Craniofacial landmark coordinates of DS mouse models

DATA Description

We make available the data used for craniofacial analysis of nine Down syndrome mouse models. For each DS model, one Zip file is available and contains

-      a minimum of four text files with all individual coordinates by genotype (DS model or wt control) for the skull or the mandibule (Mdb):

o   the first line of the text file describes the content: Cranium or mandible coordinate + wt control or DS model genotype + Sex (male or female)

o   XYZ coordinates

o   39L (how many landmarks) 3 (how many coordinates) 10A (how many samples)

o   The list of used landmarks (see figure 1 and Tables S1-S2)

o   Before the coordinate, the ID (number) of the individual is indicated 

-        Tiff files for all individuals used with the model that are in two folders, one for the DS/Dp model and one for the wt controls. All Tiff files are named with the ID number of the individual. 

Down Syndrome mouse models used

We used the Dp(16)1Yey and Tg(Dyrk1a) (official name Dp(16Lipi-Zbtb21)1Yey and Tg(Dyrk1a)189N3Yah) models (Li et al. 2007; Guedj et al. 2012) which were maintained on the C57BL/6J genetic background. We also used the SD: CRL Dp (11Lipi-Zbtb21)1Yah (short name Dp(Rno11)) rat model generated in the lab (Birling et al. 2017) that carries a duplication of the Lipi-Zbtb21, an interval similar to the mouse Dp(16)1Yey, found on rat chromosome 11.

New lines were generated via an in vivo chromosomal recombination technique, which combines a transposon system (Ruf et al. 2011) and a meiotic recombination system Cre-loxP (Hérault et al. 1998). The transposon system consists of the transposase enzyme and its substrate, the transposon. The enzyme recognizes specific repeat sequences (ITR) flanked on either side of a given DNA sequence (in this case, a vector containing a specific loxP site) (Ruf et al. 2011). Once two loxP sites bound a region of interest, successive crosses bring a transgene expressing the Cre recombinase into the same individual. In this animal, the Cre enzyme recombines the sequences of the loxP sites to produce a duplication (or partial trisomy) of the region of interest (Hérault et al. 1998; Hérault et al. 2010).

The new mouse models have been developed with the following segmental duplications in the Mmu16 (Figure 1). For Dp(16Samsn1-Cldn17)7Yah (Dp(16)7Yah) we duplicated the segment between Samsn1 and Cldn17. Dp(16Tiam1-Clic6))8Yah (Dp(16)8Yah) presents a duplication between Tiam1 and Clic6. Dp(16Cldn17-Brwd1))9Yah (Dp(16)9Yah) displays a duplication in the interval between Cldn17 and Brwd1. Dp(16Tmprss15-Setd4)10Yah (Dp(16)10Yah) has the segment between Tmprss15 and Setd4 duplicated, similar to Dp(16Tmprss15-Grik1)11Yah (Dp(16)11Yah), but this model presents a region duplicated until Grik1. Dp(16Tmprss15-Zbtb21)12Yah (Dp(16)12Yah) has the duplicated region from Tmprss15 to Zfp295, and Dp(16Cldn17-Vps26c(Dyrk1aKO))13Yah (Dp(16)13Yah) from Cldn17 to Vps26c, up to the sequence of Dyrk1a which is inactivated. All lines were maintained on C57BL/6J genetic background.

Figure 1 : Relative position of the duplicated interval in the DS mouse models is indicated by a line.

Mouse samples from the DS mouse models

To generate the data, we made cohorts of mice housed under specific pathogen-free (SPF) conditions, treated in compliance with the animal welfare policies of the French Ministry of Agriculture (law 87 848). As a major genotype effect compared to sex was previously described elsewhere independently (Redhead et al. 2023), we decided to use females. For each mouse line, about ten littermates by each genotype, DS, and wild-type (WT) were collected (n = 180).  We tried to have balanced males and females in the cohorts. For example, For the Dp(16)1Yey line, six females plus five males for the dup carrier and six males plus three females for control were used.   Nevertheless, this was not the case in all the other lines, with sometimes more female individuals collected than males, because males were used to breed the lines.

