Wdr68 requires nuclear access for craniofacial development.

Area of Science:

• Developmental Biology
• Cell Biology
• Molecular Signaling

Background:

• WD repeat domain 68 (WDR68) is a conserved scaffolding protein involved in craniofacial development and left-right asymmetry.
• A signaling pathway involving Ras, Map3k, Wdr68, and Dyrk1 is implicated in developmental processes and disease.
• The precise cellular location where WDR68 activity is required during development remains unclear.

Purpose of the Study:

• To investigate the sub-cellular localization requirements of WDR68 for craniofacial development.
• To determine the role of WDR68 in the Ras/Dyrk1 signaling pathway.
• To elucidate the function of WDR68 in gene regulation and myogenesis.

Main Methods:

• Utilized zebrafish models with GFP-WDR68 and GFPNESWDR68 fusion proteins to assess craniofacial development.
• Employed C2C12 myoblast cell lines to study protein localization and gene expression.
• Constructed transcriptional activation and repression domain fusions of WDR68 (CebpFlagWdr68, MadFlagWdr68) for functional analysis.
• Assessed myogenin (myog) promoter activity using a reporter system in C2C12 cells.

Main Results:

• A GFP-WDR68 fusion protein rescued craniofacial development in zebrafish, while a fusion with a Nuclear Export Signal (GFPNESWDR68) did not.
• GFPNESWDR68 localized to the cytoplasm and caused mislocalization of RFP-DYRK1A to the cytoplasm, disrupting the signaling relay.
• Transcriptional activation fusions of WDR68 supported craniofacial development, whereas repression fusions failed.
• WDR68 was found to be important for myogenin expression in differentiating C2C12 cells, with overexpression enhancing reporter activity and repression domain fusions interfering.

Conclusions:

• WDR68 requires nuclear localization for its essential roles in craniofacial development.
• Nuclear WDR68 is crucial for the integrity of the Ras/Dyrk1 signaling pathway.
• Findings support a critical nuclear function for WDR68-containing complexes in gene regulation and myogenesis.