The regulation of epibranchial placode development by Eya1 and Notch signaling during mouse craniofacial morphogenesis

Abstract

Craniofacial morphogenesis is a complex process that requires coordinated development of the pharyngeal arches and cranial placodes, forming structures of the face, neck and sensory organs in the head. Epibranchial placodes are a series of epithelial thickenings, generating sensory neurons for innervating the visceral organs. Eya1, a transcriptional co-activator of Six1 and phosphatase, is critical for both epibranchial neurogenesis and pharyngeal morphogenesis. The aim of this study is to investigate the roles of Eya1 during the early epibranchial specification and pharyngeal arch patterning.

Current studies suggest the epibranchial placodes arise from the dorsal-most portion of the pharyngeal ectoderm, but little is known about how the placodal cells are specified and dissected out from the broader pharyngeal ectoderm and what are the correlated molecular events. Investigation of the spatiotemporal expression of multiple placodal and pre-neural genes from E8.0 to E9.5 revealed that the anlagen of proximal pharyngeal ectoderm was initially homogeneously Eya1+/Six1+, whereas later, the proximal pharyngeal ectoderm was compartmentalized into three types of subdomains, the Vgll2+, the Sox2+ and the Neurog2+ epibranchial domains, located at the rostral, caudal and dorsal-caudal regions of each pharyngeal cleft, respectively. These molecular events preceded the onset of epibranchial neurogenesis, suggesting that the molecular compartmentalization may be a precondition for establishing the structurally segregated and functionally active epibranchial placodes.

To decipher what controls the compartmentalization during the epibranchial development, I examined Notch signaling pathway, which is central for cell fate choice and boundary formation during development. Gene expression analysis revealed that distinct Notch factors exhibited different regionalized expression profiles. By using the Pax2cre;Rosa^{N1-IC} and Actincre;RBPJ^{flox/flox} mouse models to activate Notch activity and inhibit canonical Notch activity in the anlagen of epibranchial placodes, respectively, I showed that Notch activation led to reduced rostral genes expression while increased caudal genes expression, whereas Notch blockade induced expansion of rostral genes into caudal regions. Moreover, the dorsal Neurog2 expression was inhibited, while the ventral Sox2 expression was maintained in high Notch conditions. In conclusion, these observations suggested the dosage of Notch signaling was critical for epibranchial specification in both rostrocaudal and dorsoventral axes.

To unravel the role of Eya1 during the early development of epibranchial placodes and pharyngeal arch, I took advantage of the Eya1^{-/-} and Six1^{-/-} mouse models. A specific loss of proximal pharyngeal arch and reduced FGF activity were identified in Eya1-null embryos, but not in Six1-null embryos. Interestingly, by crossing Pax2cre;Rosa^{N1-IC} with Eya1^{-/-} mice, the FGF activity and proximal arch defects were restored. Co-transfection of Eya1 and Notch1 intracellular domain (N1ICD) into 293T cells revealed that the stability of N1ICD was enhanced by Eya1 and Eya1 dephosphorylated the p-threonine-2122 of N1ICD to antagonize ubiquitin-mediated degradation of N1ICD, resulting in enhanced Notch signaling strength. These observations suggested an Eya1-Notch regulatory axis for epibranchial and pharyngeal morphogenesis.

This project characterized the spatiotemporal molecular changes for the developing epibranchial placodes, and highlighted the role of Eya1-Notch axis during the epibranchial specification and proximal-distal arch patterning. This study provided cellular and molecular insights for developing therapeutic strategies for human craniofacial defects.