This work was supported in part by grants from the Hainan Provincial Natural Science Foundation of China (824MS152).

We have complied with all relevant ethical regulations for animal testing and research. All experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the Shanghai Ninth People&#39;s Hospital, School of Medicine, Shanghai Jiaotong University.
Both the Rosa26-loxp-stop-loxp-dnRAR&#945;403 strain (R26dn/dn) and the Osterix-Cre (OsxCre) (No.006361) strain of mice used here were maintained on the C57BL/6 background. R26dn/dn mice were crossed with OsxCre mice to generate OSXCre;R26dn/dn mice. All mice were bred and maintained under specific pathogen-free (SPF) conditions.
1. Breeding of mice
NOTE: F means the number of generations of mice; N means the number of generations of mating between zygotic mice and background mice. Thus, F2+N represents the second generation as well as any subsequent mouse breeding programs. F0 means primary mice.
<ol>
	<li>Pair a sexually mature male mouse with a pair of female mice of a comparable age. Commence daily inspections for newborn pups after an initial period of 18 days. Upon identifying pregnant females, isolate them if necessary to ensure optimal maternal care. In the event that no pregnancies are observed within a month of the initial pairing, consider alternating the male mouse among different breeding setups to promote fertilization.</li>
	<li>Cross R26dn/dnmice withOSXCre mice (F0). Clip the tails for genotyping and keep the male OSXCre;R26dn/+mice in cages until sexually mature, which is ~6 weeks of age (F1).</li>
	<li>Cross six-week-old male OSXCre;R26dn/+mice with female R26dn/dnmice (F2). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F2+N).</li>
</ol>
2. Preparation
<ol>
	<li>Prepare four pairs of 4-week-old OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. Individually euthanize the mice through the administration of carbon dioxide asphyxiation. 
	NOTE: To ensure efficient euthanasia, the CO2 flow rate should be adjusted to displace approximately 30% of the cage&#39;s volume every minute. For instance, in a cage measuring 45 cm x 30 cm x 30 cm, a flow rate of 40 L/min is recommended.</li>
	<li>Cut the neck of the mouse with scissors while holding its body, leaving the skull intact. 
	NOTE: To obtain a complete skull, open the mouth of the mouse, cut along the corner of the mouth with ophthalmic scissors, pull both hands to both sides, and rotate to peel the skin of the mouse&#39;s head.</li>
	<li>Immerse the mouse skulls in 4% paraformaldehyde for 48 h, then store them in 70% ethanol. 
	CAUTION: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>Scan the collected skulls with a microCT scanner- resolution: 4.5 &#181;m; voltage: 70 kV; current: 114 &#181;A; filter: 0.5 mm Al; rotation step: 0.5&#176;.</li>
	<li>Store CT scan images. Images in DICOM format are selected for import into the software and analyzed.</li>
</ol>
3. MicroCT imaging and 3D reconstruction
NOTE: All bones utilized in this study were manually segmented.
<ol>
	<li>Click the right mouse button on the MASKs page and select New mask; a new layer named Green appears.</li>
	<li>Select the threshold range of 671-2,566 in the Thresholding page that pops up. Repeat this process for all related layers. (Figure 1A).</li>
	<li>Use the Eraser tool in the Edit menu to manually erase the bones attached to the mandible in each layer on the 2D sagittal plane (Figure 1B).</li>
	<li>Select the region of the mandible on the 2D sagittal plane after clicking on the Region Growing option (Figure 1C). 
	CAUTION: Ensure that the mandibular region is selected completely in each layer; otherwise, it will result in an incomplete mandible after reconstruction.</li>
	<li>Click on the newly created layer Mandible in the MASKS page and select Calculate 3D from the dropdown menu (Figure 1D).</li>
	<li>In the 3D objects interface, right-click Mandible and click Properties. Click to change Color. Color code all the 3D models reconstructed with different bones.</li>
	<li>Click on Measure in the upper left corner and select distance from the dropdown menu that appears. Calculate the length by marking points in the 3D image (Figure 1E). 
	CAUTION: The reference for measuring bone width is the widest part of the bone.</li>
	<li>In the 3D-objects interface, right-click Mandible and click Properties. Properties-Info-Volume module to read the reconstructed volume values for each craniomaxillofacial bone block. (Figure 1F).</li>
	<li>Measure the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone in the same way as above and mark them individually with different colors.</li>
</ol>
4. Statistical analysis
<ol>
	<li>Perform statistical analysis using the software of choice. Perform two-tailed Student&#39;s t-tests (n = 4/group).</li>
	<li>For all graphs, use error bars to represent standard deviations. Consider P-values &#60; 0.05 statistically significant.</li>
</ol>

