Primary cilia-dependent Gli processing in neural crest cells is required for early tongue development A thesis/Capstone Report submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science In the Department of Pediatrics Molecular and Developmental Biology Graduate program of the College of Medicine By Grethel Millington B.S. Oakwood University, May 2012 May 10, 2016 Committee Chair: Rashmi S. Hegde, PhD ABSTRACT Craniofacial dysmorphologies are common characteristics of the ciliopathy disease spectrum. Oral malformations, including those affecting the development of the tongue, are among the most common phenotypes of craniofacial ciliopathies. Tongue development requires contributions from cranial neural crest (CNC) cells and mesoderm-derived myoblast. The murine ciliopathic mutant, Kif3a f/f;Wnt1-Cre (Kif3a cKO), which is characterized by loss of primary cilia on CNC cells, exhibits aglossia (loss of a tongue). We found that loss of primary cilia on CNC cells rather than muscle cells results in aglossia. On a cellular level, our data revealed that aglossia in Kif3a cKO embryos is due to a loss of mesoderm-derived muscle precursors. We performed RNA-seq to elucidate the molecular mechanism for this phenotype. We found that Hedgehog (Hh) target genes, the Forkhead box genes, (Foxf1, Foxf2, Foxd1 and Foxd2), were transcriptionally downregulated in Kif3a cKO mandibles. The ratios of Hh effector transcription factors, Gli2 and Gli3 activator (GliA) to repressor (GliR) isoforms were disrupted in Kif3a cKO mutant embryos. Our data showed that the genetic removal of GliA in the CNC cells of wild- type mice recapitulated the aglossia phenotype and downregulated Fox gene expression. Furthermore, our data showed that increasing GliA activity in CNC cells partially rescues the aglossia phenotype and restores Fox gene expression in Kif3a cKO embryos. Together, our data suggests a model in which glossal development is dependent upon primary cilia-dependent GliA activity in CNC cells and the downstream activation of the mandibular Fox genes. ii Intentionally Blank iii ACKNOWLEDGEMENTS I would especially like to give thanks to my thesis advisor, Dr. Samantha Brugmann, for her tremendous support and guidance throughout my time as a graduate student in the Molecular and Developmental Biology graduate program. I could not have accomplished this milestone without Dr. Brugmann’s mentorship. I would also like to thank my thesis committee members: Dr. Rashmi Hegde (Chair), Dr. Rolf Stottmann and Dr. Saulius Summans, whose questions and comments on my thesis project were extremely insightful. This research work was made possible through the help, support and expertise of the members of the Brugmann Lab. Thank you, Ching-Fang Chang, Ya-ting Chang, Betsy Schock, Kelsey Elliot, and Jaime Struve. Thanks to Ching-Fang Chang for help with mouse husbandry and RNA-seq analysis from raw files. Thanks to Ya-ting Chang for help with western blot experiments. Additionally, I would like to thank Kelsey Elliot for help with completing some immunostaining experiments. Finally, thanks to those who supplied reagents to make this research work possible. Thanks to Yu Lan at Cincinnati Children’s Hospital Medical center for supplying ribroprobes for Foxf1, Foxf2 and Foxd2. And to Andrzej Dlugosz at the University of Michigan for supplying the CLEG2 (Gli2ΔN) mouse strain. iv TABLE OF CONTENTS Abstract--------------------------------------------------------------------------------------------------ii Acknowledgements-------------------------------------------------------------------------------------iv Introduction----------------------------------------------------------------------------------------------1-3 Results----------------------------------------------------------------------------------------------------3-11 Discussion------------------------------------------------------------------------------------------------11-15 Experimental Design and Protocols-------------------------------------------------------------------15-17 Statement of Work---------------------------------------------------------------------------------------17 Figures-----------------------------------------------------------------------------------------------------18-24 Tables------------------------------------------------------------------------------------------------------24 Supplementary Figures----------------------------------------------------------------------------------25-27 References-------------------------------------------------------------------------------------------------28-32 v INTRODUCTION Primary cilia are ubiquitous microtubule-based cellular projections that are specialized for receiving and processing