Primary Cilia-Dependent Gli Processing in Neural Crest Cells Is Required for Early Tongue Development

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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

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 of primary cilia during glossal development, we utilized the Kif3af/f; Wnt1-Cre murine ciliopathic model. Our data demonstrates that

Kif3a-mediated truncation of primary cilia in neural crest cells causes a loss of Hh function in the developing mandible. Loss of Gli2/3A results in a loss of Hh function. Thus, we hypothesized that aglossia in Kif3af/f; Wnt1-Cre embryo is due to a failure to process both Gli2FL and Gli3FL proteins into

Gli2A and Gli3A. To test our hypothesis, we employed a combination of in vivo and in vitro approaches.

Our data supports a mechanism in which primary cilia-dependent GliA processing in CNC cells is crucial for initiating the glossal development program.

RESULTS

Conditional ablation Kif3a, in neural crest cells results in aglossia

The developing tongue requires the interaction of CNC-derived mesenchymal cells and mesoderm- derived myoblasts20. We previously demonstrated that conditional loss of Kif3a in neural crest cells resulted in aglossia31; however, we did not address the cellular basis for this phenotype. To determine which mesenchymal cell population requires primary cilia for glossal development, we conditionally deleted Kif3a in myoblasts and compared the tongue phenotypes to that of Kif3af/f;Wnt1-Cre embryos.

At E13.5, the glossal phenotype of Kif3af/f;Wnt1-Cre embryos was evident by the lack of a tongue on the floor of the mandible (Fig. 3A-D; asterisk in B and D). Intriguingly, although the tongue was absent from

Kif3af/f;Wnt1-Cre embryos, we detected some lingual epithelial differentiation suggesting that lingual epithelial differentiation is partially independent of glossal development. Trichrome staining on E18.0 tongues revealed the demarcation of keratinized and non-keratinized, stratified, squamous epithelium in both control and Kif3af/f;Wnt1-Cre embryos (Fig. 3E-F). Nonetheless, we observed fewer epithelial keratinization in Kif3af/f;Wnt1-Cre embryos compared to control embryos (Fig. 3E-F, arrow heads).

Additionally, structures that are derived from the lingual epithelium and require a CNC cell contribution, such as the submandibular salivary glands and taste buds, were undetected in Kif3af/f;Wnt1-Cre embryos

3 (Fig. 3C-D; E-F, arrows). Our results indicate that although loss of primary cilia on CNC cells leads to the absence of tongue mesenchyme, partial development of the lingual epithelium occurs.

Furthermore, the aglossia phenotype in Kif3af/f;Wnt1-Cre embryos was accompanied by disruptions of glossal muscle patterning. In control E13.5 tongues, the glossal muscles were organized into intrinsic

(Fig. 3G, arrows) and extrinsic (Fig. 3G, arrow head) muscle groups. We observed that in Kif3af/f;Wnt1-

Cre embryos, the organization of the myofibers were severely disorganized resulting in a gross alteration of muscle position and pattern (Fig. 3H). Interestingly, the masticatory muscles, which are also in contact with CNC cells in the first pharyngeal arch, were unaffected in Kif3af/f;Wnt1-Cre embryos (data not shown). These data indicate a specific requirement for primary cilia in CNC-derived cells for glossal myogenesis. To efficiently demonstrate the deletion of primary cilia on CNC cells we generated

Kif3af/f;Wnt1-Cre embryos carrying the tdTomato-EGFP (mT/mG) dual reporter transgene

(Kif3af/f;mT/mG;Wnt1-Cre). We assessed Arl13b immunostaining in control and Kif3a f/f;mT/mG;Wnt1-

Cre mandibles. Arl13b is a small GTPase of the ARf/Arl family proteins that is localized to the ciliary axoneme and is used as a marker to identify the primary cilium32. Compared to control mandibles, EGFP positive neural crest-derived cells in Kif3a f/f;mT/mG;Wnt1-Cre mandibles were Arl13b-negative (Fig. 3I-

J). Together, these findings demonstrate that early tongue formation, and glossal muscle development are dependent upon the presence of primary cilia on CNC cells.

To conditionally delete primary cilia on myoblasts, we breed Kif3af/f mice with the MyoDi-Cre transgenic strain to target Kif3a gene on myoblasts 33. We found that Kif3af/f;MyoDi-Cre embryos exhibit a mild microglossia phenotype. Glossal muscle differentiation and organization were comparable to control embryos in these mutants (Fig. S1A-G). To confirm that primary cilia were deleted from myoblasts in

Kif3af/f;MyoDi-Cre embryos, we performed double immunostaining for MyoD and Arl13b. Lack of

Arl13b immunostaining on MyoD immunopositive cells of Kif3a f/f; MyoDiCre embryos confirmed the deletion of primary cilia in myoblasts (Fig. S1H-I). In sum, our results indicate that primary cilia on the

4 CNC-derived glossal mesenchyme play an instructive role in guiding early glossal development. Loss of primary cilia on CNC cells rather than myogenic progenitor cells leads to aglossia; along with a loss of mesoderm-derived glossal muscles.

Neural crest cell contribution to the glossal mesenchyme during glossal development

To understand why the glossal phenotype was more severe when primary cilia were ablated on CNC- derived cells than on myoblasts, we analyzed the developing tongue using tdTomato-EGFP

(mT/mG);Wnt1-Cre dual reporter mice. The mT/mG;Wnt1-Cre reporter mice allows for simultaneous visualization of CNC-derived cells (GFP-positive) and non-CNC-derived cell types (tdTomato-positive) in the developing tongue (Fig. 4). At E10.5, all mesenchymal cells in the paired tongue buds were almost exclusively comprised of GFP-positive cells (Fig. 4A). Although no tdTomato-positive mesenchymal cells were detected in the lateral lingual swellings, we detected punctate tdTomato-positive cells that were likely vasculature and nerve cells (Fig. 4A). By E11.5, both GFP-positive cells and tdTomato-positive cell types were present in the merged tongue buds (Fig. 4B), indicating that mesoderm-derived myoblasts migrated into the tongue anlage 17. Intriguingly, from E12.5 – E16.5, as the tongue matured; there was a marked reduction in the GFP-positive cell population accompanied by a striking increase in the tdTomato-positive cell population (Fig. 4C-E). By E14.5, the tdTomato-positive myogenic progenitor cells matured into myofibril-like structures and became organized into the extrinsic and intrinsic muscles of the tongue (Fig. 4D-E, arrows and arrow heads, respectively). Furthermore, by E18.5, fewer GFP- positive cells were observed in the mature tongue; however these CNC-derived cells encapsulated the mature glossal muscles, forming a scaffold of tendons and muscle connective tissues (Fig. 4F).

