the effect osf chlorcyclizine-induced palatal shelf ... · approximately 1, 2 and 3 days after the...

J. Embryo!, exp. Morph. Vol. 69, pp. 193-213, 1982 193Printed in Great Britain © Company of Biologists Limited 1982

The effects of chlorcyclizine-inducedalterations of glycosaminoglycans on mousepalatal shelf elevation in vivo and in vitro

LINDA L. BRINKLEY1 AND MARY M. VICKERMAN1

From the Department of Anatomy and Cell Biology,Medical School, University of Michigan

SUMMARYTo define whether glycosaminoglycans play a role in palatal shelf movement, we studied

the morphology and elevation behaviour of chlorcyclizine-treated mouse palatal shelves.Chlorcyclizine treatment was used because this agent enhances degradation of the palatalglycosaminoglycans, hyaluronate and chondroitin sulphates, with little or no effect on theirsynthesis. Use of in vitro and in vivo experiments enabled us to control the complicatingeffects of other factors on elevation.

Drug-administration resulted in a reduction in shelf size, as measured by cross-sectionalsurface area, in the posterior two-thirds of the palatal shelf. In vivo shelf reorientation wasalso inhibited. When elevation behaviour was observed in vitro, pronounced regional varia-tion was noted. The anterior third of the shelf was able to reorient, the posterior two-thirdswas not. This region also showed distinct histological changes as compared to control$.Mesenchymal cells were rounded with prominent nuclei and nucleoli and were more denselypacked than in controls.

These results suggest that for at least the posterior two-thirds of the palatal shelf, theintrinsic reorientation ability may in large part be linked to the acquisition of a specifictemporal and spatial distribution of hyaluronate and possibly other matrix components.

INTRODUCTION

The secondary palate is formed from two wedge-shaped masses of tissue, thepalatal shelves, that initially grow down vertically from the maxillary processesto occupy positions on either side of the tongue. Subsequently the shelvesundergo a reorientation or elevation to a horizontal position above the tonguewhere they meet in the midline, adhere and fuse to one another to form thesecondary palate. The palatal shelves are not passively displaced, but aregenerally acknowledged to play a direct and active role in their reorientation(Walker & Fraser, 1956). The internal 'shelf force' underlying their movementhas been the subject of considerable speculation and observation, but littledirect experimentation.

1Authors' address: Department of Anatomy and Cell Biology, Medical School, Universityof Michigan, Ann Arbor, Michigan 48109, U.S.A.

194 L. L. BRINKLEY AND M. M. VICKERMAN

We have confirmed the idea that a reorienting force clearly resides within theshelf itself by demonstrating that palatal shelves in severed heads with the brainand tongue removed are able to elevate (Brinkley, Basehoar, Branch & Avery,1975). Further, parts of shelves that have been lesioned transversely are able toreorient (Brinkley & Vickerman, 1979). Thus it seems that the shelves them-selves are in some way responsible for generating the internal force that resultsin a form change.

Examination of the shelves' composition may help to generate hypothesesabout the factors necessary for reorientation. The palatal shelves are composedof a core of mesenchymal cells surrounded by extracellular matrix and coveredby an epithelium. The extracellular matrix of shelves just prior to reorientationhas been shown to contain collagen, fibronectin and two glycosaminoglycans(GAGs): hyaluronate (HA) and chondroitin sulphates. HA is the predominantmolecule present at this time, comprising about 60 % of the extracellular GAGs(Pratt, Goggins, Wilk & King, 1973).

The presence, molecular state and interactions of HA with other extracellularmolecules have been correlated with morphogenetic changes in a variety ofembryonic systems (Toole, Underhill, Mikuni-Takagaki & Orkin, 1980). Anumber of unique physicochemical attributes of this macromolecule couldprovide the extracellular matrix with morphogenetically important properties.HA molecules occupy large domains and can enmesh with themselves and otherextracellular molecules to form networks which can sequester water and areresponsive to changes in ionic strength and tissue compression (Laurent, 1970;Comper & Laurent, 1978). Glycosaminoglycans, particularly HA, are thoughtto play a role in palatal shelf movement, but little direct evidence for thissuggestion exists (Lazzaro, 1940; Walker, 1961; Wilk, King & Pratt, 1978;Ferguson, 1978; Brinkley, 1980).

Study of the effects of disruptive agents can often clarify basic mechanisms.One such example is the potential effect of chlorcyclizine (CHLR) on palatalshelf morphology and behaviour. CHLR is an antihistaminic benzhydrylpiper-azine compound which has been shown to enhance the degradation of HA andchondroitin sulphates with little effect on their synthesis. Both chlorcyclizineadministered by gavage in vivo and exposure of isolated palatal shelves to itsmetabolite norchlorcyclizine (NORCHLR) in vitro causes the molecules to bedegraded into smaller-molecular-weight pieces with lower charge densities(Wilk, et al. 1978). In vivo treatment of pregnant mice with this compoundresults in both cleft palate and mandibular reductions in their offspring (King,1963; King, Weaver & Narrod; 1965; Wilk et al. 1978). Cleft palate could be asecondary result of the mandibular reduction, since a reduced mandible couldcause the tongue to become wedged between the palatal shelves, thus providinga physical barrier to shelf elevation (Diewert & Pratt, 1979). Another possibilityis that CHLR treatment is a direct cause of clefting through its effect on theGAGs within the shelves themselves (Wilk et al. 1978).

Chlorcydizine and palate elevation in vivo and in vitro 195

Anterior

Posterior Soft

Fig. 1. Schematic drawings of the method used to delimit the palatal shelves forcross-sectional area measurements. For anterior and mid presumptive hard palate,a horizontal line in register with the inferior surface of the nasal septum was drawn(broken line). Boundary lines for each shelf were determined by drawing a line fromthe edge of the oral cavity to the point of intersection of the horizontal line with thenasal shelf surface. Posterior presumptive hard palate and soft palate were delimitedby drawing a line from the medial edge of the tooth germ, if present, or edge of theoral cavity if no tooth germ was present, which was also tangential to the curve ofthe roof of the oral cavity. The first point of intersection of this line with the roofof the oral cavity was then used as the most superior point of the shelf. Represen-tative cross-sectional areas which were measured by this method are stippled.