<ol><li>Chai, Y., Maxson, R. E. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+craniofacial+morphogenesis%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Recent advances in craniofacial morphogenesis.</a> Dev Dyn. 235 (9), 2353-2375 (2006).</li>
<li>Mason, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Should+universal+distribution+of+high+dose+vitamin+A+to+children+cease%5BTitle%5D)+AND+%22BMJ%22%5BJournal%5D)">Should universal distribution of high dose vitamin A to children cease.</a> BMJ. 360, k927(2018).</li>
<li>Roulier, S., Rochette-Egly, C., Rebut-Bonneton, C., Porquet, D., Evain-Brion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+retinoic+acid+receptor+characterization+in+cultured+human+trophoblast+cells%3A+Effect+of+retinoic+acid+on+epidermal+growth+factor+receptor+expression%5BTitle%5D)+AND+%22Mol+Cell+Endocrinol%22%5BJournal%5D)">Nuclear retinoic acid receptor characterization in cultured human trophoblast cells: Effect of retinoic acid on epidermal growth factor receptor expression.</a> Mol Cell Endocrinol. 105 (2), 165-173 (1994).</li>
<li>Hayes, K. C., Cousins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitamin+A+deficiency+and+bone+growth.+I.+Altered+drift+patterns%5BTitle%5D)+AND+%22Calcif+Tissue+Res%22%5BJournal%5D)">Vitamin A deficiency and bone growth. I. Altered drift patterns.</a> Calcif Tissue Res. 6 (2), 120-132 (1970).</li>
<li>Ghyselinck, N. B., Duester, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinoic+acid+signaling+pathways%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Retinoic acid signaling pathways.</a> Development. 146 (13), dev167502(2019).</li>
<li>Damm, K., Heyman, R. A., Umesono, K., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+inhibition+of+retinoic+acid+response+by+dominant+negative+retinoic+acid+receptor+mutants%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants.</a> Proc Natl Acad Sci U S A. 90 (7), 2989-2993 (1993).</li>
<li>Ford-Hutchinson, A. F., Cooper, D. M., Hallgrímsson, B., Jirik, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+skeletal+pathology+in+mutant+mice+by+microcomputed+tomography%5BTitle%5D)+AND+%22J+Rheumatol%22%5BJournal%5D)">Imaging skeletal pathology in mutant mice by microcomputed tomography.</a> J Rheumatol. 30 (12), 2659-2665 (2003).</li>
<li>Brewer, S., Feng, W., Huang, J., Sullivan, S., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt1-cre-mediated+deletion+of+ap-2alpha+causes+multiple+neural+crest-related+defects%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Wnt1-cre-mediated deletion of ap-2alpha causes multiple neural crest-related defects.</a> Dev Biol. 267 (1), 135-152 (2004).</li>
<li>Wong, M. D., Dorr, A. E., Walls, J. R., Lerch, J. P., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+3d+mouse+embryo+atlas+based+on+micro-ct%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A novel 3d mouse embryo atlas based on micro-ct.</a> Development. 139 (17), 3248-3256 (2012).</li>
<li>Motch Perrine, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Craniofacial+divergence+by+distinct+prenatal+growth+patterns+in+fgfr2+mutant+mice%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Craniofacial divergence by distinct prenatal growth patterns in fgfr2 mutant mice.</a> BMC Dev Biol. 14, 8(2014).</li>
<li>Percival, C. J., Huang, Y., Jabs, E. W., Li, R., Richtsmeier, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+craniofacial+bone+volume+and+bone+mineral+density+in+fgfr2%28%2B%2Fp253r%29+and+nonmutant+mice%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Embryonic craniofacial bone volume and bone mineral density in fgfr2(+/p253r) and nonmutant mice.</a> Dev Dyn. 243 (4), 541-551 (2014).</li>
<li>Titiz, I., Laubinger, M., Keller, T., Hertrich, K., Hirschfelder, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repeatability+and+reproducibility+of+landmarks--a+three-dimensional+computed+tomography+study%5BTitle%5D)+AND+%22Eur+J+Orthod%22%5BJournal%5D)">Repeatability and reproducibility of landmarks--a three-dimensional computed tomography study.</a> Eur J Orthod. 34 (3), 276-286 (2012).</li>
