Maxilla

When analyzing growth in the antero-posterior dimension we calculated projected distances between parallel planes passing through selected landmarks. To achieve this, a best-fit mid-sagittal plane was manually constructed for each scan (Supplementary Figure 2, top), following which a transverse plane was constructed perpendicular to the mid-sagittal plane, touching the palatal cusp tips of the right and left first and second molars (for P7 animals these teeth have not yet erupted but are visible in their crypts). Finally, axial planes passing through the landmarks of interest were constructed perpendicular to the first two planes. For bilaterally paired landmarks, an average of the right and left measurements was used for the analysis An example of this is shown in Supplementary Figure 2 depicting how the palatal, maxillary, and premaxillary dimensions were measured. This method of measurement is appropriate since one of our goals was to assess the growth pattern of regions within the craniofacial skeleton (i.e., the cranial base, vault, and facial skeleton), each comprised of multiple ordered individual bones. Consequently, growth in the A-P dimension can result in dynamically altered architectural arrangement of bones within each region in multiple planes, making interpretation of Euclidian distance difficult. Additionally, our method allows us to appreciate how different regions of the craniofacial skeleton are growing in proportion to the overall A-P dimension. Since the scans of each animal were not always obtained with teeth in occlusion, projected distances could not be reliably applied to the mandible and Euclidian distances were utilized instead. For measurements in the transverse dimension, the Euclidian distance between the right and left paired set of selected landmarks was calculated. Angular measurements were obtained using mid-sagittal landmarks.
To assess significance, Students' t-tests (two-tailed, equal variance) were carried out between corresponding measurements at consecutive ages. Hence P14 was compared to P7, P21 was compared to P14 and so forth. It should be noted that significance can be influenced by the age intervals being tested. For example if we tested a larger age interval, differences would typically reach significance. However, with the additional of extra time points within the interval, the differences in growth between intervals become smaller in magnitude and, given the variability and number of animals used in this study, thus formally lose significance.

Total RNA was extracted using Trizol from freshly dissected E10.5 maxillary process tissues. Three independent RNA samples were prepared for each genotype (WT and wnt1^cre; Rosa26^Dlx2/-). We used 2 μg total RNA as input material for the library preparations for each sample. Sequencing libraries were generated using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (#E7530L, NEB, United States) following the manufacturer’s recommendations. The libraries were sequenced on an Illumina HiSeq X ten platform and 150 bp paired-end reads were generated. Sequenced reads were mapped to the mm 10 genome using STAR aligner version 2.7.3a. Comparisons between the RNA-seq datasets were performed using the DESeq2 package in R. Enrichment analyses and visualization of functional profiles of differentially expressed genes (DEGs) were performed using the clusterProfiler package in R.