DOI: 10.5061/dryad.ghx3ffbvb
title: Palatal segment contributions to midfacial anterior-posterior growth

description: 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.
description: 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
 mm<sup>3</sup> voxel size. µCT images of P1 heads were
 acquired similarly, but with 0.021 mm<sup>3</sup> 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
description: # **Palatal segment contributions to midfacial anterior-posterior growth**
 [https://doi.org/10.5061/dryad.ghx3ffbvb](https://doi.org/10.5061/dryad.ghx3ffbvb) This dataset contains all files required for morphometric measurement and analysis of palatal and midfacial structures in our ontogenetic sample of mouse specimens. ## Description of the data and file structure **Specimen Names** Embryonic and P1 specimens share a file naming convention of of *strain*_*day_partial-day_litter_specimen*. So, specimen b6_14_0_1_6 is the sixth specimen from the first litter collected at embryonic day (E) 14.0 for the C57BL/6J (b6) strain. Embryonic ages noted in the specimen names are embryonic days since copulatory plug identification rather than the limb bud-based developmental ages used throughout most of the subsequent analyses. Adult specimens have a naming convention of *strain_ad_specimen*, where "ad" refers to the adult age category and the specimen names include the standard Collaborative Cross two letter founder strain codes followed by a four digit number (see also Percival, et al., 2016). So, specimen b6_ad_bb0544 is an adult ('ad') specimen number 0544 within strain 'bb' (as defined within Collaborative Cross mouse publications), also known as the C57BL/6J inbred strain. **Micro-Computed Tomography (µCT or CT) Images** are provided as .zip compressed directories of .aim format 3D image files. This file format was developed by SCANCO Medical AG as part of their µCT analytical pipelines. These can be used to produce the minimum threshold epithelial or bone surfaces that were landmarked as the basis for morphometric analysis. * CTs_b6_Adult.zip includes µCT images for adult C57BL/6J (b6) strain mice. * CTs_b6_Embryo_1.zip includes µCT images for C57BL/6J (b6) strain mice collected between embryonic day (E)11.5 and E13.5. * CTs_b6_Embryo_2.zip includes µCT images for C57BL/6J (b6) strain mice collected between embryonic day (E)14.0 and E15.5. * CTs_b6_P1.zip includes µCT images for C57BL/6J (b6) strain mice collected at postnatal day 1 (P1) **Landmark Files** are provided within LandmarkFiles.zip. All Landmark files are in the *markups fiducial point list file format* (.fcsv) used by 3DSlicer ([https://slicer.readthedocs.io/en/latest/developer_guide/modules/markups.html](https://slicer.readthedocs.io/en/latest/developer_guide/modules/markups.html)). A P1 specimen's epithelial and skeletal landmarks are contained within a single .fcsv file. Landmarks are defined and illustrated within the text of the associated manuscript. **Visualizing Tissue Surfaces and Landmarks in 3D** These µCT images are intended for use within 3D Slicer software (Fedorov, et al., 2012; [https://www.slicer.org/](https://www.slicer.org/)). Once loaded into 3D Slicer, volume information must be changed for landmark files to line up. 1. Load the .aim image file into 3D Slicer 2. Open the Volumes Module and select the loaded image file as your Active Volume. 3. Open the Volume Information tab. 4. Set the image spacing (voxel dimensions in mm) as follows: * Embryonic specimens: 0.012 x 0.012 x 0.012 * P1 specimens: 0.021 x 0.021 x 0.021 * Adult specimens: 0.035 x 0.035 x 0.035 5. Set the image origin to 0 x 0 x 0 6. Create a minimum threshold based epithelial or skeletal surface for a specimen (depending on specimen age and tissue of interest) 7. Measured landmarks can be visualized on these surfaces after loading that specimen's .fcsv landmark file into 3D Slicer **Hindlimb Bud Images** are optical dissecting microscope images of specimen hindlimbs that were used to estimate comparable developmental ages for embryonic specimens using eMOSS (Musy et al., 2018). These .jpg images files are provided within HindlimbBudImages.zip. File names are the specimen names with a extra letter at the end of the specimen name when there are multiple images of a single specimen. **Morphometrics Analysis** was completed in R Statistical Software (R Core Team, 2021). Analysis input are provided within AnalysisInput.zip and includes the following .csv files that are intended to be read into R using the provided custom R scripts: * Landmark input files include a group of specimen's landmarks, as derived from the individual specimen landmark .fcsv files. Each row is a specimen, identified by specimen name. Each column is a single dimension coordinate for a single landmark. For example, LM1.x