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The distribution of glycogen in mouse and rat palatal processes during secondary palate formation: An ultrastructural study
Archs oral Biol. Vol. 14, pp. 385-395, 1969. Pergamon Press. Prioted in Gt. Britain.
THE DISTRIBUTION OF GLYCOGEN IN MOUSE AND RAT PALATAL PROCESSES DURING SECONDARY PALATE FORMATION: AN ULTRASTRUCTURAL STUDY V. DEANGELIS Harvard School of Dental Medicine Boston, Massachusetts 02115, U.S.A. Summary-The ultrastructure of palatal shelves in normal mouse embryos revealed the presence of glycogen reservoirs within the cytoplasm of mesenchymal cells during the shelf transposition stage of secondary palate formation. Shelves of cortisone-treated embryos, however, had considerably less mesenchymal cell glycogen through a comparable period of palatal process development. These findings suggest a relationship between glycogen and the internal shelf force responsible for shelf transposition as well as a possible local action of cortisone during cleft palate induction. In a separate investigation, ultrastructural observations of rat embryo palatal processes which normally fuse at 16 days to 163 days showed a remarkable decrease in epithelial cell glycogen between the 14+ day embryo and the 154 day embryo as well as an increase of desmosomal junctions between cells in the older group. Since the shelves of the older group have a higher fusion potential in culture, these findings suggest a positive correlation between glycogen utilization and fusion potential as well as a similar correlation between the development of desmosomes and incidence of fusion. INTRODUCTION
evidence has accumulated to suggest that mucopolysaccharides play a significant role in the embryology of the mammalian palate. In fact, one of the most perplexing phenomena in the development of the palate, i.e. the transposition of the palatal shelves from a vertical to a horizontal position, appears to be directly related to mucopolysaccharide aggregation (WALKERand FRASER,1957; LARSSON,1962; JACOBS, 1964a). Following a baseline ultrastructural study on the normal process of epithelial fusion and mesenchymal replacement during secondary palate formation (DEANGELIS and NALBANDIAN,1968) it was felt that valuable information might be gained by extending observations and correlating development at certain critical stages with the distribution of glycogen, both under normal conditions and in pharmacologically altered development leading to cleft palate. The author assumes that a correlation may exist between intracellular glycogen and mucopolysaccharide production. One part of the present study consisted of examining the ultrastructure of palatal shelves during shelf transposition in cortisone-treated mouse embryos. This specific approach was based on previously reported data. Experiments by WALKERand FRASER (1957) have indicated that corticosteroid administration delays shelf transposition sufficiently to induce clefts of the secondary palate in a high percentage of cases. The delay of horizontalization with concomitant lateral head growth may separate the processes, making ultimate contact and therefore fusion impossible. Polymerization 385
IN
RECENT years,
386
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and/or aggregation of the mucopolysaccharide structure is believed to be the principal internal force during shelf movement (LARSSON,1962; JACOBS,1964a). Walker and Fraser’s investigations have suggested that steroid inhibitsmucopolysaccharide formation and therefore reduces internal shelf force. Although most clefts of the secondary palate may be a result of faulty shelf transposition, some clefts undoubtedly form after normal shelf movement and approximation has taken place due to improper fusion (POURTOIS,1966; ANGELICI,1968). POURTOISshowed that rat embryo palatal shelves removed prior to 14 days 12 hr and placed in culture did not ultimately fuse at the normally expected time; while shelves which were left intact until 15 days 12 hr and then dissected out and placed in culture did fuse at the expected time in a high percentage of cases. Consequently, in another aspect of this’study, separate experiments were designed to compare the ultrastructure of rat embryonic shelves at 14 days and 154 days. Although the two aspects of this study initially had somewhat different objectives and were not designed to be complementary, they are being reported in one presentation, since the distribution of glycogen proved to be the most significant observation in both. However, cross comparisons between studies are neither made nor intended as there was no attempt to dissect and examine comparable palatal areas between investigations. MATERIALS AND METHODS Eight IO+ day pregnant albino CD1 mice from the Charles River Laboratory were injected IM with O-2 cm3 of solution containing 50 mg/cm” of hydrocortisone in order to induce clefts of the secondary palate. Six 104 day pregnant mice of the same strain, serving as controls, were injected with O-2 cm3 of saline IM. Six experimental and six control mice were sacrificed on the 15th day of pregnancy and their embryos were removed by caesarean section. Two experimental mice were sacrificed at 17 days and their embryos revealed clefts of the secondary palate. Fifteen day embryonic palatal shelves which were in the process of transposing from a vertical to a horizontal position in the experimental and practically in a horizontal position during transposition in the control, were dissected out and fixed for 2 hr with 4 per cent glutaraldehyde buffered to pH 7 -4 with O-1 M cacodylate buffer. Although the palatal shelves of the two groups studied were in different stages of transposition, the age of the embryos were the same as closely as could be determined. Since cortisone is believed to delay shelf movement, leading to clefts, a histological comparison between the two groups at the same age, with the untreated animal shelves rapidly transposing to the horizontal, was expected to reveal the local action of cortisone at this critical time. At the same time, an obvious lag existed in transposition of experimental shelves. Shelves prior to transposing and shelves after assuming a horizontal position were also examined from both groups in this study and revealed no remarkable differences. The specimens were washed with 0.2 M cacodylate (with sucrose) at pH 7.4 for approximately 24 hr, then postfixed for 2 hr with 1 per cent osmium tetroxide buffered to pH 7.3 with phosphate buffer (MILLONIG, 1961). Dehydration was performed with a graded series of cold ethanols, after which the
