Transcriptomic landscape of human induced pluripotent stem cell-derived osteogenic differentiation identifies a regulatory role of KLF16.

Osteogenic differentiation is essential for bone development and metabolism, but the underlying gene regulatory networks have not been well investigated. We differentiated mesenchymal stem cells, derived from 20 human induced pluripotent stem cell lines, into preosteoblasts and osteoblasts, and performed systematic RNA-seq analyses of 60 samples for differential gene expression. We noted a highly significant correlation in expression patterns and genomic proximity among transcription factor (TF) and long noncoding RNA (lncRNA) genes. We identified TF-TF regulatory networks, regulatory roles of lncRNAs on their neighboring coding genes for TFs and splicing factors, and differential splicing of TF, lncRNA, and splicing factor genes. TF-TF regulatory and gene co-expression network analyses suggested an inhibitory role of TF KLF16 in osteogenic differentiation. We demonstrate that in vitro overexpression of human KLF16 inhibits osteogenic differentiation and mineralization, and in vivo Klf16+/- mice exhibit increased bone mineral density, trabecular number, and cortical bone area. Thus, our model system highlights the regulatory complexity of osteogenic differentiation and identifies novel osteogenic genes.

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.

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.

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.

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.

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.

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.

Integration of Brain and Skull in Prenatal Mouse Models of Apert and Crouzon Syndromes.

The brain and skull represent a complex arrangement of integrated anatomical structures composed of various cell and tissue types that maintain structural and functional association throughout development. Morphological integration, a concept developed in vertebrate morphology and evolutionary biology, describes the coordinated variation of functionally and developmentally related traits of organisms. Syndromic craniosynostosis is characterized by distinctive changes in skull morphology and perceptible, though less well studied, changes in brain structure and morphology. Using mouse models for craniosynostosis conditions, our group has precisely defined how unique craniosynostosis causing mutations in fibroblast growth factor receptors affect brain and skull morphology and dysgenesis involving coordinated tissue-specific effects of these mutations. Here we examine integration of brain and skull in two mouse models for craniosynostosis: one carrying the FGFR2c C342Y mutation associated with Pfeiffer and Crouzon syndromes and a mouse model carrying the FGFR2 S252W mutation, one of two mutations responsible for two-thirds of Apert syndrome cases. Using linear distances estimated from three-dimensional coordinates of landmarks acquired from dual modality imaging of skull (high resolution micro-computed tomography and magnetic resonance microscopy) of mice at embryonic day 17.5, we confirm variation in brain and skull morphology in Fgfr2cC342Y/+ mice, Fgfr2+/S252W mice, and their unaffected littermates. Mutation-specific variation in neural and cranial tissue notwithstanding, patterns of integration of brain and skull differed only subtly between mice carrying either the FGFR2c C342Y or the FGFR2 S252W mutation and their unaffected littermates. However, statistically significant and substantial differences in morphological integration of brain and skull were revealed between the two mutant mouse models, each maintained on a different strain. Relative to the effects of disease-associated mutations, our results reveal a stronger influence of the background genome on patterns of brain-skull integration and suggest robust genetic, developmental, and evolutionary relationships between neural and skeletal tissues of the head.

Altered metabolism and resistance to obesity in long-lived mice producing reduced levels of IGF-I.

The extension of lifespan due to reduced insulin-like growth factor 1 (IGF-I) signaling in mice has been proposed to be mediated through alterations in metabolism. Previously, we showed that mice homozygous for an insertion in the Igf1 allele have reduced levels of IGF-I, are smaller, and have an extension of maximum lifespan. Here, we tested whether this specific reduction of IGF-I alters glucose metabolism both on normal rodent chow and in response to high-fat feeding. We found that female IGF-I-deficient mice were lean on a standard rodent diet but paradoxically displayed an insulin-resistant phenotype. However, these mice gained significantly less weight than normal controls when placed on a high-fat diet. In control animals, insulin response was significantly impaired by high-fat feeding, whereas IGF-I-deficient mice showed a much smaller shift in insulin response after high-fat feeding. Gluconeogenesis was also elevated in the IGF-I-deficient mice relative to controls on both normal and high-fat diet. An analysis of metabolism and respiratory quotient over 24 h indicated that the IGF-I-deficient mice preferentially utilized fatty acids as an energy source when placed on a high-fat diet. These results indicate that reduction in the circulating and tissue IGF-I levels can produce a metabolic phenotype in female mice that increases peripheral insulin resistance but renders animals resistant to the deleterious effects of high-fat feeding.

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.