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Apoptotic epithelial cell death: a prerequisite for palatal fusion. An in vivo study in rabbits
Journal of Cranio-Maxillofacial Surgery (2002) 30, 329–336 r 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd. All rights reserved doi:10.1054/jcms.2002.0323, available online at http://www.idealibrary.com on
Apoptotic epithelial cell death: a prerequisite for palatal fusion. An in vivo study in rabbits Eva-Andrea Holtgrave,1 Gisela Stoltenburg-Didinger2 1
Department of Orthodontics and Paediatric Dentistry (Head: Prof. Dr. E.A. Holtgrave), School of Dental Medicine; 2Department of Neuropathology (Head: N.N.), Benjamin Franklin University Hospital, Freie Universita¨t, Berlin, Germany SUMMARY. Objective: The aim of the present study was to investigate stage-specific changes in medial edge epithelium during in vivo fusion of embryonic palatal shelves in 25 Russian rabbits. Material and Methods: The embryos were dissected following Caesarian section at day 18. Palatal shelves of specific developmental steps (approximation, contact, fusion) were examined by light microscopy, immunohistochemistry and transmission electron microscopy. Results: Light microscopy revealed that the superfical peridermal cells underwent apoptosis prior to contact of the basal epithelial cells. Following contact, an epithelial monolayer was left on each shelf with an intact basement membrane. Apoptosis of the epithelial cells was followed by discontinuity of the basement membrane. Islands of epithelial cells remained. Conclusion: This paper presents new data on palatal development in vivo. The results support our theory that apoptotic medial edge epithelial cell death is a precondition for palatal fusion. There were no indications of epithelial mesenchymal transformation or migration. r 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd. All rights reserved.
formation of both the primary and secondary palate (Ferguson, 1988). It has been documented in rats that the basic event leading to disappearance of epithelial cells is their morphological change (Fitchett and Hay, 1989; Shuler et al., 1992; Hay, 1995; Shuler, 1995). Ultrastructural studies have found that epithelial cells change their phenotype while forming pseudopodia with subsequent transition into mesenchymal cells (epitheliomesenchymal transformation) penetrating the basement membrane. In contrast, Mori et al. (1994), using in situ staining for nuclear DNA fragments (TUNEL method), observed that the midline edge epithelial cells disappear following DNA fragmentation or apoptosis in mice. Nevertheless, these authors do not reject the epithelial–mesenchymal transformation hypothesis. Another hypothesis was developed by Ferguson (1988) as well as Sharpe and Ferguson (1988) indicating that a substantial number of epithelial cells migrate into the palatal mesenchyme carrying with them fragments of the disrupted basement membranes. Ferguson (1988) found that following this, epithelial cells are no longer distinguishable in the mesenchyme of the palatal shelves. In view of these different hypotheses, we investigated the fusion process of the palatal shelves in rabbits with special reference to epithelial cell disappearance and to the basement membrane in the midline epithelial seam.
INTRODUCTION Normal development is characterized by an equilibrium between proliferation, differentiation and cell death. Cell death occurring in organogenesis is often described as programmed cell death or apoptosis. Morphologically, apoptotic cells exhibit nuclear chromatin condensation and fragmentation as well as ‘‘blistering’’ of cell surfaces and subsequent degradation of apoptotic bodies which are phagocytosed by healthy neighbouring cells (Glu¨cksmann, 1951; Lockshin, 1997; Zakeri, 1998). Apoptosis is also described in palatal development (Taniguchi et al., 1995), but up to now seems to many authors to only be an unimportant side effect (Fitchett and Hay, 1989; Shuler et al., 1992; Hay, 1995; Shuler, 1995). The formation of the palate in man and other mammals is achieved by a coordinated sequence of events beginning with formation, growth and reorientation of the palatal shelves followed by their approximation and adhesion of their superficial epithelial cell layers. These developmental processes end in mesenchymal substitution of the epithelial cells. Prior to fusion, both palatal shelves are covered by a two-layered epithelium, an outer epithelial cell layer, called periderm, and a basal epithelial cell layer. Before contact of the shelves, the periderm is lost. During adhesion, only two epithelial cell layers come together to form the midline epithelial seam, also called the midline edge epithelium. Disappearance of this structure is the most critical step in the 329
330 Journal of Cranio-Maxillofacial Surgery
MATERIAL AND METHODS Animals Female Russian rabbits, 6 months old, were mated overnight. Embryos were assumed to be at day 0 of gestation on the following morning. Litters were collected at day 18 by Caesarean section. The foetuses (n=20) were immediately fixed in 3% buffered formalin solution. After 3 days, the heads were cut in frontal sections and embedded in paraffin. Light microscopy Paraffin sections of 5 mm were stained with haematoxylin and eosin (HE), cresyl violet (Nissl) and periodic acid–Schiff (PAS). For immunohistochemistry, the paraffin sections were mounted on pretreated glass slides and incubated overnight with primary antibodies against vimentin and Ki 67 (=MIB 1, DAKO, Hamburg, Germany). Ki 67 is an antigen which appears in the nucleus when the cell leaves the resting phase G0. In histopathology and developmental biology, KI 67 antigenicity is used as a marker of proliferation. The antigen was visualized by the APAAP technique using neofuchsin as a chromogen. For the detection of apoptosis an in situ labelling technique (BOEHRINGER-Kit, Boehringer, Mannheim, Germany) was used with diaminobenzidine (DAB) as a chromogen and counterstaining with light green (Merck, Mannheim, Germany).
Fig. 1 – Coronal section of a rabbit midpalatal region. Palatal shelves prior to fusion (embryonic day 18, HE, 10).
