Rebecca M. Green

Chapman University

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Benedikt Hallgrímsson

University of Calgary

Ralph Marcucio

University of California, San Francisco

James M. Cheverud

Loyola University Chicago

Christopher J. Percival

Stony Brook University

Nathan M. Young

University of California, San Francisco

Nils D. Forkert

Alberta Children's Hospital

Marta Vidal‐García

University of Calgary

Lucas Lo Vercio

University of Calgary

Marta Marchini

Loyola University Chicago

Charles C. Roseman

University of Illinois Urbana-Champaign

Cooperative Institutions

University of Calgary

Alberta Children's Hospital

University of Colorado Denver

Alberta Bone and Joint Health Institute

University of Colorado Anschutz Medical Campus

University of California, San Francisco

Chapman University

National Jewish Health

University of Colorado Health

University of Pittsburgh

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Exploring the Distribution and Orientation of Cell Proliferation as Drivers of Mouse Facial Development

The FASEB Journal (2022)

Andreas Dauter Rebecca M. Green Lucas Lo Vercio Elizabeth C. Barretto Samuel Robertson

During the development of the face, tissues move, change shape, and fuse in tightly orchestrated patterns to create all the parts of a normal face. These shape changes are driven by factors such as cell signaling, migration, proliferation, and apoptosis. However, the contributions of each of these drivers to morphogenesis are poorly studied. Here, we explore differential cell proliferation as a driver of mouse facial morphogenesis. We quantify patterns in both the spatial distribution and orientation of proliferation in the developing face in 3D over a critical period of murine facial development (E9.5-E11.5). We use immunostaining with light sheet microscopy (LSM) to capture total and proliferating nuclei. To compare proliferative density in facial tissues, we segment these images using a novel convolutional neural network. We then generate atlases of average proliferation at each half-day age point within our range and use these to identify relationships between morphology and cell proliferation. We show that regions with more dense proliferation tend to undergo more intensive shape changes. We then simulate outgrowth of the maxillary process using a cell simulation engine, PhysiCell, to demonstrate that differential proliferation is necessary to maintain expected morphology in growing tissues. In addition to differential proliferation, localized orientation of cell division could also affect morphology. In plants and some animal tissues, including murine limb buds, preferentially oriented cell proliferation drives shape change by causing tissue elongation in specific directions. We explore the orientation and distribution of cell proliferation using LSM: we inject pregnant dams with a synthetic nucleotide, EdU, 5 minutes before harvest to mark the daughter cells of proliferative events occurring in the interim. We then compare the angles of the proliferative axes for each pair of daughters relative to the primary direction of tissue growth. Preliminary results suggest that cell proliferation in the maxillary and nasal processes is oriented preferentially towards the axis of tissue growth. These results suggest that both the distribution and orientation of cell proliferation play a role in murine facial morphogenesis. Understanding the mechanisms underlying morphogenesis is important to guide future research that could lead to earlier and more robust diagnosis and treatment of syndromes and facial abnormalities.

Immunostaining

Cell type

10.1096/fasebj.2022.36.s1.r5695

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Fgf8 dosage regulates jaw shape and symmetry through pharyngeal‐cardiac tissue relationships

Developmental Dynamics (2022)

Nathaniel Zbasnik Katie Dolan Stephanie A. Buczkowski Rebecca M. Green Benedikt Hallgrímsson

Abstract Background Asymmetries in craniofacial anomalies are commonly observed. In the facial skeleton, the left side is more commonly and/or severely affected than the right. Such asymmetries complicate treatment options. Mechanisms underlying variation in disease severity between individuals as well as within individuals (asymmetries) are still relatively unknown. Results Developmental reductions in fibroblast growth factor 8 (Fgf8) have a dosage dependent effect on jaw size, shape, and symmetry. Further, Fgf8 mutants have directionally asymmetric jaws with the left side being more affected than the right. Defects in lower jaw development begin with disruption to Meckel's cartilage, which is discontinuous. All skeletal elements associated with the proximal condensation are dysmorphic, exemplified by a malformed and misoriented malleus. At later stages, Fgf8 mutants exhibit syngnathia, which falls into two broad categories: bony fusion of the maxillary and mandibular alveolar ridges and zygomatico‐mandibular fusion. All of these morphological defects exhibit both inter‐ and intra‐specimen variation. Conclusions We hypothesize that these asymmetries are linked to heart development resulting in higher levels of Fgf8 on the right side of the face, which may buffer the right side to developmental perturbations. This mouse model may facilitate future investigations of mechanisms underlying human syngnathia and facial asymmetry.