To distinguish between these effects both in vivo and in vitro techniques mustbe used. We have previously shown that normal palatal shelves are able toelevate, adhere and fuse in our in vitro system (Brinkley et al. 1975; Lewis,Thibault, Pratt & Brinkley, 1980). We can use this culture system to determinedirectly the effects of CHLR treatment on the shelves without the complicatingeffects of the tongue. Comparison of in vivo and in vitro behaviour of the palatalshelves of CHLR-treated embryos should contribute to a better understandingof the possible role glycosaminoglycans play in shelf elevation.

MATERIALS AND METHODS

Animals. A random-bred CD-I mouse strain was used for all experiments.Animals were maintained in quarters with controlled light cycles and fed Purinamouse chow and water ad libitum. Fertilization was assumed to occur betweenmidnight and 2 a.m. of the morning the vaginal plug was found.

Chlorcydizine administration. Sequential doses (250 mg/kg) of chlorcydizinehydrochloride (CHLR) (Burroughs-Wellcome) were administered to pregnantmice via intragastric tube on gestational days 10-5, 11-5 and 12-5, as these threedays were found to be the sensitive period for CHLR induction of cleft palate.Each dose was delivered in a total volume of 0-5 ml sterile water. Controls Weregiven intragastric feedings of equal volumes of sterile water.


Animals were sacrificed on days 13-5, 14-25, 14-5 or 15-25, times which wereapproximately 1, 2 and 3 days after the final dose of CHLR. Palate closure inthese mice normally occurs by day 14-5.

The drug treatment resulted in an 85 % survival rate of the treated mice. Halfof these mice had litters with 100% survival, a quarter had litters with a 10%resorption incidence and a quarter had litters with about 50% resorption. In alllitters, the drug dosage given resulted in 100 % cleft palate in offspring of treatedmothers despite the fact that the treatment ended two days prior to palateclosure.

Preparation of explants and culture technique. Control and CHLR-treatedpregnant females were killed by cervical dislocation at day 13-5 or 14-25.Foetuses were removed under sterile conditions, measured for crown-rumplength and assigned a morphological rating of the developmental state of foreand hindlimbs, ears, eyelids and hair follicles using the system of Walker &Crain (1960). Inter- and intralitter variability was minimized by excluding anyindividuals that differed from the group in either of these two parameters.

The brain and tongue were removed and then a small vent was cut in thefloor of the posterior oral cavity to allow for better circulation of culturemedium. The mandible was left in place, as described previously (Brinkley et al.1975). Several of the heads were then fixed in phosphate-buffered formalin toserve as time zero, unincubated controls. The remainder were hung in a gassed,circulating culture chamber that we developed (Lewis et al. 1980).

Specimens with unelevated palatal shelves were incubated in either standardmedium or standard medium containing norchlorcyclizine (NORCHLR), themetabolite of CHLR. In vivo CHLR was administered by gavage, as the animalconverts the compound to its metabolite NORCHLR. For in vitro studiesNORCHLR itself was used. This procedure provided for comparison of theelevation behaviour of the palatal shelves under recovery conditions providedby standard medium, or in the continued presence of the active drug.

Standard medium consisted of BGJb medium (Gibco) with 10 % foetal bovineserum (KC Biologicals), supplemented with 8 mM glutamine and containing50 /*g/ml gentamicin (Schering Corp.). When norchlorcyclizine (NORCHLR)was added to the medium, a final concentration of 10 /*g/ml was used. Culturescontaining NORCHLR were always kept in the dark because of the compound'slight-sensitivity.

In all cultures the medium was constantly circulated, gassed with 95% O2,5% CO2 using silicone copolymer hollow fibre devices, and maintained at34 °C. Specific details of the procedure are described in Brinkley et al. (1975)and Lewis et al. (1980). Specimens were incubated 18 h, a time span previouslyshown to permit elevation and adhesion in day-13 palates (Brinkley, Basehoar& Avery, 1978).

Histological technique. Specimens to be examined histologically were fixed for24 h in phosphate-buffered formalin, dehydrated through an alcohol series and

Control {n = 12)CHLR (n = 37)Control in = 24)CHLR (n = 20)Control in = 59)CHLR (n = 26)Control in = 27)CHLR(/z = 11)

10-4 ±0-29-8±0-3f

12-4±0-510-6 + 0-5t12-7 ±0-611-1+Olf14-6 ±0-414-2 ±0-4

4-52-3

14-156-9

14-178-9

24-2524-25

Chlorcyclizine and palate elevation in vivo and in vitro 197

Table 1. Chlorcyclizine effects on growth and morphology

Crown-rump MorphologicalGestational age and treatment length (mm)* rating

Day 13-5

Day 14-25

Day 14-5

Day 15-25

* Mean ± standard deviation.f Different from age-matched control values, P < 0005.

embedded in glycol methacryiate. Sections, 3 fim thick, were cut and stainedwith toluidine blue.

Evaluation techniques

Palatal Shelf Index (PSI). The degree of elevation of palatal shelves wasassessed using the palatal shelf index (PSI). The PSI is a five-level scale to assessdeviation of the palatal shelves from the horizontal (Wee, Wolfson & Zimmer-man, 1976). A grid with five lines from vertical (1) to horizontal (5) was placedin the ocular of a dissecting microscope. Then, with the grid in place, freehandrazor-blade sections were made transversely through the palate of each fixedspecimen at four levels along the rostral-caudal axis. The slice was exposed toa drop of toluidine blue to enhance contrast. Then the shelf position wasassessed by placing the horizontal line in register with the inferior surface of thenasal septum or cranial base and recording which of the five angular lines mostclosely corresponded to the position of the nasal surface of each shelf. This gridwas also used to assess stained, mounted sections of palates.

