A Morphological and Histochemical Study of the Developing Tongue Musculature in the Mouse : Its Relationship to Palatal Closure ' THOMAS M. HOLTZ Department of Anatomy, University of South Florida, College of Medicine, Tampa, Florida 33620
ABSTRACT Some question exists concerning the ability of the embryonic tongue to undergo reflex movements at the time of palatal closure ( 15.5 days of development). Functional motor endplates are prerequisite for such movements to occur. Light and ultrastructural cytochemical methods were employed to elucidate the morphology of neuromuscular relationships in the developing mouse tongue. The A/Jax mice used in the experiments demonstrated a 12-20% incidence (seasonal variation) of spontaneous cleft palate, allowing a correlation between normal and teratological processes. Organized myofibrils were first seen in tongues of normal and spontaneous cleft lip-cleft palate (SCL-CP) specimens a t 14.5 days of development. The thiocholine technique of Karnovsky and Roots was used to demonstrate acetylcholinesterase ( AChE) activity at the light microscope level. The Lewis and Schute method was used for ultrastructural localization of this enzyme. Tissues from normal and SCL-CP specimens from 12.5 to 20.5 days of gestation failed to show differences in amounts or distribution of AChE activity. AChE activity was seen as early as 14 day's gestation. Electron microscopic studies demonstrated reaction product in the endoplasmic reticulum and nuclear envelope of developing myoblasts. AChE activity at the developing neuromuscular junction and the occurrence of myofilaments preceded palatal closure by several days. Based on these morphological and histochemical findings the tongue of normal and SCL-CP embryos appears capable of responding to a neurogenic stimulus at the time of palatal closure. The findings suggest that the tongue of animals exhibiting a spontaneous cleft palate is not actively involved in the etiology of this condition.
Many investigations have focused on the mechanisms involved in clefts of the secondary palate. However, explanations provided for this phenomena lack general acceptance. Classically, the observations of Dursy (1869), His ('Ol), and others (Poelzl, '04; Fuchs, '10;Sicher, '15) established the concept that the tongue must descend from its position between the palatine processes before secondary palatal closure could occur. His ('01) presumed that withdrawal was accomplished by contraction of the tongue musculature, while Poelzl ('04) and Fuchs ('10) believed palatal closure was aided by a downward movement of the mandible. Sicher ('15) also believed that the mandible was responsible for removing the tongue from AM. J. ANAT., 144: 169-196.
between the palatine shelves, this task being accomplished by a n accelerated growth of the mandible just prior to the critical time of palatal closure. Experimental procedures by Poelzl ('04), Schwartz ('31), and Wragg et al. ('69), analyzed growth and orientational changes of the mandible and tongue at the time of palatal closure. Their data lend support to the suggestions of Peter ('24), Lazzaro ('40), and poswillo and Roy ('65) that deAccepted June 3, '75. 1 This investigation was supported (in part) by NIH Grant DE00241-04 and (in part) by a grant to Dr. Raymond F. Gasser from the Edward G. Schleider Foundation. 2 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy i n the Department of Anatomy of the Medical Center of the Louisiana State University and Agricultural and Mechanical College, New Orleans, Louisiana.
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scent of the tongue just prior to palatal closure releases it from between the palatine shelves and permits transposition of the shelves. Studies by Ross and Walker ('67), Walker ('69), Humphrey ('69) and Walker and Ross ('72) also tend to revive the classical concept of Dursy (1869) and His ('01) that descent of the tongue is essential for normal palatal closure. Ross and Walker ('67) first showed that upward pressure on the shelves by the tongue was necessary for complete palatal closure. In 1972 these authors concluded from their studies on rabbit embryos that the lack of an intrinsic shelf-force in the rabbit supports the hypothesis that tongue movement is a significant factor in the mechanism of palatal closure. Humphrey ('69) provided evidence that jaw depression and swallowing reflexes are present in human embryos just before palatal shelf elevation begins and continue during and after this period. She also speculated that if jaw and tongue reflexes are significant forces in withdrawing the tongue from the interpalatine space, then suppression of fetal activity through drug action could delay palatal shelf elevation, thus resulting in cleft palate. In order to test this hypothesis, Jacobs ('71) and Wragg et al. ('72) observed the effects of several neuromuscular blocking agents on palatal closure from the standpoint of inhibiting embryonic tongue movements. However, their conclusions differed. Wragg et al. ('72) noted that in order to remove the tongue from the interpalatine space it was necessary to stretch it manually far beyond the range possible by head extension or mandibular depression alone. From these observations, they felt that muscular activity of the tongue was probably involved in palatal closure and warranted further study. In order to resolve more clearly the level to which the embryonic rat tongue could respond to electrical stimulation, Wragg and his colleagues ('72) implanted electrodes in the tongue and i n the nucleus of the hypoglossal nerve. Their results showed that several hours prior to palatal closure the tongue responded to direct muscle stimulation with slight, slow, contractions resembling those of smooth muscle. Shortly after closure, the tongue responded with stronger and faster contractions and
by two days after closure its response was identical to that of the newborn. Similarly, the tongue from pre- and post-closure embryos responded to stimulation of the hypoglossal nucleus with the typical, triphasic pattern seen in newborns. From these data, they concluded that the myoneural apparatus of the embryonic rat tongue is functional during the period of palatal closure. These findings support their earlier conclusions ('69) that tongue muscular activity may be involved in palatal closure. Other investigations support the concept of early neuromuscular histogenesis in the tongue. Strauss and Weddel ('40), while electrically stimulating part of the rat brachial plexus on the fifteenth day of development, noticed an unexpected response in the tongue musculature. This induced tongue movement occurred a full day before that seen in muscles of the upper extremity and took place prior to the known time of palatal closure. This observation, coupled with Humphrey's ('69) demonstration of the complex of mouth opening, trunk and limb movements as being among the earliest reflexes in mammalian development, suggests that muscles of the tongue are among the very first to become contractile and do possess functional neuromuscular connections at the time of palatal closure. To clarify further the role of the tongue in palatal closure, the level of development of tongue musculature in the mouse during the period of closure was examined in the present study. Light and electron microscopic cytological and cytochemical approaches were used. Efforts were concentrated on tissues from prenatal specimens collected prior to, during, and after palatal closure. A comparative study was also performed to determine whether specimens with spontaneous cleft palates exhibited any morphological or histochemical variations in the pattern of tongue development from that observed in normal embryos. MATERIALS AND METHODS
A/Jax mice were housed under conditions of controlled temperature, humidity and lighting. Lights were switched on at midnight and off at noon. Adult animals were caged individually and provided with tap water and Purina mouse breeder chow
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ad libitum. When breeding was desired, males were placed into cages housing females at approximately 8 : O O PM. These animals were separated after one hour and females with a vaginal plug were considered pregnant at that time. Embryos were designated as zero hours old upon discovery of the vaginal plug and were collected at desired time points from 12 to 21 days later. The age of each embryo examined was known to within one hour. The embryos were not collected at a particular time each day. At the time of collection, the pregnant females were decapitated, laparotomized and the uterine horns exposed. In most instances only those embryos occupying the lower one-half of either uterine horn were taken for study. Each usable embryo was shelled out of its attachment site, examined and classified as either normal or having a spontaneous cleft lip-cleft palate (SCL-CP). Each specimen was then processed and compared using one of three procedures. Procedure #1 was designed to localize sites of activity of acetylcholinesterase (AChE) in tongues of sibling normal and SCL-CP embryos. Following retrieval of each embryo the tongue was rapidly dissected free, then quenched, in toto, onto a cryostat chuck by rapid immersion into a n isopentane (2-methylbutane) dry ice solution at -80°C. After exposure to the freezing mixture for several minutes, the tongue was transferred onto a cryostat and sectioned at 12-16 p. The solution used to localize sites of AChE activity within the developing tongue musculature was a modification for frozen tissues (El-Badawi and Schenk, '67) of the type first described by Karnovsky and Roots ('64). Procedure #2 was designed to reveal, at the ultrastructural level, the general cytology and state of development of the contractile elements from sibling normal and SCL-CP specimens. Each tongue was sliced very quickly with a razor blade into sections approximately 0.2 m m thick. Only sections from the central one-third of the tongue were studied. Tissue slices were rapidly immersed in a cold (2°C) fixative
composed of 2.0% glutaraldehyde in 0.1 M phosphate buffer. This recipe generally provided a fixation medium with a pH of 7.2. In those experiments where tongues from older embryos were studied, the pH was not altered. However, as progressively younger embryos were examined the pH was raised accordingly with 1 N NaOH. Table 1 summarizes the changes made in pH to facilitate tissue fixation in specimens of different ages. Folbwing a 3-hour fixation at 4"C, the sections were postfixed at room temperature using 1% OsO, in PO, buffer for one hour. Each tongue slice was then washed in buffer for 15 minutes (2-3 changes), dehydrated i n an alcohol-propylene oxide series and embedded in Maraglas. Thin sections were mounted on copper grids and doubly stained with uranyl acetate (Reynolds, '63) followed by lead citrate (Watson, '58). Procedure #3 revealed at the EM level the distribution pattern of AChE in sibling normal and SCL-CP fetuses. The tongue was dissected and sectioned as in Procedure #2. Individual tongue slices were rapidly fixed and incubated for AChE activity following the method described by Lewis and Schute ('67). OBSERVATIONS
This study confirmed previous observations that palatal shelves of normal A/Jax embryos undergo closure around 15.5 day's gestation. The incidence of spontaneous cleft lip-cleft palate averaged 16%. This figure represents observations made on at least 500 viable specimens from over 100 timed pregnancies. No detectable differences were seen in either tongue morphology or AChE distribution between normal and SCL-CP fetuses during any of the stages studied. TABLE 1
Adjustment of fixative p H to changing age Aee
18 days 17 days 16 days 15 days
14 days 13 days 12 days
DH 7.30 7.40 7.60 7.60-7.70 7.80 7.80-7.90 8.00
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A. Morphology of tongue musculature of normal specimens before palate closure Twelve and one-half days. The tongue at this stage was large and occupied much of the oral cavity. There was a general lack of tissue organization. Capillary vessels were present. However, most cellular elements showed little indication of differentiation above the level of mesenchyme. Single (and occasional multiple) nucleoli were common in these cells and were a feature seen throughout myogenesis. Ultrastructural characteristics of mesenchymal cells comprising the tongue at 12.5 days included large numbers of polyribosomes and scanty profiles of rough endoplasmic reticulum (fig. 1 ) . The cells were pleomorphic and some showed signs of mitotic activity. Thirteen and one-half days. One of the first observable changes in tissue organization occurred at approximately the thirteenth day. In tissue from normal and SCL-CP embryos there was a tendency to form regional cellular condensations. Numerous cells within areas of aggregation showed signs of elongation and a few syncytial clusters could be identified. However, undifferentiated cells still occupied a significant proportion of the tongue volume and mitotic figures were notable. Ultrastructurally, numerous free polyribosomes and some profiles of rough endoplasmic reticulum were present in the elongated cells seen at this stage. I n some cells fibrillar elements were observed which signified the first sign of differentiation toward the myoblastic cell line. Although nerve fibers coursed through the tongue at this stage, no definitive neuromuscular junctions were observed, Fourteen and one-half days. This stage was characterized by an increase in the size and number of cell clusters observed first in earlier embryos. Individual myoblasts contained a few scattered myofibrils, and accumulations of glycogen granules were usually associated with one or more lipid droplets. The syncytial nature of some cells at this stage was easily seen when viewed with the electron microscope (fig. 3 ) .
