The influence of the epithelium on palate shelf reorientation · The separation of the presumptive...

/. Embryol. exp. Morph. 88, 265-279 (1985) 2 6 5Printed in Great Britain © The Company of Biologists Limited 1985

The influence of the epithelium on palate shelfreorientation

ROBERT F. BULLEIT AND ERNEST F. ZIMMERMANDivision of Basic Science Research, Children's Hospital Research Foundation,Cincinnati, Ohio 45229, U.S.A.

SUMMARY

The intrinsic forces necessary for directing the reorientation of the secondary palate appear toreside in the anterior two thirds of the palate or presumptive hard palate. The hard palate couldreorient regardless of whether it was intact or separated from the posterior third or presumptivesoft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsicforces, within the hard palate, may be mediated by the mesenchymal cells, their extracellularmatrix, or the epithelium surrounding the shelves. This latter possibly was tested by removingthe epithelium, from either the presumptive oral or nasal surface followed by measurement ofreorientation in vitro. Only after removal of the oral epithelium was a significant inhibition inreorientation observed. The treatment used to remove the epithelium, EDTA and scraping, wasshown to remove 41 % of the oral epithelium leaving the majority of the basement membraneintact. The observed inhibition of reorientation did not appear to be a consequence of woundhealing. Creation of wounds twice the area that was observed after treatment with EDTA andscraping inhibited reorientation minimally. These results suggest that the epithelium andparticularly the anterior oral epithelium plays a major role in the reorientation of the murinesecondary palate.

INTRODUCTION

One aspect of the morphogenesis of the mammalian secondary palate involvesthe reorientation of the two apposing palate shelves. The shelves move from avertical position on either side of the tongue to a horizontal position above thetongue separating the oral and nasal cavities. The process of reorientation hasbeen studied in vitro using murine embryos (Brinkley, 1975; Wee et al. 1976).Foetal mouse heads cultured following removal of the tongue and brain showedsimilar reorientation to that observed in situ (Brinkley et al. 1975, 1978). Theseresults suggest that structures such as the tongue and brain as well as circulationare unnecessary for the process of reorientation. The observations are consistentwith an intrinsic shelf force directing reorientation (Walker & Fraser, 1956). Theintrinsic components in the shelves which could be involved in directing thismorphogenesis may be associated with the mesenchyme and its extracellularmatrix, or the epithelium surrounding the shelves (Larsson, 1960; Pourtois, 1972;Pratt et al. 1973; Babiarz et al. 1975; Ferguson, 1977; Brinkley, 1980). Theepithelium is of particular interest because epithelia define the shape and structure

Key words: palate, epithelium, reorientation, mouse embryo.

266 R. F. BULLEIT AND E. F. ZIMMERMAN

of tissues and appear to be morphogenetically active in a number of developingsystems (Grobstein, 1967; Karfunkel, 1974; Brady & Hilfer, 1982; Brun & Garson,1983). The evidence to support a role for the epithelium in palate shelfreorientation is still inconclusive. Epithelial cell shape, distribution and/orlayering changes, as well as rugae development and an increase in mitotic figures inthe presumptive oral epithelium do correlate with the timing of reorientation(Brinkley, 1984; Luke, 1984). What is uncertain is whether these changes arenecessary for reorientation to occur.

In the present study we examined the effects of epithelial removal onreorientation in vitro to determine more directly whether the epithelium plays arole in this process. Preliminary observations indicated that disruption of thepresumptive oral surface could inhibit reorientation in vitro (Bulleit &Zimmerman, 1984). The results presented in this paper substantiate those findingsand demonstrate that removal of the epithelium on the anterior oral aspect of thepalate and not wounding is primarily responsible for the observed inhibition.These findings are consistent with the epithelium, in particular the anterior oralepithelium, directing secondary palate morphogenesis.

MATERIALS AND METHODS

Animals and culture systemA/J mice (Jackson Laboratories) were used in these studies. We have also used CFW mice

(Charles River), a random-bred strain, and have obtained similar results. Matings were carriedout by placing individual females with males overnight. The presence of a vaginal plug thefollowing morning was taken as evidence for pregnancy and designated day zero. On day 14 ofgestation animals were sacrificed by cervical dislocation and gravid uteri were placed on ice. Thisis approximately 24 h prior to in situ shelf reorientation (Andrew et al. 1973). Embryos wereremoved and examined, those with cleft lip were eliminated. The remaining embryos werestaged using a morphologic rating system described by Walker & Crain (1960). Embryos with arating of 5-7 were used for culture.

