- Email: [email protected]
Palate shelf movement in mouse embryo culture: Evidence for skeletal and smooth muscle contractility
DEVELOPMENTAL
BIOLOGY
48, 91-103 (1976)
Palate Shelf Movement in Mouse Embryo Culture: Evidence for Skeletal and Smooth Muscle Contractility ELIZABETH Children’s
Hospital
L. WEE, LOIS G. WOLFSON, AND ERNEST F. ZIMMERMAN Research Foundation and Departments University of Cincinnati, Cincinnati, Accepted
August
of Pediatrics Ohio 452.29
and Pharmacology,
8,1975
Previous biochemical and morphological studies have shown the presence of contractile proteins in mouse palates at the time of shelf movement. In order to determine whether the palatal contractile proteins function in shelf rotation, an embryo culture system in which palate shelves rotate has been developed. A/J mouse fetuses with tongues removed (day 14.75) have been cultured close to the time that palatal shelves move in uiuo and pharmacological agents added. The anterior end of the palate shelf completely rotated after overnight culture in the presence or absence of drugs. However, rotation of the posterior end of the palate was only partial. Agents that stimulate skeletal and smooth muscle contractility, pyridostigmine (2 x lo-@-9 x 10eS M) and bethanechol (10-10-10-4 M), respectively, both enhanced posterior shelf rotation after overnight culture. Pyridostigmine (9 x 1OWM) increased posterior shelf rotation 74% over control; bethanechol (10m4M) 53%. Pyridostigmine effected an appreciable increase in anterior shelf movement within 60 min, while bethanechol stimulated posterior shelf rotation by 60% in that time. These results imply a cholinergic involvement in palate shelf rotation. Furthermore, contraction of “smooth muscle-like” structures previously found on the tongue side extending from top mid-palate to the posterior end may be involved in posterior palate shelf rotation; and contraction of skeletal muscle observed on the oral side posteriorly may aid both posterior and anterior shelf movement. INTRODUCTION
Studies on normal and abnormal palate development in mice have indicated that the movement of the palatine shelves from a position lateral to the tongue to a horizontal position above the tongue is a critical phase in the development of the secondary palate (Walker and Fraser, 1956, 1957; Trasler and Fraser, 1963). Horizontalization of the shelves takes place between day 14.5 and 15.5 of gestation, a.nd shortly thereafter the shelves fuse to complete palate formation. Two basic mechanisms have been proposed for the change in position of the palatine shelves. One mechanism proposed that the palate reaches the horizontal position by “remodeling” of the shelf (Polzl, 1904; Pons-Tortella, 1937). Alternatively, medial rotation of the shelf occurs in a “barndoor” fashion (Peter, 1924; Lazarro, 1940). Coleman (1965) in fact proposed that in rat the anterior portion of the shelf moves from a ventromedial position to the horizontal one by the Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
barndoor swinging of the shelf, while the posterior end moves from a vertical position by remodeling. Much controversy prevails concerning the force that causes shelf rotation. The force may actually reside inside the tissue (“intrinsic shelf force”) or may develop from external influences. Some possible external causes of shelf rotation are: straightening of the cranial base (Verrusio, 1970); and nonpalatal muscular movements such as neck flexion, swallowing, and descent of tongue and lower jaw (Walker, 1969). Alternatively, rapid synthesis of proteoglycans (mucopolysaccharides) in the palate has been proposed as an intrinsic shelf force functioning to move the shelves (Larsson, 1962). In spite of the number of studies, there remain diverging opinions on this subject. A possible candidate for an intrinsic shelf force is the molecular interaction of the contractile proteins, actin and myosin, present inside the palate. Initial studies 91
92
DEVELOPMENTAL
BIOLOGY
VOLUME
48, 1976
have shown that these contractile proteins contractility have been employed. Further are synthesized in day-14.5 mouse palates enhancement of shelf movement is thereat a rate equal to that of the tongue and fore evidence of a cholinergic involvement comprise as much as 10% of the total pro- in palate rotation. A preliminary report tein synthesized (Lessard et al., 1974). Ul- has been presented (Wee et al., 1975). trastructural studies have indicated the MATERIALS AND METHODS presence of a skeletal muscle system on the oral side of the posterior palate, a Animals. AIJ mice were purchased from “smooth muscle-like” structure on the top the Jackson Laboratory. Individual males half of the posterior shelf at the tongue and females were caged together in the side, a possible primitive (nonmuscle) con- late afternoon. The presence of a vaginal tractile system along the oral epithelium plug the following morning was taken as in the mid-palate (Babiarz et al., 1975). evidence of pregnancy and mice were desigOne way to resolve whether these contracnated day 0.5 of development. CD-l mice tile systems function as an intrinsic shelf were purchased from the Charles River force in palate shelf rotation would be to Mouse Farm. CD-l females were mated apply pharmacological agents that inhibit with males from 9 AM to 4 PM. The mice skeletal and smooth muscle activities, re- were kept on a light cycle with the lights spectively. Preliminary experiments em- on from 4 PM to 6 AM. Females with vagiploying D-tubocurarine, which blocks neu- nal plugs were considered to have conromuscular transmission in skeletal mus- ceived at 1 PM of that day. Females were cle, indicated that the fetus was about lOO- killed by cervical dislocation at day 14.0 fold more resistant to the activity of this and 14.75 of gestation for CD-l and A/J, agent than the adult (Zimmerman et al., respectively, the time just prior to shelf 1974). This is consistent with the observarotation (50% of A/J fetal palates rotated tions of Jacobs (1971) that fetuses do not by day 15.1 (Andrew et al., 1973)). The achieve complete flaccidity during curarigravid uterus was removed immediately zation of the pregnant female. The ques- and placed in a sterile dish containing cold tion whether a paralyzing concentration of Dulbecco’s modified Eagle medium that neuromuscular blocking agent can be was pregassed with 95% 02-5% CO,. The achieved in the fetus without killing the uteri were kept on ice to prevent utilizadam can be circumvented by employing in tion of the limiting supply of 0, in the vitro culture studies. Organ cultures of palfetuses. ates have been carried out (Pourtois, 1966; hhteriaZs. Nutrient mixture F-12, DulVargas, 1967). However, these systems in- becco’s modified Eagle medium, and mevolve growth and fusion of palate shelves dium 199 were purchased from Grand Island Biological Company. Gentamicin (40 and not morphogenetic rotation. Previous pg/ml) was the antibiotic added to the nustudies in mice and rats involving embryo culture have been successful only with de- trient mixture F-12 and Dulbecco’s modivelopmental times before palate shelf rotafied Eagle medium. Ascorbic acid (40 pg/ tion (New, 1971; Cockroft, 1973). This pres- ml) was also added to Dulbecco’s modified Penicillin (100 units/ml) ent work deals with the development of an Eagle medium. and streptomycin (1 pg/ml) were employed embryo culture system in which palate in medium 199. Pyridostigmine bromide shelf rotation is at least partially (Mestinon, 5 mg/ml) and bethanechol chloachieved. In these experiments the anteride (Urecholine, 5 mg/ml) were obtained rior end of the palate completely rotates. Since the posterior end of the shelf does not from Roche Laboratories and Merck, completely rotate, pharmacologic agents Sharp and Dohme, respectively. that stimulate skeletal and smooth muscle Serum. All culturing medium contained
WEE,
WOLFSON
AND
ZIMMERMAN
25% human serum. Normally prepared serum is referred to as delayed-centrifuged serum (D.C.). The blood was allowed to clot and stand overnight at 4°C. It was then centrifuged at 1OOOg for 15 min after breaking up the clot and the D.C. serum was decanted. However, Steele and New (1974) have reported that serum prepared in this manner causes impaired growth and development of double hearts in embryo culture. These difficulties were removed by preparation of an immediately centrifuged (I.C.) serum, which we also prepared. The blood was centrifuged at 1OOOg for 10 min immediately after withdrawal. The plasma was allowed to clot by standing overnight at 4°C. The clot was then broken up, recentrifuged at 1OOOgfor 15 min and the I.C. serum decanted. Serum was stored at -20°C for no longer than 2 weeks to be considered fresh. Explantation and embryo culture. The method used for explantation and culture of the embryos was a combination of that described by New et al. (1973) and Cockroft (1973). An individual conceptus was removed with fine forceps from the uterine horn and placed in a culture dish (Falcon) filled with Dulbecco’s modified Eagle medium. By using a pair of microforceps, the Reichert’s membrane was opened and a slit was cut in the yolk sac. The embryo was slipped out of the sac and special care was taken not to damage the vitelline and umbilical blood vessels (Cockroft, 1973). Preliminary experiments employing day14.5 AM embryos, in which tongues were present, allowed little palate rotation. Therefore, day-14.75 embryos were used and tongues were carefully excised with the aid of a dissecting microscope to remove a resistance to shelf rotation. Leaving the mandible intact, negligible bleeding occurred. Since some shelves will have rotated at this time, embryos were carefully observed prior to removing the tongues. Those that had their anterior shelves rotated above the tongues were not used for further study. Since cleft lip and
Palate
Rotation
in Embryo
Culture
93
cleft palate have been suggested to originate from one genetic system (Chabora and Horowitz, 19741, embryos with cleft lip were not used for culture. After measurement of their crown-rump length with an ocular scale, only single embryos with beating hearts were placed into glass vials (19 x 48 mm, Kimble Opticlear) that were filled with 2 ml of culture medium. Each vial was gassed with a mixture of 95% 0, and 5% COZ, tightly closed with a silicon rubber stopper, and sealed with tape. The explanted embryos were cultured at 37°C under sterile conditions for 16-20 hr, unless otherwise specified, by rotation of the vials at 60 rpm (New et al., 1973) in a Bellco variable speed roller drum. Assessment of growth. Viability was monitored by measuring an increase in crown-rump length and observing a beating heart. Data included in the results in the tables were embryos whose crownrump length had increased before fixation by at least 0.29 mm and whose initial length was between 10.0 and 11.86 mm. The presence of heart beat was noted after culture and the rate measured (see Fig. 6) for the time course study. At the indicated time, fetal heart rate was observed in the vial under the dissecting microscope. Initial rates were observed. At various times, embryos were collected and homogenized in 4-6 ml 0.1 N NaOH in a hand homogenizer with Teflon pestle. Aliquots of the homogenates were assayed for protein content by the method of Lowry et aZ. (1951). Assay for palate rotation. After incubation, embryos were fixed in Bouin’s solution for 24 hr at which time heads were cut off and mandibles removed to observe palate rotation and fusion. The gap between the palate shelves also was measured. Heads were then cut sagitally into blocks halfway through the anterior and posterior ends. Blocks of fixed tissue were put on end, and the angle of the anterior or posterior shelves to the nasal septa was
94
DEVELOPMENTAL
BIOLOGY
estimated with an ocular micrometer and photographed for permanent record. Photography was carried out with a Nikon macro system equipped with bellows and 55-mm lens. The position of each shelf was scored as described in Results. RESULTS
Culture
Embryo tion
and Palate Shelf Rota-
Initial culturing of A/J embryos by the combined method of New et al. (1973) and Cockroft (1973) did not produce any appreciable palate movement (Table 1, c). Since the putative “intrinsic shelf force” was so weak, tongues were removed surgically to improve movement of the palate shelves. As can be seen in the subsequent experiments, removal of the tongue allowed palate shelf rotation to varying degrees. In addition, Tanimura and Shepard (1970) found that fresh human serum substituted successfully for homologous serum in emTABLE MOUSE Conditions 25% Human serum
c. Fresh d. Fresh e. Aged, D.C.C D.C.
