Mice in the present studies were maintained and used in compliance with the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan in accordance with the National Institutes of Health Guidelines for Care and Use of Animals in research, and all experimental procedures were approved by the IACUC of the University of Michigan (PRO00009613). Generation of Evc2 mutant mice (Evc2 ^ex12/+) and Evc2 floxed mice (Evc2 ^fl/+) were as reported previously (21). The Evc2 floxed mice were bred with Sox2-Cre mice (30) to generate mice carrying a Cre-recombined allele (dE). Deletion of Evc2 in a neural crest-specific manner was achieved through crossing Evc2 ^fl/fl mice with either P0-Cre mice (31) or Wnt1-Cre mice (32). WNT signaling reporter mice were from Jaxson 004623 (33). All mice were maintained in a mixed background of C57BL6/J and 129S6 and were crossed and maintained in our semi-closed mouse colony for at least 7 years. There are no sample exclusions in our studies.

For micro-CT analysis, mouse heads at P28 were fixed in 4% paraformaldehyde in phosphate buffered saline followed by scanning at the University of Michigan using a micro-CT system (µCT100 Scanco Medical, Bassersdorf, Switzerland). Scan settings were as follows: voxel size 12 µm, 55 kVp, 109 µA, 0.5 mm AL filter, and integration time 500 ms.

To visualize the molars at postnatal day 8 (P8), the surface model for molars was generated based on the micro-CT data, using ITK-SNAP (open-source software developed by grants and contracts from the U.S. National Institutes of Health, www.itksnap.org), according to standard of segmentation using ITK-SNAP and previously described protocols (23, 34).

Mouse tooth germs were dissected from E18.5 mandibles. Under a dissection microscope, tooth germs were separated from surrounding tissues with forceps. For analysis of Hedgehog signaling and WNT signaling, total RNA was isolated from tooth germs homogenized in Trizol (Life Technology) according to standard protocols. Quantitative real-time PCR (Q-RT-PCR) was performed using Applied Biosystems ViiA7, with the following taqman probes: Axin2: Mm00443610_m1, Lef1: Mm00550265_m1, Gli1: Mm00494654_m1, Ptch1: Mm00436026_m1, and Gapdh: Mm99999915_g1. MEFs were prepared from E12.5 embryos and cultured in (DMEM with 10% Fetal bovine serum (FBS) according to previously described method (35). For arresting cell cycles and allowing growth of primary cilia, MEFs were cultured in MEF culture media (DMEM with 0.5% FBS) for at least 18 h. WNT signaling was induced by treating cells with WNT3A in concentration of 100 µg/ml for 18 h. Then total RNA was isolated from non-treated or treated cells. Then, 1 µg of total RNA was reverse transcribed using SuperScript Reverse Transcriptase (Life Technologies, Grand Island, NY, USA). Q-RT-PCR for examining induction of WNT signaling was performed in using Applied Biosystems ViiA7, with the following taqman probes: Axin2: Mm00443610_m1, Lef1: Mm00550265_m1, and Gapdh: Mm99999915_g1.

Mouse mandibles were dissected out at P8, E18.5, E16.5, E15.5 or E14.5 and fixed in 4% paraformaldehyde (PFA). Subsequently, they were embedded in paraffin, sectioned parasagittally, and stained with hematoxylin and eosin for histologic observations according to standard histology procedure. For immunohistochemistry, dissected mandibles were fixed with 4% PFA and cryo-protected by 30% sucrose in PBS before being embedded parasagittally for cryosection. Sections were incubated overnight at 4 °C with antibody against SHH (Hybridoma Bank, University of Iowa), LEF1 (Cell signaling 2230). Sections were then incubated with secondary antibody (IgG-Alexa fluor 488 (A-32731), IgG-Alexa fluor 594 (A-21203)) at room temperature for 1 h. And subsequently mounted by Prolong Gold antifade mount with DAPI (P36935, Life Technology) and imaged under a confocal microscope (Nikon C1). The intensity of signaling was quantified using Image J. For intensity quantification, at least 40 cells were quantified and the average of all quantified cells were used to represent the biological sample. There were 4 pairs of controls and mutants were used for comparisons.

A paired t test was conducted in all studies, performed in SPSS 27.0 (IBM Corp., Armonk, NY, USA). Error bars in the graph indicate standard deviation.

In contrast to alterations in tooth number reported in patients with EvC syndrome, we did not observe different numbers of molars in Evc2 mutant mice (Figures 1A–D) (26). This occurred despite the presence of previously reported dental phenotypes including hypoplastic enamel and incisor shortening (26). However, molar patterning differed between Evc2 mutants and their control counterparts at postnatal day 28 (P28). In control mice at P28, mandibular molars had a gradated pattern of decreasing crown size from anterior-to-posterior with a patterning of M1 as the biggest, then M2, and M3 as the smallest (Figures 1A,B). This is in contrast with the mandibular molars of Evc2 mutant mice at P28 which showed no regular size gradations from anterior-to-posterior, with T2 as the biggest, then T3, and T1 as the smallest (Figures 1C,D). These observations were consistent with previous reports on both Evc and Evc2 mutant mice (26, 29).

