Developmental aspects of secondary palate formation · During development, the palatal shelves of...

/ . Embryo/, exp. Morph. Vol. 36, 2, pp. 225-245, 1976 2 2 5

Printed in Great Britain

Developmental aspects of secondarypalate formation

By ROBERT M. GREENE1 AND ROBERT M. PRATTCraniofacial Anomalies Section, Laboratory of Developmental Biology and

Anomalies, National Institute of Dental Research, Bethesda, Maryland

SUMMARY

Research on development of the secondary palate has, in the past, dealt primarily withmorphological aspects of shelf elevation and fusion. The many factors thought to be involvedin palatal elevation, such as fetal neuromuscular activity and growth of the cranial base andmandible, as well as production of extracellular matrix and contractile elements in the palate,are mostly based on gross, light microscopic, morphometric or histochemical observations.Recently, more biochemical procedures have been utilized to describe palatal shelf elevation.Although these studies strongly suggest that palatal extracellular matrix plays a major role inshelf movement, interpretation of these data remains difficult owing to the complexity oftissue interactions involved in craniofacial development. Shelf elevation does not appear toinvolve a single motive factor, but rather a coordinated interaction of all of the above-mentioned developmental events. Further analysis of mechanisms of shelf elevation requiresdevelopment of new, and refinement of existing, in vitro procedures. A system that enablesone to examine shelf elevation in vitro would allow more meaningful analysis of the relativeimportance of the various components in shelf movement.

Much more is known about fusion of the palatal shelves, owing in large part to in vitrostudies. Fusion of the apposing shelves, both in vivo and in vitro, is dependent upon adhesionand cell death of the midline epithelial cells. Adhesion between apposing epithelial surfacesappears to involve epithelial cell surface macromolecules. Further analysis of palatal epi-thelial adhesion should be directed towards characterization of those cell surface com-ponents responsible for this adhesive interaction.

Midline epithelial cells cease DNA synthesis 24-36 h before shelf elevation and contact,become active in the synthesis of cell surface glycoproteins, and subsequently manifestmorphological signs of necrosis. Death of the midline epithelial cells is thought to involve aprogrammed, lysosomal-mediated autolysis. Information regarding the appearance, distri-bution and quantitation of epithelial hydrolytic enzymes is needed.

The control mechanisms which regulate adhesiveness and cell death in the palatal epi-thelium are not fully understood. Although palatal epithelial-mesenchymal recombinationexperiments have demonstrated a close relationship between the underlying mesenchyme andthe differentiating epithelium, the molecular mechanism of interaction remains unclear.Recently cyclic nucleotides have been implicated as possible mediators of palatal epithelialdifferentiation.

The developing secondary palate therefore offers a system whereby one can probe avariety of developmental phenomena. Cellular adhesion, programmed cell death and epi-thelial-mesenchymal interactions are all amenable to both morphological as well as bio-

1 Author's address: Building 30, Room 414, Laboratory of Developmental Biology &Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda,Maryland 20014, U.S.A.

226 R. M. GREENE AND R. M. PRATT

chemical analysis. Although research in the field of secondary palate development has beenextensive, there still remain many provocative questions relating to normal development ofthis structure.

I. INTRODUCTION

During development, the palatal shelves of most vertebrates undergo a com-plex reorientation from a vertical position lateral to the tongue to a horizontalposition above the tongue and fuse to form a single, continuous structure, thesecondary palate. This involves four distinct stages including growth of theindividual shelves, reorientation of the shelves and finally epithelial cell ad-hesion and autolysis.

Past research has emphasized the study of teratogens that induce cleft palate.This review is not intended to deal with all these studies, but to concentrate onnormal secondary palatal development and to discuss possible areas of futureinvestigation. Only recently has it been appreciated that a number of develop-mental events including morphogenetic movements, cellular adhesion, epithelial-mesenchymal interaction and programmed epithelial cell death can all beexamined during palatal development. Understanding of these events, which areaccessible to a variety of biochemical analyses, and occur relatively late ingestation after most organogenic interactions have been completed, may berelevant to a number of other developing tissues.

