Investigating the Role of the GLI Transcription Factors in Limb Development

Abstract

Many vertebrate organisms utilize distinct appendages to accommodate their niche within the animal kingdom— humans have hands and feet, birds have wings and claws, fish have fins. Surprisingly, these morphologically disparate limbs are patterned by the same set of signals, raising the question of how these signals achieve distinct outcomes across species? The Hedgehog (HH) pathway controls both digit specification and long bone growth during embryonic limb development, acting through the Glioma-associated (GLI) transcription factors (GLI1-3). My dissertation seeks to better understand how HH signaling promotes fundamentally distinct morphological outcomes during limb development. To assess HH/GLI signaling in limb development I pursued the following aims: 1) generation and validation of novel, epitope-tagged knock-in Gli alleles in mice; 2) visualization of endogenous GLI localization during limb development; 3) assessment of the combined contribution of Gli1-3 to long bone growth. In Chapter 2, I describe the generation and validation of endogenous epitope-tagged Gli alleles for each Gli gene (Gli1FLAG, Gli2HA, Gli3V5). We established a novel mouse model through breeding, Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5, referred to as GliFHV. Proper editing of each Gli allele was confirmed via Sanger sequencing and GliFHV animals are viable and fertile with no overt phenotypes. Similar gene expression (qPCR) and protein levels (western blot) were also confirmed between wildtype and GliFHV animals. We utilized these mice to visualize endogenous GLI localization in the developing forelimb, finding that all three GLIs localize to primary cilia with distinct distributions. Additionally, we generated immortalized GliFHV mouse embryonic fibroblasts (MEFs) and demonstrated that these cells are HH-responsive. Using the GliFHV MEFs, we found that GLIs localize to primary cilia and nuclei in a HH-dependent fashion. In Chapter 3, I assessed the combined contribution of Gli1, Gli2, and Gli3 to long bone growth through combined Gli1 germline deletion alongside conditional, limb-specific deletion of Gli2 and Gli3 (Gli1-3cKO). Gli1-3cKO animals were born, and skeletal preps were collected to assess bone length and digit number. Through analyzing these animals, we confirmed that Gli3 is the sole GLI that contributes to digit specification and demonstrated a previously unrecognized role for Gli1 in the length of long bones. Additionally, we found that Gli1, Gli2, and Gli3 variablly contribute to the length of long bones of the forelimb and hindlimb. Chapter 4 is a discussion of data chapters 1 and 2. I discuss the utility of the GliFHV mice for the field of HH signaling as well as ways the GliFHV MEFs could be used to better understand GLI dynamics in vitro. Additionally, the last section of Chapter 4 considers future directions for studying Gli1, Gli2, and Gli3 in limb development, highlighting that the tools required for studying forelimb versus hindlimb differences are now available. Lastly, the appendices include two sections (A and B) that highlight my contributions to two other publications during my Ph.D. training. Overall, this work provides significant insights into HH/GLI signaling during mouse limb development as well as a valuable resource for analysis of GLI processing, localization and function.