Collectively, these data supports that the CNC-derived mesenchyme first populate the tongue anlage and may be required to promote the survival and differentiation of the mesoderm-derived glossal muscles.

5 Ablation of primary cilia in neural crest cells reduces CNC cell and glossal muscle progenitor apoptosis in Kif3a f/f; Wnt1-Cre embryos

The aglossia phenotype in Kif3a f/f;Wnt1-Cre embryos could result from reduced proliferation and/or increase cell death of CNC and/or myogenic progenitor cells.

To determine if CNC cell proliferation was affected in Kif3a f/f;Wnt1-Cre embryos, we performed

Phosphohistone H3 (pHH3) immunostaining on E10.5 Kif3a f/f;mT/mG;Wnt1-Cre embryos. pHH3 detects the phosphorylation of histone H3, which occurs during mitosis and is used as a reliable marker to detect proliferating cells34. We found that the incidence of proliferating CNC cells was lower in Kif3a f/f;mT/mG;Wnt1-Cre embryos, however, the change was not statistically significant. The amount of proliferating CNC cell per mandibular area was statistically similar between control and Kif3a f/f;mT/mG;Wnt1-Cre embryos at E10.5 (Fig. 5A-C). Examination of cell death using cleaved caspase3 immunostaining revealed a significant increase in apoptotic CNC cells in Kif3a f/f;mT/mG;Wnt1-Cre embryos compared to control embryos (Fig. 5D-F). These results indicate that increased CNC cell apoptosis, rather than a decrease in CNC proliferation is a major contributor to the observed aglossia in

Kif3a f/f;Wnt1-Cre embryos.

To test that the aglossia phenotype in Kif3a f/f;Wnt1-Cre embryos is caused by reduced myoblast proliferation, we performed MyoD and pHH3 double immunostaining on E11.5 control and Kif3a f/f;Wnt1-Cre embryos. We found that compared to control tongues, fewer MyoD immunopositive cells were detected in the lingual regions of Kif3a f/f;Wnt1-Cre embryos. However, the amount of MyoD cells that were positive for pHH3 were statistically similar in control and Kif3a f/f;Wnt1-Cre tongue regions

(Fig. 5G-I). Examination of cell death using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) revealed a significant increase in apoptotic nuclei within the central regions of Kif3a f/f;Wnt1-Cre mandibles (Fig. 5J-L). To determine if these apoptotic regions were myogenic regions, we performed MyoD and TUNEL staining on serial sections. We found that the regions of increased cell

6 death coincided with MyoD positive regions (Fig. S2A-I). Together, our results indicate that aglossia in

Kif3a f/f;Wnt1-Cre embryos is caused by increased myoblast apoptosis in the developing tongue.

Hedgehog (Hh) loss-of-function mutants phenocopies the aglossia phenotype of Kif3a f/f;Wnt1-Cre embryos

To identify the molecular basis of aglossia in the Kif3a f/f;Wnt1-Cre ciliopathic mutant, we performed

RNA-seq on E11.5 mandibular prominences from Kif3a f/f;Wnt1-Cre and wild type embryos. We analyzed our RNA-seq data for the expression of key gene regulatory pathways that were previously shown to be required for glossal development. We quantified gene expression as the total number of mapped reads to a given reference gene or transcript35. TGF-β mediated FGF and BMP signaling pathways are required for glossal muscle proliferation and differentiation16. Based on our RNA-seq data, transcript levels of TGF-β family members and pathway read out genes such as: Tgfbr2, Tgfbr1, Fgf4,

Fgf8, Fgf10 and Bmp4 were not significantly altered in Kif3a f/f;Wnt1-Cre mandibles (Fig. 6A; Table.

1A). Secondly, Hand2, a downstream target of the Endothelin-A Receptor (Ednra) pathway in CNC cells was implicated in the patterning of the mandibular arch and subsequent initiation of glossal development36,37. Our RNA-seq analysis showed that transcript levels of Ednra pathway members:

Hand2, Dlx5, Dlx6 and Mef2C were not significantly downregulated in Kif3a f/f;Wnt1-Cre mandibles (Fig.

6B; Table. 1A). We also analyzed the expression domain of Hand2 in the developing mandible by whole mount in situ hybridization in Kif3a f/f;Wnt1-Cre embryos. We found that the expression domain of

Hand2 in Kif3a f/f;Wnt1-Cre embryos were comparable to control embryos (data not shown). Together,

f/f these results suggest that aglossia in Kif3a ;Wnt1-Cre embryos is not caused by disruptions in TGF-β or mandibular arch patterning pathways.

Given the close association between the Hh signaling pathway and the primary cilium, we examined our

RNA-seq data to determine whether Hh pathway genes were disrupted in Kif3a f/f;Wnt1-Cre mandibles.

7 We found that transcript levels of the downstream Hh effector and pathway activity readout gene, Gli1, was downregulated 2-fold in Kif3a f/f;Wnt1-Cre mandibles (Fig. 6C; Table. 1A). To further examine the requirement for Hh activity in CNC cells for glossal development, we examined several Hh pathway mutants for an aglossic phenotype. As previously reported, Smo n/c;Wnt1-Cre embryos, which lack the Hh receptor, Smoothened (Smo), in CNC cells exhibited aglossia 23 (Fig. 6D;D’-E’E’; dotted lines). When we deleted the downstream mediators of the Hh pathway, Gli2 and Gli3, from CNC cells in Gli2f/f;

Gli3f/f;Wnt1-Cre double mutants, we also observed an absent tongue (Fig. 6F-F’). Furthermore, when

Gli3 repressor (Gli3R), a repressor of the Hh pathway, was overexpressed in CNC cells in Gli3T-

Flag;Wnt1-Cre embryos, we observed the development of a bifid or cleft tongue (Fig. 6G-G’, arrows).

However, when we overexpressed the Hh pathway activator, Gli2 activator (Gli2A), in CNC cells using a constitutive active form of Gli2 (Gli2ΔN)38, we did not observe an aberrant tongue phenotype (Fig. 6H-

H). These results suggest that Hh activity in CNC cells is required to initiate the glossal development program.

Loss of Kif3a in neural crest cells disrupts the processing of Gli activator (GliA) isoforms

The primary cilium is required for the post-translational processing of Gli full-length (GliFL) transcription factors into either an activator (GliA) or repressor (GliR) isoform21,39. When the Hh pathway is off, GliFL proteins are converted into a truncated repressor form, GliR, which inhibits Gli target gene transcription. Conversely, when the Hh pathway is active, GliR production is inhibited and GliFL proteins are converted into transcriptional activators via post-translational modifications21. To determine if aglossia and the loss of Hh activity in Kif3a f/f;Wnt1-Cre mandibles were due to aberrant Gli protein processing in the developing mandible, we compared the levels of GliFL to GliR isoforms in Kif3a f/f;Wnt1-Cre embryos to that of Smo n/c;Wnt1-Cre mutants by western blot analysis.