g. Fresh
h. Fresh,
D.C.
i. Fresh,
I.C.c
j.
Fresh,
I.C.
k. Fresh,
I.C.
Embryos (No.)
15% Medium
-
;: 1
f. Fresh,
of experiment”
EMBRYO
Pooled resulta” Pooled resuw Nutrient mixture F-12 Nutrient mixture F-12 Nutrient mixture F-12 plus mouse embryo extra& (14:l) Dulbecco’s moditied Eagle modiDulbecco’s tied Eagle Medium 199 Hank’s salts Medium 199modilied Earle’s salts
12 31
11 41 8
CULTURE:
Cl-OWIb rump length bun) 10.96 * 0.17 10.91 2 0.10 12.49 -c 0.33 12.45 f 0.14 11.25 + 0.16d
VOLUME
48,
1976
bryo culture. Therefore we routinely employed human serum in our experiments since it is more readily available than mouse serum. Under these conditions complete rotation of the anterior end of the palate shelf occurred. However, little movement of the posterior end (Fig. 1C) took place. In addition, fusion of only the anterior end occurred occasionally (12%). Embryo heads prior to culture are presented for comparison (Figs. 1A and B). Since the anterior and posterior ends of the palate seemed to rotate independently of each other and the posterior end moved incompletely, a quantitative assay of movement at each end of the palate was devised. The angle of the shelf position to the nasal septum was measured at both the anterior (Fig. 2) and posterior (Fig. 3) ends of the palate. Values of 1-5 were arbitrarily assigned for the palate shelf index (P.S.I.). A number 1 was assigned for a completely vertical shelf; 5 for a com1 EFFECTS
OF MEDIA
Palate gap (mm) Anterior 0.70 0.48 0.86 0.05 0.44
* r ? -c +
Palate shelf index
Posterior
0.05 0.02 0.05 0.01 0.07
0.63
2 0.59 f 0.68 + 0.80 + 0.55 k
0.07 0.03
0.05 0.03 0.05
Anterior 1.58 2.32 1.18 4.98 2.15
+ 2 + + t
0.16 0.09 0.08 0.02 0.41
Posterior 1.21 + 0.13 1.36 + 0.08 1.09 + 0.09 2.04 -c 0.15 1.94 e 0.38
6
11.83
f 0.11
0.15 f 0.09
0.69
t 0.06
5.00 f 0.00
1.33
f 0.19
9
12.98
? 0.22
0.11 2 0.05
0.48
+ 0.08
5.00
+ 0.00
2.11
+ 0.44
5
13.37
+ 0.27
0.02
f 0.02
0.78
t 0.08
5.00
2 0.00
1.10 2 0.07
30
12.43
+ 0.16
0.08 f 0.01
0.56
-e 0.03
4.97 + 0.02
2.33
k 0.20
11
12.26 + 0.23
0.05 2 0.02
0.52
r 0.07
5.00 -c 0.00
2.00
+ 0.34
10
12.47 + 0.22
0.08 2 0.02
0.50 f 0.08
5.00 + 0.00
2.80
+ 0.37
0 All embryos have tongues removed except a and c. (a), Embryos fixed in Bouin’s solution with tongues in place without culture; (b) embryos that have tongues removed and then fixed in Bouin’s solution without culture. All values are means f SE. b Nutrient mixture F-12 and Dulbecco’s modified Eagle medium, fresh D.C. and I.C. sera. e D.C., delayed-centrifuged serum; I.C., immediately centrifuged serum. d Length after fixing with Bouin’s solution. c 300613 supernatant of day-14.5 embryo homogenates in Tyrode’s salt solution.
WEE,
WOLFSON
AND ZIMMERMAN
Palate
Rotation
in Embryo
Culture
95
FIG. 1. Heads of embryos after fixing in Bouin’s solution are shown with lower jaws removed. They are taken from (A) a day-14.75 embryo fixed in Bouin’s solution without culturing and with the tongue in place; (B) same as (A) but with tongue removed after fixing; (C) day-14.75 embryo cultured overnight with tongue x 13. removed and no drug added; (D) same as (0 but with addition of 9 x 1OF M pyridostigmine.
pletely horizontal shelf; 3 for a shelf at 45”; 2 for a shelf position between 1 and 3; and 4 for a shelf position between 3 and 5. Computing a mean position from these numerical values allowed a statistical comparison of the treatment groups. As Walker and Fraser (1956) have shown, anterior shelves start ventromedially (Figs. 2A and B) and cup the tongue between them (Fig. 1Al. The posterior shelves have the tongue ventral to the shelves which slope at an obtuse angle away from the roof of the nasal cavity (Fig. 3A). If palate shelves do rotate, there is no difficulty in showing anterior palate shelf rotation as it swings in a “barndoor” fashion (Figs. 2A to 2D). The posterior end presents much greater problems because, as Coleman (1965) has indicated, it not
only rotates but it undergoes change in tissue shape, i.e., “remodeling.” In spite of this difficulty, the same 1-5 classification was used. However, more “developmental changes” may be involved during posterior palate shelf rotation to achieve the same palate shelf index as the anterior shelf because of remodeling of the posterior shelf (Figs. 3A-E). Measurements of the distances between the shelves (gap) at the anterior and posterior ends were also made. Although the gap distance decreased as the ends of the shelves elevated, it is felt that the palate shelf index allows a more accurate assessment of rotation. Various media and various preparations of human sera have been employed. The results are shown in Table 1. Initial experiments indicated that aged human serum
96
’w I ‘I ...-. .----e DEVELOPMENTAL
VOLUME
46,
1976
5
... . ..... ....... . ..........................................1’ ... ........... ..............I.‘, :::.:.:>:.:> :.. . ~.:.::~!~~: .
A
BIOLOGY
4
\
\
I@
3
\ I ‘I. A --F ......Y. . .::::::::::::::::::I:.:::.. . .......:... :l:::::1:.~i:~:.:.~::::::::: .. ..:::: ... 1s;’ I B
‘\
;
03
\\I - -.T.rn.... ::::::::::::::::.:I:..: ..:..:.. :.:.:.:.:.:.:.:.:.,.:... .......::: .. \ :.:::: A+? 0 1
I
C
I
4
\
\
-5
0
FIG. 2. Anterior palate shelf index. Each photograph on the right shows the cross-section of a fixed embryo head taken halfway through the anterior end of the palate with lower jaw removed; x30. Each picture has a corresponding drawing on the left showing the angle between the left palate shelf and the nasal septum (heavy solid line), and the palate shelf index (P.S.I.) encircled. Reference dashed lines indicate how P.S.I. is determined. (A) and (B), The ventromedial positions of the anterior shelves when embryos have tongue excised, then head fixed without culturing; (C), a near-horizontal palate shelf for embryo cultured in 1OW M pyridostigmine for 3 hr; CD), complete flattening of anterior shelf after overnight culture in 3 x lo+ M pyridostigmine.
was not as effective as freshly prepared human serum for anterior shelf rotation (e and f). Addition of mouse embryo extract to supplement human serum (g) did not significantly improve posterior shelf rota-
tion. Human serum in which blood cells were immediately centrifuged and the plasma allowed to clot thereafter apparently allowed greater posterior shelf rotation (h, i) and have been employed in the
WEE,
WOLFSON
’ 7:y::::. +p-
E FIG. 3. Posteric heads taken halfu the angle of inclin Reference dashed
\
AND ZIMMERMAN
Palate
Rotation
in Embryo
Culture
97
I I %:::.:.I.
