Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes

Accepted Manuscript Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes Chisato Sakuma, Hideto Imura, Tomohiro Yamada, Toshio Sugahara, Azumi Hirata, Yayoi Ikeda, Nagato Natsume PII:

S1010-5182(18)30470-0

DOI:

10.1016/j.jcms.2018.09.016

Reference:

YJCMS 3102

To appear in:

Journal of Cranio-Maxillo-Facial Surgery

Received Date: 27 June 2018 Revised Date:

21 August 2018

Accepted Date: 12 September 2018

Please cite this article as: Sakuma C, Imura H, Yamada T, Sugahara T, Hirata A, Ikeda Y, Natsume N, Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes, Journal of Cranio-Maxillofacial Surgery (2018), doi: https://doi.org/10.1016/j.jcms.2018.09.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cleft palate formation after palatal fusion occurs due to the rupture of epithelial

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basement membranes

Chisato Sakumaa, Hideto Imuraa,*, Tomohiro Yamadab, Toshio Sugaharaa, Azumi

Division of Research and Treatment for Oral and Maxillofacial Congenital Anomalies,

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Hiratac, Yayoi Ikedad, Nagato Natsumea

School of Dentistry, Aichi-Gakuin University, Japan b

Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic and

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Department of Anatomy and Cell Biology, Faculty of Medicine, Osaka Medical

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College, Japan

Department of Anatomy, School of Dentistry, Aichi-Gakuin University, Japan

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Surgical Science, Faculty of Dental Science, Kyushu University, Japan

* Corresponding author.

Tel.: +81 52 751 7181; fax: +81 52 759 2151. E-mail address: [email protected]

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Funding: This research did not receive any specific grant from funding agencies in the

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public, commercial, or not-for-profit sectors.

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ABSTRACT

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and hydronephrosis in the mouse embryo. Cleft palate occurs due to failure in palatal grow, but the

underlying mechanisms are unclear. We investigated the mechanisms of cleft palate

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development in TCDD-exposed mouse embryos. We administered olive oil (control

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group) or TCDD diluted in olive oil (40 µg/kg) via gastric tubes to pregnant mice on gestational day (GD) 12. Embryos of control and TCDD-exposed groups were removed from pregnant mice on GD 14 and GD 15, respectively. One mouse embryo from the

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control group had anteroposterior palatal fusion. Palatal fusion was observed in three TCDD-exposed mouse embryos. Palates of TCDD-exposed mice fused from the interior

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to the middle of the palates, while the palates were separated in the posterior region. The middle of the embryonic palatal shelves in TCDD-exposed animals was narrow and

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split at the fusional position. At this position, palatal and blood cells were dispersed from the palatal tissue and the epithelium was split, with a discontinuous basement membrane. The results suggest that decreased intercellular adhesion or insufficient tissue strength of the palatal shelves may be involved in the development of cleft palate following palatal fusion.

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Keywords: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD); basement membrane; cell

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defect; cleft palate; post-fusional palatal split

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INTRODUCTION Cleft lip and cleft palate are one of the most common observable congenital anomalies

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in humans (Natsume, 1987; Natsume, et al., 2000). The incidence of cleft lip and cleft palate in the Japanese population is reported to be approximately one in 500 people. Patients with cleft palate constitute only 30% of all cleft lip and cleft palate cases

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palate have yet to be fully elucidated.

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(Nagase et al., 2010; Natsume and Imura, 2017). The mechanisms underlying cleft

Human palatal growth begins at embryonic week 6. At this time, bilateral palatal processes appear next to the tongue (Bush and Jiang, 2012). At embryonic week 8, the

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tongue descends, and bilateral palatal processes rise above the tongue in the form of vertical shelves. After the elevated palatal processes expand and contact each other in

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the midline, at embryonic week 10, they form the medial epithelium seam (MES) in the midline, commencing palatal fusion. Subsequently, the MES disappears with apoptosis

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of epithelial cells and mesenchymal tissues, leading to fusion and complete palatal development at embryonic week 12. Cleft palate is induced by a failure during the stage of palatal shelf development. Cleft palate seems to occur due to errors in the elevation of the palatal shelves (Murray et al., 2007), an impaired ability of the elevated shelves to grow vertically (Fitch, 1961), the inability of the bilateral palatal processes to fuse

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completely (Jin et al., 2014), or a separation of the palatal shelves after palatal fusion (Kitamura, 1966; Kitamura, 1990).

