surface coat on the epithelium of developing palatine shelves in … · fig. 1. epithelial surface...

J. Embryol. exp. Morph. Vol. 31, 3, pp. 683-692, 1974 6 8 3

Printed in Great Britain

Surface coat on theepithelium of developing palatine shelves in the

mouse as revealed by electron microscopy

By R. M. GREENE1 AND D. M. KOCHHAR1

From the Department of Anatomy,School of Medicine, University of Virginia

SUMMARY

The fine structure of the surface epithelium of developing palatine shelves in the mouse wasstudied from days 11 through 14 of gestation. Ruthenium red, a cationic stain used as anultrastructural indicator of acid mucopolysaccharides, was employed to detect the presenceof any surface coat.

Positive staining was first observed on day 12 of gestation and was seen to be presentthroughout the period of shelf elevation and fusion. It was seen over medial and lateralsurfaces as well as the inferior tic of vertical shelves. The surface coat was found to bepresent along the entire length of the shelf, extending superiorly up the medial and lateralepithelial borders until it abruptly disappeared.

Since this surface coat first appeared approximately 48 h prior to shelf elevation, it issuggested that its appearance may be associated with the ability of palatine shelves to undergofusion as shown by previous in vitro experiments. The time of acquisition by the shelves ofthis 'fusing potential' is also in the range of 48 h before shelf elevation.

INTRODUCTION

The cellular components of the palatine shelves that participate in theformation of the secondary palate have been previously described in a varietyof laboratory animals both at the light (Walker & Fraser, 1956; Coleman,1965; Walker, 1971) and electron microscopic levels (Mato, Aikawa &Katahira,1967/?; DeAngelis & Nalbandian, 1968; Farbman, 1968; Smiley & Dixon,1968; Smiley, 1970; Chaudhry & Shah, 1973). Adherence between epithelia ofadjacent shelves is one of the first events that occurs after shelf elevation(Smiley, 1972). Attempts to pull shelves apart that have been in contact resultsin tearing of the epithelial borders, demonstrating that this adherence is indeedstrong (Zeiler, Weinstein & Gibson, 1964; DeAngelis & Nalbandian, 1968;Farbman, 1968).

The basis for this tight adherence has been attributed both to desmosomes(DeAngelis & Nalbandian, 1968; Brusati, 1969; Chaudhry & Shah, 1973) and

1 Authors'' address: Department of Anatomy, Jordan Building, University of Virginia,Charlottesville, Virginia 22901, U.S.A.

684 R. M. GREENE AND D. M. KOCHHAR

to an 'ill-defined' extracellular surface coat on the epithelial cells covering thepalatine shelves (Hayward, 1969; Nanda & Kelly, 1973). The purpose of thisstudy was to establish, with the help of specific stains available for electronmicroscopy, whether or not an extracellular surface coat is present on theepithelial cells throughout the period of shelf elevation and fusion. An attemptis also made to correlate this information with that obtained by Pourtois (1966)and Vargas (1967) regarding the timing of the so called 'acquired potential' tofuse exhibited by palatine shelves in vitro.

MATERIALS AND METHODS

Random-bred TCR/DUB mice (Flow Laboratories, Dublin, Virginia) fedPurina mouse chow and tap water ad libitum were used. Females were matedbetween 8 a.m. and 12 noon so that the time of fertilization of eggs could beclosely estimated. The presence of a vaginal plug immediately afterwards wasregarded as evidence of mating. Day 1 was considered to begin at 10 a.m. thefollowing morning.

Animals were killed by cervical dislocation at various times between days11 and 14 of gestation. Fetuses were removed to a dish containing the fixativeand the heads sliced off below the mandible. Using fine dissecting knives, themandible was then sliced away from the rest of the head and the tongue care-fully removed.

The trimmed heads with exposed palatine shelves were fixed for three h atroom temperature in 3 % glutaraldehyde, buffered with 0-1 M cacodylatebuffer (pH 7-2), containing 0-05 % ruthenium red (K & K Laboratories, Inc.,Plainview, N.Y.). They were then rinsed for 15 min in 0-1 M cacodylate buffer(pH 7-2) and post-fixed in similarly buffered 2 % osmium tetroxide containing0-05 % ruthenium red, in the dark, for 3 h at room temperature (Luft, 1966).Fetal heads were then dehydrated through increasing concentrations of ethylalcohol, beginning with 30 % and concluding with two changes of absolutealcohol. Dehydration was followed by two 10 min changes of propylene oxide.The dehydrated tissues were then gently agitated for 1 h in a 1:1 mixture ofAraldite (Luft, 1961) and propylene oxide, followed by 24 h in a 3:1 Aralditeand propylene oxide mixture, and then placed in pure Araldite (Luft, 1961)overnight. The tissue was embedded in Beem capsules kept at 60 °C for 24 h.Fetal heads were oriented during embedding in such a way as to facilitatecoronal sectioning of the palatine shelves.