1
’
balate shelf index. Photographs on the right show the cross-sections of fixed embryo, through the posterior end of the palate. x 30. Corresponding drawings on the left show on of the palate shelf at the left (heavy solid line) and the palate shelf index encircled. es indicate how P.S.I. is determined.
later experiments. Nutrient mixture F-12 and Dulbecco’s modified Eagle medium both allowed approximately the same amount of shelf rotation, differences in sera accounting for the various values measured. The results of these two sera have been pooled Cc, d). It was observed that overnight embryo culture allowed
complete rotation of the anterior end of palate (P.S.I. of 4.98). However, the posterior end of the shelf only rotated to a value of about 2. Not only did the anterior and posterior ends of the shelves rotate during culture, but when the tongue was being surgically removed there was an immediate and limited shelf movement. If the
98
DEVELOPMENTAL
BIO~CY
head was fixed before removing the tongue, the P.S.I. for the anterior and posterior ends were 1.58 and 1.21, respectively (a). If the tongue was removed under medium and then the head fixed in Bouin’s solution, the shelves moved to an anterior position of 2.32 and a posterior one of 1.36 (b). This greater movement of the anterior end, under these conditions, is in accord with the results of Boss and Walker (1967). Palate shelf rotation during embryo culture was time dependent. As Fig. 4 indicates, rotation of both ends of the palate was completed by about 6 hr. In later experiments, medium 199 was employed. Since it is known that contractil-
0
2 TIME
4 - HOURS
6
FIG. 4. Time course study of the effects of lo+ M pyridostigmine and 1OmB M bethanechol on palate shelf rotation with mean +SE indicated. Palate shelf indexes are determined as described in Besuits. Number of embryos used for anterior (dashed line) and posterior (solid line) shelf rotation are: 13, 12 and 11 for 1-hr; 17, 11 and 17 for 3-hr; and 21, 17 and 25 for 6-hr cultures for controls (01, pyridostigmine- (0) and bethanechol(A) treated groups, respectively.
VOLUME
ity of muscle is a function of Ca2+ and K+ concentrations in a bathing solution, this medium containing either Hanks’ or modified Earle’s salt solutions was compared. M-199 with modified Earle’s salts seemed to produce greater shelf rotation (2.80) than that with Hanks’ (2.33), presumably because of the higher Ca2+ concentration in the modified Earle’s salt solution. However, further work is needed to verify this assumption. Growth of Embryos Table 2 reveals that average crownrump length of embryos after overnight culture increased from 10.91 mm to 12.55 mm. However, as would be expected, the increase in culture was less than that in Go. Protein content was determined for control and cultured embryos. As Table 2 indicates, there was about an eight-fold greater increase in protein of control than cultured embryos at comparable times in development. As shown in Fig. 5, the slope of the “least-squares” line for the cultured group is less than that in uiuo, indicating relatively less increase in protein than in crown-rump length during culture. The increase in elongation of the cultured embryo may be a function of the straightening of the embryo or an increase in water content. In any case, the increase was time dependent (Fig. 6B). No other changes in developmental features of the embryo during culture were observed (Fig. 7). Normally, for the overnight-culture experiments, no heart beats were observed the following morning. However, several
TABLE CROWN-RUMP Crown-rump
LENGTH
2
AND PROTEIN
length
48, 1976
CONTENT:
in Vivo
(mm)
vs in Protein
Vitro content
(mg)
Conditions In viuo In uitrd
Day 14.75
Day 15.5
10.91 + 0.20 (19)”
13.59 -c 0.30 (6)
2.68
12.55
1.64
-
e 0.18
A (24)
n Values in parentheses arc the numbers of embryos. b Embryos cultured overnight in immediately centrifuged human embryos with pyridostigmine (1O-e-1O-’ M) and 8 without drug.
Day 14.75 10.32
f 0.48
Day 15.5 (19)
serum
and Dulbecco’s
18.45
2 0.84
A (6)
11.17 + 0.48 (24) modified
8.13 0.85
Eagle medium:
16
WEE,
WOLFSON
AND
ZIMMERMAN
Palate
Rotation
in Embryo
rate decreased with the vial stopped. Effects
31 6
7 CRoviN-RUMP
8
I 9 LENGTH
I IO
I I/
mm,
between FIG. 5. Relationship crown-rump length and protein content of embryos for groups both cultured and not cultured. The solid lines are drawn by a least-squares analysis of the corresponding data points.
99
Culture
time after rotation
of Pharmacological
of
Agents
Since rotation of the posterior end of the palate shelf during embryo culture was limited, pharmacologic agents that are known to stimulate skeletal and smooth muscle contractility were added to the embryo cultures. Pyridostigmine, which stimulates skeletal muscle contraction, was first employed. Table 3 shows that 10m5M pyridostigmine partially but significantly (P
FIG. 6. Time course study of the effects of 10ms M pyridostigmine and 10e6 M bethanechol on heart rates (A) and increase in crown-rump length (B) of cultured embryos with means *SE indicated. For measuring heart rates, the numbers of embryos are: At 0 hr, control (no drug added), 22; at 1 hr, 13, 12, and 11; at 3 hr, 17,ll and 17; and at 6 hr, 14,O and 11 for control, pyridostigmineand bethanecholtreated, respectively. The numbers of embryos used for determining increase in crown-rump length are: 13, 12 and 11 for 1-hr; 17, 11 and 17 for 3-hr; 21, 17 and 25 for 6-hr, and 41 (see Table 1, d), 20 and 22 for overnight culture; for control, pyridostigmineand bethanechol-treated, respectively.
overnight-cultured embryos with beating heart or occasional limb or tail movements have been observed, especially in the more recent experiments, After 1 hr in culture, there is a significant increase (51%) in heart rate over that of the starting embryos (Fig. 6A). Thereafter, the heart rates decrease with time of culture, 107 beats/min in 3 hr, 77 beats/min in 6 hr. Initial rates were recorded because heart
FIG. 7. Photographs of a whole embryo before (A) and after (B) culture. This particular embryo grew from a crown-rump length of 11.28 mm to 12.14 mm.
x4.
100
DEVELOPMENTAL
MOUSE Conditions
-
CULTURE:
of experiment
Tongue excised 8.O
EMBRYO
Drugs
BIOLOGY 3
EFFECTS
OF PHARMACOLOGIC
Embryos (no.)
11
Cmwnrump length (mm) 12.49 + 0.33*
b.
+
41
-
12.45 2 0.14
d.
+
Pyridostigmine
(2 x 10-6M)
23
12.79 + 0.39 12.97
e.”
+
Pyridostigmine
(W5 M)
20
+ 0.22 12.68
f.
+
Bethanechol
g.
+
Pyridostigmine (10m5 M) plus bethanechol (1O-B M)
c.
Pyridostigmine
(2 x 10-eM)
6
(We M)
48, 1976
TABLE
added
-
VOLUME
Palate
Anterior
Posterior
0.86 2 0.05 0.05
1.16
1.09
+ 0.08 4.98 e 0.02
f 0.09 2.04
2 0.01 0.80 + 0.16
0.68 2 0.05 0.60 r 0.03
0.69 2 0.09
1.67
0.02
0.56
+ 0.01 0.03
2 0.21
k 0.01
20
12.29 _’ 0.18
0.03 k 0.02 0.03
line analog bethanechol, which stimulates smooth muscle contraction of the gastrointestinal tract, was employed. Bethanechol (10V6 M) also partially and significantly (P~0.05) enhanced posterior shelf movement (P.S.I. = 2.95). Since posterior shelf rotation with each pharmacological agent was not complete, both drugs were simultaneously added to the embryo culture system. The results showed no appreciable increase in rotation (3.28) compared to each individual agent (2.95). Although each individual agent increased posterior shelf movement, this effect might not be due to a cholinergic involvement in palate shelf rotation but rather due to a maintenance of heart rate for a longer time in culture or an enhancement of growth rate. Figure 6A shows that neither pyridostigmine (lop5 M) nor bethanechol (lop6 M) significantly affected heart rate. In Fig. 6B, the effect of these agents on growth, as determined by an increase in crown-rump length, is shown. Bethanechol did not affect crown-rump length while pyridostigmine caused a decrease in this parameter during the first 6 hr of culture. This effect is not considered significant because embryo length was stimulated after overnight culture.
Palate shelf index
Posterior
12.12 e 0.12
modified
gap (mm)
Anterior
22
a a-d, Pooled results using nutrient mixture F-12 and Dulbecco’s b All values are mean *SE. c e-g, Only Dulbeooo’s modified Eagle employed as medium.
AGENTS
+ 0.01
? 0.47 5.00
+ 0.05
+ 0.00
0.51 t 0.05 0.51 + 0.05 0.58 t 0.03
5.00 + 0.00 5.00 + 0.00 5.00 " 0.00
k 0.15 1.75 k 2.11 -t 2.95 2 2.95 t 3.28 ?
0.44 0.24 0.26 0.26 0.26
Eagle as media.
Figure 8 shows the effect of posterior palate shelf rotation with increasing concentrations of pyridostigmine (A) and bethanechol (B). A significant increase in posterior shelf rotation is observed at a concentration of 1O-5 M pyridostigmine (P~0.05). Although there seems to have been a greater increase in shelf rotation at the highest concentration used (74% over controls, P
ControlM.lT;,,;P,,,
$41
/co*
M-sETLHOJ~FIG. 8. Posterior palate shelf rotation dose-response curve to pyridostigmine (A) and bethanechol (B). Each point represents the mean (-tSE) of the experiments. The number of control embryos employed were 41 (no drug added, see Table 1, d). For pyridostigmine, numbers of embryos used for respective doses are: 23 for 2 x 10eB M; 17 for 6 x 1OV M; 20 for 1OW M; 34 for 3 x 1OW M; and 27 for 9 X 1OV M. For bethanechol, there are 13 embryos for lO-*O M; 16 for 1OW M; 19 for 1Om8 M; 18 for 1OW M; 22 for 1O-6 M; and 13 for lo+ M. Palate shelf indexes are determined as described in the text.