Heads were removed followed by excision of mandibles and tongues. This allowed access topalates for mechanical manipulation of the epithelial layers. A cut was also made down themidline of the cranium which allowed better access of medium to tissues. For most experimentsa transverse cut was made in the palate two thirds the distance from the anterior end to separatethe presumptive hard and soft palate (Diewert, 1978). This is indicated in Fig. 1 by the dashedline (H/S). Heads were then placed in 15 x 45 mm vials containing 3 ml Dulbecco's ModifiedEagles Medium (DMEM, Gibco) and 10 % foetal bovine serum (FBS, M.A. Bioproducts). Thevials were gassed with a mixture of 95 % O2,5 % CO2 and sealed with rubber stoppers. The vialswere placed in a rotating culture apparatus (approximately 30 r.p.m.) for up to 6 h. In one set ofexperiments cultures were maintained for 24 h.

Epithelial removal and woundingHeads were treated prior to culture in phosphate-buffered saline (PBS) without Ca2+ and

Mg2"1" containing 10mM-EDTA for lOmin at 37 °C. The tongue remained between the palateshelves during this treatment. Following treatment, the palates on either the presumptive oral ornasal surface were scraped using the edges of finely sharpened curved forceps to tease theepithelium from the underlying basement membrane.

Wounding of the palate surface was performed by making a tear in the oral surface runningthe length of the hard palate and projecting into the underlying mesenchyme. This wasaccomplished using the sharpened point of a pair of forceps. Following epithelial removal or

Epithelial influence on palate reorientation 267

Fig. 1. The dashed line H/S indicates the transverse cut made in the palate shelves toseparate the presumptive hard (H) and soft (S) palate. The solid lines indicate whererazor blade sections were cut to measure anterior (A), mid (B) or posterior (C) PSI.

wounding the tongue was removed and the presumptive hard and soft palate were separated.The heads were then cultured as described previously.

Quantitation of reorientation in vitroThe amount of reorientation occurring during culture was measured using palate shelf index,

PSI (Wee et al. 1976). Following culture, heads were fixed in Bouin's solution for 24 h. Fixedheads were cut coronally using a razor blade. Razor blade sections were made by cutting thepalate shelves in the middle of each third of the palate, allowing for measurement of anterior,mid, and posterior PSI, as indicated in Fig. 1 by the lines A, B, and C respectively. Thetransverse sections were placed on end and visualized using a dissecting microscope. Values of1-5 were assigned to express the degree of palate shelf reorientation. A number of 1 wasassigned for a completely vertical and a 5 for a completely horizontal palate. Numbers 2, 3 or 4were designated intermediate elevations of one-quarter, one-half or three-quarters of the way tothe horizontal position. Experimental results were expressed as mean PSI ± standard error.Statistical analysis of the difference between the mean PSI of treatment groups and theirrespective controls was performed using a two-tailed Student's t-test. The number of palateshelves evaluated in each treatment group is indicated in the tables.

Scanning electron and light microscopyFor SEM heads were fixed in 3% glutaraldehyde in 0-163 M-sodium cacodylate buffer for

4-5 h at 25 °C followed by washing in buffer. The heads were then dehydrated in a graded seriesof ethanol (30-100%) and critical-point dried using carbon dioxide as a transitory fluid.Thereafter heads were mounted, coated with gold, and examined using a scanning electronmicroscope (ETEC Autoscan).

For light microscopy, heads were fixed in modified Karnofsky's fixative, as modified bySingley & Solursh (1980). Isotonic sodium phosphate buffer (0-2 M) was used instead of sodiumcacodylate buffer. Heads were fixed overnight followed by dehydration in a graded series ofethanol (50-100 %) with a final passage through 100 % propylene oxide. The heads were thenembedded in Poly/Bed 812 (Polyscience, Inc.). Sections were cut at 1-2/mi, stained inRichardson's stain (Richardson et al. 1960) and examined with a Zeiss photomicroscope.