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specimens were embedded in Epon (LUFT, 1961) with orientation permitting frontal sectioning of the shelves in a serial fashion from anterior to posterior. Thick sections were cut and examined under the light microscope for general observation and ultrathin sections were prepared on the Porter Blum ultramicrotome, equipped with a diamond knife, placed on bare copper grids and double stained with uranyl acetate in 50 per cent alcohol and lead citrate (REYNOLDS,1963). Representative areas along the entire secondary palate were examined in both groups using an RCA model EMU3G electron microscope at 50 kV. Those areas in both groups undergoing movement to the horizontal were particularly selected for comparison. For the second part of this study, six 144 day and six 154 day pregnant albino rats of the Holtzmann strain were sacrificed in ether and their embryos removed by caesarean section. The embryonic palatal shelves were dissected out, processed and examined as described above. Sections from similar areas of the palate of the 14+ and 154 day animals were compared. Only comparable shelf areas not yet rotating were stressed in this investigation. RESULTS Shelf transposition study The palatal process epithelium of 15 day control mice generally consisted of two cell layers. The cells of the outer layer were relatively flat with elongated nuclei, while those of the inner layer were more cuboidal and generally less electron dense (Figs. 1 and 2). Many desmosomes were present at the cell junctions. An abundance of polyribosomes was evident throughout the cytoplasm of both cell layers and numerous mitochondria were found concentrated particularly near the basal lamina. There was a notable absence of coated endoplasmic reticulum in both cell layers. The most striking feature of this bilaminar epithelial surface was its intracellular glycogen content. Discrete reservoirs of glycogen were located within both epithelial cell layers (Fig. 3). No remarkable differences were evident when comparing the epithelium of control mice with those mice treated by cortisone. The same organelles were found and the glycogen distribution into clusters of large rosettes was also quite similar. However, differences between control and experimental animals were remarkable in the connective tissue below the basal lamina. The mesenchymal cells were composed of large central areas containing the nucleus, cytoplasm with polyribosomes, strands of endoplasmic reticulum, Golgi apparatus, coated vesicles and other organelles. Radiating from these main bodies were many organelle-containing cell processes contacting similar extensions of adjacent cells. Junctions were evident where the cell extensions contacted each other. The cytoplasm of the mesenchymal cells in control animals contained large extensive reservoirs of glycogen, which seemingly displaced organelles normally seen within the cytoplasm (Figs. 4, 5 and 7). Many lysosome-like structures were commonly found in the mesenchymal cells, also. In contrast, the mesenchyme of cortisone-treated animals was relatively free of glycogen. In these animals, mesenchymal cells contained a normal array of organelles, densely stained cytoplasm, and many polyribosome rosettes (Fig. 6). Collagen fibrils at various stages of formation were seen in the intercellular spaces of both groups.
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Fusion study
The palatal shelf epithelium of the 14i day rat was also bilaminar and the cells contained many mitochondria, polyribosomes, and other organelles. There was a notable lack of desmosomes along the cell junctions in both epithelial layers and equally remarkable was the abundance of glycogen pools dispersed throughout the cytoplasm of these cells (Fig. 8). The mesenchymal cells were morphologically similar to those of the mouse described earlier but were relatively free of glycogen. The 1%~day rat palatal epithelial surface was notably different in glycogen content. These cells contained many mitochondria and polyribosomes but had remarkably less detectable glycogen (Fig. 9). Desmosomes, however, were more commonly seen in these animals as compared to the 144 day rats. As was true in the 144 day rat embryo, the mesenchymal cells contained relatively little glycogen. This finding, in itself, appears contradictory in that, at 154 days, the rat palatal shelf is in the process of transposing; in view of the findings of comparable stages in the transposition experiment, glycogen buildup might be expected. However, in this fusion experiment concerned with only the fusion potential and not shelf transposition, no attempt was made to examine selectively the epithelial surfaces of that part of the palatal shelf which was transposing. Since the movement of the shelves is in a wave-like fashion (PETER,1924) from posterior to anterior, mesenchyme subjacent to epithelium of the shelf area not transposing might not be expected to reveal a glycogen buildup if this buildup were in fact related to an internal force during shelf movement. The basal lamina in both age groups was clearly visible and continuous along the epithelialmesenchymal junction. No other detectable differences between groups were observed. DISCUSSION Presently, many investigators agree that internal shelf force is responsible for the wave-like movements of the palatine processes from a vertical to a horizontal position prior to fusion into the secondary palate. Histochemical and autoradiographic investigations have indicated that there is an accumulation of acid mucopolysaccharides around the 14th and 15th day in the mouse, coincident with shelf movement (WALKER, 1961; LARSSON,1962 ; JACOBS,1964b). LARSSON (1960) has hypothesized that a change in the polymerization and/or aggregation of the mucopolysaccharides during this critical time results in formation of a gel which is responsible for the elasticity bringing about shelf transposition. This being the case, one would expect to find the presence of glycogen somewhere within the mesenchyme just prior to and/or during shelf movement. The glycogen would not only act as a source of potential energy during this active period but could also indirectly serve as a reservoir of building blocks for formation of mucopolysaccharides. Conversely, since cortisone is believed to inhibit the formation of sulphated mucopolysaccharides, leading to clefts of the secondary palate in mice offspring, (LARSSON,1962; JACOBS,1964b) mesenchyme in shelves treated with this teratogen might be expected to have less glycogen. Of course, the interference with mucopolysaccharide synthesis could occur somewhere beyond formation and utilization of glycogen by degradation to glucose. However, if cortisone were to act directly
