Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Integrative analysis of metabolomic, genomic, and imaging-based phenotypes identify very-low-density lipoprotein as a potential risk factor for lumbar Modic changes.

PURPOSE: Modic changes (MC) on magnetic resonance imaging (MRI) have been associated with the development and severity of low back pain (LBP). The etiology of MC remains elusive, but it has been suggested that altered metabolism may be a risk factor. As such, this study aimed to identify metabolomic biomarkers for MC phenotypes of the lumbar spine via a combined metabolomic-genomic approach. METHODS: A population cohort of 3,584 southern Chinese underwent lumbar spine MRI. Blood samples were genotyped with single-nucleotide polymorphisms (SNP) arrays (n = 2,482) and serum metabolomics profiling using magnetic resonance spectroscopy (n = 757), covering 130 metabolites representing three molecular windows, were assessed. Genome-wide association studies (GWAS) were performed on each metabolite, to construct polygenic scores for predicting metabolite levels in subjects who had GWAS but not metabolomic data. Associations between predicted metabolite levels and MC phenotypes were assessed using linear/logistic regression and least absolute shrinkage and selection operator (LASSO). Two-sample Mendelian randomization analysis tested for causal relationships between metabolic biomarkers and MC. RESULTS: 20.4% had MC (10.6% type 1, 67.2% type 2, 22.2% mixed types). Significant MC metabolomic biomarkers were mean diameter of very-low-density lipoprotein (VLDL)/low-density lipoprotein (LDL) particles and cholesterol esters/phospholipids in large LDL. Mendelian randomization indicated that decreased VLDL mean diameter may lead to MC. CONCLUSIONS: This large-scale study is the first to address metabolomics in subject with/without lumbar MC. Causality studies implicate VLDL related to MC, noting a metabolic etiology. Our study substantiates the field of "spino-metabolomics" and illustrates the power of integrating metabolomics-genomics-imaging phenotypes to discover biomarkers for spinal disorders, paving the way for more personalized spine care for patients.

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followed by comparisons with human. Leveraging on a dataset of over 2,700 proteins from 31 proteomic profiles, we identified key molecular and cellular differences between disc compartments and spine levels, but not gender. The nucleus pulposus (NP) and annulus fibrosus (AF) compartments differ the most, both in matrisome and cellularity contents. Differences in the matrisome are consistent with the fibrous nature required for tensile strength in the AF and hydration property in the NP. Novel findings for the NP cells included an enrichment in cell junction proteins for cell-cell communication (Cdh2, Dsp and Gja1) and osmoregulation (Slc12a2 and Wnk1). In NP cells, we detected heterogeneity of vacuolar organelles; where about half have potential lysosomal function (Vamp3, Copb2, Lamp1/2, Lamtor1), some contain lipid droplets and others with undefined contents. The AF is enriched in proteins for the oxidative stress responses (Sod3 and Clu). Interestingly, mitochondrial proteins are elevated in the lumbar than tail IVDs that may reflect differences in metabolic requirement. Relative to the human, cellular and structural information are conserved for the AF. Even though the NP is more divergent between mouse and human, there are similarities at the level of cell biology. Further, common cross-species markers were identified for both NP (KRT8/19, CD109) and AF (COL12A1). Overall, mouse is a relevant model to study IVD biology, and an understanding of the limitation will facilitate research planning and data interpretation, maximizing the translation of research findings to human IVDs.

Transformation of resident notochord-descendent nucleus pulposus cells in mouse injury-induced fibrotic intervertebral discs.

Intervertebral disc degeneration (IDD), a major cause of low back pain, occurs with ageing. The core of the intervertebral disc, the nucleus pulposus (NP), embedded in a proteoglycan-rich and gelatinous matrix, is derived from the embryonic notochord. With IDD, the NP becomes fibrous, containing fewer cells, which are fibroblastic and of unknown origin. Here, we used a lineage tracing strategy to investigate the origin of cells in the NP in injury-induced mouse IDD. We established a Foxa2 notochord-specific enhancer-driven Cre transgenic mouse model (Foxa2mNE-Cre) that acts only in the embryonic to foetal period up to E14.5, to genetically label notochord cells with enhanced green fluorescent protein (EGFP). When this mouse is crossed to one carrying a Cre recombinase reporter, Z/EG, EGFP-labelled NP cells are present even at 2 years of age, consistent with their notochordal origin. We induced tail IDD in Foxa2mNE-Cre; Z/EG mice by annulus puncture and observed the degenerative changes for 12 weeks. Soon after puncture, EGFP-labelled NP cells showed strong Col2a1+ expression unlike uninjured control NP. Later, accompanying fibrotic changes, EGFP-positive NP cells expressed fibroblastic and myofibroblastic markers such as Col1a1, ASMA, FAPA and FSP-1. The number of EGFP+ cells co-expressing the fibroblastic markers increased with time after puncture. Our findings suggest resident NP cells initially upregulate Col2a1+ and later transform into fibroblast-like cells during injury-mediated disc degeneration and remodelling. This important discovery concerning the cellular origin of fibrotic pathology in injury-induced IDD has implications for management in disease and ageing.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Deep-learning-assisted biophysical imaging cytometry at massive throughput delineates cell population heterogeneity.