<li>Mizutani, R., Suzuki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+microtomography+in+biology%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">X-ray microtomography in biology.</a> Micron. 43 (2-3), 104-115 (2012).</li>
<li>Salomé, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+synchrotron+radiation+microtomography+system+for+the+analysis+of+trabecular+bone+samples%5BTitle%5D)+AND+%22Med+Phys%22%5BJournal%5D)">A synchrotron radiation microtomography system for the analysis of trabecular bone samples.</a> Med Phys. 26 (10), 2194-2204 (1999).</li>
<li>Mizutani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+microtomographic+imaging+of+human+brain+cortex%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Three-dimensional microtomographic imaging of human brain cortex.</a> Brain Res. 1199, 53-61 (2008).</li>
<li>De Crespigny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+micro-CT+imaging+of+the+postmortem+brain%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">3D micro-CT imaging of the postmortem brain.</a> J Neurosci Methods. 171 (2), 207-213 (2008).</li>
<li>Johnson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Virtual+histology+of+transgenic+mouse+embryos+for+high-throughput+phenotyping%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Virtual histology of transgenic mouse embryos for high-throughput phenotyping.</a> PLoS Genet. 2 (4), e61(2006).</li>
<li>Zou, W., Hunter, N., Swain, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+polychromatic+%C2%B5ct+for+mineral+density+determination%5BTitle%5D)+AND+%22J+Dent+Res%22%5BJournal%5D)">Application of polychromatic µct for mineral density determination.</a> J Dent Res. 90 (1), 18-30 (2011).</li>
<li>Neues, F., Epple, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+microcomputer+tomography+for+the+study+of+biomineralized+endo-+and+exoskeletons+of+animals%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">X-ray microcomputer tomography for the study of biomineralized endo- and exoskeletons of animals.</a> Chem Rev. 108 (11), 4734-4741 (2008).</li>
<li>Gabner, S., Böck, P., Fink, D., Glösmann, M., Handschuh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+visible+skeleton+2.0%3A+Phenotyping+of+cartilage+and+bone+in+fixed+vertebrate+embryos+and+foetuses+based+on+x-ray+microCT%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The visible skeleton 2.0: Phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on x-ray microCT.</a> Development. 147 (11), dev187633(2020).</li>
<li>Ermakova, O., Orsini, T., Gambadoro, A., Chiani, F., Tocchini-Valentini, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+microCT+imaging+of+murine+embryonic+development+from+immediate+post-implantation+to+organogenesis%3A+Application+for+phenotyping+analysis+of+early+embryonic+lethality+in+mutant+animals%5BTitle%5D)+AND+%22Mamm+Genome%22%5BJournal%5D)">Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: Application for phenotyping analysis of early embryonic lethality in mutant animals.</a> Mamm Genome. 29 (3-4), 245-259 (2018).</li>
<li>Wong, M. D., Maezawa, Y., Lerch, J. P., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+pipeline+for+anatomical+phenotyping+of+mouse+embryos+using+micro-ct%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Automated pipeline for anatomical phenotyping of mouse embryos using micro-ct.</a> Development. 141 (12), 2533-2541 (2014).</li>
<li>Rajion, Z. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+three-dimensional+computed+tomographic+analysis+of+the+cervical+spine+in+unoperated+infants+with+cleft+lip+and+palate%5BTitle%5D)+AND+%22Cleft+Palate+Craniofac+J%22%5BJournal%5D)">A three-dimensional computed tomographic analysis of the cervical spine in unoperated infants with cleft lip and palate.</a> Cleft Palate Craniofac J. 43 (5), 513-518 (2006).</li>
</ol>