Electron microscopy The palatal portions (n=5) were excised from the tissue under a binocular microscope (Zeiss, Oberkochen, Germany). Representative areas of the palate were immersion fixed in glutaraldehyde (pH 7.2), buffered with cacodylate–HCl (pH 7.4, containing 4% sucrose), postfixed with 2% osmium tetroxide, dehydrated with a graded series of ethanol, and embedded in araldite. Sections were cut with an ultramicrotome and examined under a ZEISS electron microscope EM 10 after staining with uranyl acetate and lead citrate. RESULTS Macroscopic observations The primary palate seemed to be fused in all 25 animals. Microscopic observations The palatal shelves still separate Light microscopy revealed the fusion process to be at different developmental stages. The medial parts of
Fig. 2 – Palatal shelves still separate. Cell division in the medial edge epithelium has ceased. All nuclei stained in red present the antigen KI 67 which is present in the nucleus between phase G1 to mitosis, i.e. they are proliferating (embryonic day 18, MIB 1, 20).
both palatal shelves were covered by a two-layered epithelium underlined by a continuous basement membrane (Figs 1–4). The basal epithelium cell layer consisted of a seam of cylindrical cells with oval nuclei. The outer epithelial layer also called the periderm, consisted of flat cells with vacuolated cytoplasm. At this developmental stage, numerous peridermal cells exhibited signs of apoptosis (Figs. 3
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Immunhistochemistry, using MIB 1 reaction to find the proliferating cells, revealed complete cessation of proliferation in the nasal periderm while in the underlying epithelium a 30% proliferation rate was noted (Fig. 2). While proliferation persisted in the two-layered epithelium seam opposing the palatal shelves on the top, the epithelial cells in the midline seam exhibited an extreme reduction of the proliferation rate in the basal epithelial cell layer. This was noted by the MIB 1 reaction findings (Fig. 2). The palatal shelves meet
Fig. 3 – Two cell layers of the medial edge epithelium prior to fusion. The outer layer, ‘periderm’ with signs of single cell apoptosis (embryonic day 18, HE, 40).
When the palatal shelves met, the epithelia of both shelves consisted of only one cell layer each, plus the intact basal membranes. The most prominent feature of the nuclei was apoptosis of this basal epithelium revealed by TUNEL staining (Fig. 5A). This process also took place between the ‘‘kissing’’ epithelia of the nasal septum and nasal surfaces of the palatal shelves (Fig. 5B). In transmission electron microscopy (TEM), the continuity of the underlining basement membrane could be confirmed, together with the apoptotic process. Cell division in the attached epithelia had ceased. The palatal shelves fuse The continuous epithelial layer was dispersed into epidermal islands (Fig. 6). Mesenchymal cells penetrated and proliferated between these islands. The mitotic activity was restricted to the mesenchymal cells (Fig. 7). The basement membrane surrounded the epithelial clusters and was only preserved there (Fig. 6B). On the ultrastructural level, it became clear that the basement membrane adjacent to the apoptotic cells was disrupted and missing. In the anterior region, where fusion with the nasal septum took place simultaneously, the same disruption process of the basement membrane was observed in combination with the vanishing epithelial cells (Figs. 8–11). Transformation of resting epithelial islands into mesenchymal cells could not be verified. The apoptotic cells did not leave any traces and were replaced by mesenchymal cells.
Fig. 4 – Epithelial cell layer of the palatal process facing the nasal septum covered by PAS positive mucous material. Single cell apoptosis in the upper cell layer, see Fig. 1 (embryonic day 18, PAS, 100).
and 4). The apoptosis is characterized by chromatin condensation, and later by fragmentation into apoptotic bodies. These apoptotic cells were sloughed away while the remaining cells stayed in a form of symplasma (Figs. 3 and 4). The epithelial layer facing the nasal septum exhibited identical features although the spatial distance between both shelves was up to 5 times greater than in the midline (Figs. 1 and 4).
DISCUSSION Our main finding of the morphological analysis of palatal fusion in rabbits was the presence of apoptotic cells in the midline edge. The disappearance of the midline edge epithelium is believed to be the most critical step in the formation of the primary and secondary palates. There are three different hypotheses as to how this takes place: By epithelial–mesenchymal transformation, the epithelial cells change their phenotype whilst forming pseudopodia, with subsequent transformation to mesenchymal cells (Fitchett and Hay, 1989; Shuler
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Fig. 5 – (A) Epithelium in contact. TUNEL staining reveals abundant apoptotic cell death in nasal and medial fusion areas (embryonic day 18, 10). (B) Detail of (A). Fusion area. Apoptosis of epithelial cells (arrow), basement membrane intact (embryonic day 18, TUNEL staining, 100).
Fig. 6 – (A) Coronal section of midpalatal region. Palate fused (embryonic day 18, HE, 10). (B) Epithelial islands surrounded by basement membrane, detail of (A) (embryonic day 18, HE, 100).
et al., 1992; Mori et al., 1994; Hay, 1995; Shuler, 1995; Sun et al., 1998; Soeno et al., 1999; MartinezAlvarez et al., 2000). By migration a substantial number of epithelial cells migrate orally and usually into the epithelial triangle (Ferguson, 1988; Sharpe and Ferguson, 1988; Carette and Ferguson, 1992; Soeno et al., 1999; Martinez-Alvares et al., 2000). Only a small number of epithelial cells appear to merge into the palatal mesenchyme and are no longer distinguishable from mesenchymal cells of the palatal shelves.
By apoptosis the midline edge epithelial (MEE) cells disappear by DNA fragmentation. The authors who promote this theory do not reject the epitheliomesenchymal transformation theory (Mori et al., 1994; Taniguchi et al., 1995). As to epithelial–mesenchymal transformation: All the experiments showing this kind of transformation were performed in vitro. The palatal shelves were excised and grown as organ cultures on collagen matrices. This procedure has been performed by all authors claiming that transformation is an important
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Fig. 7 – (A) Coronal section of midpalatal area. Penetrating mesenchymal cells proliferate between the islands of non-dividing epithelial cells (embryonic day 18, MIB 1, 10). (B) Detail of (A) midline region. One of the mesenchymal cells undergoes mitosis (arrow head). The KI 67 antigen is visible in the entire cytoplasm as usual during mitosis (embryonic day 18, MIB 1, 100).
Fig. 8 – Epithelial cell layer with two apoptotic bodies adjacent to the nucleus of an epithelial cell (embryonic day 18, TEM, 4400).
Fig. 9 – Two apoptotic bodies phagocytosed by an epithelial cell (embryonic day 18, TEM, 4400).
step in palatal fusion (Fitchett and Hay, 1989; Mori et al., 1994; Hay, 1995; Shuler, 1995; Sun et al., 1998; Soeno et al., 1999; Martinez-Alvarez et al., 2000). The development of pseudopodia is behaviour typical of cultured cells, and is not comparable to in vivo conditions. The cell behaviour and differentiation in vitro is different from in vivo. The shape of the cells and the development of the cytoskeleton most closely resemble more immature stages under in vitro conditions. The differentiation of epithelial cells into mesenchymal cells does not occur in normal devel-
opment. The argument of Greenburg and Hay (1986) that, identical to the MEE transformation, the epithelial cells of lens epithelium lose polarity and are transformed with mesenchymal cells is not true for in vivo situations, since the observation has also been made in culture. Transformation into a more undifferentiated cell type is restricted to tumour development (Gruenert, 1987). According to Fitchett and Hay (1989) the periderm cells are the only ones that die, whilst basal cells remain healthy and even divide before transforma-
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2. The dead cell elicits a response in neighbouring epithelial cells that may tentatively be characterized as cannibalistic; this response is visualized as a change in cell shape resulting in a partial or complete engulfment of dead cells. 3. When the dead cell is engulfed, it is destroyed by lytic enzymes within the vacuole; its organelles lose their structural integrity and are replaced by amorphous material. Farbman (1968) clearly describes the features of apoptotic cell death although this term was only coined in 1972 by Kerr et al. (1972).