The association of the intrinsic optical and biophysical properties of cells to homeostasis and pathogenesis has long been acknowledged. Defining these label-free cellular features obviates the need for costly and time-consuming labelling protocols that perturb the living cells. However, wide-ranging applicability of such label-free cell-based assays requires sufficient throughput, statistical power and sensitivity that are unattainable with current technologies. To close this gap, we present a large-scale, integrative imaging flow cytometry platform and strategy that allows hierarchical analysis of intrinsic morphological descriptors of single-cell optical and mass density within a population of millions of cells. The optofluidic cytometry system also enables the synchronous single-cell acquisition of and correlation with fluorescently labeled biochemical markers. Combined with deep neural network and transfer learning, this massive single-cell profiling strategy demonstrates the label-free power to delineate the biophysical signatures of the cancer subtypes, to detect rare populations of cells in the heterogeneous samples (10-5), and to assess the efficacy of targeted therapeutics. This technique could spearhead the development of optofluidic imaging cell-based assays that stratify the underlying physiological and pathological processes based on the information-rich biophysical cellular phenotypes.

IRX3 and IRX5 Inhibit Adipogenic Differentiation of Hypertrophic Chondrocytes and Promote Osteogenesis.

Maintaining the correct proportions of different cell types in the bone marrow is critical for bone function. Hypertrophic chondrocytes (HCs) and osteoblasts are a lineage continuum with a minor contribution to adipocytes, but the regulatory network is unclear. Mutations in transcription factors, IRX3 and IRX5, result in skeletal patterning defects in humans and mice. We found coexpression of Irx3 and Irx5 in late-stage HCs and osteoblasts in cortical and trabecular bone. Irx3 and Irx5 null mutants display severe bone deficiency in newborn and adult stages. Quantitative analyses of bone with different combinations of functional alleles of Irx3 and Irx5 suggest these two factors function in a dosage-dependent manner. In Irx3 and Irx5 nulls, the amount of bone marrow adipocytes was increased. In Irx5 nulls, lineage tracing revealed that removal of Irx3 specifically in HCs exacerbated reduction of HC-derived osteoblasts and increased the frequency of HC-derived marrow adipocytes. ß-catenin loss of function and gain of function specifically in HCs affects the expression of Irx3 and Irx5, suggesting IRX3 and IRX5 function downstream of WNT signaling. Our study shows that IRX3 and IRX5 regulate fate decisions in the transition of HCs to osteoblasts and to marrow adipocytes, implicating their potential roles in human skeletal homeostasis and disorders.

Directed Differentiation of Notochord-like and Nucleus Pulposus-like Cells Using Human Pluripotent Stem Cells.

Intervertebral disc degeneration might be amenable to stem cell therapy, but the required cells are scarce. Here, we report the development of a protocol for directed in vitro differentiation of human pluripotent stem cells (hPSCs) into notochord-like and nucleus pulposus (NP)-like cells of the disc. The first step combines enhancement of ACTIVIN/NODAL and WNT and inhibition of BMP pathways. By day 5 of differentiation, hPSC-derived cells express notochordal cell characteristic genes. After activating the TGF-ß pathway for an additional 15 days, qPCR, immunostaining, and transcriptome data show that a wide array of NP markers are expressed. Transcriptomically, the in vitro-derived cells become more like in vivo adolescent human NP cells, driven by a set of influential genes enriched with motifs bound by BRACHYURY and FOXA2, consistent with an NP cell-like identity. Transplantation of these NP-like cells attenuates fibrotic changes in a rat disc injury model of disc degeneration.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Cellular Plasticity in Musculoskeletal Development, Regeneration, and Disease.

In this review, we highlight themes from a recent workshop focused on "Plasticity of Cell Fate in Musculoskeletal Tissues" held at the Orthopaedic Research Society's 2019 annual meeting. Experts in the field provided examples of mesenchymal cell plasticity during normal musculoskeletal development, regeneration, and disease. A thorough understanding of the biology underpinning mesenchymal cell plasticity may offer a roadmap for promoting regeneration while attenuating pathologic differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:708-718, 2020.

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes.