ImmunofluorescenceHeads were fixed for 4h in Hank's balanced salt solution without Ca2+ and Mg2+ containing

2% paraformaldehyde and 0-32M-sucrose. Heads were removed and dehydrated through agraded series of ethanol (50-100 %) followed by two changes of xylene. The heads were thenembedded in paraffin. Sections were cut at 6^m, placed on glass slides and rehydrated prior toantibody staining. Slides were incubated sequentially with rabbit antiserum and rodamine-conjugated goat anti-rabbit IgG diluted from 1:50 to 1:100. Rabbit antisera to laminin (Engvall


et al. 1983), heparan sulphate proteoglycan BM1 (Hassell et al. 1980), and type IV collagen(Timpl et al. 1978) were gifts of Erkki Ruoslahti, John Hassell and George Martin, respectively.

Morphometric analysisPalates were analysed morphometrically. The degree of epithelial removal was quantitated

from scanning electron micrographs of heads treated with EDTA and scraping of the oralsurface. From these micrographs three different areas on the oral surface were defined andmeasured using computer-assisted image analysis (Videoplan, Zeiss). The areas measured were(1) total oral surface of the palate, (2) area of epithelial removal, (3) area in which the basementmembrane had been damaged exposing the underlying mesenchyme, defined as the area ofwounding. Thirty palates were measured in this experiment and the mean, standard error, andpercent of total oral surface were determined for each defined area.

RESULTS

Reorientation of segmented palate shelves in vitro

The separation of the presumptive hard and soft palate allowed reorientation ofthe hard palate to occur while reorientation of the soft palate was inhibited. Themeasurement of palate shelf index showed that in fact the anterior and mid palatewere enhanced in their degree of reorientation following separation of the palateshelves (Table 1). This was observed at all times of culture. Particularly apparentwas a 0-6 PSI unit difference following l h of culture between controls andsegmented palates in both anterior and mid palate. By 6 h of culture the anteriorpalate had reached a PSI of 4-5 or 90 % movement toward the horizontal position.The mid palate had not yet reached the same degree of movement (75 % ofhorizontal). There was also observed in both anterior and mid palate a reducedgap between the separated palate shelf compared to the control shelf (Fig.2A,B>E),E)- in contrast the posterior palate was inhibited from reorientingfollowing separation of the hard and soft palate. By 6 h of culture there was a

Table 1. The effects of separation of the hard and soft palate on reorientation of palateshelves in vitro

Palatesegmentmeasured

Anterior

Mid

Posterior

Protocol

ControlH/S

ControlH/S

ControlH/S

2

1

1

0

•4 ± 0-09 (28)

•9 ± 0-05 (28)

•1± 0-07 (28)

PSI±S.E.M.(n)

Time in culture (h)1 3

3-4 ± 0-09 (30)4-0 ± 0-08 (34)a

2-6 ± 0-09 (30)3-2 ± 0-09 (34)a

1-4 ±0-09 (30)1-2 ±0-06 (36)

3-9 ± 0-07 (36)4-2 ± 0-07 (30)d

2-8 ± 0-07 (36)3-3±0-ll(30)b

1-7 ±0-09 (36)1-2 ± 0-09 (30)b

6

4-2 ± 0-09 (30)4-5 ± 0-07 (48)c

3-0 ±0-13 (30)3-7 ± 0-09 (48)a

1-9 ±0-10 (30)1-3 ± 0-06 (48)a

H/S: palate shelves with hard and soft palate separated.Different from control values:

aP< 0-0001b P < 0-001cP<0-02dP<0-05


difference of 0-6 PSI unit between controls and segmented palate shelves (Table1). In comparison, segmented palates had a larger gap between the posteriorshelves and they appeared more vertical (Fig. 2C,F). When heads were culturedfor 24 h, the anterior palate could fully reorient and fuse, while the soft palate wasstill markedly inhibited. However some movement of the soft palate toward thehorizontal position took place (Fig. 3B). In contrast, when the hard and soft palatewere left intact both regions of the palate could reorient and fuse (Fig. 3A).

In all further experiments, culture periods of 24 h were not employed because ofpoor survival of the inner cranial tissue. Cultures were maintained for 1 to 6hfollowing separation of the hard and soft palate. In this way the effect of epithelialremoval on reorientation of the hard palate could be examined separate frominfluences of the soft palate.

