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RESEARCH REPORT |
1 Section of Pediatric Dentistry, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan; 2 School of Dentistry, Kyushu University, Fukuoka, Japan; and 3 Department of Pathology, Nippon Dental University;
* corresponding author, sasakiy{at}dent.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: spontaneous cleft lip and palate • CL/Fr • palatal organ culture • bromodeoxyuridine (BrdU) • palatal cell proliferation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A mechanism of cleft secondary palate following cleft lip is proposed from a classic observation in A/J mice embryos, showing that the presence of a cleft lip appeared to induce mechanical obstruction by the tongue that delayed palatal elevation and fusion (Trasler and Fraser, 1963). When CL/Fr embryos were transferred into C57BL and CL/Fr dams, the severity of CLP in the affected fetuses from CL/Fr strain dams was significantly worse than that seen in the C57BL strain (Martin et al., 1995; Nonaka et al., 1997). These studies suggest that environmental factors play an important role in the production of cleft secondary palate following cleft lip. This hypothesis is confirmed by the facts that cleft lip embryos in A/J mice fused in vitro (Pourtois, 1967), and complete epithelial disruption occurred at the tip of the palatal process in the absence of any contact of palatal shelves in vivo in A/J mice embryos (Tsai and Verrusio, 1977).
Electron microscopic study (Millicovsky et al., 1982) and histologic observation (Wang et al., 1995) in CL/Fr embryos suggest that regional growth deficiency or developmental abnormality in the maxillary and nasal prominences may be a common feature in primary palatal clefting. However, palatal development subsequent to cleft lip has not been carefully investigated in either animals or humans. Spatio-temporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate (Hehn et al., 1998). To analyze regional patterns of cell proliferation in palatal shelves and to determine whether the palatal shelves have an ability to fuse after cleft lip, we conducted in vitro palatal culture and immunologic experiments with bromodeoxyuridine (BrdU) in CL/Fr embryos with or without cleft lip.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), bilateral cleft lip (CL/Fr-BCL) (Fig. 1A), and unilateral cleft lip. The unilateral cleft lip embryos were excluded from this study because the number of embryos available was insufficient. For BrdU labeling in vivo, the pregnant mice were injected intraperitoneally at E13.5 with 0.2 mL of 10 mg/mL BrdU (Sigma, St. Louis, MO, USA) and killed at 2 hrs following the injection.
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Assessment of Cell Proliferation
Palates were derived from 10 C57BL embryos (from 4 dams), 14 normal CL/Fr embryos (from 5 dams), and 8 BCL CL/Fr embryos (from 3 dams). The CL/Fr-BCL embryos were derived from the same dams that produced the CL/Fr-N embryos. The embryos’ heads were fixed in 10% formalin for 12 hrs at room temperature, and were then dehydrated with ethanol and embedded in paraffin according to standard procedures. Coronal paraffin serial sections (8 µm thick) were made of each embryo. Assessment of cell proliferation was according to the protocol of Takagi et al.(2000). De-waxed sections were treated with 1 mol/L hydrochloric acid at 37°C for 20 min. Sections were then immersed in 0.3% hydrogen peroxide/methanol for 30 min at room temperature. The sections were incubated in anti-BrdU monoclonal antibody (Amersham) for 1 hr 30 min at 37°C. Next, the sections were incubated in peroxidase-labeled antimouse IgG2a (Amersham) for 30 min at 37°C. They were then immersed in a medium containing 3-3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide for 10 min. Finally, they were counterstained with eosin, dehydrated, and mounted according to standard procedures.