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The etiology of cleft palate is multifactorial, and is affected by both genetic factors (TGFB, MSX1, TBX22, IRF6, and MEOX2) (Mijiti et al., 2015; Mori et al., 2016) and environmental factors (drinking alcohol, smoking, maternal obesity,

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2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD], and vitamin A) (Blanco et al., 2015;

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Natsume et al., 2000). TCDD is an environmental factor that is known to contribute to cleft palate. TCDD is a highly toxic dioxin and induces hydronephrosis and cleft palate in mice (Couture et al., 1990). In a recent report, Yamada et al. demonstrated that cleft

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palate development in mouse embryos could be induced in 100% of animals exposed to a critical concentration of TCDD (Yamada et al., 2006). However, Imura et al. (2010)

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reported that, in mouse embryos exposed to this critical concentration of TCDD, the incidence of non−cleft palate mouse embryos at embryonic week 14, 15, and 16 was 4%,

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17%, and 13%, respectively (Imura et al., 2010). They reported that cleft palate could occur after palatal fusion. Nevertheless, the mechanisms underlying this process are not yet clear.

We hypothesized that exposure to TCDD during palatal growth induces palatal tissue anomalies and induces palatal deformity. In the present study, we investigated how

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palatal shelves lead to cleft palate after palatal fusion.

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MATERIALS AND METHODS Animals

Female ICR, 8–10 weeks of age (CLEA, Tokyo, Japan), were used in these studies.

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Animals were housed under controlled conditions at a temperature of 22 ± 1°C,

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humidity of 50 ± 10%, and a 12-h/12-h light/dark cycle, with access to food and water ad libitum. Female mice were crossed with males of the same strain and checked for vaginal plugs the following morning (designated as GD 0). On GD 12, pregnant mice

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were administered with either 1 dose of olive oil (control group) or TCDD (Accu Standard, New Haven, CT, USA) diluted in olive oil at a dose of 40 µg/kg body weight,

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the minimum concentration to induce a 100% cleft palate rate (TCDD-exposed group). Pregnant mice of the control and TCDD-exposed groups were killed on GD 14 and GD

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15, respectively. Embryos were removed for experimental use. The TCDD-exposed embryos had a fused palate by GD 15; this was about 1 day slower than in control embryos. Therefore, the control embryos removed on GD 14 were used to compare with the TCDD-exposed embryos with a fused palate. Of the total of 83 GD 14 control embryos, 36 embryos had a fused palate. Of the total of 158 TCDD-exposed embryos

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on GD 15, only 27 embryos had a fused palate, based on stereoscopic microscope examination. One of the control embryos was selected at random from the embryos that

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demonstrated palatal fusion, and three of the TCDD-exposed embryos were selected at random from among the embryos that demonstrated palatal fusion in that group.

All experimental protocols in the present study strictly adhered to the Guidelines for

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Science of Okayama University, Okayama, Japan.

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Animal Research of the Graduate School of Medicine, Dentistry, and Pharmaceutical

Histological analysis

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One randomly selected embryo from the control group and three embryos from the TCDD-exposed group that had palatal fusion were perfused with paraformaldehyde in

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0.1 M phosphate buffer (pH 7.4) for 24 h, prior to routine embedding in paraffin. Paraffin frontal sections (6-µm thick) were cut using a microtome, mounted on glass

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slides, and stained with hematoxylin and eosin (HE).

Immunohistochemistry

The basement membranes of fusing palates and their cells from one GD 14 control embryo and three TCDD-exposed GD15 embryos were compared with those of fully

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fused palates. Histological sections were prepared and washed with phosphate-buffered saline (PBS) after removal of paraffin with xylene and subsequent dehydration. Sections

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were incubated with primary antibodies overnight at 4°C. These included anti-pan cytokeratin (CK-4, -5, -6, -8, -10, -13, -18) mouse monoclonal antibody (1:200,

#ab7753, Abcam, Cambridge, UK) to examine the localization of the epithelial cells,

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with anti-vimentin rabbit polyclonal antibody (1:2000, #ab 92547, Abcam) to examine

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the localization of the mesenchymal cells, and anti-laminin rabbit polyclonal antibody (1:500, #L9393, Sigma, St. Louis, MO, USA) to examine the localization of the basal membrane. Sections were then incubated for 30 minutes with the labeled polymer. After

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washing with PBS, sections were incubated with a peroxidase staining kit (Nova RED Substrate Kit, #SK-4800, Vector Laboratories, Burlingame, CA, USA) for 5–10 minutes

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at room temperature. Sections were examined and photographed under an all-in-one-type fluorescence microscope (BZ-X710, Keyence, Osaka, Japan) using BZ

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Analyzer Software (Keyence).

