Biology subjects

Shirai, M.

Publications and source records attributed to Shirai, M..

4 recordsLinked to original sources

bioRxiv2025

Osteochondrogenic Transdifferentiation of Vascular Smooth Muscle Cells and Microenvironmental Dynamics in Medial Arterial Calcification

BackgroundVascular calcification, particularly medial arterial calcification (MAC), considerably affects cardiovascular mortality. Current treatments are limited because of the unclear molecular mechanisms of MAC. This study aimed to establish an MAC mouse model using O-ring-induced transverse aortic constriction (OTAC) and to identify critical genes, pathways, and cellular interactions involved in MAC formation by combining lineage tracing technology and single-cell RNsA sequencing (scRNA-seq). MethodsWe developed an OTAC mouse model to mimic MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were performed at various intervals. Histological and immunohistochemical evaluations were conducted to observe changes in vascular smooth muscle cells (VSMCs). Lineage tracing was used to confirm the origin of osteochondrogenic cells in the tunica media. Additionally, scRNA-seq was performed to capture the dynamic changes in VSMCs and other cell types involved in MAC development. ResultsOsteochondrogenic cells and the subsequent MAC in OTAC mice were observed using histological evaluations. Immunohistochemistry showed time-dependent decreases in -SMA expression and increases in SOX9 and RUNX2 expression in VSMCs. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic cells originated from contractile VSMCs. scRNA-seq confirmed osteochondrogenic transdifferentiation of VSMCs and highlighted dynamic changes in surrounding cells involved in calcification-related and inflammatory processes. ConclusionThe OTAC method enabled a comprehensive understanding of the microenvironmental dynamics during MAC progression at the single-cell level. This powerful model provides a robust platform for future therapeutic intervention research. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/684711v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@c64634org.highwire.dtl.DTLVardef@12b0b9aorg.highwire.dtl.DTLVardef@106c5b4org.highwire.dtl.DTLVardef@19dd4f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Hedgehog Activity Gradient in Combination with Transcription Network Confers Multiple Hypothalamic Identities

During development, the hypothalamus emerges from the ventral diencephalon of the neural tube and is regionalised into several distinct functional domains. Each domain is characterised by different combinations of transcription factors, the expression of which is regulated by signalling molecules and downstream transcriptional networks. Transcription factors, including Nkx2.1, Nkx2.2, Pax6 and Rx, are expressed in the presumptive hypothalamus and its surrounding regions from an early developmental stage and play critical roles in the development of these areas. However, the regulation of transcription factor expression and the details of the transcriptional network among them have not been fully elucidated. As early hypothalamus development takes place in the ventral region of the forebrain where Sonic Hedgehog (Shh) is expressed, we focused on the relationship between Shh and its downstream transcription factors and investigated the transcriptional regulation along the dorsal-ventral axis. Using a reporter mouse line, in vitro neural differentiation of mouse embryonic stem cells and gene overexpression in chick embryos, we found that Pax6, Nkx2.1 and Nkx2.2 are regulated epistatically by different Shh signal intensities. Nkx2.1 and Nkx2.2 mutually repress each other; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region along the dorsal-ventral and anterior-posterior regulations. Finally, we found that the Shh signal demarcates the diencephalic region from the retinal area. Our findings suggest that Shh signalling, and its downstream transcription network, are required for hypothalamic regionalisation and establishment of diencephalic cell fate.

developmental biology↗

bioRxiv2021

Identification of transcription termination defects at DNA hypomethylated transcription termination sites in DNA methyltransferase 3a-deficient vertebrates.

CpG methylation in genomic DNA is well known as a repressive epigenetic marker in eukaryotic transcription, and DNA methylation of the promoter regions is correlated with silencing of gene expression. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study has revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions (including transcription termination sites (TTSs)) during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we employed two strategies: informatic analysis using already deposited datasets of Dnmt3a-/- mouse cells and the zebrafish model system. Bioinformatic analysis using methylome and transcriptome data showed that hypomethylated differentially methylated regions overlapping the TTSs were associated with increased read counts and chimeric transcripts downstream of TTSs in Dnmt3a-/- Agouti-related protein neurons, but not in Dnmt3a-/- embryonic stem cells and mouse embryonic fibroblasts. We experimentally detected increased read-through and chimeric transcripts downstream of hypomethylated TTSs in zebrafish maternal-zygotic dnmt3aa-/- mutant. This study is the first to identify transcription termination defects in DNA hypomethylated TTSs in Dnmt3a-/- vertebrates.

genomics↗

bioRxiv2020

Light-dependent induction of Edn2 expression and attenuation of retinal pathology by endothelin receptor antagonists in Prominin-1- deficient mice

Retinitis pigmentosa (RP) and macular dystrophy (MD) are prevalent retinal degenerative diseases associated with gradual photoreceptor death. These diseases are often caused by genetic mutations that result in degeneration of the retina postnatally after it has fully developed. The Prominin-1 gene (Prom1) is a causative gene for RP and MD, and Prom1- knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and stenosis of retinal blood vessels. The mechanisms underlying progression of such degeneration have remained unknown, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. High-throughput analysis revealed that expression of the endothelin-2 gene (Edn2) was markedly up-regulated in the Prom1-deficient retina during this period. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice that had been reared in the dark. Finally, treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis, and retinal vessel stenosis in Prom1-KO mice. Our findings suggest that inhibitors of endothelin signalling may delay the progression of RP and MD and therefore warrant further study as potential therapeutic agents for these diseases.

neuroscience↗