Genetic architecture of trait variance in craniofacial morphology.

The genetic architecture of trait variance has long been of interest in genetics and evolution. One of the earliest attempts to understand this architecture was presented in Lerner's Genetic Homeostasis (1954). Lerner proposed that heterozygotes should be better able to tolerate environmental perturbations because of functional differences between the alleles at a given locus, with each allele optimal for slightly different environments. This greater robustness to environmental variance, he argued, would result in smaller trait variance for heterozygotes. The evidence for Lerner's hypothesis has been inconclusive. To address this question using modern genomic methods, we mapped loci associated with differences in trait variance (vQTL) on 1,101 individuals from the F34 of an advanced intercross between LG/J and SM/J mice. We also mapped epistatic interactions for these vQTL in order to understand the influence of epistasis for the architecture of trait variance. We did not find evidence supporting Lerner's hypothesis, that heterozygotes tend to have smaller trait variances than homozygotes. We further show that the effects of most mapped loci on trait variance are produced by epistasis affecting trait means and that those epistatic effects account for about a half of the differences in genotypic-specific trait variances. Finally, we propose a model where the different interactions between the additive and dominance effects of the vQTL and their epistatic partners can explain Lerner's original observations but can also be extended to include other conditions where heterozygotes are not the least variable genotype.

Comparative analysis of craniofacial shape in two mouse models of Down syndrome: Ts65Dn and TcMAC21.

Mouse models are central to studying and understanding the genotypic-to-phenotypic outcomes of Down syndrome (DS), a complex condition caused by an extra copy of the long arm of human chromosome 21. The recently developed TcMAC21-a transchromosomic mouse strain with comparable gene dosage to human chromosome 21 (Hsa21)-includes more Hsa21 genes than any other model of DS. Recent studies on TcMAC21 have provided valuable insight into the molecular, physiological, and neuroanatomical aspects of the model. However, relatively little is known about the craniofacial phenotype of TcMAC21 mice, particularly as it compares to the widely studied Ts65Dn model. Here we conducted a quantitative study of the cranial morphology of TcMAC21 and Ts65Dn mice and their respective unaffected littermates. Our comparative data comprise forty three-dimensional cranial measurements taken on micro-computed tomography scans of the heads of TcMAC21 and Ts65Dn mice. Our results show that TcMAC21 exhibit similar patterns of craniofacial change to Ts65Dn. However, the DS-specific morphology is more pronounced in Ts65Dn mice. Specifically, Ts65Dn present with more medio-lateral broadening and retraction of the snout compared to TcMAC21. Our findings reveal the complexity of potential gene interaction in the production of craniofacial phenotypes.

Embryonic cranial cartilage defects in the Fgfr3Y367C /+ mouse model of achondroplasia.

Achondroplasia, the most common chondrodysplasia in humans, is caused by one of two gain of function mutations localized in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leading to constitutive activation of FGFR3 and subsequent growth plate cartilage and bone defects. Phenotypic features of achondroplasia include macrocephaly with frontal bossing, midface hypoplasia, disproportionate shortening of the extremities, brachydactyly with trident configuration of the hand, and bowed legs. The condition is defined primarily on postnatal effects on bone and cartilage, and embryonic development of tissues in affected individuals is not well studied. Using the Fgfr3Y367C/+ mouse model of achondroplasia, we investigated the developing chondrocranium and Meckel's cartilage (MC) at embryonic days (E)14.5 and E16.5. Sparse hand annotations of chondrocranial and MC cartilages visualized in phosphotungstic acid enhanced three-dimensional (3D) micro-computed tomography (microCT) images were used to train our automatic deep learning-based 3D segmentation model and produce 3D isosurfaces of the chondrocranium and MC. Using 3D coordinates of landmarks measured on the 3D isosurfaces, we quantified differences in the chondrocranium and MC of Fgfr3Y367C/+ mice relative to those of their unaffected littermates. Statistically significant differences in morphology and growth of the chondrocranium and MC were found, indicating direct effects of this Fgfr3 mutation on embryonic cranial and pharyngeal cartilages, which in turn can secondarily affect cranial dermal bone development. Our results support the suggestion that early therapeutic intervention during cartilage formation may lessen the effects of this condition.

