The role of cytoskeleton regulators in embryonic facial morphogenesis

Abstract

Small GTPases are downstream of the non‐canonical WNT JNK‐PCP pathway. The most well studied members of this group are RhoA, Rac1 and CDC42. These small GTPases regulate cell shape, orientation, and migration through regulating the actomyosin cytoskeleton. In addition, components of this pathway have been linked to non‐syndromic cleft lip with or without cleft palate. The objective of this study is to test whether small GTPase signaling is required and sufficient for facial morphogenesis at stages where lip fusion is taking place.MethodsThe faces of E4.5 chicken embryos were dissected to include the two facial processes involved in lip fusion, the frontonasal and maxillary processes (Fig. 1). After a dose‐response experiment to find the optimal concentrations, the organ cultures were treated with either an antagonist of RhoA signaling (ROCKi 20µM) or an antagonist of Rac1 (Rac1i 100µM) or an agonist (RhoA/ Rac1/CDC42 activator 0.5µg/ml). Organ cultures were photographed and measured at 0 and 24hrs. Percentage growth differences were analyzed using Prism Graphpad software (v. 9.2).ResultsIn the control cultures, frontonasal mass width decreased by 23% and increased in cranio‐caudal height by 17% over 24‐hour period. The nasal slits did not change. The maxillary prominences increased in height and width by 12% and 37% respectively over 24‐hour period similar to growth in vivo. The ROCKi treated cultures prevented the narrowing of the frontonasal mass and increased the length of the nasal slits. ROCKi treatment had no effect on the height of the frontonasal mass nor maxillary prominences. In contrast, the RAC1i had no effect on facial growth. The agonist significantly reduced frontonasal mass width compared to ROCKi. In addition, the agonist significantly decreased growth of the maxillary prominences.ConclusionsThe changes in facial morphogenesis may be explained by active cytoskeletal remodeling, particularly in the RhoA pathway. We excluded a requirement for RAC1 in facial morphogenesis in our system. Our data also suggest that different regions of the face have different requirements for specific small GTPase signaling. The frontonasal mass is dependent on RhoA signaling but not the maxillary prominences. Over activation of all GTPases shows that correct levels are necessary for normal morphogenesis. Overall, we have revealed a complex interplay between small GTPAses in midfacial morphogenesis.