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The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo

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The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo Acta Histochem. Cytochem. 52 (1): 19–26, 2019 doi: 10.1267/ahc.18038 Regular Article The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo Tadahiro Shimomura1, Masayoshi Kawakami1, Kouko Tatsumi2, Tatsuhide Tanaka2, Shoko Morita-Takemura2, Tadaaki Kirita1 and Akio Wanaka2 1Department of Oral and Maxillofacial Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634–8521, Japan and 2Department of Anatomy and Neurosciences, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634–8521, Japan Received October 22, 2018; accepted December 29, 2018; published online February 23, 2019 Cleft lip with or without cleft palate (CLP) usually results from a failure of the medial nasal prominences to fuse with the lateral and maxillary prominences. This failure inhibits facial morphogenesis regulated by several major morphogenetic signaling pathways. We hypothesized that CLP results from the failure of the Wnt signaling pathway. To examine whether Wnt signaling can influences upper jaw development, we applied beads soaked with Dickkopf-1 (Dkk-1), Alsterpaullone (AL) or Wnt3a to the right side of the maxillary prominence of the chick embryo. The embryo showed a defect of the maxilla on the treated side, and skeletal staining revealed hypoplasia of the premaxilla and palatine bone as a result of Dkk-1-soaked bead implantation. 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers in the treated maxillary prominence were significantly lower at both 24 and 48 hr after implantation. Down-regulation of the expression of Bmp4, Tbx22, Sox9, and Barx1 was confirmed in the maxillary prominence treated with Dkk-1, which indicated that the deformity of the maxillary bone was controlled by gene targets of the Wnt signaling pathway. Expression of N-cadherin was seen immunohistochemically in the maxillary prominences of embryos at 6 hr and increased at 24 hr after AL treatment. Wnt signaling enhanced by AL or Wnt3a up-regulated the expression levels of Msx1, Bmp4, Tbx22, Sox9, and Barx1. Our data suggest that the Wnt signaling pathway regulates maxillary morphogenesis and growth through Bmp4, Tbx22, Sox9, and Barx1. Wnt signaling might regulate N-cadherin expression via Msx1, resulting in cell aggregation for osteochondrogenesis. Key words: Wnt signaling, Dickkopf-1, N-cadherin, cleft lip, chick embryo I. Introduction intercellular signaling pathways and plays fundamental roles in cellular proliferation, regeneration, and differentia- Cleft lip with or without cleft palate (CLP) is one of tion. In mice, several Wnt ligands, including Wnt3 and the most common birth defects in humans [3, 25]. CLP usu- Wnt9b, are expressed in the ectoderm on the early facial ally results from a failure of the medial nasal prominences prominences [20]. Variation in Wnt signaling is tightly to fuse with the lateral and maxillary prominences, which associated with non-syndromic CLP [6]. The signaling inhibits growth and development. Facial development is activity is initiated by Wnt ligands binding to a dual recep- regulated by several major morphogenetic signaling path- tor complex, LRP5/6, resulting in the stabilization and ways, such as Wnt, FGF, and BMP signaling [11, 28, 34, 38]. nuclear translocation of β-catenin, which further binds with The Wnt signaling pathway is one of the prominent TCF/LEF to regulate the transcription of downstream target genes. WNT signaling activity is negatively regulated by secreted proteins such as Dickkopf-1 (Dkk-1). Dkk-1 Correspondence to: Masayoshi Kawakami, DDS, Ph.D., Department of dampens signaling activity by disengaging LRP from the Oral and Maxillofacial Surgery, Nara Medical University, 840 Shijo-cho, receptor complex, thereby blocking the WNT ligand- Kashihara, Nara 634–8521, Japan. E-mail: [email protected] © 2019 The Japan Society of Histochemistry and Cytochemistry 20 Shimomura et al. receptor interaction [9]. In the absence of Wnt signaling, making small incisions at stage 22 (before the fusion of free β-catenin is ubiquitinated and degraded by the protea- the frontonasal prominence and maxillary prominence) some. Mice deficient for LRP6 receptor exhibited cleft lip under a microscope (Leica MZ7.5, Wetzlar, Germany). with cleft palate, resembling the common birth defect CLP After bead implantation, the embryos were reincubated for in humans [31]. Ablation of Wnt secretion in the ectoderm additional periods ranging from 6 hr to 12 days (stage 38). leads to a failure of the frontonasal and maxillary promi- Animal procedures were approved by the Nara Medical nences to form [38]. The frontonasal and upper jaw promi- University Animal Care and Use Committee. nences do not form when β-catenin is conditionally ablated in the facial ectoderm [18, 28, 37]. However, the function Bromodeoxyuridine labeling and immunofluoresence of Wnt signaling in craniofacial development is poorly staining understood. After incubation for 6, 24, or 48 