Upon Shh reception by Ptc, Smo accumulates in the cilium to inhibit GliR formation and induces the production of GliA40. Our data showed that loss of Smo in CNC cells reduced the production of Gli2FL

8 and Gli3FL isoforms and increased the production of Gli2R and Gli3R isoforms (Fig. 7A,B). Conversely, in Kif3a f/f;Wnt1-Cre embryos, we observed an accumulation of Gli2FL and Gli3FL isoforms and reduced production of Gli2R and Gli3R in E10.5 mandibles (Fig. 7A,B). When we analyzed Hh pathway activity in Smo n/c;Wnt1-Cre and Kif3a f/f;Wnt1-Cre aglossia mutants, we found that Gli1 mRNA and protein levels were significantly downregulated (p<0.05) (Fig. 7C-D). Although we observed similar aglossic phenotype and molecular readout of Hh pathway activity in Smo n/c;Wnt1-Cre and Kif3a f/f;Wnt1-Cre aglossia mutants, our results suggest that the processing and production of Gli isoforms were different between the two aglossia mutants. The accumulation of GliFL proteins and the reduction of Hh activity in

Kif3a f/f;Wnt1-Cre mandibles suggests that the aglossia phenotype in Kif3a f/f;Wnt1-Cre embryos is due to a failure to process Gli2FL and Gli3FL into Gli2A and Gli3A isoform.

To test if aglossia in Kif3a f/f;Wnt1-Cre embryos is due to failure to produce GliA, we analyzed our RNA- seq data to determine if genes requiring Gli proteins for activation were transcriptionally downregulated.

Aglossia in the Smo n/c;Wnt1-Cre, Hh loss of function mutant, was attributed to a downregulation of

Forkhead box (Fox) genes in the mandibular prominence23. Fox genes encode a family of transcription factors that regulates craniofacial development and are direct targets of Gli proteins41,42. The expression of

Foxf1, Foxf2, Foxd1 and Foxd2 in the mandibular prominence defines the mandible, incisors and tongue.

Additionally the medial mandible, from where the tongue arises, only expresses FoxF123. When we analyzed our RNA-seq data, we found that there was greater than two-fold downregulation of Foxf1,

Foxf2, Foxd1 and Foxd2 transcripts in Kif3a f/f;Wnt1-Cre mandibles (Fig. 7E; Table. 1B). We confirmed the RNA-seq data by whole mount in situ hybridization on E10.5 control and Kif3a f/f;Wnt1-Cre mandibular prominences. In control embryos, Foxf1 was robustly expressed medially within the entire proximal to distal axis of the mandible, whereas Foxf2 was expressed mainly in the medial-distal regions of the mandible (Fig. S3A,D). In Kif3a f/f;Wnt1-Cre embryos Foxf1 and Foxf2 expression was markedly reduced relative to control embryos (Fig. S3B,E). These results show that loss of primary cilia in CNC

9 cells reduces the processing of GliFL into GliA, resulting in a significant downregulation of Gli target genes in the mandibular arch.

To test if loss of GliA was sufficient to induce an aglossic phenotype, we aimed to recapitulate aglossia by removing GliA in CNC cells of wild-type mice. Gli2 is the predominant Hh pathway activator43.

Previous studies show that Gli3 functions as a weak activator and can compensate for the loss of Gli244-46; therefore, we opted for an approach whereby we deleted both Gli2A and Gli3A in CNC cells. We generated Gli2f/f;Gli3d699/+; Wnt1-Cre embryos, in which we conditionally deleted Gli2 in CNC cells and introduced the Gli3d699 allele. Gli3d699 mouse mutant only expresses the truncated N-terminal repressor domain of Gli3, Gli3R 47. Separately, Gli2f/f; Wnt1-Cre and Gli3d699 homozygous mutants exhibited no obvious glossal defects (Fig. S4A-B; data not shown). Relative to controls, E14.5 Gli2f/f;Gli3d699/+;Wnt1-

Cre mandibles did not form a tongue (Fig. 7F-I). Furthermore, our q-PCR analysis on E10.5 mandibular prominences showed that Foxf1, Foxf2, Foxd1, and Foxd2 mRNA expression in Gli2f/f;Gli3d699/+;Wnt1-

Cre was significantly reduced compared to wild-type embryos (Fig. 7J; S3C). Taken together, these results indicate that glossal development requires GliA in neural crest cells. Our results further support that the aglossia phenotype in the Kif3a f/f;Wnt1-Cre ciliopathic mutant is caused by the failure to produce

Gli2A and Gli3A and the subsequent inactivation of mandibular Fox genes.

Overexpressing GliA in neural crest cells partially rescues the aglossia phenotype of Kif3a f/f;Wnt1-Cre embryos

To determine if GliA-deficiency in neural crest cells is the direct cause of aglossia in Kif3a f/f;Wnt1-Cre embryos, we aimed to rescue the aglossia phenotype, and restore mandibular Fox gene expression by overexpressing GliA in CNC cells. We utilized the conditional CLEG2 transgenic stain that expresses a constitutively active Gli2 which lacks the N-terminus repressor domain (Gli2ΔN)38. The processing and activation potential of Gli2ΔN is independent of the presence of primary cilia48. We generated Kif3a f/f;

CLEG2 f/+; Wnt1-Cre embryos. At E14.5, Kif3af/f;Wnt1-Cre embryos are aglossic and lack a tongue on

10 the floor of the mandible, compared to control embryos (Fig. 8A,B; asterisk in B). To our surprise, Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos formed a partial tongue-like structure on the floor of the mandible (Fig.

8B,C; black dotted lines in C). In H&E stained coronal sections, the tongue-like structure of Kif3a f/f;

CLEG2 f/+; Wnt1-Cre embryo was characterized by an increase in glossal mesenchymal tissue, relative to

Kif3af/f;Wnt1-Cre embryos (Fig. 8D-F; dotted lines). Additionally, we observed that the lingual epithelial

f/f differentiation defects in Kif3a ;Wnt1-Cre embryos were rescued with the addition of Gli2ΔN.

Trichrome staining on E18.0 tongues revealed the demarcation of keratinized and non-keratinized, stratified, squamous epithelium in control, Kif3af/f;Wnt1-Cre and Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos

(Fig. 8G-I). Fewer epithelial keratinization and a lack of taste buds were observed in Kif3af/f;Wnt1-Cre embryos compared to control embryos (Fig. 8G-H). However we observed similar epithelial keratinization and the presence taste buds in Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos (Fig. 8G;I). These results indicate that morphologically, Gli2ΔN partially restores a tongue-like structure and completely rescues the lingual epithelium defects observed in Kif3a f/f;Wnt1-Cre embryos.