WEE,
WOLFSON
Palate Rotation
AND ZIMMERMAN
many embryos and subsequent experiments employed lower concentrations of drug. Figure 1D shows shelf rotation of both the anterior and posterior ends of the palate with 9 x 10m5M pyridostigmine. At 1OV M, bethanechol produced a significant increase (53%, P
of Strains
CD-1 and AN
The mouse strain CD-l has been employed recently in studies on cleft palate (Walker and Patterson, 1974). CD-l embryos are more advanced developmentally and their palate shelves rotated and fused 1 day earlier than the A/J strain (Walker, 1974). Thus, the ability of the palate of the CD-l strain to rotate in vitro was compared with that of the A/J strain in the presence and absence of pyridostigmine and bethanechol. Table 4 shows that when CD-l embryos at day 14.0 of gestation were
cultured in Dulbecco’s modified Eagle medium with the tongues removed, similar results were obtained to that of day-14.75 A/J embryos. Embryo culture resulted in complete rotation of the anterior palate shelf and little movement of the posterior end. Pyridostigmine (lo-” M) slightly increased posterior palate shelf movement while lo-” M bethanechol increased it significantly (P
When culturing mouse embryos during late development, rotation of the anterior end of the palate was completed within about 6 hr. Palate shelf rotation of the posterior end was also assayed and was shown to have partially taken place. Since fusion does not usually occur, this system may have some usefulness in determining whether teratogens cause cleft palate by inhibiting palate shelf rotation. If the agent does not affect rotation, the block could be on the fusion process. The major finding in this work was the observation that cholinergic drugs could stimulate shelf rotation in embryo cultures. These agents could be stimulating nonpalatal muscles and hence aid in pal-
TABLE COMPARISON Conditions Cultured
Drug added
4
OF STRAINS
of experiment
CD-l,
CD-l
-
Crown-rump length (mm)
Pyridostigmine (10m5 MI Bethanechol (1O-B M)
11.71 12.42 12.71 12.76
t 2 k k
0.04 0.18 0.21 0.16
AND A/J”
day 14.0
A/J, day 14.75
Palate shelf index Anterior
+ + +
101
in Embryo Culture
2.64 5.00 5.00 5.00
k + k f
Palate shelf index
Posterior 0.20 0.00 0.00 0.00
1.65 2.02 2.21 2.75
k 2 t k
0.17 0.24 0.31 0.29
Anterior 2.32 5.00 5.00 5.00
zt f 2 +
Posterior 0.09 0.00 0.00 0.00
1.36 2.33 2.95 2.95
t -c 2 -t
0.08 0.20 0.26 0.26
’ Embryos fkom both strains were cultured in immediately centrifuged human serum and Dulbecco’s modified Eagle medium. Numbers of embryos of CD-1 are: - incubation, 7; + incubation, 22; + pyridostigmine, 12; + betbanechol, 20. A/J: - incubation, 37; + incubation, 30; + pyridostigmine, 20; + bethanechol, 22. Values are means *SE.
102
DEVELOPMENTAL
BIOLOGY
VOLUME
48, 1976
ate shelf rotation, e.g., neck flexion and mass is primarily responsible for the “reswallowing. Alternatively, these agents modeling” of the posterior palate shelf. could be stimulating a neuromuscular ap- The contractility of the smooth muscle-like paratus in the palate shelf itself, which is mass would be in accord with the waveresponsible for the “intrinsic shelf force.” like movement from the posterior end of We have previously shown that synthesis the palate towards the anterior end, first of the contractile proteins, actin and described by Walker and Fraser (1956). myosin, in the palate was equal to that in Although pyridostigmine also increased the tongue and could represent as much as the rotation of the posterior palate shelf, it 10% of the total palate proteins synthewas unexpected that it enhanced to a limsized just prior to shelf rotation (Lessard et ited degree the rotation of the anterior end al., 1974). Recently, ultrastructural stud- of the palate before that of the posterior ies (Barbiarz et al., 1975) have revealed the end; there has been no indication of skelepresence of skeletal muscle fibers on the tal muscle myofibrils at the anterior end. oral side of the far posterior end of the This effect on the anterior shelf could indipalate. In addition, a large cellular mass cate that the skeletal muscle myofibrils was found which had characteristics of de- present in the oral side of the posterior veloping smooth muscle or mesenchymal palate function to aid in the “barndoor” cells enriched with filaments. This mass rotation of the anterior shelf. Barbiarz et was found on the tongue side of the palate al. (1975) have also shown a positive histoand extends from the mid to the posterior chemical reaction for myosin ATPase end of the shelf. along the medial edge of the midpalate Although normal palatogenesis involves (region 3). Aduss (Howard Aduss, personal descent of the tongue, the tongue has been communication) has observed that in huremoved during routine culture, and par- man submucous cleft palate, skeletal mustial palate rotation is achieved. Hence de- cle, which is present along all of the mescent of tongue below the palate shelves dial edge of the palate, contracts during and subsequent forcing of the shelves up- phonation. Presumably, this skeletal musward by the tongue is not a necessary pre- cle is replaced with cartilage and bone during normal fusion of the palatal shelves. requisite for shelf rotation. On the other hand, palate shelf rotation could not occur Therefore, it is possible that region 3 of the if the tongue had not been removed. A developing palate represents a primitive similar result has been obtained by skeletal muscle or non-muscle contractile Walker and Quarles (1973) where embryos system extending from the posterior end of are placed in a special fluid bath without the palate. Contraction of the skeletal musinterrupting maternal blood circulation. cle in the posterior palate could produce a Since shelf rotation occurs only in the ab- vector of force extending along the medial sence of the tongue in our embryo cul- edge into region 3 and contribute to rotatures, this result implies that only a minition of the anterior palate by barndoor mal intrinsic shelf force exists in the pal- rotation. ate. This would be consistent with the This work was supported by a grant from the “smooth muscle-like” mass found in the National Institute of Dental Research (No. DE posterior half of the palate exerting a sus- 03469) and a Center Grant in Mental Retardation (No. HD 05221). tainable but minimal force. In the short-term culture experiment REFERENCES (Fig. 4), bethanechol enhanced rotation at ANDREW, F. D., BOWEN, D., and ZIMMERMAN, E. F. the posterior end of the palate within 1 hr. (1973). Glucocorticoid inhibition of RNA synthesis This result would be consistent with the and the critical period for cleft palate induction in hypothesis that the smooth muscle-like inbread mice. Teratology 7, 167-175.
WEE,
WOLF~ON
Palate Rotation
AND ZIMMERMAN
BABIAFU, B., ALLENSPACH, A. L., and ZIMMERMAN, E. F. (1975). Ultrastructural evidence of contractile systems in mouse palates before rotation. De-
velop. Biol. 47, 32-44. CHABORA, A. J., and HOROWITZ, S. L. (1974). lip and cleft palate: one genetic system.
Cleft
Oral
Surg. Oral Med. Oral Pathol. 38, 181-186. COCKROFT, D. L. (1973). Development in culture of rat foetuses explanted at 12.5 and 13.5 days of gestation. J. Embryol. Exp. Morphol. 29,473-483. COLEMAN, R. D. (1965). Development of the rat palate. Anat. Rec. 151, 107-118. JACOBS, R. M. (1971). Failure of muscle relaxants to produce cleft palate in mice. Teratology 4, 25-30. LARSSON, K. S. (1962). Closure of the secondary palate and its relation to sulphomucopolysaccharides. Acta Odontol. Stand. 20, Suppl. 31, l-35. LAZARRO, C. (1940). Sul meccanismo di chiusura de1 palato secondairo. Monit. 2001. Ital. 51, 249-273. LESSARD, J. L., WEE, E. L., and ZIMMERMAN, E. F. (1974). Presence of contractile proteins in mouse fetal palate prior to shelf elevation. Teratology 9, 113-126. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193,265275. NEW, D. A. T. (1971). Methods for the culture of postimplantation of rodents. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 305-319. Freeman, San Francisco. NEW, D. A. T., COPPOLA, P. T., and TERRY, S. (1973). Culture of explanted rat embryos in rotating tubes. J. Reprod. Pert. 35, 135-138. PETER, K. (1924). Die Entwicklung Des Siiugetiergaumes. Ergeb. Anat. Entwicklungsgesch. 25, 448-564. POLZL, A. (1904). Zur Entwicklungsgechichte des menchlichen Gaumens. Anat. Hefte 27, 245-281. PONS-TORTELLA, E. (1937). ijber die Bildungsweise des sekundlren Gaumens. Anat. Anz. 84, 13-17. POURTOIS, M. (1966). Onset of the acquired potentiality for fusion in the palatal shelves of rats. J. Embryol. Exp. Morphol. 16, 171-181.
Ross, L. palatine treated 522. STEELE, iants other
in Embryo Culture
103
M.,
and WALKER, B. E. (1967). Movement of shelves in untreated and teratogenmouse embryos. Amer. J. Anat. 121,509--
C. E., and NEW, D. A. T. (1974). Serum varcausing the formation of double hearts and abnormalities in explanted rat embryos. J. Embryol. Exp. Morphol. 31, 707-719. TANIMURA, T., and SHEPARD, T. H. (1970). Glucose metabolism by rat embryos in vitro. Proc. Sot
Exp. Biol. Med. 135, 51-54. TRASLER, D. G., and FRASER, F. C. (1963). Role 01 the tongue in producing cleft palate in mice with spontaneous cleft lip. Develop. Biol. 6, 45-60. VARGAS, V. I. (1967). Palatal fusion in vitro in the mouse. Arch. Oral Biol. 12, 1283-1288. VERRUSIO, A. C. (1970). A mechanism for closure of the secondary palate. Teratology 3, 17-20. WALKER, B. E. (1969). Correlation of embryonic movement with palate closure in mice. Teratology 2, 191-198. WALKER, B. E. (1974). Palate closure in strain CD-I mice. J. Dent. Res. 53, 1497. WALKER, B. E., and FRASER, F. C. (1956). Closure of the secondary palate in three strains of mice. J Embryol. Exp. Morphol. 4, 176-189. WALKER, B. E., and FRASER, F. C. (1957). The embryology of cortisone-induced cleft palate. J. Embryol. Exp. Morphol. 5, 201-209. WALKER, B. E., and PATTERSON, A. (1974). Induction of cleft palate in mice by tranquilizers and barbiturates. Teratology 10, 159-164. WALKER, B. E., and QUARLES, J. (1973). Palate closure in embryonic mice after excising the tongue. Teratology 7, A30 (Abstract). WEE, E. L., WOLFSON, L., and ZIMMERMAN, E. F. (1975). Palate shelf rotation in embryo culture: Evidence for a cholinergic response. Teratology 11, 37A (Abstract). ZIMMERMAN, E. F., PATIL, T., and CHANG, I. F. (1974). Pharmacological study of role of contractile proteins in palate shelf movement. Teratology 9, A40 (Abstract).