Fig. 2. Coronal section of palate shelves following 6 h of culture (x60). Shelves werecultured with the full shelf intact (A,B,C) or following separation of the presumptivehard and soft palate (D,E>F)- Sections were made in the anterior (A,D), mid (B,E) orposterior (C,F) palate. Bar equals 100/im.


Fig. 3. Scanning electron micrographs showing the oral surface of palate shelffollowing 24 h of culture (x35). Palate shelves were cultured with the full shelf intact(A) or following separation of the anterior two-thirds or presumptive hard palate (H)from the posterior one-third or presumptive soft palate (S). pm, premaxilla; arrow,rugae. Rugae were observed on the oral surface of all palate shelves before and afterculture. Bar equals 100[Am.

The effects of epithelial removal on reorientation in vitro

Measurements of PSI showed no consistent significant difference in the degreeof reorientation from control values following treatment with EDTA alone orEDTA followed by scraping of the nasal surface. EDTA alone inhibitedreorientation to a small degree which was only significant in the anterior palatefollowing 3h of culture. Nasal epithelial disruption showed inhibition only in themid palate after 1 h of culture.

In contrast, treatment with EDTA followed by scraping of the oral surfacesignificantly inhibited palate shelf reorientation when compared to control orEDTA treatment alone. This result was observed in both the anterior and midpalates at all time intervals measured (Table 2). However the anterior palateappeared to be more affected by this treatment. The inhibition did not appear tobe specific to the A/J strain of mouse. CFW, a random bred strain of mouse,showed a similar pattern of inhibition. When treated with EDTA alone the PSI inthe anterior end was 4-5 ±0-11 and the mid palate was 3-7 ±0-11 after 6h ofculture. In contrast, if they were treated with EDTA followed by scraping of theoral epithelium, inhibition was observed in both anterior (PSI = 3-0 ± 040) andmid (2-7 ± 0-13) palates. In all of these studies the posterior or soft palate did notreorient and was unaffected by these treatments.

The morphology of the palate shelves after 6h of culture in control, EDTAtreatment alone, or EDTA treatment followed by nasal epithelial disruption, wasvery similar (Fig. 4B-D). After nasal surface disruption, removal of theepithelium was observed as well as some minor disruption of the underlying


structures (Figs 4D, 5C). In contrast the morphology of palate shelves treated withEDTA followed by disruption of the oral epithelium was altered (Fig. 4E). Theshape of the palates appeared expanded compared to controls or EDTA treatmentalone. Also observed was an increase in the gap between the two apposing palateshelves. At higher magnification, the oral surface was devoid of epithelium andthe mesenchyme directly beneath this removed epithelium appeared relativelyundisturbed (Fig. 5D).

Analysis of epithelial removal

The degree of epithelial removal and wounding following treatment with EDTAand scraping was quantitated using computer-assisted image analysis. It wasobserved that on the average 41 % of the oral epithelium had been removed whileonly 4% of the oral surface showed evidence of wounding (Table 3). Scanningelectron micrographs showed areas in which the epithelium was still intact andother areas where the epithelium had been teased away exposing a non-cellularstructure (Fig. 6A,B). In some places the non-cellular structure had been tornaway to expose underlying mesenchymal cells, which indicate some wounding(Fig. 6B). These observations suggested that the non-cellular structure wasbasement membrane apposed between epithelium and mesenchyme. This

Table 2. The effect of EDTA treatment and mechanical disruption of the palateepithelium on reorientation of palate shelves in vitro

~ PSI±s.B.M.(n)

segment Time in culture (h)measured Treatment 0 1 3 6

Anterior Control 2-4 ±0-09 (28) 4-0 ±0-08 (34) 4-2 ±0-07 (30) 4-5 ±0-07 (48)EDTA - 3-8 ±0-10 (18) 3-9 ± 0-14 (20)d 4-3 ±0-11 (30)

EDTA,N/E - 4-0 ±0-11 (18) 4-2 ±0-11 (20) 4-4 ±0-13 (20)EDTA,O/E - 2-8 ± 0-13 (18)a 2-9 ± 0-15 (20)a 3-3 ± 0-10 (32)a