RESULTS

Histological observations Representative histological sections of the palates of mouse embryos in the control

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group, stained with HE, are shown in Figures 1A–C. Complete fusion of the palate was observed, and the MES was observed in the middle of the palatal fusion. Histological

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sections of three palates of mouse embryos exposed to TCDD (case Nos. 1, 2, and 3), stained with HE, are shown in Figures 2A–I. All three of the TCDD-exposed palates

were fused in the anterior region, but the palates remained cleft in the posterior region.

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TCDD-exposed palates No. 1 and No. 2 were split in the hard palates, whereas case No.

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3 was split in the soft palate.

In control palate sections, mesenchymal cells in the anterior palatal shelf were ubiquitously distributed. In contrast, in TCDD-exposed palate sections, mesenchymal

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cells were sparse in the center of the anterior palatal shelves. The palatal shelves were thin and split in the middle of the palates. In this region in case No. 1, cells were

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dispersed from the palatal shelf. Defective palatal cells and blood corpuscles were

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released from the palatal shelf in case No. 3.

Immunohistological observations Immunostaining for cytokeratin, vimentin, and laminin of the palates from the anterior fused region to the posterior split region in TCDD-exposed mouse embryos were compared with those of control mouse embryos (Figs. 3 and 4). In both control and

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TCDD-exposed embryos, cells immunopositive for cytokeratin were localized in the oral and nasal mucosal epithelium, as well as in the MES. Cells immunopositive for

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vimentin were distributed throughout the palatal shelves, but not in the MES, at the anterior palatal shelves. Laminin-immunopositive cells were localized in the

submucosal tissue of the anterior palatal shelves in both control and TCDD-exposed

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embryos. The anterior palatal shelves of control and TCDD-exposed embryos

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demonstrated continuity of the epithelium, mesenchymal tissue, and basement membrane (Fig. 3A–L). In TCDD-exposed embryos, at the narrow posterior part of the palates, cytokeratin-immunopositive cells and vimentin-immunopositive cells were

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detected partially. The epithelium and mesenchyme of the palates were observed to be discontinuous. In this region in these embryos, the basement membrane demonstrated a

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(Fig. 4A–L).

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split, with less localization of laminin-positive cells in the palatal submucosal tissue

DISCUSSION

TCDD is an endocrine disruptor and has been reported to have several toxic biological effects, such as acute toxicity, immunotoxicity, genotoxicity, developmental toxicity, and carcinogenicity (Fujimaki et al., 2002). Maternal exposure to TCDD is

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known to cause fetal hydronephrosis and cleft palate in terms of macro-developmental toxicity, and to cause epithelial dysplasia in terms of micro-developmental toxicity

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(Couture et al., 1990; Puhvel and Sakamoto, 1988). In a study that examined the mechanisms underlying TCDD-induced formation of cleft palate, 100% of embryos exposed to TCDD on GD12.5 developed cleft palate, but 0% of aryl hydrocarbon

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receptor (AhR)−knockout embryos exposed to TCDD on GD12.5 developed cleft palate

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(Mimura et al., 1997). This study suggests that AhR may be a molecular substrate underpinning the effects of TCDD on cleft palate development. Regarding the etiology of TCDD-induced cleft palate, it is considered that the lateral

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development of both palatine processes is obstructed by TCDD and that they fail to contact each other (Mimura et al., 1997), that TCDD inhibits palatal fusion by reducing

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the disappearance of MES cells (Takagi et al., 2000), or that TCDD leads to a palatal split after fusion of the palatine processes (Yamada et al., 2014). Findings by Jin and

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Ding corroborated these proposed mechanisms (Jin and Ding, 2006). However, Jin and Ding observed 35.5% cleft palate embryos among Meox-2-/- embryos, and observed that none of the clefts in Meox-2 mutants demonstrated epithelium along the medial edge. The authors hypothesized that the cleft palates in Meox-2 mutant embryos may be caused by a post-fusion defect.