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on the mesenchymal cells, as is suggested by some investigators, (MEYER, 1960; BCXXROM and RODEN,1961) alteration in glycogen synthesis might be expected. The findings reported here reveal that the mesenchymal cells of normal animals were heavily ladened with glycogen during the shelf transposition stage, whereas those mesenchymal cells under the influence of cortisone were relatively free of this polysaccharide. Whether the decrease of glycogen in the mesenchymal cells of cortisone treated shelves is a reflection of the inactivity of the shelf and therefore of the lessened energy requirements or whether its absence results in less availability of glucose for incorporation into mucopolysaccharide production, thereby leading to reduction of internal shelf force, is still a matter of conjecture. In the second investigation based on Pourtois’ conclusions that the rat palatal process is endowed at approximately 144 to 15Q days with a fusion potential, any morphologic changes at the ultrastructure level taking place at this critical time could be of importance. Although Pourtois infers the importance of mesenchymal influence upon the epithelium during the fusion process, embryos studied at age groups spanning this time period revealed no morphologic differences in mesenchymal cells. Absence of visible differences in the mesenchyme in our studies does not rule out the possibility that differences do exist, so further experimentation on this problem is under way. However, two rather obvious discrepancies in the epithelium itself are evident between animals of the 14Q day and the 154 day age groups, the most striking being the reduction of glycogen during the one day period from the younger to the older animals. The glycogen is possibly utilized, after a signal is received in uivo,to produce a change in the epithelium which ultimately makes it suitable for fusion. Should the extensive amount of glycogen still be present in the epithelial cells when palatal processes are dissected out and placed in culture, as is the case in the 144 day cultured shelves reported by Pourtois, glycogen utilization and possibly fusion potentiality may never be transferred. Shelves which have been in culture for the period of time after 14+ days would have to be examined at the ultrastructure level as a logical next step to test this hypothesis. The disappearance of glycogen prior to 15g days may be simply a reflection of the maturation process of epithelial cells in this area. Therefore, another explanation for the inability of the younger shelves to fuse is that the shelf which is removed prior to glycogen utilization may not be differentiated sufficiently to complete the epithelial autolytic process necessary in secondary palate formation (SMILEYand DIXON, 1967; FARBMAN,1967). Nonetheless, as in the case of the mouse experiments, the exact function of glycogen during this critical period remains unknown. The other major differences between the epithelial surfaces of the two age groups is the quantity of desmosomes joining the cells. More desmosomes were seen in the older age group. The importance of the desmosome in the fusion process has been elaborated upon in a previous paper which shows that, on contact of the palatine processes, a desmosome union is made (DEANGELISand NALBANDIAN,1968). This desmosome chain, along the junction of the shelves, was shown to be a tenacious bond during completion of the fusion process and ultimate mesenchymal replacement.
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Acknowledgements-The author is greatly indebted to Dr. J. NALBANDIAN for his critical review of the manuscript and to Miss R. GILLESPIE and Mr. A. L. PEELER,II for their technical assistance. This work was supported by research grant no. DE 01766 from the National Institute of Dental Research, U.S.P.H.S. R&n&--L’etude ultrastructurale des lames palatines d’embryons de souris demontre la presence de reserves de glycogene dans le cytoplasme des cellules mesenchymateuses, pendant le stade de la formation du palais secondaire. Des lames d’embryons, trait& par la cortisone, presentent moins de glycogene cellulaire a un stade similaire. Ces resultats suggerent l’existence d’un rapport entre le glycogene et la force intrinseque, responsable de la transposition des lames ainsi qu’une action locale possible de la cortisone pendant la formation d’une division palatine. Au cours d’une autre etude, ii apparait, au point de vue ultrastructural, que les lames palatines d’embryons de rat, qui se fusionnent normalement entre le 16cme et Ie 16&me jour et demi, presentent une diminution caracteristique du glycogene des cellules epitheliales ainsi qu’une augmentation des jonctions desmosomiques intercellulaires dans le groupe d%ge plus avance. Etant donne que les lames de ce demier groupe se fusionnent plus facilement en culture de tissus, il semble que ces resultats indiquent un rapport positif entre l’utilisation de glycogene et le pouvoir de fusion, ainsi qu’un rapport entre le developpement des desmosomes et la frequence de la fusion. Znsammenfassung-Die Ultrastruktur der Gaumenfortsltze normaler Mauseembryonen deckte die Gegenwart von Glykogen-Reservoiren im Zytoplasma mesenchymaler Z&en wlhrend des Stadiums der Gaumenfortsatz-Transposition auf. Die Gaumenfortsatze von cortisonbehandelten Embryonen besal3en dagegen wahrend eines vergleichbaren Zeitraumes der Entwicklung deutlich weniger Zellglykogen im Mesenchym. Diese Beobachtungen deuten aufeine Beziehung zwischen Glykogen und den inneren Kraftenhin, die flir die Fortsatztransposition wie such flir eine mijgliche lokale Wirkung von Cortison bei der Induktion von Gaumenspalten verantwortlich sind. Weitere ultrastrukturelle Untersuchungen ilber die Gaumenfortsltze von Rattenembryonen, die sich normalerweise im Alter von 16 bis 164 Tagen vereinigen, lieBen einen bemerkenswerten Verlust des epithelialen Glykogengehaltes im Embryonalalter zwischen 14+ und 154 Tagen erkennen. In gleicher Weise war bei der Ilteren Gruppe eine Steigerung der desmosomalen Bindungen zwischen den Zellen zu bemerken. Da die Fortsltze der alteren Gruppe in der Kultur ein griiberes Fusionspotential besitzen, deuten diese Befunde eine positive Korrelation zwischen der Glykogenverwertung und dem Fusionspotential wie such eine Bhnliche Korrelation zwischen der Entwicklungvon Desmosomen und dem Zustandekommen der Fusion an.