is the x dimension coordinate of landmark 1, LM1.y is the y dimension coordinate for the same landmark. Each landmark has an x, y, and z coordinate, so each is defined with three consecutive columns. * Embryo_Landmarks.csv includes embryo specimen landmarks * P1_Epi_Landmarks.csv includes P1 specimen landmarks collected on an epithelial palate surface * Skel_Landmarks.csv includes P1 and adult specimen landmarks collected on a skeletal palatal surface * Reliability_LMs.csv includes landmarks from two landmark collection trials for 9 embryonic specimens. This data serves as the basis for a landmark collection intraobserver error study, but are not precisely the same coordinates used for the published morphometric analysis * Covariate input files include the covariates for a group of specimens. Each row of a covariate file are single specimens in the same order that they are found in the associated landmark input file, identified by specimen name. Column 'X' is a specimen's row index within the file. Column 'Geno' is the mouse strain with three possible values (b6, nod, pwk). The 'PWK.Specs', 'C57.Specs', and 'NOD.Specs' are TRUE/FALSE columns where TRUE indicates that the specimen is a given strain within the 'Geno' column, where PWK==PWK/PhJ, C57==C57BL/6J, and NOD==NOD/ShiLtJ. Column 'Ages' is a text column that indicates the age of each specimen at euthanasia as either embryonic day since copulatory plug (between 11_5 and 15_5), postnatal day 1 (p1) or adult (ad). Other columns are defined for the files they are found within. * Embryo_Covar.csv is the covariate data for specimens in Embryo_Landmarks.csv. Column 'Ages.Num' is the numeric embryonic day value from text column 'Ages'. Column 'Limb.Ages' is the limb bud derived developmental age estimate for each specimen, as estimated using eMOSS (Musy et al., 2018). Column 'LimbAges.Simple' are the developmental ages from 'Limb.Ages' after binning the values to the closest whole or half day. * P1_Epi_Covar.csv is the covariate data for specimens in P1_Epi_Landmarks.csv. Column 'Ages.Num' is the numeric embryonic day value from text column 'Ages', where we assume that birth occurs at embryonic day 18.5 and therefore postnatal day 1 is the equivalent of embryonic day 19.5. This column was intended for direct quantitative comparison of P1 epithelial measures to those collected for embryonic samples. * Skel_Covar.csv is the covariate data for specimens in Skel_Landmarks.csv. Morphometric Analysis R scripts are provided through Zenodo. * Embyro_Morpho.R represents the morphometric analysis epithelial landmarks collected on the embryonic sample, as described in the associated manuscript. * Postnatal_Morpho.R represents the morphometric analysis of epithelial and skeletal landmarks collected on the P1 and skeletal landmarks collected on adult sample, as described in the associated manuscript. * Reliability_Study.R represents the intraobserver landmark placement error study based on two data collection trials from 9 embryonic specimens, as described in the associated manuscript R scripts were run in R Statistical Software (R Core Team, 2021) version 4.3.1 The following R libraries are used within these scripts: * *geomorph* version 4.0.5 * *ggplot2* version3.4.2 * *abind* version 1.4-5 * *segmented* version 1.6-4 * *RRPP* version 1.3.1 * *rgl* version 1.2.1 * *Matrix* 1.6-1 ## Sharing/Access information Adult landmark coordinate data was previously analyzed and described in the following manuscript: * 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](https://doi.org/10.1111/joa.12382) Other publicly accessible data related to the adult specimens in this analysis can be found in the MusMorph dataset: * Devine, J., Vidal-García, M., Liu, W., Neves, A., Lo Vercio, L.D., Green, R.M., Richbourg, H.A., Marchini, M., Unger, C.M., Nickle, A.C., Radford, B., Young, N.M., Gonzalez, P.N., Schuler, R.E., Bugacov, A., Rolian, C., Percival, C.J., Williams, T., Niswander, L., Calof, A.L., Lander, A.D., Visel, A., Jirik, F.R., Cheverud, J.M., Klein, O.D., Birnbaum, R.Y., Merrill, A.E., Ackermann, R.R., Graf, D., Hemberger, M., Dean, W., Forkert, N.D., Murray, S.A., Westerberg, H., Marcucio, R.S., Hallgrímsson, B., 2022. MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses. Sci. Data 9, 1–18. [https://doi.org/10.1038/s41597-022-01338-x](https://doi.org/10.1038/s41597-022-01338-x) * [https://www.facebase.org/](https://www.facebase.org/), [https://doi.org/10.25550/3-HXMC](https://doi.org/10.25550/3-HXMC) ## References 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](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](https://doi.org/10.1111/joa.12382) R Core Team, 2021. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria [WWW Document]. URL [https://www.R-project.org/](https://www.R-project.org/)