To understand the mechanism leading to abnormal patterning of molars in Evc2 mutant mice, we then evaluated the molars at postnatal day 8 (P8), a stage when M1 and M2 are almost ready to erupt, but M3 is still with minimal levels of mineralization in control mice. As expected, in control mice at postnatal day 8 (P8), the M1 and M2 were mineralized, present, and nearing eruption while M3 had minimal levels of mineralization and were not present in scans (Figures 1E–G). In contrast, we observed three mineralized molar bodies, T1, T2, and T3, in the maxillae and mandibles of Evc2 mutants (Figures 1H–J). Volumetric quantification of P8 molars in each arch corroborated P28 molar pattern abnormalities and indicated that M1 was larger than either T1 or T2 and that M2 was larger than T3 (Figure 1K). The observation of 3 molars in Evc2 mutants at P8, however, led us to question the actual identity of each molar and the role of Evc2 on molar pattern and morphology. We henceforth used neural-crest specific Evc2 mutant mice to investigate these questions as these mice presented with identical molar phenotypes (see next section) to Evc2 global mutants while circumventing problems associated with postnatal lethality and delayed growth (21).

Although tooth development involves coordinated epithelial-mesenchymal interactions, we previously reported that dental epithelia-specific Evc2 deletion did not lead to overt dental abnormalities (26). We therefore investigated the role of Evc2 in molar patterning using two Cre lines, P0-Cre and Wnt1-Cre, that specifically deleted Evc2 within neural crest cells that give rise to dental mesenchymal tissues. At P28, we observed no apparent body weight differences, suggesting that there is no apparent delay in development due to Evc2 loss of function within neural crest derived tissues. P0-Cre mediated deletion of Evc2 (referred to as Evc2 P0 mutant hereafter) resulted in mice that recapitulated the abnormal molar pattern of Evc2 global mutants only 20% of the time (2 out of 10 mutant mice examined) (Figures 2A–C vs. 2D–F). This is in direct contrast to the 100% penetrance (12 out of 12 mutant mice examined) of abnormal molar patterning observed in both P8 (Figures 3A–C vs. 3D–F) and P28 (Figures 3H–J vs. 3K–M) mice with Wnt1-Cre mediated deletion of Evc2 (referred to as Evc2 Wnt1 mutant hereafter).

Volumetric quantification of maxillary and mandibular molars in Evc2 Wnt1 mutants corroborated molar pattern abnormalities and indicated that M1 was larger than either T1 or T2 and that M2 was larger than T3 at both the P8 and P28 timepoints (Figures 3G,N, respectively). Similar trends were found for crown volume, root volume, crown height, and root height in Evc2 Wnt1 mutants at P28 (Figures 3O–R). Most importantly, phenotypic similarities between mice with mesenchyme-specific loss of Evc2 function and Evc2 global mutants suggest that Evc2 in mesenchymal tissues plays a critical role in determining molar patterning and morphology.

Crown formation precedes root formation and elongation during tooth development. Because the process of tooth maturation in mice predictably occurs in an anterior to posterior fashion, we therefore investigated the identity of accessory molar buds in Evc2 global mutants via histological means. Roots were fully formed in M1 but rudimentary in M2 of control mice at P8 (Figure 4A brackets and arrows, respectively). By contrast, roots were fully formed in both T1 and T2 but rudimentary in T3 of Evc2 global mutants at P8 (Figure 4B brackets and arrows, respectively). This suggests that molars M1, T1, and T2 were at a similar and more advanced stage of development than molars M2 and T3.

Postnatal observations regarding tooth developmental progression were corroborated via further analyses at embryonic day E18.5. Not only was M1 predictably more developed (bell stage) than M2 (late cup/early bell) in control mice (Figure 4C), but T1 and T2 in Evc2 global mutants were at the same stage, albeit slightly delayed compared to M1 (Figure 4D). The T3 molar in Evc2 global mutants was also at a comparable, though slightly delayed, stage of development to M2 in control mice (Figure 4D). When analyzed at the earlier E16.5 timepoint, M1, which was at late cup stage/early bell stage was still more developed than M2, which was at the cup stage in control mice (Figure 4E). Both T1 and T2 (cup stage) in Evc2 global mutants were a bit delayed than M1, and T3 was a bit delayed than M2 (Figure 4F). Because anterior molars are known inducers of posterior molar formation (36), the presence of similarly staged structures in both Evc2 global and Evc2 Wnt1 mutants (Figures 4G,H) suggests the presence of a supernumerary anterior tooth. We did not see evidence of T4 formation (corresponding to M3 in controls) in Evc2 global mutants or Evc2 Wnt1 mutants. These observations are consistent with micro-CT analysis of Evc2 Wnt1-Cre mutants that Evc2 loss of function within dental mesenchyme phenocopies the molar pattern abnormalities in Evc2 global mutants.