From among the serial sections for each embryo, one section was selected to represent the middle one-third of the palate along the rostro-caudal axis. These sections were photographed, and the following procedure was performed on the photographs. On each section, the medial tip of the shelf was defined as the point where the radius of the epithelial curvature was the smallest (asterisk in Figs. 2B, 2C, 2D). For the assessment of mesenchymal proliferation, an arc was drawn on the photograph with a radius of 200 µm (actual length on the sections) and centered at the point of smallest curvature. The labeled and unlabeled nuclei were counted in the area surrounded by the arc, and the ratio of cell proliferation was defined as the number of labeled cells divided by the total number of labeled and unlabeled cells in mesenchyme and epithelia, respectively. The data were analyzed by unpaired Student’s t test for comparison among the three groups (C57BL, CL/Fr-N, CL/Fr-BCL) and between left- and right-side palatal shelves.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Nasal and oral epithelial triangles had formed and seam degeneration had commenced, but some epithelial islands were still observed after 48 hrs. Fusion of pairs of the CL/Fr-BCL palatal shelves progressed as well as those from CL/Fr-N (Fig. 1F). Fifteen out of 20 pairs of palatal shelves dissected from CL/Fr-N and 7 out of 10 pairs of palatal shelves from CL/Fr-BCL underwent mesenchymal fusion and resorption of epithelial barriers or epithelial islands. Failure of palatal fusion (5 out of 20 pairs of CL/Fr-N and 3 out of 10 pairs of CL/Fr-BCL) was caused by the lack of initial contact of medial edge epithelia due to a technical problem of placement on the filter paper. There were no apparent differences between normal embryos and embryos with bilateral cleft lip.
Cell Proliferation
Representative frontal sections (right palatal shelf) from E13.5 embryos are shown in Figure 2. At E13.5 the palatal shelves were still in a vertical orientation. Many BrdU-positive cells were observed in the mesenchyme and epithelium.
Differences between left- and right-side palatal shelves were analyzed for the number of positive cells and the ratio of positive to total number of cells (Fig. 3). In the epithelia, the left side had a greater number and a higher ratio of positive cells in the three groups (Figs. 3A, 3B). A similar tendency was seen in the mesenchyme except for the CL/Fr-N palates, which had a greater number and a higher ratio of positive cells in the right side (Figs. 3C, 3D). However, these differences were statistically significant only in the epithelium of C57BL palates. Therefore, our statistical comparison of the palatal shelves among the three groups included equal sample numbers of left- and right-side palatal shelves in each group, to exclude any effects from the side-difference. Inter-group differences of the labeling index for palatal mesenchyme and epithelium are given in the Table. There were significant group differences in the absolute numbers and the ratios of positive cells in the mesenchyme (C57BL > CL/Fr-N > CL/Fr-BCL). The epithelium of CL/Fr embryos with or without cleft lip had both fewer and a smaller ratio of positive cells than C57BL embryos, but there was no difference between CL/Fr-N and CL/Fr-BCL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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For visceral and external abnormalities in CL/Fr mice, only congenital heart anomalies, such as ASD and patent ductus arteriosus (PDA), have been reported (Fraser and Rosen, 1975). The clefting and defect of conotruncal lesions in the heart occur together with abnormalities of neural crest cell proliferation and migration (Wyse et al., 1990). Kadowaki et al.(1997b) examined newborn hearts of CL/Fr embryos histologically and indicated that the septum primum in CLP(+) mice tended to be less developed than that in CLP(–) mice, while a significant difference in body weight was not detected between CLP(+) and CLP(–). Accordingly, our BrdU data on palatal shelves reflect not the overall growth of CL/Fr-BCL, but rather a stage-specific palatal growth spurt delay in CL/Fr-BCL.
Orofacial observations in 79 CL/Fr embryos in 18-day-old fetuses and newborns with complete cleft lip demonstrated that several types of palatal development were found. The most frequent was bilateral shelf elevation with or without palatal fusion, followed by unilateral palatal and complete failure of palatal elevation, and the development of palatal shelves was more advanced in cases with mild cleft lip than in those with severe cleft lip (Kadowaki et al., 1997a). Our analysis of cell kinetics for proliferation indicates that palatal shelves from CL/Fr-BCL have less capability for mesenchymal cell proliferation compared with CL/Fr-N, and suggests that, in addition to environmental factors reported in the past, some aspects of cell kinetics for the proliferation of palatal shelf mesenchyme may also be directly associated with the production of cleft secondary palate in CL/Fr embryos with cleft lip. Epithelial cell proliferation was similar between CL/Fr-N and CL/Fr-BCL. This result is consistent with our culture experiment where the amount of epithelial fusion at E13.5 in CL/Fr-BCL was similar to that of CL/Fr-N.