The authors have no conflicts of interest to disclose.

MicroCT is a powerful tool for obtaining realistic and isotropic 3D information from dense and opaque biological samples with micrometer resolution. The data obtained from microCT are calibrated for geometry and intensity, making it especially useful for quantitative studies13,14,15,16,17. It is used to study bone and dental microstructure18,19. In recent decades, researchers have used Alcian Blue and Alizarin Red to clean and stain experimental animal bones, which is considered the gold standard for studying bone development.
In the investigation of craniofacial abnormalities, a sole focus on individual tissues or 2D sections falls short in providing a holistic view of craniofacial bone anomalies in mutant mice. Integrating defined anatomical markers with 3D microCT imagery of craniofacial bone structures offers a potent approach to studying craniofacial anomalies and anatomical deviations. Nevertheless, alternative visualization techniques utilizing X-ray microCT imaging have been suggested, enabling the acquisition of precise 3D data of the entire developing skeleton at micrometer-level resolution, thereby enhancing our understanding of bone and cartilage development20. MicroCT is a non-destructive method that is widely used to study morphological and structural changes in mouse embryos and neonates during development. The 3D volumes obtained can be used for morphological analysis and provide clear histological definitions for identifying anatomical features through 2D virtual slices21,22.
In this paper, we describe in detail a research methodology aimed at visualizing and quantifying the mouse cranium, building upon a previous MicroCT study. In stark contrast to other methods, our 3D model enables researchers to precisely measure a wide range of cranial parameters, encompassing length, width, height, and volume, as well as angles and distances between various skeletal structures. These meticulous measurements form a solid, quantitative basis for scientific research, providing researchers with unprecedented insight into cranial morphology.
Moreover, the 3D digital data serve as a versatile platform, allowing researchers to customize analysis tools and scripts tailored to their specific research needs. This flexibility fosters a more comprehensive and in-depth analysis of the skull, enabling researchers to define anatomical landmarks, conduct volume analysis, and compare morphological differences between different experimental groups or across time points. Overall, our methodology represents a significant advancement in the field, offering researchers a powerful tool for quantifying and interpreting cranial morphology with unparalleled precision.
MicroCT allows researchers to analyze the effects of genetic mutations on individual craniofacial bones and assess how these defects may impact other parts of the skull. In this study, we employed computer software to analyze and reconstruct the craniofacial bones of four-week-old OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. This approach enabled us to obtain a 3D image of each craniomaxillofacial bone, thereby facilitating more accurate analysis and study of craniomaxillofacial bone growth and development. Furthermore, it allowed us to elucidate the regulatory mechanisms controlling craniofacial morphogenesis.
Craniofacial bones pose a challenge due to their irregular shapes and connections, except for the temporomandibular joints, which are not connected by sutures. First, the integrity of the mouse cranial sampling is a critical aspect of the preparation. This method requires the researcher to be knowledgeable about the anatomical models of craniomaxillofacial bones and able to isolate each bone to visualize and assess phenotypic details. Manual erasure tools were used to remove irrelevant bone fragments. Manual erasure minimizes segmentation errors associated with other bones. While manually segmenting the skull, it is easy to miss some layers, so special attention must be paid to avoid confusion with the surrounding bones. After 3D reconstruction, each component of the skeleton becomes an independent entity. Consequently, researchers can conduct detailed, individual studies on each bone structure as a standalone 3D model. Additionally, they can explore the anatomical relationships between individual bones and other structures, rotating them for observation along any axis. Furthermore, it becomes feasible to measure volumes and distances within the reconstructed image data, providing valuable insights into skeletal morphology and anatomy.
Due to the importance of shape and size in the classification of craniofacial birth defects, 3D micro-CT analysis is essential for studying structural malformations in mice as a model for human disease23.
As imaging technologies and data analysis methodologies continue to progress, so do the approaches for dissecting and measuring mouse crania. Notably, innovative imaging techniques such as optical coherence tomography (OCT) and photoacoustic imaging (PAI) are progressively being integrated into biomedical research, offering diverse avenues for the analysis of cranial structures. Furthermore, the advancement of artificial intelligence and machine learning techniques has facilitated the emergence of automated segmentation and quantitative analysis methods for cranial structures. These advancements significantly enhance the efficiency and accuracy of research, ushering in a new era of cranial analysis.
This approach could be applied in clinical settings in the future, enabling the separation of human skulls in software to assist craniomaxillofacial surgeons in preoperative planning, surgical simulation, and patient-specific intraoperative guidance. Furthermore, it could offer valuable visualization of 2D and 3D models, benefiting less experienced surgeons and serving as a useful tool for effective communication between surgeons and patients. However, these potential applications require further investigation and validation.

