Recent advances in primary palate and midface morphogenesis research.

Critical Reviews http://cro.sagepub.com/ in Oral Biology & Medicine

Recent Advances in Primary Palate and Midface Morphogenesis Research Virginia M. Diewert and Kang-Yee Wang CROBM 1992 4: 111 DOI: 10.1177/10454411920040010201 The online version of this article can be found at: http://cro.sagepub.com/content/4/1/111

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Critical Reviews in Oral Biology and Medicine,

4(1): 111-130 (1992)

Recent Advances in Primary Palate and Midface Morphogenesis Research* Virginia M.

Diewert and Kang-Yee

Wang

Departments of Clinical Dental Sciences, and Oral Biology, University of British Columbia,

Vancouver, British Columbia

Symposium: Current Concepts in Craniofacial Embryology, Dr. G. Symposium on Anatomical Sciences, Toronto, Canada, July 1-4, 1991:

*Presented at the

ABSTRACT:

H.

Sperber, Chairman, Xth International

During the sixth week of human development, the primary palate develops as facial prominences

enlarge around the nasal pits to form the premaxillary region. Growth of craniofacial components changes facial

morphology and affects the extent of contact between the facial prominences. Our recent studies have focused developing methods to analyze growth of the primary palate and the craniofacial complex to define morphological phases of normal development and to determine alterations leading to cleft lip malformation. Analysis of human embryos in the Carnegie Embryology Collection and mouse embryos of cleft lip and noncleft strains showed that human and mouse embryos have similar phases of primary palate development: first, an epithelial seam, the nasal fin, forms; then a mesenchymal bridge develops through the nasal fin and enlarges rapidly. A robust mesenchymal bridge must form between the facial prominences before advancing midfacial growth patterns tend to separate the facial components as the medial nasal region narrows and elongates, the nasal pits narrow, and the primary choanae (posterior nares) open posterior to the primary palate. In mouse strains with cleft lip gene, maxillary growth, nasal fin formation, and mesenchymal replacement of the nasal fin were all delayed compared with noncleft strains of mice. Successful primary palate formation involves a sequence of local cellular events that are closely timed with spatial changes associated with craniofacial growth that must occur within a critical developmental period.

on

KEY WORDS:

I.

embryology, craniofacial growth, palatogenesis, cleft palate.

INTRODUCTION

The primary palate develops as facial prominences enlarge around the nasal pit and coalesce to form the premaxillary region, the future upper lip, alveolus, and hard palate tissues anterior to the incisive foramen. Further posteriorly, the secondary palate, which gives rise to the hard and soft palate regions, develops later than the primary palate. The primary and secondary palatal regions are recognized as different embryologic entities (Trasler and Fraser, 1963; Fraser, 1970; Sperber, 1989), and genetic and environmental factors that influence their closures are different. Abnormal development of the primary palate, leading to a cleft lip, may interfere secondarily

with secondary palate closure to cause cleft palate. Thus, on both embryologic and genetic grounds, congenital cleft lip and cleft lip with cleft palate (CLP) appear to be etiologically related and are designated as CL(P). Isolated clefting of the secondary palate (CP) is an etiologically independent entity (Fraser and Baxter 1954; Trasler and Fraser, 1963; Woolf et al, 1963;

Fraser, 1970, 1989). Cleft lip with or without cleft palate [CL(P)] is one of the most common craniofacial congenital malformations in human embryos. A large number of syndromes exist in which CL(P) may be one of the features. For most of these, the cause is unidentified. A few are associated with recognizable chromosomal aberrations, and about

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a third are caused by major mutant genes. Each of these syndromes is rare, and together they may account for perhaps 5% of all cases (Nora and Fraser, 1974). Most cases of CL(P) without associated malformations in humans appear to be multifactorially determined (Fraser, 1970, 1989). There are striking differences in the CL(P)

frequency between races: North American Indians in British Columbia have very high frequencies (2.75 per 1000 births) (Lowry and Renwick, 1969); Orientals have relatively high frequencies (1.7 per 1000 births) (Kobayashi, 1958; Neel, 1958). Caucasians are intermediate (1 per 1000); Blacks tend to have low frequencies (0.4 per 1000) (Chung and Myrianthopoulos, 1968; Khoury et al, 1983). Although the cause of nonsyndromic CL(P) remains unknown (Fraser, 1989), these differences persist in different geographic regions, suggesting that they do not result from environmental alternations. They are thought to be associated with differences in face shape (Fraser and Pashayan, 1970). Because the multifactorial threshold model has been applied to cleft lip (Carter, 1969; Fraser, 1970, 1980), it would be useful to identify some biological attribute of liability such as face shape that could be an indicator of increased risk. II. NORMAL DEVELOPMENT OF THE PRIMARY PALATE A. The Origin of Facial

Mesenchyme Before primary palate formation occurs, neural crest cells migrate from the neural tube to the craniofacial region to form mesenchyme of the facial prominences. The importance of the role of the neural crest cells in forming the mesenchyme of the facial prominences was shown in experiments with chick embryos by Johnston

(1964, 1966). Explantation of small segments of neural crest labeled with tritiated 3H-thymidine from either the mid- or forebrain to the corresponding region of host embryos showed that these cells made a significant contribution to the formation of the facial mesenchyme (Johnston, 1964). Removal of segments of the midbrain neural crest before cell migration resulted in severe facial malformations, including absence of

a pronounced frontonasal prominence and a mandibular prominence, whereas removal of the forebrain neural crest, which normally makes a lesser contribution to facial mesenchyme, frequently resulted in the clefting of the primary palate only

(Johnston, 1966).

