Retinoic acid delays murine palatal shelf elevation by inhibiting Wnt5a-mediated noncanonical Wnt signaling and downstream Cdc-42/F-actin remodeling in mesenchymal cells

Yan-Qing Ma; Xin-Yu Zhang; Shi-Wei Zhao; Dou Li; Min-Qin Cai; Hui Yang; Xiao-Ming Wang; Hui Xue

doi:10.1002/bdr2.2244

Retinoic acid delays murine palatal shelf elevation by inhibiting Wnt5a-mediated noncanonical Wnt signaling and downstream Cdc-42/F-actin remodeling in mesenchymal cells

Birth Defects Res. 2023 Oct 15;115(17):1658-1673. doi: 10.1002/bdr2.2244. Epub 2023 Sep 7.

Authors

Yan-Qing Ma^{1

2}, Xin-Yu Zhang^{1

2}, Shi-Wei Zhao^{1

2}, Dou Li^{1

2}, Min-Qin Cai^{1

2}, Hui Yang^{1

2}, Xiao-Ming Wang^{1

2}, Hui Xue³

Affiliations

¹ Key Laboratory of Dental Maxillofacial Reconstruction and Biological Intelligence Manufacturing (No: 20JR10RA653 - ZDKF20210401), School of Stomatology, Lanzhou University, Lanzhou, Gansu Province, People's Republic of China.
² Department of Orthodontics, School of Stomatology, Lanzhou University, Lanzhou, Gansu Province, People's Republic of China.
³ Department of Stomatology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, Jiangsu Province, People's Republic of China.

PMID: 37675882
DOI: 10.1002/bdr2.2244

Abstract

Background: Mammalian palatal shelves erupted from maxillary prominences undergo vertical extention, transient elevation, and horizontal growth to fuse. Previous studies in mice reported that the retinoic acid (RA) contributed to cleft palate in high incidence by delaying the elevating procedure, but little was known about the underlying biological mechanisms.

Methods: In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the RA-treated mice model. In situ hybridization and RT-qPCR were used to detect the expression of genes involved in Wnt signaling pathway. The palatal mesenchymal cells were cultured in vitro, and stimulated with RA or CASIN, and co-treated with Foxy5. Wnt5a and Ccd42 expression were evaluated by immunofluorescence staining. Phalloidin was used to label the microfilament cytoskeleton (F-actin) in cultured cells.

Results: We revealed that RA resulted in 100% incidence of cleft palate in mouse embryos, and the expression of genes responsible for Wnt5a-mediated noncanonical Wnt signal transduction were specifically downregulated in mesenchymal palatal shelves. The in vitro study of palatal mesenchymal cells indicated that RA treatment disrupted the organized remodeling of cytoskeleton, an indicative structure of cell migration regulated by the small Rho GTPase Cdc42. Moreover, we showed that the suppression of cytoskeleton and cell migration induced by RA was partially restored using the small molecule Foxy-5-mediated activation of Wnt5A, and this restoration was attenuated by CASIN (a selective GTPase Cdc42 inhibitor) again.

Conclusions: These data identified a crucial mechanism for Wnt5a-mediated noncanonical Wnt signaling in acting downstream of Rho GTPase Cdc42 to regulate cytoskeletal remodeling and cell migration during the process of palate elevation. Our study provided a new explanation for the cause of cleft palate induced by RA.

Keywords: Rho GTPase; Wnt signaling; cleft palate; cytoskeleton; retinoic acid.

Abstract

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