Palatal closure stage Fifteen and one-half days. Further differentiation of myoblasts and organization of the cell clusters into regions of presumptive muscle fascicles characterized this stage. Elongated syncytial cells were directed in several planes, with less differentiated cells occupying the interf asciculcar regions, Ultrastructurally, typical myoblasts revealed an increase in the number of myofibrils over those seen previously. Large glycogen deposits were also common around the nuclear poles. I n addition to the paranuclear deposits, glycogen granules were almost universally distributed between the myofibrils (fig. 4 ) . Stages after palatal closure Sixteen a n d one-half days. Maturation of the differentiating myoblasts continued and there was consolidation into more highly organized fascicles. Electron microscopy of the myoblasts revealed a dramatic increase in the number of myofibrils (fig. 5). A few collagen fibrils cculd be identified between the cells. Eighteen and one-half days. After 18.5 days the primitive fascicles became organized into highly elaborate patterns. The sarcoplasm contained numerous well-developed myofibrils (fig. 6 ) . Also present were smaller cells resembling fibroblasts. Myofiber nuclei generally assumed a more peripheral position.
B . Morphology of tongue musculature of SCL-CP specimens Tongue tissues were examined at timepoints identical to those used for tissues from normal specimens. Ultrastructural study of cells from embryos exhibiting spontaneous cleft lipcleft palate revealed that cell morphology in these tongues was similar to that seen in normal specimens. For example, cells in early stages (12.5 days) exhibited polyribosomes, little endoplasmic reticulum, and pleomorphic cell shapes (fig. 2).3 In later stages (e.g., 17 days) SCL-CP embryos revealed a continued similarity in 31t was assumed that specimens exhibiting a cleft lip at stages prior to normal palatal closure would also have developed a cleft palate had they been allowed to continue development. Rationale for this view is based on the,high incidence in A/Jax mice of spontaneous combined cleft lip-cleft palate malformations. (Trader and Fraser, '63).
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE
cytoarchitectural features to those already described for normal specimens. The distribution and organization of myofibrils, glycogen, and polyribosomes resembled the pattern observed in cells from normal tongues. Cell sizes and shapes were also similar (figs. 7, 8). C. AChE localization - light microscopy
Stage before palatal closure Fourteen and one-half days. The first visualization of acetylcholinesterase in frozen tissue samples occurred at approximately 14.5 days. At this stage, the reaction product was observed a s a finely distributed, nonlocalized deposit. This precipitate characteristically exhibited a brownish color (Hatchett’s brown) at the sites of enzyme activity. Only those tissue regions containing elongated myoblasts displayed any evidence of the reaction product (fig. 9 ) . Palate closure stage Fifteen and one-half days. During the time of palatal closure, more precise localization of the reaction product was evident. Sites of reaction product were seen in both normal and SCL-CP specimens a s dark, hook-shaped structures i n association with one side of the myotube surface (fig. 11). No noticeable differences i n either number or structural complexity of enzyme localization sites were observed between normal and SCL-CP littermates. Stages after palatal closure Sixteen and one-half days. During the 24 hours after palatal closure the acetylcholinesterase localization sites increased in number and size (fig. 1 3 ) . More of the developing muscle cell surface was covered by the cap-like deposits of reaction product, signifying areas of AChE localization. In some areas, almost half of the cell surface was covered with deposits of reaction product. A gradual increase in the number of AChE sites was apparent as development proceeded. In this regard, it appeared that clusters of adjacent developing muscle cells acquired the staining reaction for AChE at the same time. Seventeen and one-half days. Continued elaboration of surface areas exhibiting AChE was observed in both normal and
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SCL-CP specimens. Also demonstrable at this stage was a n increase in the number of clusters of myotubes exhibiting surface sites of AChE activity (fig. 1 4 ) . Eighteen and one-half days. Only subtle changes occurred i n the pattern of AChE distribution between this stage and earlier ones. Myotube diameter increased and, consequently, resulted in a decrease in the cell surface area covered by the caps of AChE reaction product (figs. 15, 16). D. AChE localization - electron microscopy
Stages before palatal closure Fourteen and one-half days. After 14.5 days those cells which had unquestionably differentiated into myotubes showed large amounts of acetylcholinesterase activity when incubated according to the Lewis and Schute technique. Foci of enzyme activity could be identified a t varying points along the nuclear membrane and within the developing sarcoplasmic reticulum (fig. 10). At this stage less differentiated cell types exhibited no reaction product. Fifteen days. Little increase in the overall distribution of acetylcholinesterase activity occurred between 14.5 and 15 days in normal and SCL-CP embryos. The foci of activity along the nuclear envelopes displayed a distribution pattern similar to that seen in the earlier stage, although possibly more widespread in distribution along the nuclear membrane. A few less differentiated cell types could be identified, which lacked AChE activity, while occasional heavy deposits of reaction product could be seen in the intercellular spaces between adjacent myofibers and axons. These “C” shaped areas of enzyme activity may mark the sites of initial development of myoneural junctions (fig, 17). Several significant features of this putative primitive myoneural structure were evident at this stage. These included : ( 1) the distribution of AChE activity around the entire myotube nuclear envelope, ( 2 ) a n increase in the amount of sarcoplasmic reticulum, with AChE activity, ( 3 ) the appearance of nerve fibers in extremely close apposition to the myotube and ( 4 ) the presence of large amounts of reaction product in the area of myoneural apposition. Other note-
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THOMAS
worthy observations included the presence of AChE activity within Schwann cells and in scattered vesicles within the smaller more terminal nerve processes.