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It is possible that cleft palate formation after palatal fusion can be induced by factors other than TCDD exposure. In this study, we observed several TCDD-exposed

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embryonic palates with anterior fusion, a palate split in the middle, and a posterior palatal cleft. Several mechanisms for the post-fusional cleft palate include: a)

disappearance of mesenchymal cells by apoptosis caused palatal defects, b) decreased

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tissue strength due to decreased intercellular adhesion leading to a split after palatal

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fusion, and c) separation of palatal tissue by structural disorder of the basement membrane. In this study, we discovered that vimentin-immunopositive cells were absent in the middle of the narrow palate in TCDD-exposed animals. This result implies that

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apoptosis of mesenchymal cells at the palatal fusion region may induce post-fusional defects. Future studies should investigate cell death from the anterior palatal shelves to

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the posterior cleft palates after exposure to TCDD. The cells in the middle of the palatal shelves in TCDD-exposed embryos were less densely distributed. Furthermore, there

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were dispersed cells and cellular defects at the narrow palatal shelves. This suggests that tissue strength was decreased, due to decreased intercellular adhesion, and may have caused a split of the plates after fusion. Imura et al. reported that E-cadherin, an adhesion factor, was not expressed in the epithelial and mesenchymal tissue of the nasal mucosa, suggesting that the mechanisms of cleft palate after palatal fusion are related to

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intercellular factors (Imura et al., 2010). Future studies should examine the expression of intercellular adhesion elements from the palatal shelves to the defective palates to

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elucidate the development of cleft palate. The phenomenon of defective tissue fusion has parallels with cancer infiltration or

metastasis (Imura et al., 2018). Cancer cells can infiltrate and metastasize by repeatedly

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disrupting the basement membrane (Sato et al., 1994). The basement membrane is

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composed of extracellular matrix proteins. When cancer cells disrupt the basement membrane to infiltrate another organ, they produce matrix metalloproteases, such as MT1-MMP and MMP-2, as well as invadopodia containing actin filaments (Gligorijevic

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et al., 2012). Given the finding of a ruptured epithelium and basement membrane in the narrow palatal shelves of embryos exposed to TCDD, we suggest that the factors

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contributing to cleft palate after palatal fusion may be related to structural anomalies of the basement membrane. It will therefore be necessary to investigate the potential

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involvement of MT1-MMP or MMP-2 in formation of cleft palate after palatal fusion.

CONCLUSION

TCDD-exposed palatal shelves were observed to be thin in the middle region. In this area, mesenchymal cells were sparse, and several palatal cells were dispersed or

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defective in certain sections. Therefore, decreased intercellular adhesion or a reduction in the tissue strength of the palatal shelves due to apoptosis may be involved in the

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had a fused palate at a stage prior to cleft palate observation.

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development of cleft palate after palatal fusion, although few TCDD-exposed embryos

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Conflict of interest

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The authors have no conflict of interest to declare.

Acknowledgements

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This work was supported by JSPS KAKENHI (No.16K11772, No.16K11773).

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Figure 1. Hematoxylin and eosin−stained image of the palate of the control group embryo on gestational day 14 (bar: 100 µm). The palate is fused from the anterior to the

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posterior region (A, B, C), and the epithelial seam (A, B, C arrow) can be recognized in the middle of the palate. Cells are uniformly distributed throughout the palatal shelf (A,

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B, C).

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Figure 2. Hematoxylin and eosin−stained image of the palatal fusion seen in three embryos from the TCDD-exposed group on the 15th gestational day (bar: 100 µm). All three embryos show fusion in the anterior part of the palate (A, D, G), whereas the

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posterior parts of the palate are separated (C, F, I). Cells are sparse in the median part of the palatal shelf (A, D arrow). Furthermore, in the narrowed part of the palatal shelves,

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cell dropout (B arrow head), cell defect (H arrow), and blood cell component dropout

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(arrow H arrowhead) were recognized.

Figure 3. Immunohistochemically stained images of the anterior part of the palate (control group: gestational day 14, TCDD-exposed group: gestational day 15) (bar: 100 µm). Cytokeratin-positive cells were found in the mucosal epithelium of the nasal and oral cavities in both the control and TCDD-exposed groups, and in the epithelial seam

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(A, D, G, J). Vimentin-positive cells were found across the entire palatal shelf, except in the epithelial seam (B, E, H, K). Both the control and TCDD-exposed groups had

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laminin-positive cells under the epithelium (C, F, I, L).

Figure 4. Immunohistochemically stained images of the middle section of the palate of

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the control group (embryonic day 14) and the TCDD-exposed group (embryonic day

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15) (bar: 100 µm). In the middle section of the palate (A–C) of the control group, cytokeratin-positive cells (A) were observed in the mucosal epithelium and epithelial coad. Vimentin-positive cells (B) and laminin-positive cells were observed under the

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epithelium throughout the palatal shelf except at the epithelial seam (C arrow). The disappearance of cytokeratin and vimentin positive cells was observed in the palatal

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shelf constriction (D , E, G, H, J, K) in the TCDD-exposed group (G, H, K arrow). The disappearance of laminin-positive cells (F, I, L) was also observed in the

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TCCD-exposed group (F, I arrow).

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