Fig. 10 – Several apoptotic bodies adjacent to the nucleus of a freefloating cell (embryonic day 18, TEM, 3000).
Fig. 11 – Intracytoplasmic apoptotic body in the fusion area (embryonic day 18, TEM, 12 000).
tion into mesenchymal cells. Our observation indicates that periderm cells disappeared completely prior to contact of the palatal shelves. MIB 1 staining revealed that the basal cell layer stops mitotic activity. Transformation to mesenschymal cells is unlikely when the epithelial cells stop dividing. In contrast, our morphological findings revealed apoptotic cell death of the epithelial seam. Thus, we can support the findings of Farbman (1968), who described the sequence of events as follows: 1. Certain epithelial cells in the seam die for unknown reasons.
As to migration: Migration of epithelial cells into the palatal mesenchyma is said to occur either before (Fitchett and Hay, 1989) or after transformation into mesenchymal cells (Sharpe and Ferguson, 1988). Migration includes two different mechanisms: First, the epithelial cells of the midline migrate to the oral and nasal side and thus contribute to the thinning of palatal epithelium. In our experiment, the thinning of the epithelium in the midline is a consequence of apoptotic cell death of peridermal cells prior to fusion (they slough off) and to apoptotic cell death of the medial edge epithelium after contact. When apoptotic cell death includes the suprabasal epithelial cells, they are no longer able to support the basement membrane, which consequently breaks down and allows mesenchymal cells to cross from one shelf to the other. In our study, the cluster of epithelial cells with a surrounding basement membrane make up some of the persisting epithelia. Second, Sharpe and Ferguson (1988) reported migration of epithelial cells into the mesenchyma together with attached basement molecules. We have never observed this process. We found the thinning of the midline epithelial seam to be due to apoptotic cell death and not due to migration. Migration of epithelial cells into the mesenchyme was said to be followed by transformation into mesenchymal cells (Ferguson, 1988; Mori et al., 1994). Transformation was only observed in organ cultures suggesting that epithelio-mesenchymal transformation may be a side effect of the in vitro system (Fitchett and Hay, 1989; Griffit and Hay, 1992; Shuler et al., 1992; Shuler, 1995). As to apoptosis: In view of the different theories mentioned and derived from animal observations in mice, we elucidated the role of apoptosis in a rabbit model with particular reference to the disappearance of the epithelial cells during normal development. This study has confirmed that the basis of palatal fusion was apoptosis of the superficial and later of the basal midline epithelium. This has already been reported by Farbman in 1968 who observed widespread evidence of cell death in the midline epithelial seam immediately following palatal fusion. Dead epithelial cells were phagocytosed by neighbouring epithelial cells. In the electron micrographs of Farbman (1968), the ultrastructural morphology of apoptosis and apoptotic bodies is similar to that in our figures. Similar ultrastructure features were
Apoptotic epithelial cell death
described by Shapiro and Sweney (1969) and Smiley (1970). The process of palate fusion was also examined by Mori et al. (1994) in 13- and 14-day-old mouse foetuses. Taniguchi et al. (1995) found the same in rats using in situ staining for nuclear DNA-fragmentation (TUNEL method) and immunofluoresce staining for keratin, paying special attention to disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palatal fusion, but not in the palatal shelves prior to contact, nor in the intact midline epithelial seam. These results suggest that MEE cells undergo apoptosis during palatal fusion (Taniguchi et al., 1995). In our own animals TUNEL-positive staining was only present in epithelial cells, especially at the fusion site, where the midline edges of the palatal shelves met and where the nasal septum met the upper surfaces of the palatal shelves. The earliest study proposing programmed cell death for MEE was that of Shapiro and Sweney in 1969. In 1976, a review article (Greene and Pratt, 1976) stated that, ‘Fusion of the opposing shelves, both in vivo and in vitro, is dependent upon adhesion and cell death of the MEE cells’. By the mid-1970s, the hypothesis on programmed cell death for MEE was generally accepted (Farbman, 1968; Hayward, 1969; Mato et al., 1975; Pratt and Hassel, 1975). Prior to apoptotic cell death, the MEE stops proliferating. The arrest of mitotic activity is specially related to the medial edge epithelium. It is well known that in mice and rats, the intrauterine development is very short, and this is also true for the process of formation and involution of the epithelial sheet of the secondary palate. Since apoptosis is a transitory event we have therefore chosen rabbits as a species with a longer duration of palatogenesis in order to catch the different steps of apoptotic cell disappearance. The results of a gene trap study with mice lacking the human homology of CED-4 provide the most convincing evidence for the crucial role of apoptosis in secondary palate fusion. The phenotype had palatal clefts (Cecconi et al., 1998). The basic mechanism of apoptosis found to be present in the nematode Caenorhabditis elegans led to the isolation of genes that are specifically required for inducing programmed cell death. By these means, apoptotic cell death was amenable to molecular analysis and led to the identification of at least 14 genes implicated in developmental cell death (Villa et al., 1997). Three of these CED genes are of special importance: the proapoptotic death-promoting genes CED-3 and CED-4, and the anti-apoptotic gene CED-9. The cloning of the c-DNA for these genes led to the realization that all three had human homologues. The CED-4 gene encodes a 63-kD protein with no significant similarity to other known peptides (Yuan and Horowitz, 1992). CED-4 participates in the caspase 9-dependent activation of caspase 3 in the general apoptotic pathway. The human CED-4 homologue has been
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isolated and termed APAF 1 (apoptotic proteases activating factor 1; Zou et al., 1997). In situ immunodetection found that the absence of APAF-1 protein prevents activation of caspase 3 in vivo. It remains to be determined to what extent the protein is involved in developing programmed cell death. To address this question, Gruss (1997) and Cecconi et al. (1998) have taken advantage of a gene trap study (Chowdhury et al., 1997) that has provided the means to isolate a null allele of the murine APAF1 gene. Foetuses homozygous for APAF-1 mutation have a characteristic craniofacial phenotype whose major traits are midline facial cleft and cleft palate. Apoptosis has been postulated as playing a key role in palatal fusion by eliminating the medial edge epithelium of the secondary palatal shelves after they have contacted in the midline (Ferguson, 1988). Accordingly, late and imperfect palatal fusion with persistence of midline epithelium is a key component of the APAF-1 mutant phenotype. The authors came to the conclusion that apoptosis is essential in midline fusion of craniofacial structures (Cecconi et al., 1998). CONCLUSION The importance of apoptosis in palatal development has been confirmed in this study by conventional histology, immunohistochemistry and TEM microscopy. The results underline the importance of morphological studies and re-evaluate the primary conclusions drawn by some authors as early as the late 1960s and 1970s (Farbman, 1968; Shapiro and Sweney, 1969; Smiley, 1970) who have postulated that apoptosis is a major event in palatal development.