II. GROWTH AND ELEVATION OF THE PALATAL SHELVES

After formation of the first pharyngeal arch, the maxillary, frontonasal andmandibular processes form the boundaries of the oral-nasal cavity. Cells of thecranial neural crest are known to migrate into and contribute significantly to thecell population of the face, including the maxillary process (Johnston &Listgarten, 1972). It is reasonable to assume that much of the original palatalshelf mesenchyme originates from the neural crest. The palatal shelves initiallyappear as small outgrowths at the medial surface of the maxillary processeswhich subsequently grow down on either side of the tongue (Fig. 1 A). At thisstage, each shelf consists of mesenchymal cells surrounded by extracellularmatrix and covered by a two- to three-cell layered epithelium (Fig. 2).

The palatal shelves begin movements which will bring them from a verticalposition on either side of the tongue into a horizontal position, superior to thetongue (Fig. 1B). These movements occur rather late in gestation, well aftermost organogenic processes have been completed, both in man (7th week ofgestation (Fulton, 1957)) and in the rat (day 16 of a 21-day gestational period(Coleman, 1965)). Early investigators envisaged the shelves rotating, as if on ahinge, from a vertical to a horizontal position (Peter, 1924; Lazzaro, 1940) andrecently, Walker & Ross (1972) have supported this concept.

This interpretation is dependent on the location along the anterior-posteriorlength of the shelf, since movement occurs in different fashions in different

Secondary palate development 227

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Fig. 1. Schematic illustration of frontal sections taken through the anterior hardpalate of the fetal rat at various times during gestation. (A) Day 15 ( + 8 h) - thepalatal shelves have grown from the maxillary processes and assume a position oneither side of the tongue. (B) Day 16 ( + 8 h) - the shelves have rotated to a positionabove the tongue, and medial-edge epithelial surfaces will make contact. (C) Day 17(+8h) - the medial-edge and nasal epithelial surfaces have adhered, and begunautolysis with some mesenchymal penetration of the epithelial seam (dotted line).Legend: N, nasal epithelium; O, oral epithelium; M, medial-edge epithelium.

Fig. 2. Photomicrograph of a single, vertically orientated mouse palatal shelf (PS)just prior to elevation. T, Tongue; M, maxilla, x 250.

regions. Coleman (1965) has observed in the rat fetus that rostrally (anteriorly)the palatal shelves elevate via rotation, as implied in Fig. 1A and IB, whilecaudally (posteriorly) shelf elevation proceeds by means of a 'remodeling' ofthe vertical shelf. Remodeling presumably involves a shift of the palatalmesenchyme resulting in a medially directed protrusion (Fig. 3) which meets theopposite shelf in the midline above the tongue. Similar observations in otherspecies have been reported (Walker & Fraser, 1956; Larsson, 1962; Kochhar &Johnson, 1965; Harris, 1967; Greene & Kochhar, 19736).

(a) Coordinated tissue movements during shelf elevationAfter growth from the maxillary processes, the palatal shelves are confronted

with a major obstacle between them, the growing tongue (Fig. 1 A). The tongue


MP

Fig. 3. Photomicrograph of a single palatal shelf (PS) remodeling from a verticalposition lateral to the tongue (T) to a horizontal position superior to the tongue.Medial protrusion (MP) of the palatal shelf, x 250.

has long been thought actively to withdraw from between the shelves prior topalatal shelf elevation. Several of the intrinsic muscles of the tongue have dif-ferentiated into fibers, responsive to both direct electrical as well as hypoglossalnerve nucleus stimulation, at least 4 h prior to palatal shelf elevation (Wragg,Smith &Boden, 1972). Recently, acetylcholinesterase activity was demonstratedat developing motor end plates in the tongue of fetal mice prior to shelf elevation(Holt, 1974). The myoneural apparatus of the tongue is therefore functional atthe time of palatal shelf elevation, suggesting that the tongue has the capacityfor voluntary movement at this time. The close synchrony between the onsetof tongue movements and shelf elevation suggests a relationship between thesetwo phenomena, but it is unlikely that such movement of the tongue is directlyresponsible for shelf elevation. Walker & Patterson (1974«) have demonstratedthat palatal shelves will elevate despite prior removal of the tongue. The obstruc-tive influence of the tongue is further supported by recent studies on shelf move-ment in vitro (Brinkley Basehoar & Avery, 1974; Brinkley, Basehoar, Branch& Avery 1975), in which fetal mouse heads cultured prior to in vivo shelf eleva-tion showed the most progress in attaining shelf elevation when the tongue wasremoved.

