Project description:Mutations in the RMRP gene are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in the RMRP gene lead to skeletal dysplasia. Viperin is a known substrate of RMRP. Since chondrogenic differentiation of the growth plate is required for development of the long bones, we hypothesized that viperin functions as a chondrogenic regulator downstream of RMRP. Viperin protein is expressed throughout the stages of chondrogenic differentiation in vivo. Viperin gene expression is increased during knockdown of Rmrp RNA in the ATDC5 model for chondrogenic differentiation. Viperin is expressed during ATDC5 chondrogenic differentiation. Viperin knockdown reduces, while viperin overexpression increases overall protein secretion, with CXCL10 identified as a potential target via mass spectrometry-proteomics. CXCL10 protein expression is reduced during knockdown and increased during overexpression of viperin and CXCL10 protein expression coincides with viperin expression in ATDC5 chondrogenic differentiation. Viperin knockdown induces, while viperin overexpression reduces TGFβ activity. Furthermore, viperin knockdown conditioned media increases, while viperin overexpression conditioned media reduces chondrogenic differentiation of ATDC5 cells. TGFβ target genes Pai1 and Smad7 are increased during knockdown and reduced during overexpression of viperin. Moreover, TGFβ activity is reduced when differentiating ATDC5 cells are exposed to CXCL10 and, acting as a viperin overexpression mimic, CXCL10 similarly reduces chondrogenic differentiation of ATDC5. Lastly, we show that in CHH patient cells, RMRP expression is reduced and viperin expression is increased, coinciding with reduced chondrogenic differentiation and increased CXCL10 expression, possibly explaining the CHH phenotype. Together our data show that viperin may play a pivotal role in chondrogenic differentiation, with potential consequences for cartilage-hair hypoplasia pathobiology.

Project description:Purpose: We have succeeded in the generation and long-term expansion of SOX9-expressing CD271+PDGFRa+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1-CD271hiPDGFRaloCD73- neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/TGFb inhibitor (SB) and FGF2 (hereafter called FSB). When M-bM-^@M-^\primedM-bM-^@M-^] with TGFb, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. Under the FSB condition, ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages, maintained normal karyotype for at least 10 passages, which any conditions deviated from it (e.g. FGF2 alone or SB alone) failed to support. In order to address the molecular basis of such effects of FSB, a series of RNA-seq experiments were carried out. Methods: We generated and compared the transcriptome profiles of human ectomesenchymal cells expanded under FSB with those cultured under FSB first then under FGF2 alone (F). As a control, we also generated transcriptome of ectomesenchymal cells expanded from the begining under F conditions. RNA-sequencing libraries were prepared using a SureSelect Strand Specific RNA Library Preparation kit (Agilent technologies, Santa Clara, CA). Sequencing was performed on an Illumina HiSeq 1500 using a TruSeq Rapid SBS kit (Illumina, San Diego, CA) in a 50-base single-end mode. Sequenced reads were mapped against the human reference genome (GRCh37), using TopHat v2.0.12 (http://ccb.jhu.edu/software/tophat/index.shtml). Expression levels were calculated as fragments per kilobase of exon per million mapped fragments (FPKMs) using Cufflinks v2.1.1 (http://cole-trapnell-lab.github.io/cufflinks). Results: Ectomesenchymal cells maintained under FSB conditions preferentially expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B, MYCN), cranial mesenchymes (ALX1/3/4) and chondroprogenitors (SOX9, COL2a1) of the neural crest origin (SOX8/9, NGFR, NES). In contrast, those cultured under FSB then F, still expressed SOX4/11/12, but lost LIN28, MYCN, ALX1/3/4, NGFR, COL2a1 expression. Interestingl it enhances expresion ofTGFM-NM-2-inducible genes such as THBS1/2 and DCN and osteogenesis-related genes such as COL1a1/2 and RUNX1/2. Conclusions: The CD271+CD73+ ectomesenchymal cells accumulated under FSB conditions possess an mRNA profile of proliferating primitive neural crest/ectomesenchymal cells, although they lacked SOX10 expression, which is critical for neural and melanocytic lineage commitment. Transition from FSB to F conditions supressed the proliferation-related genes expression and enhanced the ossification/mineralization, vasculogenesis/angiogenesis, and cardiac myogenesis-related gene expression. Thus, suppression of TGFM-NM-2 signaling by SB does not seem to freeze the developmental stage of the hPSC-derived neural crest during expansion. Such suppression may instead simply support the high proliferative potential of the cells as well as the expression of SOX9 (and COL2a1), and thereby maintain chondrogenic activity. SOX9 expression initiated at the specification and pre-migratory stages is transient in trunk neural crest but persists in cranial neural crest. The chondrogenic CD271+CD73+ ectomesenchymal cells that maintain SOX9 transcription and translation may therefore represent proliferating cranial neural crest, with a slight commitment to non-neural lineages. Examination of human ES-derived neural crest-like progenies expanded in 3 different culture media. Each group contains three biological replicates.

Project description:The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape. Disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2'704 putative chondrogenic enhancer regions. Most of these enhancers (74%) showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Moreover, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci each exhibited a significant reduction of cognate gene expression, therefore demonstrating their functional importance. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.

Project description:Introduction: In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation. Materials: Sox9 expression in ATDC5 cells was knocked-down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 hours, and 7 days of differentiation. The mRNA sequencing library was generated using TruSeq mRNA sample preparation kit (Illumina, Eindhoven, the Netherlands). In short, mRNA was enriched using magnetic beads coated with poly-dT, followed by fragmentation. The fragmented mRNA enriched samples were subjected to cDNA synthesis by reverse transcriptase, followed by dA-tailing and ligation of specific double-stranded bar-coded adapters. Next, the library was amplified and following cleanup the sizes of the libraries were determined on an Agilent 2100 Bioanalyzer via a DNA 1000 chip according manufacturer’s protocol. Pooled libraries consisting of equal molar samples were sequenced on a high-output 75bp single read on the NextSeq500 (Illumina). For each sample, the number of reads covering one or more exons of a given transcript were extracted. Triplicates of samples that were treated with either Scrambled or Sox9 siRNAs, at two different time points, were grouped separately. A transcript was defined as expressed when all replicates of a group had at least 5 reads extracted within the transcript's region. The grouped data were then compared to one another. The fold-change difference and the p-value were calculated using R-package edgeR, after which the p-value was corrected for multiple testing (false discovery rate (FDR)-corrected). The transcriptomes using a RNA-seq approach was analyzed using pathway and network analyses. Results: Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown.

Project description:The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape. Disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2'704 putative chondrogenic enhancer regions. Most of these enhancers (74%) showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Moreover, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci each exhibited a significant reduction of cognate gene expression, therefore demonstrating their functional importance. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.

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Transcription profiling of mouse induced chondrogenic cells

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