Stages after palatal closure Sixteen and me-half days. The pattern of AChE distribution observed ultrastructurally in the tongue during the time of palatal closure remained relatively unchanged (fig. 5 ) . Less differentiated cell types, some of which showed slight levels of AChE activity, were still observed. A comparison between normal and SCLCP specimens revealed no apparent ultrastructural differences in enzyme patterns. Seventeen days. The features of AChE distribution observed in the younger tissue samples remained prominent. AChE activity was present along the nuclear membrane and throughout the sarcoplasmic reticulum of normal (fig. 7 ) and SCLCP (fig 8) specimens. An interesting feature of both groups of animals was the presence of less differentiated cell types devoid of AChE activity. The Schwann cells in both groups continued to show AChE activity. The activity pattern observed in the Schwann cells was identical to that seen i n the developing muscle cells (i.e., along the nuclear envelope and throughout the endoplasmic reticulum). Large concentrations of the end product were seen within the presumptive synaptic gutters. A few vesicles were also scattered within the neural components closest to the junctional region. DISCUSSION
For many years a controversy has existed over the role of the tongue in normal palatal development. Early studies by His ('Ol), Poelzl ('04), and Sicher ('15) established the concept that the tongue must descend from its position between the palatine processes before closure can occur. More recent investigations by Wragg ('69), Humphrey ('69), Walker and Ross ('72) and Walker and Patterson ('74) have strengthened this concept and suggested that active descent of the tongue just prior to palatal closure permits transposition of the shelves. On the other hand, observations by Walker and Fraser ('56), Wragg et al. ('72) and Green and Kochhar ('73)
M. HOLT
suggest that the tongue is only passively involved, if at all, in the mechanism of palatal closure. Several approaches were utilized in this study including light microscopy, electron microscopy and localization of acetylcholinesterase activity, to provide data on myogenesis and the development of neuromuscular junctions. The A/Jax mouse strain was used because these animals exhibit a high incidence of spontaneously formed cleft palates. Specimens from this strain not exhibiting clefts were used as controls. If the tongue normally responds to reflexogenic neural stimulation during or prior to the period of shelf closure, then one would expect to see developing muscle cells exhibiting myofibrillar differentiation at that time. Syncytial myotubes with well developed sarcomeres were observed in the present study a full 24 hours prior to palatal closure. If tongue reflex activity is prerequisite to normal palatal closure, then a n embryo which spontaneously develops a cleft palate might show less well developed myofibrillar patterns prior to and during the time of palatal closure. I n this investigation, tongue tissue from embryos likely to develop a cleft palate, on the basis of possessing a cleft lip at stages prior to actual closure (Trader, '63) were observed to have syncytial myotubes and sarcomere patterns identical to those of cells from normal specimens. In fact, syncytial myotubes were present in the tongues of both normal and SCL-CP embroys 24 hours prior to palatal closure. The presence of myotubes, although necessary for contraction of the tongue, does not in itself insure that muscle contraction occurs. Some authors have suggested (Humphrey, '69; Wragg et al., '72) that the tongue does respond to neurogenic stimulation around the time of palatal closure, which indicates that well developed nerves are present during this period, as well as differentiated muscle cells. The results of this study substantiate such a notion, since elaborate nerve fibers were observed prior to palatal closure. In addition, many instances were observed where nerve fibers were closely apposed to developing muscle cells. This observation agrees closely with that of Schweichel and Seinsch ('73) who observed bundles of neurites
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE
around the time of palatal closure, approaching developing rat tongue muscle fibers at right angles to their longitudinal axes. The morphological findings here also support the electrophysiological evidence for the presence of functional neuromuscular junctions in the developing rat tongue reported by Wragg et al. ('72). This group recorded typical triphasic responses from the tongue before and after palatal closure following electrical stimulation of the hypoglossal nucleus. On the basis of these findings the question arises as to whether the distribution of nerve fibers in the developing tongue of SCL-CP specimens reflected a pattern similar to that observed in normal animals. A comparative study of the nerve fiber distribution in the normal and SCL-CP specimens confirmed the similarity between these two prenatal groups. A new approach was incorporated to further strengthen the morphological data which suggested that the tongue musculature is capable of contraction prior to palatal closure. This approach determined the level of differentiation of acetylcholinesterase prior to, during, and after palatal closure. It is generally accepted that some degree of organization between the neurotransmitter substance, acetylcholine, and its degradative enzyme, acetylcholinesterase, must exist before a coordinated reflex movement can occur. Thus, a series of comparative studies at the light level was undertaken to determine the level of activity and localization of this enzyme in normal and SCL-CP prenatal specimens. Data collected in this phase of the investigation show that 24 hours prior to the time of palatal closure diffuse enzymatic activity existed. More important, however, was the observation that sharply localized regions of activity existed around the sarcolemma of some myotubes at the time of palatal closure. The areas of AChE activity clearly exhibited the same structure as that observed in older fetuses. Thus, at the light microscopic level, the enzymatic conditions present support the thesis that the embryonic tongue is capable of responding to neurogenic stimulation at the time of palatal closure. An equivalent level of enzymatic differentiation existed in the tissue samples from normal and SCL-CP fetuses.
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This finding weakens the idea that cleft palate formation in these animals is caused by retarded neuromuscular development. I n order to assess the development of AChE activity within the myotubes, a study was undertaken to localize this enzyme at the ultrastructural level. Results from this phase of the investigation c o n h the presence of AChE within myotubes 24 hours prior to the time of palatal closure. Furthermore, this approach revealed the presence of what appeared to be primitive myoneural junctions a full 12 hours before the period of palatal closure. It should be emphasized that at those timepoints where samples from both groups were observed ( 1 2 hours prior to the predicted normal closure and 24-48 hours after closure), the appearance, distribution, and concentration of AChE was identical. Tennyson et al. ('71), investigated the initial appearance of acetylcholinesterase within developing muscle cells and described the presence of sarcoplasmic AChE within the myoblasts of rabbit myotomes. They demonstrated localization of the enzyme in the nuclear envelope, in the endoplasmic reticulum and in some elements of the Golgi complex. Data from the present study reflect a similar AChE distribution pattern even though the tissue sources were different. Tennyson et al. ('73) recently described the reticulum-bound AChE as persisting throughout much of myogenesis but gradually decreasing as the muscle cell reaches maturity. In the present study, no noticeable decrease in the concentration of AChE occurred during the developmental stage studied. However, the latest timepoint observed for ultrastructural AChE localization was at 17 days of development. Diamond and Miledi ('61), using iontophoretic applications of acetylcholine on fetal rat diaphragm muscle cells, observed that the entire sarcolemma was sensitive to this drug throughout prenatal development. By birth, however, they reported that the sensitivity had begun to recede from the ends of the fiber and eventually becomes restricted to the neurojunctional region, These authors concluded that establishment of neuromuscular connections is responsible for restricting the sarcolem-
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ma1 chemo-sensitivity. They observed nerve fibers in junctional contact with developing muscle fibers before the seventeenth day of development and concluded that cholinesterases were formed as a result of neural activity. I n the present study, acetylcholinesterase appears to be synthesized by developing myoblasts and myotubes prior to any observed contact by nerve fibers. Tennyson et al. ('73) agree that this enzyme, plus another cholinesterase (butarylcholinesterase), appear many days prior to any neuromuscular contact. Her group speculated that the decrease in the enzyme-containing reticulum observed in later stages of myogenesis can be correlated with the shrinking chemo-sensitivity to AChE reported by Diamond and Miledi. Tennyson et al. ('73) observed a n increase in the diameter of the myotubes from rabbit trunk muculature during the course of myogenesis. In the present investigation developing muscle cells from the mouse tongue underwent similar growth changes, Tennyson's group also reported that Schwann cells near the sites of developing neuromuscular junctions contained a soluble non-membrane bound form of AChE. Data from the ultrastructural AChE localization procedures employed in the present study revealed instead a typical, reticulumbound form of this enzyme. No diffuse, non-localized AChE was observed i n the Schwann cells. Rather, it filled the endoplasmic reticulum and lined the nuclear envelope, as was observed in developing myotubes. A study by Schweichel and Seinsch ('73) concentrated on the development of the motor system in the rat tongue. These authors reported the presence of muscle fibers, primitive myoneural contacts complete with acetylcholine vesicles and a typical acetylcholinesterase enzyme pattern at the time of palatal closure ( 1 7 days). They concluded that the structural and enzymatic pre-conditions exist for a coordinated movement of the tongue prior to closure of the palate. The present study demonstrated morphologically mature neuromuscular junctions in the mouse tongue after 18.5 days of development. Primitive myoneural contacts were observed around the time of palatal closure. However, they
