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.

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 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 quantitative method for staging mouse embryos based on limb morphometry.

To determine the developmental stage of embryonic mice, we apply a geometric morphometric approach to the changing shape of the mouse limb bud as it grows from embryonic day 10 to embryonic day 15 post-conception. As the ontogenetic sequence results in the de novo emergence of shape features not present in the early stages, we have created a standard ontogenetic trajectory for limb bud development - a quantitative characterization of shape change during limb morphogenesis. This trajectory of form as a function of time also gives us the reverse function: the ability to infer developmental stage from form, with a typical uncertainty of 2 h. We introduce eMOSS (embryonic mouse ontogenetic staging system) as a fast, reliable, convenient and freely available online tool for staging embryos from two-dimensional images of their limb buds, and illustrate its use in phenotyping early limb abnormalities.

Midface and upper airway dysgenesis in FGFR2-related craniosynostosis involves multiple tissue-specific and cell cycle effects.

Midface dysgenesis is a feature of more than 200 genetic conditions in which upper airway anomalies frequently cause respiratory distress, but its etiology is poorly understood. Mouse models of Apert and Crouzon craniosynostosis syndromes exhibit midface dysgenesis similar to the human conditions. They carry activating mutations of Fgfr2, which is expressed in multiple craniofacial tissues during development. Magnetic resonance microscopy of three mouse models of Apert and Crouzon syndromes revealed decreased nasal passage volume in all models at birth. Histological analysis suggested overgrowth of the nasal cartilage in the two Apert syndrome mouse models. We used tissue-specific gene expression and transcriptome analysis to further dissect the structural, cellular and molecular alterations underlying midface and upper airway dysgenesis in Apert Fgfr2+/S252W mutants. Cartilage thickened progressively during embryogenesis because of increased chondrocyte proliferation in the presence of Fgf2 Oral epithelium expression of mutant Fgfr2, which resulted in a distinctive nasal septal fusion defect, and premature facial suture fusion contributed to the overall dysmorphology. Midface dysgenesis in Fgfr2-related craniosynostosis is a complex phenotype arising from the combined effects of aberrant signaling in multiple craniofacial tissues.

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.

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.

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.

Additive genetic variation in the craniofacial skeleton of baboons (genus Papio) and its relationship to body and cranial size.

OBJECTIVES: Determining the genetic architecture of quantitative traits and genetic correlations among them is important for understanding morphological evolution patterns. We address two questions regarding papionin evolution: (1) what effect do body and cranial size, age, and sex have on phenotypic (VP ) and additive genetic (VA ) variation in baboon crania, and (2) how might additive genetic correlations between craniofacial traits and body mass affect morphological evolution? MATERIALS AND METHODS: We use a large captive pedigreed baboon sample to estimate quantitative genetic parameters for craniofacial dimensions (EIDs). Our models include nested combinations of the covariates listed above. We also simulate the correlated response of a given EID due to selection on body mass alone. RESULTS: Covariates account for 1.2-91% of craniofacial VP . EID VA decreases across models as more covariates are included. The median genetic correlation estimate between each EID and body mass is 0.33. Analysis of the multivariate response to selection reveals that observed patterns of craniofacial variation in extant baboons cannot be attributed solely to correlated response to selection on body mass, particularly in males. DISCUSSION: Because a relatively large proportion of EID VA is shared with body mass variation, different methods of correcting for allometry by statistically controlling for size can alter residual VP patterns. This may conflate direct selection effects on craniofacial variation with those resulting from a correlated response to body mass selection. This shared genetic variation may partially explain how selection for increased body mass in two different papionin lineages produced remarkably similar craniofacial phenotypes.

The Influence of trisomy 21 on facial form and variability.

Triplication of chromosome 21 (trisomy 21) results in Down syndrome (DS), the most common live-born human aneuploidy. Individuals with DS have a unique facial appearance that can include form changes and altered variability. Using 3D photogrammatic images, 3D coordinate locations of 20 anatomical landmarks, and Euclidean Distance Matrix Analysis methods, we quantitatively test the hypothesis that children with DS (n = 55) exhibit facial form and variance differences relative to two different age-matched (4-12 years) control samples of euploid individuals: biological siblings of individuals with DS (n = 55) and euploid individuals without a sibling with DS (n = 55). Approximately 36% of measurements differ significantly between DS and DS-sibling samples, whereas 46% differ significantly between DS and unrelated control samples. Nearly 14% of measurements differ significantly in variance between DS and DS sibling samples, while 18% of measurements differ significantly in variance between DS and unrelated euploid control samples. Of those measures that showed a significant difference in variance, all were relatively increased in the sample of DS individuals. These results indicate that faces of children with DS are quantitatively more similar to their siblings than to unrelated euploid individuals and exhibit consistent, but slightly increased variation with most individuals falling within the range of normal variation established by euploid samples. These observations provide indirect evidence of the strength of the genetic underpinnings of the resemblance between relatives and the resistance of craniofacial development to genetic perturbations caused by trisomy 21, while underscoring the complexity of the genotype-phenotype map.