BIOLOGY
48, 91-103 (1976)
Palate Shelf Movement in Mouse Embryo Culture: Evidence for Skeletal and Smooth Muscle Contractility ELIZABETH Children’s
Hospital
L. WEE, LOIS G. WOLFSON, AND ERNEST F. ZIMMERMAN Research Foundation and Departments University of Cincinnati, Cincinnati, Accepted
August
of Pediatrics Ohio 452.29
and Pharmacology,
8,1975
Previous biochemical and morphological studies have shown the presence of contractile proteins in mouse palates at the time of shelf movement. In order to determine whether the palatal contractile proteins function in shelf rotation, an embryo culture system in which palate shelves rotate has been developed. A/J mouse fetuses with tongues removed (day 14.75) have been cultured close to the time that palatal shelves move in uiuo and pharmacological agents added. The anterior end of the palate shelf completely rotated after overnight culture in the presence or absence of drugs. However, rotation of the posterior end of the palate was only partial. Agents that stimulate skeletal and smooth muscle contractility, pyridostigmine (2 x lo-@-9 x 10eS M) and bethanechol (10-10-10-4 M), respectively, both enhanced posterior shelf rotation after overnight culture. Pyridostigmine (9 x 1OWM) increased posterior shelf rotation 74% over control; bethanechol (10m4M) 53%. Pyridostigmine effected an appreciable increase in anterior shelf movement within 60 min, while bethanechol stimulated posterior shelf rotation by 60% in that time. These results imply a cholinergic involvement in palate shelf rotation. Furthermore, contraction of “smooth muscle-like” structures previously found on the tongue side extending from top mid-palate to the posterior end may be involved in posterior palate shelf rotation; and contraction of skeletal muscle observed on the oral side posteriorly may aid both posterior and anterior shelf movement. INTRODUCTION
Studies on normal and abnormal palate development in mice have indicated that the movement of the palatine shelves from a position lateral to the tongue to a horizontal position above the tongue is a critical phase in the development of the secondary palate (Walker and Fraser, 1956, 1957; Trasler and Fraser, 1963). Horizontalization of the shelves takes place between day 14.5 and 15.5 of gestation, a.nd shortly thereafter the shelves fuse to complete palate formation. Two basic mechanisms have been proposed for the change in position of the palatine shelves. One mechanism proposed that the palate reaches the horizontal position by “remodeling” of the shelf (Polzl, 1904; Pons-Tortella, 1937). Alternatively, medial rotation of the shelf occurs in a “barndoor” fashion (Peter, 1924; Lazarro, 1940). Coleman (1965) in fact proposed that in rat the anterior portion of the shelf moves from a ventromedial position to the horizontal one by the Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
barndoor swinging of the shelf, while the posterior end moves from a vertical position by remodeling. Much controversy prevails concerning the force that causes shelf rotation. The force may actually reside inside the tissue (“intrinsic shelf force”) or may develop from external influences. Some possible external causes of shelf rotation are: straightening of the cranial base (Verrusio, 1970); and nonpalatal muscular movements such as neck flexion, swallowing, and descent of tongue and lower jaw (Walker, 1969). Alternatively, rapid synthesis of proteoglycans (mucopolysaccharides) in the palate has been proposed as an intrinsic shelf force functioning to move the shelves (Larsson, 1962). In spite of the number of studies, there remain diverging opinions on this subject. A possible candidate for an intrinsic shelf force is the molecular interaction of the contractile proteins, actin and myosin, present inside the palate. Initial studies 91
92
DEVELOPMENTAL
BIOLOGY
VOLUME
48, 1976
have shown that these contractile proteins contractility have been employed. Further are synthesized in day-14.5 mouse palates enhancement of shelf movement is thereat a rate equal to that of the tongue and fore evidence of a cholinergic involvement comprise as much as 10% of the total pro- in palate rotation. A preliminary report tein synthesized (Lessard et al., 1974). Ul- has been presented (Wee et al., 1975). trastructural studies have indicated the MATERIALS AND METHODS presence of a skeletal muscle system on the oral side of the posterior palate, a Animals. AIJ mice were purchased from “smooth muscle-like” structure on the top the Jackson Laboratory. Individual males half of the posterior shelf at the tongue and females were caged together in the side, a possible primitive (nonmuscle) con- late afternoon. The presence of a vaginal tractile system along the oral epithelium plug the following morning was taken as in the mid-palate (Babiarz et al., 1975). evidence of pregnancy and mice were desigOne way to resolve whether these contracnated day 0.5 of development. CD-l mice tile systems function as an intrinsic shelf were purchased from the Charles River force in palate shelf rotation would be to Mouse Farm. CD-l females were mated apply pharmacological agents that inhibit with males from 9 AM to 4 PM. The mice skeletal and smooth muscle activities, re- were kept on a light cycle with the lights spectively. Preliminary experiments em- on from 4 PM to 6 AM. Females with vagiploying D-tubocurarine, which blocks neu- nal plugs were considered to have conromuscular transmission in skeletal mus- ceived at 1 PM of that day. Females were cle, indicated that the fetus was about lOO- killed by cervical dislocation at day 14.0 fold more resistant to the activity of this and 14.75 of gestation for CD-l and A/J, agent than the adult (Zimmerman et al., respectively, the time just prior to shelf 1974). This is consistent with the observarotation (50% of A/J fetal palates rotated tions of Jacobs (1971) that fetuses do not by day 15.1 (Andrew et al., 1973)). The achieve complete flaccidity during curarigravid uterus was removed immediately zation of the pregnant female. The ques- and placed in a sterile dish containing cold tion whether a paralyzing concentration of Dulbecco’s modified Eagle medium that neuromuscular blocking agent can be was pregassed with 95% 02-5% CO,. The achieved in the fetus without killing the uteri were kept on ice to prevent utilizadam can be circumvented by employing in tion of the limiting supply of 0, in the vitro culture studies. Organ cultures of palfetuses. ates have been carried out (Pourtois, 1966; hhteriaZs. Nutrient mixture F-12, DulVargas, 1967). However, these systems in- becco’s modified Eagle medium, and mevolve growth and fusion of palate shelves dium 199 were purchased from Grand Island Biological Company. Gentamicin (40 and not morphogenetic rotation. Previous pg/ml) was the antibiotic added to the nustudies in mice and rats involving embryo culture have been successful only with de- trient mixture F-12 and Dulbecco’s modivelopmental times before palate shelf rotafied Eagle medium. Ascorbic acid (40 pg/ tion (New, 1971; Cockroft, 1973). This pres- ml) was also added to Dulbecco’s modified Penicillin (100 units/ml) ent work deals with the development of an Eagle medium. and streptomycin (1 pg/ml) were employed embryo culture system in which palate in medium 199. Pyridostigmine bromide shelf rotation is at least partially (Mestinon, 5 mg/ml) and bethanechol chloachieved. In these experiments the anteride (Urecholine, 5 mg/ml) were obtained rior end of the palate completely rotates. Since the posterior end of the shelf does not from Roche Laboratories and Merck, completely rotate, pharmacologic agents Sharp and Dohme, respectively. that stimulate skeletal and smooth muscle Serum. All culturing medium contained
WEE,
WOLFSON
AND
ZIMMERMAN
25% human serum. Normally prepared serum is referred to as delayed-centrifuged serum (D.C.). The blood was allowed to clot and stand overnight at 4°C. It was then centrifuged at 1OOOg for 15 min after breaking up the clot and the D.C. serum was decanted. However, Steele and New (1974) have reported that serum prepared in this manner causes impaired growth and development of double hearts in embryo culture. These difficulties were removed by preparation of an immediately centrifuged (I.C.) serum, which we also prepared. The blood was centrifuged at 1OOOg for 10 min immediately after withdrawal. The plasma was allowed to clot by standing overnight at 4°C. The clot was then broken up, recentrifuged at 1OOOgfor 15 min and the I.C. serum decanted. Serum was stored at -20°C for no longer than 2 weeks to be considered fresh. Explantation and embryo culture. The method used for explantation and culture of the embryos was a combination of that described by New et al. (1973) and Cockroft (1973). An individual conceptus was removed with fine forceps from the uterine horn and placed in a culture dish (Falcon) filled with Dulbecco’s modified Eagle medium. By using a pair of microforceps, the Reichert’s membrane was opened and a slit was cut in the yolk sac. The embryo was slipped out of the sac and special care was taken not to damage the vitelline and umbilical blood vessels (Cockroft, 1973). Preliminary experiments employing day14.5 AM embryos, in which tongues were present, allowed little palate rotation. Therefore, day-14.75 embryos were used and tongues were carefully excised with the aid of a dissecting microscope to remove a resistance to shelf rotation. Leaving the mandible intact, negligible bleeding occurred. Since some shelves will have rotated at this time, embryos were carefully observed prior to removing the tongues. Those that had their anterior shelves rotated above the tongues were not used for further study. Since cleft lip and
Palate
Rotation
in Embryo
Culture
93
cleft palate have been suggested to originate from one genetic system (Chabora and Horowitz, 19741, embryos with cleft lip were not used for culture. After measurement of their crown-rump length with an ocular scale, only single embryos with beating hearts were placed into glass vials (19 x 48 mm, Kimble Opticlear) that were filled with 2 ml of culture medium. Each vial was gassed with a mixture of 95% 0, and 5% COZ, tightly closed with a silicon rubber stopper, and sealed with tape. The explanted embryos were cultured at 37°C under sterile conditions for 16-20 hr, unless otherwise specified, by rotation of the vials at 60 rpm (New et al., 1973) in a Bellco variable speed roller drum. Assessment of growth. Viability was monitored by measuring an increase in crown-rump length and observing a beating heart. Data included in the results in the tables were embryos whose crownrump length had increased before fixation by at least 0.29 mm and whose initial length was between 10.0 and 11.86 mm. The presence of heart beat was noted after culture and the rate measured (see Fig. 6) for the time course study. At the indicated time, fetal heart rate was observed in the vial under the dissecting microscope. Initial rates were observed. At various times, embryos were collected and homogenized in 4-6 ml 0.1 N NaOH in a hand homogenizer with Teflon pestle. Aliquots of the homogenates were assayed for protein content by the method of Lowry et aZ. (1951). Assay for palate rotation. After incubation, embryos were fixed in Bouin’s solution for 24 hr at which time heads were cut off and mandibles removed to observe palate rotation and fusion. The gap between the palate shelves also was measured. Heads were then cut sagitally into blocks halfway through the anterior and posterior ends. Blocks of fixed tissue were put on end, and the angle of the anterior or posterior shelves to the nasal septa was
94
DEVELOPMENTAL
BIOLOGY
estimated with an ocular micrometer and photographed for permanent record. Photography was carried out with a Nikon macro system equipped with bellows and 55-mm lens. The position of each shelf was scored as described in Results. RESULTS
Culture
Embryo tion
and Palate Shelf Rota-
Initial culturing of A/J embryos by the combined method of New et al. (1973) and Cockroft (1973) did not produce any appreciable palate movement (Table 1, c). Since the putative “intrinsic shelf force” was so weak, tongues were removed surgically to improve movement of the palate shelves. As can be seen in the subsequent experiments, removal of the tongue allowed palate shelf rotation to varying degrees. In addition, Tanimura and Shepard (1970) found that fresh human serum substituted successfully for homologous serum in emTABLE MOUSE Conditions 25% Human serum
c. Fresh d. Fresh e. Aged, D.C.C D.C.
