TGF Signaling Regulates Decorin and Biglycan Expression and Distribution During Murine Palatal Fusion
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abstract
Orofacial clefts are the most prevalent craniofacial birth defects. The secondary palate in humans and mice forms from maxillary process shelves that are mesenchyme covered with epithelium. The shelves adhere to form the midline epithelial seam (MES). MES cells then either proceed through epithelial to mesenchymal transition (EMT) or apoptosis to yield a fused palate. The fusion of opposing palatal shelves is a critical event, and alteration may cause cleft secondary palates. Transforming growth factor beta 3 (TGF3) signaling is essential for normal palatogenesis. Previous studies showed that the large family of chondroitin sulphate proteoglycans (CSPG) on the apical surface of the medial edge epithelia (MEE) were necessary for palatal shelf adhesion using an antibody directed to the sugar chain (Gato et al, 2002 Dev Biol). However, the identity of the CSPGs involved in palate fusion remains to be elucidated. The objective of this study was to investigate the expression of two specific proteoglycans with chondroitin (CS) or dermatan sulfate (DS) side chains, decorin and biglycan, in palatal shelf adhesion. Decorin is in the small leucinerich proteoglycan (SLRP) family. It consists of a protein core containing leucine repeats with a glycosaminoglycan (GAG) chain consisting of either CS or DS. Biglycan is also a SLRP with two GAG chains consisting of either CS or DS, with DS being more abundant in most connective tissues. We also asked if TGF signaling was necessary for their expression and activity.MethodsMouse palatal shelves from three stages of palatal shelf development (embryonic days 13.514.5) were used for immunohistochemistry and laser capture microdissection (LCM) to collect MEE cells for realtime polymerase chain reaction (RTPCR). Immunohistochemistry on mouse palates treated with TGF RI kinase inhibitor (SB 431542) for 48 hours were used to investigate the effects on blocking TGF signaling.ResultsThe expression of biglycan was detected on the lateral surface MEE cells as early as E13.5, when the palatal shelves have elevated but not approached each other. As palatal shelves approached, a thin layer of decorin and biglycan proteins were distributed on the apical and lateral surfaces of the MEE cells. Palatal shelves in close contact had abundant expression of both proteins on the apical and lateral surfaces of MEE. Furthermore, the expression continued on the lateral surfaces of the MES cells after fusion. The immunohistochemical staining for biglycan was more intense than decorin at all palatal stages. Biglycan mRNA levels, in contrast to decorin, coincided with the protein expression. Palatal shelves treated with TGF RI kinase inhibitor failed to fuse and had a persistent MES. Decorin and biglycan proteins were not expressed in the MEE/MES cells. In conclusion, the expression pattern of decorin and biglycan in the MEE and MES during palatal adhesion indicates that these proteoglycans have a role in normal palatal fusion. Furthermore, they are dependent on TGF signaling.Support or Funding InformationSupported by Baylor Oral Health Foundation and the Department of Biomedical Sciences.