Cross-Sectional Area (CSA) measurements. Sections of anterior, mid andposterior presumptive hard palate and presumptive soft palate were selectedfrom each specimen. With the aid of a microscope equipped with a drawingtube, both shelves were traced. Anatomical landmarks visible in each regionwhich were used to assign a given section to a palate region were those ofDiewert (1978). The landmarks were: (1) anterior hard palate: nasal septum,vomeronasal organs, nasal cavity and palatal shelves; (2) mid hard palate: nasalseptum posterior to vomeronasal organ, nasal cavity and palatal shelves; (3)posterior hard palate: posterior aspects of nasal cavity or immediately posteriorto it, maxillary and mandibular toothbuds; (4) soft palate: cranial structures,the cranial base and palatal shelves. The shelf area measured for each regiondescribed is shown in Fig. 1.

o CO

Tab

le 2

. E

leva

tion

beh

avio

ur o

f co

ntro

l an

d C

HL

R-t

reat

ed p

alat

al s

helv

es in

vit

ro

Ges

tati

onal

age

and

tre

atm

ent

Ant

erio

rM

idPo

ster

ior

Soft

r

Pal

atal

she

lf i

ndex

of

shel

f re

gion

s (m

ean

± st

anda

rd d

evia

tion)

®

Tim

e ze

ro (

n =

14)

2-

7 ±

0-7

2-6

±0-

5 2-

3 ±

0-6

1-4

±0-

5C

ultu

red,

sta

ndar

d m

ediu

m (

n =

20)

5

0±

0*

4-1

±0-

7*

4-0±

0-7*

3-

7±0-

6*T

ime

zero

(n

= 1

2)

2-5

±0-

6 2-

9 ±

0-2

1-9

±0-6

1-

7 ±

0-4

Cul

ture

d, s

tand

ard

med

ium

(n

= 1

6)

4-6±

0-5*

3-

6±O

-7*

2-2±

0-6

2-0±

0-4

Cul

ture

d, N

OR

CH

LR

med

ium

(n

= 1

0)

4-3±

0-5*

3-

5±O

-5f

2-2±

0-6

l-0±

0-2

Tim

e ze

ro (

n =

8)

50

±0

50

±0

50

±0

50

±0

Tim

e ze

ro (

n =

14)

4-

2±0-

7*,$

3-

3±O

-5*,

t 2-

7±0-

5*,}

2-

l±0-

5*,:

Cul

ture

d, s

tand

ard

med

ium

(n

= 1

4)

4-5±

0-8

4-2±

0-9

3-9±

l-0f

3-

8±l-

2fC

ultu

red,

NO

RC

HL

R m

ediu

m (

« =

8)

4-8±

0-7

41

±1-

0 4-

1 ±

l-0f

40

± l-2

f

* D

iffer

ent

from

age

-mat

ched

, ti

me

zero

, con

trol

s, P

< 0

01

.t

Dif

fere

nt f

rom

age

-mat

ched

, tim

e ze

ro,

CH

LR

s, P

< 0

01

.i

Diff

eren

t fr

om d

ay-1

3-5

time

zero

, CH

LR

s, P

< 0

05.

Day

Day

13-5

14-2

5

Con

trol

s

CH

LR

Con

trol

sC

HL

R

D S < i—i o m

Tab

le 3

. Cha

nges

in

shel

f cr

oss-

sect

iona

l are

a ov

er t

he ti

me

ofm

vi

vo p

alat

e cl

osur

e

Ges

tati

onal

Day

13-

5

Day

14-

25

Day

14-

5D

ay 1

5-25

age

and

trea

tmen

t

Con

trol

s («

= 8

)C

HL

R i

n =

12)

Con

trol

s (n

= 8

)C

HL

R i

n =

14)

Con

trol

s («

= 1

0)C

HL

R i

n =

12)

Cro

ss-s

ectio

nal

Ant

erio

r

61-7

±10

-959

-4 +

22-

711

0-9±

33-7

t97

-7±3

5-4f

167-

4±49

-3J,

§11

9-0±

26-8

§

area

of

shel

f re

gion

s i/

im2

Mid

97-9

+10-

794

-9 ±

17-8

194-

2 ±8

0-0f

127-

8 +4

2-5*

, f25

1-5

+ 5

8-3J

199-

2 ±3

1-6§

x 10

3 ) (m

ean

± st

anda

rd

Post

erio

r

156-

2 +

25-

311

4-9

±2

80

*18

2-3

±38-

914

8-3

+ 4

4-9*

, t20

2-9+

30-

4f24

2-3

±40

0§

devi

atio

n)

Soft

110-

6 ±4

4-4

78-1

±34

-1*

174-

9 ±3

5-8f

122-

7 +3

6-8*

, f18

1-3

±47-

2f18

2-6

±40-

4

• 3 OS a late elevatior

* D

iffer

ent

from

age

-mat

ched

con

trol

s, P

< 0

-05.

t D

iffe

rent

fro

m d

ay-1

3-5

trea

tmen

t-m

atch

ed g

roup

s, P

< 0

-01.

% D

iffe

rent

fro

m d

ay-1

3-5

and

day

14-2

5 tr

eatm

ent-

mat

ched

gro

ups,

P <

000

4.§

Dif

fere

nt f

rom

day

-14-

5 co

ntro

ls, P

< 0

-005

.o s I—

>• p 3


The anterior and mid regions each represent about 20% of overall shelflength, while the posterior and soft regions comprise about 30% each (Brinkley& Vickerman, 1979).

Sampling and statistical analysis. Because of possible individual variability inall evaluation parameters samples were taken by randomly selecting two ormore foetuses from each of 4-12 litters. Crown-rump length, PSI and CSA datawere analysed by a univariate analysis of variance. Only differences found to besignificant with P < 0-05 were accepted.