REFERENCES
ANGELICI,D. R. 1969. Reopening of fused palatal shelves at epithelial interfaces in A/Jax mouse embryos. Cleft Palate Journal in press.
BOSTROM, H. and RODEN,L. 1961. On the metabolism of mucupolysaccharides.
Biochem. Pharmac.
6, 100-123.
BURSTON, W. R. 1959. The development of cleft lip and palate. Ann. R. Coil. Surg. 25, 225-238. DEANGELIS, V. and NALBANDIAN, J. 1968. Ultrastructure of mouse and rat palatal processes prior to and during secondary palate formation.
Arch. oral Biol. 13, 60-608.
FARBMAN, A. I. 1967. Electron microscope study of palatal fusion in mouse embryos. Internat. Ass. for Dent. Res., Preprinted abstracts, 45th General Meeting, Abstract 336. JACOBS, R. M. 1964a. Histochemieal study of morphogenesis and teratogenesis in the palate in mouse embryos. Anat. Rec. 149, 691-698. JACOBS,R. M. 1964b. Ss5-liquid-scintillation count analysis of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec. 150, 271-278.
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LARSSON,K. S. 1960. Studies on the closure of the secondary palate. II. Occurrence of the sulphomucopolysaccharides in the palatine processes of the normal mouse embryo. Expl cell Res. 21, 498-503. LARSSON,K. S. 1962. Closure of the secondary palate and its relation to sulpho mucopolysaccharides. Acta odont. stand. 20, 5-35. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409414. MEYER, K. 1960. Nature and function of mucopolysaccharides of connective tissue. In: Molecular Biology (Edited by NACHMANSOHN, D.) pp. 69-76, Academic Press, New York. MILLONIG,G. 1961. Advantage of a phosphate buffer for OsO., solutions in fixation. J. appl. Phys. 32. 1637. PETER,‘K. 1924. Die Entwicklung des Saugetiergaumens. Ergebn. Anat. Entw. Gesch. 25, 448-564. POURTOIS,M. 1966. Onset of the acouired uotentialitv for fusion in the nalatal shelves of rats. _ J. EmbryoI. exp. Morph. 16, 171-82. _ REYNOLDS,E. S. 1962. The use of lead citrate at high pH as on electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SMILEY,G. R. and DD(ON, A. D. 1967. Fine structure of embryonic midline palatal epithelium. Internat. Ass. for Dent. Res., Preprinted abstracts, 45th General Meeting, Abstract 258. WALKER, B. E. and FRASER,F. C. 1956. Closure of the secondary palate in three strains of mice. J. Embryol. exp. Morph. 4, 176-89. WALKER,B. E. and FRASER,F. C. 1957. The embryology of cortisone induced cleft palate. J. Embryol. exp. Morph. 5,201-209. WALKER,B. E. 1961. The association of mucopolysaccharides with morphogenesis of the palate and other structures in mouse embryos. J. Embryol. exp. Morph. 9, 22-31.
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PLATE1 FIG. 1. Mouse palatal process in vertical position lateral to tongue prior to its transposition to the horizontal plane. Haematoxylin and eosin. x 148. FIG. 2. Outlined shelf area from Fig. 1 shows two epithelial cell layers with underlying mesenchyme separated by a thin continuous basal lamina. (a) Flattened surface cell in outer epithelial cell layer, (b) cubiodal inner epithelial surface layer, (c) basal lamina, (d) mesenchymal cell process. x 12,000. FIG. 3. Palatal shelf surface of a 144 day mouse embryo illustrates areas of glycogen within the cuboidal epithelial cell layer during transposition to the horizontal. (a) Glycogen, (b) mitochondrion, (c) desmosome. x 31,150.
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f.p. 392
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PLATF
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DlSTRIBUT¶ON
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PLATE 2 FIG. 4. Epithelial-mesenchymal junction at the shelf tip of a 144 day mouse embryo shows glycogen reservoirs within the cells on both sides of the basal lamina. (See arrow Fig. 1) (a) Basal lamina, (b) glycogen in epithelial cell, (c) glycogen in mesenchymal cells, (d) collagen fibrils of various sizes dispersed throughout the extra-cellular spaces within the mesenchyme. x 11,900. Fro. 5. Mesenchymal cells in the shelf of 144 day mouse embryo are laden with glycogen. (a) Glycogen, (b) lysosomes, (c) mitochondrion, (d) collagen fibrils in longitudinal and cross section. x 21,500.
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PLATE 3 FIG. 6. Mesenchymal cell at shelf tip of a 14) day cortisone treated mouse depicts the marked decrease of glycogen within the cytoplasm. (a) Mitochondria, (b) network of coated endoplasmic reticulum, (c) golgi x 41,300. FIG. 7. Mesenchymal cell of an untreated 144 day mouse embryo from a comparable area as in Fig. 6. Note abundance of glycogen rosettes displacing cell organelles. (a) Glycogen, (b) coated endoplasmic reticulum, (c) mitochondria. x 41,000.
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PLATF 4
GLYCGGEN DISTRIBUTION DURING SECONDARY PALATE FORMATION
PLATE 4 FIG. 8. Palatal shelf tip of a 149 day rat embryo illustrates reservoirs of glycogen within the cells of the epithelial surface destined to fuse at approximately 16 days. (a) Glycogen, (b) lysosome, (c) basal lamina. x 19,110. FIG.