FGF8

10.1002/dvdy.501

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Citations (10)

Nonlinear gene expression‐phenotype relationships contribute to variation and clefting in the A/WySn mouse

Developmental Dynamics (2019)

Rebecca M. Green Courtney L. Leach Virginia M. Diewert J. David Aponte Eric J. Schmidt

Cleft lip and palate is one of the most common human birth defects, but the underlying etiology is poorly understood. The A/WySn mouse is a spontaneously occurring model of multigenic clefting in which 20% to 30% of individuals develop an orofacial cleft. Recent work has shown altered methylation at a specific retrotransposon insertion downstream of the Wnt9b locus in clefting animals, which results in decreased Wnt9b expression.Using a newly developed protocol that allows us to measure morphology, gene expression, and DNA methylation in the same embryo, we relate gene expression in an individual embryo directly to its three-dimensional morphology for the first time. We find that methylation at the retrotransposon relates to Wnt9b expression and morphology. IAP methylation relates to shape of the nasal process in a manner consistent with clefting. Embryos with low IAP methylation exhibit increased among-individual variance in facial shape.Methylation and gene expression relate nonlinearly to nasal process morphology. Individuals at one end of a continuum of phenotypic states display a clinical phenotype and increased phenotypic variation. Variable penetrance and expressivity in this model is likely determined both by among-individual variation in methylation and changes in phenotypic robustness along the underlying liability distribution for orofacial clefting.

Variation (astronomy)

Expression (computer science)

10.1002/dvdy.110

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Citations (22)

Morphometrics, 3D Imaging, and Craniofacial Development

Current topics in developmental biology/Current Topics in Developmental Biology (2015)

Benedikt Hallgrímsson Christopher J. Percival Rebecca M. Green Nathan M. Young Washington Mio

Morphometrics

Variation (astronomy)

Morphology

10.1016/bs.ctdb.2015.09.003

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Citations (81)

Factors affecting craniofacial development in a mouse model of Crouzon Syndrome

The FASEB Journal (2016)

Katherine A. Brakora Christopher J. Percival Rebecca M. Green Siew‐Ging Gong Benedikt Hallgrímsson

Crouzon Syndrome (CS) is characterized by premature fusion of calvarial bones (craniosynostosis) as well facial malformations including midfacial hypoplasia and hypertelorism. The phenotype is variable, even when caused by the same mutation within the same family. The cause(s) of this variability remain unknown. Using a mouse model of CS, caused by a missense mutation in FGF Receptor 2, we investigate the role of age, sex, and parental genotype in the development of the craniofacial phenotype. Specimens were collected at E10.5–13.5, E17.5, P0, and at 12 weeks or more, andgenotyped. Specimens younger than 3 weeks were sexed using PCR for the SRY gene. 2D and 3D geometric morphometrics was used to compare craniofacial size and shape, using surface landmarks for embryos, and bones after birth. Wildtype and heterozygous groups were compared at each age, controlling for all other variables, to create a detailed picture of the ontogenetic progression of the CS phenotype caused by this mutation. Then, specimens were grouped by genotype and tested for differences in size and shape based on sex or parental genotypes. We identify when in development homozygous mutants, heterozygous mutants, and wildtype phenotyeps diverge in size and shape, and to what degree sex and parental genotypes affect the timing, severity, or variability of heterozygous and wildtype phenotypes at each age. Support or Funding Information Supported by NIH‐NIDCR grants F32DE024939 to KB and 2R01DE018234‐05A1 to RM and BH.