A variety of other fetal movements occur during the time of shelf elevation.The initiation of fetal mouth-opening reflexes (Humphrey, 1969) as well as' swallowing' movements (Walker, 1969) is closely followed by palatal elevation.To test if these were necessary for elevation of the palatal shelves, Jacobs (1971)


administered muscle relaxants to mice prior to and during the time of palateelevation. Such treatments failed to produce cleft palate and these studies wereinterpreted as indicating that spontaneous or reflexogenic fetal muscularactivity is not essential for normal shelf elevation. It was not, however, de-monstrated that these agents actually crossed the placenta and were active in thefetus. Recently, Walker & Patterson (19746) reported cleft palate in the off-spring of pregnant mice treated with barbiturates and tranquillizers, indicatingthat the developing fetus is affected, possibly by direct action on the fetalneuromuscular system, by the action of various central nervous system depress-ing agents.

It still remains, however, to be clearly demonstrated that fetal movementsplay a direct role in palatal development. The evidence available to date sup-ports the concept that a moderate downward growth of the tongue anteriorlyallows elevation to proceed by rotation. In the posterior region, the tongue issituated firmly in the common oral-nasal cavity so that a moderate movementdownward will not allow for rotation, thereby indirectly forcing the shelf toremodel around the tongue to a horizontal position.

Growth and straightening of the cranial base has also been proposed as play-ing a role in palatal elevation. In rodent fetuses, the flexed cartilagenous cranialbase straightens prior to and during shelf elevation and various authors havesuggested that this straightening is necessary for shelf elevation (Harris, 1964,1967; Verrusio, 1970; Wragg, Klein, Steinvorth & Warpeha, 1970; Smiley,Hart & Dixon, 1971; Hart, Smiley & Dixon, 1972; Larsson, 1972; Long,Larsson & Lohmander, 1973; Taylor & Harris, 1973). This movement, com-bined with growth of the lower jaw (Harris, 1967; Humphrey, 1971) results inan increased vertical dimension of the anterior one-half of the oral cavity.Lifting of the anterior portion of the cranial base could facilitate the rotationof the anterior region of the palatal shelves (Harris, 1967). However, measure-ments of cranial base changes are usually derived from fixed, embeddedmaterial, subject to shrinkage and other artifacts inherent in tissue preparation.A study of cranial base changes on frozen sections should prove interesting. Therole of the cranial base in shelf elevation remains controversial, as elevationin vitro does not require an intact cranial base (Brinkley et al. 1975).

Mandibular growth has also been implicated in palatal elevation (Zeiler,Weinstein & Gibson, 1964). The forward growth of the mandible presumablycarries the base of the tongue from between the shelves posteriorly, and down-ward growth of the mandible creates room above the tongue anteriorly for theshelves to elevate. The evidence, however, for a differential mandibular growthspurt prior to shelf elevation is presently inconclusive (Zeiler et al. 1964; Hart,Smiley & Dixon, 1969).

The preceding discussion (II a) points out the fact that the role these externalstructures play in palatal elevation is complex and as yet unclear. It seemsreasonable that a highly coordinated interaction of many of these structures


will prove to be necessary for elevation, but at the present state of our knowledge,it is impossible to say which of these factors is most important.

(b) Role of cellular proliferation and extracellular matrix

An 'internal shelf force' developing from within the shelf was proposed byWalker & Fraser (1956) to explain elevation of the palatal shelves. While it isconceivable that rapid proliferation of mesenchymal cells in certain areas of thepalate could induce movement, Walker & Fraser (1956) and Hughes, Furstman& Berdick (1967) found no evidence of an unusually high mitotic rate in theshelf mesenchyme at the time of elevation. Jelinik & Dostal (1973, 1974) injectedcolchicine into the amniotic fluid of the mouse fetus at various times prior to aswell as during elevation and subsequently counted metaphase figures in themesenchymal cells. They found that the peak of proliferation in the mesenchymepreceded elevation by 24 to 48 h. Similar conclusions have been reached bymeasuring the accumulation of DNA with time in the rat palate (Hassell, Pratt& King, 1974).

