(A–F) Comparative 3D morphology of the epithelial part of developing upper molars in DUHi (A, C) and FVB (B, D) strains at cap transition. A, B are side views and C, D upper views. The variable anterior region including R2 rudimentary bud is denoted by the bracket. Expression of Shh at the same timepoint is shown below for DUHi (E) and FVB (F), for comparative purposes. The red arrowhead in E points to R2 signaling center. In F, the faint staining anterior to the mature M1 signaling center might correspond to re-expression of Shh in cells that formed R2 signaling center. (G–J) panel G shows the three measurements taken from all epithelial samples of a computed age of 14.25–15 cpdc (between 180 and 250 mg weight), comparing DUHi with FVB samples. The measures were taken between the anterior or posterior limit of dental epithelium and the anterior or posterior limit of the M1 signaling center, as shown on the cartoon. Boxplots H-J show the results in upper molars (see Figure 4—figure supplement 1 for lower molars). Pre-M1 region and Post-M1 regions are significantly different in DUHi versus FVB mice (t-test; p<0.01, see Supplementary file 4). (K–M) The developing molars of DUHi and FVB mice react differently when cultured in vitro at 13.0 dpc (p=0.015 in an exact Fisher test, see Supplementary file 4): R2 bud tends to form a clear individualized bud (M) in most DUHi tooth cultures (n = 18), whereas a single developing tooth (L) is seen in most FVB tooth cultures (n = 18). (N) Target genes of the Bmp4 pathway differentially expressed between the two strains at the cap transition. Differential expression analysis was performed on both lower and upper molar samples, taking molar type into account in the statistical treatment by DEseq2. Genes were classified as targets activated or repressed by the pathway based on O'Connell et al. (2012). The log2 fold change in DUHi as compared to FVB is shown (positive: expression level increased in DUHi tooth germs; negative: expression level decreased in DUHi tooth germs). Depending if the gene is an activated or a repressed target, and is increased or decreased in DUHi tooth germs, it may suggest that the pathway is more active (pink) or less active (blue) in DUHi tooth germs.