9. Area comparable to Fig. 8 of palatal shelf tip in 15) day rat embryo shows that glycogen is much less evident within these more differentiated epithelial cells. (a) Polyribosomes, (b) mitochondria, (c) desmosome. x 22,190.
395
THE DISTRIBUTION OF GLYCOGEN IN MOUSE AND RAT PALATAL PROCESSES DURING SECONDARY PALATE FORMATION: AN ULTRASTRUCTURAL STUDY V. DEANGELIS Harvard School of Dental Medicine Boston, Massachusetts 02115, U.S.A. Summary-The ultrastructure of palatal shelves in normal mouse embryos revealed the presence of glycogen reservoirs within the cytoplasm of mesenchymal cells during the shelf transposition stage of secondary palate formation. Shelves of cortisone-treated embryos, however, had considerably less mesenchymal cell glycogen through a comparable period of palatal process development. These findings suggest a relationship between glycogen and the internal shelf force responsible for shelf transposition as well as a possible local action of cortisone during cleft palate induction. In a separate investigation, ultrastructural observations of rat embryo palatal processes which normally fuse at 16 days to 163 days showed a remarkable decrease in epithelial cell glycogen between the 14+ day embryo and the 154 day embryo as well as an increase of desmosomal junctions between cells in the older group. Since the shelves of the older group have a higher fusion potential in culture, these findings suggest a positive correlation between glycogen utilization and fusion potential as well as a similar correlation between the development of desmosomes and incidence of fusion. INTRODUCTION
evidence has accumulated to suggest that mucopolysaccharides play a significant role in the embryology of the mammalian palate. In fact, one of the most perplexing phenomena in the development of the palate, i.e. the transposition of the palatal shelves from a vertical to a horizontal position, appears to be directly related to mucopolysaccharide aggregation (WALKERand FRASER,1957; LARSSON,1962; JACOBS, 1964a). Following a baseline ultrastructural study on the normal process of epithelial fusion and mesenchymal replacement during secondary palate formation (DEANGELIS and NALBANDIAN,1968) it was felt that valuable information might be gained by extending observations and correlating development at certain critical stages with the distribution of glycogen, both under normal conditions and in pharmacologically altered development leading to cleft palate. The author assumes that a correlation may exist between intracellular glycogen and mucopolysaccharide production. One part of the present study consisted of examining the ultrastructure of palatal shelves during shelf transposition in cortisone-treated mouse embryos. This specific approach was based on previously reported data. Experiments by WALKERand FRASER (1957) have indicated that corticosteroid administration delays shelf transposition sufficiently to induce clefts of the secondary palate in a high percentage of cases. The delay of horizontalization with concomitant lateral head growth may separate the processes, making ultimate contact and therefore fusion impossible. Polymerization 385
IN
RECENT years,
386
V. DEANCELIS
and/or aggregation of the mucopolysaccharide structure is believed to be the principal internal force during shelf movement (LARSSON,1962; JACOBS,1964a). Walker and Fraser’s investigations have suggested that steroid inhibitsmucopolysaccharide formation and therefore reduces internal shelf force. Although most clefts of the secondary palate may be a result of faulty shelf transposition, some clefts undoubtedly form after normal shelf movement and approximation has taken place due to improper fusion (POURTOIS,1966; ANGELICI,1968). POURTOISshowed that rat embryo palatal shelves removed prior to 14 days 12 hr and placed in culture did not ultimately fuse at the normally expected time; while shelves which were left intact until 15 days 12 hr and then dissected out and placed in culture did fuse at the expected time in a high percentage of cases. Consequently, in another aspect of this’study, separate experiments were designed to compare the ultrastructure of rat embryonic shelves at 14 days and 154 days. Although the two aspects of this study initially had somewhat different objectives and were not designed to be complementary, they are being reported in one presentation, since the distribution of glycogen proved to be the most significant observation in both. However, cross comparisons between studies are neither made nor intended as there was no attempt to dissect and examine comparable palatal areas between investigations. MATERIALS AND METHODS Eight IO+ day pregnant albino CD1 mice from the Charles River Laboratory were injected IM with O-2 cm3 of solution containing 50 mg/cm” of hydrocortisone in order to induce clefts of the secondary palate. Six 104 day pregnant mice of the same strain, serving as controls, were injected with O-2 cm3 of saline IM. Six experimental and six control mice were sacrificed on the 15th day of pregnancy and their embryos were removed by caesarean section. Two experimental mice were sacrificed at 17 days and their embryos revealed clefts of the secondary palate. Fifteen day embryonic palatal shelves which were in the process of transposing from a vertical to a horizontal position in the experimental and practically in a horizontal position during transposition in the control, were dissected out and fixed for 2 hr with 4 per cent glutaraldehyde buffered to pH 7 -4 with O-1 M cacodylate buffer. Although the palatal shelves of the two groups studied were in different stages of transposition, the age of the embryos were the same as closely as could be determined. Since cortisone is believed to delay shelf movement, leading to clefts, a histological comparison between the two groups at the same age, with the untreated animal shelves rapidly transposing to the horizontal, was expected to reveal the local action of cortisone at this critical time. At the same time, an obvious lag existed in transposition of experimental shelves. Shelves prior to transposing and shelves after assuming a horizontal position were also examined from both groups in this study and revealed no remarkable differences. The specimens were washed with 0.2 M cacodylate (with sucrose) at pH 7.4 for approximately 24 hr, then postfixed for 2 hr with 1 per cent osmium tetroxide buffered to pH 7.3 with phosphate buffer (MILLONIG, 1961). Dehydration was performed with a graded series of cold ethanols, after which the
