Palate Development after Fetal Tongue Removal in Cortisone-treated Mice BRUCE E WALKER AND ANITA PATTERSON Department o f d n a t o m y , Mtchrgan State Uniuersity, East Lansing, Michigan 48824
ABSTRACT
Morphological studies of cortisone-induced cleft palate have shown retardation in the rotation of palatine shelves from a sagittal to a transverse plane. Cortisone also reduces fetal muscular movements, which may explain why displacement of the tongue from between the palatine shelves is delayed. Previous work with extrauterine development of control fetuses demonstrated t h a t fetal membranes and tongue were major obstacles to shelf rotation. Thus, removal of these obstacles might permit rotation and fusion of palatine shelves in cortisone-treated fetuses. In t h e present experiment, fetuses from cortisone-treated strain CD-1 mice were released from uterus and membranes and allowed to develop for eight hours in a fluid medium with the umbilical cord left intact. Compared to 4% fusion in utero, there was palatal fusion in 20% of fetuses released from membranes. When t h e fetal tongue was removed during extrauterine development, the frequency of fusions increased to 61%.Fusion appeared normal by the criteria applicable through light microscopy. Thus, cortisone induces cleft palate primarily through interference with shelf rotation. The palatine shelves of treated fetuses retain their ability to fuse when they can come in contact during the normal time for palate closure.
Transposition of palatine shelves from a sagittal to a transverse plane is delayed in fetuses of cortisone-treated strain A/J mice (Walker and Fraser, '57). The resulting cleft palates consist of unfused palatine shelves in the various possible combinations of shelf position relative to these two planes (Walker and Grain, '60). Since both shelves eventually do reach a transverse plane in some fetuses yet do not fuse, the possibility t h a t cortisone interferes with the events involved in fusion has been studied (Greene and Kochhar, '73, '75). Thus, cortisone has a known effect of delaying shelf transposition and a possible additional effect involving fusion. One hypothesis proposed to explain the former effect is t h a t the cortisone acts adversely on fetal skeletal muscle (Walker, '71). Fetal movements are decreased by anti-inflammatory steroids (Walker and Quarles, '75) and this could retard displacement of the tongue from between t h e palatine shelves, thus blocking transposition of t h e shelves. If t h e fetal tongue could be removed after cortisone treatment, this hypothesis could be tested. A method has been devised to permit tongue removal TERATOLOGY (1978)17: 51-56.
with a sufficient survival time after the operation for palatine shelves to move from a sagittal to a transverse plane and fuse in fetuses not treated with a teratogen (Walker and Quarles, '76). Application of this technique to cortisone-treated fetuses should permit testing of the ability of their palatine shelves to move and to fuse in the absence of a tongue (Walker and Patterson, '74). MATERIALS AND METHODS
Strain CD-1 mice were given free access to water and food (Wayne Mouse Breeder Blox) and maintained on a reverse light cycle with t h e dark period extending from 3 A.M. to 1 P.M. Ovulation was calculated to occur at 10 A.M. (Snell e t al., '40), so mice were mated between 8 A.M. and 12 noon. Females with vaginal plugs were assumed to have conceived at the time of ovulation, but were palpated 11 days later to confirm pregnancy. One hundred and two pregnant mice were injected intramuscularly with 2.5 mg cortisone acetate daily for three Received June 20, '17.Accepted Sept. 23, '17. I This investigation was supported by the National lnstitute of Dental Research, NIH, Research Grant R01 DE 2852.
51
52
BRUCE E. WALKER AND ANITA PATTERSON
successive days starting 11 days postconception. Ten litters were collected at 17 days to estimate the frequency of cleft palate produced by this dose in the CD-1 strain. The remaining mice were used to study the effect of fetal tongue removal. At 13 days, 22 hours each pregnant mouse was etherized briefly to perform a spinal cord section between the third and fourth lumbar vertebrae. The uterine wall and fetal membranes were incised to deliver t h e fetuses into the fluid medium without transecting the umbilical cord (Walker and Quarles, '76). Litters in which all fetuses had a n estimated MR (morphological rating: Walker and Crain, '60) of less t h a n 6 were discarded. In remaining litters, fetuses a t palate stage 1 (Walker and Fraser, '56) and an estimated MR6 or greater were subjected to tongue excision, or identified as tongue-intact controls. Heartbeat was monitored at intervals of about 30 minutes and each fetus was removed and fixed in Bouin's fluid when its heart was no longer beating. After eight hours in the fluid medium, all fetuses still alive were removed and fixed. After fixation, each fetus was weighed and studied with a dissecting microscope to establish a n MR and palate stage. In some fetuses subject to tongue excision, a blood clot obscured the medial edges of shelves in a transverse plane. Such fetuses were evaluated further by cutting through the head with a scalpel in a coronal plane a t a level passing through the rostra1 edge of each eye. The cut edges were viewed to decide whether the shelves were in contact. Selected heads were embedded in a butoxyethanol-glycol methacrylate mixture (Polysciences), sectioned at 2 on a JB-4-microtome (Sorvall), and stained with hematoxylin and eosin.
