycnotic nuclei, longitudinal FIG. 11. 18%day mdg/mdg section. Note nail-shaped nucleus partly outside the cell (arrow). Hematoxylin and eosin. Magnification: X 370. section. de Castro. MagnificaFIG. 12. Newborn normal muscle, longitudinal tion: X 370. section. Fragmented deFIG. 13. Newborn mdg/mdg muscle, longitudinal generating cells; no cross striations. de Castro. Magnification: x370.
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cells is observed. Tongue musculature in the mutant newborn shows many of the abnormalities characteristic of other mutant skeletal musculature, e.g., fragmentation, chains of nuclei, floccular degeneration. Motor end plate studies. Normal and mutant embryos were studied at 15-19 days for acetylcholinesterase activity at the motor end plates in skeletal muscle. Whereas enzyme activity at the myoneural junction is present in normal muscles at all stages studied, no activity which might indicate the presence of normal end plates can be seen in mutant skeletal muscle at any stage (Figs. 14 and 15). Control sections of normal and mutant muscle incubated in medium without substrate are completely unstained; sections incubated in medium with lo-” M eserine salicylate show light background staining in tissues such as cartilage, but not the black concentration of staining at the motor end plates. Peripheral nervous system. Peripheral nerves of mdg homozygotes appear morphologically normal at the time of onset of skeletal muscle abnormalities. No differences have been observed between cranial nerves of normal and mutant embryos at 14X days when traced in serial cross sections from their origins to their terminations. Nerve endings are visible even on extremely abnormal skeletal muscle cells of 17%day mutant embryos. Some of the nerve endings appear swollen (Fig. 16), others are found on loose cells which may be connective tissue or fragmented muscle cells (Fig. 17). Central nervous system. The brain and spinal cord of mutant fetuses, studied in serial cross sections at different stages, appear to be FIG. 14. 18-day normal muscles with motor end plates (MEP) in the middle of the muscle fibers. Thiolacetic acid method for cholinesterase. Magnification: x 22.5. muscle showing the absence of staining at motor FIG. 15. 18-day mclg/m& end plates. N, nerve fibers. Thiolacetic acid method for cholinesterase. Magnification: X 110. FIG. 16. 17%day mdg/mdg muscle with swollen nerve ending (arrow). de Castro. Magnification: X 480. FIG. 17. 17%day mdg/mdg muscle with nerve endings between muscle cells (arrow). de Castro. Magnification: x 810. 17%day normal joint cavity (arrow) between humerus (H) and FIG. 18. scapula ( S ) . Hematoxyhn and eosin. Magnification: X 143. FIG. 19. 17%day mdg/mdg humerus (H) and scapula (S). Note absence of joint cavity (arrow). Hematoxylin and eosin. Magnification: x 110.
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morphologically normal. There is some indication of occasional degeneration of anterior horn motor and spinal ganglia neurons at late stages in gestation; however, most of the neurons appear normal. Edema. The subcutaneous edema grossly distinguishing mdg homozygotes from normal sibs at 13% days appears to have developed very rapidly, since no signs of edema are observed grossly or histologically at 12% days. As the edema progresses, large spaces resembling expanded lymphatic vessels are seen in the subcutaneous connective tissues (Fig. 8). When traced serially, however, the spaces soon disappear in the connective tissue. At 18% days, when the edema subsides, these spaces are no longer seen. No morphological abnormalities were found in the major blood vessels of the head and neck of the mutants at 14% days. At no time during development did the viscera appear edematous and swollen. Joint cavity development. The development of joint cavities between limb and girdle primordia is abnormal in mutant fetuses. Although the process of cavitation begins in the mesenchyme between cartilage primordia in both normal and mutant embryos at 14% days of gestation, it is not completed in the mutants. At 16% days no joint cavities are found in mdg homozygotes in contrast to normal sibs (Figs. 18 and 19). Brown fat. Brown adipose tissue appears first at 15% days in the dorsal mesenchyme of normal and mutant embryos. By 16% days this tissue has become hyperplastic in mutants. The fat bodies do not show as much lobulation as the normal, and appear larger and denser (Figs. 7 and 8). DISCUSSION
The specificity of effect of the mutation muscular dysgenesis ( mdg ) , indicated by studies of newborn mutants, was confirmed by studies of muscle histogenesis in homozygous embryos: abnormalities were confined to the differentiating skeletal musculature; in contrast, cardiac and smooth muscles were normal. It could be shown that the deficiency of muscles in newborn mutant mice resulted from abnormality of differentiation and subsequent degeneration of embryonic skeletal muscle cells. Abnormalities in mutants make their appearance at a stage of histogenesis when myoblasts differentiate into myotubes with cross stria-
