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

by Selleckchem | Dec 10 2023

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.

Comments:

Your study presents a comprehensive investigation into the mechanisms underlying cleft palate formation induced by retinoic acid (RA) treatment in mice. The research method involved a series of analyses, including histological staining, immunofluorescence, in situ hybridization, RT-qPCR, and in vitro cell culture experiments. Here's a breakdown of your findings and their significance:

### 1. **RA-Induced Cleft Palate:**
- **Incidence Rate:** RA treatment led to a 100% incidence of cleft palate in mouse embryos, suggesting a strong correlation between RA exposure and this congenital condition.

### 2. **Downregulation of Wnt5a-Mediated Noncanonical Wnt Signaling:**
- **Gene Expression Analysis:** Genes involved in Wnt5a-mediated noncanonical Wnt signaling were found to be specifically downregulated in mesenchymal palatal shelves following RA exposure.
- **Role of Wnt5A:** This indicates the involvement of Wnt5a in the disruption caused by RA, suggesting a potential key player in palate development.

### 3. **RA-Induced Disruption of Cytoskeleton and Cell Migration:**
   - **In Vitro Studies:** RA treatment disrupted the organized remodeling of the cytoskeleton in palatal mesenchymal cells. This disruption is critical as the cytoskeleton is essential for cell migration.
   - **Role of Cdc42:** The study highlighted the role of the small Rho GTPase Cdc42, a regulator of cytoskeletal dynamics.
   - **Foxy-5 Treatment:** Foxy-5, a small molecule, partially restored the disruption induced by RA. Foxy-5 activated Wnt5A, emphasizing the importance of Wnt5a-mediated noncanonical Wnt signaling in cytoskeletal regulation.

### 4. **Cdc42 Inhibition and Attenuation of Restoration:**
- **CASIN Treatment:** The selective GTPase Cdc42 inhibitor, CASIN, attenuated the restoration of cytoskeletal organization and cell migration induced by Foxy-5. This emphasizes the specificity of Cdc42 involvement in this process and highlights a potential target for future studies.

### 5. **Conclusions and Implications:**
- **Mechanistic Insight:** Your research provides a crucial mechanistic insight into the relationship between RA exposure, Wnt5a-mediated noncanonical Wnt signaling, Cdc42, cytoskeletal remodeling, and cell migration during palate development.
- **Clinical Implications:** Understanding these mechanisms could lead to potential therapeutic interventions or preventive strategies for cleft palate induced by RA exposure, contributing significantly to the field of developmental biology and congenital disorder research.

In summary, your study delineates a novel pathway involving RA, Wnt5a-mediated noncanonical Wnt signaling, and Cdc42 in the context of cleft palate development. This research not only sheds light on the biological processes underlying this congenital condition but also opens avenues for further exploration into potential treatments and preventive measures.