RESULTSIn vivo

Growth and morphology. Beginning on day 13-5, and continuing to day 14-5,the day of usual palate closure, GHLR-treated individuals were smaller asjudged by crown-rump length and were less well developed as evidenced bylower morphological ratings (Table 1). By day 15-25, almost three days afterthe last drug treatment, CHLR-treated foetuses had apparently recovered andattained the same crown-rump length and morphological rating as controlindividuals. However, at all ages examined, CHLR-treated foetuses had bothcleft palate and reduced mandibles.

Palatal shelf elevation. On day 13-5 no difference in shelf position was foundbetween control and CHLR palates (Table 2). By day 14-25 palates of controlfoetuses had closed whereas only the anterior, and to a lesser degree the posteriorof the CHLR-treated foetuses showed changes in shelf positions. The anteriorhad essentially reoriented, but despite some change in the position of theposterior shelves, it was insufficient to achieve elevation of this portion.

Changes in shelf CSA. Cross-sectional area of control and CHLR-treatedshelves was measured at several times over the span of expected palate closure(Table 3). On day 13-5, one day prior to palate closure, a comparison of CSAvalues of control and CHLR shelves shows that for this parameter the posteriorand soft palate regions are significantly affected by CHLR treatment. These tworegions were reduced to 74% and 71 % of control values, respectively.

After palate elevation, day 14-25, controls showed significant CSA increasesin anterior (80 %), mid (98 %) and soft (58 %) palate regions over that seen atday 13-5. But no significant change in the posterior region was seen. By the timeadhesion had taken place (day 14-5), the anterior and mid shelf showed anadditional increase in CSA.

During the same period, day 13-5 to day 14-25, only the anterior region ofCHLR-treated palates had elevated (Table 2). The CSA values of control andCHLR shelves were similar in the anterior, but the CHLR values were signi-ficantly lower than those of controls in the remainder of the palate. CHLR-treated mid palate achieved only 66% of control surface area, whereas posterior

Tab

le 4

. In

vit

ro c

hang

es i

n cr

oss-

sect

iona

l are

a of

day

-13-

5 pa

lata

l sh

elve

s

In v

ivo

trea

tmen

t an

d cu

ltur

e co

ndit

ions

Cro

ss-s

ectio

nal

area

of

shel

f re

gion

s (/*

m2 x

103 )

(mea

n ±

stan

dard

dev

iatio

n)

Ant

erio

rM

idPo

ster

ior

Soft

Con

trol

Tim

e ze

ro (

n =

8)

61-7

± 1

0-9

Cul

ture

d, s

tand

ard

med

ium

(n

= 1

2)

84-9

± 1

6-3

CH

LR

Tim

e ze

ro (

n =

12)

59

-4 ±

22-7

Cul

ture

d, s

tand

ard

med

ium

(n

= 2

2)

75-0

±22

-5C

ultu

red,

NO

RC

HL

R m

ediu

m (

n =

20)

66

-1 ±

20-

8

97-9

±10

-715

1-1

±36-

6*

94-9

±17

-812

1-3

±32

-4*,

t97

-2±3

7-0§

* D

iffe

rent

fro

m t

he tr

eatm

ent-

mat

ched

tim

e ze

ro g

roup

, P <

002

.t

Dif

fere

nt f

rom

con

trol

s, t

ime

zero

, P <

0-0

2.t

Dif

fere

nt f

rom

con

trol

s, c

ultu

red

in s

tand

ard

med

ium

, P <

0-0

3.§

Dif

fere

nt f

rom

CH

LR

, cu

lture

d in

sta

ndar

d m

ediu

m, P

< 0

01

.

156-

3 ±2

5-3

131-

7±18

-3

114-

9±28

0t16

9-4

± 32

-3*,

t13

1-2±

22-8

§

110-

6 ±4

4-4

129-

5 ±3

1-8

78-l

±34

-lf

114-

8 ±

30-8

*, t

83-3

±32

-5

a" 5^ s.


Table 5. In vitro changes in cross-sectional area of CHLR-treatedday-14-25 palatal shelves

Cross-sectional area of shelf regions Cam2 x 103)(mean ± standard deviation)

Treatment Anterior Mid Posterior Soft

Time zero (n = 14) 97-7 ±35-4 127-8 ±42-5 148-3 ±44-9 122-7 ±36-8Cultured, standard

medium (n = 14) 119-3±30-6 118-8±43-3* 186-7 + 25-1* 127-3±16-4

* Different from time zero, P < 0003.

and soft palate had CSAs which were 8 1 % and 70% of control values, re-spectively.

Since no in vivo elevation of CHLR shelves had taken place by day 14-25, anadditional day of in vivo development was allowed to determine what changesif any, would occur in shelf position and area. By day 15-25, three days afterthe last in vivo CHLR treatment, 5 of 12 individuals (42%) had shelves whichwere elevated, but all had large gaps between them. No statistically significantdifference in CSA was found between elevated and unelevated day-15-25 shelves.Thus, for purposes of comparison with controls, the mean of all values was used.

No significant increase in CSA over that seen at day 14-25 was observed forthe anterior, but the remaining shelf regions had increased. The cross-sectionalareas achieved by the anterior, mid and soft palate regions of day-15-25 CHLRshelves were approximately those of day-14-25 controls, that had all elevated.However, the posterior region of day-15-25 CHLR shelves attained areasgreater than those seen in either day-14-25 or -14-5 controls.

In vitro

Palatal shelf elevation. Palates of day-13-5 control specimens were able toelevate along the entire length of the shelf, and adhere in the posterior two-thirds of the shelf in 67% of the cases. When day-13-5 CHLR individuals wereincubated in standard medium, elevation was achieved in the anterior and tosome degree the mid regions, but no change in shelf position was observed ineither the posterior or soft region. CHLR specimens cultured in medium con-taining NORCHLR exhibited the same regional pattern of shelf-elevationbehaviour (Table 2).

Day-14-25 CHLR-treated palates were cultured to determine whether theposterior and soft regions of the shelves would now be able to elevate in vitro.By this age these regions had achieved CSAs similar to day-13-5 controls.CHLR-treated palates were indeed able to elevate in both regions whethercultured in standard medium or NORCHLR medium (Table 2).