To determine if Gli2ΔN activity in Kif3af/f;Wnt1-Cre embryos rescued the glossal defects at the molecular level, we examined the expression of mandibular Fox genes in wild-type, Kif3af/f;Wnt1-Cre, and Kif3a f/f;

CLEG2 f/+; Wnt1-Cre mandibles at E10.5 by qPCR. We observed that the expression of Foxf1, Foxf2,

Foxd1 and Foxd2 in Kif3af/f;Wnt1-Cre embryos were reduced to 12%, 32%, 13%, and 12% of wild-type levels, respectively (Fig. 8G). When Gli2A was introduced into Kif3af/f;Wnt1-Cre embryos, we observed that the expression of Foxf1, Foxf2, Foxd1 and Foxd2 were increased to 26%, 46%, 56%, and 34% of wild-type levels, respectively (Fig. 8J). Concurrent with the partial morphological glossal rescue, these results suggest that Gli2ΔN partially rescues the ciliopathic glossal defect at the molecular level. Further experiments are required to determine the mechanisms of Gli2ΔN-mediated partial glossal phenotype in

Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos.

11 DISCUSSION

It is well established that the Hh pathway plays a crucial role in the early stages of glossal development23,49. However, the mechanisms by which Hh signaling in CNC cells pattern the early glossal mesenchyme and the role primary cilia play in these processes remained to be elucidated.

Loss of primary cilia on CNC cells results in aglossia

We report that loss of primary cilia, via conditional loss of the ciliary anterograde IFT protein, KIF3A, in

CNC cells rather than myogenic progenitor cells results in an absent tongue with severely disorganized glossal muscles (Fig. 3;S1). In hand with the finding that CNC cells first populate the tongue anlage17

(Fig. 4), these results support that primary cilia are solely required on CNC cells for glossal development.

However, recent studies demonstrate that primary cilia are extended during early myogenesis and are required to regulate myogenic differentiation50. Therefore, it is possible that primary cilia on myoblasts may also be required to promote glossal myogenesis. During embryonic myogenesis, specified presumptive myoblasts express myogenic regulatory factors, Myf5 or MyoD51,52. At E10.5, Myf5 expressing myoblasts were first detected in the pharyngeal arches followed by MyoD expressing myoblasts53; thus it is possible that we deleted primary cilia after glossal myogenesis began. To fully understand the role of primary cilia on the mesoderm-derived mesenchyme, it will be important to determine if loss of cilia on Myf5 expressing myoblasts can produce a glossal phenotype.

On the cellular level, we found that aglossia in Kif3af/f;Wnt1-Cre embryos was accompanied by increased cell death in both the CNC and mesoderm-derived glossal mesenchyme, a severely disorganized glossal muscles, and a partially differentiated lingual epithelium (Fig. 3 and Fig. 5). Additionally, the mandibles of Kif3af/f;Wnt1-Cre embryos were reduced by 30% relative to control littermates due to a loss of proximal mandibular structures31. These results were largely consistent with another aglossia mutant,

Hand2 f/f;Wnt1-Cre, in which the transcription factor Hand2 was deleted from neural crest cells36. Hand2 is a basic helix-loop-helix (bHLH) transcription factor expressed in the mandibular prominence and

12 regulates the activity of Dlx5 and Dlx6 in neural crest cells54. Dlx5 and Dlx6 define and pattern the mandibular arch55. Deletion of both genes results in a homeotic transformation of the mandible to the maxilla55. Hand2 f/f;Wnt1-Cre embryos exhibited no change in cell death within the glossal mesenchyme, but displayed severely disorganized glossal muscles, and a partially differentiated lingual epithelium.

Furthermore, in contrast to Kif3af/f;Wnt1-Cre embryos, the mandibles of Hand2 f/f;Wnt1-Cre mutants were significantly shorter than control embryos due to a loss of distal rather than proximal mandibular arch structures36. Aglossia in Hand2 f/f;Wnt1-Cre mutants was attributed to disruptions in mandibular patterning due to the distal expansion of Dlx5 and Dlx6 rather than an absence of mandibular arch tissue54. Our RNA-seq data showed no significant change in Hand2, Dlx5 or Dlx6 transcript levels between wild-type and Kif3af/f;Wnt1-Cre mandibles (Fig. 6B). Based on these findings, it does appear that aglossia in Kif3af/f;Wnt1-Cre embryos is not caused by disruptions is mandibular patterning, but by the absence of sufficient arch tissue and increased cell death of mesoderm-derived muscle precursors.

Previous studies demonstrate that CNC cells play a crucial role in head muscle development56. Genetic ablation of CNC cells results in severely reduced and disorganized jaw muscles57. Thus, primary cilia- dependent signaling in the CNC-derived mesenchyme may be involved in transmitting environmental signals to the myogenic progenitors to coordinate tongue development. Together, these results support that there is a cilia-dependent and a mandibular patterning-dependent pathway, which may function in parallel or synergistically to coordinate the initiation of glossal development.

Glossal development requires GliA production in CNC cells

We determined that the aglossia phenotype in Kif3af/f;Wnt1-Cre ciliopathic model was due to a loss of

GliA in the developing mandible (Fig. 6). We further showed that genetic deletion of GliA in CNC cells of wild-type mice was sufficient to induce an aglossic phenotype and downregulation of mandibular Fox genes (Fig. 7F-J). Unexpectedly, introducing Gli2A via Gli2ΔN was not sufficient to completely rescue the aglossia in Kif3af/f;Wnt1-Cre embryos (Fig. 8). Several factors could account for the partial glossal rescue in Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos. GliR has an inhibitory effect on Gli2ΔN-mediated Hh

13 pathway activation48 and glossal development. When an extra copy of GliR was introduced into

Kif3af/f;Wnt1-Cre observed that the aglossia and mandibular phenotype were exacerbated (data not shown). Since only one copy of Gli2ΔN was introduced in Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos, it is possible that the partial glossal rescue was due to a repressive effect of wild-type GliR on Gli2ΔN- mediated Hh pathway activation in the absence of the cilium. Secondly, the partial glossal rescue in Kif3a f/f; CLEG2 f/+; Wnt1-Cre embryos could result from insufficient Gli2ΔN occupying Gli target genes.

These would support a model in which a high threshold of GliA in CNC cells would be required to initiate the glossal development program (Fig. 9). Further experiments will be required to determine if

f/f additional cilia-independent mechanisms inhibit Gli2ΔN-mediated Hh activation in Kif3a ;Wnt1-Cre embryos.