lacked most of the features normally associated with mature motor endplates. Only scattered terminal vesicles were observed, yet a relatively high concentration of AChE was present. No typical synaptic clefts were evident. The observations of myotubes well endowed with myofibrils, the myoneural association, even though immature in appearance, and the presence of organized AChE a t sites of myoneural contact all support the contention that the mouse tongue is capable of responding to neurogenic stimulation during the time of palatal closure. There are similarities in the myogenesis, the pattern of nerve fiber distribution and acetylcholinesterase localization in tongue samples from normal and SCL-CP specimens. These similarities support the hypothesis that tongues from animals with spontaneously formed cleft palates are normal with respect to their morphogenesis. Furthermore, they should be equally capable of reflex contraction. Thus, if lack of tongue movement is a prime etiological factor in development of spontaneous cleft palate, then this aberration cannot be attributed to any abnormal development of the structural or enzymatic conditions necessary for muscle contraction. However, this does not eliminate the possibility, in the SCL-CP embryos, of abnormal development or functioning of higher centers such as the hypoglossal nucleus. Neither does it preclude the possibility of physiological blockage in the functioning of either the muscle cells or the neuromuscular junctions, even though morphologically and histochemically all components appear normal. ACKNOWLEDGMENT
The author wishes to thank Dr. Raymond F. Gasser for so willingly contributing his time and efforts to help complete this project. His suggestions and advice are greatly appreciated. LITERATURE CITED Diamond, J., and R. Miledi 1961 A study of foetal and newborn rat muscle fibers. J. Physiol., 162: 393-408. Dursy, E. 1969 Zur Entwicklungsgeschichte des Kopfes des Menschen und der hoheren Wirbelthiere. Thesis, Tubingen. H. Laupp' schen Buchhandlung.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE El-Badawi, A., and E. A. Schenk 1967 Histochemical methods for separate, consecutive and simultaneous demonstration of AChE and norepinephrine in cryostat sections. J. Histochem. Cytochem., 15: 580. Fuchs, H. 1910 uber Korrelative Beziehungen zwischen Zungen-und Gaumenentwicklung bie Saugerembryonen. Z. Morph. Anthrop., 13: 97-130. Green, R. M., and D. M. Kochhar 1973 Palatal closure i n the mouse as demonstrated i n frozen sections (1). Am. J. Anat., 137: 477-482. His, W. 1901 Beobachtungen zur Geschichte der Nasen- und Gaumenbildung beim menschlichen Embryo. Abh. Sach. Akad. Wiss. Leipzig, Math-Phys. IU.,27: 349-389. Humphrey, T. 1969 The relation between human fetal mouth opening reflexes and closure of the palate. Am. J. Anat., 125: 317344. Jacobs, R. M. 1971 Failure of muscle relaxants to produce cleft palate in mice. Teratology, 4: 25-30. Karnovsky, M. J., and L. Roots 1964 A “directcoloring” thiocholine method for cholinesterases. J. Histochem. Cytochem., 12: 219-221. Lazarro, C. 1940 Sul Meccanismo Di chiusara del Palato Secondario. Monit. Zool. Ital., 51: 249-273. Lewis, P. R., and C. C. D. Shute 1966 The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell Sci., 1: 381-390. Peter, K. 1924 Die Entwicklung des Saeugetiergamens. Ergebn. Anat. Entwgesch., 25: 448564. Poelzl, A. 1904 Zur Entwicklungsgeschichte des menschlichen Gaumens. Anat. Hefte, 27: 243-284. Poswillo, C., and L. J. Roy 1965 The pathogenesis of cleft palate -an animal study. Br. J. Surg., 52: 902-913. Reynolds, E. S. 1963 The use of lead citrate at a high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208. Ross, L. M., and B. E. Walker 1967 Movement of palatine shelves in untreated and teratogen treated mouse embryos. Am. J. Anat., 121: 509-522. Schwartz, A. M. 1931 Die Ontogenese des menchlichen Gebisses in ihren Bezihungen zur
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Orthodontik I Teil: Untersuchungen ueber die Embryoanle Progenie. Fortschr. Orthod., I: 821. Schweichel, J., and W. Seinsch 1973 Die Entiwicklung des Bewegungsapparates der Rattenzunge im Licht-und Elektronenmikroskop. Z. Anat. Entwick1.-Gesch., 140: 153-171. Sicher, H. 1915 Die Entwickelung des Sekundaeren Gaumens biem Menschen. Anat. Anz., 47: 513-523, 545-562. Strauss, W. L., and G. Weddel 1940 Nature of the first visible contractions of the forelimb musculature i n rat fetuses. J. Neurophysiol., 3: 358-369. Tennyson, V. M., M. Brzin and L. T. Kremzner 1973 Acetylcholinesterase activity in the myotube and muscle satellite cell of the fetal rabbit. An electron microscopic-cytochemical and biochemical study. J. Histochem. Cytochem., 21: 634-652. Tennyson, V. M., M. Brzin and P. Slotwinder 1971 The appearance of acetylcholinesterase in the myotome of the embryonic rabbit. An electron microscopic-cytochemical and biochemical study. J. Cell. Biol., 51: 703. T r a s h , D. C., and F. C. Fraser 1963 Role of the tongue in producing cleft palate in mice with spontaneous cleft lip. Dev. Biol., 6: 45-60. Walker, B. E. 1969 Correlation of embryonic movement with palate closure in mice. TeratolOgy, 2: 191-198. Walker, B. E., and F. C. Fraser 1956 Closure of the secondary palate in three strains of mice. J. Embryol. Exp. Morph., 4: 176-189. Walker, B. E., and A. Patterson 1974 The mechanism of cortisone-induced cleft palate. J. Dent. Res., 53: 63. Walker, B. E., and L. M. Ross 1972 Observation of palatine shelves i n living rabbit embryos. Teratology, 5: 97-102. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4 : 475-478. Wragg, L. E., J. A. Smith, and C. S. Borden 1969 Tongue muscle morphology and function at the time of secondary palate closure in the rat. Anat. Rec., 163; 288. 1972 Myoneural maturation and function of the foetal rat tongue at the time of secondary plate closure. Archs. Oral Biol., 17: 673-682.
PLATE 1 EXPLANATION O F FIGURES
1 Tissue from a normal 12-day, 15.5-hour specimen approximately three days prior to palatal closure. Cells exhibit mesenchymal characteristics and occasional mitoses ( M ) . Scanty profiles of rough endoplasmic reticulum (ER) are notable. Numerous polyribosomes ( P ) can be identified throughout the cytoplasm. X 7,750. 2
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Tissue from a 12-day, 15.5-hour specimen which exhibited spontaneous cleft lip formation. Note the close morphological resemblance to tissue from a normal specimen seen in figure 1. Endoplasmic reticulum ( E R ) , polyribosomes ( P ). x 7,750.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 1
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PLATE 2 EXPLANATION O F FIGURES
180
3
Tissue from a normal 14-day, 13.5-hour specimen approximately one day prior to palatal closure. Note the syncytial nature of the cells. Myofibrillar development is significant ( M ) . Glycogen ( G ) and lipid ( L ) deposits are also apparent. x 6,589.
4
Tissue from a normal ls-day, 13-hour specimen. Palatal shelves are in various stages of closure and/or fusion in specimens at this stage. Myofibrils ( M ) , glycogen deposits (G), and lipid droplets ( L ) are increased from :hose observed at 14.5-days (fig. 3 ) . x 6,589.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 2
181
PLATE 3 EXPLANATION O F FIGURES
182
5
Tissue from a normal 16-day, 12-hour specimen approximately one day after palatal closure. These cells exhibit an abundance of myofibrils ( M ) . AChE activity is localized on the nuclear membrane ( N ? and in the sarcoplasmic reticulum ( S R ) . x 9,496.
6
Tissue from a normal 18-day, 12-hour specimen approximately three days after palatal closure. Features typical of cells at this stage include peripherally placed nuclei ( N ) , myofibrils in register ( M ) , considerable quantities of glycogen ( G ) and lipid (L). x 5,426.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 3
183
PLATE 4 EXPLANATION OF FIGURES
7
Tissue from a normal l7-day, 0-hour specimen approximately one and one-half days after palatal closure. Note the close proximity of the small nerve bundle to the well-developed muscle cell. The Schwann cells surrounding the nerve fibers exhibit localized AChE activity along the endoplasmic reticulum and nuclear membrane (Arrows). Less differentiated cells lack AChE activity. x 5,426.
8
Tissue from a 17-day, 0-hour specimen which exhibited spontaneous cleft lip-cleft palate formation. AChE reaction product can be seen between the closely apposed muscle cell and nerve (Arrow). Other features of this tissue resemble those observed in normal animals.
x 9,496.
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DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 4
185
PLATE 5 EXPLANATION OF FIGURES
9 Frozen tissue from a normal 14-day, 12-hour specimen approximately one day prior to palatal closure. Areas of cellular elongation demonstrate diffuse granular foci of AChE activity (Arrows). B a r = 20 p . 10
186
Tissue from a normal 14-day, 13.5-hour specimen approximately one day prior to palatal closure. Note sites of AChE activity along portions of the myoblast nuclear envelope ( N ) and throughout the sarcoplasmic reticulum ( S R ) . x 7,750.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 5
187
PLATE 6 EXPLANATION OF FIGURES
11 Frozen tissue from a normal 15-day, 12-hour specimen. Palatal shelves were closed and partially fused in this specimen. Sites of AChE activity appear as cap-like deposits of copper ferrocyanide (Arrows). Bar = 20 p. 12 Frozen tissue from a normal 16-day, 0-hour specimen. Sites of AChE activity (Arrows) are increased in number and complexity from those observed earlier (fig. 11). Bar = 20 p.
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DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 6
189
PLATE 7 EXPLANATION OF FIGURES
13
Frozen tissue from a normal 16-day, 12-hour specimen approximately one day after palatal closure. Note the increased development of individual myotubes and their regions of AChE activity (Arrow). Bar .= 20 p.
14 Frozen tissue from a normal 17-day, 12-hour specimen approximately two days after palatal closure. Note the increased diameter of individual myotubes ( M ) over that observed at earlier stages. Also note the continued cap-like appearance of AChE activity along regions of the myotube surface (Arrow). Bar = 20 p.
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DEVELOPING TONGUE MUSCLE PALATAL CLOSURE T h o m a s M. Holt
PLATE 7
191
PLATE 8 EXPLANATION OF FIGURES
192
15
Frozen tissue from a normal 18-day, 12-hour specimen approximately three days after palatal closure. Note the pattern of increasing myotube diameter with age. Arrow marks site of AChE localization. Bar = 25 p .
16
Frozen tissue from a normal 18-day, 12-hour specimen. Sagitally sectioned myotubes reveal the limited extent of the surface regions possessing AChE activity (Arrows). Bar = 25 p.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 8
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PLATE 9 EXPLANATIOH OF FIGURE
17 Tissue from a normal 15-day, 1-hour specimen. Palatal shelves of this specimen were still open. AChE activity is localized within the space between developing nerve fibers and muscle cells (arrow), along the muscle cell nuclear membrane ( N ) , and within its sarcoplasmic reticulum ( S R ) . Myofibrils (M). x 13,759.