Mid Control 1-9 ±0-05 (28) 3-2 ±0-09 (34) 3-3 ±0-11 (30) 3-7 ±0-09 (48)EDTA - 3-2 ±0-10 (18) 3-3 ±0-12 (20) 3-5 ±0-14 (30)

EDTA,N/E - 2-8 ± 0-10 (18)c 3-3 ±0-14 (20) 3-5 ±0-11 (20)EDTA,O/E - 2-7 ± 0-11 (18)c 2-6 ± 0-11 (20)b 2-8 ± 0-11 (32)a

Posterior Control 1-1 ±0-07 (28) 1-2 ±0-06 (34) 1-2 ±0-09 (30) 1-3 ±0-06 (48)EDTA - 1-1 ±0-06 (18) 1-2 ±0-10 (20) 1-2 ±0-07 (30)

EDTA,N/E - 1-2 ±0-10 (18) 1-3 ±0-10 (20) 1-2 ±0-09 (20)EDTA,O/E - 1-2 ±0-09 (18) 1-3 ±0-10 (20) 1-1 ±0-11 (32)a

Palate shelves were pretreated with 10mM-EDTA for lOmin. Mechanical disruption wasperformed by scraping the presumptive nasal (N/E) or oral (O/E) surface with finely sharpenedforceps. Following this treatment the palate shelves were separated into hard and soft palate andcultured in DMEM plus 10 % FBS.

Different from values for EDTA treatment alone:a P < 0-0001b P < 0-001CP< 0-005

Different from control values:dP<0-05


conclusion was confirmed by performing immunofluorescence studies usingantisera to two basement membrane antigens, type IV collagen and heparansulphate proteoglycan. Both antisera showed immunoreactivity with a structureapposed between epithelium and mesenchyme, as well as remaining attached toareas of mesenchyme after epithelial removal (Fig. 7A,B)- A similar pattern ofimmunoreactivity was also observed with antisera to the basement membraneglycoprotein laminin (result not shown).

The effects of wounding on reorientation in vitro

Wounds were made on the oral surface running the length of the hard palate andcovering approximately 9 % of the oral surface, or greater than twice the area of

Fig. 4. Coronal section through palate shelves following Oh (A) or 6h (B,C,D,E) ofculture (x60). Palate shelves were cultured following treatment with PBS with Ca2+

and Mg2+ (B), PBS without Ca2+ and Mg2+ containing 10mM-EDTA alone (C),followed by scraping the presumptive nasal surface (D), or scraping the presumptiveoral surface (E). Bar equals 100 ̂ m.


Fig. 5. The epithelial surface of palate shelves cultured for 6h (xl50). Oral epithelialsurface of a control palate shelf following treatment in PBS with Ca2+ and Mg2+ (A).Oral epithelial surface of a palate shelf treated in PBS without Ca2+ and Mg2*containing 10mM-EDTA (B). Nasal epithelial surface of a palate shelf treated in PBSwithout Ca2+ and Mg2* containing 10 mM-EDTA followed by scraping of the nasalsurface (C). Oral epithelial surface of a palate shelf treated in PBS without Ca2+ andMg2+ containing 10 mM-EDTA following by scraping of the oral surface (D). Barequals 100 fim.

wounding observed after EDTA and oral surface disruption (Fig. 8A). Thewounds protruded deep into the underlying mesenchyme and the surface of thewound appeared devoid of epithelium (Fig. 8B). Following 6h of culture, woundhealing could be observed in the palate shelves (Fig. 8C). The mesenchyme in thearea of the wound was condensed and contracted with some areas of themesenchyme still remaining separated. The epithelium at this time had not yetcovered the wound. The palate shelves also showed substantial movement toward

Table 3. Morphometric analysis of the oral surf ace following EDTA and mechanicaltreatment

Area measuredMean area of % of total oral

micrographs (mm2) ± S.E. surface area

Total oral surfaceArea of epithelial removalArea of basement membrane disruption

3323-3 ±89-11363-8 ± 67-8130-6 ± 20-7

100414

Analysis was performed on scanning electron micrographs of 30 palates immediatelyfollowing treatment with EDTA for lOmin and mechanical scraping of the oral surface.Micrographs were magnified x85.