g. Fresh
h. Fresh,
D.C.
i. Fresh,
I.C.c
j.
Fresh,
I.C.
k. Fresh,
I.C.
Embryos (No.)
15% Medium
-
;: 1
f. Fresh,
of experiment”
EMBRYO
Pooled resulta” Pooled resuw Nutrient mixture F-12 Nutrient mixture F-12 Nutrient mixture F-12 plus mouse embryo extra& (14:l) Dulbecco’s moditied Eagle modiDulbecco’s tied Eagle Medium 199 Hank’s salts Medium 199modilied Earle’s salts
12 31
11 41 8
CULTURE:
Cl-OWIb rump length bun) 10.96 * 0.17 10.91 2 0.10 12.49 -c 0.33 12.45 f 0.14 11.25 + 0.16d
VOLUME
48,
1976
bryo culture. Therefore we routinely employed human serum in our experiments since it is more readily available than mouse serum. Under these conditions complete rotation of the anterior end of the palate shelf occurred. However, little movement of the posterior end (Fig. 1C) took place. In addition, fusion of only the anterior end occurred occasionally (12%). Embryo heads prior to culture are presented for comparison (Figs. 1A and B). Since the anterior and posterior ends of the palate seemed to rotate independently of each other and the posterior end moved incompletely, a quantitative assay of movement at each end of the palate was devised. The angle of the shelf position to the nasal septum was measured at both the anterior (Fig. 2) and posterior (Fig. 3) ends of the palate. Values of 1-5 were arbitrarily assigned for the palate shelf index (P.S.I.). A number 1 was assigned for a completely vertical shelf; 5 for a com1 EFFECTS
OF MEDIA
Palate gap (mm) Anterior 0.70 0.48 0.86 0.05 0.44
* r ? -c +
Palate shelf index
Posterior
0.05 0.02 0.05 0.01 0.07
0.63
2 0.59 f 0.68 + 0.80 + 0.55 k
0.07 0.03
0.05 0.03 0.05
Anterior 1.58 2.32 1.18 4.98 2.15
+ 2 + + t
0.16 0.09 0.08 0.02 0.41
Posterior 1.21 + 0.13 1.36 + 0.08 1.09 + 0.09 2.04 -c 0.15 1.94 e 0.38
6
11.83
f 0.11
0.15 f 0.09
0.69
t 0.06
5.00 f 0.00
1.33
f 0.19
9
12.98
? 0.22
0.11 2 0.05
0.48
+ 0.08
5.00
+ 0.00
2.11
+ 0.44
5
13.37
+ 0.27
0.02
f 0.02
0.78
t 0.08
5.00
2 0.00
1.10 2 0.07
30
12.43
+ 0.16
0.08 f 0.01
0.56
-e 0.03
4.97 + 0.02
2.33
k 0.20
11
12.26 + 0.23
0.05 2 0.02
0.52
r 0.07
5.00 -c 0.00
2.00
+ 0.34
10
12.47 + 0.22
0.08 2 0.02
0.50 f 0.08
5.00 + 0.00
2.80
+ 0.37
0 All embryos have tongues removed except a and c. (a), Embryos fixed in Bouin’s solution with tongues in place without culture; (b) embryos that have tongues removed and then fixed in Bouin’s solution without culture. All values are means f SE. b Nutrient mixture F-12 and Dulbecco’s modified Eagle medium, fresh D.C. and I.C. sera. e D.C., delayed-centrifuged serum; I.C., immediately centrifuged serum. d Length after fixing with Bouin’s solution. c 300613 supernatant of day-14.5 embryo homogenates in Tyrode’s salt solution.
WEE,
WOLFSON
AND ZIMMERMAN
Palate
Rotation
in Embryo
Culture
95
FIG. 1. Heads of embryos after fixing in Bouin’s solution are shown with lower jaws removed. They are taken from (A) a day-14.75 embryo fixed in Bouin’s solution without culturing and with the tongue in place; (B) same as (A) but with tongue removed after fixing; (C) day-14.75 embryo cultured overnight with tongue x 13. removed and no drug added; (D) same as (0 but with addition of 9 x 1OF M pyridostigmine.
pletely horizontal shelf; 3 for a shelf at 45”; 2 for a shelf position between 1 and 3; and 4 for a shelf position between 3 and 5. Computing a mean position from these numerical values allowed a statistical comparison of the treatment groups. As Walker and Fraser (1956) have shown, anterior shelves start ventromedially (Figs. 2A and B) and cup the tongue between them (Fig. 1Al. The posterior shelves have the tongue ventral to the shelves which slope at an obtuse angle away from the roof of the nasal cavity (Fig. 3A). If palate shelves do rotate, there is no difficulty in showing anterior palate shelf rotation as it swings in a “barndoor” fashion (Figs. 2A to 2D). The posterior end presents much greater problems because, as Coleman (1965) has indicated, it not
only rotates but it undergoes change in tissue shape, i.e., “remodeling.” In spite of this difficulty, the same 1-5 classification was used. However, more “developmental changes” may be involved during posterior palate shelf rotation to achieve the same palate shelf index as the anterior shelf because of remodeling of the posterior shelf (Figs. 3A-E). Measurements of the distances between the shelves (gap) at the anterior and posterior ends were also made. Although the gap distance decreased as the ends of the shelves elevated, it is felt that the palate shelf index allows a more accurate assessment of rotation. Various media and various preparations of human sera have been employed. The results are shown in Table 1. Initial experiments indicated that aged human serum
96
’w I ‘I ...-. .----e DEVELOPMENTAL
VOLUME
46,
1976
5
... . ..... ....... . ..........................................1’ ... ........... ..............I.‘, :::.:.:>:.:> :.. . ~.:.::~!~~: .
A
BIOLOGY
4
\
\
I@
3
\ I ‘I. A --F ......Y. . .::::::::::::::::::I:.:::.. . .......:... :l:::::1:.~i:~:.:.~::::::::: .. ..:::: ... 1s;’ I B
‘\
;
03
\\I - -.T.rn.... ::::::::::::::::.:I:..: ..:..:.. :.:.:.:.:.:.:.:.:.,.:... .......::: .. \ :.:::: A+? 0 1
I
C
I
4
\
\
-5
0
FIG. 2. Anterior palate shelf index. Each photograph on the right shows the cross-section of a fixed embryo head taken halfway through the anterior end of the palate with lower jaw removed; x30. Each picture has a corresponding drawing on the left showing the angle between the left palate shelf and the nasal septum (heavy solid line), and the palate shelf index (P.S.I.) encircled. Reference dashed lines indicate how P.S.I. is determined. (A) and (B), The ventromedial positions of the anterior shelves when embryos have tongue excised, then head fixed without culturing; (C), a near-horizontal palate shelf for embryo cultured in 1OW M pyridostigmine for 3 hr; CD), complete flattening of anterior shelf after overnight culture in 3 x lo+ M pyridostigmine.
was not as effective as freshly prepared human serum for anterior shelf rotation (e and f). Addition of mouse embryo extract to supplement human serum (g) did not significantly improve posterior shelf rota-
tion. Human serum in which blood cells were immediately centrifuged and the plasma allowed to clot thereafter apparently allowed greater posterior shelf rotation (h, i) and have been employed in the
WEE,
WOLFSON
’ 7:y::::. +p-
E FIG. 3. Posteric heads taken halfu the angle of inclin Reference dashed
\
AND ZIMMERMAN
Palate
Rotation
in Embryo
Culture
97
I I %:::.:.I.
1
’
balate shelf index. Photographs on the right show the cross-sections of fixed embryo, through the posterior end of the palate. x 30. Corresponding drawings on the left show on of the palate shelf at the left (heavy solid line) and the palate shelf index encircled. es indicate how P.S.I. is determined.
later experiments. Nutrient mixture F-12 and Dulbecco’s modified Eagle medium both allowed approximately the same amount of shelf rotation, differences in sera accounting for the various values measured. The results of these two sera have been pooled Cc, d). It was observed that overnight embryo culture allowed
complete rotation of the anterior end of palate (P.S.I. of 4.98). However, the posterior end of the shelf only rotated to a value of about 2. Not only did the anterior and posterior ends of the shelves rotate during culture, but when the tongue was being surgically removed there was an immediate and limited shelf movement. If the
98
DEVELOPMENTAL
BIO~CY
head was fixed before removing the tongue, the P.S.I. for the anterior and posterior ends were 1.58 and 1.21, respectively (a). If the tongue was removed under medium and then the head fixed in Bouin’s solution, the shelves moved to an anterior position of 2.32 and a posterior one of 1.36 (b). This greater movement of the anterior end, under these conditions, is in accord with the results of Boss and Walker (1967). Palate shelf rotation during embryo culture was time dependent. As Fig. 4 indicates, rotation of both ends of the palate was completed by about 6 hr. In later experiments, medium 199 was employed. Since it is known that contractil-
0
2 TIME
4 - HOURS
6
FIG. 4. Time course study of the effects of lo+ M pyridostigmine and 1OmB M bethanechol on palate shelf rotation with mean +SE indicated. Palate shelf indexes are determined as described in Besuits. Number of embryos used for anterior (dashed line) and posterior (solid line) shelf rotation are: 13, 12 and 11 for 1-hr; 17, 11 and 17 for 3-hr; and 21, 17 and 25 for 6-hr cultures for controls (01, pyridostigmine- (0) and bethanechol(A) treated groups, respectively.