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DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 9
195
ABSTRACT Some question exists concerning the ability of the embryonic tongue to undergo reflex movements at the time of palatal closure ( 15.5 days of development). Functional motor endplates are prerequisite for such movements to occur. Light and ultrastructural cytochemical methods were employed to elucidate the morphology of neuromuscular relationships in the developing mouse tongue. The A/Jax mice used in the experiments demonstrated a 12-20% incidence (seasonal variation) of spontaneous cleft palate, allowing a correlation between normal and teratological processes. Organized myofibrils were first seen in tongues of normal and spontaneous cleft lip-cleft palate (SCL-CP) specimens a t 14.5 days of development. The thiocholine technique of Karnovsky and Roots was used to demonstrate acetylcholinesterase ( AChE) activity at the light microscope level. The Lewis and Schute method was used for ultrastructural localization of this enzyme. Tissues from normal and SCL-CP specimens from 12.5 to 20.5 days of gestation failed to show differences in amounts or distribution of AChE activity. AChE activity was seen as early as 14 day's gestation. Electron microscopic studies demonstrated reaction product in the endoplasmic reticulum and nuclear envelope of developing myoblasts. AChE activity at the developing neuromuscular junction and the occurrence of myofilaments preceded palatal closure by several days. Based on these morphological and histochemical findings the tongue of normal and SCL-CP embryos appears capable of responding to a neurogenic stimulus at the time of palatal closure. The findings suggest that the tongue of animals exhibiting a spontaneous cleft palate is not actively involved in the etiology of this condition.
Many investigations have focused on the mechanisms involved in clefts of the secondary palate. However, explanations provided for this phenomena lack general acceptance. Classically, the observations of Dursy (1869), His ('Ol), and others (Poelzl, '04; Fuchs, '10;Sicher, '15) established the concept that the tongue must descend from its position between the palatine processes before secondary palatal closure could occur. His ('01) presumed that withdrawal was accomplished by contraction of the tongue musculature, while Poelzl ('04) and Fuchs ('10) believed palatal closure was aided by a downward movement of the mandible. Sicher ('15) also believed that the mandible was responsible for removing the tongue from AM. J. ANAT., 144: 169-196.
between the palatine shelves, this task being accomplished by a n accelerated growth of the mandible just prior to the critical time of palatal closure. Experimental procedures by Poelzl ('04), Schwartz ('31), and Wragg et al. ('69), analyzed growth and orientational changes of the mandible and tongue at the time of palatal closure. Their data lend support to the suggestions of Peter ('24), Lazzaro ('40), and poswillo and Roy ('65) that deAccepted June 3, '75. 1 This investigation was supported (in part) by NIH Grant DE00241-04 and (in part) by a grant to Dr. Raymond F. Gasser from the Edward G. Schleider Foundation. 2 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy i n the Department of Anatomy of the Medical Center of the Louisiana State University and Agricultural and Mechanical College, New Orleans, Louisiana.
169
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THOMAS M. HOLT
scent of the tongue just prior to palatal closure releases it from between the palatine shelves and permits transposition of the shelves. Studies by Ross and Walker ('67), Walker ('69), Humphrey ('69) and Walker and Ross ('72) also tend to revive the classical concept of Dursy (1869) and His ('01) that descent of the tongue is essential for normal palatal closure. Ross and Walker ('67) first showed that upward pressure on the shelves by the tongue was necessary for complete palatal closure. In 1972 these authors concluded from their studies on rabbit embryos that the lack of an intrinsic shelf-force in the rabbit supports the hypothesis that tongue movement is a significant factor in the mechanism of palatal closure. Humphrey ('69) provided evidence that jaw depression and swallowing reflexes are present in human embryos just before palatal shelf elevation begins and continue during and after this period. She also speculated that if jaw and tongue reflexes are significant forces in withdrawing the tongue from the interpalatine space, then suppression of fetal activity through drug action could delay palatal shelf elevation, thus resulting in cleft palate. In order to test this hypothesis, Jacobs ('71) and Wragg et al. ('72) observed the effects of several neuromuscular blocking agents on palatal closure from the standpoint of inhibiting embryonic tongue movements. However, their conclusions differed. Wragg et al. ('72) noted that in order to remove the tongue from the interpalatine space it was necessary to stretch it manually far beyond the range possible by head extension or mandibular depression alone. From these observations, they felt that muscular activity of the tongue was probably involved in palatal closure and warranted further study. In order to resolve more clearly the level to which the embryonic rat tongue could respond to electrical stimulation, Wragg and his colleagues ('72) implanted electrodes in the tongue and i n the nucleus of the hypoglossal nerve. Their results showed that several hours prior to palatal closure the tongue responded to direct muscle stimulation with slight, slow, contractions resembling those of smooth muscle. Shortly after closure, the tongue responded with stronger and faster contractions and
by two days after closure its response was identical to that of the newborn. Similarly, the tongue from pre- and post-closure embryos responded to stimulation of the hypoglossal nucleus with the typical, triphasic pattern seen in newborns. From these data, they concluded that the myoneural apparatus of the embryonic rat tongue is functional during the period of palatal closure. These findings support their earlier conclusions ('69) that tongue muscular activity may be involved in palatal closure. Other investigations support the concept of early neuromuscular histogenesis in the tongue. Strauss and Weddel ('40), while electrically stimulating part of the rat brachial plexus on the fifteenth day of development, noticed an unexpected response in the tongue musculature. This induced tongue movement occurred a full day before that seen in muscles of the upper extremity and took place prior to the known time of palatal closure. This observation, coupled with Humphrey's ('69) demonstration of the complex of mouth opening, trunk and limb movements as being among the earliest reflexes in mammalian development, suggests that muscles of the tongue are among the very first to become contractile and do possess functional neuromuscular connections at the time of palatal closure. To clarify further the role of the tongue in palatal closure, the level of development of tongue musculature in the mouse during the period of closure was examined in the present study. Light and electron microscopic cytological and cytochemical approaches were used. Efforts were concentrated on tissues from prenatal specimens collected prior to, during, and after palatal closure. A comparative study was also performed to determine whether specimens with spontaneous cleft palates exhibited any morphological or histochemical variations in the pattern of tongue development from that observed in normal embryos. MATERIALS AND METHODS
A/Jax mice were housed under conditions of controlled temperature, humidity and lighting. Lights were switched on at midnight and off at noon. Adult animals were caged individually and provided with tap water and Purina mouse breeder chow
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DEVELOPING TONGUE MUSCLE PALATAL CLOSURE
ad libitum. When breeding was desired, males were placed into cages housing females at approximately 8 : O O PM. These animals were separated after one hour and females with a vaginal plug were considered pregnant at that time. Embryos were designated as zero hours old upon discovery of the vaginal plug and were collected at desired time points from 12 to 21 days later. The age of each embryo examined was known to within one hour. The embryos were not collected at a particular time each day. At the time of collection, the pregnant females were decapitated, laparotomized and the uterine horns exposed. In most instances only those embryos occupying the lower one-half of either uterine horn were taken for study. Each usable embryo was shelled out of its attachment site, examined and classified as either normal or having a spontaneous cleft lip-cleft palate (SCL-CP). Each specimen was then processed and compared using one of three procedures. Procedure #1 was designed to localize sites of activity of acetylcholinesterase (AChE) in tongues of sibling normal and SCL-CP embryos. Following retrieval of each embryo the tongue was rapidly dissected free, then quenched, in toto, onto a cryostat chuck by rapid immersion into a n isopentane (2-methylbutane) dry ice solution at -80°C. After exposure to the freezing mixture for several minutes, the tongue was transferred onto a cryostat and sectioned at 12-16 p. The solution used to localize sites of AChE activity within the developing tongue musculature was a modification for frozen tissues (El-Badawi and Schenk, '67) of the type first described by Karnovsky and Roots ('64). Procedure #2 was designed to reveal, at the ultrastructural level, the general cytology and state of development of the contractile elements from sibling normal and SCL-CP specimens. Each tongue was sliced very quickly with a razor blade into sections approximately 0.2 m m thick. Only sections from the central one-third of the tongue were studied. Tissue slices were rapidly immersed in a cold (2°C) fixative
composed of 2.0% glutaraldehyde in 0.1 M phosphate buffer. This recipe generally provided a fixation medium with a pH of 7.2. In those experiments where tongues from older embryos were studied, the pH was not altered. However, as progressively younger embryos were examined the pH was raised accordingly with 1 N NaOH. Table 1 summarizes the changes made in pH to facilitate tissue fixation in specimens of different ages. Folbwing a 3-hour fixation at 4"C, the sections were postfixed at room temperature using 1% OsO, in PO, buffer for one hour. Each tongue slice was then washed in buffer for 15 minutes (2-3 changes), dehydrated i n an alcohol-propylene oxide series and embedded in Maraglas. Thin sections were mounted on copper grids and doubly stained with uranyl acetate (Reynolds, '63) followed by lead citrate (Watson, '58). Procedure #3 revealed at the EM level the distribution pattern of AChE in sibling normal and SCL-CP fetuses. The tongue was dissected and sectioned as in Procedure #2. Individual tongue slices were rapidly fixed and incubated for AChE activity following the method described by Lewis and Schute ('67). OBSERVATIONS
This study confirmed previous observations that palatal shelves of normal A/Jax embryos undergo closure around 15.5 day's gestation. The incidence of spontaneous cleft lip-cleft palate averaged 16%. This figure represents observations made on at least 500 viable specimens from over 100 timed pregnancies. No detectable differences were seen in either tongue morphology or AChE distribution between normal and SCL-CP fetuses during any of the stages studied. TABLE 1
Adjustment of fixative p H to changing age Aee
18 days 17 days 16 days 15 days
14 days 13 days 12 days
DH 7.30 7.40 7.60 7.60-7.70 7.80 7.80-7.90 8.00
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A. Morphology of tongue musculature of normal specimens before palate closure Twelve and one-half days. The tongue at this stage was large and occupied much of the oral cavity. There was a general lack of tissue organization. Capillary vessels were present. However, most cellular elements showed little indication of differentiation above the level of mesenchyme. Single (and occasional multiple) nucleoli were common in these cells and were a feature seen throughout myogenesis. Ultrastructural characteristics of mesenchymal cells comprising the tongue at 12.5 days included large numbers of polyribosomes and scanty profiles of rough endoplasmic reticulum (fig. 1 ) . The cells were pleomorphic and some showed signs of mitotic activity. Thirteen and one-half days. One of the first observable changes in tissue organization occurred at approximately the thirteenth day. In tissue from normal and SCL-CP embryos there was a tendency to form regional cellular condensations. Numerous cells within areas of aggregation showed signs of elongation and a few syncytial clusters could be identified. However, undifferentiated cells still occupied a significant proportion of the tongue volume and mitotic figures were notable. Ultrastructurally, numerous free polyribosomes and some profiles of rough endoplasmic reticulum were present in the elongated cells seen at this stage. I n some cells fibrillar elements were observed which signified the first sign of differentiation toward the myoblastic cell line. Although nerve fibers coursed through the tongue at this stage, no definitive neuromuscular junctions were observed, Fourteen and one-half days. This stage was characterized by an increase in the size and number of cell clusters observed first in earlier embryos. Individual myoblasts contained a few scattered myofibrils, and accumulations of glycogen granules were usually associated with one or more lipid droplets. The syncytial nature of some cells at this stage was easily seen when viewed with the electron microscope (fig. 3 ) .