The blocks were trimmed on an LKB 11800 Pyramitome and sectionedwith glass knives on an LKB-Huxley Mark 2 Ultramicrotome. One //m sec-tions were stained with a 1 % toluidine blue solution (Trump, Smuckler &Bonditt, 1961) for tissue orientation and identification. Both thick (1 /im)and thin (60-80 nm) sections were taken from various regions of the secondarypalate, ranging from the most anterior area to the most posterior. In appropriate

Surface coat on palatine shelves 685

regions, 30 //m high mesas were formed on the block face using the pyramitome.Thin sections were cut from these mesas with glass knives and transferred topre-cleaned, Formvar and carbon-coated 75-mesh copper grids. Thin sectionsthus obtained were not stained, for maximal ruthenium red contrast, andexamined on an RCA EMU-3H electron microscope operating at an accelerat-ing voltage of 50 kV.

Other fetal heads were prepared as above with the omission of rutheniumred from the glutaraldehyde and osmium tetroxide solutions. Thin sectionsobtained from these blocks were stained with alcoholic uranyl acetate (Watson,1958) and lead citrate (Reynolds, 1963) before examination in the electronmicroscope.

RESULTS

On day 11 of gestation the palatine processes are represented by a longi-tudinally oriented extension of the maxillary arch on either side of the tongue.Each process consists of loosely distributed mesenchymal tissue covered witha double-layered epithelium. At this stage the outer epithelial surface of theentire shelf remained unstained with ruthenium red (Fig. 1). Positive stainingwas first observed on day 12 of gestation. It was most evident over the inferiortip of the vertical shelf, extended over microvillous processes of the epithelialcells and for a short distance on both the medial and lateral surfaces of theshelf (Figs. 2-4). This surface coat was found to be present along the entirelength of the shelf. At higher magnification the epithelial surface appeared tobe covered with a layer of electron-dense granules matted together to form amore or less continuous coating (Figs. 5, 6).

At intervals along the epithelial covering of the palatine shelves, surfacecells of greater electron density were frequently seen (Fig. 7). These cells weregenerally found in the tip or medial margin of the vertical palatine shelvesoccupying the outer of the double-layered epithelial jacket around the shelves.This electron density was never seen in the deeper cuboidal cells of the palatineepithelium. It is questionable whether these cells represent an expression of theprogrammed cell death thought to occur in this region (Morgan & Harris,1972; Smiley & Koch, 1972; Hudson & Shapiro, 1973), as they are found asearly as day 12 of gestation and, when seen near the time of shelf elevationand fusion, demonstrate normal morphology except for their increased electrondensity. These cells also are not easily defined in terms of the morphology ofphysiological cell death (Schweichel & Merker, 1973).

The surface coating found in the inferior region of vertical shelves extendedsuperiorly up the medial and lateral epithelial borders for some distance andabruptly disappeared (Fig. 8). The sudden termination of the surface coatingwas more evident on the lateral than the medial epithelial surface. On themedial surface the coat merely became irregular and discontinuous as oneprogressed superiorly.


s-f

Fig. 1. Epithelial surface of the tip of a vertically oriented palatine shelf on day 11of gestation. Note the absence of any continuous surface coating on the cell mem-brane. Ruthenium red en bloc staining, x 55800.Fig. 2. Palatal shelf epithelium on day 12 of gestation from an area correspondingto that shown in Fig. 1. Note positive staining at the cell membrane extending overmicrovillous processes of epithelial cells. Ruthenium red en bloc staining,x 38300.Fig. 3. Epithelial surface of the medial border of a vertically oriented palatineshelf at 13 days 12 h of gestation. Ruthenium red en bloc staining, x 38300.

Surface coat on palatine shelves

Fig. 4. The lateral border of a vertically oriented palatal shelf at 13 days, 12 h ofgestation. Ruthenium red en bloc staining, x 35200.Figs. 5 and 6. A higher magnification of the epithelial surface of a palatine shelfat day 12 of gestation. The surface coat appears to be made up of a layer of electron-dense granules matted together to form a continuous coating. Ruthenium red enbloc staining. Fig. 5, x 88100; Fig. 6, x 62400.

43 EMB 31


Fig. 7. Note a cell of greater electron density than adjacent and subjacent palatalepithelial cells. Ruthenium red en bloc staining, x 22000.Fig. 8. Lateral epithelial border of day 13 vertical palatal shelf (tip is towards theleft of the micrograph). Note abrupt disappearance of surface coating (arrow).Ruthenium red en bloc staining, x 15000.Fig. 9. Epithelial borders of two adjacent palatine shelves just prior to contact.Note the thin epithelial microvillous process extending from one shelf toward theopposite shelf. Also note the fine coating (arrows) on both shelves. Uranyl acetateand lead citrate staining, x 72700.


Sections not stained with ruthenium red also demonstrated some sort ofsurface substance on palatal epithelial cells. Figure 9 illustrates the epithelialborders of two adjacent palatine shelves just prior to contact. A thin epithelialmicrovillous process from one shelf is seen extending toward the oppositeshelf. The epithelial surface of both shelves demonstrates a fine fuzz-likecoating.

DISCUSSION

After elevation and contact of the two developing palatine shelves, a tightadherence of one shelf to the other has been demonstrated (Zeiler et al. 1964;Farbman, 1968). This adherence is so strong that attempts to pull the shelvesapart after initial contact produced artifactual cell tearing (Farbman, 1968;DeAngelis & Nalbandian, 1968).