hr, the bead- Interaction between Wnt signaling and other signaling implanted embryos were injected with 10 μl of 5-bromo-2'- pathways may be important for craniofacial development. deoxyuridine (BrdU) (Sigma-Aldrich, St Louis, MO, USA) Bone morphogenetic protein (BMP) signaling is known to 2 hr before euthanization. They were then fixed in 4% par- be important for growth of facial prominences by control- aformaldehyde and processed in paraffin. Paraffin sections ling the expression of Sox9 and Runx2 in the maxillofacial were pretreated with 2N HCl for 5 min after antigen activa- region [5, 23, 36]. While BMP signaling mainly affects tion in 0.1 M sodium citrate buffer at 95°C for 10 min. The proliferation, fibroblast growth factor (FGF) signaling in slides were incubated with rat anti-BrdU monoclonal anti- the cranial frontonasal mass is required to maintain cell body (1:100). proliferation and cell survival [33]. BMP and FGF in the Another set of embryos were treated with Dkk-1- or frontonasal mass regulate Tbx22 expression, which can AL-soaked beads at stage 20 and collected 24 or 48 hr after contribute to CLP [12]. Wnt9b gene expression positively implantation. Antigen retrieval for anti-N-cadherin anti- regulates FGF gene expression in the ectoderm of the max- body was performed by incubating the beads in 0.1 M illary prominence, and is required for the proliferation of sodium citrate buffer for 10 min at 95°C. Rabbit polyclonal mesenchymal cells of the maxillary prominence in mice antibody to N-cadherin (1:200; Abcam, Cambridge, UK) [14]. was applied overnight at 4°C. For immunohistochemical Wnt signaling plays crucial roles in embryonic devel- staining, biotinylated anti-rat IgG (H+L) (BA-9400, Vector opment, but mechanisms of Wnt signaling function in the Laboratories, Burlingame, CA, USA) and were applied for facial prominence are not well characterized. It is likely 1 hr at room temperature. Immunofluorescence sections that decreased signaling function can explain all of the were incubated with Alexa Flour 488-conjugated goat anti- mechanisms underlying CLP. We hypothesize that CLP rabbit IgG (H+L) (Invitrogen, CA). The sections were results from a failure in development and growth of the coverslipped in VECTASHIELD Hard Set Mounting facial prominence due to reduced Wnt signaling. In this Medium with DAPI (Vector Laboratories, Burlingame, CA, study, we used chick embryos and examined whether the USA). Images were acquired using a confocal fluorescence Wnt signaling pathway is required for maxillofacial devel- microscope (C2/Ni-E, Nikon, Tokyo, Japan). opment. To test the hypothesis, we used Dkk-1 as an antagonist and investigated morphological changes and Real-time RT-PCR gene expression in the developing upper jaw and lip. After incubation for 6 hr, the maxillary prominence of the bead-treated side was dissected and stored in RNA later II. Materials and Methods (Ambion, Austin, TX, USA) (N = 6–8 for each experi- ment). Total RNA was isolated with Sepasol-RNA I Super Embryos and bead implantation G (Nacalai Tesque, Kyoto, Japan) from the dissected Fertilized white leghorn eggs were obtained from processes. We synthesized cDNA using a QuantiTect Takeuchi Farm (Nara, Japan) and incubated at 38°C until Reverse Transcription Kit (Qiagen, Mainz, Germany), and the embryos reached the appropriate stage [10]. Affi-Gel chicken cDNA was prepared as previously described [18]. Blue beads (Bio-Rad, Hercules, CA, USA) were soaked in The polymerase chain reaction (PCR) primers used were as 0.1 mg/ml Dkk-1 (Abcam, Cambridge, UK), 0.5 mg/ml previously described [12, 18, 20, 29, 32, 38]. Semi- Noggin (R&D Systems, Minneapolis, MN, USA) or 1.0 quantitative RT-PCR was performed according to the man- mg/ml Wnt3a (R&D Systems, Minneapolis, MN, USA) ufacturer’s protocol. At least six processes were included in with 2% bovine serum albumin (BSA). AG1-X2 beads one sample, and three samples were examined in each (Bio-Rad, Hercules, CA, USA) was in 10 mg/ml Alster- quantitative analysis for statistical significance. paullone (AL) (Sigma-Aldrich, St Louis, MO, USA) in DMSO for 1 hr with a drop of 0.01% Fast Green added for Skeletal staining bead visualization. Sham operations were performed using To study morphological changes of bone and cartilage, beads soaked in 2% BSA. In all cases, the beads were embryos were fixed at stage 38 (after 12 days’ incubation) inserted on right side of the maxillary prominence by and kept in 100% ethanol for 4 days. The embryos were Wnt Signaling Pathway in Upper Jaw Development 21 then infiltrated with acetone for 4 days, stained with Alcian blue and Alizarin red for up to 10 days, and cleared with KOH as previously described [18]. Statistical analysis The delta-delta CT method and ANOVA using Micro- soft Excel were carried out for statistical comparisons, and a value of P < 0.05 was considered significant. III. Results External and skeletal effects of Dkk-1-soaked bead implantation Dkk-1 is an antagonist of the canonical Wnt signaling pathway by reducting in β-catenin [7, 18, 22].
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