BACKGROUND: The growth plate is a special region of the cartilage that drives longitudinal growth of long bones. Proliferating chondrocytes in the growth plate, arranged in columns, divide perpendicular to the long axis of the growth plate then intercalate to re-align with parental columns. Which molecular partners maintain growth plate columnar structures and chondrocyte cytokinesis has not been fully revealed. It is reported that kinesin family member 3A (KIF3A), a subunit of kinesin-2, plays an important role in maintaining columnar organization in growth plates via controlling primary cilia formation and cell proliferation. RESULT: Here we identify kinesin family member 5B (KIF5B), the heavy chain of kinesin-1, a ubiquitously expressed motor protein for anterograde intracellular transport along the microtubule network, as a key modulator of cytokinesis in chondrocytes via maintenance of central spindle organization. We show that KIF5B is concentrated in the central spindle during cytokinesis in both primary chondrocytes and chondrogenic ATDC5 cells. CONCLUSION: The failure of cytokinesis in KIF5B null chondrocytes leads to incomplete cell rotation, disrupting proliferation and differentiation, and results in a disorganized growth plate.

Sox2 and FGF20 interact to regulate organ of Corti hair cell and supporting cell development in a spatially-graded manner.

The mouse organ of Corti, housed inside the cochlea, contains hair cells and supporting cells that transduce sound into electrical signals. These cells develop in two main steps: progenitor specification followed by differentiation. Fibroblast Growth Factor (FGF) signaling is important in this developmental pathway, as deletion of FGF receptor 1 (Fgfr1) or its ligand, Fgf20, leads to the loss of hair cells and supporting cells from the organ of Corti. However, whether FGF20-FGFR1 signaling is required during specification or differentiation, and how it interacts with the transcription factor Sox2, also important for hair cell and supporting cell development, has been a topic of debate. Here, we show that while FGF20-FGFR1 signaling functions during progenitor differentiation, FGFR1 has an FGF20-independent, Sox2-dependent role in specification. We also show that a combination of reduction in Sox2 expression and Fgf20 deletion recapitulates the Fgfr1-deletion phenotype. Furthermore, we uncovered a strong genetic interaction between Sox2 and Fgf20, especially in regulating the development of hair cells and supporting cells towards the basal end and the outer compartment of the cochlea. To explain this genetic interaction and its effects on the basal end of the cochlea, we provide evidence that decreased Sox2 expression delays specification, which begins at the apex of the cochlea and progresses towards the base, while Fgf20-deletion results in premature onset of differentiation, which begins near the base of the cochlea and progresses towards the apex. Thereby, Sox2 and Fgf20 interact to ensure that specification occurs before differentiation towards the cochlear base. These findings reveal an intricate developmental program regulating organ of Corti development along the basal-apical axis of the cochlea.

Quantitative Phase Imaging Flow Cytometry for Ultra-Large-Scale Single-Cell Biophysical Phenotyping.

Cellular biophysical properties are the effective label-free phenotypes indicative of differences in cell types, states, and functions. However, current biophysical phenotyping methods largely lack the throughput and specificity required in the majority of cell-based assays that involve large-scale single-cell characterization for inquiring the inherently complex heterogeneity in many biological systems. Further confounded by the lack of reported robust reproducibility and quality control, widespread adoption of single-cell biophysical phenotyping in mainstream cytometry remains elusive. To address this challenge, here we present a label-free imaging flow cytometer built upon a recently developed ultrafast quantitative phase imaging (QPI) technique, coined multi-ATOM, that enables label-free single-cell QPI, from which a multitude of subcellularly resolvable biophysical phenotypes can be parametrized, at an experimentally recorded throughput of >10,000 cells/s-a capability that is otherwise inaccessible in current QPI. With the aim to translate multi-ATOM into mainstream cytometry, we report robust system calibration and validation (from image acquisition to phenotyping reproducibility) and thus demonstrate its ability to establish high-dimensional single-cell biophysical phenotypic profiles at ultra-large-scale (>1,000,000 cells). Such a combination of throughput and content offers sufficiently high label-free statistical power to classify multiple human leukemic cell types at high accuracy (~92-97%). This system could substantiate the significance of high-throughput QPI flow cytometry in enabling next frontier in large-scale image-derived single-cell analysis applied in biological discovery and cost-effective clinical diagnostics. © 2019 International Society for Advancement of Cytometry.

Mechanistic insights into skeletal development gained from genetic disorders.