References Carette MJM, Ferguson MWJ: The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy. Development 114: 379–388, 1992 Cecconi F, Alvarez-Bolando G, Meyer BI, Roth KA, Gruss P: Apaf1 (CED-4 homologue) regulates programmed cell death in mammalian development. Cell 94: 727–737, 1998 Chowdhury K, Bonaldo P, Torres M, Stoykova A, Gruss P: Evidence for the stochastic integration of gene trap vectors into the mouse germline. Nucl Acids Res 25: 1531–1536, 1997 Farbman AI: Electron microscopic study of palate fusion in mouse embryos. Dev Biol 18: 93–116, 1968 Ferguson MJW: Palate development. Development 103 (Suppl): 41–60, 1988 Fitchett JE, Hay ED: Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev Biol 119: 455–478, 1989 Glu¨cksmann A: Cell death in normal vertebral ontogeny. Biol Rev 26: 59–86, 1951 Greenburg G, Hay ED: Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev Biol 115: 363–379, 1986 Greene RM, Pratt RM: Developmental aspects of secondary palate formation. J Embryol Exp Morphol 36: 225–245, 1976 Griffit CM, Hay ED: Epithelial–mesenchymal transformation during palate fusion: carboxyfluorescein traces cells at light
336 Journal of Cranio-Maxillofacial Surgery and electron microscopic levels. Development 116: 1087–1099, 1992 Gruenert DC: Differentiated properties of human epithelial cells transformed in vitro. Bio Techniques 5: 740–745, 1987 Gruss P: Evidence for stochastic integration of gene trap vectors into the mouse germline. Nucl Acids Res 25: 1531–1536, 1997 Hay ED: An overview of epithelio-mesenchymal transformation. Acta Anat 154: 8–20, 1995 Hayward AF: Ultrastructural changes in the epithelium during fusion of the palatal processes in rats. Arch Oral Biol 14: 661–678, 1969 Kerr JFR, Wyllie AH, Currie AR: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972 Lockshin RA: The early modern period of cell death. Cell Death Diff 4: 347–351, 1997 Martinez-Alvarez C, Tudela C, Perez-Miguelsanz J, O0 Kane S, Puerta J, Ferguson MW: Medial edge epithelial cell fate during fusion. Dev Biol 15: 343–357, 2000 Mato M, Uchiyama Y, Aikawa E, Smiley GR: Ultrastructural changes in rat palatal epithelium after b-aminopropionitrile. Teratology 11: 153–168, 1975 Mori C, Nakamura N, Okamoto Y, Osawa M, Shiota K: Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation. Anat Embryol (Berl) 190: 21–28, 1994 Pratt RM, Hassel JR: Appearance and distribution of carbohydrate rich macromolecules on the epithelial surface of the rat palatal shelf. Dev Biol 45: 192–198 Shapiro BL, Sweney L: Electron microscopic and histochemical examination of oral epithelium–mesenchymal interaction (programmed cell death). J Dent Res 48: 652–660, 1969 Sharpe PM, Ferguson MWJ: Mesenchymal influence on epithelial differentiation in developing systems. J Cell Sci Suppl 10: 195–230, 1988 Shuler CF: Programmed cell death and cell transformation in craniofacial development. Crit Rev Oral Biol Med 6: 202–217, 1995 Shuler CF, Halpern DE, Guo AC, Sank AC: Medial edge epithelium fate traced by cell lineage analysis during epithelial– mesenchymal transformation. Dev Biol 154: 318–330, 1992 Smiley GR: Fine structure of mouse embryonic palatal epithelium prior to and after midline fusion. Arch Oral Biol 15: 287–296, 1970
Soeno Y, Shimazu Y, Taya Y, Aoba T: Ultrastructure and fates of medial edge epithelial cells during murine palatal fusion in vitro. J Dent Res 78: 529, 1999 Sun D, McAlmon KR, Davies JA, Bernsfield M, Hay D: Simultaneous loss of expression of syndecan-1 and E-cadherin in the embryonic palate during epithelial–mesenchymal transformation. Intern J Dev Bio 42: 733–736, 1998 Taniguchi K, Sato N, Uchiyama Y: Apoptosis and heterophagy of medial edge epithelial cells of the secondary palatine shelves during fusion. Arch Histol Cytol 58: 191–203, 1995 Villa P, Kaufmann SH, Earnshaw WC: Caspases and caspase inhibitors. TIBS 22: 388–392, 1997 Yuan J, Horovitz HR: The Caenorhabdidis elegans all death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development 116: 309–320, 1992 Zakeri Z: The study of cell death by the use of cellular and developmental models. In: Lockshin RA, Zakeri Z, Toilly JL. (Eds), When Cells Die. A Comprehensive Evaluation of Apoptosis and Progammed Cell Death. New York, Chichester, Weinheim. Brisbane, Singapore and Toronto: Wiley-Liss, 1998; 97–129. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X: Apaf, a human protein homologous to C. elegans CED-4 , participates in cytochrome c-dependent activation of caspase –3. Cell 90: 405–413, 1997
Prof. Dr. E.-A. Holtgrave Department of Orthodontics and Paediatric Dentistry School of Dental Medicine Benjamin Franklin Hospital Amannshauser Strasse 4-6 14197, Berlin Germany Tel: +++49 41 30 8445 6241 Fax: +++49 41 030 8445 6242 E-mail: holtgrave @medizin.fu-berlin.de Paper received 1 June 2001 Accepted 4 July 2002
Apoptotic epithelial cell death: a prerequisite for palatal fusion. An in vivo study in rabbits Eva-Andrea Holtgrave,1 Gisela Stoltenburg-Didinger2 1
Department of Orthodontics and Paediatric Dentistry (Head: Prof. Dr. E.A. Holtgrave), School of Dental Medicine; 2Department of Neuropathology (Head: N.N.), Benjamin Franklin University Hospital, Freie Universita¨t, Berlin, Germany SUMMARY. Objective: The aim of the present study was to investigate stage-specific changes in medial edge epithelium during in vivo fusion of embryonic palatal shelves in 25 Russian rabbits. Material and Methods: The embryos were dissected following Caesarian section at day 18. Palatal shelves of specific developmental steps (approximation, contact, fusion) were examined by light microscopy, immunohistochemistry and transmission electron microscopy. Results: Light microscopy revealed that the superfical peridermal cells underwent apoptosis prior to contact of the basal epithelial cells. Following contact, an epithelial monolayer was left on each shelf with an intact basement membrane. Apoptosis of the epithelial cells was followed by discontinuity of the basement membrane. Islands of epithelial cells remained. Conclusion: This paper presents new data on palatal development in vivo. The results support our theory that apoptotic medial edge epithelial cell death is a precondition for palatal fusion. There were no indications of epithelial mesenchymal transformation or migration. r 2002 European Association for Cranio-Maxillofacial Surgery. Published by Elsevier Science Ltd. All rights reserved.
