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Abstract Visual observing muscle tissue regeneration is used to measure experimental effect size in biological research to discover the mechanism of muscle strength decline due to illness or aging. Quantitative computer imaging analysis for support evaluating the recovery phase has not been established because of the localized nature of recovery and the difficulty in selecting image features for cells in regeneration. We constructed MyoRegenTrack for segmenting cells and classifying their regeneration phase in hematoxylin–eosin (HE) stained images. A straightforward approach to classification is supervised learning. However, obtaining detailed annotations for each fiber in a whole slide image is impractical in terms of cost and accuracy. Thus, we propose to learn individual recovery phase classification utilizing the proportions of cell class depending on the days after muscle injection to induce regeneration. We extract implicit multidimensional features from the HE-stained tissue images and train a classifier using weakly supervised learning, guided by their class proportion for elapsed time on recovery. We confirmed the effectiveness of MyoRegenTrack by comparing its results with expert annotations. A comparative study of the recovery relation between two different muscle injections shows that the analysis result using MyoRegenTrack is consistent with findings from previous studies.

Abstract Runt-related transcription factor 2 (Runx2), a regulator of osteoblast differentiation, is pathologically involved in vascular calcification; however, the significance of Runx2 in cardiac homeostasis remains unclear. Here, we investigated the roles of Runx2 in cardiac remodeling after myocardial infarction (MI). The expression of Runx2 mRNA and protein was upregulated in murine hearts after MI. Runx2 was expressed in heart-infiltrating myeloid cells, especially in macrophages, at the border zone of post-infarct myocardium. To analyze the biological functions of Runx2 in cardiac remodeling, myeloid cell-specific Runx2 deficient (CKO) mice were exposed to MI. After MI, ventricular weight/tibia length ratio was increased in CKO mice, concomitant with severe cardiac dysfunction. Cardiac fibrosis was exacerbated in CKO mice, consistent with the upregulation of collagen 1a1 expression. Mechanistically, immunohistochemical analysis using anti-CD31 antibody showed that capillary density was decreased in CKO mice. Additionally, conditioned culture media of myeloid cells from Runx2 deficient mice exposed to MI induced the tube formation of vascular endothelial cells to a lesser extent than those from control mice. RNA-sequence showed that the expression of pro-angiogenic or anti-angiogenic factors was altered in macrophages from Runx2-deficient mice. Collectively, Runx2 + myeloid cells infiltrate into post-infarct myocardium and prevent adverse cardiac remodeling, at least partially, by regulating endothelial cell function.

Abstract Bidirectional transcription has been proposed to play a role associated with enhancer activity. Transcripts called enhancer RNAs (eRNAs) play important roles in gene regulation; however, their roles in osteoclasts are unknown. To analyse eRNAs in osteoclasts comprehensively, we used cap-analysis of gene expression (CAGE) to detect adjacent transcription start sites (TSSs) that were distant from promoters for protein-coding gene expression. When comparing bidirectional TSSs between osteoclast precursors and osteoclasts, we found that bidirectional TSSs were located in the 5′-flanking regions of the Nrp2 and Dcstamp genes. We also detected bidirectional TSSs in the intron region of the Nfatc1 gene. To investigate the role of bidirectional transcription in osteoclasts, we performed loss of function analyses using the CRISPR/Cas9 system. Targeted deletion of the DNA regions between the bidirectional TSSs led to decreased expression of the bidirectional transcripts, as well as the protein-coding RNAs of Nrp2, Dcstamp, and Nfatc1, suggesting that these transcripts act as eRNAs. Furthermore, osteoclast differentiation was impaired by targeted deletion of bidirectional eRNA regions. The combined results show that eRNAs play important roles in osteoclastogenic gene regulation, and may therefore provide novel insights to elucidate the transcriptional mechanisms that control osteoclast differentiation.

Abstract During antral folliculogenesis, developmental competence of prospective oocytes is regulated in large part by the follicular somatic component to prepare the oocyte for the final stage of maturation and subsequent embryo development. The underlying molecular mechanisms are poorly understood. Oocytes reaching the advanced stage of follicular growth by administration of exogenous follicle-stimulating hormone (FSH) possess higher developmental competence than oocytes in FSH-untreated smaller follicles. In this study, the transcriptomic profile of the cumulus cells from cows receiving FSH administration (FSH-priming) was compared, as a model of high oocyte competence, with that from untreated donor cows (control). Ingenuity Pathway Analysis showed that cumulus cells receiving FSH-priming were rich in down-regulated transcripts associated with cell movement and migration, including the extracellular matrix-related transcripts, probably preventing the disruption of cell-to-cell contacts. Interestingly, the transcriptomic profile of up-regulated genes in the control group was similar to that of granulosa cells from atretic follicles. Interferon regulatory factor 7 was activated as the key upstream regulator of FSH-priming. Thus, acquisition of developmental competence by oocytes can be ensured by the integrity of cumulus cells involved in cell-to-cell communication and cell survival, which may help achieve enhanced oocyte-somatic cell coupling.