Using interspecific grafts of the neural primordium between quail and chick embryos, Le Lievre and Le Douarin (1975) have shown that the mesenchyme of the maxillary prominence and the branchial arches is composed of mesectodermal cells. Noden (1975, 1983) demonstrated that the avian crest cells from the posterior mesencephalon migrate en masse away from the neural tube between the epidermis and the underlying mesoderm. Then this neural crest population migrates and proliferates throughout both the regions ventrolateral to the mesencephalon and the future maxillary prominence. Neural crest cells migrating from anterior mesencephalon and posterior diencephalon move rostrally, and at later stages, they overlap with those derived from the posterior mesencephalon, contributing to the formation of the maxillary prominence. Nichols (1986) has shown that in mouse embryos, neural crest formation is complete at the midbrain-rostral hindbrain neural fold at 4 + to 5 somites of development. The neural crest leaves behind overlying squamous epithelium. At approximately 10 somites of development, this neural crest mesenchyme is distributed dorsolateral to the pharynx and displaced ventromedially in a narrow, transient subectodermal space functionally similar to that observed in the chick embryo. If the fate of the crest mesenchyme in the mouse is similar to that in birds and amphibians, this mesenchyme will form bone, cartilage, and connective tissue of the first branchial arch. B. Craniofacial Growth

Formation

during Palate

Induction of the nasal organ starts primary palate formation. At about 33 d (stage 15) after fertilization in humans, the area around the future nose is induced to elevate, forming the medial and lateral nasal prominences (Streeter, 1948; O'Rahilly, 1967; O'Rahilly and Miller, 1987).


The mesenchyme underlying the ectoderm of the facial region originates from the neural crest cell population (Johnston, 1964, 1966; Le Lievre and Le Douarin, 1975). Its high rate of proliferation (Minkoff and Kuntz, 1977, 1978) is thought to be maintained by an epithelial-mesenchymal interaction. From an implant-labeling technique using sable-hair probes as carriers for 3H-thymidine, studies have indicated that lateral movement of medial nasal prominence mesenchyme into the base of the nasal groove and medial area of the lateral nasal prominence had occurred (Patterson et al., 1984; Patterson and Minkoff, 1985).

A

B

During formation of the primary palate, major morphogenetic changes occur in the embryonic craniofacial region (Figures 1 to 4). Although overall patterns of change have been observed and described (Streeter, 1948; Patten, 1961; Vermeij-Keers, 1972, 1990; Johnston, 1966; Desmond and O'Rahilly, 1981; Gribnau and Geijsberts, 1981, 1985; Vermeij-Keers et al.,

1983; Hinrichsen, 1985; Burdi etal., 1988), little definitive information is available about midfacial growth during the embryonic period when the primary palate develops. A quantitative study of growth in the human brain (Desmond and

C

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FIGURES 1 to 4. Composite of facial photographs, histological sections, and computer graphic images of FEM analysis. FIGURE 1. Facial photographs of human embryos of stages 16 to 19. FIGURE 2. (A,B) Frontal sections through the anterior nasal region of S16 and S19 embryos showing landmarks and elements. (C,D) FEM results showing major and minor strains required to change average S16 morphology to S19 morphology. FIGURE 3. (A,B) Frontal sections through the ocular regions of S16 and S18 embryos showing landmarks and elements. (C,D) FEM results showing major and minor strains required to change S16 morphology to achieve average S18 morphology. FIGURE 4. (A,B) Medial sections of S15 and S19 embryos showing location of landmarks and elements studied. (C,D) FEM results showing major and minor strains required to change average S15 morphology to achieve average S19 morphology.


growth changes that are associated with changes in craniofacial morphology and formation of the primary palate (Diewert and Lozanoff, 1993b; Diewert et al., 1993). Photographs of frontal sections of 31 human embryos of stages 16 to 19 in the Carnegie Embryology Collection were selected at serial planes through the head, enlarged,

O'Rahilly, 1981) showed regional differential growth patterns in the brain in embryos of stages 11 to 23, at crown-rump lengths (CRL) of 3 to 39 mm. In a recent study of human midfacial patterning, Burdi et al., (1988) showed that patterns of midfacial growth in the fetal period were

similar to those established in the last week of embryonic development. However, the earlier embryonic period in which these patterns are first developed was not studied. Differential growth patterns during the late embryonic period when the secondary palate develops were shown to involve predominantly sagittal and vertical growth of the craniofacial complex in rodent and human embryos and fetuses (Diewert, 1974, 1978, 1982,

and craniofacial landmarks were located for morphometric measurements (see Figures 2,3). The finite element modeling (FEM) provided graphic displays of regional growth change between stages 16 and 19 (see Figures 2,3) (Lozanoff and Diewert, 1986, 1989; Diewert and Lozanoff, 1993a, 1993b). Extensive changes in midfacial morphology occurred as the frontonasal prominence elongated vertically (height increased by seven times) and narrowed to approximately half the original width (see Figures 5 to 8), with changes more pronounced in the anterior nasal region. The brain and the face became vertically separated, and the brain became more superiorly positioned relative to the face as the craniofacial

1983, 1985, 1986).

