Palatal segment contributions to midfacial anterior-posterior growth

Following facial prominence fusion, anterior-posterior (A-P) elongation of the palate is a critical aspect of palatogenesis and integrated midfacial elongation. Reciprocal epithelial-mesenchymal interactions drive secondary palate elongation and periodic signaling center formation within the rugae growth zone (RGZ). However, the relationship between RGZ dynamics and the morphogenetic behavior of underlying palatal bone mesenchymal precursors has remained enigmatic. As part of a broader multifaceted study of these interactions within C57BL/6J mice, we completed a morphometrics analysis of 1) ontogenetic shape change of the palate and midface between embryonic day (E) 11.0 and E15.0 and 2) embryonic and postnatal longitudinal growth and proportional contributions of primary palate, anterior secondary palate, and posterior secondary palate to overall hard palate length.

Our overall shape analysis identifed the major ontogenetic trends in palatal and midfacial shape change between E11.0 and E15.0, a critical early period of facial development. Our ontogenetic analysis of palatal segment lengths indicated that the three major palatal segments significantly elongated during the same embryonic period, between E15 and postnatal day (P) 1, and between P1 and adulthood. The anterior secondary palate contributed proportionally more than the primary palate or posterior secondary palate to overall embryonic and perinatal hard palate elongation. However, the primary palate contributed proportionally more to longitudinal hard palate growth between P1 and adult samples. These results indicate the major importance of anterior secondary palate growth during the earliest period of midfacial outgrowth. Changes to the rate or timing of anterior secondary palate elongation, potentially by modifying associated RGZ gene expression dynamics, may contribute to intraspecies or interspecies differences in upper jaw morphology. However, postnatal growth processes, including the significant postnatal growth of the primary palate derived premaxillary bone, also contribute to variation in adult upper jaw morphology.

Methods
Animal breeding, specimen collection, and tissue fixation were performed in accordance with the protocols of the University of California, San Francisco Institutional Animal Care and Use Committee under protocol approval number AN192776-01F. Mice were socially housed under a twelve-hour light-dark cycle with food and water ad libitum. Additional enrichment was provided when single housing was required for breeding purposes. Mice were euthanized by CO2 inhalation followed by cervical dislocation or decapitation. C57BL/6J (RRID:IMSR_JAX:000664; Jackson Labs, Bar Harbor, ME) embryos were collected between gestational days E11.5 and E15.5, as determined from copulatory plug occurrence. Ten postnatal day one (P1) specimens were collected. Embryo and P1 specimens were fixed in 4% PFA and stored in 1x PBS for micro-computed tomography (μCT) imaging.

Specimens were received, stored, and imaged at the University of Calgary in accordance with the protocols of the University of Calgary Institutional Care and Use Committee under approval number AC13-0268. After approximately an hour of soaking in Cysto-Conray II (Liebel-Flarsheim Canada), embryo heads were placed upside down on cheese wax and immediately µCT scanned in air, a method that leads to minimal dehydration and good tissue surface quality of surface anatomical structures (Schmidt, et al. 2010).  These µCT images were acquired with a Scanco µ35 with 45kV/177µA for images of 0.012 mm3 voxel size. µCT images of P1 heads were acquired similarly, but with 0.021 mm3 voxel size. Photographs of embryo hindlimb buds were collected using a dissecting microscope for developmental age estimation. Adult specimens were previously collected and µCT imaged as described by Percival et al., 2016. 

Developmental age was estimated for each embryonic specimen using eMOSS, an application that predicts developmental age from hindlimb bud outlines, based on a previous analysis of C57BL/6J mice (Musy et al., 2018). The resulting limb-based estimates of developmental age were reported as days since conception, up to two decimal places. We combined similar developmental age estimates within whole- or half-day developmental age categories, which include specimens within 0.25 days of their initial eMOSS estimate.

All embryo midfacial and palate landmarks were collected within Meshlab (Cignoni et al., 2008) on minimum threshold-based epithelial tissue surfaces (downsampled x2) produced from the µCT images. These epithelial landmarks are defined in the associated publication. A subset of epithelial palatal landmarks were collected on epithelial surfaces produced from ten P1 specimen 𝜇CT scans. Minimum threshold-based skeletal surfaces of the same ten P1 and twenty adult (70-73 days old; 9 male and 11 female) specimens were produced using 3D Slicer (Fedorov et al., 2012) after Gaussian blur image filtering (sigma set to 0.01 for P1; sigma set to 0.02 for adult). Great care was taken to identify skeletal anatomical landmarks that closely and homologously matched the palate segment landmarks defined on surface epithelium. 

A reliability study was completed to quantify intraobserver error in landmark placement. Two trials were completed by a single epithelial landmark observer on three E11.5, three E12.5, and three E14.5 specimens. The second trials were completed more than one year after the first trials were completed, providing a realistic estimate of error across the entire period of landmark data collection.

All landmark and µCT files found within this Dryad dataset have been reformatted to be loaded into 3D Slicer (Fedorov, et al., 2012) rather than Meshlab, based on current Percival Lab practice.

References

Cignoni, P., Callieri, M., Corsini, M., Dellepiane, M., Ganovelli, F., Ranzuglia, G., 2008. Meshlab: an open-source mesh processing tool., in: Eurographics Italian Chapter Conference. pp. 129–136. https://doi.org/10.2312/LocalChapterEvents/ItalChap/ItalianChapConf2008/129-136

Fedorov, A., Beichel, R., Kalpathy-Cramer, J., Finet, J., Fillion-Robin, J.-C., Pujol, S., Bauer, C., Jennings, D., Fennessy, F., Sonka, M., Buatti, J., Aylward, S., Miller, J.V., Pieper, S., Kikinis, R., 2012. 3D Slicer as an image computing platform for the Quantitative Imaging Network. Magnetic Resonance Imaging 30, 1323–1341. https://doi.org/10.1016/j.mri.2012.05.001

Musy, M., Flaherty, K., Raspopovic, J., Robert-Moreno, A., Richtsmeier, J.T., Sharpe, J., 2018. A quantitative method for staging mouse embryos based on limb morphometry. Development 145, 1–7.

Percival, C.J., Liberton, D.K., Pardo-Manuel de Villena, F., Spritz, R., Marcucio, R., Hallgrímsson, B., 2016. Genetics of murine craniofacial morphology: diallel analysis of the eight founders of the Collaborative Cross. Journal of Anatomy 228, 96–112. https://doi.org/10.1111/joa.12382