Micro-computed tomography scan of the skull and mandibule of mutant and control mouse lines

Animals were euthanized with the standard procedure at 14 weeks old.  Briefly, the mouse heads were dissected apart from the body. A polystyrene section was interposed between the mandible and maxilla to separate the jaws. After dissection, samples were fixed in a 4% paraformaldehyde solution (PFA), washed with water, and stored in 70% ethanol. The mouse heads were scanned using the Quantum FX micro-computed tomography imaging system (Caliper Life Sciences, Hopkinton, MA, USA) to evaluate the morphology of the skull and mandible. The images obtained were delivered in DICOM format. The scan parameters used to carry out the scanning of the samples correspond to 2 scans of every sample, anterior part, and posterior part using the mode Scan Technique Fine of 2 minutes, with a field of view (FOV) of 40 mm, the voltage 90 kV, CT 160 μA, resolution pixel size 10 µm and the capture size for live mode viewing in small, live current 80kV.

Imaging Processing

For each sample, two scans were obtained, one from the anterior area of the skull and one from the posterior region. FIJI software was used to unite these two scans and create a single file, performing the plugin “Stitching” and saving one file in TIFF format for each individual per model (WTs and Duplication or transgenic genotype). This format can be opened using different image processors. We make the level of raw data available here in the folder TIFF of each model.

Then, Stratovan Checkpoint software (Stratovan Corporation, Sacramento, USA, Version 2018.08.07. Aug 07, 2018.) was used to place the landmarks (Table S1 and S2; Figure 2) and extract the 3D coordinates for all the landmarks in all the samples. So, for each DS model, you will find an additional text file with individuals' landmark coordinates. You will find four text files for each model, 2 for the skull and 2 for the mandible, divided into WT and DS models.

Morphometrics is the quantification and statistical analysis of form. Form is the combination of size and shape of a geometric object in an arbitrary orientation and location (shape is what remains of the geometry of such an object once it is standardized for size). Various approaches can be employed when conducting morphometric analysis. The method of interest in this study is the landmark-based method, which is a conventional approach that relies on phenotypic measurements such as linear distances, angles, weights, and areas. In this case, we used 61 landmarks, 39 in the skull and 22 in the mandible (Figure 2), to obtain the 3D coordinates of the structure (Hallgrimsson et al. 2015).

Figure 2. Landmark positions for the skull and mandibule analysis.

Based on 3D coordinates, Euclidean Distance Matrix Analysis (EDMA) is one of the principal tools for analyzing landmark-based morphometric data (Lele et Richtsmeier 2001). This method builds a matrix of linear distances between all possible pairs of landmarks for each specimen (Lele et Richtsmeier 1991). Morphological differences between groups can be pinpointed to specific linear distances on an object through pairwise comparisons of mean form or shape matrices, followed by bootstrapping to estimate the significance of these differences (Lele et Richtsmeier 2001). In this study, two tests were done for each group of samples, first to analyze the form of the skull and mandibles with form difference matrix (FDM) and then the shape with the shape difference matrix (SDM).

In addition, to track the landmarks associated with a significant change and understand where they are located in the CF structures, “EDMA FORM or SHAPE Influence landmark analysis” was performed (Cole et Richtsmeier 1998). The purpose of this test is to search which landmarks present a Relative Euclidean distance (RED) > 1.05 or < 0.95 (outside of the confidence interval 97,8%), meaning, which landmarks show a bigger difference in linear distances between every landmark and in what direction.

Another way to handle landmark-based data is using a multivariate statistical analysis of form, geometric morphometric. This method relies on the superimposition of landmark coordinate data to place individuals into a common morpho-space. The most used superimposition form is the Generalized Procrustes (GP) method and Principal Component Analysis (PCA). This method places multiple individual specimens into the same shape space by scaling, translating, and rotating the landmark coordinates around the centroid of every sample (Rohlf et Slice 1990). As an alternative, we took advantage of Stratovan Checkpoint (Stratovan Corporation, Sacramento, USA) to create population average models and perform a voxel-based analysis, where we can observe directly in 3D models the changes between populations.

Finally, using the 3dMD Vultus® software, we created Procrustes average models created in Checkpoint to perform a landmarking calculation.