The society for craniofacial genetics and developmental biology 39th annual meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) aims to promote education, research, and communication, about normal and abnormal development of the tissues and organs of the head. Membership of the SCGDB is broad and diverse-including clinicians, orthodontists, scientists, and academics-but with all members sharing an interest in craniofacial biology. Each year, the SCGDB hosts a meeting where members can share their latest research, exchange ideas and resources, and build on or establish new collaborations. © 2017 Wiley Periodicals, Inc.

Chronic up-regulation of sonic hedgehog has little effect on postnatal craniofacial morphology of euploid and trisomic mice.

BACKGROUND: In Ts65Dn, a mouse model of Down syndrome (DS), brain and craniofacial abnormalities that parallel those in people with DS are linked to an attenuated cellular response to sonic hedgehog (SHH) signaling. If a similarly reduced response to SHH occurs in all trisomic cells, then chronic up-regulation of the pathway might have a positive effect on development in trisomic mice, resulting in amelioration of the craniofacial anomalies. RESULTS: We crossed Ts65Dn with Ptch1(tm1Mps/+) mice and quantified the craniofacial morphology of Ts65Dn;Ptch(+/-) offspring to assess whether a chronic up-regulation of the SHH pathway rescued DS-related anomalies. Ts65Dn;Ptch1(+/-) mice experience a chronic increase in SHH in SHH-receptive cells due to haploinsufficiency of the pathway suppressor, Ptch1. Chronic up-regulation had minimal effect on craniofacial shape and did not correct facial abnormalities in Ts65Dn;Ptch(+/-) mice. We further compared effects of this chronic up-regulation of SHH with acute pathway stimulation in mice treated on the day of birth with a SHH pathway agonist, SAG. We found that SHH affects facial morphology differently based on chronic vs. acute postnatal pathway up-regulation. CONCLUSIONS: Our findings have implications for understanding the function of SHH in craniofacial development and for the potential use of SHH-based agonists to treat DS-related abnormalities.

The society for craniofacial genetics and developmental biology 38th annual meeting.

The mission of the Society for Craniofacial Genetics and Developmental Biology (SCGDB) is to promote education, research, and communication about normal and abnormal development of the tissues and organs of the head. The SCGDB welcomes as members undergraduate students, graduate students, post doctoral researchers, clinicians, orthodontists, scientists, and academicians who share an interest in craniofacial biology. Each year our members come together to share their novel findings, build upon, and challenge current knowledge of craniofacial biology. © 2016 Wiley Periodicals, Inc.

Facing up to the challenges of advancing Craniofacial Research.

Craniofacial anomalies are among the most common human birth defects and have considerable functional, aesthetic, and social consequences. The early developmental origin as well as the anatomical complexity of the head and face render these tissues prone to genetic and environmental insult. The establishment of craniofacial clinics offering comprehensive care for craniofacial patients at a single site together with international research networks focused on the origins and treatment of craniofacial disorders has led to tremendous advances in our understanding of the etiology and pathogenesis of congenital craniofacial anomalies. However, the genetic, environmental, and developmental sources of many craniofacial disorders remain unknown. To overcome this problem and further advance craniofacial research, we must recognize current challenges in the field and establish priority areas for study. We still need (i) a deeper understanding of variation during normal development and within the context of any disorder, (ii) improved genotyping and phenotyping and understanding of the impact of epigenetics, (iii) continued development of animal models and functional analyses of genes and variants, and (iv) integration of patient derived cells and tissues together with 3D printing and quantitative assessment of surgical outcomes for improved practice. Only with fundamental advances in each of these areas will we be able to meet the challenge of translating potential therapeutic and preventative approaches into clinical solutions and reduce the financial and emotional burden of craniofacial anomalies.