Finally, loss of primary cilia-dependent Gli processing in CNC cells might also interfere with other signaling pathways that contribute to early glossal development. Gli proteins can cooperate with a combination of transcription factors to control tissue specific gene expression58. Gli proteins bind to a consensus Gli binding region (GBR) that frequently occurs with E-box sequences59. bHLH transcription factors such as Hand2 binds to E-box motifs to activate gene transcription60. Although Hand2 transcript levels were comparable to wild-type in Kif3af/f;Wnt1-Cre embryos (Fig. 6B), on the protein level, it may be possible that loss of primary cilia-dependent Gli processing disrupts the ability of Gli proteins to interact with Hand2 to initiate glossal development. Our preliminary findings show that Gli3 immuno- precipitates with Hand2 in the mandibular prominence. Future work will determine if Gli-hand2 interaction is disrupted in ciliopathic aglossia and if this interaction is required to promote glossal development.

In summary, we proposed a model in which during normal glossal development, GliA binds the promoter regions of mandibular Fox genes, thereby, promoting their transcription and the initiation of the glossal

14 development program. In the absence of cilia the accumulation of aberrant GliFL proteins occupies the promoter regions of mandibular Fox genes, preventing their transcription and inhibiting glossal initiation.

Furthermore, we propose that Gli2ΔN partially outcompetes aberrant GliFL in the ciliopathic mutant; thus resulting in partial activation of mandibular Fox genes and a partial rescue of the ciliopathic aglossia phenotype. Moreover, genetic removal of Gli2 and Gli3A photocopies the ciliopathic aglossia. Absence of GliA results in inactivation of mandibular Fox genes and consequently an aglossic phenotype (Fig. 9)

Our study revealed a novel mechanism in which primary cilia-dependent GliFL to GliA post-translational processing in CNC cells initiates the glossal development program via the activation of downstream mandibular Fox genes.

EXPERIMENTAL DESIGN AND PROTOCOLS

Mouse strains

The Wnt1-Cre, Gli3f/f, Smof/f, Smonull, Gli3T-Flag and ROSAmT/mG mouse strains were purchased from

Jackson Laboratory. MyoDi-Cre mice were acquired from Dr. David Goldhamer at University of

Connecticut; Kif3af/f, from Dr. Bradley Yoder at University of Alabama at Birmingham; Gli2f/f from Dr.

Alexandra Joyner at Memorial Sloan-Kettering Cancer Center, Gli3Δ699 from Dr. Chi-Chung Hui at the

Hospital for Sick Kids, Canada; and CLEG2 (Gli2ΔN) strain from Andrzej Dlugosz at University of

Michigan. Animal usage was approved by the Institutional Animal Care and Use Committee (IACUC) and maintained by Veterinary Services at Cincinnati Children’s Hospital Medical Center.

Mandible and tongue morphology analysis

For whole mandible images, E13.5 and E14.5 embryos were fixed in Bouin’s Fix at 40 Celsius overnight with shaking. The mandibles were dissected and imaged using Leica imaging software in gray scale mode.

15 Histological analysis and immunostaining

Hematoxylin and eosin (H&E), and immunohistochemistry and immunofluorescence experiments were performed according to standard protocols. Immunostainings were performed using primary antibodies against the following: anti-Arl13b (Proteintech), anti-MyoD1 (clone 5.8A, from Novus bio), myosin heavy chain, anti-PHH3 (clone MoBU-1, Santa Cruz), and anti-active Caspase3 (clone Asp175, from Cell

Signaling). Secondary antibodies with fluorescence tags were applied and incubated at room temperature for 1 hour. Slides were then stained with 4’,6-diamino-2-phenylindone (DAPI; 5 µg/mL; Invitrogen) and mounted with Prolong gold mounting media from Invitrogen. Cell proliferation and death counts were performed using ImageJ. The Student’ t test was used for statistical analysis.

In situ hybridization

Whole mount In Situ hybridizations were performed following procedures modified Gallus Expression in situ hybridization Analysis (GEISHA) protocols. The Digoxigenin-labeled anti-sense riboprobes for

Foxf1, Foxf2 and Foxd2 were generated from plasmid DNA provided by Yu Lan at Cincinnati Children’s

Hospital Medical Center. Anti-sense riboprobes for Foxd1 were created from wild-type mouse cDNA templates that were amplified from primer sequences acquired from Gene Paint.

Western blot analysis

Combined E10.5 mandibular prominences were sonicated in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1 %

NP-40, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease inhibitors

(Roche), phosphatase inhibitors (1 mM PMSF, 1 mM Na3VO4, 10 mM NaF, 60 mM b-glycerophosphate).

Protein concentration was measured by BCA protein assay (Thermo Scientific, # 23227). 10µg protein was loaded to a 6% SDS-PAGE gel for Gli proteins and 5ug protein on a 10% SDS-PAGE gel for

GAPDH. Goat polyclonal anti-Gli2 antibody (R&D, AF3635) and goat polyclonal anti-Gli3 antibody

(R&D, AF3690) were used to detect both full-length and processed repressor forms of Gli2 and Gli3.

Rabbit polyclonal anti-GAPDH antibody (Santa Cruz) was used to detect GAPDH. An

16 electrochemiluminescence (ECL) assay (ECL prime, Amersham, Pittsburg, PA, USA) was performed to develop the chemiluminescence signals.

qPCR and RNA-seq analysis

RNA was extracted from mandibular prominences using the PureLink RNA Micro Scale (Thermo Fisher) and cDNA was made by RT-PCR. SYBR® Green Supermix (Bio- Rad) and Bio-Rad CFX96 TouchTM real-time PCR detection system (Bio-Rad) were used to perform quantitative real-time PCR. All the genes were normalized to GAPDH expression. For RNA-seq analysis, RNAs were processed according to recommended procedures. RNA-seq was carried by the DNA sequencing and Genotyping core at

Cincinnati Children’s Hospital Research Foundation according to standard protocols. BAM files underwent whole genome RNA-seq analysis using Strand-NGS software. Quality inspection was run for the data set and revealed that the data was acceptable for further analysis. We identified differentially expressed genes between wild-type and Kif3af/f;Wnt1-Cre mandibular prominences with greater than 2 fold change in expression (n=1).

STATEMENT OF WORK

This work is funded by R01DE023804 from NIDCR/NIH. My thesis mentor, Dr. Samantha Brugmann designed all experiments in this Masters thesis/ capstone report. I performed all experiments including mouse husbandry and embryo collection with the exception of the following: RNA-seq analysis from raw

BAM files, western blot analysis and Arl13b immunostaining on Kif3a f/f;TomGFP;Wnt1-Cre embryos.

Post-doctoral fellows, Ching-Fang Chang and Ya-ting Chang, conducted the RNA-seq analysis and western blot experiments respectively. First year MDB graduate student Kelsey Elliot completed the

Arl13b immunostaining on Kif3a f/f;TomGFP;Wnt1-Cre embryos.