formation of both the primary and secondary palate (Ferguson, 1988). It has been documented in rats that the basic event leading to disappearance of epithelial cells is their morphological change (Fitchett and Hay, 1989; Shuler et al., 1992; Hay, 1995; Shuler, 1995). Ultrastructural studies have found that epithelial cells change their phenotype while forming pseudopodia with subsequent transition into mesenchymal cells (epitheliomesenchymal transformation) penetrating the basement membrane. In contrast, Mori et al. (1994), using in situ staining for nuclear DNA fragments (TUNEL method), observed that the midline edge epithelial cells disappear following DNA fragmentation or apoptosis in mice. Nevertheless, these authors do not reject the epithelial–mesenchymal transformation hypothesis. Another hypothesis was developed by Ferguson (1988) as well as Sharpe and Ferguson (1988) indicating that a substantial number of epithelial cells migrate into the palatal mesenchyme carrying with them fragments of the disrupted basement membranes. Ferguson (1988) found that following this, epithelial cells are no longer distinguishable in the mesenchyme of the palatal shelves. In view of these different hypotheses, we investigated the fusion process of the palatal shelves in rabbits with special reference to epithelial cell disappearance and to the basement membrane in the midline epithelial seam.
INTRODUCTION Normal development is characterized by an equilibrium between proliferation, differentiation and cell death. Cell death occurring in organogenesis is often described as programmed cell death or apoptosis. Morphologically, apoptotic cells exhibit nuclear chromatin condensation and fragmentation as well as ‘‘blistering’’ of cell surfaces and subsequent degradation of apoptotic bodies which are phagocytosed by healthy neighbouring cells (Glu¨cksmann, 1951; Lockshin, 1997; Zakeri, 1998). Apoptosis is also described in palatal development (Taniguchi et al., 1995), but up to now seems to many authors to only be an unimportant side effect (Fitchett and Hay, 1989; Shuler et al., 1992; Hay, 1995; Shuler, 1995). The formation of the palate in man and other mammals is achieved by a coordinated sequence of events beginning with formation, growth and reorientation of the palatal shelves followed by their approximation and adhesion of their superficial epithelial cell layers. These developmental processes end in mesenchymal substitution of the epithelial cells. Prior to fusion, both palatal shelves are covered by a two-layered epithelium, an outer epithelial cell layer, called periderm, and a basal epithelial cell layer. Before contact of the shelves, the periderm is lost. During adhesion, only two epithelial cell layers come together to form the midline epithelial seam, also called the midline edge epithelium. Disappearance of this structure is the most critical step in the 329
330 Journal of Cranio-Maxillofacial Surgery
MATERIAL AND METHODS Animals Female Russian rabbits, 6 months old, were mated overnight. Embryos were assumed to be at day 0 of gestation on the following morning. Litters were collected at day 18 by Caesarean section. The foetuses (n=20) were immediately fixed in 3% buffered formalin solution. After 3 days, the heads were cut in frontal sections and embedded in paraffin. Light microscopy Paraffin sections of 5 mm were stained with haematoxylin and eosin (HE), cresyl violet (Nissl) and periodic acid–Schiff (PAS). For immunohistochemistry, the paraffin sections were mounted on pretreated glass slides and incubated overnight with primary antibodies against vimentin and Ki 67 (=MIB 1, DAKO, Hamburg, Germany). Ki 67 is an antigen which appears in the nucleus when the cell leaves the resting phase G0. In histopathology and developmental biology, KI 67 antigenicity is used as a marker of proliferation. The antigen was visualized by the APAAP technique using neofuchsin as a chromogen. For the detection of apoptosis an in situ labelling technique (BOEHRINGER-Kit, Boehringer, Mannheim, Germany) was used with diaminobenzidine (DAB) as a chromogen and counterstaining with light green (Merck, Mannheim, Germany).
Fig. 1 – Coronal section of a rabbit midpalatal region. Palatal shelves prior to fusion (embryonic day 18, HE, 10).
Electron microscopy The palatal portions (n=5) were excised from the tissue under a binocular microscope (Zeiss, Oberkochen, Germany). Representative areas of the palate were immersion fixed in glutaraldehyde (pH 7.2), buffered with cacodylate–HCl (pH 7.4, containing 4% sucrose), postfixed with 2% osmium tetroxide, dehydrated with a graded series of ethanol, and embedded in araldite. Sections were cut with an ultramicrotome and examined under a ZEISS electron microscope EM 10 after staining with uranyl acetate and lead citrate. RESULTS Macroscopic observations The primary palate seemed to be fused in all 25 animals. Microscopic observations The palatal shelves still separate Light microscopy revealed the fusion process to be at different developmental stages. The medial parts of
Fig. 2 – Palatal shelves still separate. Cell division in the medial edge epithelium has ceased. All nuclei stained in red present the antigen KI 67 which is present in the nucleus between phase G1 to mitosis, i.e. they are proliferating (embryonic day 18, MIB 1, 20).