References

Birling, Marie-Christine, Laurence Schaeffer, Philippe André, Loic Lindner, Damien Maréchal, Abdel Ayadi, Tania Sorg, Guillaume Pavlovic, et Yann Hérault. 2017. « Efficient and Rapid Generation of Large Genomic Variants in Rats and Mice Using CRISMERE ». Scientific Reports 7 (1): 43331. https://doi.org/10.1038/srep43331.

Cole, T. M., et J. T. Richtsmeier. 1998. « A Simple Method for Visualization of Influential Landmarks When Using Euclidean Distance Matrix Analysis ». American Journal of Physical Anthropology 107 (3): 273‑83. https://doi.org/10.1002/(SICI)1096-8644(199811)107:3<273::AID-AJPA4>3.0.CO;2-1.

Guedj, Fayçal, Patricia Lopes Pereira, Sonia Najas, Maria-Jose Barallobre, Caroline Chabert, Benoit Souchet, Catherine Sebrie, et al. 2012. « DYRK1A: A master regulatory protein controlling brain growth ». Neurobiology of Disease 46 (1): 190‑203. https://doi.org/10.1016/j.nbd.2012.01.007.

Hallgrimsson, Benedikt, Christopher J. Percival, Rebecca Green, Nathan M. Young, Washington Mio, et Ralph Marcucio. 2015. « Chapter Twenty - Morphometrics, 3D Imaging, and Craniofacial Development ». In Current Topics in Developmental Biology, édité par Yang Chai, 115:561‑97. Craniofacial Development. Academic Press. https://doi.org/10.1016/bs.ctdb.2015.09.003.

Hérault, Y., M. Rassoulzadegan, F. Cuzin, et D. Duboule. 1998. « Engineering Chromosomes in Mice through Targeted Meiotic Recombination (TAMERE) ». Nature Genetics 20 (4): 381‑84. https://doi.org/10.1038/3861.

Hérault, Yann, Arnaud Duchon, Damien Maréchal, Matthieu Raveau, Patricia L. Pereira, Emilie Dalloneau, et Véronique Brault. 2010. « Controlled Somatic and Germline Copy Number Variation in the Mouse Model ». Current Genomics 11 (6): 470‑80. https://doi.org/10.2174/138920210793176038.

Lele, Subhash R., et Joan T. Richtsmeier. 2001. An Invariant Approach to Statistical Analysis of Shapes. CRC Press.

Li, Zhongyou, Tao Yu, Masae Morishima, Annie Pao, Jeffrey LaDuca, Jeffrey Conroy, Norma Nowak, Sei-Ichi Matsui, Isao Shiraishi, et Y. Eugene Yu. 2007. « Duplication of the Entire 22.9 Mb Human Chromosome 21 Syntenic Region on Mouse Chromosome 16 Causes Cardiovascular and Gastrointestinal Abnormalities ». Human Molecular Genetics 16 (11): 1359‑66. https://doi.org/10.1093/hmg/ddm086.

Redhead, Yushi, Dorota Gibbins, Eva Lana-Elola, Sheona Watson-Scales, Lisa Dobson, Matthias Krause, Karen J. Liu, Elizabeth M. C. Fisher, Jeremy B. A. Green, et Victor L. J. Tybulewicz. 2023. « Craniofacial dysmorphology in Down syndrome is caused by increased dosage of Dyrk1a and at least three other genes ». Development 150 (8): dev201077. https://doi.org/10.1242/dev.201077.

Rohlf, F. James, et Dennis Slice. 1990. « Extensions of the Procrustes Method for the Optimal Superimposition of Landmarks ». Systematic Biology 39 (1): 40‑59. https://doi.org/10.2307/2992207.

Ruf, Sandra, Orsolya Symmons, Veli Vural Uslu, Dirk Dolle, Chloé Hot, Laurence Ettwiller, et François Spitz. 2011. « Large-Scale Analysis of the Regulatory Architecture of the Mouse Genome with a Transposon-Associated Sensor ». Nature Genetics 43 (4): 379‑86. https://doi.org/10.1038/ng.790.