17 FIGURES

receptors (Smoothened)

axoneme

IFT proteins (Kif3a)

Primary Cilia BA” C

Figure 1. (A) Schematic of the primary cilium. KIF3A is an intraflagellar transport protein (IFT), which is localized to the ciliary axoneme. KIF3A mediates anterograde

transport, the trafficking of proteins from the cytoplasm to the cilium. Loss of KIF3A results in a truncated primary cilium and causes glossal abnormalities such as lobulated and bifid tongue presented in (B) OFD syndrome and (C) Joubert syndrome, respectively.

18 A E10.5 B E11.5 C E13.5

PS A’ PS OS

D PRESUMPTIVE MYOBLASTS MYOBLASTS MYOTUBES

Pax3/ Pax7 MyoD/ Myf5 MHC

Figure 2. Schematic of murine glossal development. (A-A’) Glossal development requires contributions from CNC cells (blue) and mesodermal derived muscles (red). (B) Presumptive myoblasts migrate from the occipital somites (OS) and occupy the tongue primordia by E11.5. (C-D) Pax3/7 expressing Presumptive myoblasts proliferate, becomes committed and differentiates into MyoD or Myf5 expressing myoblasts. These myoblasts undergo maturation

intoFigure myotubes 1. , expressing myosin heavy chain (MHC).

Control Kif3a f/f ;Wnt1-Cre A HB Figure 3. Truncation of primary cilia in neural crest cells result in aglossia. Dorsal view of the mnp mnp * * tongue (dotted black lines) and mandible (mn) in E13.5 (A) control and (B) tongue remnant (asterisk) in f/f E13.5 Kif3a ;Wnt1-Cre embryo. (C,D) Hematoxylin and C D eosin-stained coronal section illustrate aglossia (asterisk) on the lingual surface (dotted black lines) of

H&E * the mandible and underdeveloped submandibular salivary glands in Kif3a f/f;Wnt1-Cre embryo (black E13.5 arrows). Trichrome staining on E18.0 tongues E F illustrates non-keratinized to keratinized stratified squamous epithelium transition and the presence of keratin (arrow heads) in both control (E) and Kif3a f/f;Wnt1-Cre embryo (F). Taste buds (arrows in E) Trichrome were not detected the lingual epithelium in Kif3a E18.0 f/f;Wnt1-Cre embryo. (G) Anti-MHC immunostaining G H (red) in control E13.5 tongues and (H) Kif3a f/f;Wnt1- Cre tongue remnant. Anti-arl13b marks the ciliary axoneme. (I) Arl13b (red) expression in control DAPI mandibular CNC cells marked by EGFP (green). (J) f/f MHC Arl13b expression is lost in Kif3a ;Wnt1-Cre mandibular CNC cells. Cell nuclei appear blue with E13.5 DAPI counterstain. Scale bars: (A-D, G,H) = 250um; I J (F) = 100um; (J) = 10um. DAPI Arl13b

EGFP EGFP E11.5 19 Fig2. Cranial neural crest cell contribution to the developing tongue. Scale bars=250um A B C

E10.5 E11.5 E12.5 D E F

E14.5 E16.5 E18.5 Figure 4. Neural crest contribution to the glossal mesenchyme. (A-F) mT/mG reporter analysis on coronal sections of mT/mG; Wnt1-Cre embryos marks neural crest (green) and non-neural crest (red) contribution to the glossal mesenchyme from E10.5-E18.5. (A) White arrows indicate majority neural crest-derived mesenchymal cells in the tongue buds at E10.5. (B-C) non-neural crest-derived presumptive myoblasts were detected in the tongue anlage by E11.5 (dotted white lines). (D-F) by E14.5-E18.5 we detected majority non-neural crest derived muscles in the mature tongue and a striking reduction in neural crest-derived cells. Scale bars = 250um. p

Fig. 3 Figure 5. Loss of primary cilia in neural Control Kif3a f/f ;Wnt1-Cre A B C crest cells affects CNC cell and myoblast AA B Control 0.80 n.s Kif3a f/f ;Wnt1-Cre apoptosis. (A-B) Anti-pHH3 (red) 0.70

DAPI 0.60 immunostaining on sagittal sections of 0.50 f/f a 0.40 E10.5 control and Kif3a ;mT/mG;Wnt1- mnp mnp 0.30 E10.5 p EGFP PHH3 EGFP 0.20 Cre embryos. (C) Quantification of

PHH3 (+) CNCC/ Area (mm ) PHH3 (+) CNCC/ 0.10 0.00 proliferation in EGFP positive CNC cells E10.5 (green) (p > 0.05); n.s (not significant). (D-

D E F Control E) Anti-cleaved caspase3 (red) f/f 0.05 * Kif3a ;Wnt1-Cre immunostaining on sagittal sections of 0.04 f/f DAPI E10.5 control and Kif3a ;mT/mG;Wnt1- 0.03 Cre embryos. (F) Quantification of 0.02 EGFP CC3 EGFP apoptosis in EGFP positive CNC cells (p < 0.01 CC3 (+) CNCC/ Area (mm ) CC3 (+) CNCC/ 0.00 0.05); significant (*). (G-H) Anti-pHH3 E11.5 E10.5 (green) and anti-MyoD1 (red) double A B G H I Control f/f immunostaining on sagittal sections of n.s Kif3a ;Wnt1-Cre f/f 0.30 E11.5 control and Kif3a ;Wnt1-Cre 0.25

PHH3 0.20 embryos. Arrowheads (lingual) and arrows

0.15

MyoD (aboral) surface of the mandible. Solid white 0.00 0.10

0.05 line outlines glossal muscle region. (I) PHH3 (+) MyoD/ MyoD (mm ) E11.5 0.00 Quantification of proliferation in MyoD1 J K L immuno-positive myogenic cells (p > 0.05); * Control 20 Kif3a f/f ;Wnt1-Cre n.s. (J-K) TUNEL staining (green) on 15

12 sagittal sections of E11.5 control and Kif3a

DAPI f/f 9 ;Wnt1-Cre embryos. (L) Quantification of f/f 6

TUNEL apoptosis in control and Kif3a ;Wnt1-Cre 3 embryos (p < 0.05); significant (*). White TUNEL positive cells/ Area (AU) positive cells/ TUNEL 0 E11.5 dotted lines outline the tongue and mandible. 20 Figure 4. Add scale bars 250um

TGF-beta Pathway Mandibular Patterning Hh Pathway

A wild-type Kif3a f/f ;Wnt1-Cre B wild-type Kif3a f/f ;Wnt1-Cre C

Tgfbr2 Dlx5 wild-type Kif3a f/f ;Wnt1-Cre Tgfbr1 Dlx6 Shh Fgf10 Ednra Gli1 RNA-seq Fgf8 Hand2