Tab

le 6

. Su

mm

ary

of c

hang

es i

n sh

elf

Pal

atal

She

lf I

ndex

an

d cr

oss-

sect

iona

l ar

ea d

urin

g p

alat

e cl

osur

e

Tre

atm

ent

Shel

f re

gion

(Pa

lata

l Sh

elf

Inde

x/cr

oss-

sect

iona

l ar

ea)

Ant

erio

rM

idPo

ster

ior

Soft

, 1

In v

ivo

Con

trol

sC

HL

RIn

vit

roC

ontr

ols

CH

LR

, st

anda

rd m

ediu

mC

HL

R,

NO

RC

HL

OR

med

ium

0/+

+

+ +

+ +

/0+

+ +

/0+

+ +

/0+

+/0

+ +

+ +

/0

+ +

+/0

0/+

+

0/+

+ +

+ +

/00/

+ +

0/0

PS

I: N

o ch

ange

= 0

; sig

nifi

cant

inc

reas

e to

fina

l PSI

of:

2-2

5-3-

0 =

+ ;

3-0

-3-7

5 =

+

+ ;

3-7

5-4-

25 =

+ +

+ ;

4-2

5-5-

0 =

+ +

+ +

CS

A:

No

chan

ge =

0; s

igni

fican

t in

crea

se o

f: 0

-25

% =

+ ;

25-

50 %

= +

+ ;

50-

75 %

= +

+ +

; 7

5-10

0 %

= +

+ +

+.

o I a. 3


Fig. 2. Day-13-5 control (A and B) and CHLR-treated (C, D and E) shelves beforeand after incubation. (A) Time-zero controls, prior to incubation. (B) After culture,shelves have elevated and adhered. Areas of lowered cell density are obvious (-*).(C) Unincubated, time-zero CHLR-treated shelves. (D) Incubated in standardmedium. Note enlargement of vascular spaces (*). (E) Incubated in NORCHLRmedium. Vascular spaces are not enlarged.

Changes in shelf CSA. Only the mid region of control day-13-5 palates in-creased significantly in surface area during the in vitro culture period (Table 4).CHLR specimens cultured in standard medium showed significant CSA in-creases in the mid, posterior and soft palate regions. Despite this increase,neither the mid nor soft palate of those specimens achieved CSA values attainedby the same regions of cultured controls. The CSA increase found in theposterior brought the CHLR shelves to a size similar to that of unculturedcontrol shelves that showed no CSA change during their culture period.

CHLR specimens cultured in NORCHLR-containing medium increased in


Fig. 3. Comparison of mesenchymal cells of day-13-5 control (A and B), andCHLR-treated (C, D and E) shelves. (A) Mesenchymal cells of time zero, un-incubated shelves. (B) After in vitro elevation and adhesion. (C) Mesenchymalcells of time zero, unincubated CHLR shelves. (D) After incubation in standardmedium. (E) Cells of shelves incubated in NORCHLR medium. Although denselypacked, several mitotic figures were observed in each field (->).

surface area only in the posterior, achieving a size similar to controls, butsmaller than that of CHLR shelves cultured in standard medium.

When day-14-25 CHLR-treated palates were cultured in standard mediuiti,the CSA of these shelves increased significantly in the posterior regions (Table5), and achieved a CSA similar to the posterior region of day-14-25 controls bythe end of the culture period (Table 3).

Summary ofPSI and CSA changes in vivo and in vitro. Table 6 contains atabulation of the significant changes observed in both elevation behaviour aridshelf surface area in vivo and in vitro. In vivo, shelf elevation and expansion incross-sectional area of controls occurred concurrently in the anterior, mid afld


Fig. 4. Comparison of day-14-25 control (A) and CHLR-treated (B and C) shelves.(A) Control shelves. (B) Unincubated CHLR shelves. (C) CHLR shelves elevatedin vitro, o, Areas of osteogenesis; t, tooth germ.

soft shelf regions, but elevation without expansion was seen in the posteriorshelf. In vitro, only mid shelf of control specimens expanded during elevation.Thus, in normal palates, elevation of the anterior, posterior and soft palateregions does not appear to be linked to overall shelf expansion as measured bychanges in CSA.

CHLR treatment effectively blocked shelf elevation in vivo in the mid and softpalate and retarded it to a large degree in the posterior region. However,increases in CSA of these regions was observed. In vitro behaviour of CHLRshelves reinforced the findings from controls that changes in overall shelf CSAare not necessarily linked to elevation in the anterior, posterior and soft palateregions. This may not, however, be the case for the mid-shelf region. It showeda pronounced divergence of behaviour in vitro as compared to in vivo. In vitromid region was able both to elevate and to expand to some degree in standardmedium, whereas in vivo only CSA expansion is observed.

The presence of NORCHLR in the culture medium permitted the same degreeof shelf reorientation seen in standard medium, but effectively blocked orseverely reduced CSA expansion in all shelf regions.

It is clear from both in vivo and in vitro studies that CHLR treatment renderedthe caudal two-thirds of the palate, the posterior and soft palatal regions, unableto elevate. These were also the regions whose day-13-5 CSA was significantlyreduced as compared to control shelves (Table 3).


Fig. 5. Comparison of mesenchymal cells of day-14-25 control (A and B) andCHLR-treated (C and D) shelves. (A) Medial region of adhered control shelves.(B) Control mesenchymal cells still show similar morphology and density to thatseen at day 13-5. (C) Medial region of adhered CHLR shelves. (D) Cells of CHLRshelves are now more similar to controls in morphology, but are still smaller.