Come together over me: Cells that form the dermatocranium and chondrocranium in mice.

Most bone develops either by intramembranous ossification where bone forms within a soft connective tissue, or by endochondral ossification by way of a cartilage anlagen or model. Bones of the skull can form endochondrally or intramembranously or represent a combination of the two types of ossification. Contrary to the classical definition of intramembranous ossification, we have previously described a tight temporo-spatial relationship between cranial cartilages and dermal bone formation and proposed a mechanistic relationship between chondrocranial cartilage and dermal bone. Here, we further investigate this relationship through an analysis of how cells organize to form cranial cartilages and dermal bone. Using Wnt1-Cre2 and Mesp1-Cre transgenic mice, we determine the derivation of cells that comprise cranial cartilages from either cranial neural crest (CNC) or paraxial mesoderm (PM). We confirm a previously determined CNC-PM boundary that runs through the hypophyseal fenestra in the cartilaginous braincase floor and identify four additional CNC-PM boundaries in the chondrocranial lateral wall, including a boundary that runs along the basal and apical ends of the hypochiasmatic cartilage. Based on the knowledge that as osteoblasts differentiate from CNC- and PM-derived mesenchyme, the differentiating cells express the transcription factor genes RUNX2 and osterix (OSX), we created a new transgenic mouse line called R2Tom. R2Tom mice carry a tdTomato reporter gene joined with an evolutionarily well-conserved enhancer sequence of RUNX2. R2Tom mice crossed with Osx-GFP mice yield R2Tom;Osx-GFP double transgenic mice in which various stages of osteoblasts and their precursors are detected with different fluorescent reporters. We use the R2Tom;Osx-GFP mice, new data on the cell derivation of cranial cartilages, histology, immunohistochemistry, and detailed morphological observations combined with data from other investigators to summarize the differentiation of cranial mesenchyme as it forms condensations that become chondrocranial cartilages and associated dermal bones of the lateral cranial wall. These data advance our previous findings of a tendency of cranial cartilage and dermal bone development to vary jointly in a coordinated manner, promoting a role for cranial cartilages in intramembranous bone formation.

Meckel's Cartilage in Mandibular Development and Dysmorphogenesis.

The Fgfr2c C342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in the fibroblast growth factor receptor 2 (Fgfr2) gene equivalent to a FGFR2 mutation commonly associated with Crouzon and Pfeiffer syndromes in humans. The Fgfr2c C342Y mutation results in constitutive activation of the receptor and is associated with upregulation of osteogenic differentiation. Fgfr2cC342Y/+ Crouzon syndrome mice show premature closure of the coronal suture and other craniofacial anomalies including malocclusion of teeth, most likely due to abnormal craniofacial form. Malformation of the mandible can precipitate a plethora of complications including disrupting development of the upper jaw and palate, impediment of the airway, and alteration of occlusion necessary for proper mastication. The current paradigm of mandibular development assumes that Meckel's cartilage (MC) serves as a support or model for mandibular bone formation and as a template for the later forming mandible. If valid, this implies a functional relationship between MC and the forming mandible, so mandibular dysmorphogenesis might be discerned in MC affecting the relationship between MC and mandibular bone. Here we investigate the relationship of MC to mandible development from the early mineralization of the mandible (E13.5) through the initiation of MC degradation at E17.7 using Fgfr2c C342Y/+ Crouzon syndrome embryos and their unaffected littermates (Fgfr2c +/+ ). Differences between genotypes in both MC and mandibular bone are subtle, however MC of Fgfr2c C342Y/+ embryos is generally longer relative to unaffected littermates at E15.5 with specific aspects remaining relatively large at E17.5. In contrast, mandibular bone is smaller overall in Fgfr2c C342Y/+ embryos relative to their unaffected littermates at E15.5 with the posterior aspect remaining relatively small at E17.5. At a cellular level, differences are identified between genotypes early (E13.5) followed by reduced proliferation in MC (E15.5) and in the forming mandible (E17.5) in Fgfr2c C342Y/+ embryos. Activation of the ERK pathways is reduced in the perichondrium of MC in Fgfr2c C342Y/+ embryos and increased in bone related cells at E15.5. These data reveal that the Fgfr2c C342Y mutation differentially affects cells by type, location, and developmental age indicating a complex set of changes in the cells that make up the lower jaw.