In our laboratory, quantitative methods have been developed and applied to the study of changes in craniofacial morphology in human embryos during primary palate formation (Figures 2 to 13). Frontal sections of human embryonic heads were studied to identify regional

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FIGURES 5 to 8. Summary of morphogenetic changes in frontal plane of human embryos. FIGURE 5. Outlines of stages 17 and 18 human embryonic sections through the nasal region showing frontal element numbers. FIGURE 6. The areas of the upper brain (12), the maxilla (6,7), and the lateral nasal prominences (10,11) increased most rapidly, whereas the lower brain area between facial regions (13) decreased. FIGURES 7,8. Linear measurements showing changes in width and height in the embryos revealed narrowing of the nasal septal region at all levels and a decrease in height of the brain between facial structures (51 to 61). Measurements of other widths and heights showed similar increases.


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FIGURES 9 to 11. Summary of morphogenetic changes in the medial plane of human embryos. FIGURE 9. Linear measurements between craniofacial landmarks shown in Figure 4 revealed that rates of growth were similar in most regions except in the posterior cranial base (landmarks L1 to L7) and the anterior cranial base (L1 to L4). In the posterior cranial region, a 15.5% increase in height (L1 to L6) and only a 5.5% increase in length produced a significant decrease in cranial base angle. As the posterior cranial angle (P) decreased, the oropharyngeal angle (O) increased reciprocally. The middle cranial angle (M) was unchanged, whereas the anterior angle (A) increased a little. FIGURE 11. A composite of stages 15 to 18 superimposed on the posterior cranial base registered at the base of the hypophysis (L1) illustrates overall changes as the posterior cranial angle decreases and the anterior cranial base rotates up and back. The face

develops in the region above the thorax that is created as these cranial morphogenetic changes occur.


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FIGURES 12 and 13. FIGURE 12. Computer reconstructions of frontally sectioned embryos showing changes in regional morphology and changes in spatial relations of the brain (B), eye (E), fronto-nasal region (FN), and the maxillary prominences (Mx) at stages 16, 17 (early, mid, and late), and 18. Section outlines of similar stages are shown to the right in Figure 13. During this period, a vertical separation of the brain and facial tissues occurs. FIGURE 13. Graph showing mean areas of the maxillary prominences at seven different frontal planes measured through the face. The maxillary prominences increased in size and grew forward to overlap the nasal cavity as the primary palate formed.

region developed. The size of the maxillary region increased rapidly, particularly in the most anterior regions, as the maxillary prominences grew forward to contribute to the primary palate (see Figures 6,12,13). The lateral nasal prominences increased in size with a predominantly horizontal growth pattern (see Figure 2). These patterns of vertical growth of the midfacial tissues with narrowing of the frontonasal prominence, forward growth of the maxilla, and relative sep-

aration of the brain and face were identified as predominant features of embryonic craniofacial growth during midfacial pattern development (see

Figures 12,13).

Because the facial prominences are essentially attached to the brain, changes in brain morphology would be expected to have an impact on the developing face. A study of medial sections was undertaken to identify regional changes that are associated with changes in overall morphol-


ogy during palatal development (Diewert and Lozanoff, 1993a). Photographs of sagittal sections of 35 human embryos of stages 15 to 19 in the Carnegie Embryology Collection were enlarged, and landmarks were located for angular and linear measurements and for FEM analysis (see Figure 4) (Lozanoff and Diewert, 1986, 1989). Anatomical form change was illustrated with FEM by showing deformation required to change the average form for the stage 15 group into the corresponding elements at later stages.

The results showed that as the facial and cranial components increased in size, shape change was most pronounced in the posterior cranial and orofacial regions (see Figure 4). Cranial linear dimensions increased at a more rapid rate than those in the anterior and posterior cranial base regions (see Figure 9). These differential growth rates of craniofacial components were associated with changes in cranial base flexure. With the stalk of Rathke's pouch viewed as the central point, analysis of angular components showed that between stages 15 and 18, the posterior cranial angulation decreased by 21°, the orofacial angle increased by 22°, but the anterior and middle cranial angles remained unchanged (see Figure 10). Simultaneously with these changes in the posterior cranial region, the anterior and midbrain rotated up and back toward the hindbrain and the orofacial angle increased (see Figure 11). The results suggest that a direct relationship exists between growth changes in the posterior cranial and facial regions during primary palate formation. The morphometric studies and reconstructions showed changes in shape of the ventral portion of the brain above the face, elongation and ventral positioning of the facial prominences relative to the brain, and forward positioning of the face relative to the brain. Because the cranial flexion changes during this period and the forebrain lifts superiorly and posteriorly, this superior positioning of the brain appears to be a factor in withdrawal of the brain from between the facial tissues. These growth movements appear to have an important role in establishing early midfacial morphology before the chondrocranium develops later in the embryonic period. In addition, these normal growth patterns appear to be important in primary palate formation, first by facilitating contact between facial prominences and later by

moving components apart as the medial nasal region narrows. If a robust mesenchymal bridge has not formed in the primary palate, failure of palatal closure can be expected. C. Development of Internal Palate Components

Primary

Epithelial Fusion and Mesenchymal Ingrowth 1.

