All ETDs from UAB

Advisory Committee Chair

Bradey K Yoder

Advisory Committee Members

Rosa Serra

Robert A Kesterson

Jianbo Wang

Stuart Frank

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Hedgehog (Hh) signaling is required for many developmental processes, and causes several diseases and cancers. Described in chapter one below, vertebrate Hh sig-naling is dependent on the primary cilium (PC), a small organelle that extends from the surface of most mammalian cell types. A better understanding of how the PC modulates Hh signaling is important, as defects in the structure or function of PC result in severe human disorders termed the ciliopathies. For example, patients with Meckel-Gruber Syndrome have extra digits and neural tube defects, which are Hh both related pheno-types. In chapter two we sought to further understand the relationship between Hh medi-ated limb patterning and cilia loss on different cell populations in the limb. Using multi-ple Cre lines to delete the ciliogenic gene Ift88 in the limb, no patterning defects were observed. This was due to the fact that cilia remained on many cells days after Cre in-duction in vivo. Follow up in vitro studies revealed prompt genetic recombination of Ift88 and Kif3a, however these proteins were detectable for more than 96 hours. These data indicate that protein and PC loss were prolonged beyond the window of Hh pattern-ing. To complement this study, in chapter three, a Cre line was generated using the Alx4 promoter. This is the first Cre line known to exclusively affect the anterior limb. We used it to develop a fate map of Alx4 expressing cells and noted that they do not contrib-ute to the ulna, and that Alx4 is expressed in the developing kidney. Finally, in chapter four, we developed a mouse expressing a fluorescent ciliary protein, Sstr3::GFP, for di-rect visualization of PC in vivo. Interestingly, with this model we were able to observe ciliary movement within live renal tubules, and robust labeling of PC in many organs. The CiliaGFP mouse will allow for real-time evaluation of cilia in live samples, and will facilitate the evaluation of ciliary integrity over time. In sum, these studies have provided insights into the temporal requirements of cilia during limb patterning and have yielded useful tools for in vivo analysis of cilia function in multiple tissues.

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