Palatal closure stage Fifteen and one-half days. Further differentiation of myoblasts and organization of the cell clusters into regions of presumptive muscle fascicles characterized this stage. Elongated syncytial cells were directed in several planes, with less differentiated cells occupying the interf asciculcar regions, Ultrastructurally, typical myoblasts revealed an increase in the number of myofibrils over those seen previously. Large glycogen deposits were also common around the nuclear poles. I n addition to the paranuclear deposits, glycogen granules were almost universally distributed between the myofibrils (fig. 4 ) . Stages after palatal closure Sixteen a n d one-half days. Maturation of the differentiating myoblasts continued and there was consolidation into more highly organized fascicles. Electron microscopy of the myoblasts revealed a dramatic increase in the number of myofibrils (fig. 5). A few collagen fibrils cculd be identified between the cells. Eighteen and one-half days. After 18.5 days the primitive fascicles became organized into highly elaborate patterns. The sarcoplasm contained numerous well-developed myofibrils (fig. 6 ) . Also present were smaller cells resembling fibroblasts. Myofiber nuclei generally assumed a more peripheral position.
B . Morphology of tongue musculature of SCL-CP specimens Tongue tissues were examined at timepoints identical to those used for tissues from normal specimens. Ultrastructural study of cells from embryos exhibiting spontaneous cleft lipcleft palate revealed that cell morphology in these tongues was similar to that seen in normal specimens. For example, cells in early stages (12.5 days) exhibited polyribosomes, little endoplasmic reticulum, and pleomorphic cell shapes (fig. 2).3 In later stages (e.g., 17 days) SCL-CP embryos revealed a continued similarity in 31t was assumed that specimens exhibiting a cleft lip at stages prior to normal palatal closure would also have developed a cleft palate had they been allowed to continue development. Rationale for this view is based on the,high incidence in A/Jax mice of spontaneous combined cleft lip-cleft palate malformations. (Trader and Fraser, '63).
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE
cytoarchitectural features to those already described for normal specimens. The distribution and organization of myofibrils, glycogen, and polyribosomes resembled the pattern observed in cells from normal tongues. Cell sizes and shapes were also similar (figs. 7, 8). C. AChE localization - light microscopy
Stage before palatal closure Fourteen and one-half days. The first visualization of acetylcholinesterase in frozen tissue samples occurred at approximately 14.5 days. At this stage, the reaction product was observed a s a finely distributed, nonlocalized deposit. This precipitate characteristically exhibited a brownish color (Hatchett’s brown) at the sites of enzyme activity. Only those tissue regions containing elongated myoblasts displayed any evidence of the reaction product (fig. 9 ) . Palate closure stage Fifteen and one-half days. During the time of palatal closure, more precise localization of the reaction product was evident. Sites of reaction product were seen in both normal and SCL-CP specimens a s dark, hook-shaped structures i n association with one side of the myotube surface (fig. 11). No noticeable differences i n either number or structural complexity of enzyme localization sites were observed between normal and SCL-CP littermates. Stages after palatal closure Sixteen and one-half days. During the 24 hours after palatal closure the acetylcholinesterase localization sites increased in number and size (fig. 1 3 ) . More of the developing muscle cell surface was covered by the cap-like deposits of reaction product, signifying areas of AChE localization. In some areas, almost half of the cell surface was covered with deposits of reaction product. A gradual increase in the number of AChE sites was apparent as development proceeded. In this regard, it appeared that clusters of adjacent developing muscle cells acquired the staining reaction for AChE at the same time. Seventeen and one-half days. Continued elaboration of surface areas exhibiting AChE was observed in both normal and
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SCL-CP specimens. Also demonstrable at this stage was a n increase in the number of clusters of myotubes exhibiting surface sites of AChE activity (fig. 1 4 ) . Eighteen and one-half days. Only subtle changes occurred i n the pattern of AChE distribution between this stage and earlier ones. Myotube diameter increased and, consequently, resulted in a decrease in the cell surface area covered by the caps of AChE reaction product (figs. 15, 16). D. AChE localization - electron microscopy
Stages before palatal closure Fourteen and one-half days. After 14.5 days those cells which had unquestionably differentiated into myotubes showed large amounts of acetylcholinesterase activity when incubated according to the Lewis and Schute technique. Foci of enzyme activity could be identified a t varying points along the nuclear membrane and within the developing sarcoplasmic reticulum (fig. 10). At this stage less differentiated cell types exhibited no reaction product. Fifteen days. Little increase in the overall distribution of acetylcholinesterase activity occurred between 14.5 and 15 days in normal and SCL-CP embryos. The foci of activity along the nuclear envelopes displayed a distribution pattern similar to that seen in the earlier stage, although possibly more widespread in distribution along the nuclear membrane. A few less differentiated cell types could be identified, which lacked AChE activity, while occasional heavy deposits of reaction product could be seen in the intercellular spaces between adjacent myofibers and axons. These “C” shaped areas of enzyme activity may mark the sites of initial development of myoneural junctions (fig, 17). Several significant features of this putative primitive myoneural structure were evident at this stage. These included : ( 1) the distribution of AChE activity around the entire myotube nuclear envelope, ( 2 ) a n increase in the amount of sarcoplasmic reticulum, with AChE activity, ( 3 ) the appearance of nerve fibers in extremely close apposition to the myotube and ( 4 ) the presence of large amounts of reaction product in the area of myoneural apposition. Other note-
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THOMAS
worthy observations included the presence of AChE activity within Schwann cells and in scattered vesicles within the smaller more terminal nerve processes.