Fig. 1 Palate area in a cortisone-treated fetus kept in a fluid medium for eight hours after removal of the tongue. The shelves are fused together throughout most of their length. However, the caudal ends of the shelves are vertical, presumably because the remaining stub of the excised tongue was wedged between them. X 14.
RESULTS
The ten litters collected 17 days postconception to assess cleft palate frequency contained 5 fetuses with fused palates and 109 fetuses with open palates. Among t h e latter, 6 had both shelves in a sagittal plane (stage l),56 had both shelves in a transverse plane with a gap between them (stage 4), and t h e remaining 47 had one shelf in each plane (stage 3). Among the remaining 92 cortisone-treated mice, 11 had resorbed litters, 17 died during the operative procedures, 6 died while partially submerged in the fluid medium, and 30 were discarded because their fetuses all had a n MR < 6. Thus, 28 pregnant mice had litters
Fig. 2 Triangle area and midline seam of a palate in a plastic embedded fetal head. The shelves had been in a sagittal plane (stage 1) when the fetus was released from the uterus and subjected to tongue excision. Eight hours later, the shelves were in contact throughout most of their length. This section demonstrates that the opposing epithelial layers had fused and the resulting midline seam had become discontinuous in some place. X 470.
53
PALATE DEVELOPMENT AFTER CORTISONE TABLE 1
Correlation ofpalate development with development ofexternal features (MR) in cortisone-treated fetuses after eight hours ofertrauterine deuelopment. All fetuses listed had an M R above 5 when first released from the uterus Condition of fetal tongue
Intact
No. of open palates (by stages) Development of fetus (MR)
1
3
4
5
No. of fused palates (stage 61
6
7 8 9 10 11 12 13 Excised
1 19 1 3
2
1
2 3
5
6
1
1
1 1
1 9 1 1
2 3 1
1 10
6
7 8
9 10 11 12 13
with a t least some fetuses suitable for study. Among these fetuses, 291 met the criteria of MR > 5 and palates a t stage 1 when first released into the fluid medium. Tongue excision was performed on 141 fetuses. At the end of 8 hours extrauterine development, 46 of these were still alive, and 46 of the 150 fetuses used as tongue-intact controls were alive. Palate fusion (figs. l, 2) had occurred in 28 of the former, but only 9 of the latter (x2 = 16.3, p < 0.001). However, even this lower number in the tongue-intact group represents a higher frequency of fusion than occurred in cortisone-treated fetuses developing in utero (xz = 9.58, p < 0.01). Designating a palate as fused was based on two criteria. While observing the palate through a dissecting microscope, light pressure was exerted laterally on each cheek to make sure the shelves would not separate. Secondly, a ridge of lighter colored tissue was sought at the midline junction of the two shelves as evidence for an aggregation of epithelial cells (fig. 2) produced by the fusing epithelia (Smiley and Dixon, '68). In doubtful cases, as when a blood clot obscured the junction, a coronal cut was made through the head to see whether the shelves were in contact. When a blood clot was present, it usually was found to extend between the shelves so t h a t there was no fusion. Examination of 85 stage 4 palates from tongue-excised fetuses revealed t h a t 71 had blood clots between the shelves.