Embryonic craniofacial bone volume and bone mineral density in Fgfr2(+/P253R) and nonmutant mice.

BACKGROUND: Quantifying multiple phenotypic aspects of individual craniofacial bones across early osteogenesis illustrates differences in typical bone growth and maturation and provides a basis for understanding the localized and overall influence of mutations associated with disease. We quantify the typical pattern of bone growth and maturation during early craniofacial osteogenesis and determine how this pattern is modified in Fgfr2(+/P253R) Apert syndrome mice. RESULTS: Early differences in typical relative bone density increase are noted between intramembranous and endochondral bones, with endochondral bones normally maturing more quickly during the prenatal period. Several craniofacial bones, including the facial bones of Fgfr2(+/P253R) mice, display lower volumes during the earliest days of osteogenesis and lower relative densities until the perinatal period relative to unaffected littermates. CONCLUSIONS: Estimates of bone volume and linear measures describing morphology do not necessarily covary, highlighting the value of quantifying multiple facets of gross osteological phenotypes when exploring the influence of a disease causing mutation. Differences in mechanisms of osteogenesis likely underlie differences in intramembranous and endochondral relative density increase. The influence of the FGFR2 P253R mutation on bone volume changes across the prenatal period and again after birth, while its influence on relative bone density is more stable.

Craniofacial divergence by distinct prenatal growth patterns in Fgfr2 mutant mice.

BACKGROUND: Differences in cranial morphology arise due to changes in fundamental cell processes like migration, proliferation, differentiation and cell death driven by genetic programs. Signaling between fibroblast growth factors (FGFs) and their receptors (FGFRs) affect these processes during head development and mutations in FGFRs result in congenital diseases including FGFR-related craniosynostosis syndromes. Current research in model organisms focuses primarily on how these mutations change cell function local to sutures under the hypothesis that prematurely closing cranial sutures contribute to skull dysmorphogenesis. Though these studies have provided fundamentally important information contributing to the understanding of craniosynostosis conditions, knowledge of changes in cell function local to the sutures leave change in overall three-dimensional cranial morphology largely unexplained. Here we investigate growth of the skull in two inbred mouse models each carrying one of two gain-of-function mutations in FGFR2 on neighboring amino acids (S252W and P253R) that in humans cause Apert syndrome, one of the most severe FGFR-related craniosynostosis syndromes. We examine late embryonic skull development and suture patency in Fgfr2 Apert syndrome mice between embryonic day 17.5 and birth and quantify the effects of these mutations on 3D skull morphology, suture patency and growth. RESULTS: We show in mice what studies in humans can only infer: specific cranial growth deviations occur prenatally and worsen with time in organisms carrying these FGFR2 mutations. We demonstrate that: 1) distinct skull morphologies of each mutation group are established by E17.5; 2) cranial suture patency patterns differ between mice carrying these mutations and their unaffected littermates; 3) the prenatal skull grows differently in each mutation group; and 4) unique Fgfr2-related cranial morphologies are exacerbated by late embryonic growth patterns. CONCLUSIONS: Our analysis of mutation-driven changes in cranial growth provides a previously missing piece of knowledge necessary for explaining variation in emergent cranial morphologies and may ultimately be helpful in managing human cases carrying these same mutations. This information is critical to the understanding of craniofacial development, disease and evolution and may contribute to the evaluation of incipient therapeutic strategies.

Overlapping trisomies for human chromosome 21 orthologs produce similar effects on skull and brain morphology of Dp(16)1Yey and Ts65Dn mice.

Trisomy 21 results in gene-dosage imbalance during embryogenesis and throughout life, ultimately causing multiple anomalies that contribute to the clinical manifestations of Down syndrome. Down syndrome is associated with manifestations of variable severity (e.g., heart anomalies, reduced growth, dental anomalies, shortened life-span). Craniofacial dysmorphology and cognitive dysfunction are consistently observed in all people with Down syndrome. Mouse models are useful for studying the effects of gene-dosage imbalance on development. We investigated quantitative changes in the skull and brain of the Dp(16)1Yey Down syndrome mouse model and compared these mice to Ts65Dn and Ts1Cje mouse models. Three-dimensional micro-computed tomography images of Dp(16)1Yey and euploid mouse crania were morphometrically evaluated. Cerebellar cross-sectional area, Purkinje cell linear density, and granule cell density were evaluated relative to euploid littermates. Skulls of Dp(16)1Yey and Ts65Dn mice displayed similar changes in craniofacial morphology relative to their respective euploid littermates. Trisomy-based differences in brain morphology were also similar in Dp(16)1Yey and Ts65Dn mice. These results validate examination of the genetic basis for craniofacial and brain phenotypes in Dp(16)1Yey mice and suggest that they, like Ts65Dn mice, are valuable tools for modeling the effects of trisomy 21 on development.