The intricate development of the human skull and face encompasses a sophisticated 3D morphogenetic process, intricately orchestrated by numerous genes. These genes play a pivotal role in regulating the intricate patterns, proliferation, and differentiation of tissues derived from diverse embryonic sources. This highly coordinated process underscores the complexity of human craniofacial growth and development. Craniofacial malformations (including cleft lip and palate, cranial suture closure, and facial hypoplasia) occurring as a result of developmental abnormalities account for more than one-third of all congenital birth defects. As a commonly used model animal in biomedical research, the mouse has a complex and delicate craniomaxillofacial bone structure that is very similar to the human craniomaxillofacial bone in terms of anatomy and physiology. The study of craniomaxillofacial developmental biology has come a long way in recent years with the advent of new techniques in mouse genetics, especially in malformations1.
Retinoic acid (RA) is the in vivo metabolite of vitamin A2. Vitamin A deficiency (VAD) is associated with a range of serious multisystem disorders, such as poor bone remodeling, fractures, as well as craniofacial malformations and skeletal malformations characterized by dwarfism3,4 . Retinoid receptors (RARs) are crucial transcription factors in retinoid signaling5. A dominant-negative RAR&#945;403 mutant (dnRAR&#945;) was designed6 and a mouse model established in which osteoblasts expressed dnRAR&#945;. This resulted in the mice exhibiting dwarfism, craniofacial deformities, incomplete cortical bone formation, and increased but poorly remodeled trabecular bone.
Microcomputed tomography (microCT) has great potential for the study of craniomaxillofacial malformations. It possesses the capability to detect and track the evolution of both innate and acquired skeletal abnormalities in rodent models. MicroCT imaging analysis offers an in-depth exploration of craniofacial growth disturbances in genetically modified mouse models7,8 .Furthermore, 3D imaging emerges as a vital tool for delineating morphological traits, facilitating tailored analysis and visualization approaches9. Micro-CT has been used in several studies to analyze craniofacial phenotypes, including defining anatomical landmarks in humans and mice and volumetric analysis of each craniofacial bone10,11,12. Here, we describe in detail a method based on microCT technology to separate and measure 3D models of mouse craniomaxillofacial bones to enable better evaluation and analysis of mouse craniomaxillofacial skeletal development than is possible with current methods.

<table><tbody><tr><td>GraphPad Prism 6.01 Software</td><td>GraphPad Software Inc., La Jolla, CA, USA</td><td>/</td><td></td></tr><tr><td>Micro-CT</td><td>Quantum GX micro CT, PerkinElmer,&lt;br/&gt; Waltham, MA, USA</td><td>/</td><td></td></tr><tr><td>Mimics Medical 19.0&amp;nbsp;</td><td>Materialise, Leuven, Belgium</td><td>/</td><td></td></tr><tr><td>Osterix-Cre (OsxCre)&amp;nbsp;</td><td>/</td><td>/</td><td>from the Jackson Laboratory</td></tr><tr><td>Rosa26-loxp-stop-loxp-dnRAR&amp;alpha;403 strain</td><td>/</td><td>/</td><td>from the Columbia University, USA</td></tr></tbody></table>