4 4 5 3
Among fetuses surviving eight hours after tongue removal (table l ) ,there was a greater proportion of fused palates with an MR > 9 than with a n MR S 9 ( x 2 = 7.4, p < 0.01).The effect of survival time on fusion in the same group of fetuses (table 2) was pronounced (r = f 0 . 6 5 , p < 0.001), with the majority of fusions occurring in fetuses still alive a t eight hours. Whereas 124 out of 150 fetuses were a t palate stage 1 after varying periods in the fluid medium without tongue removal, only one fetus was a t stage 1 after tongue removal (table 2). The latter fetus was found to have undergone relatively incomplete tongue removal, so t h a t a long remnant of tongue was still wedged between the palatine shelves. In many fetuses with the intended extensive tongue removal, the remaining stub lodged between the caudal ends of the shelves. The latter were still vertical even though the rostral portions had fused (fig. 1). In histologic sections of fused palates, the midline seam typically consisted of a n epithelial layer ranging from one to two cells thick, with a n occasional gap (fig. 2). Towards the rostral end of the palate, where it normally fuses with the nasal septum, there was a substantial gap between the palate and nasal septum as described previously (Walker and Quarles, '76). This was accompanied by a flat ventral surface contour of the palate, instead
54
BRUCE E. WALKER AND ANITA PATTERSON TABLE 2
Correlation of palate stage with hours of development outside uterus and membranes in cortisone-treated fetuses. In the upper group designated T i the tongue w a s left intact. In the lower group, T; the fetal tongue had been removed 2
6
3
9
5
1
CJ M
a
2
4
1
I
21
1
34
19
1
1
1
6
8
10
2
29
4
1
3
6
9
4
2
12
7
8
I
1 5
3
w
2 4
1
1
2
37
10
3 1 1
I
1
2
3
4
5
6
Hours released
of the arched condition seen when the tongue was present. DISCUSSION
Jacobs ('64a) reported a 90% frequency of cleft palate among offspring of CD-1 mice treated with cortisone, and the incidence of clefts in the work presented here is consistent with his report. The high frequency of intermediate stages in palate closure persisting until 17 days postconception indicates that cortisone treatment interferes with shelf rotation, as proposed by Jacobs ('64b) for this strain. However, compared to cortisone treated strain A/J mice (Walker and Fraser, '57), the effect was a little less severe both in terms of cleft palate frequency (96% in CD-1 vs. 100% in AIJ) and of interference with shelf rotation (average palate stage of 0.63 in CD-1 vs. 0.51 in AIJ reported by Walker and Crain, '60). Extrauterine survival rate was similar in
cortisone-treated fetuses with, and without tongue removal (33% and 31%, respectively). These rates are comparable to the extrauterine survival rate of control fetuses after tonge incision (32%), but less than control fetuses without tongue removal (Walker and Quarles, '76). Thus, cortisone did not have a pronounced deleterious effect on extrauterine survival. The tongue is an obstacle to shelf rotation in cortisone-treated fetuses, since significantly more palates fused after tongue excision than when the tongue was left intact. Since fusion was significantly more frequent in tongue-intact fetuses after release from the uterus and fetal membranes than after development in utero, the confining properties of the membranes also provide an obstacle to palate closure. Even when released from the limiting effects of tongue and membranes, the cortisone-treated fetuses may have a less effective closure mechanism than normal. In a previous study of control fetuses, there were 84%fused palates in extrauterine development after tongue removal (Walker and Quarles, '761, compared to 61% in cortisone-treated fetuses (x2 = 6.6, p < 0.02). Although this difference is of borderline significance, the difference between tongue-intact fetuses in these two studies (56%fused in controls vs. 20% in cortisonetreated fetuses; 'x = 13.5, p < 0.001) demonstrates a pronounced handicap for the cortisone-treated fetuses. This observed deficiency is consistent with the hypothesis that muscular movements are necessary to displace the tongue from between the palatine shelves (Walker, '691, and that the deficiency of muscular movements observed in cortisonetreated fetuses (Walker and Quarles, '75) results in delayed shelf rotation. However, it does not rule out comparable hypotheses involving active participation by the palatine shelves (Lessard et al., '74). Evidence for involvement of the neuromuscular system in normal palate development has been circumstantial and controversial (Greene and Pratt, '76; Walker and Quarles, '76). Although these results with cortisone lend further support to the hypothesis of neuromuscular involvement, the evidence remains inconclusive. Fusion in cortisone-treated fetuses developing outside the uterus is relatively complete, including the merging and thinning of opposing epithelial layers. The normal nature of this fusion is further supported by preliminary electron microscopic observations
PALATE DEVELOPMENT AFTER CORTISONE
(Walker and Cunningham, '76). These results are consistent with observations in vitro which indicated ability of palatine shelves from hydrocortisone or cortisone-treated fetuses to fuse when they were brought in contact (Chaudry and Siar, '67; Lahti e t al., '72; Saxen, '73). Greene and Kochhar ('73) reported lack of fusion between shelves in contact after cortisone treatment. However, they described contact at 12-14 hours after t h e normal time of fusion, whereas the fusion described here occurred during a developmental stage (mainly, MR 9-12) corresponding to t h a t of control fetuses undergoing palate closure in utero (Walker, '74). The question of how long palatine shelves maintain a capacity to fuse is still controversial for control fetuses, although there is evidence indicating t h a t a capacity for fusion extends about 24 hours beyond normal fusion time (Goss e t al., '70; Ross and Gibson, '75; Walker and Sohn, '75; Morgan, '76). Considering t h e t i m e span over which fetuses were collected during extrauterine development, the lack of stage 1 palates after tongue excision shows t h a t the shelves moved towards a transverse plane relatively quickly. Manipulation of the shelves during tongue removal cannot be ruled out a s a factor in this rapid conversion from stage 1to stage 4. Subsequent flattening towards the midline and eventual contact may have required considerably more time, since the majority of fusions occurred in fetuses still alive at seven to eight hours. Another possible reason for the large number of s t a g e 4 palates among tongue-excised fetuses is t h a t the blood clot seen in 84% of them may have blocked t h e shelves from coming together and fusing. The abnormal contour of the palate (flattened instead of arched) i n t h e absence of a tongue reflects t h e molding action of the tongue (Walker and Quarles, '76). Whether the rotation and flattening of t h e shelves is brought about by upward pressure from the mandible in the absence of a tongue, or by some other force, is not resolved by these experiments.