At about 37 d after fertilization in humans (stage 16), after the facial prominences are induced to form the nasal placodes, the lateral walls of the nasal placodes are formed caudo-occipitally by the maxillary prominences and craniofrontally by the lateral nasal prominences (Streeter, 1948; O'Rahilly, 1967; O'Rahilly and Muller, 1987). Their medial boundaries consist of the medial nasal prominences, which contact the maxillary prominences in the frontal portion of the nasal grooves, and the lateral nasal prominences in the caudal portion. As the prominences enlarge, the area of contact is called the epithelial plate or nasal fin (Streeter, 1948; Vermeij-Keers, 1972, 1990; Sperber, 1989) which establishes continuity between the nasal cavity and the roof of the mouth. This continuity between the nasal sac and the roof of the mouth is interrupted and replaced by mesenchymal tissues of the maxillary and nasal prominences during stage 17 (Streeter,

1948; Warbrick, 1960; Vermeij-Keers, 1972, 1990; Sperber, 1989; Diewert and Shiota, 1990; Diewert and Van der Meer, 1991).

In the mouse embryo, development of the primary palate starts at about 10 d and 18 h (Reed, 1933; Trasler, 1968). Fusion of lateral nasal and medial nasal prominences commences from the posterior (dorsal) portion of the mouth and proceeds ventrally. Trasler (1968) showed that at the "crescent" face stage, the epithelia of the medial and lateral nasal prominences begin to make contact in a posterior to anterior direction, forming a flat plate of double epithelium called the nasal fin. A "zipping up" process follows this fusion of epithelia giving the nasal opening a comma shape. The initial contact between the cells of the medial and lateral nasal swellings is made by short projections from one superficial cell to the 117


opposing superficial cell in mouse embryos (Gaare and Langman, 1977a). Trasler surface of an

and Ohannessian (1983) have also shown that cells approaching or in contact with opposing cells form cell projections, intercellular junctions, desmosomes, and microfilaments, demonstrating firm contact between the opposing epithelia. Millicovsky and Johnston (1981) have shown that in the mouse embryo, epithelial cells lose their surface microvilli before contact. After a brief period of quiescence, they begin to fill the groove separating the facial prominences by producing a series of surface projections that increase in size and complexity as the process of fusion progresses. However, observing external changes in the epithelial tissues has provided limited understanding of internal changes occurring simultaneously with these as the palate forms. A carbohydrate surface coat has been suggested as an essential factor in mediating adhesion between opposing palatal shelves of the secondary palate (Greene and Pratt, 1976). When these investigators inhibited surface coat production by means of diazo-oxo-norleucine (DON) in vitro, the palatal shelves failed to fuse. A cell surface coat is also found over the epithelial linings of the nasal prominences in the region of presumptive fusion using ruthenium red and radioactive precursors (Gaare and Langman, 1977a; Figueroa and Pratt, 1979). Using 3-H Concanavalin A, Burk et al. (1979) found the concentration of surface coat material on the epithelium of the presumed fusion area to be higher than other regions of the nasal folds. These findings support the hypothesis that the cell surface coat is associated with the ability of epithelial shelves or folds to adhere and fuse. In fact, complex carbohydrates have for some time been implicated in cell aggregation and intercellular adhesion in various in vitro cell systems (Pessac and

Defendi, 1972; Oppenheimer, 1973; Roseman, 1974; Greig and Jones, 1977).

At about 41 d after conception in humans (stage 17), a portion of the nasal fin is replaced by mesenchyme from the medial nasal, lateral nasal, and maxillary prominences (Figure 14). This mesenchyme connects the medial and lateral walls of the nasal groove and establishes the primordium of the palate (Streeter, 1948; Warbrick, 1960). There are two concepts about mechanisms

of formation of the primary palate. One of these is fusion between the facial prominences similar to that in the secondary palate in which the two covering epithelial layers of the palatal processes are brought into contact. An epithelial seam forms and shortly thereafter the epithelial seam begins to fragment as the cells either degenerate (Greene and Pratt, 1976) or transform into mesenchyme (Fitchett and Hay, 1989). In contrast to fusion, a series of events take place that were named "merging" by Patten (1961). In merging, mesenchymal growth and migration of the mensenchyme underlying the epithelium of the prominences eliminates the intervening epithelium. The epithelium is pushed out from between the elevations instead of being apposed and then disintegrating or transforming as in fusion. Tondury (1950) noticed in the anterior part of the epithelial plate degenerative processes expressed by the occurrence of pyknotic nuclei and nuclear fragments followed by destruction of the basement membrane. In contrast, Anderson and Matthiessen (1967) interpreted these nuclear fragments as peripherally sectioned mitotic figures. In addition, they did not detect histiocytes, which they postulate to be present wherever em-

bryonic epithelium disappears. Therefore, they were inclined to agree with Patten (1961) who explains the disappearance of the epithelial plate by a process called merging, in which the epithelium between two swellings is squeezed out by pressure exerted by the underlying mesenchyme on the epithelium of the groove. Vermeij-