both palatal shelves were covered by a two-layered epithelium underlined by a continuous basement membrane (Figs 1–4). The basal epithelium cell layer consisted of a seam of cylindrical cells with oval nuclei. The outer epithelial layer also called the periderm, consisted of flat cells with vacuolated cytoplasm. At this developmental stage, numerous peridermal cells exhibited signs of apoptosis (Figs. 3
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Immunhistochemistry, using MIB 1 reaction to find the proliferating cells, revealed complete cessation of proliferation in the nasal periderm while in the underlying epithelium a 30% proliferation rate was noted (Fig. 2). While proliferation persisted in the two-layered epithelium seam opposing the palatal shelves on the top, the epithelial cells in the midline seam exhibited an extreme reduction of the proliferation rate in the basal epithelial cell layer. This was noted by the MIB 1 reaction findings (Fig. 2). The palatal shelves meet
Fig. 3 – Two cell layers of the medial edge epithelium prior to fusion. The outer layer, ‘periderm’ with signs of single cell apoptosis (embryonic day 18, HE, 40).
When the palatal shelves met, the epithelia of both shelves consisted of only one cell layer each, plus the intact basal membranes. The most prominent feature of the nuclei was apoptosis of this basal epithelium revealed by TUNEL staining (Fig. 5A). This process also took place between the ‘‘kissing’’ epithelia of the nasal septum and nasal surfaces of the palatal shelves (Fig. 5B). In transmission electron microscopy (TEM), the continuity of the underlining basement membrane could be confirmed, together with the apoptotic process. Cell division in the attached epithelia had ceased. The palatal shelves fuse The continuous epithelial layer was dispersed into epidermal islands (Fig. 6). Mesenchymal cells penetrated and proliferated between these islands. The mitotic activity was restricted to the mesenchymal cells (Fig. 7). The basement membrane surrounded the epithelial clusters and was only preserved there (Fig. 6B). On the ultrastructural level, it became clear that the basement membrane adjacent to the apoptotic cells was disrupted and missing. In the anterior region, where fusion with the nasal septum took place simultaneously, the same disruption process of the basement membrane was observed in combination with the vanishing epithelial cells (Figs. 8–11). Transformation of resting epithelial islands into mesenchymal cells could not be verified. The apoptotic cells did not leave any traces and were replaced by mesenchymal cells.
Fig. 4 – Epithelial cell layer of the palatal process facing the nasal septum covered by PAS positive mucous material. Single cell apoptosis in the upper cell layer, see Fig. 1 (embryonic day 18, PAS, 100).
and 4). The apoptosis is characterized by chromatin condensation, and later by fragmentation into apoptotic bodies. These apoptotic cells were sloughed away while the remaining cells stayed in a form of symplasma (Figs. 3 and 4). The epithelial layer facing the nasal septum exhibited identical features although the spatial distance between both shelves was up to 5 times greater than in the midline (Figs. 1 and 4).
DISCUSSION Our main finding of the morphological analysis of palatal fusion in rabbits was the presence of apoptotic cells in the midline edge. The disappearance of the midline edge epithelium is believed to be the most critical step in the formation of the primary and secondary palates. There are three different hypotheses as to how this takes place: By epithelial–mesenchymal transformation, the epithelial cells change their phenotype whilst forming pseudopodia, with subsequent transformation to mesenchymal cells (Fitchett and Hay, 1989; Shuler
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Fig. 5 – (A) Epithelium in contact. TUNEL staining reveals abundant apoptotic cell death in nasal and medial fusion areas (embryonic day 18, 10). (B) Detail of (A). Fusion area. Apoptosis of epithelial cells (arrow), basement membrane intact (embryonic day 18, TUNEL staining, 100).
Fig. 6 – (A) Coronal section of midpalatal region. Palate fused (embryonic day 18, HE, 10). (B) Epithelial islands surrounded by basement membrane, detail of (A) (embryonic day 18, HE, 100).
et al., 1992; Mori et al., 1994; Hay, 1995; Shuler, 1995; Sun et al., 1998; Soeno et al., 1999; MartinezAlvarez et al., 2000). By migration a substantial number of epithelial cells migrate orally and usually into the epithelial triangle (Ferguson, 1988; Sharpe and Ferguson, 1988; Carette and Ferguson, 1992; Soeno et al., 1999; Martinez-Alvares et al., 2000). Only a small number of epithelial cells appear to merge into the palatal mesenchyme and are no longer distinguishable from mesenchymal cells of the palatal shelves.
By apoptosis the midline edge epithelial (MEE) cells disappear by DNA fragmentation. The authors who promote this theory do not reject the epitheliomesenchymal transformation theory (Mori et al., 1994; Taniguchi et al., 1995). As to epithelial–mesenchymal transformation: All the experiments showing this kind of transformation were performed in vitro. The palatal shelves were excised and grown as organ cultures on collagen matrices. This procedure has been performed by all authors claiming that transformation is an important
Apoptotic epithelial cell death
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Fig. 7 – (A) Coronal section of midpalatal area. Penetrating mesenchymal cells proliferate between the islands of non-dividing epithelial cells (embryonic day 18, MIB 1, 10). (B) Detail of (A) midline region. One of the mesenchymal cells undergoes mitosis (arrow head). The KI 67 antigen is visible in the entire cytoplasm as usual during mitosis (embryonic day 18, MIB 1, 100).
Fig. 8 – Epithelial cell layer with two apoptotic bodies adjacent to the nucleus of an epithelial cell (embryonic day 18, TEM, 4400).
Fig. 9 – Two apoptotic bodies phagocytosed by an epithelial cell (embryonic day 18, TEM, 4400).
step in palatal fusion (Fitchett and Hay, 1989; Mori et al., 1994; Hay, 1995; Shuler, 1995; Sun et al., 1998; Soeno et al., 1999; Martinez-Alvarez et al., 2000). The development of pseudopodia is behaviour typical of cultured cells, and is not comparable to in vivo conditions. The cell behaviour and differentiation in vitro is different from in vivo. The shape of the cells and the development of the cytoskeleton most closely resemble more immature stages under in vitro conditions. The differentiation of epithelial cells into mesenchymal cells does not occur in normal devel-
opment. The argument of Greenburg and Hay (1986) that, identical to the MEE transformation, the epithelial cells of lens epithelium lose polarity and are transformed with mesenchymal cells is not true for in vivo situations, since the observation has also been made in culture. Transformation into a more undifferentiated cell type is restricted to tumour development (Gruenert, 1987). According to Fitchett and Hay (1989) the periderm cells are the only ones that die, whilst basal cells remain healthy and even divide before transforma-
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2. The dead cell elicits a response in neighbouring epithelial cells that may tentatively be characterized as cannibalistic; this response is visualized as a change in cell shape resulting in a partial or complete engulfment of dead cells. 3. When the dead cell is engulfed, it is destroyed by lytic enzymes within the vacuole; its organelles lose their structural integrity and are replaced by amorphous material. Farbman (1968) clearly describes the features of apoptotic cell death although this term was only coined in 1972 by Kerr et al. (1972).