Fig. 6. Scanning electron micrographs of palate shelves immediately followingtreatment in PBS without Ca2+ and Mg2+ containing 10 mM-EDTA and scraping of theoral surface A (x 143), B (x715). e, epithelial cells; m, mesenchymal cells; b, basementmembrane; arrow, wounded surface. Bar equals 10/mi.

the horizontal position. Measurement of PSI showed that wounding could inhibitreorientation only to a small degree and this inhibition was significantly less thanthat observed following treatment with EDTA and oral epithelial removal (Table4).

DISCUSSION

The process of palate shelf reorientation may be directed by forces intrinsic tothe palate shelves (Walker & Fraser, 1956). Consistent with this idea is the abilityof the palate shelves to reorient in culture in the absence of the tongue, brain andcirculation (Brinkley, 1975, 1978). Experiments presented in this paper as well asobservations made previously (Brinkley & Vickerman, 1979) suggest that thisintrinsic force is particularly associated with the anterior two thirds or presumptivehard palate. The hard palate could reorient whether intact or separated from theposterior third or presumptive soft palate, while the soft palate failed to reorient(Table 1). This result was also observed even after an extended culture period,although some minimal movement was apparent (Fig. 3). This result might implythat soft palate reorientation requires extrinsic factors provided by the hardpalate. This process could be accomplished by an anterior to posterior 'zippering'effect of adhesion and fusion along the medial edge (Brinkley & Vickerman,1979). Alternatively, hard palate reorientation may provide a pulling force fordirecting the soft palate to a horizontal position. This latter interpretation issupported by observations that the soft palate provides resistance to reorientationof the hard palate. When the two segments were separated, hard palate


reorientation was accelerated while the soft palate failed to reorient. Thereorientation of the isolated soft palate may require factors that cannot beexpressed in this culture system. The soft palate has been described as remodellingaround the tongue. This remodelling may require the presence of the tongue forfull expression. However, the importance of the tongue and these other factorsappear to be minimal in this culture system since the intact shelf showedsubstantial reorientation and fusion of the soft palate (Fig. 3).

The major emphasis of this paper was to examine the role of the epithelium ingenerating the intrinsic force for reorientation. The results observed suggest thatepithelium on the oral aspect of the hard palate is a major factor in generating thisintrinsic force. Removal of the oral epithelium significantly inhibited the pattern ofreorientation in vitro. In contrast, disruption of the epithelium on the presumptivenasal surface had no effect on the degree of movement. The treatment used toremove the epithelium, EDTA and scraping, removed primarily epitheliumleaving the majority of the basement membrane intact. This observation was

7A

m

Fig. 7. Immunofluorescence photographs of palate shelves treated in PBS withoutCa2+ and Mg2+ containing lOmM-EDTA and scraping of the oral surface (x500).Palate shelf was fixed immediately following treatment for histology (A) orimmunofluorescence (B,C,D). Immunofluorescence was carried out using rabbitantiserum to type IV collagen (B), antiserum to heparan sulphate proteoglycan (C), orwith a control rabbit serum (D). e, epithelium; m, mesenchyme. Bar equals O


Fig. 8. Palate shelves following wounding of the oral surfaces. The scanning electronmicrograph taken immediately following wounding shows the wound (wd) running thelength of the hard palate, mag. x35. Light micrograph of coronal section through thepalate immediately following wounding shows the wound (wd) running deep into themesenchyme, mag. X60 (B). Coronal section through wounded palate shelf following6h of culture. Arrows indicate area of wound healing, mag. x60 (C). Bar equals100 ̂ m.

Table 4. The effect of wounding the oral surface of the palate on reorientation of palateshelves in vitro

Palatesegmentmeasured

Anterior

Mid

Posterior

Treatment

ControlWounded

ControlWounded

ControlWounded

PSI-SE (n)

Time in <0

2-4 ± 0-09 (28)

1-9 ±0-05 (28)

1-1 ±0-07 (28)

;ulture (h)6

4-5 ± 0-07 (48)4-3 ± 0-08 (52)a

3-7 ± 0-09 (48)3-4 ± 0-07 (52)b

1-3 ±0-06 (48)1-2 ± 0-06 (52)

Palate shelves were wounded on the presumptive oral surface using finely sharpened forceps.Following this treatment the palate shelves were separated into hard and soft palate andcultured in DMEM plus 10 % FBS.

