Craniofacial abnormality with skeletal dysplasia in mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase-1

Hiroko Ida-Yonemochi,Shunichi Shibata,Kosei Takeuchi,Yuka Takeuchi,Yuki Morioka,Michihiro Igarashi,Nobuo Sugiura,Hideto Watanabe,Ryosuke Kawakami,Wataru Morita,Takeshi Imamura,Toshiya Sato,Hayato Ohshima

doi:10.1038/s41598-018-35412-5

Abstract

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

Highlights

Craniofacial skeletal development is a complex series of events composed of two distinct ossification modes: intramembranous ossification and endochondral ossification
Connective tissue surrounding osteopontin (OPN) and collagen type 1-positive neonatal bones was thinning in T1KO mice, and immunostaining intensity of Chondroitin sulfate (CS), Wnt3a and β-catenin was attenuated in the palatal mesenchyme in the osteogenic front of T1KO mice on postnatal day 0 (P0) (Fig. 1B, Supplementary Fig. S2)
Collagen fibers visualized by second harmonic generation (SHG) microscopy were irregular, thick and aggregated in the calvaria (Fig. 2D) and scalp (Fig. 2E) of T1KO mice on P0

Summary

Introduction

Craniofacial skeletal development is a complex series of events composed of two distinct ossification modes: intramembranous ossification and endochondral ossification. CS-related signaling molecules, such as Wnts, hedgehog and fibroblast growth factors (FGFs), regulate palatogenesis[15,16], cranial base development[17] and cranial suture morphogenesis[18,19], and these mutant mice exhibited skull deformation including cleft lip and palate[20]. There is little information about the importance of CS and T1 in intramembranous ossification It is well worth analyzing the role of CS and associated signaling molecules in membrane bone formation to understand craniofacial development. A reduced amount of collagen type 1 and disarranged collagen fibers in the connective tissues were observed in T1KO mice These results suggest that CS and T1 play important roles in connective tissue organization and following intramembranous ossification of the craniofacial skeleton, and it is conceivable that a marked CS reduction may cause connective tissue disorders in T1KO mice

Methods

Results

Conclusion