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specimens were embedded in Epon (LUFT, 1961) with orientation permitting frontal sectioning of the shelves in a serial fashion from anterior to posterior. Thick sections were cut and examined under the light microscope for general observation and ultrathin sections were prepared on the Porter Blum ultramicrotome, equipped with a diamond knife, placed on bare copper grids and double stained with uranyl acetate in 50 per cent alcohol and lead citrate (REYNOLDS,1963). Representative areas along the entire secondary palate were examined in both groups using an RCA model EMU3G electron microscope at 50 kV. Those areas in both groups undergoing movement to the horizontal were particularly selected for comparison. For the second part of this study, six 144 day and six 154 day pregnant albino rats of the Holtzmann strain were sacrificed in ether and their embryos removed by caesarean section. The embryonic palatal shelves were dissected out, processed and examined as described above. Sections from similar areas of the palate of the 14+ and 154 day animals were compared. Only comparable shelf areas not yet rotating were stressed in this investigation. RESULTS Shelf transposition study The palatal process epithelium of 15 day control mice generally consisted of two cell layers. The cells of the outer layer were relatively flat with elongated nuclei, while those of the inner layer were more cuboidal and generally less electron dense (Figs. 1 and 2). Many desmosomes were present at the cell junctions. An abundance of polyribosomes was evident throughout the cytoplasm of both cell layers and numerous mitochondria were found concentrated particularly near the basal lamina. There was a notable absence of coated endoplasmic reticulum in both cell layers. The most striking feature of this bilaminar epithelial surface was its intracellular glycogen content. Discrete reservoirs of glycogen were located within both epithelial cell layers (Fig. 3). No remarkable differences were evident when comparing the epithelium of control mice with those mice treated by cortisone. The same organelles were found and the glycogen distribution into clusters of large rosettes was also quite similar. However, differences between control and experimental animals were remarkable in the connective tissue below the basal lamina. The mesenchymal cells were composed of large central areas containing the nucleus, cytoplasm with polyribosomes, strands of endoplasmic reticulum, Golgi apparatus, coated vesicles and other organelles. Radiating from these main bodies were many organelle-containing cell processes contacting similar extensions of adjacent cells. Junctions were evident where the cell extensions contacted each other. The cytoplasm of the mesenchymal cells in control animals contained large extensive reservoirs of glycogen, which seemingly displaced organelles normally seen within the cytoplasm (Figs. 4, 5 and 7). Many lysosome-like structures were commonly found in the mesenchymal cells, also. In contrast, the mesenchyme of cortisone-treated animals was relatively free of glycogen. In these animals, mesenchymal cells contained a normal array of organelles, densely stained cytoplasm, and many polyribosome rosettes (Fig. 6). Collagen fibrils at various stages of formation were seen in the intercellular spaces of both groups.
388
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Fusion study
The palatal shelf epithelium of the 14i day rat was also bilaminar and the cells contained many mitochondria, polyribosomes, and other organelles. There was a notable lack of desmosomes along the cell junctions in both epithelial layers and equally remarkable was the abundance of glycogen pools dispersed throughout the cytoplasm of these cells (Fig. 8). The mesenchymal cells were morphologically similar to those of the mouse described earlier but were relatively free of glycogen. The 1%~day rat palatal epithelial surface was notably different in glycogen content. These cells contained many mitochondria and polyribosomes but had remarkably less detectable glycogen (Fig. 9). Desmosomes, however, were more commonly seen in these animals as compared to the 144 day rats. As was true in the 144 day rat embryo, the mesenchymal cells contained relatively little glycogen. This finding, in itself, appears contradictory in that, at 154 days, the rat palatal shelf is in the process of transposing; in view of the findings of comparable stages in the transposition experiment, glycogen buildup might be expected. However, in this fusion experiment concerned with only the fusion potential and not shelf transposition, no attempt was made to examine selectively the epithelial surfaces of that part of the palatal shelf which was transposing. Since the movement of the shelves is in a wave-like fashion (PETER,1924) from posterior to anterior, mesenchyme subjacent to epithelium of the shelf area not transposing might not be expected to reveal a glycogen buildup if this buildup were in fact related to an internal force during shelf movement. The basal lamina in both age groups was clearly visible and continuous along the epithelialmesenchymal junction. No other detectable differences between groups were observed. DISCUSSION Presently, many investigators agree that internal shelf force is responsible for the wave-like movements of the palatine processes from a vertical to a horizontal position prior to fusion into the secondary palate. Histochemical and autoradiographic investigations have indicated that there is an accumulation of acid mucopolysaccharides around the 14th and 15th day in the mouse, coincident with shelf movement (WALKER, 1961; LARSSON,1962 ; JACOBS,1964b). LARSSON (1960) has hypothesized that a change in the polymerization and/or aggregation of the mucopolysaccharides during this critical time results in formation of a gel which is responsible for the elasticity bringing about shelf transposition. This being the case, one would expect to find the presence of glycogen somewhere within the mesenchyme just prior to and/or during shelf movement. The glycogen would not only act as a source of potential energy during this active period but could also indirectly serve as a reservoir of building blocks for formation of mucopolysaccharides. Conversely, since cortisone is believed to inhibit the formation of sulphated mucopolysaccharides, leading to clefts of the secondary palate in mice offspring, (LARSSON,1962; JACOBS,1964b) mesenchyme in shelves treated with this teratogen might be expected to have less glycogen. Of course, the interference with mucopolysaccharide synthesis could occur somewhere beyond formation and utilization of glycogen by degradation to glucose. However, if cortisone were to act directly