55
Greene, R. M., and D. M. Kochhar 1973 Spatial relations in the oral cavity of cortisone-treated mouse fetuses during the time of secondary palate closure. Teratology, 8: 153-162. 1975 Some aspects of corticosteroid-induced cleft palate: a review. Teratology, 11: 47-56. Greene, R. M., and R. M. Pra tt 1976 Developmental aspects of secondary palate formation. J. Embryol. exp. Morph., 36: 225-245. Jacobs, R. M. 1964a Preliminary survey of the effects of cortisone upon palate formation, litter size, and fetal weight in CD-1 strain of mice. J. Dent. Res., 43; 715. 1964b Histochemical study of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec., 149: 691-697. Lahti, A., E. Antila and L. Saxen 1972 The effect of hydrocortisone on the closure of the palatal shelves in two inbred strains of mice in vivo and in vitro. Teratology, 6: 37-42. Lessard, J. L., E. L. Wee and E. F. Zimmerman 1974 Presence of contractile proteins in mouse fetal palate prior to shelf elevation. Teratology, 9: 113-126. Morgan, P. R. 1976 The fate of the expected fusion zone in rat fetuses with experimentally-induced cleft palate - an ultrastructural study. Devel. Biol., 51: 225-240. Ross, L. M., and M. H. L. Gibson 1975 An S. E.M. study of cleft palate after amniotic sac puncture in mice. Teratology, 11: 31A (Abstract). Saxen, I. 1973 Effects of hydrocortisone on the development in vitro of the secondary palate in two inbred strains of mice. Archs. oral Biol., 18: 1469-1479. Smiley, G . R., and A. D. Dixon 1968 Fine structure of midline epithelium in the developing palate of the mouse. Anat. Rec., 161: 293-310. Snell, G. D., E. Fekete, K. P. Hummel and L. W. Law 1940 The relation of mating, ovulation and the estrous smear in the house mouse to time of day. Anat. Rec.. 76: 39-54. Walker, B. E. 1969 Correlation of embryonic movement with palate closure in mice. Teratology, 2: 191-198. - 1971 Induction of cleft palate in rats with antiinflammatory drugs. Teratology, 39-42. Walker, B. E. 1974 Palate closure in strain CD-1 mice. J. Dent. Res., 53: 1497. Walker, B. E., and B. Crain, Jr. 1960 Effects of hypervitaminosis A on palate development in two strains of mice. Am. J. Anat., 107: 49-58. Walker, B. E., and D. Cunningham 1976 Palate ultrastructure in extrauterine development of control and cortisone-treated fetuses. J. Dent. Res., 55: B202 (Abstract). Walker, B. E., and F. C. Fraser 1956 Closure of secondary palate in three strains of mice. J. Embryol. exp. Morph., 4: 176-189. 1957 The embryology of cortisone-induced cleft palate. J. Embryol. exp. Morph., 5: 201-209. Walker, B. E., and A. Patterson 1974 The mechanism of cortisone-induced cleft palate. J. Dent. Res., 53: 63 (Abstract). Walker, B.E., and J. Quarles 1975 Effects of anti-inflammatory steroids on mouse embryonic movements during LITERATURE CITED palatal development. J. Dent. Res., 54: 1200-1206. 1976 Palate development in mouse foetuses Chaudhrey, A. P., and S. Siar 1967 In vitro study of fusion after tongue removal. Archs. oral Biol., 21: 405-412. of palatal shelves in A/Jax mouse embryos. J. Dent. Res., 46: 257-260. Walker.. B. E... and R. Sohn 1975 Palatine shelf fusion after Goss, A. N., J. W. Bodner and J. K. Avery 1970 In vitro amniotic sac puncture and tongue displacement. J. Dent. fusion of cleft palate shelves. Cleft Palate J., 7: 737-747. Res., 54 (NSIA): 82 (Abstract).
