GliFHV mice: a tool to investigate GLI processing and localization

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• GLI proteins (GLI1-3) are key transcriptional effectors of mammalian hedgehog (HH) signaling pathways.
• Existing research on GLI function is limited by the lack of specific and robust GLI antibodies.

Purpose of the Study:

• To generate a novel mouse model with endogenously epitope-tagged GLI proteins (GLI1, GLI2, GLI3) to facilitate GLI studies.
• To characterize the utility of this model for investigating GLI localization and function in HH signaling.

Main Methods:

• CRISPR-based gene editing was used to create epitope-tagged Gli alleles (Gli1FLAG, Gli2HA, Gli3V5).
• A novel mouse model (GliFHV) was generated by breeding tagged alleles.
• Sanger sequencing, long-range DNA sequencing, qPCR, and Western blot were used for validation.
• GliFHV mouse embryonic fibroblasts (MEFs) were generated and tested for HH responsiveness.

Main Results:

• The GliFHV mouse model is viable, fertile, and exhibits no overt phenotypes.
• Gene editing was confirmed, and expression/protein levels were comparable to wild-type.
• All three GLI proteins were observed to localize to primary cilia in developing limbs with distinct distributions.
• GliFHV MEFs demonstrated HH responsiveness, with GLI proteins localizing to primary cilia and nuclei in an HH-dependent manner.

Conclusions:

• The developed GliFHV mouse model and associated cell lines are valuable resources for studying GLI protein processing, localization, and function.
• This model overcomes previous antibody limitations, enabling detailed investigation of GLI roles in embryogenesis, development, and adult physiology.
• The findings provide new insights into GLI localization dynamics within primary cilia and nuclei during HH signaling.