VOLUME
ity of muscle is a function of Ca2+ and K+ concentrations in a bathing solution, this medium containing either Hanks’ or modified Earle’s salt solutions was compared. M-199 with modified Earle’s salts seemed to produce greater shelf rotation (2.80) than that with Hanks’ (2.33), presumably because of the higher Ca2+ concentration in the modified Earle’s salt solution. However, further work is needed to verify this assumption. Growth of Embryos Table 2 reveals that average crownrump length of embryos after overnight culture increased from 10.91 mm to 12.55 mm. However, as would be expected, the increase in culture was less than that in Go. Protein content was determined for control and cultured embryos. As Table 2 indicates, there was about an eight-fold greater increase in protein of control than cultured embryos at comparable times in development. As shown in Fig. 5, the slope of the “least-squares” line for the cultured group is less than that in uiuo, indicating relatively less increase in protein than in crown-rump length during culture. The increase in elongation of the cultured embryo may be a function of the straightening of the embryo or an increase in water content. In any case, the increase was time dependent (Fig. 6B). No other changes in developmental features of the embryo during culture were observed (Fig. 7). Normally, for the overnight-culture experiments, no heart beats were observed the following morning. However, several
TABLE CROWN-RUMP Crown-rump
LENGTH
2
AND PROTEIN
length
48, 1976
CONTENT:
in Vivo
(mm)
vs in Protein
Vitro content
(mg)
Conditions In viuo In uitrd
Day 14.75
Day 15.5
10.91 + 0.20 (19)”
13.59 -c 0.30 (6)
2.68
12.55
1.64
-
e 0.18
A (24)
n Values in parentheses arc the numbers of embryos. b Embryos cultured overnight in immediately centrifuged human embryos with pyridostigmine (1O-e-1O-’ M) and 8 without drug.
Day 14.75 10.32
f 0.48
Day 15.5 (19)
serum
and Dulbecco’s
18.45
2 0.84
A (6)
11.17 + 0.48 (24) modified
8.13 0.85
Eagle medium:
16
WEE,
WOLFSON
AND
ZIMMERMAN
Palate
Rotation
in Embryo
rate decreased with the vial stopped. Effects
31 6
7 CRoviN-RUMP
8
I 9 LENGTH
I IO
I I/
mm,
between FIG. 5. Relationship crown-rump length and protein content of embryos for groups both cultured and not cultured. The solid lines are drawn by a least-squares analysis of the corresponding data points.
99
Culture
time after rotation
of Pharmacological
of
Agents
Since rotation of the posterior end of the palate shelf during embryo culture was limited, pharmacologic agents that are known to stimulate skeletal and smooth muscle contractility were added to the embryo cultures. Pyridostigmine, which stimulates skeletal muscle contraction, was first employed. Table 3 shows that 10m5M pyridostigmine partially but significantly (P
FIG. 6. Time course study of the effects of 10ms M pyridostigmine and 10e6 M bethanechol on heart rates (A) and increase in crown-rump length (B) of cultured embryos with means *SE indicated. For measuring heart rates, the numbers of embryos are: At 0 hr, control (no drug added), 22; at 1 hr, 13, 12, and 11; at 3 hr, 17,ll and 17; and at 6 hr, 14,O and 11 for control, pyridostigmineand bethanecholtreated, respectively. The numbers of embryos used for determining increase in crown-rump length are: 13, 12 and 11 for 1-hr; 17, 11 and 17 for 3-hr; 21, 17 and 25 for 6-hr, and 41 (see Table 1, d), 20 and 22 for overnight culture; for control, pyridostigmineand bethanechol-treated, respectively.
overnight-cultured embryos with beating heart or occasional limb or tail movements have been observed, especially in the more recent experiments, After 1 hr in culture, there is a significant increase (51%) in heart rate over that of the starting embryos (Fig. 6A). Thereafter, the heart rates decrease with time of culture, 107 beats/min in 3 hr, 77 beats/min in 6 hr. Initial rates were recorded because heart
FIG. 7. Photographs of a whole embryo before (A) and after (B) culture. This particular embryo grew from a crown-rump length of 11.28 mm to 12.14 mm.
x4.
100
DEVELOPMENTAL
MOUSE Conditions
-
CULTURE:
of experiment
Tongue excised 8.O
EMBRYO
Drugs
BIOLOGY 3
EFFECTS
OF PHARMACOLOGIC
Embryos (no.)
11
Cmwnrump length (mm) 12.49 + 0.33*
b.
+
41
-
12.45 2 0.14
d.
+
Pyridostigmine
(2 x 10-6M)
23
12.79 + 0.39 12.97
e.”
+
Pyridostigmine
(W5 M)
20
+ 0.22 12.68
f.
+
Bethanechol
g.
+
Pyridostigmine (10m5 M) plus bethanechol (1O-B M)
c.
Pyridostigmine
(2 x 10-eM)
6
(We M)
48, 1976
TABLE
added
-
VOLUME
Palate
Anterior
Posterior
0.86 2 0.05 0.05
1.16
1.09
+ 0.08 4.98 e 0.02
f 0.09 2.04
2 0.01 0.80 + 0.16
0.68 2 0.05 0.60 r 0.03
0.69 2 0.09
1.67
0.02
0.56
+ 0.01 0.03
2 0.21
k 0.01
20
12.29 _’ 0.18
0.03 k 0.02 0.03
line analog bethanechol, which stimulates smooth muscle contraction of the gastrointestinal tract, was employed. Bethanechol (10V6 M) also partially and significantly (P~0.05) enhanced posterior shelf movement (P.S.I. = 2.95). Since posterior shelf rotation with each pharmacological agent was not complete, both drugs were simultaneously added to the embryo culture system. The results showed no appreciable increase in rotation (3.28) compared to each individual agent (2.95). Although each individual agent increased posterior shelf movement, this effect might not be due to a cholinergic involvement in palate shelf rotation but rather due to a maintenance of heart rate for a longer time in culture or an enhancement of growth rate. Figure 6A shows that neither pyridostigmine (lop5 M) nor bethanechol (lop6 M) significantly affected heart rate. In Fig. 6B, the effect of these agents on growth, as determined by an increase in crown-rump length, is shown. Bethanechol did not affect crown-rump length while pyridostigmine caused a decrease in this parameter during the first 6 hr of culture. This effect is not considered significant because embryo length was stimulated after overnight culture.
Palate shelf index
Posterior
12.12 e 0.12
modified
gap (mm)
Anterior
22
a a-d, Pooled results using nutrient mixture F-12 and Dulbecco’s b All values are mean *SE. c e-g, Only Dulbeooo’s modified Eagle employed as medium.
AGENTS
+ 0.01
? 0.47 5.00
+ 0.05
+ 0.00
0.51 t 0.05 0.51 + 0.05 0.58 t 0.03
5.00 + 0.00 5.00 + 0.00 5.00 " 0.00
k 0.15 1.75 k 2.11 -t 2.95 2 2.95 t 3.28 ?
0.44 0.24 0.26 0.26 0.26
Eagle as media.
Figure 8 shows the effect of posterior palate shelf rotation with increasing concentrations of pyridostigmine (A) and bethanechol (B). A significant increase in posterior shelf rotation is observed at a concentration of 1O-5 M pyridostigmine (P~0.05). Although there seems to have been a greater increase in shelf rotation at the highest concentration used (74% over controls, P
ControlM.lT;,,;P,,,
$41
/co*
M-sETLHOJ~FIG. 8. Posterior palate shelf rotation dose-response curve to pyridostigmine (A) and bethanechol (B). Each point represents the mean (-tSE) of the experiments. The number of control embryos employed were 41 (no drug added, see Table 1, d). For pyridostigmine, numbers of embryos used for respective doses are: 23 for 2 x 10eB M; 17 for 6 x 1OV M; 20 for 1OW M; 34 for 3 x 1OW M; and 27 for 9 X 1OV M. For bethanechol, there are 13 embryos for lO-*O M; 16 for 1OW M; 19 for 1Om8 M; 18 for 1OW M; 22 for 1O-6 M; and 13 for lo+ M. Palate shelf indexes are determined as described in the text.