Histological effects of chlorcyclizine

Day 13-5. The mid, posterior and soft palate regions of the shelf exhibitedhistological changes after CHLR treatment. Examined at low magnification,control shelves (Fig. 2 A) had less densely packed mesenchymal cells than doCHLR-treated specimens of the same age (Fig. 2C). Local differences in cellpacking were also apparent. An area of lower cell density medial to the toothgerm was observed in control shelves, which was less extensive in the CHLR-treated specimen. After in vitro elevation this area of lowered cell density wftspronounced in control specimen (Fig. 2B), less so in CHLR shelves cultured instandard medium (Fig. 2D), and NORCHLR-cultured specimens showed little


change in this area (Fig. 2E). CHLR-standard-medium-cultured specimens alsoshowed an area of decreased cell density on the nasal side of the shelf, whichwas not seen in controls or NORCHLR-cultured individuals. It was also con-sistently noted that the lumens of the blood vessels of CHLR-standard-culturedshelves were considerably expanded when compared to control animals or toNORCHLR-cultured specimen.

A qualitative comparison of cell density, size and morphology at highermagnifications revealed substantial differences between uncultured controltissue (Fig. 3 A) and that of uncultured CHLR-treated individuals (Fig. 3C).Mesenchymal cells of control shelves appeared less densely packed, smaller,more elongate or stellate, and with relatively indistinct nuclei. In contrast, cellsof uncultured CHLR-treated tissue (Fig. 3 C) were more densely packed, larger,more rounded and exhibited distinct nuclei with prominent nucleoli. Afterculture (Fig. 3B), cells of control tissue appeared even more elongate and in factboth cells and nuclei exhibited wavy perimeters. Culture of CHLR-treatedspecimens in standard medium (Fig. 3D) resulted in shelves with mesenchymalcells which appear to be smaller than those of uncultured CHLR individuals,but which had a similar rounded shape with prominent nuclei and nucleoli.However, they remained larger and more densely packed as compared to in-cubated controls (Fig. 3B). Those cultured in NORCHLR medium (Fig. 3E)exhibited extremely dense mesenchymal cell packing, and cells which were aboutthe same size as those seen in uncultured CHLR specimens. However, thenuclei were lighter staining with several prominent nucleoli; little cytoplasm wasvisible around them. Although densely packed with cells, several mitotic figureswere observed in each field.

Day 14-25. All control specimens were elevated and adhered (Fig. 4 A) withvisible epithelial breakdown and areas of osteogenesis. CHLR individuals(Fig. 4B), however, showed no elevation, retarded tooth-germ development andless-developed areas of osteogenesis. If such treated individuals were culturedin standard medium (Fig. 4C), 70% elevated and adhered and ossificationproceeded.

High-power views of control shelves (Fig. 5 A, B) revealed mesenchymal cellmorphologies similar to those seen in day-13-5 controls. Whereas mesenchymalcells of CHLR shelves which had elevated in vitro (Fig. 5 C, D), remained moredensely packed than controls and still exhibited the prominent nuclei andnucleoli observed in day-13-5 CHLR specimen. Cells of day-14-25 CHLRshelves (Fig. 5D) also appeared smaller than those of controls (Fig. 5B).

DISCUSSION

Nature and distribution of CHLR effect

Direct effect on shelves. Our experiments demonstrate that CHLR adminis-tration results in changes in shelf size and loss of the inherent ability to elevate.

Chlorcyclizine and palate elevation in vivo and in vitro 209Other investigators have demonstrated direct effects of CHLR on GAGs in thepalatal shelves, but have also shown an effect on mandibular growth (Wilk et al.1978; King, 1963). Therefore, in our studies either GAG effects or mandibularreduction could be key elements in the observed clefting. The latter alternativecan be eliminated. We have shown that the elevation ability of the palatalshelves is directly affected by CHLR treatment using in vitro techniques, whereinthe tongue is removed and the behaviour of the shelves monitored directly.

Distribution of effects. CHLR effects on shelves were also observed to beregional, with the severity of response to drug treatment graded in an anterior-posterior manner. A similar gradation in in vitro elevation behaviour has beenpreviously observed (Brinkley & Vickerman, 1979). The anterior shelf appearedto be unaffected by CHLR treatment. The loss of elevation ability and reductionof shelf CSA were evident in the mid to soft regions. The greatest effects werefound in the posterior and soft areas.

There are two possible explanations for a lack of effect in the anterior. Eitherformation and elevation of the anterior palate are not dependent on productionof an extensive extracellular matrix, and thus are unaffected by CHLR treat*ment, or the anterior recovers more rapidly and to a much greater extent thanother regions. The former explanation seems most likely, not only because of theconsistent lack of any drug effect on the CSA and elevation changes in this area>but also because the anterior is the most highly cellular region of the palate,with little extracellular space visible (Brinkley, 1980). Likewise, the pronouncedeffects observed in the posterior portion of the shelves suggest that these area$may be richer in the two CHLR-affected GAGs: HA and the chondroitifisulphates.

Shelf expansion and elevation ability

One of the oldest proposed mechanisms for the motive force underlyingpalatal shelf movement is a rapid increase in shelf volume due to increasedintercellular volume in the connective tissue (Lazzaro, 1940; Walker, 1961). Invivo, shelf expansion appears to occur over the course of rat palate closure whenobserved in frozen sections (Diewert & Tait, 1979). Our in vivo findings of shelfcross-sectional area expansion are in agreement with those of Diewert & Taitfor all shelf regions except the posterior, where we observed no significant shelfexpansion. It is interesting that both the frozen tissue used by them and thefixed tissue used in the present study reveal a fairly similar pattern of in vivoexpansion despite the tissue shrinkage associated with fixation.

Comparison of both in vivo and in vitro studies permits us to determinewhether shelf expansion is actually necessary for shelf elevation. Our studiesdemonstrate that overt shelf expansion, as measured by change in shelf cross-sectional surface area, is not required for shelf reorientation in the anterior,posterior and soft palate, given the degree of maturity of a day-13-5 palatal shelf.In the mid palate though, elevation and expansion appear to be linked to some


degree. Present results also suggest that the in vivo condition is somehow relatedto shelf expansion. Given the physical properties of an HA-rich extracellularnetwork it seems possible that in vivo shelf expansion is a response to the ex-ternal compression of the shelves caused by their wedged position between thetongue and mandible. This expansion may be necessary for initiating the move-ment of the shelves around the tongue or for stimulating the tongue to moveout of the way of the elevating shelves.