It was thought that even after elevation, the shelves had to grow extensivelytowards each other to make contact. However, recent studies using freeze-dried(Wragg, Diewert & Klein, 1972) or freeze-sectioned material (Greene & Kochhar,1973 a) have shown that the palatal shelves are in contact along at least part oftheir medial edges immediately upon elevation. This is most evident in theanterior region of the shelves where elevation occurs by rotation. Growth doesnot appear to play a direct role in elevation of the shelves, although a criticalexamination of mesenchymal growth rates in different regions of the shelf bothprior to and during elevation is needed.

Walker & Fraser (1956) postulated that an elastic fiber network in the palatalmesenchyme provided the necessary force for elevation, although it has sincebeen demonstrated that no such network is seen in the palate at the time ofelevation (Stark & Ehrman, 1958; Loevy, 1962; Frommer, 1968; Frommer &Monroe, 1969). Attention thus turned to the role of other extracellular matrixcomponents in elevation, since the matrix is a prominent constituent of theshelf mesenchyme and a variety of teratogens alter the synthesis or metabolismof its components.

Prior to and during elevation the palatal shelves actively synthesize sulfatedproteoglycans (see discussion below) (Larsson, Bostrom & Carlsoo, 1959;Larsson, 1961; Walker, 1961) and there is also a rapid accumulation of extra-cellular metachromatic material in the palatal mesenchyme (Larsson, 1961;Jacobs, 1962). This metachromatic material was degradable by testicularhyaluronidase (Anderson & Mathiessen, 1967) and based on the knownspecificity of this enzyme was presumed to be chondroitin sulfate and/orhyaluronic acid. Pratt, Goggins, Wilk & King (1973) labeled the glycosamino-glycans synthesized by palatal shelves at the time of elevation in vivo and also invitro with[ 3H]-glucosamine and [35S]-sulfate. Subsequently the tissue was treated


with papain to remove the protein portions and the various polysaccharidespecies were resolved by chromatography on DEAE-cellulose. Tissue levels werefound to reflect synthetic activity and showed that hyaluronic acid accountedfor 65% of the total glycosaminoglycans and sulfated glycosaminoglycansaccounted for the remainder.

Hyaluronate is an extremely hydrated high molecular weight unbranchedpolysaccharide (Laurent, 1970), and its production and accumulation is oftenassociated with swelling of embryonic tissues and morphogenetic movements(Toole, Jackson & Gross, 1972; Pratt, Larsen & Johnston, 1975). Prior toelevation, the presumptive oral half of the palatal mesenchyme contains largerextracellular spaces than the presumptive nasal portion of the shelf (Anderson& Mathiesson, 1967). The accumulation of hyaluronate may be especially highin this region of the shelf. A number of teratogens, which induce cleft palate byinhibiting or delaying palatal elevation, alter glycosaminoglycan metabolism inthe palate (cortisone, Larsson, 1962; chlorcyclizine, Wilk, Steffek & King, 1970;diazo-oxo-nor Leucine, DON, Pratt, Coggins, Wilk & King 1973). These studiessuggest that glycosaminoglycans, especially hyaluronate, play an important rolein shelf elevation.

Another component of the palatal shelf mesenchyme which may play a rolein elevation is collagen. Pratt & King (1971) found a significant increase in theamount of palatal collagen, estimated to be 4-6% of the total palatal protein,at the time of shelf elevation (day 15-16 in the rat). /?-aminopropionitrile(BAPN) appears to inhibit shelf elevation and cause cleft palate by preventingthe crosslinking of collagen in the palate as well as the whole embryo (Steffek,Verrusio & Watkins, 1972; Wilk, King, Horigan & Steffek, 1972; Pratt & King,1972#, b). BAPN specifically inactivates lysyl oxidase which ordinarily oxidizeslysine residues in collagen to aldehydes that react to form the actual crosslinks(Pinnel & Martin, 1968). The teratogenic action of BAPN implies that collagenfibers play a structural role in shelf elevation since no change in synthesis ordeposition is noted and presumably only the tensile strength of collagen fibers isaffected.

Collagen fibrils have been observed in the palate of the mouse (Smiley &Dixon, 1968; Smiley, 1970). In the palate of the rat, collagen fibrils are observedthroughout the mesenchyme, but are particularly abundant in the area adjacentto the basal lamina of the presumptive oral epithelium. Here, the fibres areoriented uniformly along the anterior (rostral) to posterior (caudal) axis of theshelf (Hassell & Orkin, 1974). This location in the palate suggests that thesefibers may serve as attachment or anchorage sites by which a force could betransmitted to the rest of the mesenchyme during elevation.