Fig. 10 – Several apoptotic bodies adjacent to the nucleus of a freefloating cell (embryonic day 18, TEM, 3000).
Fig. 11 – Intracytoplasmic apoptotic body in the fusion area (embryonic day 18, TEM, 12 000).
tion into mesenchymal cells. Our observation indicates that periderm cells disappeared completely prior to contact of the palatal shelves. MIB 1 staining revealed that the basal cell layer stops mitotic activity. Transformation to mesenschymal cells is unlikely when the epithelial cells stop dividing. In contrast, our morphological findings revealed apoptotic cell death of the epithelial seam. Thus, we can support the findings of Farbman (1968), who described the sequence of events as follows: 1. Certain epithelial cells in the seam die for unknown reasons.
As to migration: Migration of epithelial cells into the palatal mesenchyma is said to occur either before (Fitchett and Hay, 1989) or after transformation into mesenchymal cells (Sharpe and Ferguson, 1988). Migration includes two different mechanisms: First, the epithelial cells of the midline migrate to the oral and nasal side and thus contribute to the thinning of palatal epithelium. In our experiment, the thinning of the epithelium in the midline is a consequence of apoptotic cell death of peridermal cells prior to fusion (they slough off) and to apoptotic cell death of the medial edge epithelium after contact. When apoptotic cell death includes the suprabasal epithelial cells, they are no longer able to support the basement membrane, which consequently breaks down and allows mesenchymal cells to cross from one shelf to the other. In our study, the cluster of epithelial cells with a surrounding basement membrane make up some of the persisting epithelia. Second, Sharpe and Ferguson (1988) reported migration of epithelial cells into the mesenchyma together with attached basement molecules. We have never observed this process. We found the thinning of the midline epithelial seam to be due to apoptotic cell death and not due to migration. Migration of epithelial cells into the mesenchyme was said to be followed by transformation into mesenchymal cells (Ferguson, 1988; Mori et al., 1994). Transformation was only observed in organ cultures suggesting that epithelio-mesenchymal transformation may be a side effect of the in vitro system (Fitchett and Hay, 1989; Griffit and Hay, 1992; Shuler et al., 1992; Shuler, 1995). As to apoptosis: In view of the different theories mentioned and derived from animal observations in mice, we elucidated the role of apoptosis in a rabbit model with particular reference to the disappearance of the epithelial cells during normal development. This study has confirmed that the basis of palatal fusion was apoptosis of the superficial and later of the basal midline epithelium. This has already been reported by Farbman in 1968 who observed widespread evidence of cell death in the midline epithelial seam immediately following palatal fusion. Dead epithelial cells were phagocytosed by neighbouring epithelial cells. In the electron micrographs of Farbman (1968), the ultrastructural morphology of apoptosis and apoptotic bodies is similar to that in our figures. Similar ultrastructure features were
Apoptotic epithelial cell death
described by Shapiro and Sweney (1969) and Smiley (1970). The process of palate fusion was also examined by Mori et al. (1994) in 13- and 14-day-old mouse foetuses. Taniguchi et al. (1995) found the same in rats using in situ staining for nuclear DNA-fragmentation (TUNEL method) and immunofluoresce staining for keratin, paying special attention to disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palatal fusion, but not in the palatal shelves prior to contact, nor in the intact midline epithelial seam. These results suggest that MEE cells undergo apoptosis during palatal fusion (Taniguchi et al., 1995). In our own animals TUNEL-positive staining was only present in epithelial cells, especially at the fusion site, where the midline edges of the palatal shelves met and where the nasal septum met the upper surfaces of the palatal shelves. The earliest study proposing programmed cell death for MEE was that of Shapiro and Sweney in 1969. In 1976, a review article (Greene and Pratt, 1976) stated that, ‘Fusion of the opposing shelves, both in vivo and in vitro, is dependent upon adhesion and cell death of the MEE cells’. By the mid-1970s, the hypothesis on programmed cell death for MEE was generally accepted (Farbman, 1968; Hayward, 1969; Mato et al., 1975; Pratt and Hassel, 1975). Prior to apoptotic cell death, the MEE stops proliferating. The arrest of mitotic activity is specially related to the medial edge epithelium. It is well known that in mice and rats, the intrauterine development is very short, and this is also true for the process of formation and involution of the epithelial sheet of the secondary palate. Since apoptosis is a transitory event we have therefore chosen rabbits as a species with a longer duration of palatogenesis in order to catch the different steps of apoptotic cell disappearance. The results of a gene trap study with mice lacking the human homology of CED-4 provide the most convincing evidence for the crucial role of apoptosis in secondary palate fusion. The phenotype had palatal clefts (Cecconi et al., 1998). The basic mechanism of apoptosis found to be present in the nematode Caenorhabditis elegans led to the isolation of genes that are specifically required for inducing programmed cell death. By these means, apoptotic cell death was amenable to molecular analysis and led to the identification of at least 14 genes implicated in developmental cell death (Villa et al., 1997). Three of these CED genes are of special importance: the proapoptotic death-promoting genes CED-3 and CED-4, and the anti-apoptotic gene CED-9. The cloning of the c-DNA for these genes led to the realization that all three had human homologues. The CED-4 gene encodes a 63-kD protein with no significant similarity to other known peptides (Yuan and Horowitz, 1992). CED-4 participates in the caspase 9-dependent activation of caspase 3 in the general apoptotic pathway. The human CED-4 homologue has been
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isolated and termed APAF 1 (apoptotic proteases activating factor 1; Zou et al., 1997). In situ immunodetection found that the absence of APAF-1 protein prevents activation of caspase 3 in vivo. It remains to be determined to what extent the protein is involved in developing programmed cell death. To address this question, Gruss (1997) and Cecconi et al. (1998) have taken advantage of a gene trap study (Chowdhury et al., 1997) that has provided the means to isolate a null allele of the murine APAF1 gene. Foetuses homozygous for APAF-1 mutation have a characteristic craniofacial phenotype whose major traits are midline facial cleft and cleft palate. Apoptosis has been postulated as playing a key role in palatal fusion by eliminating the medial edge epithelium of the secondary palatal shelves after they have contacted in the midline (Ferguson, 1988). Accordingly, late and imperfect palatal fusion with persistence of midline epithelium is a key component of the APAF-1 mutant phenotype. The authors came to the conclusion that apoptosis is essential in midline fusion of craniofacial structures (Cecconi et al., 1998). CONCLUSION The importance of apoptosis in palatal development has been confirmed in this study by conventional histology, immunohistochemistry and TEM microscopy. The results underline the importance of morphological studies and re-evaluate the primary conclusions drawn by some authors as early as the late 1960s and 1970s (Farbman, 1968; Shapiro and Sweney, 1969; Smiley, 1970) who have postulated that apoptosis is a major event in palatal development.