analysis of craniomaxillofacial malformations in mice using three-dimensional microcomputed tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Extensive research underscores the multifaceted impact of genetic mutations on mouse growth, development, and organ systems. A comprehensive evaluation of craniofacial bones in mutant mice necessitates methods beyond single-tissue or 2D image analysis due to their limitations. Therefore, elucidating craniofacial bone development holds paramount importance for investigating human craniofacial disorders.
This method provides a useful tool for assessing the growth and development of craniomaxillofacial bones in mice (Figure 1). The skulls of 4-week-old OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice were analyzed by micro-CT using the method described above. Three-dimensional reconstruction of the craniofacial bones was performed. The results demonstrated a dose-dependent association between skeletal defects and dnRAR&#945; expression. These defects included reduced length of the mandible, frontal, and parietal bones (Figure 2 and Figure 3).
We quantified the volumes of multiple bones, including the mandible, frontal bone, parietal bone, nasal bone, premaxilla, interparietal bone, and occipital bone (Figure 2 and Figure 3). Furthermore, we observed that the frontal and parietal bones exhibited significant dose-dependent ossification insufficiency, as evidenced by an increase in the surface area of the defects (Figure 2). Consequently, it can be postulated that the inhibition of RAR in osteoblasts may result in craniofacial deformities, characterized by abnormalities in the shape, size, and ossification of the craniofacial skeleton.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67340/67340fig1.jpg" /> 
Figure 1: Method overview. (A) Create a new layer. (B) Use the Eraser tool to erase irrelevant skeletal areas. (C) Select the area of the mandible to be reconstructed. (D) Change the color after 3D reconstruction. (E) Measure the distance between the mandibles bilaterally. (F) Measure the volume of the mandible bilaterally. <a href="https://www.jove.com/files/ftp_upload/67340/67340fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67340/67340fig2.jpg" /> 
Figure 2: MicroCT and 3D reconstruction of craniofacial bones. (A) 3D reconstruction of craniofacial bones from 4-week-old male OsxCre, OSXCre;R26dn/, and OSXCre;R26dn/dn mice. (B) 3D reconstruction of the mandibles from 4-week-old male OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. (C) Statistical analysis of the width and volume of the mandibles from 4-week-old male OsxCre (n = 4), OSXCre;R26dn/+ (n = 4), and OSXCre;R26dn/dn (n = 4) mice. Error bars are represented as mean &#177; S.D., *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/67340/67340fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67340/67340fig3.jpg" /> 
Figure 3: 3D reconstruction of the nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone from OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. (A) 3D reconstruction of the frontal bone from 4-week-old male OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. (B) Top view of the parietal bone, side view of the premaxilla, top view of the nasal bone, top view of the interparietal bone, and back view of the occipital bone from OsxCre, OSXCre;R26dn/+, and OSXCre;R26dn/dn mice. (C) Statistical analysis of the width and volume of the frontal bone from 4-week-old male OsxCre (n = 4), OSXCre;R26dn/+ (n = 4), and OSXCre;R26dn/dn (n = 4) mice. Error bars are represented as mean &#177; S.D., *P &#60; 0.05, **P &#60; 0.01, ***P &#60;0.001, ****P &#60;0.0001. (D) Statistical analysis of the width and volume of the parietal bone from 4-week-old male OsxCre (n = 4), OSXCre;R26dn/+ (n = 4), and OSXCre;R26dn/dn (n = 4) mice. Error bars are represented as mean &#177; S.D., *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. (E) Volume of the premaxilla, nasal bone, interparietal bone, and occipital bone from 4-week-old male OsxCre (n = 4), OSXCre;R26dn/+ (n = 4), and OSXCre;R26dn/dn (n = 4) mice. Error bars are represented as mean &#177; S.D., *P &#60; 0.05, **P &#60; 0.01, ***P &#60; 0.001, ****P &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/67340/67340fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

analysis-craniomaxillofacial-malformations-mice-using-three

Universidad Científica del Sur

Research

JoVE Journal

Developmental Biology

230 Views.  Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Video: Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Medicine

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Biology

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Mouse Neonatal Brain Micro-CT Scanning and Image Processing

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Author Spotlight: High-Resolution Imaging of Mouse Neonate Brains &#8211; A Micro-CT Protocol with Lugol&#39;s Solution Contrast Agent

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard ...

