Eya1/Six1 and Notch signaling in branchial arch development

Abstract

Branchio-Oto-Renal (BOR) syndrome patients, who have mutations in the Eya1 and Six1 genes, exhibit craniofacial defects. Craniofacial anomalies can also be identified in the 〖Eya1〗^(-/-) and 〖Six1〗^(-/-) mutant mice. During early embryogenesis, both 〖Eya1〗^(-/-) and 〖Six1〗^(-/-) mutant embryos showed hypoplastic second branchial arches (BA2) and impeded neural crest cell migration. Additionally, the 〖Eya1〗^(-/-) mutant embryos showed branchial epithelial abnormalities. Interestingly, Notch signaling was downregulated in the 〖Eya1〗^(-/-) mutant branchial epithelium but upregulated in the 〖Six1〗^(-/-) mutant branchial mesenchyme. I hypothesized that Eya1 and Six1 may interact with the Notch signaling pathway to regulate branchial epithelium and neural crest development. The aim of this study is to investigate the molecular mechanisms underlying the craniofacial abnormalities in BOR syndrome using Eya1 and Six1 mutant mouse as disease models.

By cell lineage tracing experiments using the Sox2-CreER mice, I identified a group of Sox2-positive branchial epithelial progenitors in E8.5 embryos. These progenitor cells could be distinguished from the Sox3-positve epibranchial placodes at E9.5. In BA2, these Sox2-positive branchial epithelial progenitors expressed Fgf3 and Fgf8 and functioned as signaling centres. Analysis of Eya1-/- mutants suggested that these progenitors failed to form branchial clefts, nor to synthesize signaling molecules such as Fgf3 and Fgf8. On the other hand, in the 〖Six1〗^(-/-) mutant embryos, the Sox2-positive progenitors could express Fgfs and BA segmentation was unaffected, indicating that Eya1 and Six1 have independent functions during branchial arch development. As the reduction of Notch signaling activity could be found in the 〖Eya1〗^(-/-) mutant ectodermal branchial epithelium, I hypothesized that Eya1 and Notch signaling pathway may cooperate to specify the branchial epithelial progenitor cell fate, and/or to maintain the proliferation and differentiation capacities, thereby control the development of the branchial arches. To address this hypothesis, I overexpressed a stable and activated form of the Notch1 receptor (NICD) in 〖Eya1〗^(-/-) mutants by genetics approach. I found that over-expression of NICD resulted in ectopic epithelial progenitors. Furthermore, the branchial cleft defects of the 〖Eya1〗^(-/-) mutants were rescued. Using cell transfection system, co-immunoprecipitation assays showed that Eya1 could physically interact with NICD. In the presence of Eya1, the phosphorylation level of NICD was reduced. Moreover, cycloheximide treatment experiments showed that Eya1 could stabilize the NICD protein, indicating that Eya1 can maintain the Notch signaling activity through dephosphorylating and stabilizing NICD. In the 〖Six1〗^(-/-) mutant embryos, the migrating neural crest cells displayed increased cell death and post-migratory patterning defects. Moreover, the expression of Notch1 and Hes1 were elevated in the branchial arch mesenchyme, indicating that Six1 is essential for maintaining cell survival and suppressing Notch signaling activity during neural crest cell development in the branchial arches.

In summary, this study unravels the distinct functions of Eya1 and Six1 during craniofacial development. The craniofacial abnormalities of BOR syndrome patients could be resulted from abnormal Notch signaling activities, which have to be precisely controlled in different branchial arch components. The molecular mechanisms underlying the regulatory processes of Six1/Eya1 and Notch signaling pathway may provide novel therapeutic strategies for craniofacial defects of BOR syndrome patients.