A dysmorphic mouse model reveals developmental interactions of chondrocranium and dermatocranium.

The cranial endo and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete 3D reconstructions of the laboratory mouse chondrocranium from embryonic day (E) 13.5 through E17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D micro-computed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D, and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on their association with forming dermal bones using the Fgfr2cC342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically, we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2cC342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2cC342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2cC342Y/+ mice reduced in most dimensions compared to Fgfr2c+/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology.

Embryonic and early postnatal cranial bone volume and tissue mineral density values for C57BL/6J laboratory mice.

BACKGROUND: Laboratory mice are routinely used in craniofacial research based on the relatively close genetic relationship and conservation of developmental pathways between humans and mice. Since genetic perturbations and disease states may have localized effects, data from individual cranial bones are valuable for the interpretation of experimental assays. We employ high-resolution microcomputed tomography to characterize cranial bones of C57BL/6J mice at embryonic day (E) 15.5 and E17.5, day of birth (P0), and postnatal day 7 (P7) and provide estimates of individual bone volume and tissue mineral density (TMD). RESULTS: Average volume and TMD values are reported for individual bones. Significant differences in volume and TMD during embryonic ages likely reflect early mineralization of cranial neural crest-derived and intramembranously forming bones. Although bones of the face and vault had higher TMD values during embryonic ages, bones of the braincase floor had significantly higher TMD values by P7. CONCLUSIONS: These ontogenetic data on cranial bone volume and TMD serve as a reference standard for future studies using mice bred on a C57BL/6J genetic background. Our findings also highlight the importance of differentiating "control" data from mice that are presented as "unaffected" littermates, particularly when carrying a single copy of a cre-recombinase gene.

A transchromosomic rat model with human chromosome 21 shows robust Down syndrome features.

Progress in earlier detection and clinical management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available as a result of technical challenges. We developed a transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein-coding genes. RNA-seq of neonatal forebrains demonstrates that TcHSA21rat expresses HSA21 genes and has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, which is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

Single-cell analysis identifies a key role for Hhip in murine coronal suture development.

Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone, and suture mesenchyme separates adjacent bones. Here, we perform single-cell RNA-seq analysis of the embryonic, wild type murine coronal suture to define its population structure. Seven populations at E16.5 and nine at E18.5 comprise the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog signaling, marks a mesenchymal population distinct from those of other neurocranial sutures. Tracing of the neonatal Hhip-expressing population shows that descendant cells persist in the coronal suture and contribute to calvarial bone growth. In Hhip-/- coronal sutures at E18.5, the osteogenic fronts are closely apposed and the suture mesenchyme is depleted with increased hedgehog signaling compared to those of the wild type. Collectively, these data demonstrate that Hhip is required for normal coronal suture development.

An Analysis of Anatomy Education Before and During Covid-19: May-August 2020.