-
ABSTRACT
Morphological studies of cortisone-induced cleft palate have shown retardation in the rotation of palatine shelves from a sagittal to a transverse plane. Cortisone also reduces fetal muscular movements, which may explain why displacement of the tongue from between the palatine shelves is delayed. Previous work with extrauterine development of control fetuses demonstrated t h a t fetal membranes and tongue were major obstacles to shelf rotation. Thus, removal of these obstacles might permit rotation and fusion of palatine shelves in cortisone-treated fetuses. In t h e present experiment, fetuses from cortisone-treated strain CD-1 mice were released from uterus and membranes and allowed to develop for eight hours in a fluid medium with the umbilical cord left intact. Compared to 4% fusion in utero, there was palatal fusion in 20% of fetuses released from membranes. When t h e fetal tongue was removed during extrauterine development, the frequency of fusions increased to 61%.Fusion appeared normal by the criteria applicable through light microscopy. Thus, cortisone induces cleft palate primarily through interference with shelf rotation. The palatine shelves of treated fetuses retain their ability to fuse when they can come in contact during the normal time for palate closure.
Transposition of palatine shelves from a sagittal to a transverse plane is delayed in fetuses of cortisone-treated strain A/J mice (Walker and Fraser, '57). The resulting cleft palates consist of unfused palatine shelves in the various possible combinations of shelf position relative to these two planes (Walker and Grain, '60). Since both shelves eventually do reach a transverse plane in some fetuses yet do not fuse, the possibility t h a t cortisone interferes with the events involved in fusion has been studied (Greene and Kochhar, '73, '75). Thus, cortisone has a known effect of delaying shelf transposition and a possible additional effect involving fusion. One hypothesis proposed to explain the former effect is t h a t the cortisone acts adversely on fetal skeletal muscle (Walker, '71). Fetal movements are decreased by anti-inflammatory steroids (Walker and Quarles, '75) and this could retard displacement of the tongue from between t h e palatine shelves, thus blocking transposition of t h e shelves. If t h e fetal tongue could be removed after cortisone treatment, this hypothesis could be tested. A method has been devised to permit tongue removal TERATOLOGY (1978)17: 51-56.
with a sufficient survival time after the operation for palatine shelves to move from a sagittal to a transverse plane and fuse in fetuses not treated with a teratogen (Walker and Quarles, '76). Application of this technique to cortisone-treated fetuses should permit testing of the ability of their palatine shelves to move and to fuse in the absence of a tongue (Walker and Patterson, '74). MATERIALS AND METHODS
Strain CD-1 mice were given free access to water and food (Wayne Mouse Breeder Blox) and maintained on a reverse light cycle with t h e dark period extending from 3 A.M. to 1 P.M. Ovulation was calculated to occur at 10 A.M. (Snell e t al., '40), so mice were mated between 8 A.M. and 12 noon. Females with vaginal plugs were assumed to have conceived at the time of ovulation, but were palpated 11 days later to confirm pregnancy. One hundred and two pregnant mice were injected intramuscularly with 2.5 mg cortisone acetate daily for three Received June 20, '17.Accepted Sept. 23, '17. I This investigation was supported by the National lnstitute of Dental Research, NIH, Research Grant R01 DE 2852.
51
52
BRUCE E. WALKER AND ANITA PATTERSON
successive days starting 11 days postconception. Ten litters were collected at 17 days to estimate the frequency of cleft palate produced by this dose in the CD-1 strain. The remaining mice were used to study the effect of fetal tongue removal. At 13 days, 22 hours each pregnant mouse was etherized briefly to perform a spinal cord section between the third and fourth lumbar vertebrae. The uterine wall and fetal membranes were incised to deliver t h e fetuses into the fluid medium without transecting the umbilical cord (Walker and Quarles, '76). Litters in which all fetuses had a n estimated MR (morphological rating: Walker and Crain, '60) of less t h a n 6 were discarded. In remaining litters, fetuses a t palate stage 1 (Walker and Fraser, '56) and an estimated MR6 or greater were subjected to tongue excision, or identified as tongue-intact controls. Heartbeat was monitored at intervals of about 30 minutes and each fetus was removed and fixed in Bouin's fluid when its heart was no longer beating. After eight hours in the fluid medium, all fetuses still alive were removed and fixed. After fixation, each fetus was weighed and studied with a dissecting microscope to establish a n MR and palate stage. In some fetuses subject to tongue excision, a blood clot obscured the medial edges of shelves in a transverse plane. Such fetuses were evaluated further by cutting through the head with a scalpel in a coronal plane a t a level passing through the rostra1 edge of each eye. The cut edges were viewed to decide whether the shelves were in contact. Selected heads were embedded in a butoxyethanol-glycol methacrylate mixture (Polysciences), sectioned at 2 on a JB-4-microtome (Sorvall), and stained with hematoxylin and eosin.