Although the components of the palatal matrix have thus far been consideredindividually, it is likely that they form functional macromolecular complexes.In other systems, specific interaction between collagen and sulfated GAG (Toole& Lowther, 1968; Lowther, Toole & Herrington, 1970; Greenwald, Schwartz &


Cantor, 1975) and also between hyaluronate and proteoglycans (Hardingham &Muir, 1972) have been observed. The hyaluronate-proteoglycan interaction wassubsequently shown to be essential in the organization of proteoglycan aggre-gates in cartilage matrices (Hascall & Heinegard, 1974). Additionally, smallmolecular weight proteins were shown to be critical for stabilizing the hyaluron-ate-proteoglycan interaction (Gregory, 1973; Hardingham & Muir, 1974;Hascall & Heinegard, 1974; Rosenberg, Hellman & Kleinschmidt, 1915a). Amodel for these interactions has been discussed recently (Hascall & Heinegard,1975; Rosenberg et al. 1975 b; Wiebkin, Hardingham & Muir, 1975). Suchcomplexes have not yet been defined in other tissues but if they occur in palatalshelves they could be of structural importance during elevation.

Rapid movements in biological systems are usually brought about by con-tractile elements. The contractile nature of wound granulation tissue hasrecently been demonstrated (Gabbiani, Majno & Ryan; 1973), and actin andmyosin have been reported in a variety of tissues and cells other than muscle(Pollard & Weihing, 1974). Lessard, Wee & Zimmerman (1974) have shownthat actin and myosin are synthesized in the mouse palate at the time of shelfmovement. A calcium-dependent ATPase, presumably myosin, has been ob-served in the posterior area of the palate and it has been suggested that thisrepresents smooth muscle which along with striated muscle in the extremeposterior soft palate, might be involved in shelf elevation (Babiarz, Allenspach& Zimmerman, 1975).

The relative importance of the extrinsic, as well as various intrinsic, factorsinvolved in palatal shelf elevation can be further clarified by the developmentof new and the refinement of existing in vitro methods. The in vitro procedures ofBrinkley et al. (1975) and of Walker & Patterson (1974a) may offer techniquesby which the process of shelf elevation may be directly observed.

The most widely held concept of palatal elevation supports a direct andactive involvement of the palatal shelves in their own morphogenetic movement.This view, however, does not rule out the possible involvement of variousexternal factors previously discussed.

III. FUSION OF THE PALATAL SHELVES

(a) Epithelial cell adhesion

Electron microscopic studies of mouse (Farbman, 1968), rat (Hayward, 1969),hamster (Chaudhry & Shah, 1973) and human palatal tissue (Matthiessen &Anderson, 1972; Mato, Smiley & Dixon, 1972) have demonstrated that the finestructure of the palatal epithelium both before and during epithelial adhesionand seam formation is remarkably similar in these species. Prior to contact, theepithelium of the medial edge of the elevating palatal shelves is two to three celllayers thick with a surface squamous cell layer and underlying cuboidal celllayer(s). The epithelial-mesenchymal border exhibits an intact basal lamina


MV

M

Fig. 4. Electron micrograph of a squamous epithelial cell on the surface of a mousepalatal shelf just prior to contact with the opposite shelf. Note the microvilli (MV)present at the surface. D, desmosome; G, Golgi apparatus; M, mitochondrion;RER, rough endoplasmic reticulum. x 36400.

separating the epithelial cells from the underlying mesenchymal cells (DeAngelis& Nalbandian, 1968; Smiley, 1970; Chaudhry & Shah, 1973). The surfaceepithelial cells exhibit prominent microvilli, as cytoplasmic extensions (Fig. 4)which flatten and subsequently disappear upon contact with the oppositepalatal shelf (DeAngelis & Nalbandian, 1968; Matthiessen & Anderson, 1972;Waterman & Meller, 1974).

234 R M. GREENE AND R. M. PRATT

Once contact between the two apposing shelves has been made, a tightadhesion develops, as attempts to separate the shelves after contact producetearing of epithelial cell membranes (Zeiler, Weinstein & Gibson, 1964; Farb-man, 1968). Although desmosomes may support contiguity between apposingepithelial surfaces (DeAngelis &Nalbandian, 1968; Brusati, 1969; Chaudhry &Shah, 1973), Farbman (1968) found no evidence of desmosomes betweenadhering palatal epithelial surfaces in the mouse.