Coronavirus disease 2019 (Covid-19) created unparalleled challenges to anatomy education. Gross anatomy education has been particularly impacted given the traditional in-person format of didactic instruction and/or laboratory component(s). To assess the changes in gross anatomy lecture and laboratory instruction, assessment, and teaching resources utilized as a result of Covid-19, a survey was distributed to gross anatomy educators through professional associations and listservs. Of the 67 survey responses received for the May-August 2020 academic period, 84% were from United States (US) institutions, while 16% were internationally based. Respondents indicated that in-person lecture decreased during Covid-19 (before: 76%, during: 8%, P < 0.001) and use of cadaver materials declined (before: 76 ± 33%, during: 34 ± 43%, P < 0.001). The use of cadaver materials in laboratories decreased during Covid-19 across academic programs, stand-alone and integrated anatomy courses, and private and public institutions (P ≤ 0.004). Before Covid-19, cadaveric materials used in laboratories were greater among professional health programs relative to medical and undergraduate programs (P ≤ 0.03) and among stand-alone relative to integrated anatomy courses (P ≤ 0.03). Furthermore, computer-based assessment increased (P < 0.001) and assessment materials changed from cadaveric material to images (P < 0.03) during Covid-19, even though assessment structure was not different (P > 0.05). The use of digital teaching resources increased during Covid-19 (P < 0.001), with reports of increased use of in-house created content, BlueLink, and Complete Anatomy software (P < 0.05). While primarily representing US institutions, this study provided evidence of how anatomy educators adapted their courses, largely through virtual mediums, and modified laboratory protocols during the initial emergence of the Covid-19 pandemic.

Phenotypes, Developmental Basis, and Genetics of Pierre Robin Complex.

The phenotype currently accepted as Pierre Robin syndrome/sequence/anomalad/complex (PR) is characterized by mandibular dysmorphology, glossoptosis, respiratory obstruction, and in some cases, cleft palate. A causative sequence of developmental events is hypothesized for PR, but few clear causal relationships between discovered genetic variants, dysregulated gene expression, precise cellular processes, pathogenesis, and PR-associated anomalies are documented. This review presents the current understanding of PR phenotypes, the proposed pathogenetic processes underlying them, select genes associated with PR, and available animal models that could be used to better understand the genetic basis and phenotypic variation of PR.

Cartilage Segmentation in High-Resolution 3D Micro-CT Images via Uncertainty-Guided Self-training with Very Sparse Annotation.

Craniofacial syndromes often involve skeletal defects of the head. Studying the development of the chondrocranium (the part of the endoskeleton that protects the brain and other sense organs) is crucial to understanding genotype-phenotype relationships and early detection of skeletal malformation. Our goal is to segment craniofacial cartilages in 3D micro-CT images of embryonic mice stained with phosphotungstic acid. However, due to high image resolution, complex object structures, and low contrast, delineating fine-grained structures in these images is very challenging, even manually. Specifically, only experts can differentiate cartilages, and it is unrealistic to manually label whole volumes for deep learning model training. We propose a new framework to progressively segment cartilages in high-resolution 3D micro-CT images using extremely sparse annotation (e.g., annotating only a few selected slices in a volume). Our model consists of a lightweight fully convolutional network (FCN) to accelerate the training speed and generate pseudo labels (PLs) for unlabeled slices. Meanwhile, we take into account the reliability of PLs using a bootstrap ensemble based uncertainty quantification method. Further, our framework gradually learns from the PLs with the guidance of the uncertainty estimation via self-training. Experiments show that our method achieves high segmentation accuracy compared to prior arts and obtains performance gains by iterative self-training.

It takes two: Building the vertebrate skull from chondrocranium and dermatocranium.

