In mammals the primary cilium is considered a hub for

In mammals, the primary cilium is considered a hub for signal transduction. The cilium has previous been associated with transduction of several pathways including Wnt (Corbit et al., 2008, McDermott et al., 2010, Tran et al., 2014), PDGF (Clement et al., 2013, Schneider et al., 2010, Schneider et al., 2005, Schneider et al., 2009), FGF (Hong and Dawid, 2009, Neugebauer et al., 2009, Tabler et al., 2013) and BMP (Xie et al., 2016). Of these pathways, however; the strongest association has been with the Hh pathway (Briscoe and Therond, 2013). Hh activity is mediated by the GLI transcription factors, GLI1-3. GLI1 is a transcriptional activator which primarily acts to potentiate the pathway (Ingham and McMahon, 2001), whereas GLI2 and GLI3 can act as either a transcriptional activator or repressor of Hh target genes (Hui and Angers, 2011, Niewiadomski et al., 2014). Primary DMPO are necessary for the post-translational modification which transform GLI2/3 full-length (GLI2/3FL) proteins into GLI activator (GLIA) or GLI repressor (GLIR) isoforms (Sasai and Briscoe, 2012). When the Hh ligand is absent, GLI2/3FL proteins are cleaved into truncated repressor forms (GLI2/3R) via proteolytic cleavage. When the Hh ligand is present, Patched suppression of Smoothened (SMO) is alleviated and SMO translocates into the primary cilium (Corbit et al., 2005). SMO inhibits GLI2/3R production and GLI2/3FL proteins are converted into transcriptional activators (GLI2/3A) via sequential phosphorylation, acetylation and sumoylation events (Kim et al., 2009). Interestingly, when SMO is deleted from NCCs, the resulting embryos lack Hh responsiveness on NCCs and the tongue does not form (Jeong et al., 2004). We have previously reported a similar aglossia phenotype when the intraflagellar protein (IFT), KIF3A is conditionally ablated on NCCs using the Wnt1-Cre driver (Kif3a;Wnt1-Cre) (Brugmann et al., 2010). KIF3A is a subunit of the kinesin-2 motor protein, which is responsible for moving molecular cargo towards the plus ends of microtubules (anterograde direction) (Yamazaki et al., 1995). Loss of KIF3A prevents ciliogenesis (Kondo et al., 1994). Despite previously reporting this phenotype, we did not examine the mechanisms behind the onset of the aglossia. Here, we examined the cellular behaviors and molecular changes associated with ciliopathic aglossia. Taken together, our data add mechanistic insights into glossal development under normal and disease conditions.
Results
Discussion Despite being a vital organ for vertebrate communication and feeding, little is known about the development of the tongue. We previously reported that loss of cilia, via conditional knock-out of the kinesin motor protein KIF3a, resulted in micrognathia and aglossia (Brugmann et al., 2010). Herein we characterized the glossal deficit (Fig. 1) and explored the cellular (Fig. 2, Fig. 3, Fig. 4) and molecular (Fig. 5, Fig. 6, Fig. 7) basis for this phenotype. From a cellular perspective, the loss of cilia on NCCs, resulted in increased apoptosis in both NCCs and mesodermally-derived muscle precursors. Additionally, the mesodermally-derived muscle precursors failed to invade the tongue anlage. Molecularly, NCCs that lacked cilia failed to potentiate a Hh signal due to a loss of GLIA activity. The loss of GLIA activity subsequently prevented the transcription of direct GLI targets important for mandibular and glossal development, including members of the Fox gene family. Taken together, these data established the foundation for several interesting hypotheses regarding the role of different signaling pathways during mandibular patterning and glossal outgrowth, as well as possibility that facial prominences required unique input from GLIA and GLIR isoforms for proper development.
Materials and methods
Acknowledgements We thank members of the Brugmann lab for helpful comments and suggestions, Jaime Struve for genotyping, Bradley Yoder, Alex Joyner and C.C. Hui for sharing transgenic mouse lines, and Jill Murphy for critical reading of the manuscript. This research was supported by National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grant R01DE023804 (S.A.B). G.M. was partially funded by T32 ES007051.