Much of the present emphasis on the study of palatal adhesion can be attri-buted to the earlier work of Pourtois (1966, 1968«, b, 1970) on epithelial celladhesion and death during palatal development. Initial adhesion of one shelfto another may be explained by the presence of an extracellular carbohydrate-rich surface coat on the developing epithelium of the palatal shelves. Epithelialcell membranes of apposing shelves are separated by a 10-20 nm. Intercellularspace of uniform width (Hay ward, 1969), presumably containing surface glyco-proteins. Initial adhesion mediated by cell-surface carbohydrates may serve tokeep the shelves in contact until more permanent, desmosomal connections canbe made. Farbman (1968) and Matthiessen & Anderson (1972) were unable,utilizing various histochemical stains, to demonstrate a surface coat, althoughHay ward (1969) using transmission electron microscopy described an 'ill-defined fuzzy material' on medial-edge epithelial cell surfaces prior to contact.

A carbohydrate-rich, surface coat has been shown to increase dramaticallyon the epithelial surface (medial-edge) prior to contact. This has been shown atthe ultrastructural level by the binding of the plant lectin concanavalin A (CONA) (Pratt, Gibson & Hassell, 1973a; Pratt & Hassell, 1975) and by staining withruthenium red (Greene & Kochhar, 1974). The chemical nature of this surfacecoat is probably complex since CON A binds to mannosyl and glucosyl residuesof glycolipids and glycoproteins and ruthenium red binds to glycosaminoglycansand glycoproteins. Scanning electron microscopic observations of the medial-edge epithelial surface prior to contact in several species show the progressiveappearance of a fine filamentous material (Waterman, Ross & Meller, 1973;Meller, personal communication, 1974), although this material is not observedin the human (Waterman & Meller, 1974).

Surface glycoproteins and glycosyltransferases have been implicated in cell-to-cell adhesion in a variety of systems (Roseman, 1970; McGuire, 1972).DON (diazo-oxo-norleucine), a glutamine analog, has been shown to preventadhesion of mouse teratoma cells (Oppenheimer, 1973) and epithelial adhesionbetween homotypic palatal shelves (Pratt, Greene, Hassell & Greenberg,1975). D-glucosamine reversed the action of DON on both the teratoma cellsand palatal epithelial cells. Shelves cultured in the presence of DON bind muchless labeled CON A at the epithelial surface than shelves cultured without DON(Greene & Pratt, 1975). These data suggest that amino sugars are incorporatedinto macromolecules which are necessary for intercellular adhesion. The ob-servation (Pourtois, 1970, 1972) that adhesion of palatal shelves in vitro was not


inhibited by proteolytic enzymes could be explained by rapid resynthesis of cell-surface components (Collins, Holland & Sanchez, 1973) or the resistance ofessential surface components to proteolysis.

The presence of a carbohydrate surface coat may explain the phenomenon ofan 'acquired potential' to fuse exhibited by palatal shelves of rats (Pourtois,1966) and mice (Vargas, 1967) in vitro. It must be pointed out that the term'fusion' refers only to the merging of mesenchyme of adjacent palatal shelvesafter midline epithelial seam breakdown. Epithelial cells from adjacent shelvesadhere to one another, but there is no plasma membrane fusion during forma-tion of the midline seam. Pourtois (1966) and Vargas (1967) have shown thatpalatal explants from early mouse and rat fetuses do not fuse in vitro, althoughexplants from older fetuses show an increasing ability to fuse. Recently, how-ever, Tyler & Koch (1975), using different culture conditions, have shown thatfetal mouse palates, explanted 2-5,- days prior to contact in vivo, w/7/fuse after 72 hin culture. This suggests that the activating changes necessary for shelf-fusioncan occur in vitro. The acquisition of particular cell-surface properties is not anuncommon occurrence as cell-surface alterations during development have beendemonstrated in a variety of embryonic systems (Moscona, 1971; Gottlieb,Merrell & Glaser, 1974; Krach, Green, Nicholson & Oppenheimer, 1974). Anincreasing number of observations indicate a functional role for palatal epithelialcell-surface carbohydrates, and it is likely that their programmed appearanceis necessary for adhesion between adjacent palatal shelves.