Keers (1972) found that the basement membrane of the epithelial plate had disintegrated locally. Between the normal epithelial cells in this plate, nuclear fragments were found. The limited number of human embryos of the relevant stages available for these studies limited definite conclusions. In a recent detailed study of growth of the internal components of the human primary palate, embryos of stages 16 to 19 (37 to 48 d postfertilization), with CRL 8 to 20 mm, were examined (Figures 14 to 17, Diewert and Van der Meer, 1991; Diewert, 1992). The purpose of this study was to provide more definitive information about morphological development by measuring growth of the epithelial and mesenchymal components during normal palatal formation. Serial


S17M

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FIGURE 14. Histologic sections of Carnegie human embryos of stages (A) 16, (B) early 17, (C,D) mid-17, and (E,F) late 17. The nasal fin (NF) first formed between the medial nasal (MN) and maxillary (Mx) prominences at stage 16. The lateral nasal (LN) prominence formed part of the primary palate later. At stage 17, only one of the twelve embryos did not have mesenchyme through the NF. (C,D) Mid and (E,F) late stage 17 embryos had mesenchymal bridges and the MN region became elongated and narrower.

photographs of 37 serially sectioned embryos of stages 16 to 19 in the Carnegie Embryology Collection were enlarged, and the heights and depths of tissue components were measured. The areas were calculated and regression lines were generated to show growth relative to CRL. The results showed that during stage 16, an epithelial seam, the nasal fin, formed between the medial nasal and maxillary prominences (see Figure 14).

the nasal fin enlarged rapidly as the seam formed between the medial nasal prominence and the maxillary prominences and now also the lateral nasal prominence (see Figure 14). During stage 17, a mesenchymal bridge developed through the seam and the size of the mesenchymal component increased rapidly to occupy up to 50% of the total area (see Figures 14 and 17). During stages 18 and 19, the total pri-

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FIGURES 15 to 17. Growth of the primary palate was measured in frontal sections of 39 Carnegie embryos of stages 16 to 19. FIGURE 15. The total area increased exponentially relative to crown-rump length (CRL). FIGURE 16. At a similar CRL, the embryos of advanced stages had larger right and left areas (TRA,TLA) of the palate. FIGURE 17. Graphic plots of height and depth of epithelial (black) and mesenchymal (dotted) components illustrate the NF at (A,B) stage 16 and (C-E) development of a mesenchymal bridge during stage 17 with an increased proportion of mesenchyme (17 to 50%) and further enlargement of the primary palate and mesenchyme at stages 18 and 19.

mary palate area increased and the mesenchymal bridge enlarged to 65 to 85% of the total area. During the period studied, total primary palate area increased exponentially relative to CRL (see Figure 15). Similar CRL embryos of more advanced stages had larger palatal areas than earlier

staged embryos (see Figure 16).

Gaare and Langman (1977b) reported that in embryos, shortly before the epithelial linof ings the opposing nasal prominences make contact, cell degeneration characterized by condensation and fragmentation occurs in the epithelial linings of the prospective fusion areas. After fusion has established the nasal fin, epi-

mouse


thelial cells continue to degenerate in thp same manner. However, cell degeneration cannot account for complete regression of the nasal fin, because many morphologically healthy epithelial cells are always present. They suggested that these surviving epithelial cells incorporate into the adjacent epithelial linings of the expanding primary nasal and oral cavities. The interchange of tissue phenotype, especially epithelial to mesenchymal, is a common

phenomenon during early embryogenesis (Hay, 1968). It is possible that a readily triggered mechanism exists in most epithelia that turns on the mesenchymal genetic program. Greenburg and Hay (1982, 1986, 1988) demonstrated that a variety of adult and embryonic epithelia that normally do not give rise to mesenchyme do so when the isolated tissue is immersed inside hydrated type I collagen gels. Confronted with collagen

fibrils in close contact on all sides, these wellestablished epithelia express the potential for tissue-type conversion in response to an abnormal extracellular matrix environment. Fitchett and Hay (1989) showed that palatal medial edge epithelium is an ectoderm that retains the ability to transform into mesenchymal cells. They report that cell death is not the major mechanism leading to removal of the midline epithelial seam created by contact of the two palatal shelves. Rather, opposing basal cells adhere, after sloughing of the periderm, proliferate, and then transform into mesenchyme. The basal lamina disappears as basal cells extend filopodia and then pseudopodia into the adjacent connective tissue compartment. The glycogen-rich basal cells have euchromatic, vesicular nuclei and abundant rough endoplasmic reticulum. Before they begin to elongate and move into the extracellular matrix, they acquire a vimentin-rich cytoskeleton and lose keratin expression. Vimentin filaments are the characteristic intermediate filament type of mesenchymal cells. The changes in cell shape and cytoskeleton are similar to those reported in already established epithelial mesenchymal transformations (Hay, 1968: Bernanke and Markwald, 1979, 1982; Nichols, 1981; Franke etal., 1982).

