**Background:** Teeth are the most mineralized vertebrate tissues and key to studying mammalian evolution because subtle crown shape changes often mark speciation events. The Notch signaling pathway, including its ligand Jagged1, is known to regulate cell fate and tooth development. Jagged1 is expressed from early odontogenesis, but its germline deletion is embryonically lethal. This study used a tissue-specific knockout (K14Cre;Jagged1^fl/fl^) to investigate Jagged1's role in dental epithelium and its contribution to evolutionary tooth shape variation.
**Methods:** K14Cre;Jagged1^fl/fl^ mice were generated by crossing K14:Cre and Jagged1^flox^ lines. E18.5 embryos and adult specimens were analyzed. High-resolution X-ray synchrotron microtomography (ESRF beamline BM5, 20 keV, 7.45 µm voxel) was used for 3D imaging. Length and width of each molar were measured, and five additional distances (d1–d5) quantified mesial cusp variations. Elliptic Fourier Transform (EFT) outline analysis was performed on M^1^ and M~1~ using 64 equally spaced points; the first 9 harmonics for M^1^ and first 5 for M~1~ were retained. Principal component analysis (PCA) and MANOVA tested shape differences. RNA sequencing (Illumina HiSeq 4000, single-end 125 bp) was performed on lower first molars from 4 mutant and 4 WT E18.5 embryos. Differential expression was assessed with edgeR (p ≤ 0.05, fold change ≥ 0.5, ≥10 counts in half of samples in one group). RT-PCR validated key genes (n = 8 per group). Immunohistochemistry detected Notch1-ICD, Hes1, Hes5, β-catenin, Ki67, and Amelogenin. A 3D morphing algorithm (ANSA by BETA CAE Systems) was applied to compute transformation vectors from WT to mutant mouse M^1^ and transfer them to an average human M^1^ (derived from 246 stone replicas of West European 16–20 year-olds, scanned at 50 µm × 50 µm resolution).
**Key Results:** Mutant molars showed a U-shaped c^1^-c^2^ connection in ~60% of M^1^ specimens versus the V-shaped profile in all WT mice. The c^1^ cusp was positioned more linguo-distally, increasing spacing from c^2^ (d2 significantly greater). On M^2^, the c^1^ spur was absent in ~60% of mutants. On M^3^, c^1^ was reduced and merged with c^4^ in 30% of mutants. Lower molars showed partial fusion of c~1~ and c~2~ (d3–d5 significantly different; p = 0.003, <0.001, 0.001). PCA on M^1^ outlines showed partial separation on the third axis; MANOVA confirmed significant differences (Wilk's λ = 0.340, F = 7.427, p < 0.001). Mean molar sizes (L and W) were significantly lower in mutants (t-test), with no variance difference (F-test). RNA-seq identified >2000 differentially expressed genes. Upregulated pathways included mineralization, autophagy, ion transport, and cell cycle arrest. Enamel genes (Amelx, Ambn, Enam, Mmp20, Klk4) and odontoblast genes (Dspp, Dmp1) were upregulated. Mesenchymal patterning genes (Pax9, Barx1, Dlx1, Dlx2) were downregulated. Wnt pathway showed downregulation of Wnt11, Wnt10b, Wnt9b, Frizzled receptors, Tcf factors, and upregulation of Wnt3a, Wnt6, Wnt7a, Wnt10a, Apc. FGF inhibitors Spry1, Spry2, Spry4 were downregulated. Notch pathway members Jagged1, Hes5, Hes6, Lfng, Maml2 were downregulated; Hey1 and Dll4 were upregulated. RT-PCR confirmed Jag1, Notch1, Notch2, Hes5, Lef1, Tcf3, Fzd2 downregulation and Amelx, Ambn, Dspp, Dmp1, Wnt10a, Apc upregulation. Immunohistochemistry showed loss of Notch1-ICD and Hes1 in stratum intermedium of mutant cusps, reduced Hes5 in inner dental epithelium and odontoblasts, expanded Amelogenin distribution, and increased Ki67 in stratum intermedium and preameloblasts. The 3D morphing model predicted that in humans carrying Jagged1 mutations, the M^1^ would show a mesial-lingual/palatal shift of the hypocone, mesial displacement of the metacone, a deeper and narrower distal ridge, and a slightly widened distal-lingual/palatal groove, with minimal changes to the protocone and paracone.
**Clinical Implications:** This study identifies Jagged1-mediated Notch signaling as a fine-tuned regulator of tooth crown morphology, with disruption producing subtle changes reminiscent of microevolutionary variation seen in murid rodents. The computational model provides a testable prediction for dental phenotypes in humans with JAGGED1 mutations (e.g., Alagille syndrome). The findings suggest that premature cytodifferentiation of ameloblasts and odontoblasts, driven by deregulation of Notch, Wnt, and FGF pathways, may stabilize altered crown shapes during development. Understanding these molecular mechanisms could inform future studies of dental anomalies and evolutionary developmental biology.