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Cons, regardless of the gestational age at which this occurs in the various muscles. Tongue musculature, for example, normally differentiates 3 days later than thoracic muscle, and shows the first signs of degeneration in mutant fetuses correspondingly later, indicating an abnormality of the very process of muscle differentiation in the mutants. Further support for the assumption of an inherent abnormality of muscle cell differentiation may be seen in the almost complete absence of fully differentiated skeletal muscle cells in homozygous newborn animals. Even a few exceptional cells found to be striated were not normal, and had centrally located nuclei in contrast to normal muscle fibers. The failure of any mutant embryonic skeletal muscles to develop normally, and the subsequent degeneration of the undifferentiated cells distinguish the myopathy caused by the mdg mutation from all known hereditary muscular dystrophies, which show degeneration of fully differentiated muscle fibers only (Adams et al,, 1962). The normal appearance of the embryonic nervous system at the time of onset of muscle abnormalities, supports the previous interpretation ( Pai, 1965)) which excluded a neuropathy as the cause of degeneration of skeletal musculature in mdg homozygotes. Furthermore, since the differentiation of skeletal muscles has been shown by Harrison (1904) and Hamburger (1939) to be independent of innervation, the degeneration of embryonic muscle cells in mutant homozygotes cannot be ascribed to an abnormality of innervation and appears to be due to a primary deficiency of muscle differentiation. The inability of skeletal muscle cells of mdg homozygous embryos to differentiate is further reflected in the absence of various products of normal cell differentiation. During the course of normal myogenesis, fine structures such as cross striations of myofibrils are formed (Holtzer, 1961). Also, in the middle third of each cell the cell membrane forms the subneural folds of motor end plates in which the enzyme acetylcholinesterase, believed to be a product of the muscle cell itself (Koelle, 1963; Shen, 1958), is concentrated. Abnormal differentiation of cross striations in the muscle cells of mutant fetuses was apparent even at the earliest stages. Not only was the number of cells showing any striation decreased strikingly, but in any particular cell the areas of striation appear to be reduced. The other product of muscle cell differentiation, the motor end
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plate, may be visualized normally by staining its active enzyme, acetylcholinesterase. The failure to demonstrate motor end plates in mdg homozygotes at all stages studied is most probably due to the inability of mutant myotubes to produce active enzyme. The hypothesis of a myogenic source of acetylcholinesterase at myoneural junctions therefore derives support from the absence of the enzyme in mutant muscle cells unable to form normal differentiation products. The deficiency of differentiated skeletal musculature may be expected to affect other developmental systems of the mutant. An example of this is the syndrome of skeletal anomalies described in newborn homozygous mutant mice (Pai, 1965). In addition to these, histological studies revealed a failure of development of joint cavities in mutant fetuses indicating a possible causal role of muscle function in normal joint development. Joint abnormalities have also been reported in humans (Banker et al., 1957) and in sheep ( Hadlow, 1961) afflicted with congenital myopathies. Although the development of the nervous system is known to be affected by the peripheral field (Hamburger, 1958): the methods used did not reveal any obvious degeneration of motor neurons in homozygous mutants even at birth, in spite of the general abnormality of skeletal muscles and the loss of some muscle masses. The hyperplasia of brown fat bodies in mutant animals cannot at present be related directly to the muscle degeneration. A possible interpretation might refer to the lowered energy requirements of these mutant fetuses and decreased utilization of fat stores. The etiology of the edema in md,0 mutant fetuses is unclear, although the occurrence of this anomaly simultaneously with the first appearance of abnormalities in differentiating skeletal muscles suggests a relationship between these events. The loss of edema shortly before birth may be due to the activation of water-regulating mechanisms more efficient in the elimination of excess fluids than excretion via placental circulation alone. Fetal kidneys, in rats for example, begin to function 2 days before term (McCance, 1948 ) . It is not possible to ascertain from the present study whether the processes of muscle cell differentiation themselves are defective in mdg homozygotes, or whether the death of mutant myoblasts prevents them from undergoing differentiation. It seems significant, however,