Fig. 1 Palate area in a cortisone-treated fetus kept in a fluid medium for eight hours after removal of the tongue. The shelves are fused together throughout most of their length. However, the caudal ends of the shelves are vertical, presumably because the remaining stub of the excised tongue was wedged between them. X 14.
RESULTS
The ten litters collected 17 days postconception to assess cleft palate frequency contained 5 fetuses with fused palates and 109 fetuses with open palates. Among t h e latter, 6 had both shelves in a sagittal plane (stage l),56 had both shelves in a transverse plane with a gap between them (stage 4), and t h e remaining 47 had one shelf in each plane (stage 3). Among the remaining 92 cortisone-treated mice, 11 had resorbed litters, 17 died during the operative procedures, 6 died while partially submerged in the fluid medium, and 30 were discarded because their fetuses all had a n MR < 6. Thus, 28 pregnant mice had litters
Fig. 2 Triangle area and midline seam of a palate in a plastic embedded fetal head. The shelves had been in a sagittal plane (stage 1) when the fetus was released from the uterus and subjected to tongue excision. Eight hours later, the shelves were in contact throughout most of their length. This section demonstrates that the opposing epithelial layers had fused and the resulting midline seam had become discontinuous in some place. X 470.
53
PALATE DEVELOPMENT AFTER CORTISONE TABLE 1
Correlation ofpalate development with development ofexternal features (MR) in cortisone-treated fetuses after eight hours ofertrauterine deuelopment. All fetuses listed had an M R above 5 when first released from the uterus Condition of fetal tongue
Intact
No. of open palates (by stages) Development of fetus (MR)
1
3
4
5
No. of fused palates (stage 61
6
7 8 9 10 11 12 13 Excised
1 19 1 3
2
1
2 3
5
6
1
1
1 1
1 9 1 1
2 3 1
1 10
6
7 8
9 10 11 12 13
with a t least some fetuses suitable for study. Among these fetuses, 291 met the criteria of MR > 5 and palates a t stage 1 when first released into the fluid medium. Tongue excision was performed on 141 fetuses. At the end of 8 hours extrauterine development, 46 of these were still alive, and 46 of the 150 fetuses used as tongue-intact controls were alive. Palate fusion (figs. l, 2) had occurred in 28 of the former, but only 9 of the latter (x2 = 16.3, p < 0.001). However, even this lower number in the tongue-intact group represents a higher frequency of fusion than occurred in cortisone-treated fetuses developing in utero (xz = 9.58, p < 0.01). Designating a palate as fused was based on two criteria. While observing the palate through a dissecting microscope, light pressure was exerted laterally on each cheek to make sure the shelves would not separate. Secondly, a ridge of lighter colored tissue was sought at the midline junction of the two shelves as evidence for an aggregation of epithelial cells (fig. 2) produced by the fusing epithelia (Smiley and Dixon, '68). In doubtful cases, as when a blood clot obscured the junction, a coronal cut was made through the head to see whether the shelves were in contact. When a blood clot was present, it usually was found to extend between the shelves so t h a t there was no fusion. Examination of 85 stage 4 palates from tongue-excised fetuses revealed t h a t 71 had blood clots between the shelves.