Primary Choana Formation As the primary palate is developing, the medial nasal region is narrowing and elongating. At

2.

about 44 d after conception (stage 18), the width of nasal septum and medial nasal prominence decreases to 0.5 to 0.7 times that of stage 16 (Diewert et al., 1989; Diewert and Lozanoff, 1989, 1990, 1993b). At the same stage, the nasal fin dorsal to the zone of mesenchymal replacement persists and cavitates, forming the oronasal membrane that separates the nasal pit from the stomodeal cavity. Rupture of the oronasal membrane is brought about by disintegration of the cells that form it. This results in the opening of a respiratory passage from the nostril (posterior nares) through the primary choana to the pharynx

(Streeter, 1948; Warbrick, 1960).

In the mouse embryo, after disintegration of the nasal fin and penetration of mesenchyme, a portion of the nasal fin remains at the back of the nasal pit, forming the oronasal membrane with the formation of interstitial gaps occurring on the 11th day after conception (Tamarin, 1982; Trasler, 1968). The gaps enlarge and coalesce so that a completely patent opening between nasal passage and stomodeum is established by 13 d. The membrane consists of two layers of simple squamous epithelium, which become separated as involution progresses. The form of the choanal antrum changes from a simple funnel-shaped ellipse early in the 13th day to a complex slitlike opening within the following 24 h. This coincides with the completion of a definitive primary palate and enlargement and elevation of the secondary palatal shelves.

III. ABNORMAL DEVELOPMENT OF THE PRIMARY PALATE AND CLEFT LIP MALFORMATION A. Morphogenesis of Cleft Lip in the Human Embryo Cleft lip is a result of failure of fusion between the medial nasal prominence and the max-


illary prominence or lateral nasal prominence or both. One possible cause of this defect is that the ventral ends of the prominences fail to come into actual contact with each other for fusion to occur. This can occur if growth is defective in either or both prominences. Tondury (1964) describes an embryo with unilateral complete cleft lip where the primary cause for the faulty development appears to be defective growth of the lateral nasal prominences. Another explanation is that the nasal fin persists throughout the developmental stages preventing the mesenchyme of the maxillary and frontonasal prominences from making contact. Subsequently, when the dorsal part of the nasal fin undergoes the normal cavitation and cleavage, resulting in the formation of the primary choana, the ventral part of the nasal fin also undergoes cleavage resulting in the formation of cleft lip (Stark, 1954; Warbrick, 1960). Anderson and Matthiessen (1967) suggested that complete cleft lip will also appear if mesenchymal proliferation is retarded in the medial nasal and maxillary prominences and incomplete cleft lip will appear in cases with a less marked retardation of the mesenchymal proliferation in the previously mentioned mesenchymal centers. Furthermore, by investigating human embryos with primary palatal clefting in the Kyoto Collection, Diewert and Shiota (1990) showed deficient mesenchymal bridge growth and a visible deficiency of tissue in the cleft areas. The results also showed regional growth deficiency or developmental abnormality in the palatal tissues during the critical time of rapid mesenchymal bridge enlargement in cases of partial or incom-

plete clefting.

B. Morphometric Human Cleft Lip

Study of Postnatal

Fraser and Pashayan (1970) have shown that parents of children with cleft lip tend to differ from the general population in certain dimensions of facial topography. There was a significant tendency for the anterior surface of the maxilla to be flatter in the defective group compared with the normal. In addition, the mean dizygomatic and intraocular chin measurements were larger in the defective group, the frequency of rectan-

gular and trapezoid shapes was higher, and the upper lip was less protuberant relative to the lower lip than normal. Coccaro et al. (1972) have also shown that parents who lack facial deformities but have children with cleft lip and palate, exhibit faces that are less convex, with a tendency toward mandibular prognathism. Vertical and horizontal measurements of the upper face and the nose length were found to be shorter for parents of children with cleft lip and palate. In monozygotic twins, CL(P) occurs in only one of the twins in 56.5% of cases (Ross and Johnston, 1972). A study of facial morphology in radiographs of monozygotic twins discordant for CL(P) showed that approximately two thirds of the unaffected twins of cleft lip cases had narrower nasal cavity widths than a control population (Johnston and Hunter, 1989). In the remaining one third of twins of cleft lip cases, the derivatives of the maxillary prominences appeared underdeveloped compared with controls. Thus, Johnston and Hunter (1989) proposed the existence of two major CL(P) groups: one with small medial nasal prominences that result in narrower nasal cavities and the other with small

maxillary prominences. C. Morphogenesis of the in the Mouse Model

Primary Palate

Reed (1933) proposed that harelip (cleft lip) in animals was primarily due to failure of fusion between the lateral nasal prominence and the medial nasal prominence that was probably caused by a retarded growth rate of the maxillary prominence. Trasler (1968) has shown that formation of the normal lip requires the posterior portions of the medial and lateral nasal prominences to remain continuous with each other and with the medial portion of the maxillary prominence. In an embryo genetically predisposed to clefting, the medial nasal prominences do not diverge laterally as much as they do in an embryo that is not predisposed. This results in a decrease or failure of epithelial fusion between the medial and lateral nasal prominences and, consequently, a lack of consolidation of the isthmus then occurs. The hypothesis that face shape is a causal factor in genetic predisposition to cleft lip in mice


