Primary cilia are microtubule-based organelles that serve as cellular antennae and coordinate the transduction of multiple signaling pathways. Individuals with ciliopathies, a class of diseases characterized by loss of primary cilia, frequenty present with orofacial defects such as oral hamartomas and dental abnormalities. Primary cilia are important regulators of the post-translational processing of Gli proteins into either their activator (GliA) or repressor (GliR) isoforms. Gli2 primarily functions as a transcriptional activator (Gli2A), while Gli3 is primarily a transcriptional repressor (Gli3R). Gli proteins have many transcriptional targets, including genes important for proliferation, Shh signaling, and canonical-Wnt signaling. Orofacial development depends upon tight spatial regulation of proliferation and Shh/Wnt signaling pathways. This proposed work focuses on understanding the requirement of ectodermal primary cilia in orofacial development and utilizes the conditional mouse knockout Kif3afl/fl;Crect. Kif3a is an anterograde intraflagellar protein that is required fo ciliogenesis, and Crect drives Cre recombinase activity specifically in the surface ectoderm of the developing embryo. My preliminary data indicates that Kif3afl/fl;Crect have ectopic Shh and Lef1 expression in the oral ectoderm and mesenchyme, respectively. I hypothesize ectodermal primary cilia regulate Gli-processing in the underlying mesenchyme to restrict spatial domains of Shh and canonical-Wnt pathway activity.
In Aim 1, I will determine if ectodermal loss of primary cilia causes Gli2A-dependent hyperproliferation in the oral mesenchyme. Shh pathway activity is associated with increased proliferation and tumorigenesis. Kif3afl/fl;Crect mutants ectopically express Shh in the oral ectoderm. For this aim, I hypothesize that ectopic Shh expression due to loss of ectodermal primary cilia causes Gli2A-dependent hyperproliferation in the oral mesenchyme.
For Aim 2, I will test if primary cilia on the oral ectoderm are required for Gli3R-dependent restriction of canonical-Wnt signaling in dental mesenchyme. Kif3afl/fl;Crect mutants develop ectopic tooth buds, which are accompanied by an expansion of Lef1, a readout for canonical-Wnt signaling, in the dental mesenchyme. Similarly, canonical-Wnt signaling gain-of-function mutants develop supernumerary teeth. Gli3R is known to restrict canonical-Wnt signaling. I hypothesize that loss of ectodermal primary cilia results in increased canonical-Wnt signaling due to loss of Gli3R regulation of pathway antagonists. As a whole, this study is important for advancing our understanding of the cellular and molecular etiology of orofacial defects in ciliopathic patients. Currently, the only avenue these patients have to alleviate severe orofacial defects is surgery. A deeper understanding of the developmental mechanisms of orofacial ciliopathies is necessary for the development of alternate therapeutic opportunities.
Primary cilia are organelles that extend out from the cell and sense the molecular environment. Defects in primary cilia results in a class of disease called ciliopathies, which are characterized by a broad range of defects. In this application, we will study the cellular and molecular basis of the craniofacial phenotypes commonly seen in ciliopathic patients.
| Schock, Elizabeth N; Brugmann, Samantha A (2017) Neural crest cells utilize primary cilia to regulate ventral forebrain morphogenesis via Hedgehog-dependent regulation of oriented cell division. Dev Biol 431:168-178 |
| Schock, Elizabeth N; Struve, Jaime N; Chang, Ching-Fang et al. (2017) A tissue-specific role for intraflagellar transport genes during craniofacial development. PLoS One 12:e0174206 |