Previous studies have indicated that elevated WNT signaling and decreased Hedgehog signaling lead to abnormal molar patterning and supernumerary tooth formation in mice (37, 38). We therefore investigated WNT signaling changes in E16.5 Evc2 global mutant molars using LEF1 as a readout of active WNT signaling. Compared to controls, we observed more cells labeled with LEF1 within the dental mesenchyme of Evc2 global mutant molars (Figures 5A,C,D,F). Additionally, the distribution pattern of SHH corroborates our earlier histologic assessment wherein M1 is in the bell stage, while M2, T1, and T2 are in the cup stage, respectively, at E16.5 (Figures 5B,E). Changes in overall Hedgehog signaling and WNT signaling levels were assessed from dissected M1 and T1 tooth germs at E18.5. qRT-PCR analyses indicated that Hedgehog signaling levels measured by expression of Gli1 and Ptch1 were lower in T1 than M1 (Figure 5G), despite comparable levels of SHH between two genotypes (Figures 5B,E) and that WNT signaling measured by levels of Axin2 and Lef1 was elevated in T1 than M1 (Figure 5H).

To understand at which development stage the compromised Hedgehog signaling and elevated WNT signaling leads to supernumerary anterior tooth formation, we then examined the Hedgehog and WNT signaling at E15.5. Comparing to controls, we observed lower intensity of PTCH1 immuno-signals and higher intensity of LEF1 immuno-signals in both dental epithelia and dental mesenchyme (Figures 6A–D,N), suggesting that compromised Hedgehog signaling and elevated WNT signaling are already present at this stage. In contrast to the controls that show only 1 enamel knot at this stage (Figure 6E), we detected two enamel knots in Evc2 mutants (4 out of 4 mutants). In addition to the one found in the middle of tooth germ (Figure 6G), the second one was identified at an anterior location (Figure 6F, F and G are from the same tooth germ but different plain). Associated with the additional enamel knot, we observed abnormal shape of dental epithelia in Evc2 mutant (Figure 6F), suggesting that the supernumerary tooth starts to form anterior to the M1 at E15.5 in Evc2 mutants.

Similar to E15.5, we detected both compromised Hedgehog signaling and elevated WNT signaling in Evc2 mutants at E14.5 (Figures 6H–K,O). However, we detected only 1 enamel knot in both controls and Evc2 mutants (Figures 6L,M). These observations suggest that compromised Hedgehog signaling and elevated WNT signaling induce abnormal molar patterning between E14.5 and E15.5. The observation of abnormal shaped epithelial tissue at E15.5, not at E14.5 support the idea that initial abnormal patterning occurs between E14.5 and E15.5.

Previous studies have demonstrated that primary cilia play a negative role in regulating WNT signaling (39). Since EVC2 is a ciliary-located protein, we evaluated whether Evc2 loss of function could lead to elevated cellular response to WNT ligands. Using isolated mouse embryonic fibroblasts (MEFs) cultured in a low serum condition to prompt ciliogenesis (ciliated cells), we quantified both Lef1 and Axin2 as readouts of the levels of WNT signaling induced by WNT3A. Under these conditions, we observed elevated WNT signaling in ciliated MEFs with an Evc2 mutation (Figure 7A). In contrast, for cells cultured in under normal serum conditions (non-ciliated cells), we did not observe any changes in response to WNT ligand between Evc2 mutants and controls. These studies indicate that primary cilia function to negatively regulate WNT signaling and that EVC2 protein within primary cilia is responsible, at least in part, for this function.

Finally, we then examined if there are other tissues in Evc2 mutant mice with elevated WNT signaling. Using TOP-gal reporter mice, we detected an increased number of X-gal-stained cells within Evc2 mutant growth plates compared to controls (Figures 7C–E). Similarly, WNT signaling was elevated within the perichondrium of the growth plates in Evc2 mutants. In E16.5 tibia, we observed an elevated number of cells with nuclear located beta-catenin in the perichondrium of Evc2 mutants (Figures 7F–K). This observation of elevated WNT signaling in multiple tissues of Evc2 mutant mice supports the idea that Evc2 loss of function potentially leads elevated response to WNT ligand in developing tooth, thereby contributing to abnormal molar patterning found in Evc2 mutant mice.