Neuroscience

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

3D Cephalometric Landmark Annotation Demonstration on Human CBCT Scans

Three-Dimensional Cephalometric Landmark Annotation Demonstration on Human Cone Beam Computed Tomography Scans

Craniofacial cephalometric analysis is a diagnostic tool used for the assessment of the relationship of various bones and soft tissues in the head and face. Cephalometric analysis has been traditionally conducted with the use of 2D radiographs and landmark sets and restricted to size, linear and angular measurements, and 2D relationships. The increasing use of 3D cone beam computed tomography (CBCT) scans in the dental field dictates the need for the evolution to 3D cephalometric analysis, which incorporates shape and a more realistic analysis of longitudinal development in all three planes. This study is a demonstration of 3D cephalometric analysis with the use of a validated set of skeletal tissue landmarks on human CBCT scans. Detailed instructions for the annotation of each landmark on a 3D volume are provided as part of a step-by-step protocol. The generated measurements and 3D coordinates of the landmarks can be exported and used both for clinical and research purposes. The introduction of 3D cephalometric analysis in basic and clinical craniofacial studies will lead to future advancements in the field of craniofacial growth and development.

Author Spotlight: Three-Dimensional Cephalometric Landmark Annotation Demonstration on Human Cone Beam Computed Tomography Scans  

Presented here is a detailed protocol for the conduction of three-dimensional cephalometric analysis with the use of human cone beam computed tomography scans.

Craniofacial cephalometric analysis is a diagnostic tool used for the assessment of the relationship of various bones and soft tissues in the head and ...

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

Studying Orthodontic Tooth Movement in Mice

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at ...

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the ...

Intravital Longitudinal Imaging of Vascular Dynamics in the Calvarial Bone Marrow

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the context of tissue regeneration, tumor development, and therapeutic responses. Particularly, the calvarial bone marrow is a highly dynamic tissue, where the hematopoietic fate is interconnected with the surrounding microenvironment, with specialized vessels responding to normal and pathologic hematopoiesis. Traditional imaging of fixed tissues offers static information, often limiting a comprehensive understanding of these processes. 
The integration of transgenic animals expressing cell-specific markers, live cell tracers, advancements in imaging equipment, and the use of specialized chambers has elevated intravital microscopy to a pivotal tool for gaining insights into dynamic biological phenomena. One application of intravital imaging is the investigation of tumor vessel behavior and therapeutic effects. A newly designed 3D-printed titanium head fixation implant can be stably connected to the mouse skull and is suitable for longitudinal imaging during multiple sessions. 
The proposed protocol allows for the spatial and temporal examination of vascular dynamics in the calvarial bone marrow, including visualization and quantification of vascular heterogeneity, interaction with stromal and hematopoietic cells, and measurement of vascular functional parameters. Additionally, the technique enables the visualization of established vascular beds and the monitoring of therapeutic effects, stem cell mobilization, and the localization of chemotherapeutic compounds over time using two-photon microscopy. Overall, this intravital longitudinal imaging protocol provides a comprehensive platform for investigating both tumor vessel behavior and hematopoietic cell dynamics, offering valuable insights into the intricate processes governing these biological phenomena.

Intravital microscopy allows the study of dynamic biological processes such as tissue regeneration and tumor development. The calvarial bone marrow, a highly dynamic tissue, offers insights into hematopoiesis and vascular function. Using a biocompatible 3D-printed head fixation implant allows for repetitive longitudinal imaging, enhancing our understanding of tissue dynamics and the tumor microenvironment.

Intravital longitudinal fluorescence microscopy imaging has emerged as a crucial technique for studying dynamic biological processes, notably in the ...