4 4 5 3
Among fetuses surviving eight hours after tongue removal (table l ) ,there was a greater proportion of fused palates with an MR > 9 than with a n MR S 9 ( x 2 = 7.4, p < 0.01).The effect of survival time on fusion in the same group of fetuses (table 2) was pronounced (r = f 0 . 6 5 , p < 0.001), with the majority of fusions occurring in fetuses still alive a t eight hours. Whereas 124 out of 150 fetuses were a t palate stage 1 after varying periods in the fluid medium without tongue removal, only one fetus was a t stage 1 after tongue removal (table 2). The latter fetus was found to have undergone relatively incomplete tongue removal, so t h a t a long remnant of tongue was still wedged between the palatine shelves. In many fetuses with the intended extensive tongue removal, the remaining stub lodged between the caudal ends of the shelves. The latter were still vertical even though the rostral portions had fused (fig. 1). In histologic sections of fused palates, the midline seam typically consisted of a n epithelial layer ranging from one to two cells thick, with a n occasional gap (fig. 2). Towards the rostral end of the palate, where it normally fuses with the nasal septum, there was a substantial gap between the palate and nasal septum as described previously (Walker and Quarles, '76). This was accompanied by a flat ventral surface contour of the palate, instead
54
BRUCE E. WALKER AND ANITA PATTERSON TABLE 2
Correlation of palate stage with hours of development outside uterus and membranes in cortisone-treated fetuses. In the upper group designated T i the tongue w a s left intact. In the lower group, T; the fetal tongue had been removed 2
6
3
9
5
1
CJ M
a
2
4
1
I
21
1
34
19
1
1
1
6
8
10
2
29
4
1
3
6
9
4
2
12
7
8
I
1 5
3
w
2 4
1
1
2
37
10
3 1 1
I
1
2
3
4
5
6
Hours released
of the arched condition seen when the tongue was present. DISCUSSION
Jacobs ('64a) reported a 90% frequency of cleft palate among offspring of CD-1 mice treated with cortisone, and the incidence of clefts in the work presented here is consistent with his report. The high frequency of intermediate stages in palate closure persisting until 17 days postconception indicates that cortisone treatment interferes with shelf rotation, as proposed by Jacobs ('64b) for this strain. However, compared to cortisone treated strain A/J mice (Walker and Fraser, '57), the effect was a little less severe both in terms of cleft palate frequency (96% in CD-1 vs. 100% in AIJ) and of interference with shelf rotation (average palate stage of 0.63 in CD-1 vs. 0.51 in AIJ reported by Walker and Crain, '60). Extrauterine survival rate was similar in
cortisone-treated fetuses with, and without tongue removal (33% and 31%, respectively). These rates are comparable to the extrauterine survival rate of control fetuses after tonge incision (32%), but less than control fetuses without tongue removal (Walker and Quarles, '76). Thus, cortisone did not have a pronounced deleterious effect on extrauterine survival. The tongue is an obstacle to shelf rotation in cortisone-treated fetuses, since significantly more palates fused after tongue excision than when the tongue was left intact. Since fusion was significantly more frequent in tongue-intact fetuses after release from the uterus and fetal membranes than after development in utero, the confining properties of the membranes also provide an obstacle to palate closure. Even when released from the limiting effects of tongue and membranes, the cortisone-treated fetuses may have a less effective closure mechanism than normal. In a previous study of control fetuses, there were 84%fused palates in extrauterine development after tongue removal (Walker and Quarles, '761, compared to 61% in cortisone-treated fetuses (x2 = 6.6, p < 0.02). Although this difference is of borderline significance, the difference between tongue-intact fetuses in these two studies (56%fused in controls vs. 20% in cortisonetreated fetuses; 'x = 13.5, p < 0.001) demonstrates a pronounced handicap for the cortisone-treated fetuses. This observed deficiency is consistent with the hypothesis that muscular movements are necessary to displace the tongue from between the palatine shelves (Walker, '691, and that the deficiency of muscular movements observed in cortisonetreated fetuses (Walker and Quarles, '75) results in delayed shelf rotation. However, it does not rule out comparable hypotheses involving active participation by the palatine shelves (Lessard et al., '74). Evidence for involvement of the neuromuscular system in normal palate development has been circumstantial and controversial (Greene and Pratt, '76; Walker and Quarles, '76). Although these results with cortisone lend further support to the hypothesis of neuromuscular involvement, the evidence remains inconclusive. Fusion in cortisone-treated fetuses developing outside the uterus is relatively complete, including the merging and thinning of opposing epithelial layers. The normal nature of this fusion is further supported by preliminary electron microscopic observations
PALATE DEVELOPMENT AFTER CORTISONE
(Walker and Cunningham, '76). These results are consistent with observations in vitro which indicated ability of palatine shelves from hydrocortisone or cortisone-treated fetuses to fuse when they were brought in contact (Chaudry and Siar, '67; Lahti e t al., '72; Saxen, '73). Greene and Kochhar ('73) reported lack of fusion between shelves in contact after cortisone treatment. However, they described contact at 12-14 hours after t h e normal time of fusion, whereas the fusion described here occurred during a developmental stage (mainly, MR 9-12) corresponding to t h a t of control fetuses undergoing palate closure in utero (Walker, '74). The question of how long palatine shelves maintain a capacity to fuse is still controversial for control fetuses, although there is evidence indicating t h a t a capacity for fusion extends about 24 hours beyond normal fusion time (Goss e t al., '70; Ross and Gibson, '75; Walker and Sohn, '75; Morgan, '76). Considering t h e t i m e span over which fetuses were collected during extrauterine development, the lack of stage 1 palates after tongue excision shows t h a t the shelves moved towards a transverse plane relatively quickly. Manipulation of the shelves during tongue removal cannot be ruled out a s a factor in this rapid conversion from stage 1to stage 4. Subsequent flattening towards the midline and eventual contact may have required considerably more time, since the majority of fusions occurred in fetuses still alive at seven to eight hours. Another possible reason for the large number of s t a g e 4 palates among tongue-excised fetuses is t h a t the blood clot seen in 84% of them may have blocked t h e shelves from coming together and fusing. The abnormal contour of the palate (flattened instead of arched) i n t h e absence of a tongue reflects t h e molding action of the tongue (Walker and Quarles, '76). Whether the rotation and flattening of t h e shelves is brought about by upward pressure from the mandible in the absence of a tongue, or by some other force, is not resolved by these experiments.