Stages after palatal closure Sixteen and me-half days. The pattern of AChE distribution observed ultrastructurally in the tongue during the time of palatal closure remained relatively unchanged (fig. 5 ) . Less differentiated cell types, some of which showed slight levels of AChE activity, were still observed. A comparison between normal and SCLCP specimens revealed no apparent ultrastructural differences in enzyme patterns. Seventeen days. The features of AChE distribution observed in the younger tissue samples remained prominent. AChE activity was present along the nuclear membrane and throughout the sarcoplasmic reticulum of normal (fig. 7 ) and SCLCP (fig 8) specimens. An interesting feature of both groups of animals was the presence of less differentiated cell types devoid of AChE activity. The Schwann cells in both groups continued to show AChE activity. The activity pattern observed in the Schwann cells was identical to that seen i n the developing muscle cells (i.e., along the nuclear envelope and throughout the endoplasmic reticulum). Large concentrations of the end product were seen within the presumptive synaptic gutters. A few vesicles were also scattered within the neural components closest to the junctional region. DISCUSSION
For many years a controversy has existed over the role of the tongue in normal palatal development. Early studies by His ('Ol), Poelzl ('04), and Sicher ('15) established the concept that the tongue must descend from its position between the palatine processes before closure can occur. More recent investigations by Wragg ('69), Humphrey ('69), Walker and Ross ('72) and Walker and Patterson ('74) have strengthened this concept and suggested that active descent of the tongue just prior to palatal closure permits transposition of the shelves. On the other hand, observations by Walker and Fraser ('56), Wragg et al. ('72) and Green and Kochhar ('73)
M. HOLT
suggest that the tongue is only passively involved, if at all, in the mechanism of palatal closure. Several approaches were utilized in this study including light microscopy, electron microscopy and localization of acetylcholinesterase activity, to provide data on myogenesis and the development of neuromuscular junctions. The A/Jax mouse strain was used because these animals exhibit a high incidence of spontaneously formed cleft palates. Specimens from this strain not exhibiting clefts were used as controls. If the tongue normally responds to reflexogenic neural stimulation during or prior to the period of shelf closure, then one would expect to see developing muscle cells exhibiting myofibrillar differentiation at that time. Syncytial myotubes with well developed sarcomeres were observed in the present study a full 24 hours prior to palatal closure. If tongue reflex activity is prerequisite to normal palatal closure, then a n embryo which spontaneously develops a cleft palate might show less well developed myofibrillar patterns prior to and during the time of palatal closure. I n this investigation, tongue tissue from embryos likely to develop a cleft palate, on the basis of possessing a cleft lip at stages prior to actual closure (Trader, '63) were observed to have syncytial myotubes and sarcomere patterns identical to those of cells from normal specimens. In fact, syncytial myotubes were present in the tongues of both normal and SCL-CP embroys 24 hours prior to palatal closure. The presence of myotubes, although necessary for contraction of the tongue, does not in itself insure that muscle contraction occurs. Some authors have suggested (Humphrey, '69; Wragg et al., '72) that the tongue does respond to neurogenic stimulation around the time of palatal closure, which indicates that well developed nerves are present during this period, as well as differentiated muscle cells. The results of this study substantiate such a notion, since elaborate nerve fibers were observed prior to palatal closure. In addition, many instances were observed where nerve fibers were closely apposed to developing muscle cells. This observation agrees closely with that of Schweichel and Seinsch ('73) who observed bundles of neurites
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE
around the time of palatal closure, approaching developing rat tongue muscle fibers at right angles to their longitudinal axes. The morphological findings here also support the electrophysiological evidence for the presence of functional neuromuscular junctions in the developing rat tongue reported by Wragg et al. ('72). This group recorded typical triphasic responses from the tongue before and after palatal closure following electrical stimulation of the hypoglossal nucleus. On the basis of these findings the question arises as to whether the distribution of nerve fibers in the developing tongue of SCL-CP specimens reflected a pattern similar to that observed in normal animals. A comparative study of the nerve fiber distribution in the normal and SCL-CP specimens confirmed the similarity between these two prenatal groups. A new approach was incorporated to further strengthen the morphological data which suggested that the tongue musculature is capable of contraction prior to palatal closure. This approach determined the level of differentiation of acetylcholinesterase prior to, during, and after palatal closure. It is generally accepted that some degree of organization between the neurotransmitter substance, acetylcholine, and its degradative enzyme, acetylcholinesterase, must exist before a coordinated reflex movement can occur. Thus, a series of comparative studies at the light level was undertaken to determine the level of activity and localization of this enzyme in normal and SCL-CP prenatal specimens. Data collected in this phase of the investigation show that 24 hours prior to the time of palatal closure diffuse enzymatic activity existed. More important, however, was the observation that sharply localized regions of activity existed around the sarcolemma of some myotubes at the time of palatal closure. The areas of AChE activity clearly exhibited the same structure as that observed in older fetuses. Thus, at the light microscopic level, the enzymatic conditions present support the thesis that the embryonic tongue is capable of responding to neurogenic stimulation at the time of palatal closure. An equivalent level of enzymatic differentiation existed in the tissue samples from normal and SCL-CP fetuses.
175
This finding weakens the idea that cleft palate formation in these animals is caused by retarded neuromuscular development. I n order to assess the development of AChE activity within the myotubes, a study was undertaken to localize this enzyme at the ultrastructural level. Results from this phase of the investigation c o n h the presence of AChE within myotubes 24 hours prior to the time of palatal closure. Furthermore, this approach revealed the presence of what appeared to be primitive myoneural junctions a full 12 hours before the period of palatal closure. It should be emphasized that at those timepoints where samples from both groups were observed ( 1 2 hours prior to the predicted normal closure and 24-48 hours after closure), the appearance, distribution, and concentration of AChE was identical. Tennyson et al. ('71), investigated the initial appearance of acetylcholinesterase within developing muscle cells and described the presence of sarcoplasmic AChE within the myoblasts of rabbit myotomes. They demonstrated localization of the enzyme in the nuclear envelope, in the endoplasmic reticulum and in some elements of the Golgi complex. Data from the present study reflect a similar AChE distribution pattern even though the tissue sources were different. Tennyson et al. ('73) recently described the reticulum-bound AChE as persisting throughout much of myogenesis but gradually decreasing as the muscle cell reaches maturity. In the present study, no noticeable decrease in the concentration of AChE occurred during the developmental stage studied. However, the latest timepoint observed for ultrastructural AChE localization was at 17 days of development. Diamond and Miledi ('61), using iontophoretic applications of acetylcholine on fetal rat diaphragm muscle cells, observed that the entire sarcolemma was sensitive to this drug throughout prenatal development. By birth, however, they reported that the sensitivity had begun to recede from the ends of the fiber and eventually becomes restricted to the neurojunctional region, These authors concluded that establishment of neuromuscular connections is responsible for restricting the sarcolem-
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THOMAS M. HOLT
ma1 chemo-sensitivity. They observed nerve fibers in junctional contact with developing muscle fibers before the seventeenth day of development and concluded that cholinesterases were formed as a result of neural activity. I n the present study, acetylcholinesterase appears to be synthesized by developing myoblasts and myotubes prior to any observed contact by nerve fibers. Tennyson et al. ('73) agree that this enzyme, plus another cholinesterase (butarylcholinesterase), appear many days prior to any neuromuscular contact. Her group speculated that the decrease in the enzyme-containing reticulum observed in later stages of myogenesis can be correlated with the shrinking chemo-sensitivity to AChE reported by Diamond and Miledi. Tennyson et al. ('73) observed a n increase in the diameter of the myotubes from rabbit trunk muculature during the course of myogenesis. In the present investigation developing muscle cells from the mouse tongue underwent similar growth changes, Tennyson's group also reported that Schwann cells near the sites of developing neuromuscular junctions contained a soluble non-membrane bound form of AChE. Data from the ultrastructural AChE localization procedures employed in the present study revealed instead a typical, reticulumbound form of this enzyme. No diffuse, non-localized AChE was observed i n the Schwann cells. Rather, it filled the endoplasmic reticulum and lined the nuclear envelope, as was observed in developing myotubes. A study by Schweichel and Seinsch ('73) concentrated on the development of the motor system in the rat tongue. These authors reported the presence of muscle fibers, primitive myoneural contacts complete with acetylcholine vesicles and a typical acetylcholinesterase enzyme pattern at the time of palatal closure ( 1 7 days). They concluded that the structural and enzymatic pre-conditions exist for a coordinated movement of the tongue prior to closure of the palate. The present study demonstrated morphologically mature neuromuscular junctions in the mouse tongue after 18.5 days of development. Primitive myoneural contacts were observed around the time of palatal closure. However, they