References Carette MJM, Ferguson MWJ: The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy. Development 114: 379–388, 1992 Cecconi F, Alvarez-Bolando G, Meyer BI, Roth KA, Gruss P: Apaf1 (CED-4 homologue) regulates programmed cell death in mammalian development. Cell 94: 727–737, 1998 Chowdhury K, Bonaldo P, Torres M, Stoykova A, Gruss P: Evidence for the stochastic integration of gene trap vectors into the mouse germline. Nucl Acids Res 25: 1531–1536, 1997 Farbman AI: Electron microscopic study of palate fusion in mouse embryos. Dev Biol 18: 93–116, 1968 Ferguson MJW: Palate development. Development 103 (Suppl): 41–60, 1988 Fitchett JE, Hay ED: Medial edge epithelium transforms to mesenchyme after embryonic palatal shelves fuse. Dev Biol 119: 455–478, 1989 Glu¨cksmann A: Cell death in normal vertebral ontogeny. Biol Rev 26: 59–86, 1951 Greenburg G, Hay ED: Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev Biol 115: 363–379, 1986 Greene RM, Pratt RM: Developmental aspects of secondary palate formation. J Embryol Exp Morphol 36: 225–245, 1976 Griffit CM, Hay ED: Epithelial–mesenchymal transformation during palate fusion: carboxyfluorescein traces cells at light
336 Journal of Cranio-Maxillofacial Surgery and electron microscopic levels. Development 116: 1087–1099, 1992 Gruenert DC: Differentiated properties of human epithelial cells transformed in vitro. Bio Techniques 5: 740–745, 1987 Gruss P: Evidence for stochastic integration of gene trap vectors into the mouse germline. Nucl Acids Res 25: 1531–1536, 1997 Hay ED: An overview of epithelio-mesenchymal transformation. Acta Anat 154: 8–20, 1995 Hayward AF: Ultrastructural changes in the epithelium during fusion of the palatal processes in rats. Arch Oral Biol 14: 661–678, 1969 Kerr JFR, Wyllie AH, Currie AR: Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972 Lockshin RA: The early modern period of cell death. Cell Death Diff 4: 347–351, 1997 Martinez-Alvarez C, Tudela C, Perez-Miguelsanz J, O0 Kane S, Puerta J, Ferguson MW: Medial edge epithelial cell fate during fusion. Dev Biol 15: 343–357, 2000 Mato M, Uchiyama Y, Aikawa E, Smiley GR: Ultrastructural changes in rat palatal epithelium after b-aminopropionitrile. Teratology 11: 153–168, 1975 Mori C, Nakamura N, Okamoto Y, Osawa M, Shiota K: Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation. Anat Embryol (Berl) 190: 21–28, 1994 Pratt RM, Hassel JR: Appearance and distribution of carbohydrate rich macromolecules on the epithelial surface of the rat palatal shelf. Dev Biol 45: 192–198 Shapiro BL, Sweney L: Electron microscopic and histochemical examination of oral epithelium–mesenchymal interaction (programmed cell death). J Dent Res 48: 652–660, 1969 Sharpe PM, Ferguson MWJ: Mesenchymal influence on epithelial differentiation in developing systems. J Cell Sci Suppl 10: 195–230, 1988 Shuler CF: Programmed cell death and cell transformation in craniofacial development. Crit Rev Oral Biol Med 6: 202–217, 1995 Shuler CF, Halpern DE, Guo AC, Sank AC: Medial edge epithelium fate traced by cell lineage analysis during epithelial– mesenchymal transformation. Dev Biol 154: 318–330, 1992 Smiley GR: Fine structure of mouse embryonic palatal epithelium prior to and after midline fusion. Arch Oral Biol 15: 287–296, 1970
Soeno Y, Shimazu Y, Taya Y, Aoba T: Ultrastructure and fates of medial edge epithelial cells during murine palatal fusion in vitro. J Dent Res 78: 529, 1999 Sun D, McAlmon KR, Davies JA, Bernsfield M, Hay D: Simultaneous loss of expression of syndecan-1 and E-cadherin in the embryonic palate during epithelial–mesenchymal transformation. Intern J Dev Bio 42: 733–736, 1998 Taniguchi K, Sato N, Uchiyama Y: Apoptosis and heterophagy of medial edge epithelial cells of the secondary palatine shelves during fusion. Arch Histol Cytol 58: 191–203, 1995 Villa P, Kaufmann SH, Earnshaw WC: Caspases and caspase inhibitors. TIBS 22: 388–392, 1997 Yuan J, Horovitz HR: The Caenorhabdidis elegans all death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development 116: 309–320, 1992 Zakeri Z: The study of cell death by the use of cellular and developmental models. In: Lockshin RA, Zakeri Z, Toilly JL. (Eds), When Cells Die. A Comprehensive Evaluation of Apoptosis and Progammed Cell Death. New York, Chichester, Weinheim. Brisbane, Singapore and Toronto: Wiley-Liss, 1998; 97–129. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X: Apaf, a human protein homologous to C. elegans CED-4 , participates in cytochrome c-dependent activation of caspase –3. Cell 90: 405–413, 1997
Prof. Dr. E.-A. Holtgrave Department of Orthodontics and Paediatric Dentistry School of Dental Medicine Benjamin Franklin Hospital Amannshauser Strasse 4-6 14197, Berlin Germany Tel: +++49 41 30 8445 6241 Fax: +++49 41 030 8445 6242 E-mail: holtgrave @medizin.fu-berlin.de Paper received 1 June 2001 Accepted 4 July 2002