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on the mesenchymal cells, as is suggested by some investigators, (MEYER, 1960; BCXXROM and RODEN,1961) alteration in glycogen synthesis might be expected. The findings reported here reveal that the mesenchymal cells of normal animals were heavily ladened with glycogen during the shelf transposition stage, whereas those mesenchymal cells under the influence of cortisone were relatively free of this polysaccharide. Whether the decrease of glycogen in the mesenchymal cells of cortisone treated shelves is a reflection of the inactivity of the shelf and therefore of the lessened energy requirements or whether its absence results in less availability of glucose for incorporation into mucopolysaccharide production, thereby leading to reduction of internal shelf force, is still a matter of conjecture. In the second investigation based on Pourtois’ conclusions that the rat palatal process is endowed at approximately 144 to 15Q days with a fusion potential, any morphologic changes at the ultrastructure level taking place at this critical time could be of importance. Although Pourtois infers the importance of mesenchymal influence upon the epithelium during the fusion process, embryos studied at age groups spanning this time period revealed no morphologic differences in mesenchymal cells. Absence of visible differences in the mesenchyme in our studies does not rule out the possibility that differences do exist, so further experimentation on this problem is under way. However, two rather obvious discrepancies in the epithelium itself are evident between animals of the 14Q day and the 154 day age groups, the most striking being the reduction of glycogen during the one day period from the younger to the older animals. The glycogen is possibly utilized, after a signal is received in uivo,to produce a change in the epithelium which ultimately makes it suitable for fusion. Should the extensive amount of glycogen still be present in the epithelial cells when palatal processes are dissected out and placed in culture, as is the case in the 144 day cultured shelves reported by Pourtois, glycogen utilization and possibly fusion potentiality may never be transferred. Shelves which have been in culture for the period of time after 14+ days would have to be examined at the ultrastructure level as a logical next step to test this hypothesis. The disappearance of glycogen prior to 15g days may be simply a reflection of the maturation process of epithelial cells in this area. Therefore, another explanation for the inability of the younger shelves to fuse is that the shelf which is removed prior to glycogen utilization may not be differentiated sufficiently to complete the epithelial autolytic process necessary in secondary palate formation (SMILEYand DIXON, 1967; FARBMAN,1967). Nonetheless, as in the case of the mouse experiments, the exact function of glycogen during this critical period remains unknown. The other major differences between the epithelial surfaces of the two age groups is the quantity of desmosomes joining the cells. More desmosomes were seen in the older age group. The importance of the desmosome in the fusion process has been elaborated upon in a previous paper which shows that, on contact of the palatine processes, a desmosome union is made (DEANGELISand NALBANDIAN,1968). This desmosome chain, along the junction of the shelves, was shown to be a tenacious bond during completion of the fusion process and ultimate mesenchymal replacement.
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Acknowledgements-The author is greatly indebted to Dr. J. NALBANDIAN for his critical review of the manuscript and to Miss R. GILLESPIE and Mr. A. L. PEELER,II for their technical assistance. This work was supported by research grant no. DE 01766 from the National Institute of Dental Research, U.S.P.H.S. R&n&--L’etude ultrastructurale des lames palatines d’embryons de souris demontre la presence de reserves de glycogene dans le cytoplasme des cellules mesenchymateuses, pendant le stade de la formation du palais secondaire. Des lames d’embryons, trait& par la cortisone, presentent moins de glycogene cellulaire a un stade similaire. Ces resultats suggerent l’existence d’un rapport entre le glycogene et la force intrinseque, responsable de la transposition des lames ainsi qu’une action locale possible de la cortisone pendant la formation d’une division palatine. Au cours d’une autre etude, ii apparait, au point de vue ultrastructural, que les lames palatines d’embryons de rat, qui se fusionnent normalement entre le 16cme et Ie 16&me jour et demi, presentent une diminution caracteristique du glycogene des cellules epitheliales ainsi qu’une augmentation des jonctions desmosomiques intercellulaires dans le groupe d%ge plus avance. Etant donne que les lames de ce demier groupe se fusionnent plus facilement en culture de tissus, il semble que ces resultats indiquent un rapport positif entre l’utilisation de glycogene et le pouvoir de fusion, ainsi qu’un rapport entre le developpement des desmosomes et la frequence de la fusion. Znsammenfassung-Die Ultrastruktur der Gaumenfortsltze normaler Mauseembryonen deckte die Gegenwart von Glykogen-Reservoiren im Zytoplasma mesenchymaler Z&en wlhrend des Stadiums der Gaumenfortsatz-Transposition auf. Die Gaumenfortsatze von cortisonbehandelten Embryonen besal3en dagegen wahrend eines vergleichbaren Zeitraumes der Entwicklung deutlich weniger Zellglykogen im Mesenchym. Diese Beobachtungen deuten aufeine Beziehung zwischen Glykogen und den inneren Kraftenhin, die flir die Fortsatztransposition wie such flir eine mijgliche lokale Wirkung von Cortison bei der Induktion von Gaumenspalten verantwortlich sind. Weitere ultrastrukturelle Untersuchungen ilber die Gaumenfortsltze von Rattenembryonen, die sich normalerweise im Alter von 16 bis 164 Tagen vereinigen, lieBen einen bemerkenswerten Verlust des epithelialen Glykogengehaltes im Embryonalalter zwischen 14+ und 154 Tagen erkennen. In gleicher Weise war bei der Ilteren Gruppe eine Steigerung der desmosomalen Bindungen zwischen den Zellen zu bemerken. Da die Fortsltze der alteren Gruppe in der Kultur ein griiberes Fusionspotential besitzen, deuten diese Befunde eine positive Korrelation zwischen der Glykogenverwertung und dem Fusionspotential wie such eine Bhnliche Korrelation zwischen der Entwicklungvon Desmosomen und dem Zustandekommen der Fusion an.