lacked most of the features normally associated with mature motor endplates. Only scattered terminal vesicles were observed, yet a relatively high concentration of AChE was present. No typical synaptic clefts were evident. The observations of myotubes well endowed with myofibrils, the myoneural association, even though immature in appearance, and the presence of organized AChE a t sites of myoneural contact all support the contention that the mouse tongue is capable of responding to neurogenic stimulation during the time of palatal closure. There are similarities in the myogenesis, the pattern of nerve fiber distribution and acetylcholinesterase localization in tongue samples from normal and SCL-CP specimens. These similarities support the hypothesis that tongues from animals with spontaneously formed cleft palates are normal with respect to their morphogenesis. Furthermore, they should be equally capable of reflex contraction. Thus, if lack of tongue movement is a prime etiological factor in development of spontaneous cleft palate, then this aberration cannot be attributed to any abnormal development of the structural or enzymatic conditions necessary for muscle contraction. However, this does not eliminate the possibility, in the SCL-CP embryos, of abnormal development or functioning of higher centers such as the hypoglossal nucleus. Neither does it preclude the possibility of physiological blockage in the functioning of either the muscle cells or the neuromuscular junctions, even though morphologically and histochemically all components appear normal. ACKNOWLEDGMENT
The author wishes to thank Dr. Raymond F. Gasser for so willingly contributing his time and efforts to help complete this project. His suggestions and advice are greatly appreciated. LITERATURE CITED Diamond, J., and R. Miledi 1961 A study of foetal and newborn rat muscle fibers. J. Physiol., 162: 393-408. Dursy, E. 1969 Zur Entwicklungsgeschichte des Kopfes des Menschen und der hoheren Wirbelthiere. Thesis, Tubingen. H. Laupp' schen Buchhandlung.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE El-Badawi, A., and E. A. Schenk 1967 Histochemical methods for separate, consecutive and simultaneous demonstration of AChE and norepinephrine in cryostat sections. J. Histochem. Cytochem., 15: 580. Fuchs, H. 1910 uber Korrelative Beziehungen zwischen Zungen-und Gaumenentwicklung bie Saugerembryonen. Z. Morph. Anthrop., 13: 97-130. Green, R. M., and D. M. Kochhar 1973 Palatal closure i n the mouse as demonstrated i n frozen sections (1). Am. J. Anat., 137: 477-482. His, W. 1901 Beobachtungen zur Geschichte der Nasen- und Gaumenbildung beim menschlichen Embryo. Abh. Sach. Akad. Wiss. Leipzig, Math-Phys. IU.,27: 349-389. Humphrey, T. 1969 The relation between human fetal mouth opening reflexes and closure of the palate. Am. J. Anat., 125: 317344. Jacobs, R. M. 1971 Failure of muscle relaxants to produce cleft palate in mice. Teratology, 4: 25-30. Karnovsky, M. J., and L. Roots 1964 A “directcoloring” thiocholine method for cholinesterases. J. Histochem. Cytochem., 12: 219-221. Lazarro, C. 1940 Sul Meccanismo Di chiusara del Palato Secondario. Monit. Zool. Ital., 51: 249-273. Lewis, P. R., and C. C. D. Shute 1966 The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell Sci., 1: 381-390. Peter, K. 1924 Die Entwicklung des Saeugetiergamens. Ergebn. Anat. Entwgesch., 25: 448564. Poelzl, A. 1904 Zur Entwicklungsgeschichte des menschlichen Gaumens. Anat. Hefte, 27: 243-284. Poswillo, C., and L. J. Roy 1965 The pathogenesis of cleft palate -an animal study. Br. J. Surg., 52: 902-913. Reynolds, E. S. 1963 The use of lead citrate at a high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208. Ross, L. M., and B. E. Walker 1967 Movement of palatine shelves in untreated and teratogen treated mouse embryos. Am. J. Anat., 121: 509-522. Schwartz, A. M. 1931 Die Ontogenese des menchlichen Gebisses in ihren Bezihungen zur
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Orthodontik I Teil: Untersuchungen ueber die Embryoanle Progenie. Fortschr. Orthod., I: 821. Schweichel, J., and W. Seinsch 1973 Die Entiwicklung des Bewegungsapparates der Rattenzunge im Licht-und Elektronenmikroskop. Z. Anat. Entwick1.-Gesch., 140: 153-171. Sicher, H. 1915 Die Entwickelung des Sekundaeren Gaumens biem Menschen. Anat. Anz., 47: 513-523, 545-562. Strauss, W. L., and G. Weddel 1940 Nature of the first visible contractions of the forelimb musculature i n rat fetuses. J. Neurophysiol., 3: 358-369. Tennyson, V. M., M. Brzin and L. T. Kremzner 1973 Acetylcholinesterase activity in the myotube and muscle satellite cell of the fetal rabbit. An electron microscopic-cytochemical and biochemical study. J. Histochem. Cytochem., 21: 634-652. Tennyson, V. M., M. Brzin and P. Slotwinder 1971 The appearance of acetylcholinesterase in the myotome of the embryonic rabbit. An electron microscopic-cytochemical and biochemical study. J. Cell. Biol., 51: 703. T r a s h , D. C., and F. C. Fraser 1963 Role of the tongue in producing cleft palate in mice with spontaneous cleft lip. Dev. Biol., 6: 45-60. Walker, B. E. 1969 Correlation of embryonic movement with palate closure in mice. TeratolOgy, 2: 191-198. Walker, B. E., and F. C. Fraser 1956 Closure of the secondary palate in three strains of mice. J. Embryol. Exp. Morph., 4: 176-189. Walker, B. E., and A. Patterson 1974 The mechanism of cortisone-induced cleft palate. J. Dent. Res., 53: 63. Walker, B. E., and L. M. Ross 1972 Observation of palatine shelves i n living rabbit embryos. Teratology, 5: 97-102. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4 : 475-478. Wragg, L. E., J. A. Smith, and C. S. Borden 1969 Tongue muscle morphology and function at the time of secondary palate closure in the rat. Anat. Rec., 163; 288. 1972 Myoneural maturation and function of the foetal rat tongue at the time of secondary plate closure. Archs. Oral Biol., 17: 673-682.
PLATE 1 EXPLANATION O F FIGURES
1 Tissue from a normal 12-day, 15.5-hour specimen approximately three days prior to palatal closure. Cells exhibit mesenchymal characteristics and occasional mitoses ( M ) . Scanty profiles of rough endoplasmic reticulum (ER) are notable. Numerous polyribosomes ( P ) can be identified throughout the cytoplasm. X 7,750. 2
178
Tissue from a 12-day, 15.5-hour specimen which exhibited spontaneous cleft lip formation. Note the close morphological resemblance to tissue from a normal specimen seen in figure 1. Endoplasmic reticulum ( E R ) , polyribosomes ( P ). x 7,750.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 1
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PLATE 2 EXPLANATION O F FIGURES
180
3
Tissue from a normal 14-day, 13.5-hour specimen approximately one day prior to palatal closure. Note the syncytial nature of the cells. Myofibrillar development is significant ( M ) . Glycogen ( G ) and lipid ( L ) deposits are also apparent. x 6,589.
4
Tissue from a normal ls-day, 13-hour specimen. Palatal shelves are in various stages of closure and/or fusion in specimens at this stage. Myofibrils ( M ) , glycogen deposits (G), and lipid droplets ( L ) are increased from :hose observed at 14.5-days (fig. 3 ) . x 6,589.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 2
181
PLATE 3 EXPLANATION O F FIGURES
182
5
Tissue from a normal 16-day, 12-hour specimen approximately one day after palatal closure. These cells exhibit an abundance of myofibrils ( M ) . AChE activity is localized on the nuclear membrane ( N ? and in the sarcoplasmic reticulum ( S R ) . x 9,496.
6
Tissue from a normal 18-day, 12-hour specimen approximately three days after palatal closure. Features typical of cells at this stage include peripherally placed nuclei ( N ) , myofibrils in register ( M ) , considerable quantities of glycogen ( G ) and lipid (L). x 5,426.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 3
183
PLATE 4 EXPLANATION OF FIGURES
7
Tissue from a normal l7-day, 0-hour specimen approximately one and one-half days after palatal closure. Note the close proximity of the small nerve bundle to the well-developed muscle cell. The Schwann cells surrounding the nerve fibers exhibit localized AChE activity along the endoplasmic reticulum and nuclear membrane (Arrows). Less differentiated cells lack AChE activity. x 5,426.
8
Tissue from a 17-day, 0-hour specimen which exhibited spontaneous cleft lip-cleft palate formation. AChE reaction product can be seen between the closely apposed muscle cell and nerve (Arrow). Other features of this tissue resemble those observed in normal animals.
x 9,496.
184
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 4
185
PLATE 5 EXPLANATION OF FIGURES
9 Frozen tissue from a normal 14-day, 12-hour specimen approximately one day prior to palatal closure. Areas of cellular elongation demonstrate diffuse granular foci of AChE activity (Arrows). B a r = 20 p . 10
186
Tissue from a normal 14-day, 13.5-hour specimen approximately one day prior to palatal closure. Note sites of AChE activity along portions of the myoblast nuclear envelope ( N ) and throughout the sarcoplasmic reticulum ( S R ) . x 7,750.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 5
187
PLATE 6 EXPLANATION OF FIGURES
11 Frozen tissue from a normal 15-day, 12-hour specimen. Palatal shelves were closed and partially fused in this specimen. Sites of AChE activity appear as cap-like deposits of copper ferrocyanide (Arrows). Bar = 20 p. 12 Frozen tissue from a normal 16-day, 0-hour specimen. Sites of AChE activity (Arrows) are increased in number and complexity from those observed earlier (fig. 11). Bar = 20 p.
188
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 6
189
PLATE 7 EXPLANATION OF FIGURES
13
Frozen tissue from a normal 16-day, 12-hour specimen approximately one day after palatal closure. Note the increased development of individual myotubes and their regions of AChE activity (Arrow). Bar .= 20 p.
14 Frozen tissue from a normal 17-day, 12-hour specimen approximately two days after palatal closure. Note the increased diameter of individual myotubes ( M ) over that observed at earlier stages. Also note the continued cap-like appearance of AChE activity along regions of the myotube surface (Arrow). Bar = 20 p.
190
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE T h o m a s M. Holt
PLATE 7
191
PLATE 8 EXPLANATION OF FIGURES
192
15
Frozen tissue from a normal 18-day, 12-hour specimen approximately three days after palatal closure. Note the pattern of increasing myotube diameter with age. Arrow marks site of AChE localization. Bar = 25 p .
16
Frozen tissue from a normal 18-day, 12-hour specimen. Sagitally sectioned myotubes reveal the limited extent of the surface regions possessing AChE activity (Arrows). Bar = 25 p.
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 8
193
PLATE 9 EXPLANATIOH OF FIGURE
17 Tissue from a normal 15-day, 1-hour specimen. Palatal shelves of this specimen were still open. AChE activity is localized within the space between developing nerve fibers and muscle cells (arrow), along the muscle cell nuclear membrane ( N ) , and within its sarcoplasmic reticulum ( S R ) . Myofibrils (M). x 13,759.
194
DEVELOPING TONGUE MUSCLE PALATAL CLOSURE Thomas M. Holt
PLATE 9
195
