Gas1 Regulates Patterning of the Murine and Human Dentitions through Sonic Hedgehog

The mammalian dentition is a serially homogeneous structure that exhibits wide numerical and morphological variation among multiple different species. Patterning of the dentition is achieved through complex reiterative molecular signaling interactions that occur throughout the process of odontogenesis. The secreted signaling molecule Sonic hedgehog (Shh) plays a key role in this process, and the Shh coreceptor growth arrest-specific 1 (Gas1) is expressed in odontogenic mesenchyme and epithelium during multiple stages of tooth development. We show that mice engineered with Gas1 loss-of-function mutation have variation in number, morphology, and size of teeth within their molar dentition. Specifically, supernumerary teeth with variable morphology are present mesial to the first molar with high penetrance, while molar teeth are characterized by the presence of both additional and absent cusps, combined with reduced dimensions and exacerbated by the presence of a supernumerary tooth. We demonstrate that the supernumerary tooth in Gas1 mutant mice arises through proliferation and survival of vestigial tooth germs and that Gas1 function in cranial neural crest cells is essential for the regulation of tooth number, acting to restrict Wnt and downstream FGF signaling in odontogenic epithelium through facilitation of Shh signal transduction. Moreover, regulation of tooth number is independent of the additional Hedgehog coreceptors Cdon and Boc, which are also expressed in multiple regions of the developing tooth germ. Interestingly, further reduction of Hedgehog pathway activity in Shhtm6Amc hypomorphic mice leads to fusion of the molar field and reduced prevalence of supernumerary teeth in a Gas1 mutant background. Finally, we demonstrate defective coronal morphology and reduced coronal dimensions in the molar dentition of human subjects identified with pathogenic mutations in GAS1 and SHH/GAS1, suggesting that regulation of Hedgehog signaling through GAS1 is also essential for normal patterning of the human dentition.

For the analysis of erupted tooth morphology in post-natal (P) mice, Gas1 +/mice in a 129sv/CD1 background were used to generate single mutants (these mice present with a milder craniofacial phenotype and those without a cleft palate survive beyond birth). For histological and molecular analysis, Gas1 +/mice in a mixed 129sv/C57BL/6 background were used to generate single mutants (these mice have a more severe craniofacial phenotype, which includes cleft palate and do not survive beyond birth). All analysis of tooth number in these mice was conducted on embryonic material (E15.5 and beyond). All mice examined for supernumerary tooth prevalence (n=16) had evidence of at least two supernumerary teeth with 88% in the maxilla and 63% in the mandible. Timed-matings were set up such that noon of the day on which vaginal plugs were detected was considered as embryonic day (E) 0.5. In a mixed 129sv/C57BL/6 background Gas1;Shh GFP homozygous mutants have severe developmental disruptions and essentially do not survive to E15.5.

Imaging of murine tooth rows
The sample set for tooth row imaging consisted of 13 WT, 10 Gas1 +/and 15 Gas1 -/mice (a total of 152 dental rows or quadrants). The specimen age ranged from P2 weeks to 1 year, which meant that M3 analysis was only possible for mice >3 weeks of age (M3 is only mineralised from P20) (Cohn 1957). All non-mineralized tissues were removed to allow observation of the dental rows. Tooth rows were imaged using conventional X-ray microtomography with a Pheonix Nanotom (General Electrics) using the following parameters: 70 kV tensions, 100 mA intensity, 3000 images with time exposure of 500 ms and a voxel size of 3 µm. 3D renderings were performed using VG Studiomax software (Volume Graphics, Germany). The length, width and area of relevant tooth crowns were generated using ImageJ opensource software from occlusal-oriented pictures of the scanned volumes. More specifically, the crown surface area was calculated by drawing the outline of the molars. Since our measurements are based on the line of greatest contour of each molar, this allows us to disregard the varying effects of attrition depending on the age of the specimens. Kruskal Wallis test was used to verify the significance of differences in tooth size. A threshold p value of 0.05 was used to assess the significance of the observed differences.