55
Greene, R. M., and D. M. Kochhar 1973 Spatial relations in the oral cavity of cortisone-treated mouse fetuses during the time of secondary palate closure. Teratology, 8: 153-162. 1975 Some aspects of corticosteroid-induced cleft palate: a review. Teratology, 11: 47-56. Greene, R. M., and R. M. Pra tt 1976 Developmental aspects of secondary palate formation. J. Embryol. exp. Morph., 36: 225-245. Jacobs, R. M. 1964a Preliminary survey of the effects of cortisone upon palate formation, litter size, and fetal weight in CD-1 strain of mice. J. Dent. Res., 43; 715. 1964b Histochemical study of morphogenesis and teratogenesis of the palate in mouse embryos. Anat. Rec., 149: 691-697. Lahti, A., E. Antila and L. Saxen 1972 The effect of hydrocortisone on the closure of the palatal shelves in two inbred strains of mice in vivo and in vitro. Teratology, 6: 37-42. Lessard, J. L., E. L. Wee and E. F. Zimmerman 1974 Presence of contractile proteins in mouse fetal palate prior to shelf elevation. Teratology, 9: 113-126. Morgan, P. R. 1976 The fate of the expected fusion zone in rat fetuses with experimentally-induced cleft palate - an ultrastructural study. Devel. Biol., 51: 225-240. Ross, L. M., and M. H. L. Gibson 1975 An S. E.M. study of cleft palate after amniotic sac puncture in mice. Teratology, 11: 31A (Abstract). Saxen, I. 1973 Effects of hydrocortisone on the development in vitro of the secondary palate in two inbred strains of mice. Archs. oral Biol., 18: 1469-1479. Smiley, G . R., and A. D. Dixon 1968 Fine structure of midline epithelium in the developing palate of the mouse. Anat. Rec., 161: 293-310. Snell, G. D., E. Fekete, K. P. Hummel and L. W. Law 1940 The relation of mating, ovulation and the estrous smear in the house mouse to time of day. Anat. Rec.. 76: 39-54. Walker, B. E. 1969 Correlation of embryonic movement with palate closure in mice. Teratology, 2: 191-198. - 1971 Induction of cleft palate in rats with antiinflammatory drugs. Teratology, 39-42. Walker, B. E. 1974 Palate closure in strain CD-1 mice. J. Dent. Res., 53: 1497. Walker, B. E., and B. Crain, Jr. 1960 Effects of hypervitaminosis A on palate development in two strains of mice. Am. J. Anat., 107: 49-58. Walker, B. E., and D. Cunningham 1976 Palate ultrastructure in extrauterine development of control and cortisone-treated fetuses. J. Dent. Res., 55: B202 (Abstract). Walker, B. E., and F. C. Fraser 1956 Closure of secondary palate in three strains of mice. J. Embryol. exp. Morph., 4: 176-189. 1957 The embryology of cortisone-induced cleft palate. J. Embryol. exp. Morph., 5: 201-209. Walker, B. E., and A. Patterson 1974 The mechanism of cortisone-induced cleft palate. J. Dent. Res., 53: 63 (Abstract). Walker, B.E., and J. Quarles 1975 Effects of anti-inflammatory steroids on mouse embryonic movements during LITERATURE CITED palatal development. J. Dent. Res., 54: 1200-1206. 1976 Palate development in mouse foetuses Chaudhrey, A. P., and S. Siar 1967 In vitro study of fusion after tongue removal. Archs. oral Biol., 21: 405-412. of palatal shelves in A/Jax mouse embryos. J. Dent. Res., 46: 257-260. Walker.. B. E... and R. Sohn 1975 Palatine shelf fusion after Goss, A. N., J. W. Bodner and J. K. Avery 1970 In vitro amniotic sac puncture and tongue displacement. J. Dent. fusion of cleft palate shelves. Cleft Palate J., 7: 737-747. Res., 54 (NSIA): 82 (Abstract).
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