In most modern bony vertebrates, a considerable portion of the chondrocranium remains cartilaginous only during a relatively small window of embryonic development, making it difficult to study this complex structure. Yet, the transient nature of some chondrocranial elements is precisely why it is so intriguing. Since the chondrocranium has never been lost in any vertebrate, its function is critical to craniofacial development, disease, and evolution. Experimental evidence for the various roles of the chondrocranium is limited, and though snapshots of chondrocranial development in various species at isolated time points are valuable and informative, these cannot provide the data needed to determine the functions of the chondrocranium, or its relationship to the dermatocranium in evolution, in development, or in disease. Observations of the spatiotemporal associations of chondrocranial cartilage, cartilage bone, and dermal bone over early developmental time are available for many vertebrate species and these observations represent the data from which we can build hypotheses. The testing of those hypotheses requires precise control of specific variables like developmental time and molecular signaling that can only be accomplished in a laboratory setting. Here, we employ recent advances in contrast-enhanced micro computed tomography to provide novel 3D reconstructions of the embryonic chondrocranium in relation to forming dermal and cartilage bones in laboratory mice across three embryonic days (E13.5, E14.5, and E15.5). Our observations provide support for the established hypothesis that the vertebrate dermal (exo-) skeleton and endoskeleton evolved as distinct structures and remain distinct. Additionally, we identify spatiotemporal patterning in the development of the lateral wall, roof, and braincase floor of the chondrocranium and the initial mineralization and growth of the bones associated with these cartilages that provides support for the hypothesis that the chondrocranium serves as a scaffold for developing dermatocranial bones. The experimental protocols described and data presented provide tools for further experimental work on chondrocranial development.

Identifying the Misshapen Head: Craniosynostosis and Related Disorders.

Pediatric care providers, pediatricians, pediatric subspecialty physicians, and other health care providers should be able to recognize children with abnormal head shapes that occur as a result of both synostotic and deformational processes. The purpose of this clinical report is to review the characteristic head shape changes, as well as secondary craniofacial characteristics, that occur in the setting of the various primary craniosynostoses and deformations. As an introduction, the physiology and genetics of skull growth as well as the pathophysiology underlying craniosynostosis are reviewed. This is followed by a description of each type of primary craniosynostosis (metopic, unicoronal, bicoronal, sagittal, lambdoid, and frontosphenoidal) and their resultant head shape changes, with an emphasis on differentiating conditions that require surgical correction from those (bathrocephaly, deformational plagiocephaly/brachycephaly, and neonatal intensive care unit-associated skill deformation, known as NICUcephaly) that do not. The report ends with a brief discussion of microcephaly as it relates to craniosynostosis as well as fontanelle closure. The intent is to improve pediatric care providers' recognition and timely referral for craniosynostosis and their differentiation of synostotic from deformational and other nonoperative head shape changes.

A non-mosaic transchromosomic mouse model of down syndrome carrying the long arm of human chromosome 21.

Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.

Phosphotungstic acid-enhanced microCT: Optimized protocols for embryonic and early postnatal mice.

BACKGROUND: Given the need for descriptive and increasingly mechanistic morphological analyses, contrast-enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro-level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA-enhanced microCT. RESULTS: Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. CONCLUSION: These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

Mandibular dysmorphology due to abnormal embryonic osteogenesis in FGFR2-related craniosynostosis mice.

One diagnostic feature of craniosynostosis syndromes is mandibular dysgenesis. Using three mouse models of Apert, Crouzon and Pfeiffer craniosynostosis syndromes, we investigated how embryonic development of the mandible is affected by fibroblast growth factor receptor 2 (Fgfr2) mutations. Quantitative analysis of skeletal form at birth revealed differences in mandibular morphology between mice carrying Fgfr2 mutations and their littermates that do not carry the mutations. Murine embryos with the mutations associated with Apert syndrome in humans (Fgfr2+/S252W and Fgfr2+/P253R ) showed an increase in the size of the osteogenic anlagen and Meckel's cartilage (MC). Changes in the microarchitecture and mineralization of the developing mandible were visualized using histological staining. The mechanism for mandibular dysgenesis in the Apert Fgfr2+/S252W mouse resulting in the most severe phenotypic effects was further analyzed in detail and found to occur to a lesser degree in the other craniosynostosis mouse models. Laser capture microdissection and RNA-seq analysis revealed transcriptomic changes in mandibular bone at embryonic day 16.5 (E16.5), highlighting increased expression of genes related to osteoclast differentiation and dysregulated genes active in bone mineralization. Increased osteoclastic activity was corroborated by TRAP assay and in situ hybridization of Csf1r and Itgb3 Upregulated expression of Enpp1 and Ank was validated in the mandible of Fgfr2+/S252W embryos, and found to result in elevated inorganic pyrophosphate concentration. Increased proliferation of osteoblasts in the mandible and chondrocytes forming MC was identified in Fgfr2+/S252W embryos at E12.5. These findings provide evidence that FGFR2 gain-of-function mutations differentially affect cartilage formation and intramembranous ossification of dermal bone, contributing to mandibular dysmorphogenesis in craniosynostosis syndromes.This article has an associated First Person interview with the joint first authors of the paper.