was further tested by Juriloff and Trasler (1976). Their results from measuring photographs of embryos support this hypothesis. However, it is suggested that the susceptibility to another type of cleft lip in other genotypes could arise, for example, through hypoplasia of one of the facial prominences. Trasler and Machado (1979) found that a particular facial complex is associated with cleft lip predisposition. Premaxillary length is significantly shorter in newborns and adults in mouse cleft lip lines CL/Fr, A/J, and L than in noncleft lines M and C57BL/6J. Premaxilla width also tended to be narrower in the adults, and gum length in newborns and adults tended to be shorter in cleft lip lines. Developmental alterations associated with spontaneous cleft lip and palate in CL/Fr mice (Millicovsky et al., 1982; Forbes et al., 1989) include: altered facial geometry, in which the orientation of the medial nasal prominences is almost parallel to the midsagittal plane; depressed ability of the surface epithelium of primary palate primordia to participate in the fusion process; and hypoplasia of the lateral nasal prominences. Ohbayashi and Eto (1986) suggested that the medial nasal prominences play a critical role in normal facial development and cleft lip formation based on in vitro experiments done using rat embryos with microsurgically removed portions of facial prominences. They found that cleft lip-like malformations were observed only in the group with excised medial nasal prominences. Either the maxillary or the lateral nasal prominences were able to form the lateral component to contact with the medial nasal prominence. In our laboratory, we are studying development of internal palatal structures analyzed morphometrically in different strains of mice. In one study, we compared growth of internal structures in the primary palate of CL/Fr mice with a frequency of 20 to 30% cleft lip and C57 mice with 0% cleft lip (Wang and Diewert, 1990). In mice, body somite or tail somite number has been found to reflect body development more accurately than chronologic age or body weight (Trasler, 1968; Wang, 1992). Therefore, somite numbers are used to compare embryos of different strains. Heads of 41 CL/Fr and 31 C57 embryos with 9 to 20 tail somites were serially sectioned for study (Wang and Diewert, 1990). Three stages of pri-

were identified: formation of the nasal fin at 9 to 12 tail somites; formation of the mesenchymal bridge formed by 14 tail somites in C57 and at 16 tail somites in CL/Fr embryos (Figure 18); and enlargement of the primary palate mesenchymal bridge. Measurements were made on serial sections to determine the heights and depths of the nasal fin epithelial seam and mesenchymal and epithelial components of the primary palate (see Figure 18). At 14 tail somites, all the C57 palates had large areas with mesenchymal bridges present, whereas the CL/Fr had smaller palatal areas without mesenchyme (see Figure 18). At 16 tail somites, approximately half of the CL/Fr embryos had mesenchyme in their palates. The depths of the primary palate, the mesenchymal bridge, and the maxillary prominence were analyzed to determine rates of growth relative to body development (Figures 18 to 21). Regression line analysis of growth changes showed that the depths of the maxillary prominence and primary palate increased at similar rates in the two strains but were retarded relative to tail somites in the cleft lip strain (see Figures 19 and 20). The mean depth of the mesenchymal bridge was also significantly smaller in CL/Fr embryos (see Figures 19 and 20). The depth of the primary palate, however, was found to be proportional to the depth of the maxillary prominence in both strains (see Figure 21), which suggests that growth of the maxilla contributes directly to the increase in depth of the primary palate. The size of the mesenchymal bridge, however, was smaller in the cleft lip strain at the same depth of maxilla or primary palate or both (see Figure 21), which suggests that the cleft lip strain has an additional delay in mesenchymal replacement of the nasal fin that is not directly associated with maxillary growth. Later in development, CL/Fr embryos with successful primary palate closure had larger mesenchymal bridge depths, but most remained smaller than in C57 embryos at similar somite numbers. These results suggest that retarded facial growth and delayed ingrowth of mesenchymal tissue into the primary palate during forward growth of the maxillary prominences may be the morphological basis for strain differences in primary palate closure (Figure 22). A threshold model for timing of mesenchymal bridge formation for the two strains

mary palate development


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shows that delayed mesenchymal bridge formation places the CL/Fr strain very near the threshold (see Figure 22). In our laboratory, craniofacial growth was also studied in two strains of mice with different clefting frequencies: A/WySn with 28% cleft lip and C57BL/6J without cleft lip (Wang and Diewert, 1992). The A/WySn strain has a clefting frequency similar to that of CL/Fr (Juriloff, 1982). Both strains have the same cleft lip gene on different genetic backgrounds. Standardized photographs of 27 A/WySn and 25 C57BL/6J embryos with 8 to 22 tail somites (34 to 46 somites)

taken in the superior, frontal, and lateral views. Landmarks were located and digitized for computerized analysis of growth change relative to somite number and at stages of face development before, during, and after primary palate closure. The results showed that both strains had similar overall growth patterns with increases in head width and face width and decreases in nasal pit width. During early palatal closure in C57BL/ 6J mice, the nasal pit width was unchanged as brain width increased rapidly; then later, the nasal pit width decreased as brain width increased slowly. However, during early closure in A/WySn

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FIGURES 19 to 21. Regression lines of measurements of depth of the primary palate (PP), the mesenchymal bridge (MB), and the maxillary prominence (MxP) in the C57 and CL/Fr strains. FIGURE 19. Depths of the PP and MB increased at the same rates in both strains but the CL strain was smaller in size. FIGURE 20. The depth of the MxP increased at a similar rate but was smaller in the CL strain. FIGURE 21. In both strains, the depths of the PP and the MxP were proportional, however, the MB was smaller in the CL strain.