GAGs and shelf elevation

HA is the predominant GAG species present in the shelves around the timeof elevation (Pratt et ah 1973). Thus CHLR effects on shelf morphology andelevation behaviour must in large part be attributable to degradation of thismolecule into smaller pieces, resultant changes in its ionic responsiveness and/oraltered interactions with other extracellular molecules. To understand how thesechanges might affect shelf elevation it is necessary to examine some of theproperties of HA.

HA in extracellular matrices. The hyaluronate content of the extracellularmatrix is known to be important in the morphogenesis of that tissue (Toole et ah1977; Toole et ah 1980). HA is a very large, highly charged polyanion with alarge molecular domain that tends to sequester water (Laurent, 1970). Itsmolecular weight, length and the presence of other molecules including proteinsor cations can influence the viscosity of a hyaluronate solution (Balazs & Gibbs,1970; Comper & Laurent, 1978; Morriss, Rees & Welsh, 1980). Hyaluronateoften enmeshes with sulphated proteoglycans and collagen to form a gel-fibrenetwork characteristic of extracellular matrices (Bernfield, 1981). Cellularbehaviour is known to be directly influenced by its association with the extra-cellular matrix. For instance, anchorage of cells to a substratum determines cellshape and mobility and can thus alter cell metabolism (Bernfield, 1981).Indirectly, the gel-fibre matrix could simply contribute to a complex frameworkwhose shape is the primary determinant of tissue form (Nakamura & Manasek,1978).

These networks not only have specific physical configurations due to theirmolecular components, but also have unusual physiological properties. Suchnetworks are capable of generating a 'swelling pressure' that is composed of anosmotic contribution from the hyaluronate portion's tendency to sequesterwater and an elastic contribution from the contractility of the fibres. If theequilibrium between osmotic and elastic components is disturbed the networkwill expand or shrink, depending on which components are affected (Comper &Laurent, 1978). Morphogenetic changes could then be brought about in tissuesas a result of these physicochemical properties. Physical disturbance such asexternal compression of a tissue containing the network, or chemical changessuch as local or systemic variations in the ionic environment, could alter theconfiguration of the network and in turn that of surrounding tissue. Such

Chlorcyclizine and palate elevation in vivo and in vitro 211remodelling could passively displace or translocate cells to areas of least re-sistance (Solursh, Fisher, Meier & Singley, 1979; Brinkley, 1980), or providespaces for actual cell migration (Pratt, Larsen & Johnston, 1975; Tosney, 1978;Erickson, Tosney & Weston, 1980).

Consequences of altered HA. If one major component, for example HA, isabnormal, it is reasonable to expect an alteration in extracellular matrix organ-ization. Such a disturbance could affect cell attachment to the matrix as well asthe physicochemical properties of the matrix itself. If the cells were unable toattach properly a rounded rather than stellate appearance could result, and achange in cellular metabolism might occur which could preclude active cellmigration. An altered gel-fibre network in the extracellular matrix might also beunable to undergo conformational changes necessary to displace cells passivelyand create local expansions and compressions.

Both the elevation behaviour of CHLR-treated shelves and the histologicalappearance of their mesenchymal compartment provide strong support for theidea that HA plays a central role in shelf elevation through its spatial distribu-tion'and macromolecular interactions in an extracellular network. The regionalnature of CHLR effects on elevation suggests that there is an underlying spatialor temporal sequence of either HA and chondroitin sulphate production in theshelves or of differential recovery from the effects of CHLR. Histologically themesenchymal cells of CHLR shelves appear large and rounded with prominentnuclei and nucleoli which may reflect direct metabolic alterations due to theCHLR. Their morphology might also be the result of an inability to adhereproperly to the surrounding abnormal extracellular matrix. Another histologicalfinding that may be related to the degradation of hyaluronate is that, followingin vitro culture in standard medium, the vascular spaces of CHLR-treatedshelves are enlarged as compared to controls. This result is comparable to thatof Singley & Solursh (1981), who found similar vascular inflation after degrada-tion of hyaluronate by local injection of Streptomyces hyaluronidase in chickwing buds.

Culture in NORCHLR medium, however, did not elicit this response, but didresult in a noticeable increase'in the number of dividing cells. It seems possiblethat further exposure to the drug may have resulted in sufficient additionalalterations in the gel portion of the matrix both to change its osmotic properties,precluding vascular enlargement, and to alter the relationship of cells to matrixwhich may have resulted in mitotic stimulation.

Possible role of HA in shelf elevation. Our studies do not rule out the possibilitythat some subtle regional shelf expansion may indeed play a role in shelf eleva-tion, but do preclude overall shelf swelling as the motive force. Alteration of theshape of a structure such as a shelf, with no change in surface area, could beaccomplished by local expansions compensated for by other local compressionsBoth the present results and changes previously observed in cell patterning oyerthe course of shelf elevation (Brinkley, 1980), suggest that the internal compo-


nents must be rearranged but not expanded. Given the wide range of propertiesof gel-fibre matrices a highly patterned distribution of an HA-rich extracellularmatrix with specific local variations in the nature of the matrix could be theunderlying physical basis for such local tissue expansions and contractions. Thedevelopment of 'internal shelf force', i.e. the intrinsic ability of shelves toelevate, may in large part be the acquisition of a specific temporal and spatialdistribution of HA and other matrix components.

This research was supported by NIH-NIDR grant DE02774 to L.L.B.

REFERENCES

BALAZS, E. A. & GIBBS, D. A. (1970). The rheological properties and biological functionof hyaluronic acid. In Chemistry and Molecular Biology of the Intercellular Matrix.New York.

BERNFIELD, M. (1981). Organization and remodelling of the extracellular matrix in morpho-genesis. In Morphogenesis and Pattern Formation (ed. T. G. Connelly, L. L. Brinkley &B. M. Carlson), pp. 139-162. New York: Raven Press.