Different from control values:aP<0-05bP<0-02


supported by both morphological (Hall & McSween, 1984) and immuno-histochemical criteria (Table 3, Fig. 7). However this treatment also created somewounding. It is possible that contraction of such wounds during wound healingmay have created a force to overcome the force necessary for reorientation andwas responsible for the observed inhibition. This interpretation appears unlikelyfor two reasons. First, the degree of wounding observed after treatment withEDTA and scraping was minimal, covering only on the average 4 % of the oralsurface. Secondly, when wounds were created covering two times this area only asmall degree inhibition of palate reorientation was observed. It could not bedetermined whether this small degree of inhibition was due to wound healing orthe removal of the. epithelium over the wound. These results are consistent withthe removal of the oral epithelium and not wounding as being responsible for theobserved inhibition of palate shelf reorientation.

Brinkley & Vickerman (1979) have previously suggested that reorientation ofthe hard palate may actually occur by expression of a pre-existing infrastructure.The oral epithelium or an interaction between the oral epithelium and themesenchyme may help define this infrastructure. Alternative mechanisms forcontrolling the process of reorientation include the expression of an activecontractile system within the mesenchyme (Babiarz et al. 1975) or the accumu-lation of a hyaluronic acid (HA)-rich extracellular matrix (Larsson, 1960; Pratt etal. 1973; Furguson, 1977; Brinkley, 1980). It appears most likely that HA plays arole in the reorientation of the mid palate. Enhanced degradation of glycos-aminoglycans including HA by chlorcyclizine was able to inhibit reorientation inthis area while reorientation of the anterior palate was unaffected (Brinkley,1982). This is in contrast to removal of the oral epithelium which inhibitedreorientation in the anterior palate more effectively (Table 2). It is possible thatthe intrinsic force for reorientation is generated not by simply one element but bythe interaction of a number of elements including the oral epithelium, mes-enchyme and a HA-rich extracellular matrix.

Our results suggest that the oral epithelium is particularly important ingenerating the force for reorientation. How this is accomplished is unclear. Thecontrol of growth and differentiation of this epithelium may be responsible forgenerating a particular structure which is necessary for expression of this force.Luke (1984) has observed an unequal cell division within palate epithelium. Theoral epithelium appears to be dividing more rapidly than the nasal epithelium. Hehas suggested that this differential in cell division and development of rugae on theoral surface may be responsible for palate shelf reorientation. The rugae may beparticularly important since they are exclusively associated with the hard palateand their development correlate with reorientation in a number of mammals(Waterman & Meller, 1974; Meller et al 1980). The formation of rugae on the oralsurface may stiffen the structure of the oral epithelium forcing the palate shelftoward the horizontal position. Interference with the development of theepithelium and rugae may then lead to the induction of cleft palate.


This work represents part of the Ph.D. dissertation of Robert Bulleit to the GraduateProgram in Developmental Biology, University of Cincinnati.

This study was supported by a NIEHS Training Grant (ESO7051).

REFERENCES

ANDREW, F. D., BOWEN, D. & ZIMMERMAN, E. F. (1973). Glucocorticoid inhibition of RNAsynthesis and the critical period for cleft palate induction in inbred mice. Teratology 7,167-175.

BABIARZ, B. S., ALLENSPACH, A. L. & ZIMMERMAN, E. F. (1975). Ultrastructural evidence ofcontractile systems in mouse palate prior to rotation. Devi Biol. 47, 32-44.

BRADY, R. C. & HILFER, S. R. (1982). Optic cup formation: A calcium-regulated process. Proc.natn. Acad. Sci., U.S.A. 79, 5587-5591.

BRINKLEY, L. L. (1980). In vitro studies of palatal shelf elevation. In Current Research Trends inPrenatal Craniofacial Development (ed. R. M. Pratt & R. L. Christiansen), pp. 203-220.Amsterdam: Elsevier North Holland Inc.

BRINKLEY, L. L. (1984). Changes in cell distribution during mouse secondary palate closure invivo or in vitro. I. Epithelial cells. Devi Biol. 102, 216-227.

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

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

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

BRINKLEY, L. L. & VICKERMAN, M. M. (1982). The effects of chlorcyclizine-induced alterations ofglycosaminoglycans on mouse palate shelf elevation in vivo and in vitro. J. Embryol. exp.Morph. 69, 193-213.

