Macrophage TGF-ß signaling is critical for wound healing with heterotopic ossification after trauma.

Transforming growth factor-ß1 (TGF-ß1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-ß1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-ß1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-ß1-stimulated genes at binding sites specific for transcription factors of activated TGF-ß1 (SMAD2/3). Genetic deletion of TGF-ß1 receptor type 1 (Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-ß1/3 ligand trap TGF-ßRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-ß1/ALK5 signaling pathway in HO.

The SEQC2 epigenomics quality control (EpiQC) study.

BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.

Early-life social experience affects offspring DNA methylation and later life stress phenotype.

Studies in rodents and captive primates suggest that the early-life social environment affects future phenotype, potentially through alterations to DNA methylation. Little is known of these associations in wild animals. In a wild population of spotted hyenas, we test the hypothesis that maternal care during the first year of life and social connectedness during two periods of early development leads to differences in DNA methylation and fecal glucocorticoid metabolites (fGCMs) later in life. Here we report that although maternal care and social connectedness during the den-dependent life stage are not associated with fGCMs, greater social connectedness during the subadult den-independent life stage is associated with lower adult fGCMs. Additionally, more maternal care and social connectedness after den independence correspond with higher global (%CCGG) DNA methylation. We also note differential DNA methylation near 5 genes involved in inflammation, immune response, and aging that may link maternal care with stress phenotype.

High Frequency Spectral Ultrasound Imaging Detects Early Heterotopic Ossification in Rodents.

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Rods Contribute to Visual Behavior in Larval Zebrafish.

Purpose: Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown. Methods: We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf. Results: Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs. Conclusions: Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

Immobilization after injury alters extracellular matrix and stem cell fate.

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

The role of the histone H3 variant CENPA in prostate cancer.

Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.