BRINKLEY, L. L. (1980). In vitro studies of palatal shelf elevation. In Current ResearchTrends in Prenatal Craniofacial Development (ed. R. M. Pratt & R. L. Christiansen), pp.203-220. New York: Elsevier/North-Holland.

BRINKLEY, L., BASEHOAR, G., BRANCH, A. & AVERY, J. (1975). A new in vitro system forstudying secondary palatal development. / . Embryol. exp. Morph. 34, 485-495.

BRINKLEY, L., BASEHOAR, G. & AVERY, J. (1978). Effects of craniofacial structures on mousepalatal closure in vitro. J. dent. Res. 57, 402-411.

BRINKLEY, L. L. & VICKERMAN, M. M. (1979). Elevation of lesioned palatal shelves in vitro.J. Embryol. exp. Morph. 54, 229-240.

COMPER, W. D. & LAURENT, T. C. (1978). Physiological function of connective tissue poly-saccharides. Physiol. Rev. 58, 255-315.

DIEWERT, V. M. (1978). A quantitative coronal plane evaluation of craniofacial growth andspatial relations during secondary palate development in the rat. Archs oral Biol. 23,607-629.

DIEWERT, V. M. & PRATT, R. M. (1979). Selective inhibition of mandibular growth andinduction of cleft palate by diazo-oxo-norleucine (DON) in the rat. Teratology 20, 37-52.

DIEWERT, V. M. & TAIT, B. (1979). Palatal process movement in the rat as demonstrated infrozen sections. / . Anat. 128, 609-618.

ERICKSON, C. A., TOSNEY, K. W. & WESTON, J. A. (1980). Analysis of migratory behavior ofneural crest and fibroblastic cells in embryonic tissues. Devi Biol. 11, 142-156.

FERGUSON, M. W. J. (1978). Palatal elevation in the Wistar rat fetus. / . Anat. 125, 555-577.KING, C. T. G. (1963). Teratogenic effects of meclizine hydrochloride in the rat. Science 141,

353-355.KING, C. T. G., WEAVER, S. A. & NARROD, S. A. (1965). Antihistamines and teratogenicity

in the rat. / . Pharmacol, exp. Ther. 147, 391-398.LAURENT, T. C. (1970). Structure of hyaluronic acid. In Chemistry and Molecular Biology of

Intercellular Matrix, vol. 2 (ed. E. A. Balazs), pp. 703-732. New York: Academic Press.LAZZARO, C. (1940). Sul meccanismo di chiusura del palato secondario. Monitore zool. Ital.

51, 249-273.LEWIS, C. A., THIBAULT, L., PRATT, R. M. & BRINKLEY, L. L. (1980). An improved culture

system for secondary palatal elevation. In Vitro 16(6), 453-460.MORRISS, E. R., REES, D. A. & WELSH, E. J. (1980). Conformation and dynamic interactions

in hyaluronate solutions. / . molec. Biol. 138, 383-400.NAKAMURA, A. & MANASEK, F. J. (1978). Experimental studies of the shape and structure of

isolated cardiac jelly. J. Embryol. exp. Morph. 43, 167-183.

Chlorcyclizine and palate elevation in vivo and in vitro 213PRATT, R. M., GOGGINS, J. R., WILK, A. L. & KING, C. T. G. (1973). Acid mucopolysao

charide synthesis in the secondary palate of the developing rat at the time of rotation andfusion. Devi Biol. 32, 230-237.

PRATT, R. M., LARSEN, M. A. & JOHNSTON, M. C. (1975). Migration of cranial neural crestcells in a cell-free hyaluronate-rich matrix. Devi Biol. 44, 298-305.

SINGLEY, C. T. & SOLURSH, M. (1981). The spatial distribution of hyaluronic acid andmesenchymal condensation in the embryonic chick wing. Devi Biol. 84, 102-120.

SOLURSH, M., FISHER, M., MEIER, S. & SINGLEY, C. T. (1979). The role of extracellularmatrix in the formation of the sclerotome. J. Embryol. exp. Morph. 54, 75-98.

TOOLE, B. P., OKAYAMA, M., ORKIN, R. W., YOSHIMURA, M., MUTO, M. & KAJI, A. (1977),Developmental roles of hyaluronate and chondroitin sulfate proteoglycans. In Cell andTissue Interactions (ed. J. W. Lash & M. M. Burger). New York: Raven Press.

TOOLE, B., UNDERHILL, C. B., MIKUNI-TAKAGAKI, Y. & ORKIN, R. (1980). Hyaluronate inmorphogenesis. In Current Research Trends in Prenatal Craniofacial Development (ed>R. M. Pratt & R. L. Christiansen), pp. 263-275. New York: Elsevier/North-Holland.

TOSNEY, K. W. (1978). The early migration of neural crest cells in the trunk region of theavian embryo: an electron microscopic study. Devi Biol. 62, 317-333.

WALKER, B. E. (1961). The association of mucopolysaccharides with morphogenesis of thesecondary palate and other structures in mouse embryos. / . Embryol. exp. Morph. 9, 22-31.

WALKER, B. E. & FRASER, C. F. (1956). Closure of the secondary palate in three strains ofmice. / . Embryol. exp. Morph. 4, 176-189.

WALKER, B. E. & CRAIN, B. (1960). Effects of hypervitaminosis A on palate development intwo strains of mice. Am. J. Anat. 107, 49-58.

WEE, E. L., WOLFSON, L. G. & ZIMMERMAN, E. F. (1976). Palate shelf movement in mouseembryo culture: evidence for skeletal and smooth muscle contractility. Devi Biol. 48,91-103.

WILK, A. L., KING, C. T. G. & PRATT, R. M. (1978). Chlorcyclizine induction of cleft palatein the rat: degradation of palatal glycosaminoglycans. Teratology 18, 199-210.

(Received 9 September 1981, revised 7 December 1981)

the effect osf chlorcyclizine-induced palatal shelf ... · approximately 1, 2 and 3 days after the...

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