BRUN, R. B. & GARSON, J. A. (1983). Neurulation in the mexican salamander (Ambystomamexicaonum): a drug study and cell shape analysis of the epidermis and the neural plate./. Embryol. exp. Morph. 74, 275-295.

BULLEIT, R. F. & ZIMMERMAN, E. F. (1984). Role of the oral epithelium in secondary palatemorphogenesis. Teratology 29, 21 A.

BURNSIDE, B.(1973). Microtubules and microfilaments in amphibian neurulation. Arner. Zool.13, 989-1006.

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.

ENGVALL, E., KRUSIUS, T., WEWER, U. & RUOSLAHTI, E. (1983). Laminin from rat yolk sactumor: Isolation, partial characterization, and comparison with mouse laminin. ArchsBiochem. Biophys. 222, 649-656.

FERGUSON, M. W. J. (1977). The mechanism of palatal shelf elevation and the pathogenesis ofcleft palate. Virchows Arch. path. Anat. Histol. 375, 97-113.

GROBSTEIN, C. (1967). Mechanisms of organogenetic tissue interaction. Natn. Cancer Inst.Monogr. 26, 279-299.

HALL, B. K. & MACSWEEN, M. C. (1984). A SEM analysis of the epithelial-mesenchymalinterface in the mandible of the embryonic chick. J. Craniofac. Genetic devl Biol. 4, 59-76.

HASSELL, J. R., ROBEY, P. G., BARRACH, H., WILCZEK, J., RENNARD, S.I. & MARTIN, G. R. (1980).Isolation of a heparan sulfate-containing proteoglycan from basement membrane. Proc. natn.Acad. Sci., U.S.A. 77, 4494-4498.

KARFUNKEL, P. (1974). The mechanisms of neural tube formation. Ml Rev. Cytol. 38, 245-272.LARSSON, K. S. (1960). Studies on the closure of the secondary palate. II. Occurrence of

sulphomucopolysaccharides in the palatine processes of normal mouse embryos. Expl CellRes. 21, 498-503.

LUKE, D. A. (1984). Epithelial proliferation and development of rugae in relation to palate shelfelevation in the mouse. J. Anat. 138, 251-258.

MELLER, S. M., DEPAOLA, D., BARTON, L. H. & MANDELLA, R. D. (1980). Secondary palataldevelopment in the New Zealand white rabbit: A scanning electron microscopic study. Anat.Rec. 198, 229-244.

Epithelial influence on palate reorientation 279POURTOIS, M. (1972). Morphogenesis of the primary and secondary palate. In Developmental

Aspects of Oral Biology, (ed. H. C. Slavkin & L. A. Bavetta), pp. 81-108. London: AcademicPress.

PRATT, R. M., GOGGINS, J. F., WILK, A. L. & KING, C. T. (1973). Acid mucopolysaccharidesynthesis in the secondary palate of the developing rat at the time of rotation and fusion. DeviBiol. 32, 230-237.

RICHARDSON, K. C , JARETT, L. & FINK, E. (1960). Embedding in expoxy resins for ultrathinsectioning in electron microscopy. Stain Technol. 35, 313-323.

SINGLEY, C. T. & SOLURSH, M. (1980). The use of tannic acid for the ultrastructural visualizationof hyaluronic acid. Histochemistry 65, 93-101.

TIMPL, R., MARTIN, G. R., BRUCKNER, P., WICK, G. & WIEDEMANN, H. (1978). Nature of thecollagenous protein in a tumor basement membrane. Eur. J. Biochem. 84, 43-52.

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

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

WATERMAN , R. E. & MELLER, S. M. (1974). Alterations in the epithelial surface of human palatalshelves prior to and during fusion: A scanning electron microscope study. Anat. Rec. 180,111-136.

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.

WEE, E. L., BABIARZ, B. S., ZIMMERMAN, S. & ZIMMERMAN, E. F. (1979). Palatemorphogenesis. IV. Effects of serotonin and its antagonists on reorientation in embryoculture. /. Embryol. exp. Morph. 53, 75-90.

(Accepted 21 February 1985)

The influence of the epithelium on palate shelf reorientation · The separation of the presumptive...

Documents

Transcript of The influence of the epithelium on palate shelf reorientation · The separation of the presumptive...