A coupled reaction-diffusion-strain model predicts cranial vault formation in development and disease.

How cells utilize instructions provided by genes and integrate mechanical forces generated by tissue growth to produce morphology is a fundamental question of biology. Dermal bones of the vertebrate cranial vault are formed through the direct differentiation of mesenchymal cells on the neural surface into osteoblasts through intramembranous ossification. Here we join a self-organizing Turing mechanism, computational biomechanics, and experimental data to produce a 3D representative model of the growing cerebral surface, cranial vault bones, and sutures. We show how changes in single parameters regulating signaling during osteoblast differentiation and bone formation may explain cranial vault shape variation in craniofacial disorders. A key result is that toggling a parameter in our model results in closure of a cranial vault suture, an event that occurred during evolution of the cranial vault and that occurs in craniofacial disorders. Our approach provides an initial and important step toward integrating biomechanics into the genotype phenotype map to explain the production of variation in head morphology by developmental mechanisms.

Craniofacial skeletal response to encephalization: How do we know what we think we know?

Dramatic changes in cranial capacity have characterized human evolution. Important evolutionary hypotheses, such as the spatial packing hypothesis, assert that increases in relative brain size (encephalization) have caused alterations to the modern human skull, resulting in a suite of traits unique among extant primates, including a domed cranial vault, highly flexed cranial base, and retracted facial skeleton. Most prior studies have used fossil or comparative primate data to establish correlations between brain size and cranial form, but the mechanistic basis for how changes in brain size impact the overall shape of the skull resulting in these cranial traits remains obscure and has only rarely been investigated critically. We argue that understanding how changes in human skull morphology could have resulted from increased encephalization requires the direct testing of hypotheses relating to interaction of embryonic development of the bones of the skull and the brain. Fossil and comparative primate data have thoroughly described the patterns of association between brain size and skull morphology. Here we suggest complementing such existing datasets with experiments focused on mechanisms responsible for producing the observed patterns to more thoroughly understand the role of encephalization in shaping the modern human skull.

Nonsyndromic craniosynostosis: novel coding variants.

BACKGROUND: Craniosynostosis (CS), the premature fusion of one or more neurocranial sutures, is associated with approximately 200 syndromes; however, about 65-85% of patients present with no additional major birth defects. METHODS: We conducted targeted next-generation sequencing of 60 known syndromic and other candidate genes in patients with sagittal nonsyndromic CS (sNCS, n = 40) and coronal nonsyndromic CS (cNCS, n = 19). RESULTS: We identified 18 previously published and 5 novel pathogenic variants, including three de novo variants. Novel variants included a paternally inherited c.2209C>G:p.(Leu737Val) variant in BBS9 of a patient with cNCS. Common variants in BBS9, a gene required for ciliogenesis during cranial suture development, have been associated with sNCS risk in a previous genome-wide association study. We also identified c.313G>T:p.(Glu105*) variant in EFNB1 and c.435G>C:p.(Lys145Asn) variant in TWIST1, both in patients with cNCS. Mutations in EFNB1 and TWIST1 have been linked to craniofrontonasal and Saethre-Chotzen syndrome, respectively; both present with coronal CS. CONCLUSIONS: We provide additional evidence that variants in genes implicated in syndromic CS play a role in isolated CS, supporting their inclusion in genetic panels for screening patients with NCS. We also identified a novel BBS9 variant that further shows the potential involvement of BBS9 in the pathogenesis of CS.