Quantification of facial skeletal shape variation in fibroblast growth factor receptor-related craniosynostosis syndromes.

BACKGROUND: fibroblast growth factor receptor (FGFR) -related craniosynostosis syndromes are caused by many different mutations within FGFR-1, 2, 3, and certain FGFR mutations are associated with more than one clinical syndrome. These syndromes share coronal craniosynostosis and characteristic facial skeletal features, although Apert syndrome (AS) is characterized by a more dysmorphic facial skeleton relative to Crouzon (CS), Muenke (MS), or Pfeiffer syndromes. METHODS: Here we perform a detailed three-dimensional evaluation of facial skeletal shape in a retrospective sample of cases clinically and/or genetically diagnosed as AS, CS, MS, and Pfeiffer syndrome to quantify variation in facial dysmorphology, precisely identify specific facial features pertaining to these four syndromes, and further elucidate what knowledge of the causative FGFR mutation brings to our understanding of these syndromes. RESULTS: Our results confirm a strong correspondence between genotype and facial phenotype for AS and MS with severity of facial dysmorphology diminishing from Apert FGFR2(S252W) to Apert FGFR2(P253R) to MS. We show that AS facial shape variation is increased relative to CS, although CS has been shown to be caused by numerous distinct mutations within FGFRs and reduced dosage in ERF. CONCLUSION: Our quantitative analysis of facial phenotypes demonstrate subtle variation within and among craniosynostosis syndromes that might, with further research, provide information about the impact of the mutation on facial skeletal and nonskeletal development. We suggest that precise studies of the phenotypic consequences of genetic mutations at many levels of analysis should accompany next-generation genetic research and that these approaches should proceed cooperatively.

Angiogenesis and intramembranous osteogenesis.

BACKGROUND: Angiogenesis is likely critical for the process of intramembranous osteogenesis; however, the developmental relationship between blood vessels and bone mineralization is not well studied within intramembranous bones. Given its importance, changes in angiogenesis regulation are likely to contribute to evolutionarily and medically relevant craniofacial variation. RESULTS: We summarize what is known about the association between angiogenesis and intramembranous osteogenesis, supplementing with information from the better-studied processes of endochondral ossification and distraction osteogenesis. Based on this review, we introduce a model of angiogenesis during early intramembranous osteogenesis as well as a series of null hypotheses to be tested. CONCLUSIONS: This model can serve as a basis of future research on the spatio-temporal association and regulatory interactions of mesenchymal, vascular, and bone cells, which will be required to illuminate the potential effects of angiogenesis dysregulation on craniofacial skeletal phenotypes.

Tissue-specific responses to aberrant FGF signaling in complex head phenotypes.

BACKGROUND: The role of fibroblast growth factor and receptor (FGF/FGFR) signaling in bone development is well studied, partly because mutations in FGFRs cause human diseases of achondroplasia and FGFR-related craniosynostosis syndromes including Crouzon syndrome. The FGFR2c C342Y mutation is a frequent cause of Crouzon syndrome, characterized by premature cranial vault suture closure, midfacial deficiency, and neurocranial dysmorphology. Here, using newborn Fgfr2c(C342Y/+) Crouzon syndrome mice, we tested whether the phenotypic effects of this mutation go beyond the skeletal tissues of the skull, altering the development of other non-skeletal head tissues including the brain, the eyes, the nasopharynx, and the inner ears. RESULTS: Quantitative analysis of 3D multimodal imaging (high-resolution micro-computed tomography and magnetic resonance microscopy) revealed local differences in skull morphology and coronal suture patency between Fgfr2c(C342Y/+) mice and unaffected littermates, as well as changes in brain shape but not brain size, significant reductions in nasopharyngeal and eye volumes, and no difference in inner ear volume in Fgfr2c(C342Y/+) mice. CONCLUSIONS: These findings provide an expanded catalogue of clinical phenotypes in Crouzon syndrome caused by aberrant FGF/FGFR signaling and evidence of the broad role for FGF/FGFR signaling in development and evolution of the vertebrate head.