Tables

Landmarks Cranium

1

Nasale: Intersection of nasal bones, rostral point

2

Nasion: Intersection of nasal bones, caudal point   

3

Bregma: intersection of frontal bones and parietal bones at midline

4

Intersection of parietal bones with anterior aspect of interparietal bone at midline

5

Intersection of interparietal bones with squamous portion of occipital bone at midline

6

Opisthion, midsagittal point on the posterior margin of the foramen magnum

7

Center of alveolar ridge over maxillary incisor, right side

8

Anterior Intersection of frontal process of maxilla with frontal bone, right side.

9

Anterior notch on frontal process lateral to infraorbital fissure, right side

10

Intersection of frontal process of maxilla with frontal and lacrimal bones, right side

11

Frontal-squasmosal intersection at temporal crest, right side

12

Intersection of zygoma (jugal) with zygomatic process of temporal, superior aspect, left side

13

Intersection of zygoma (jugal) with zygomatic process of temporal, inferior aspect, left side

14

Most posteroinferior point on the superior portion of the tympanic ring, right side

15

Center of alveolar ridge over maxillary incisor, left side

16

Anterior Intersection of frontal process of maxilla with frontal bone, left side.

17

Anterior notch on frontal process lateral to infraorbital fissure, left side

18

Intersection of frontal process of maxilla with frontal and lacrimal bones, left side

19

Frontal-squasmosal intersection at temporal crest, left side

20

Intersection of zygoma (jugal) with zygomatic process of temporal, superior aspect, right side

21

Intersection of zygoma (jugal) with zygomatic process of temporal, inferior aspect, right side

22

Most poteroinferior point on the superior portion of the tympanic ring, left side

23

Most anterior point of the anterior palatine foramen, right side

24

Most posterior point of the anterior palatine foramen, right side

25

Most infero lateral point on premaxilla-maxilla suture, right side

26

The anterior most point on the central ant/post axis of the right molar alveolus

27

Intersection of zygomatic process of maxilla with zygoma (jugal), inferior surface, right side

28

Lateral intersection of maxilla and palatine bone posterior to the third molar, right side

29

Joining of squasmosal body to zygomatic process of squasmosal, right side

30

Most inferior aspect of posterior tip of medial pterygoid process, right side

31

Most anterior point of the anterior palatine foramen, left side

32

Most posterior point of the anterior palatine foramen, left side

33

Most infero lateral point on premaxilla-maxilla suture, left side

34

The anterio most point on the central ant/post axis of the left molar alveolus

35

Intersection of zygomatic process of maxilla with zygoma (jugal), inferior surface, left side

36

Lateral intersection of maxilla and palatine bone posterior to the third molar, left side

37

Joining of squasmosal body to zygomatic process of squasmosal, left side

38

Most inferior aspect of posterior tip of medial pterygoid process, left side

39

Basion, midsagittal point on the anterior margin of the foramen magnum

 Table S1: 39 Skull Landmarks.

Landmarks Mandible

1

Apex of coronoid process, Right side

2

Intersection of molar alveolar rim and base of coronoid process, Right side

3

Anterior edge of alveolar process where first molar hits alveolus at the midline, Right side

4

Superior-most point on incisor alveolar rim at midline (at bone-tooth junctions), Right side

5

Inferior-most point on incisor alveolar rim at midline (at bone-tooth junction), Right side

6

Inferior point on mandibular symphysis, Right side

7

Anterior edge of the coalescence of curve of masseteric ridge with post-symphyseal rugged area, Right side

8

Tip of mandibular angle, Right side

9

Posterior midline point on condyle, Right side

10

Anterior midline point on condyle, Right side

11

Anterior edge of the mental foramen, Right side

12

Apex of the coronoid process, left side

13

Intersection of molar alveolar rim and base of coronoid process, left side

14

Anterior edge of alveolar process where first molar hits alveolus at the midline, left side

15

Superior-most point on incisor alveolar rim at midline (at bone-tooth junctions), left side

16

Inferior-most point on incisor alveolar rim at midline (at bone-tooth junction), left side

17

Inferior point on mandibular symphysis, left side

18

Anterior edge of the coalescence of curve of masseteric ridge with post-symphyseal rugged area, left side

19

Tip of mandibular angle, left side

20

Posterior midline point on condyle, left side

21

Anterior midline point on condyle, left side

22

Anterior edge of the mental foramen, left side

 Table S2: 22 mandible Landmarks.