mice, the nasal pit width decreased rapidly as brain width increased slowly; then later, the nasal pit width was unchanged as brain width increased more rapidly. During early palatal closure, the narrower nasal pit width in A/WySn mice appeared to result from delayed growth of the supporting forebrain as the nasal pits became more

with normal face development. Previous work by Juriloff and Trasler (1976) showed that at the initial stage of lip formation, the crescent stage, embryos of a cleft lip line had a smaller distance between the nasal pits than mice of noncleft strains. Our study extended the stage studied to late comma stage and ex-

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plained the narrower nasal pit width by the delayed growth of the forebrain shown in the superior, frontal, and lateral view. In the lateral view, increases in maxillary prominence depth of A/WySn embryos were

smaller than C57BL/6J at 38 to 39 somites (crescent stage) (Wang and Diewert, 1992). Formation of the normal primary palate requires that the posterior ends of the medial and lateral nasal prominences remain continuous with each other and with the medial end of the maxillary prominence (Trasler, 1968). Reed (1933) summarized evidence that cleft lip is primarily due to the failure of the lateral and medial nasal prominences to fuse. This failure, he hypothesized, is probably due to a retarded growth rate of the maxillary prominences, which fail to exert needed lateral pressure at the critical time. Our results showing deficient maxillary prominence depth in A/WySn support this hypothesis. The angular measurements in the lateral view of A/WySn and C57BL/6J had similar growth patterns. The posterior cranial region rotated upward and backward, allowing the facial prominences to grow downward and forward. These results are comparable to the study of human sagittal craniofacial growth (Diewert et al., 1989; Diewert and Lazanoff, 1993a) in which the posterior cranial angle decreases with the oropharyngeal angle increasing during the human primary palate for-

mation. In this study, changes in posterior cranial base flexion did not differ in noncleft and cleft lip strains (Wang and Diewart, 1992). The factors contributing to differences in primary palate closure caused by face growth were observed: earlier narrowing of nasal pit width and deficient maxillary prominence growth at the corresponding developmental stages (Wang and Diewert, 1992). Further studies of craniofacial growth in cleft lip and noncleft lip mouse genotypes are in progress to determine the relative role of maxillary prominence growth and nasal pit position in normal and abnormal primary palate formation.

IV. SUMMARY Our recent studies of primary palate formation have focused on defining phases of primary palate development and developing methods to analyze and quantify relevant changes within the

primary palate and in the craniofacial complex. The results provide new approaches to studying primary palate formation that have enhanced the understanding of palatogenesis and craniofacial growth, how the two are related, and created new directions for experimental model testing. Studies of human craniofacial growth (Carnegie Collection) provided the first quantitative analyses of human facial growth and primary pal-


ate formation. Major growth changes shown were narrowing and elongation of the fronto-nasal region, forward growth of the maxillary prominences, straightening of the cranial base, and withdrawal of the brain from between facial structures. Analysis of internal palate growth in the same Carnegie embryos showed that primary palate area increased exponentially relative to CRL. At stage 16, the nasal fin formed; at stage 17, the nasal fin enlarged and mesenchyme replaced the nasal fin and increased up to 50% of the total area; at stages 18 and 19, the total area increased and the mesenchymal bridge occupied up to 80% of the primary palate. In human embryos with partial clefts in the Kyoto Collection, reduced mesenchymal growth in the areas of the epithelial seam was observed. Measurements showed deficient mesenchyme and excessive epithelial tissue in the primary palate. As maxillary mesenchyme grew into the primary palate, greater cell dispersion and ingrowth of the neurovascular bundle were observed. In mice, narrower nasal pit width is a recognized feature of face shape that contributes to cleft lip. Our study of craniofacial growth in a noncleft and a cleft lip strain showed that pit width decreased with face development. In the cleft lip strain, a lag in frontal brain growth appeared to contribute to more convergent medial nasal prominences. All strains had similar patterns but different timing of primary palate formation. As the maxilla grew forward, the rapid increases in nasal fin size were highly correlated with maxillary depth. At similar tail somite numbers (reflecting developmental status), cleft lip strains had smaller nasal fin sizes and maxillary prominence depths than normal strains. The time of mesenchyme replacement of the nasal fin was also later in cleft lip strains. Therefore, regional delays of primary palate development relative to head and body development appear central to

clefting. These studies confirmed that human and mouse embryos have similar phases of primary palate formation and craniofacial growth and have led to the proposed multifactorial developmental threshold model for further testing of morphological mechanisms of clefting. In both human and mouse embryos, a small area of fusion and mesenchymal replacement begins primary palate

formation. Thereafter, merging of the facial prominences appears to be the mechanism by which the mesenchymal bridge enlarges. The molecular mechanisms by which these are controlled remain to be identified.

ACKNOWLEDGMENTS This work was supported by Medical Research Council of Canada Operating Grant MT4543 to Virginia M. Diewert. The authors thank Dr. Ronan O'Rahilly, former Director of the Carnegie Laboratories of Embryology, California Primate Research Center (CPRC), for availability of the embryonic collection; Dr. Andrew Hendrickx, Director of the CPRC for providing facilities and equipment; Mrs. Barbara Tait for providing technical assistance; and Ms. Emily Robertson for statistical assistance.

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