B. Epithelial Cell Autolysis

Elimination of certain cells occurs during the development of a variety oftissues. This process is frequently referred to as programmed cell death, sincethe changes occur at precise stages of development (Lockshin & Williams, 1965;Saunders & Fallon, 1966; Dorgan & Schultz, 1971). Such changes are seen inpalatal medial edge epithelial cells (Mato, Aikawa & Katahira, 1966; Smiley,1970; Smiley & Koch, 1971; Matthiessen & Anderson, 1972; Chaudhry &Shah, 1973). Preautolytic alterations include mitochondrial swelling (Sweeny &Shapiro, 1970; Mato, Smiley & Dixon, 1972) and the appearance of lysosomalbodies in medial-edge epithelial cells (Mato, Aikawa & Katahira, 1966;Hayward, 1969).

Studies in the rat in vivo (Hudson & Shapiro, 1973) and in vitro (Pratt &Martin, 1975) have shown by measuring the incorporation of [3H]-thymidinethat medial-edge epithelial cells cease DNA synthesis as early as 36 h priorto contact. These cells are not at a local nutritional disadvantage since theadjacent oral and nasal epithelial cells, as well as the mesenchymal cells,continue to divide (Fig. 5). Although DNA synthesis ceases, medial-edgecells incorporate [3H]-uridine into RNA (Pratt & Greene, 1975) and labeledglucosamine and fucose into macromolecules, some of which appear to be

236 R. M.GREENE AND R. M. PRATT

f'.fi%-:.»m*** \(«) <̂* o

Fig. 5. Autoradiograms of palatal shelves cultured for 4 h submerged in growthmedium containing [3H]-thymidine at 40 /iCi/ml. (a) Day 14(+12 h) (x 350) and (b)Day 15(+12 h) (x 350). The various areas of the epithelium are: M, medial edge;O, oral; N, nasal epithelium; mes, mesenchyme.

transported to the cell surface (Pratt & Hassell, 1974; DePaola, Drummond,Lorent & Miller, 1974).

Smiley & Koch (1972) demonstrated that selective death occurs in the medial-edge epithelial cells of palatal shelves explanted onto Millipore filters. Recently,Tyler & Koch (1974) demonstrated that isolated palatal epithelium explantedonto Millipore filters also displays selective cell death in the presumptive medial-edge region. Selective death of medial-edge cells was also observed in vivo infetuses of the A/Jax mouse that displayed spontaneous cleft lip and palate(Smiley & Koch, 1972) and in rat fetuses after amniotic sac puncture (Goss,Bodner & Avery, 1970; Morgan, 1975). In both these cases inhibition ofelevation due to the obstructive position of the tongue prevented epithelialcontact between shelves.

Palatal epithelial-mesenchymal recombination experiments (Pourtois, 1969;Tyler & Koch, 1975) support the hypothesis that autolysis in medial-edgeepithelial cells is dependent on a prior interaction between these tissues. Thequestion of the actual time when the medial-edge cells become irreversiblycommitted to cell death remains unanswered, although several studies have


Fig. 6. Photomicrograph of a plastic-embedded secondary palate illustrating earlyseam formation between adjacent palatal shelves. S, midline epithelial seam; NS,nasal septum, x 250.

shown that certain compounds may inhibit fusion in vitro by preventing celldeath in the medial-edge epithelium (/?-thienylalanine, Baird & Verrusio, 1973;hadacidin, Fairbanks & Kollar, 1974; epidermal growth factor, Hassell, King& Cohen, 1974; DON, Pratt & Greene, 1975).