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that no evidence of cell death can be detected before the time that differentiation products normally become apparent. Furthermore, large muscles such as the supraspinatus muscle in mutant homozygotes appear to increase in size during development, indicating that some mutant muscle cells grow, a fact not reconcilable with cell death as the basic effect of the mutation. It therefore appears more likely that the mdg mutation interferes directly with differentiation of the muscle cell, by affecting, for example, the formation of striations. This may be due to a specific inherent defect of skeletal muscle protein synthesis, in which case the mdg gene would be considered a cell lethal with autonomous effects, as discussed by Hadorn (1961) and by Gluecksohn-Waelsch (1963). On the other hand, it is possible that the mutation is responsible for the presence of an extrinsic detrimental factor to which skeletal muscle cells are particularly susceptible and which interferes with the differentiation process. In this case, the effect would be nonautonomous, and removal of cells from the detrimental environment by explantation before they have become affected might permit normal differentiation. Studies with in &TO techniques of mutant myoblasts may be able to answer this question. Further investigation of the cellular nature of the effects of the mdg mutation may be expected to contribute to knowledge of mechanisms of normal myogenesis and their genetic control. SUMhlARY
The developmental analysis of the lethal mutation, muscular dysgenesis (mdg), reveals that the myopathy results from a genetically determined specific interference with skeletal muscle cell differentiation. A description of the morphogenesis of mutants is presented, with particular emphasis on the histogenesis of skeletal muscle. Abnormalities are first apparent in embryos homozygous for the mdg mutation at the time when myoblasts differentiate into striated myotubes, regardless of the embryonic age at which this occurs in the various muscles. Subsequently, all skeletal muscle cells degenerate. Other abnormalities of the rn,dg syndrome, such as the skeletal anomalies, appear to result from the muscle deficiency, demonstrating interaction between skeletal musculature and other systems during embryogenesis.
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The developmental effects of the mdg mutation illustrate gene action on the cellular level by the specific interference with differentiation of cells of a particular histotype, the skeletal myoblasts. 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-BROWN, D., and PEARSON, C. ( 1962). “Diseases of Muscle, A Study in Pathology.” Harper & Row, New York. BANKER, B. Q., VICTOR, M., and ADAMS, R. D. ( 1957). Arthrogryposis multiplex due to congenital muscular dystrophy. Brain 80, 319-334. BAHRNETT, R. J. (1962). The fine structural localization of acetylcholinesterase at the myoneural junction. J. Cell Bid. 12, 247-262. CREVIER, M., and B~LANGER, L. F. (1955). Simple method for histochemical detection of esterase activity. Science 122, 556. GLUECKSOHN-WAELSCH, S. (1963). Lethal genes and analysis of differentiation. Science 142, 1269-1276. HADLOW, W. J. ( 1961). Genetic muscular diseases in animals. Z’roc. 2nd Intern. Congr. Human Genet., Rome, 1961, E81. Excerpta Medica Foundation, New York. HADORN, E. (1961). “Developmental Genetics and Lethal Factors.” Wiley, New York. HAMBURGER, V. ( 1939). The development and innervation of transplanted limb primordia of chick embryos. J. Exptl. 2001. SO, 347-389. HAMBURGER, V. ( 1958). Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anat. 102, 365-410. HARRISON, R. G. ( 1904). An experimental study of the relation of the nervous system to the developing musculature in the embryo of the frog. Am. .I. Anut. 3, 197-220. HOLTZER, H. ( 1961). Chondrogenesis and myogenesis. In “Synthesis of Molecular and Cellular Structure” (D. Rudnick, ed.), pp. 233-243. Ronald Press, New York. KOELLE, G. B. ( 1963). Cytological distributions and physiological functions of cholinesterase. In “Handbuch der Experimentellen Pharmakologie,” Vol. 15, pp. 189-298. MCCANCE, R. A. ( 1948). Renal function in early life. Physiol. Reo. 28, 331-348. MUMENTHALER, M., and ENGEL, W. K. ( 1961). Cytological localization of cholinesterase in developing chick embryo skeletal muscle. Actu Anut. 47, 274-299.
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PAI, A. ( 1965). Developmental genetics of a lethal mutation, muscular dysgenesis (mdg) in the mouse. I. Genetic analysis and gross morphology. Develop. Biol. 11, 82-92. SHEN, S. C. (1958). Changes in enzymic patterns during development. In “The Chemical Basis of Development” ( W. D. McElroy and B. Glass, eds. ), pp. 416432. The Johns Hopkins Press, Baltimore.