The source of this firm adhesion remains unknown. The presence of desmo-somes have been suggested by some to have the mechanical function of main-taining contact between the fusing epithelia of adjacent shelves until disruptionof the epithelial seam and mesenchymal interpenetration occurs (DeAngelis &Nalbandian, 1968; Brusati, 1969; Chaudhry & Shah, 1973).

The occurrence of an extracellular surface layer, although controversial(Farbman, 1968; Matthiessen & Anderson, 1972), has been suggested (Matoet al. 1967a; Hayward, 1969; Nanda & Kelly, 1973). Mato et al. (1967a) havedescribed what they term a 'cell-reaction', essential for fusion, that takes placeat the contact sites between the nasal septum and the palatine shelves. Usingthe light microscope, they observe a thin dark cell surface layer on the surfaceswhich make contact. This layer could very likely be composed of the electron-dense surface cells described here (Fig. 7). Others have variously described an'ill-defined' extracellular surface layer (Hayward, 1969) and the presence ofelectron-transparent areas (Nanda & Kelly, 1973) as perhaps playing a rolein holding adjacent palatine shelves together. The nature of this surface coat,however, remains obscure.

Recently, the first positive evidence of a definitive surface coat on palatalshelf epithelium has emerged. Using concanavalin A, a carbohydrate-bindingprotein bound to horseradish peroxidase for electron microscopic study, Pratt,Gibson & Hassell (1973) have demonstrated reaction product at the surfaceof epithelia undergoing fusion. They concluded that a carbohydrate coatappears on the surface of palatal epithelial cells just prior to palatal fusion.Waterman, Ross & Meller (1973) utilizing the scanning electron microscope,have also exhibited the progressive accumulation of a filamentous materialalong the medial edge of shelves prior to contact.

Ruthenium red (ruthenium oxychloride) is a polyvalent cation which whenused with osmium tetroxide results in an electron-opaque material easilyobserved ultrastructurally. Prior treatment with substances which bind tomucopolysaccharides(Martinez-Palomo, 1970) prevents ruthenium-red staining.

43-2


Although the mechanism by which this metallic dye acts can only be hypo-thesized (Luft, 1971 a), it has been reliably used as an ultrastructural indicatorof acid mucopolysaccharides (Luft, 19716).

To confirm the presence of a surface coat, we have also attempted to detectaldehyde groups at the ultrastructural level by using metallic bismuth as asubstitute for the SchifT reagent (Ainsworth, Ito & Karnovsky, 1972). This, wereasoned, would afford us another method to confirm the presence of a surfacecoat. This method, however, has not provided unequivocal results since theyare difficult to reproduce.

The presence of a definitive carbohydrate surface coat may help to explainthe phenomenon of an 'acquired potential' to fuse exhibited by palatine shelvesof mice (Vargas, 1967) and rats (Pourtois, 1966) in vitro. Preparative changeshave been reported to occur in the epithelium of organ-cultured palatal shelvesprior to fusion (Morgan, 1969). Vargas (1967) has shown that palatal explantsfrom early 12-day mouse fetuses did not fuse in vitro, while explants fromolder mouse fetuses fused normally. The potentiality to fuse in this species isacquired at least 40 h prior to actual closure of the secondary palate. Thoseexplants that fused in vitro have been suggested as acquiring some propertiesin vivo which gave the explant the ability to fuse in vitro (Vargas, 1967). Sincea carbohydrate surface coat first appeared on day 12 of gestation in our study,approximately 48 h prior to shelf elevation, it seems reasonable to speculatethat it may be associated with the acquisition of potential to fuse which some-how does not occur if shelves are cultured from earlier embryos (Pourtois,1972). Although fusion of palatal shelves in organ culture is not inhibited bythe presence of proteolytic or saccharolytic enzymes (Pourtois, 1972), the timeof acquisition by the shelves of a 'potential to fuse' in vitro has an intriguingparallel in the acquisition of a surface coat by the palatal epithelium.

Once the surface coat appeared, it was generally found at the inferior tip ofvertically oriented shelves (Figs. 5, 6). A distinct lack of surface coat could bedetected as one progressed superiorly on both the lateral and medial marginsof vertically oriented shelves (Fig. 8). It is tempting to speculate with Watermanet al. (1973) that this inferior area might represent the future area of contactand fusion once the shelves have elevated.

The absence of surface coating on palatal epithelial cells may be of directconsequence to the production of cleft palate. Lack of normal adhesion be-tween adjacent palatal shelves may prevent continued contact and result inseparation of the shelves. Indeed, reopening of fused epithelial interfaces ofpalatal shelves has been suggested both in rodents (Buresh & Urban, 1967;Angelici, 1968; Greene & Kochhar, 1973) and man (Kitamura, 1966) as con-tributing to cleft palate.

This work was supported by NIH grant HD0655O.


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{Received 17 September 1973)

surface coat on the epithelium of developing palatine shelves in … · fig. 1. epithelial surface...

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