Results from our study suggest another mechanism for inducing spontaneous cleft palate in CL/Fr embryos. The cell kinetics for proliferation in palatal shelves in CL/Fr embryos is slower than that in C57BL embryos, meaning that cell proliferation in the palatal shelf at the time of palatal elevation in CL/Fr embryos occurs later compared with proliferation in CLP-resistant C57BL embryos. Histologic analysis in CLP-susceptible strains of mice suggests that palatal fusion in CL/Fr-N (Hamachi et al., 2003) and A/J embryos with or without cleft lip (Walker and Fraser, 1956) occurs later than in C57BL/6 embryos. The cleft-lip-susceptible strains of A/J, A/WySn, and CL/Fr reach the tail somite stage (TS) interval of 8 to 18 TS at a later chronological age (day 11/hour 2 to day 11/hour 18) than does a normal strain of C57BL/6 (day 10/hour 17 to day 11/hour 11), and lip formation in the CL/Fr embryos is delayed relative to their somite stage (Wang et al., 1995). These studies suggest that lip and palate formation is delayed in CL/Fr embryos relative to their general body growth, and this, in addition to the tongue obstruction, may affect the production of cleft palate. We hypothesize that the decrease in cell proliferation in the CL/Fr strain is associated with that strain’s susceptibility to cleft secondary palate subsequent to cleft lip.
Some factors that control cell proliferation in palatal mesenchyme include: EGF and TGFbeta1 for DNA synthesis in embryonic hamster palate mesenchymal cells (Izadnegahdar et al., 1999); EGF and MAP kinase during morphogenesis of the quail secondary palate (Hehn et al., 1998); retinoic acid and TGFbeta in embryonic murine palate mesenchymal cells (Nugent et al., 1998); and the Msx1 homeobox gene involving BMP and Shh signals regulating the growth of anterior palate during murine palatogenesis (Zhang et al., 2002). In a qualitative analysis of mRNA in whole-mount in situ hybridization of CL/Fr embryos (Hamachi et al., 2003), Pax9 (which enhances mesenchymal cell proliferation throughout the body in vertebrates) was observed at pre-fusion of the palatal shelves along the mesial epithelial edge in C57BL, CL/Fr-N, and CL/Fr-BCL. However, the expression pattern in CL/Fr-BCL was similar to that in C57BL and CL/Fr-N. Additional quantitative analysis of the molecules associated with cell proliferation in the palatal shelf is crucial for determination of which molecules are responsible for secondary cleft palate in CL/Fr embryos.
Interestingly, BrdU labeling indices for palatal epithelium on the left side had a significantly greater number and a higher ratio of positive cells than the right side in C57BL embryos. In complete bilateral cleft lip and palate in CL/Fr embryos, unilateral palatal elevation occurs more frequently on the left side than on the right side (Kadowaki et al., 1997a). The presence of more positive cells in the left-side palatal epithelium suggests that there may be growth asymmetry between the right- and left-side palatal shelves regardless of facial clefting.