REFERENCES
ANGELICI,D. R. 1969. Reopening of fused palatal shelves at epithelial interfaces in A/Jax mouse embryos. Cleft Palate Journal in press.
BOSTROM, H. and RODEN,L. 1961. On the metabolism of mucupolysaccharides.
Biochem. Pharmac.
6, 100-123.
BURSTON, W. R. 1959. The development of cleft lip and palate. Ann. R. Coil. Surg. 25, 225-238. DEANGELIS, V. and NALBANDIAN, J. 1968. Ultrastructure of mouse and rat palatal processes prior to and during secondary palate formation.
Arch. oral Biol. 13, 60-608.
FARBMAN, A. I. 1967. Electron microscope study of palatal fusion in mouse embryos. Internat. Ass. for Dent. Res., Preprinted abstracts, 45th General Meeting, Abstract 336. JACOBS, R. M. 1964a. Histochemieal study of morphogenesis and teratogenesis in the palate in mouse embryos. Anat. Rec. 149, 691-698. JACOBS,R. M. 1964b. Ss5-liquid-scintillation count analysis of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec. 150, 271-278.
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LARSSON,K. S. 1960. Studies on the closure of the secondary palate. II. Occurrence of the sulphomucopolysaccharides in the palatine processes of the normal mouse embryo. Expl cell Res. 21, 498-503. LARSSON,K. S. 1962. Closure of the secondary palate and its relation to sulpho mucopolysaccharides. Acta odont. stand. 20, 5-35. LUFT, J. H. 1961. Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409414. MEYER, K. 1960. Nature and function of mucopolysaccharides of connective tissue. In: Molecular Biology (Edited by NACHMANSOHN, D.) pp. 69-76, Academic Press, New York. MILLONIG,G. 1961. Advantage of a phosphate buffer for OsO., solutions in fixation. J. appl. Phys. 32. 1637. PETER,‘K. 1924. Die Entwicklung des Saugetiergaumens. Ergebn. Anat. Entw. Gesch. 25, 448-564. POURTOIS,M. 1966. Onset of the acouired uotentialitv for fusion in the nalatal shelves of rats. _ J. EmbryoI. exp. Morph. 16, 171-82. _ REYNOLDS,E. S. 1962. The use of lead citrate at high pH as on electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SMILEY,G. R. and DD(ON, A. D. 1967. Fine structure of embryonic midline palatal epithelium. Internat. Ass. for Dent. Res., Preprinted abstracts, 45th General Meeting, Abstract 258. WALKER, B. E. and FRASER,F. C. 1956. Closure of the secondary palate in three strains of mice. J. Embryol. exp. Morph. 4, 176-89. WALKER,B. E. and FRASER,F. C. 1957. The embryology of cortisone induced cleft palate. J. Embryol. exp. Morph. 5,201-209. WALKER,B. E. 1961. The association of mucopolysaccharides with morphogenesis of the palate and other structures in mouse embryos. J. Embryol. exp. Morph. 9, 22-31.
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PLATE1 FIG. 1. Mouse palatal process in vertical position lateral to tongue prior to its transposition to the horizontal plane. Haematoxylin and eosin. x 148. FIG. 2. Outlined shelf area from Fig. 1 shows two epithelial cell layers with underlying mesenchyme separated by a thin continuous basal lamina. (a) Flattened surface cell in outer epithelial cell layer, (b) cubiodal inner epithelial surface layer, (c) basal lamina, (d) mesenchymal cell process. x 12,000. FIG. 3. Palatal shelf surface of a 144 day mouse embryo illustrates areas of glycogen within the cuboidal epithelial cell layer during transposition to the horizontal. (a) Glycogen, (b) mitochondrion, (c) desmosome. x 31,150.
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PLATE 2 FIG. 4. Epithelial-mesenchymal junction at the shelf tip of a 144 day mouse embryo shows glycogen reservoirs within the cells on both sides of the basal lamina. (See arrow Fig. 1) (a) Basal lamina, (b) glycogen in epithelial cell, (c) glycogen in mesenchymal cells, (d) collagen fibrils of various sizes dispersed throughout the extra-cellular spaces within the mesenchyme. x 11,900. Fro. 5. Mesenchymal cells in the shelf of 144 day mouse embryo are laden with glycogen. (a) Glycogen, (b) lysosomes, (c) mitochondrion, (d) collagen fibrils in longitudinal and cross section. x 21,500.
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PLATE 3 FIG. 6. Mesenchymal cell at shelf tip of a 14) day cortisone treated mouse depicts the marked decrease of glycogen within the cytoplasm. (a) Mitochondria, (b) network of coated endoplasmic reticulum, (c) golgi x 41,300. FIG. 7. Mesenchymal cell of an untreated 144 day mouse embryo from a comparable area as in Fig. 6. Note abundance of glycogen rosettes displacing cell organelles. (a) Glycogen, (b) coated endoplasmic reticulum, (c) mitochondria. x 41,000.
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PLATE 4 FIG. 8. Palatal shelf tip of a 149 day rat embryo illustrates reservoirs of glycogen within the cells of the epithelial surface destined to fuse at approximately 16 days. (a) Glycogen, (b) lysosome, (c) basal lamina. x 19,110. FIG.
9. Area comparable to Fig. 8 of palatal shelf tip in 15) day rat embryo shows that glycogen is much less evident within these more differentiated epithelial cells. (a) Polyribosomes, (b) mitochondria, (c) desmosome. x 22,190.
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