A complex cascade of highly regulated processes of cell fate determination, differentiation, proliferation and transdifferentiation dictate the patterning, morphogenesis and growth of the vertebrate skeleton, perturbation of which results in malformation. In humans over 450 different dysplasias involving the skeletal system constitute a significant fraction of documented Mendelian disorders. The combination of clinical, phenotypic characterization of rare human skeletal dysmorphologies, the discovery of causative mutations and functional validation in animal models has contributed enormously to the understanding of molecular control of skeletal development. These studies revealed a myriad of genes and pathways, such as WNT, Hedgehog (HH), planar cell polarity and primary cilia, as key regulators for skeletal patterning, growth and homeostasis. The generation of mouse models recapitulating human congenital skeletal dysplasia has provided mechanistic insights into the diverse pathologies caused by single gene mutations, integrated action of developmental pathways such as WNT and HH and the role of stress responses. Technological developments in whole genome and exome sequencing have accelerated the discovery of disease-causing mutations and are changing approaches for diagnosis. The discovery that non-coding variants and disorganization of the 3D genome are associated with limb patterning disorders has revealed an additional level of complexity in the regulatory framework of skeletal development and disease mechanisms. This chapter focuses on a selection of human skeletal pathologies which illustrate how new findings about the coding and noncoding genome, combined with functional modeling, are contributing to deeper understanding of skeletal development, mechanisms of disease, with therapeutic potential for chondrodysplasias.

Multi-ATOM: Ultrahigh-throughput single-cell quantitative phase imaging with subcellular resolution.

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost-effective and label-free cellular assays, which provides useful insights into understanding the biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single-cell precision to define cell identities in a large and heterogeneous population of cells-hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric-detection time-stretch optical microscopy (multi-ATOM) that captures and processes quantitative label-free single-cell images at ultrahigh throughput without compromising subcellular resolution. We show that multi-ATOM, based upon ultrafast phase-gradient encoding, outperforms state-of-the-art QPI in permitting robust phase retrieval at a QPI throughput of >10 000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi-ATOM for large-scale, label-free, multivariate, cell-type classification (e.g. breast cancer subtypes, and leukemic cells vs peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi-ATOM could empower new strategies in large-scale biophysical single-cell analysis with applications in biology and enriching disease diagnostics.

Unique and overlapping GLI1 and GLI2 transcriptional targets in neoplastic chondrocytes.

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.

Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells.

Previous studies have demonstrated the ability of reprogramming endochondral bone into induced pluripotent stem (iPS) cells, but whether similar phenomenon occurs in intramembranous bone remains to be determined. Here we adopted fluorescence-activated cell sorting-based strategy to isolate homogenous population of intramembranous calvarial osteoblasts from newborn transgenic mice carrying both Osx1-GFP::Cre and Oct4-EGFP transgenes. Following retroviral transduction of Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), enriched population of osteoblasts underwent silencing of Osx1-GFP::Cre expression at early stage of reprogramming followed by late activation of Oct4-EGFP expression in the resulting iPS cells. These osteoblast-derived iPS cells exhibited gene expression profiles akin to embryonic stem cells and were pluripotent as demonstrated by their ability to form teratomas comprising tissues from all germ layers and also contribute to tail tissue in chimera embryos. These data demonstrate that iPS cells can be generated from intramembranous osteoblasts.

Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions.

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.

Early onset of disc degeneration in SM/J mice is associated with changes in ion transport systems and fibrotic events.

Intervertebral disc degeneration (IDD) causes back pain and sciatica, affecting quality of life and resulting in high economic/social burden. The etiology of IDD is not well understood. Along with aging and environmental factors, genetic factors also influence the onset, progression and severity of IDD. Genetic studies of risk factors for IDD using human cohorts are limited by small sample size and low statistical power. Animal models amenable to genetic and functional studies of IDD provide desirable alternatives. Despite differences in size and cellular content as compared to human intervertebral discs (IVDs), the mouse is a powerful model for genetics and assessment of cellular changes relevant to human biology. Here, we provide evidence for early onset disc degeneration in SM/J relative to LG/J mice with poor and good tissue healing capacity respectively. In the first few months of life, LG/J mice maintain a relatively constant pool of notochordal-like cells in the nucleus pulposus (NP) of the IVD. In contrast, chondrogenic events are observed in SM/J mice beginning as early as one-week-old, with progressive fibrotic changes. Further, the extracellular matrix changes in the NP are consistent with IVD degeneration. Leveraging on the genomic data of two parental and two recombinant inbred lines, we assessed the genetic contribution to the NP changes and identified processes linked to the regulation of ion transport systems. Significantly, "transport" system is also in the top three gene ontology (GO) terms from a comparative proteomic analysis of the mouse NP. These findings support the potential of the SM/J, LG/J and their recombinant inbred lines for future genetic and biological analysis in mice and validation of candidate genes and biological relevance in human cohort studies. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD008784.