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ACKNOWLEDGMENTS |
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Received January 10, 2004; Last revision July 27, 2004; Accepted July 28, 2004
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Brunet CL, Sharpe PM, Ferguson MWJ (1993). The distribution of epidermal growth factor binding sites in the developing mouse palate. Int J Dev Biol 37:451–458.[Medline]
Fraser FC, Rosen J (1975). Association of cleft lip and atrial septal defect in mice: a preliminary report. Teratology 11:321–324.[Medline]
Hamachi T, Sasaki Y, Hidaka K, Nakata M (2003). Association between palatal morphogenesis and Pax9 expression pattern in CL/Fr embryo with clefting during palatal development. Arch Oral Biol 48:581–587.[ISI][Medline]
Hehn BM, Izadnegahdar MF, Young AV, Sanghera JS, Pelech SL, Shah RM (1998). In vivo and in vitro assessment of mitogen activated protein kinase involvement during quail secondary palate formation. Anat Rec 252:194–204.[Medline]
Izadnegahdar MF, Rathanaswami P, Shah RM (1999). Effect of EGF and TGFbeta1 on c-myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells. Anat Rec 254:453–464.[Medline]
Jones MC (1988). Etiology of facial clefts: prospective evaluation of 428 patients. Cleft Palate J 25:16–20.[Medline]
Kadowaki S, Sakamoto M, Kamiishi H, Tanimura T (1997a). Embryologic features of term fetuses and newborns in CL/Fr mice with special reference to cyanosis. Cleft Palate Craniofac J 34:211–217.[Medline]
Kadowaki S, Sakamoto M, Kamiishi H, Tanimura T (1997b). Atrial septal defect in cyanotic CL/Fr mice. Cleft Palate Craniofac J 35:58–64.
Martin DA, Nonaka K, Yanagita K, Nakata M (1995). The effect of dam strain on the craniofacial morphogenesis of CL/Fr mouse fetuses. J Craniofac Genet Dev Biol 15:117–124.[Medline]
Millicovsky G, Ambrose LJH, Johnston MC (1982). Developmental alterations associated with spontaneous cleft lip and palate in CL/Fr mice. Am J Anat 164:29–44.[Medline]
Nagata M, Amin N, Kannari Y, Hayatsu M, Ohashi Y, Oguro A (1997). Isolated maxillary bending in CL/FR strain mice: observation of craniofacial deformity and inheritance pattern. Cleft Palate Craniofac J 34:101–105.[Medline]
Nonaka K, Sasaki Y, Watanabe Y, Yanagita K, Nakata M (1997). Effects of fetus weight, dam strain, dam weight, and litter size on the craniofacial morphogenesis of CL/Fr mouse fetuses affected with cleft lip and palate. Cleft Palate Craniofac J 34:325–330.[Medline]
Nugent P, Ma L, Greene RM (1998). Differential expression and biological activity of retinoic acid-induced TGFbeta isoforms in embryonic palate mesenchymal cells. J Cell Physiol 177:36–46.[ISI][Medline]
Pourtois M (1967). Influence of cleft lip upon palatal closure in A/Jax mice. Cleft Palate J 4:120–123.[Medline]
Takagi TN, Matsui KA, Yamashita K, Ohmori H, Yasuda M (2000). Pathogens of cleft palate in mouse embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Teratogen Carcinogen Mutagen 20:73–86.[ISI][Medline]
Taya Y, O’Kane S, Ferguson MWJ (1999). Pathogenesis of cleft palate in TGF-ß3 knockout mice. Development 126:3869–3879.[Abstract]
Trasler DG, Fraser FC (1963). Role of the tongue in producing cleft palate in mice with spontaneous cleft lip. Dev Biol 6:45–60.[ISI][Medline]
Tsai HM, Verrusio AC (1977). Epithelial breakdown in the palatal processes of mouse fetuses with spontaneous cleft lip and palate. Teratology 15:121–126.[Medline]
Walker BE, Fraser FC (1956). Closure of the secondary palate in three strains of mice. J Embryol Exp Morphol 4:176–189.
Wang KY, Juriloff DM, Diewert VM (1995). Deficient and delayed primary palatal fusion and mesenchymal bridge formation in cleft lip-liable strains of mice. J Craniofac Genet Dev Biol 15:99–116.[Medline]
Wyse RKH, Mars M, Al-Mahdawi S, Russell-Eggitt IM, Blake KD (1990). Congenital heart anomalies in patients with clefts of the lip and/or palate. Cleft Palate J 27:258–264.[Medline]
Zhang Z, Song Y, Zhao X, Zhang X, Fermin C, Chen Y (2002). Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129:4135–4146.
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