Fgf4 Mef2c -0.5 2.1 Bmp4 -2.7 0 2.7 -3.4 0 3.4 Normal Loss of Hh Receptor Loss of Hh Mediators Increased Hh Repressor Increased Hh Activator Control Smo n/c ;Wnt1-Cre Gli2 f/f ;Gli3 f/f ;Wnt1-Cre Gli3T-Flag;Wnt1-Cre CLEG2 f/+ ;Wnt1-Cre C D A EB F G D H

* * mn mn mn mn mn E14.5 E14.5 E14.5 E14.5 E13.5 D’ E’ F’ G’ H’ mc * * *

H&E * mc mc mc mc E14.5 E14.5 E14.5 E14.5 E13.5 Figure 6. Hedgehog (Hh) loss of function mutants phenocopies aglossia in Kif3a f/f; Wnt1-Cre embryos. RNA-seq results for (A) TGF-, (B) endothelin-A receptor, and (C) Hh signaling pathway expression in E11.5 Wild type and Kif3a f/f; Wnt1-Cre mandibular prominences . Heat map scale indicates expression level. (D) Dorsal and coronal view (H&E staining) of the tongue (dotted black lines) and mandible (mn) in E14.5 control embryo. (E,E’) Loss of the Hh receptor, Smoothened (Smo) on CNC cells of E14.5 Smo n/c;Wnt1-Cre embryos resulted in aglossia (asterisk) and nodules in place of the tongue (dotted yellow lines and arrows). Loss of the downstream effectors for the Shh pathway Gli2 and Gli3 on CNC cells in E14.5 Gli2 f/f;Gli3 f/f;Wnt1-Cre embryos results in aglossia (asterisk). (G,G’) Overexpressing the repressor of the Shh pathway, Gli3 repressor (Gli3R) in CNC cells in E14.5 Gli3T-Flag;Wnt1-Cre embryos results in aglossia with a bifid tongue remnant (dotted black lines and asterisk). (H,H’) Overexpressing the major activator of the Shh pathway, Gli2 activator (Gli2A) in CNC cells in E14.5 Gli2deltaN f/+;Wnt1-Cre embryos does not result in aglossia, dotted black lines outlines the tongue.

null null f/f d699/+ Smo Kif3a Smo Kif3a Control Gli2 ; Gli3 ;Wnt1-Cre A WT B cKO cKO WT cKO cKO F G Gli2FL Gli3FL T

* mnp mnp Gli2R Gli3R mnp E14.5 GAPDH GAPDH H I e10.5 C D 1.2 * * Smonull Kif3a 1.0 * H&E WT cKO cKO 0.8 0.6 Gli1 n.s 0.4 0.2 E14.5 Gli1 mRNA expression Gli1 mRNA 0.0 WT Smo null Kif3a J cKO cKO Wild-type Gli2 f/f ;Gli3 d699 ;Wnt1-Cre 1.2 100% 100% E 1.0 100% 100% Foxf1 Foxf2 Foxd1 Foxd2 2.1 0.8

WT 0.6 0.4 37% 19% 20% 24% 0.2 0 Kif3a cKO -0.5 Fold change of Wild-type 0.0 Foxf1 Foxf2 Foxd1 Foxd2

Figure 7. Post-translational processing of Gli2 and Gli3 in aglossia mutants. (A,B) The levels of full-length Gli2 and Gli3 proteins (Gli2FL and Gli3FL) are reduced in Smo n/c;Wnt1- Cre (Smon/c cKO) MNP and increased in Kif3a f/f;Wnt1-Cre (Kif3a cKO) MNP compared to wild type. The amounts of Gli2 and Gli3 repressor (Gli2R and Gli3R) are increase in Smo n/c;Wnt1-Cre MNP, but reduced in Kif3a f/f;Wnt1-Cre MNP. (C-D) Gli1 protein expression and mRNA expression was reduced in Smo n/c;Wnt1-Cre (p<0.05) and Kif3a f/f;Wnt1-Cre (p<0.05) MNP. (E) RNA-seq results for Gli target gene expressions in wild type and Kif3a f/f;Wnt1-Cre MNP. Heat map scale indicates expression level. (F-I) Loss of Gli2 and Gli3 activator proteins (Gli2A and Gli3A) recapitulates aglossia phenotype. (F,G) Dorsal view of the tongue (dotted black lines) in control E14.5 mandible (mn) and tongue remnant (asterisk) in Gli2 f/f;Gli3d699/+;Wnt1-Cre embryos. (H,I) Hematoxylin and eosin-stained coronal section illustrate aglossia (asterisk) in Gli2 f/f;Gli3d699/+;Wnt1-Cre mn. (J) q-PCR analysis of Fox genes expression in E10.5 wild type and Gli2 f/f;Gli3d699/+;Wnt1-Cre MNP (n=3; p<0.05)

Control Kif3a f/f ;Wnt1-Cre Kif3a f/f ; CLEG2 f/+ ;Wnt1-Cre A B C T *

mnp mnp mnp E14.5 mnp E14.5 E14.5 D E F

H&E *

E14.5 E14.5 E14.5 G H I Trichrome E18.0 E18.0 E18.0 J WT 1.2 Kif3a f/f ;Wnt1-Cre 100% 100% 100% 100% Kif3a f/f ; CLEG2 f/+ ;Wnt1-Cre 1.0

0.8 56%

0.6 46% q-PCR 32% 32% 0.4 26% Fold change of Wild Type Fold change of Wild 0.2 12% 13% 12%

0.0 Foxf1 Foxf2 Foxd1 Foxd2

Figure 8. Constitutively active Gli2 (Gli2N) partially rescues aglossia in Kif3a f/f;Wnt1-Cre embryos. (A-C) Dorsal view of the tongue (dotted black lines) in E14.5 control, Kif3a f/f;Wnt1-Cre and Kif3a f/f;CLEG2 f/+;Wnt1-Cre mandibles (mn). (A-B;D-E) At E14.5, the aglossia phenotype is evident in Kif3a f/f;Wnt1-Cre mandibles (asterisk). (C and F) In E14.5 Kif3a f/f;CLEG2 f/+;Wnt1- Cre mandibles, a partial tongue-like structure was observed when Gli2N was introduced into Kif3a f/f;Wnt1-Cre embryos. (G) Fox q-PCR analysis on E10.5 mnp confirmed downregulation of Fox genes in Kif3a f/f;Wnt1-Cre. Fox gene expression was partially restored in Kif3a f/f;CLEG2 f/+;Wnt1-Cre mandibles,

