Regulation of Jaw Morphogenesis in Avian Models

Objectives: The morphogenesis of beak involves multiple signaling pathways, which may either be dependent or independent to each other. However, it is generally believed that the morphogenesis of a complex organ requires signaling circuits forming networks. According to the previous studying beaks and mandibles, several signaling pathways which involves key genes make cross talks during morphogenesis. In this study, well focus more on the RA-ALXs regulation durinh chicken beak development.
Methods: In order to trace the cell and the key genetic expressions in the time window, a culturing system and reporter systems are newly established by lentiviral system and live traced by confocal microscope. Next, to evaluate the genetic expression and regulation of ALXs during beak morphogenesis, several genetic tools (including lentivirus to overespression, shRNA, and dCAS9 systems) will be applied on the genes of interest. Last, to confirm the regulation of RA circuits in regulating beak morphogenesis, RNA-seq will be performed in RA treated beak comparing to the untreated controls, and ChIP-seq would also be applied using H3K27ac and H3K4me1 antibodies in beak tissues for predicting potential promoters or enhancers, followed by the comparison of RA-upregulated transcripts.
Results: The infected ectomesenchymes at the front of signaling zones (around mandibular symphysis area) are detected by the 4D confocal imaging. The infected cells could be traced and analyzed individually for the moving speed and tracks. Cells are expected to migrate more in anterior than ventral or dorsal directions. Alx1 and Alx4 showed different expressing patterns duing E5 to E9 in the beak areas. The phenotypic changes of those with RA treatment are similar to those infected with ALX1 overexpressed virus: the beak become sharper, while the palate keeps either unchanged or thinner in the medial-lateral direction. ALXs coexpress with some of the RARE. ALX1 is also an active transcription factor showing significantly enriched TFBS in beak during morphogenesis by analyzing ChIP-seq result.
Conclusions: The RA-ALXs signaling plays an important role during beak morphogenesis.