Crouzon syndrome

Hypertelorism

Morphometrics

Synostosis

10.1096/fasebj.30.1_supplement.1038.5

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Understanding the development of variable craniofacial phenotypes caused by altered methylation affecting Wnt9b in A‐strain mice

The FASEB Journal (2018)

Rebecca M. Green Courtney L. Leach Eric J. Schmidt Virginia M. Diewert Ralph Marcucio

Variable clinical presentation is common in many structural birth defects, two children with the same genetic mutation often have different clinical presentation. Therefore, understanding the developmental origin of such variability is of fundamental importance to developing patient targeted treatment, or precision medicine. A/WySn mice are genetically identical, inbred mice, but about 20% of them develop cleft lip and palate. This has been linked to loss of methylation at an IAP retrotransposon near Wnt9b (Wnt9bIAP). Using a newly developed protocol for generating RNA and DNA as well as a 3D microCT scan that can be used to quantify shape, we show that loss of methylation at this locus directly correlates with facial shape in precleft embryos. Line1 methylation is unaffected in embryos with abnormal Wnt9bIAP methylation levels. Wnt9bIAP methylation levels are bimodal, but continuous, as are facial shape, Wnt9b and Axin2 levels. We use all of these factors to build a model of how multiple continuous bimodal inputs lead to a discontinuous phenotype (clefting). We also determine how this model relates to underlying changes in the cell biology in the affected tissue. Support or Funding Information NIH R01 2R01DE019638 to RSM and BH. NSERC 238992‐17 to BH. Alberta Children's Hospital Research Institute Fellowship to RG. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

10.1096/fasebj.2018.32.1_supplement.776.14

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The Developmental Basis for Evolvability

The MIT Press eBooks (2023)

Benedikt Hallgrímsson J. David Aponte Marta Vidal‐García Heather A. Richbourg Rebecca M. Green

Evolvability

Basis (linear algebra)

10.7551/mitpress/14126.003.0011

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Citations (5)

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses

Scientific Data (2022)

Jay Devine Marta Vidal‐García Wei Liu Amanda Neves Lucas Lo Vercio

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

Morphometrics

File format

10.1038/s41597-022-01338-x

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Citations (5)

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Jay Devine Marta Vidal‐García Wei Liu Amanda Neves Lucas Lo Vercio

Abstract Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen ( N =10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , doi.org/10.25550/3-HXMC) and GitHub ( https://github.com/jaydevine/MusMorph ).

Morphometrics

10.1101/2021.11.11.468142

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Citations (2)

Developmental constraint through negative pleiotropy in the zygomatic arch

EvoDevo (2018)

Christopher J. Percival Rebecca M. Green Charles C. Roseman Daniel M. Gatti Judith L. Morgan

Previous analysis suggested that the relative contribution of individual bones to regional skull lengths differ between inbred mouse strains. If the negative correlation of adjacent bone lengths is associated with genetic variation in a heterogeneous population, it would be an example of negative pleiotropy, which occurs when a genetic factor leads to opposite effects in two phenotypes. Confirming negative pleiotropy and determining its basis may reveal important information about the maintenance of overall skull integration and developmental constraint on skull morphology. We identified negative correlations between the lengths of the frontal and parietal bones in the midline cranial vault as well as the zygomatic bone and zygomatic process of the maxilla, which contribute to the zygomatic arch. Through gene association mapping of a large heterogeneous population of Diversity Outbred (DO) mice, we identified a quantitative trait locus on chromosome 17 driving the antagonistic contribution of these two zygomatic arch bones to total zygomatic arch length. Candidate genes in this region were identified and real-time PCR of the maxillary processes of DO founder strain embryos indicated differences in the RNA expression levels for two of the candidate genes, Camkmt and Six2. A genomic region underlying negative pleiotropy of two zygomatic arch bones was identified, which provides a mechanism for antagonism in component bone lengths while constraining overall zygomatic arch length. This type of mechanism may have led to variation in the contribution of individual bones to the zygomatic arch noted across mammals. Given that similar genetic and developmental mechanisms may underlie negative correlations in other parts of the skull, these results provide an important step toward understanding the developmental basis of evolutionary variation and constraint in skull morphology.

Pleiotropy

Zygomatic bone

Zygomatic arch

10.1186/s13227-018-0092-3

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Citations (10)