extracellular signaling cues (Fig. 1A). Disruptions in primary cilia function are associated with a spectrum of complex human genetic disorders known as ciliopathies1-4. Craniofacial dysmorphologies are common characteristics of the ciliopathy disease spectrum5. Among the more consistent craniofacial phenotype in ciliopathies is the presence of oral malformations, including those affecting the development of the tongue. Ciliopathies such as Oro-facial digital syndrome and Joubert syndrome frequently present with (tongue) glossal abnormalities including: microglossia, an abnormally small tongue; bifid or cleft tongue; and tumors of the tongue6-9 (Fig.1B-C). These developmental glossal defects can impair suckling, speech, mastication, and produce severe dental malocclusions10. Furthermore, tongue squamous carcinoma is one of the most prevalent malignant cancers affecting the oral cavity11. Surgical methods are the main basis of treatment, which often results in impaired glossal functions and quality of life12. Therefore, understanding both normal and ciliopathy-associated glossal development will contribute to therapeutic approaches to facilitate the treatment of ciliopathy-associated and non-ciliopathy-associated glossal defects. This project aims to elucidate the cellular and molecular roles of primary cilia during normal and ciliopathic glossal development. The tongue has a substantial contribution from neural crest cells13. Neural crest cells are a migratory, multipotent cell population that migrates from the dorsal neural tube and populates the first pharyngeal arch from where the tongue is derived 14. In the craniofacial region, neural crest (cranial neural crest) cells give rise to the connective tissues, and bones in the face and ventral neck15. The tongue is composed of cranial neural crest (CNC)-derived connective tissues and mesoderm-derived muscles covered by an ectoderm-derived lingual epithelium16. Developmentally, the tongue begins with the emergence of bi- lateral elevations called the lateral lingual swellings on the floor of the first pharyngeal arch around 4 weeks of gestation in humans (E10.5 in mice)10,16. During this stage, the tongue anlage is composed of CNC-derived mesenchymal cells16-18 (Fig. 2A-A’). At E11.5, the lateral lingual swellings enlarge and fuse 1 forming the tongue bud (Fig. 2B) Simultaneously, the mesoderm-derived myoblasts that migrate from the occipital somites invade the tongue bud and give rise to the glossal muscles 17,19 (Fig.2B-D). Although the morphological events that take place during glossal development are well defined, the molecular mechanisms that regulate these processes are less understood. Several studies implicate that CNC cells initiate and direct mechanisms that regulate glossal development17. Non-canonical and canonical Transforming Growth Factor-β (TGF-β) signaling in CNC cells control the proliferation and organization of glossal muscles, after the formation of the tongue bud20. While these studies have led to significant insights into the mechanisms of glossal development, the identities of other factors that regulate the early aspects of glossal development remain poorly understood. The Hedgehog (Hh) signaling pathway regulates cell, proliferation, differentiation and organogenesis during embryonic development21,22. Interestingly, Hh signaling has been implicated as a pathway critical for the initiation of the glossal development program23. In mammals, the primary cilium is known to potentiate and house several key components of the Hh signaling pathway24. The Hh gene expression program is mediated by Gli transcription factors: Gli1, Gli2 and Gli3. Gli1 is a transcriptional activator25, whereas, Gli2/3 can act as either transcriptional activators or repressors at Hh target promoters26,27. The post-translational modification of Gli2/3 full-length (Gli2/3FL) proteins into transcriptional activators or repressors requires primary cilia28. Gli2/3FL proteins are converted into truncated repressor forms (Gli2/3R) via proteolytic cleavage when the Hh pathway is inactive. When the Hh pathway is active, the Hh receptor, Smoothened (Smo), translocates to the primary cilium29 (Fig.1A). Smo inhibits Gli2/3R production and Gli2/3FL proteins are converted into transcriptional activators (Gli2/3A) via post-translational modifications30. Interestingly, when Smo, is deleted from CNC cells, the resulting embryos lack Hh responsiveness on CNC cells and the tongue does not form (aglossia)23. Similarly, loss of the ciliary intraflagellar transport protein (IFT), KIF3A (Fig. 1A) on CNC cells (Kif3af/f; Wnt1-Cre) results in a truncated and non-functional primary cilium and the 2 resulting embryos exhibited aglossia31. To understand the role