The first signs of programmed cell death in the medial-edge cells is a cessationof DNA synthesis. Numerous studies with various cultured cell lines have shownthat elevated levels of cyclic adenosine 3'-5'monophosphate (CAMP) areassociated with reduced proliferative activity (Otten, Johnston & Pasten, 1972;Bombik & Burger, 1973). In the rat palatal shelf, levels of cyclic AMP have beenshown to increase dramatically approximately 18 to 24 h prior to epithelialcontact (Pratt & Martin, 1975). Whether or not this increase occurs in theepithelium or is related to cessation of DNA synthesis in the medial-edge cellsis not known. The addition of dibutyryl cyclic AMP to the immature day-14 ratshelf in vitro causes a precocious decrease in medial-edge epithelial DNAsynthesis and an increase in glycoprotein synthesis. These induced changesappear to mimic those that normally occur on days 15 to 16. These resultssuggest that activation of medial-edge cells to mature in vivo, involving bothdecreased proliferation and increased adhesiveness, may in part be mediatedthrough cyclic nucleotides. This is further supported by the ability of exogenousdibutyryl cyclic AMP to prevent EGF-induced inhibition of epithelial cell death

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(Pratt, Green, Hassel & Greenberg 1975). There are undoubtably other factorsinvolved in this activation and maturation process, although they remainunknown at the present time.

The developing palatal shelves in the chick embryo may provide an interestingsystem in which to investigate these programmed changes. Chick palatalshelves do not appear to maintain epithelial contact and therefore formation ofa fused secondary palate never occurs here. A comparison between the pro-grammed changes involved in palatal adhesion and fusion of most vertebratesand the events which bring about the cleft palate of the chick should proveinteresting. The developing eyelids (Anderson, Ehlers & Matthiessen, 1965;Anderson et al. 1967) may provide another system to investigate epithelialadhesion, and control of these developmental events.

After midline epithelial adhesion and the onset of autolysis, the epithelium ofone shelf soon becomes indistinguishable from that derived from the adjacentshelf, as an epithelial seam is formed in the midline of the future secondarypalate. The midline epithelial seam changes rapidly from the original 4-5 cell-layered seam (Fig. 6) to a 1-2 cell-layered strip of cells (Fig. 7) (Smiley & Dixon,1968; Hayward, 1969). Superiorly, at the nasal surface of the secondary palate,the seam expands into a triangular area (Smiley & Dixon, 1968), and inferiorly,at the oral surface, the seam also expands into a triangular area. Both of theseareas are characterized by large intercellular spaces with cells containingnumerous electron-dense granular structures, presumably lysosomal in naturesince they have been shown to be positive for acid phosphatase (Mato, Aikawa& Katahira, 1967).

Lysosomal bodies appear throughout the palatal epithelial midline seam andare rare in the epithelium covering the oral and nasal palatal surfaces (Mato,Aikawa & Katahira, 1967). In a study of the role of acid phosphatase in fusionof the secondary palate, Angelici & Pourtois (1968) localized all enzyme re-actions in the epithelial cytoplasm. Autolysis of the midline seam is accomplishedby lysosomal enzymes produced by the midline epithelial cells, indicating aprimary lysosomal involvement in this type of programmed cell death. This is incontrast to programmed cell death in the posterior necrotic and interdigital zoneof the developing chick limb, where cells are first fragmented by unknownmechanisms and these fragments taken up and degraded by macrophagescontaining lysosomal enzymes (secondary lysosomal involvement) (Saunders &Fallon, 1966).

As autolytic breakdown of the midline seam and the superior and inferiortriangular areas proceeds, the epithelial cells become irregular in outline and areseparated by increasingly larger extracellular spaces. The epithelial cells, how-ever, remain connected throughout this process by desmosomes, and initialepithelial degeneration is not directly associated with apparent breakdown ofthe basal lamina (Hayward, 1969) (Fig. 8). The final phases of breakdown of themidline epithelial seam in both hard and soft palate involve disruption of the

16-2


basal lamina interposed between epithelial and mesenchymal cells, and migra-tion of macrophages into this region with subsequent phagocytosis of degenerat-ing epithelial cells (Koziol & Steffek, 1969; Matthiessen & Anderson, 1972;Shah & Chaudhry, 1974). Epithelial seam breakdown has been assumed toallow extensive intermixing of the mesenchymal cells of adjacent palatal shelves.However, Koch & Smiley (1973) have combined in vitro colchicine-treatedshelves labeled with [3H]thymidine and unlabeled shelves, and have thus shownthat little intermixing takes place between mesenchymal cells derived fromoriginally separate shelves.

We wish to thank Dr George Martin, Laboratory of Developmental Biology and Anoma-lies, National Institute of Dental Research, for numerous helpful suggestions during the pre-paration of this manuscript. We are also indebted to Mrs Barbara Stoops for her excellentsecretarial assistance.

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{Received 12 January 1976)

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