WEE,
WOLFSON
Palate Rotation
AND ZIMMERMAN
many embryos and subsequent experiments employed lower concentrations of drug. Figure 1D shows shelf rotation of both the anterior and posterior ends of the palate with 9 x 10m5M pyridostigmine. At 1OV M, bethanechol produced a significant increase (53%, P
of Strains
CD-1 and AN
The mouse strain CD-l has been employed recently in studies on cleft palate (Walker and Patterson, 1974). CD-l embryos are more advanced developmentally and their palate shelves rotated and fused 1 day earlier than the A/J strain (Walker, 1974). Thus, the ability of the palate of the CD-l strain to rotate in vitro was compared with that of the A/J strain in the presence and absence of pyridostigmine and bethanechol. Table 4 shows that when CD-l embryos at day 14.0 of gestation were
cultured in Dulbecco’s modified Eagle medium with the tongues removed, similar results were obtained to that of day-14.75 A/J embryos. Embryo culture resulted in complete rotation of the anterior palate shelf and little movement of the posterior end. Pyridostigmine (lo-” M) slightly increased posterior palate shelf movement while lo-” M bethanechol increased it significantly (P
When culturing mouse embryos during late development, rotation of the anterior end of the palate was completed within about 6 hr. Palate shelf rotation of the posterior end was also assayed and was shown to have partially taken place. Since fusion does not usually occur, this system may have some usefulness in determining whether teratogens cause cleft palate by inhibiting palate shelf rotation. If the agent does not affect rotation, the block could be on the fusion process. The major finding in this work was the observation that cholinergic drugs could stimulate shelf rotation in embryo cultures. These agents could be stimulating nonpalatal muscles and hence aid in pal-
TABLE COMPARISON Conditions Cultured
Drug added
4
OF STRAINS
of experiment
CD-l,
CD-l
-
Crown-rump length (mm)
Pyridostigmine (10m5 MI Bethanechol (1O-B M)
11.71 12.42 12.71 12.76
t 2 k k
0.04 0.18 0.21 0.16
AND A/J”
day 14.0
A/J, day 14.75
Palate shelf index Anterior
+ + +
101
in Embryo Culture
2.64 5.00 5.00 5.00
k + k f
Palate shelf index
Posterior 0.20 0.00 0.00 0.00
1.65 2.02 2.21 2.75
k 2 t k
0.17 0.24 0.31 0.29
Anterior 2.32 5.00 5.00 5.00
zt f 2 +
Posterior 0.09 0.00 0.00 0.00
1.36 2.33 2.95 2.95
t -c 2 -t
0.08 0.20 0.26 0.26
’ Embryos fkom both strains were cultured in immediately centrifuged human serum and Dulbecco’s modified Eagle medium. Numbers of embryos of CD-1 are: - incubation, 7; + incubation, 22; + pyridostigmine, 12; + betbanechol, 20. A/J: - incubation, 37; + incubation, 30; + pyridostigmine, 20; + bethanechol, 22. Values are means *SE.
102
DEVELOPMENTAL
BIOLOGY
VOLUME
48, 1976
ate shelf rotation, e.g., neck flexion and mass is primarily responsible for the “reswallowing. Alternatively, these agents modeling” of the posterior palate shelf. could be stimulating a neuromuscular ap- The contractility of the smooth muscle-like paratus in the palate shelf itself, which is mass would be in accord with the waveresponsible for the “intrinsic shelf force.” like movement from the posterior end of We have previously shown that synthesis the palate towards the anterior end, first of the contractile proteins, actin and described by Walker and Fraser (1956). myosin, in the palate was equal to that in Although pyridostigmine also increased the tongue and could represent as much as the rotation of the posterior palate shelf, it 10% of the total palate proteins synthewas unexpected that it enhanced to a limsized just prior to shelf rotation (Lessard et ited degree the rotation of the anterior end al., 1974). Recently, ultrastructural stud- of the palate before that of the posterior ies (Barbiarz et al., 1975) have revealed the end; there has been no indication of skelepresence of skeletal muscle fibers on the tal muscle myofibrils at the anterior end. oral side of the far posterior end of the This effect on the anterior shelf could indipalate. In addition, a large cellular mass cate that the skeletal muscle myofibrils was found which had characteristics of de- present in the oral side of the posterior veloping smooth muscle or mesenchymal palate function to aid in the “barndoor” cells enriched with filaments. This mass rotation of the anterior shelf. Barbiarz et was found on the tongue side of the palate al. (1975) have also shown a positive histoand extends from the mid to the posterior chemical reaction for myosin ATPase end of the shelf. along the medial edge of the midpalate Although normal palatogenesis involves (region 3). Aduss (Howard Aduss, personal descent of the tongue, the tongue has been communication) has observed that in huremoved during routine culture, and par- man submucous cleft palate, skeletal mustial palate rotation is achieved. Hence de- cle, which is present along all of the mescent of tongue below the palate shelves dial edge of the palate, contracts during and subsequent forcing of the shelves up- phonation. Presumably, this skeletal musward by the tongue is not a necessary pre- cle is replaced with cartilage and bone during normal fusion of the palatal shelves. requisite for shelf rotation. On the other hand, palate shelf rotation could not occur Therefore, it is possible that region 3 of the if the tongue had not been removed. A developing palate represents a primitive similar result has been obtained by skeletal muscle or non-muscle contractile Walker and Quarles (1973) where embryos system extending from the posterior end of are placed in a special fluid bath without the palate. Contraction of the skeletal musinterrupting maternal blood circulation. cle in the posterior palate could produce a Since shelf rotation occurs only in the ab- vector of force extending along the medial sence of the tongue in our embryo cul- edge into region 3 and contribute to rotatures, this result implies that only a minition of the anterior palate by barndoor mal intrinsic shelf force exists in the pal- rotation. ate. This would be consistent with the This work was supported by a grant from the “smooth muscle-like” mass found in the National Institute of Dental Research (No. DE posterior half of the palate exerting a sus- 03469) and a Center Grant in Mental Retardation (No. HD 05221). tainable but minimal force. In the short-term culture experiment REFERENCES (Fig. 4), bethanechol enhanced rotation at ANDREW, F. D., BOWEN, D., and ZIMMERMAN, E. F. the posterior end of the palate within 1 hr. (1973). Glucocorticoid inhibition of RNA synthesis This result would be consistent with the and the critical period for cleft palate induction in hypothesis that the smooth muscle-like inbread mice. Teratology 7, 167-175.
WEE,
WOLF~ON
Palate Rotation
AND ZIMMERMAN
BABIAFU, B., ALLENSPACH, A. L., and ZIMMERMAN, E. F. (1975). Ultrastructural evidence of contractile systems in mouse palates before rotation. De-
velop. Biol. 47, 32-44. CHABORA, A. J., and HOROWITZ, S. L. (1974). lip and cleft palate: one genetic system.
Cleft
Oral
Surg. Oral Med. Oral Pathol. 38, 181-186. COCKROFT, D. L. (1973). Development in culture of rat foetuses explanted at 12.5 and 13.5 days of gestation. J. Embryol. Exp. Morphol. 29,473-483. COLEMAN, R. D. (1965). Development of the rat palate. Anat. Rec. 151, 107-118. JACOBS, R. M. (1971). Failure of muscle relaxants to produce cleft palate in mice. Teratology 4, 25-30. LARSSON, K. S. (1962). Closure of the secondary palate and its relation to sulphomucopolysaccharides. Acta Odontol. Stand. 20, Suppl. 31, l-35. LAZARRO, C. (1940). Sul meccanismo di chiusura de1 palato secondairo. Monit. 2001. Ital. 51, 249-273. LESSARD, J. L., WEE, E. L., and ZIMMERMAN, E. F. (1974). Presence of contractile proteins in mouse fetal palate prior to shelf elevation. Teratology 9, 113-126. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193,265275. NEW, D. A. T. (1971). Methods for the culture of postimplantation of rodents. In “Methods in Mammalian Embryology” (J. C. Daniel, ed.), pp. 305-319. Freeman, San Francisco. NEW, D. A. T., COPPOLA, P. T., and TERRY, S. (1973). Culture of explanted rat embryos in rotating tubes. J. Reprod. Pert. 35, 135-138. PETER, K. (1924). Die Entwicklung Des Siiugetiergaumes. Ergeb. Anat. Entwicklungsgesch. 25, 448-564. POLZL, A. (1904). Zur Entwicklungsgechichte des menchlichen Gaumens. Anat. Hefte 27, 245-281. PONS-TORTELLA, E. (1937). ijber die Bildungsweise des sekundlren Gaumens. Anat. Anz. 84, 13-17. POURTOIS, M. (1966). Onset of the acquired potentiality for fusion in the palatal shelves of rats. J. Embryol. Exp. Morphol. 16, 171-181.
Ross, L. palatine treated 522. STEELE, iants other
in Embryo Culture
103
M.,
and WALKER, B. E. (1967). Movement of shelves in untreated and teratogenmouse embryos. Amer. J. Anat. 121,509--
C. E., and NEW, D. A. T. (1974). Serum varcausing the formation of double hearts and abnormalities in explanted rat embryos. J. Embryol. Exp. Morphol. 31, 707-719. TANIMURA, T., and SHEPARD, T. H. (1970). Glucose metabolism by rat embryos in vitro. Proc. Sot
Exp. Biol. Med. 135, 51-54. TRASLER, D. G., and FRASER, F. C. (1963). Role 01 the tongue in producing cleft palate in mice with spontaneous cleft lip. Develop. Biol. 6, 45-60. VARGAS, V. I. (1967). Palatal fusion in vitro in the mouse. Arch. Oral Biol. 12, 1283-1288. VERRUSIO, A. C. (1970). A mechanism for closure of the secondary palate. Teratology 3, 17-20. WALKER, B. E. (1969). Correlation of embryonic movement with palate closure in mice. Teratology 2, 191-198. WALKER, B. E. (1974). Palate closure in strain CD-I mice. J. Dent. Res. 53, 1497. WALKER, B. E., and FRASER, F. C. (1956). Closure of the secondary palate in three strains of mice. J Embryol. Exp. Morphol. 4, 176-189. WALKER, B. E., and FRASER, F. C. (1957). The embryology of cortisone-induced cleft palate. J. Embryol. Exp. Morphol. 5, 201-209. WALKER, B. E., and PATTERSON, A. (1974). Induction of cleft palate in mice by tranquilizers and barbiturates. Teratology 10, 159-164. WALKER, B. E., and QUARLES, J. (1973). Palate closure in embryonic mice after excising the tongue. Teratology 7, A30 (Abstract). WEE, E. L., WOLFSON, L., and ZIMMERMAN, E. F. (1975). Palate shelf rotation in embryo culture: Evidence for a cholinergic response. Teratology 11, 37A (Abstract). ZIMMERMAN, E. F., PATIL, T., and CHANG, I. F. (1974). Pharmacological study of role of contractile proteins in palate shelf movement. Teratology 9, A40 (Abstract).