Histological analysis and 3D reconstruction of embryonic tooth-germs
For histological analysis, embryos were fixed in 4% paraformaldehyde (PFA) at 4°C, dehydrated through a graded ethanol series, embedded in paraffin wax, sectioned at 7 µm and stained with haematoxylin and eosin. Continuous para-sagittal sections of the developing maxillary and mandibular molar regions were imaged and photographed using an Axioskop 2plus microscope, AxioCam HRC camera and Axiovision 3.0 software (Zeiss, Germany).
Images were imported into Adobe Photoshop CS6 (Adobe, USA) software, edited to highlight the epithelial component of the developing tooth-germs and saved. Images were imported into DeltaViewer 2.1 3D Imaging software (freeware) and computationally stacked and aligned using the boundary of the oral epithelium and dental lamina as vertical and horizontal plane reference lines. In addition, continuous cross-referencing against the original histology was carried out throughout the process. The software reconstructed these images to produce a three-dimensional image of the developing molar tooth-germs whilst excluding the background. The reconstructed surface was then smoothened and saved as a QuickTime 7.7.5 (Apple Corp, USA) movie file. Static images were also taken from different views for each developing tooth (top, oral, anterior, buccal and palatal views) and finally montaged in Adobe Photoshop CS6 to facilitate ease of comparison between control and mutant molars.
For Shh gene expression superimposition, continuous light-field images of 35 S radioactive in situ hybridisation were taken and included with the relevant histology. Images were imported into Fiji Life-Line version 2015 software, stacked and manually aligned. Those regions corresponding to Shh expression were coloured red whilst grey was used to outline the toothgerm. A total of n=5 Gas1 -/mutant embryos at E13.5 (n=1), E14.5 (n=3) and E15.5 (n=1) and stage-matched WT controls were collected. Each embryo allowed 3D analysis of n=4 quadrants, equating to a total of (n=20) quadrants for 3D analysis.

In situ hybridization
Section in situ hybridization using 35 S-UTP riboprobes was carried out as previously described (Gaete et al. 2015;Wilkinson 1992

Proliferation and TUNEL assays
Bromodeoxyuridine (BrdU) labeling for cell proliferation was carried out on histological sections using a Zymed BrdU Labeling and Detection Kit (Invitrogen) according to the manufacturer's instructions. Mouse embryos were labelled with BrdU via intra-peritoneal injection into pregnant females (5mg/100g body weight) 2 hours prior to sacrifice. BrdU labelling in epithelium and mesenchyme at E13.5 and E14.5 was quantified manually by counting the proportion of BrdU-labelled cells within an area 100 µm 2 in 5 consecutive 7 µm para-sagittal sections through the R2 region of maxillary tooth-germs (n=4 WT and mutant animals, respectively).
Immunohistochemical detection of apoptotic cell death was carried out on histological sections using Terminal deoxynucleotidyl transferase-mediated deoxyUridine triphosphate Nick End Labeling (TUNEL). TUNEL was carried out using an APOPTag® Plus Fluorescein In Situ Apoptosis Detection Kit (Chemicon International) according to the manufacturer's instructions. TUNEL staining in the epithelium at E14.5 was quantified manually by counting the number of TUNEL-labelled cells within an area of 100 µm 2 in 5 consecutive 7 µm parasagittal sections through the R2 region of maxillary tooth-germs (n=4 WT and mutant animals, respectively). All P-values were calculated using a Student's two-tailed t-test in Microsoft Excel.

Analysis of human subjects
The analysis of human subjects was previously approved by the Institutional Review Board of the Hospital de Reabilitaeao de Anomalias Craniofaciais, Bauru, Brazil (Ribeiro et al. 2010).
Written informed consent was obtained from all parents and individuals included in the study.
The human observational study conforms to STROBE guidelines. Human subjects with GAS1 The sequence analysis has been previously published with all identified GAS1 variants considered to be damaging (Ribeiro et al. 2010). Dental panoramic radiographs and study casts were taken as part of routine dental care for these patients. Human maxillary and mandibular dental arches were imaged using 3Shape TRIOS scanners (3Shape, Denmark) from stone casts poured from maxillary and mandibular alginate impressions (n=3 per group).
The length and width of M1 was measured using ImageJ software from occlusal-oriented pictures. Kruskal Wallis test was used to verify the significance of differences in tooth size. A threshold p value of 0.05 was used to assess the significance of the observed differences. E15.5 (palatal view above, aboral view below). In WT at E13.5, there was restricted Shh in R1 and a larger domain of in R2, whilst in the mutant R1 Shh was more prominent and a clear R2 domain was also present. At E14.5, the R2 domain had been lost in WT, whilst a large M1 domain was present in the enamel knot of the cap-stage tooth-germ. In the mutant at E14.5, there was evidence of prolonged R2 survival through continued Shh expression and delayed M1 development marked by an absence of Shh in the future enamel knot region. In addition, there was a small region of ectopic Shh expression spanning the oral side of the epithelial cap ( Fig. 4D, red arrows). At E15.5, in WT the M1 domain began to regress whilst Shh expression appeared in secondary enamel knots; in the mutant, there was M1-associated Shh expression noted for the first time in the primary enamel knot and sustained expression in R2, which was now established as a cap stage supernumerary tooth-germ. M1=first molar; SN=supernumerary tooth; 2ek=secondary enamel knot. a=aboral; o=oral; m=mesial; d=distal; p=palatal; b=buccal. Scale bar in F=250 µm for A-F.