23 WILD-TYPE MUTANT PARTIAL RESCUE PHENOCOPY

PARTIAL

ACTIVATION INACTIVATION ACTIVATION INACTIVATION

GLI Foxf1, Foxf2, Foxf1, Foxf2, Foxf1, Foxf2, Foxf1, Foxf2, Gli2dN GliFL GliFL GliFL GliFL Foxd1,Foxd2 GliFL Gli2dN GliFL Foxd1,Foxd2 Foxd1,Foxd2

BINDING GliA GliA GliA GliA Foxd1,Foxd2 GLI ACTIVITY

T PS PS PS PS

PHENOTYPE Kif3afl/fl;Wnt1-Cre Kif3afl/fl;CLEG2 f/+ ;Wnt1-Cre Gli2fl/fl;Gli3 d699/+ ;Wnt1-Cre fl/fl fl/fl NORMAL LOSS OF CILIA EXTRA GLI ACTIVATOR LOSSGli2 OF ;Gli3GLI ACTIVATOR;Wnt1-Cre

Figure 9. Working model of primary cilia-dependent Gli processing during glossal development.

TABLES:

Glossal Development Pathway Genes

Gene Fold Change of WT Regulation Tgfbr1 0.12642479 Upregulated (n.s) TGF-beta Pathway Tgfbr2 0.22354126 Upregulated (n.s) Fgf10 0.8469219 Upregulated (n.s) Fgf8 0.18664455 Upregulated (n.s) Fgf4 0 No Change Bmp4 0.18291283 Upregulated (n.s) Dlx5 -0.13149834 Downregulated (n.s) Dlx6 0 No Change Mandibular Patterning Endnra 0 No Change Hand2 -0.40994382 Downregulated (n.s) Mef2C 0.60511017 Upregulated (n.s)

Shh -2.517698 Upregulated* RNA-seq Hh Pathway Gli1 -2.0231116 Downregulated*

GliA Target Genes B

Gene Fold Change of WT Regulation Foxf1 -8.334625 Downregulated* Gli Target genes Foxf2 -5.782424 Downregulated* Foxd1 -4.370502 Downregulated* Foxd2 -4.501634 Downregulated*

* (> 2 fold regulation) Table 1. Table of normalized raw transcript reads in Kif3a f/f;Wnt1-Cre mandibles. (A) Fold change of glossal development pathway genes between wild-type and Kif3a f/f;Wnt1-Cre mandibles. (B) Fold change of GliA target genes between wild- type and Kif3a f/f;Wnt1-Cre mandibles. (*) denotes > 2 fold gene expression difference between wild-type and Kif3a f/f;Wnt1-Cre mandibles. 24 SUPPLEMENTARY FIGURES Fig S1

f/f Control Kif3a ;MyoDi-Cre Control F A B

E13.5 E13.5 DAPI C D f/f G Kif3a ;MyoDi-Cre MHC H&E

E13.5

E Control n.s 2.5 Kif3a f/f ;MyoDi-Cre 2.0 H Control I Kif3a f/f ;MyoDi-Cre 1.5 DAPI

1.0

0.5 Arl13b Tongue length (mm) Tongue p=0.18, n=3

0.0 MyoD E11.5 Figure S1. Loss of primary cilia on MyoD expressing myoblasts does not result in aglossia. Dorsal view of the tongue (dotted black lines) and mandible in E13.5 (A) control and (B) Kif3a f/f;MyoDi-Cre embryo. (C,D) H&E stained coronal sections illustrates a mild microglossia in Kif3a f/f;MyoDi-Cre embryo. (E) Quantification of tongue length shows an insignificant reduction in the tongue lengths of Kif3a f/f;MyoDi-Cre embryos, n=3;p>0.05; n.s (not significant). Anti-MHC immunostaining (red) in (F) control and (G) Kif3a f/f;MyoDi-Cre embryos depicts comparable muscle organization and density between control and mutant tongues. Anti-Arl13b marks the ciliary axoneme. (H) Arl13b (red) expression in control glossal MyoD expressing myoblasts (green). (I) Arl13b expression is lost in Kif3a f/f;MyoDi-Cre myoblasts. Scale bars: (A- G) =250um; (H-I) = 10um

f/f Control Kif3a ;Wnt1-Cre A B

TUNEL TUNEL DAPI DAPI C D e11.5

MyoD MyoD E F

TUNEL TUNEL MyoD MyoD

Figure S2. Increase cell apoptosis is localized to MyoD expressing glossal regions. TUNEL staining (green) on sagittal sections of E11.5 (A) control and (B) Kif3a f/f;Wnt1- Cre embryos. MyoD immunohistochemistry on sections adjacent to TUNEL staining in (C) control and (D) Kif3a f/f;Wnt1-Cre embryos. (E-F) Overlay of TUNEL and MyoD staining depicts localized apoptosis to MyoD expressing myoblasts in Kif3a f/f;Wnt1-Cre embryos.

26 Fig. 6

Control Kif3a f/f ;Wnt1-Cre Kif3a f/f ; CLEG2 f/+ ;Wnt1-Cre Control Kif3af/f ;Wnt1-Cre Gli2 f/f ; Gli3 d699/+ ;Wnt1-Cre

A B C

* mnp mnp mnp Foxf1 mnp Foxf1 Foxf1 Foxf1

D E

H&E * Foxf2 Foxf2 Foxf2 Figure S3: Mandibular Fox gene expression. Whole mount in situ hybridization showing the oral view of Foxf1 and Foxf2 expression in E10.5 mandibular prominences of (A-B) control and (D-E) Kif3a f/f;Wnt1-Cre embryos. (D-E) Foxf1 and Foxf2 expression is lost in Kif3a f/f;Wnt1-Cre embryos. Oral view of E10.5 Gli2 f/f;Gli3d699/+;Wnt1-Cre mandibular prominence shows that Foxf1 expression is lost, when GliA was removed from CNC cells. Foxf1 Foxf2 Foxd1 Foxd2 2.1 Fig. S2

WT Control Gli2 f/f ;Wnt1-Cre

A B

Gli2 f/f;Gli3d699 fox data? 0

Kif3a cKO -0.5

1.2 mnpmnp WT mnp 1.0 Kif3a f/f ;Wnt1-Cre Kif3a f/f ; CLEG2 f/+ ;Wnt1-Cre 0.8 E14.5 E14.5 0.6 Figure S4: Conditional deletion of Gli2 in CNC cells does not produce glossal defects. Dorsal view of the tongue (dotted black lines) and mandible in E14.5 (A) control and (B) 0.4 Gli2 f/f;Wnt1-Cre embryos Fold change of Wild Type Fold change of Wild 0.2

0.0 Foxf1 Foxf2 Foxd1 Foxd2 27 REFERENCES

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