Denise Bonsu
Pronouns: they/them
Research Mentor(s): Benjamin Allen
Research Mentor School/College/Department: Cell and Developmental Biology / Medicine
Program: UROP
Session: Session 1 (9:00am – 9:50am)
Authors: Denise Bonsu, Hannah Schrader, Benjamin Allen
Abstract
The Hedgehog signaling pathway (HH) plays critical roles in directing mammalian embryonic development. GLI proteins (GLI1-3) are key transcriptional effectors of HH signaling. These transcription factors regulate cellular responses through context-specific activation and/or repression of gene expression. Investigating the mechanisms of GLI function have been hindered by the lack of suitable, commercially available antibodies. To address this, we have used CRISPR-editing to create mice carrying novel, epitope-tagged Gli alleles (Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5). From these animals, we have generated mouse embryonic fibroblast (MEF) cell lines to study GLI processing, localization, and target gene binding. This study focuses on using Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5 MEFs to study GLI protein localization before and after HH stimulation in vitro. HH signaling is activated using a Smoothened agonist (SAG) and then the cells are fixed and stained with antibodies directed against different epitope tags (i.e., FLAG, HA, and V5). These MEFs are also stained with markers to identify both nuclei (DAPI), and primary cilia (ARL13B), known sites of GLI localization. The MEFs are imaged using confocal microscopy to define GLI localization in the absence and presence of HH pathway activation. Using this novel approach, we hope to be able to gather more information about the subcellular localization dynamics of the GLI transcription factors.
