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Deficient Cell Proliferation in Palatal Shelf Mesenchyme of CL/Fr Mouse Embryos1 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; Correspondence: * corresponding author, sasakiy{at}dent.kyushu-u.ac.jp
How secondary palate formation is affected in the cleft lip genotype remains poorly understood. The purpose of this study was to analyze regional patterns of cell proliferation in CL/Fr mouse embryos with or without cleft lip. Pairs of palatal shelves were dissected at E13.5 from CL/Fr normal embryos (CL/Fr-N), CL/Fr embryos with bilateral cleft lip (CL/Fr-BCL), and a control strain of C57BL embryos (C57BL). The explants were examined histologically after 48 hrs of organ culture. Cell kinetics for proliferation in the palatal shelves was examined at E13.5 by the bromodeoxyuridine method in vivo. The CL/Fr-BCL palates fused as well as the CL/Fr-N palates in vitro. There were inter-group differences in the absolute number of BrdU-positive cells and the ratio of positive/(positive+negative) cells in the palate’s mesenchyme (C57BL > CL/Fr-N > CL/Fr-BCL) and epithelium (C57BL > CL/Fr-N = CL/Fr-BCL). These findings indicate that a cleft palate follows reduced cell proliferation of secondary palatal mesenchyme in CL/Fr mice.
Key Words: spontaneous cleft lip and palate • CL/Fr • palatal organ culture • bromodeoxyuridine (BrdU) • palatal cell proliferation
Cleft lip with cleft palate (CLP) is a major congenital craniofacial birth defect that has a complex etiology of genetic and environmental factors, and in humans is notable for significant morbidity. Animal studies of CLP provide a valuable model for complex craniofacial disorders in general, because most studies suggest that 70% of cases with cleft lip (with or without cleft palate) are non-syndromic (Jones, 1988). The CL/Fr mouse has been established as a strain in which 15% to 40% of newborns spontaneously develop CLP (Millicovsky et al., 1982; Wang et al., 1995; Nagata et al., 1997). Therefore, study of the developing palate in CL/Fr embryos might elucidate the mechanism of cleft palate following cleft lip. More than 96% of CL/Fr embryos with cleft lip subsequently develop cleft secondary palate (Brown et al., 1985), suggesting that production of cleft lip in CL/Fr embryos is an important factor in the succeeding cleft secondary palate. 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.
Embryos An original pair of CL/Fr mice was kindly provided by the 2nd Department of Oral and Maxillofacial Surgery, Niigata University School of Dentistry, Japan. Cleft lip/palate-resistant C57BL/6 mice were obtained from Japan SLC, Inc. Our Animal Use Protocol was reviewed and approved by the Institutional Review Board in the Faculty of Dentistry at Kyushu University. For collection of embryos in each strain, females cohabited with one fertile male overnight, and the morning when a copulatory plug was detected was designated as embryonic day 0 (E0). On the evening of embryonic day 13 (E13.5), females were killed by cervical dislocation, and live embryos were removed from the placental membranes. Under a dissecting microscope, CL/Fr embryos were classified into normal (CL/Fr-N) (Fig. 1B  ), 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.
Organ Culture Procedures Palatal organ culture was established as described in Brunet et al.(1993) and Taya et al.(1999). Twenty pairs of palatal shelves from CL/Fr-N and 10 pairs of palatal shelves from CL/Fr-BCL were dissected from the embryos’ heads and placed on 0.8-µm Millipore filters (Millipore Corporation, Billerica, MA, USA) with their medial edges in contact. The filters were positioned on grids and placed in cold DMEM/F-12 (Gibco, Paisley, Scotland, UK) supplemented with 1% penicillin/streptomycin, and fresh ascorbic acid (40 µg/mL) was added. The cultures were maintained at 37°C and 100% humidity in an air incubator for up to 72 hrs. The medium was replaced every 12 hrs, and the cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline at 48 hrs for histologic paraffin sections. The 5-µm sections were stained with Harris’ hematoxylin and eosin and photographed.
Assessment of Cell Proliferation
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
Palatal Organ Culture In culture, the bilateral palatal shelves from CL/Fr-BCL began to fuse up to 48 hrs after being explanted at E13.5 (Fig. 1E  ). 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
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 our study, palatal shelves dissected from CL/Fr-BCL fused histologically in vitro. This result is consistent with a previous report showing that pairs of palatal shelves from cleft lip embryos in A/Jax mice, which have spontaneous cleft lip and palate, also fused in vitro (Pourtois, 1967). The success of palatal fusion in CL/Fr-BCL in vitro means that the presence of cleft lip alone obviously did not affect the propensity of the palatal processes to fuse. 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.
We thank Dr. Masaki Nagata (The 2nd Department of Oral and Maxillofacial Surgery, Niigata University School of Dentistry) for providing a pair of CL/Fr mice. We also thank Prof. Minoru Nakata and Dr. Kazuaki Nonaka, who were generous with their help in the preparation of experimental facilities, and for encouragement. This work was supported by a Grant (No.13771266) from the Japanese Society for Promotion of Science. Received for publication January 10, 2004. Revision received July 27, 2004. Accepted for publication July 28, 2004.
Journal of Dental Research, Vol. 83, No. 10,
797-801 (2004)
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