Satoshi Yamashita

Sox9 directly promotes Bapx1 gene expression to repress Runx2 in chondrocytes.

The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood. In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect. Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.

Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification

BackgroundChondrogenesis and subsequent endochondral ossification are processes tightly regulated by the transcription factor Sox9 (SRY-related high mobility group-Box gene 9), but molecular mechanisms underlying this activity remain unclear. Here we report that coactivator-associated arginine methyltransferase 1 (CARM1) regulates chondrocyte proliferation via arginine methylation of Sox9.ResultsCARM1-null mice display delayed endochondral ossification and decreased chondrocyte proliferation. Conversely, cartilage development of CARM1 transgenic mice was accelerated. CARM1 specifically methylates Sox9 at its HMG domain in vivo and in vitro. Arg-methylation of Sox9 by CARM1 disrupts interaction of Sox9 with beta-catenin, regulating Cyclin D1 expression and cell cycle progression of chondrocytes.ConclusionThese results establish a role for CARM1 as an important regulator of chondrocyte proliferation during embryogenesis.

Indocyanine Green Angiography of Retrobulbar Vascular Structures in Severe Myopia

PURPOSEnTo evaluate angiographic findings of retrobulbar arteries and veins in severely myopic patients.nnnMETHODSnWe examined 416 severely myopic eyes (213 patients) with refractive errors greater than -8.25 diopters using indocyanine green videoangiography. A control group of 74 eyes (37 patients) had refractive errors within plano +/- 3 diopters. Four severely myopic patients underwent computed tomographic angiography to identify the entire intraorbital course of retrobulbar veins.nnnRESULTSnOf 416 severely myopic eyes, 231 (55.5%) exhibited retrobulbar arteries, which were tortuous and pulsatile behind the posterior pole of the globe. Retrobulbar arteries connected directly with choroidal arteries temporal to the macular area. In 17 of these 231 eyes, retrobulbar arteries were also observed nasal to the optic nerve head, continuous with the Zinn-Haller ring around the optic nerve head and directly connected with choroidal arteries. In 39 severely myopic eyes (31 patients), indocyanine green angiography disclosed retrobulbar veins, most of which coursed vertically just behind the posterior pole of the globe. These retrobulbar veins originated as an inferior vascular network of the inferior orbital vein and drained into the superior orbital vein in the upper orbit.nnnCONCLUSIONnRetrobulbar arteries observed in this study were temporal and nasal short posterior ciliary arteries. Only the lateral collateral vein, which was one of the collateral channels between the superior and inferior orbital veins, was visible in severely myopic eyes. Indocyanine green angiography is useful in evaluating retrobulbar vascular structure in severely myopic eyes.

Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis

Significance Molecular mechanisms of tendon development and homeostasis are not well understood. Generation and analysis of Mkx−/− rats revealed new functions of Mohawk (Mkx) in mediating cellular responses to mechanical stress. An Mkx-ChIP assay in rat tendon-derived cells with Mkx expression suggested that this factor may associate with both tendon- and cartilage-related genes to orchestrate tendon cell differentiation and maintenance. These findings advance our understanding of tendon physiology and pathology. Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)−/− rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx−/− mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx−/− mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx−/− TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx+/+ TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9. Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.

Zinn-Haller arterial ring observed by ICG angiography in high myopia

AIMS To delineate the entire Zinn–Haller arterial ring angiographically in vivo. METHODS 382 highly myopic eyes (210 patients) with refractive errors greater than −8.25 D were examined using indocyanine green (ICG) videoangiography. A control group of 80 eyes (40 patients) had refractive errors within plano +/− 3 D. RESULTS The Zinn–Haller ring was visible in 206 of 382 highly myopic eyes (53.9%) by ICG angiography. Although only a part of the Zinn–Haller ring was visible in 162 of 206 eyes, in the remaining 44 eyes it was observed almost completely around the optic nerve head. No anastomotic channels between lateral and medial short posterior ciliary arteries were filled by ICG angiography. In 22 of the 44 eyes (50.0%) the Zinn–Haller ring was supplied by branches of the lateral and medial short posterior ciliary arteries; in seven eyes, it was supplied only by the lateral short posterior ciliary artery; and in seven eyes, it was supplied only by the medial short posterior ciliary artery. In none of the control subjects was the Zinn–Haller ring visible by ICG angiography. CONCLUSIONS The Zinn–Haller ring observed by ICG angiography was not a complete collateral circle between lateral and medial posterior ciliary arteries. Also, the patterns in supply vessels to the Zinn–Haller ring varied. ICG angiography made possible the detailed observation of the Zinn–Haller ring in human eyes in vivo.

Posterior routes of choroidal blood outflow in high myopia.

Purpose: A few reports in the ophthalmic literature have described choroidal blood outflow through posterior routes. Most of the patients reported were highly myopic; therefore, a correlation between such posterior routes and high myopia has been suspected. The authors examined highly myopic eyes using indocyanine green (ICG) videoangiography and investigated the prevalence and clinical significance of posterior routes in them. Methods: The authors examined 255 highly myopic eyes (146 patients) using ICG videoangiography. All had refractive errors greater than -8.25 diopters (D). They also examined a control group consisting of 42 eyes (26 patients) that had refractive errors within ±3 D. Results: Of 255 highly myopic eyes, 61 (23.9%) had choroidal blood outflow through posterior routes. These routes were classified by type of vein according to its penetration site. One drained into the margin of the optic nerve head, and the other penetrated the sclera near the macula. However, only 1 of the 42 eyes (2.4%) in the control group showed choroidal outflow by a posterior route. The prevalence of posterior routes was significantly higher in the highly myopic eyes than in the control group (P < 0.05). Conclusion: Posterior routes of choroidal blood outflow were observed in nearly 25% of highly myopic eyes. These vessels appear to be one of the major routes of posterior choroidal outflow in highly myopic eyes.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

Posterior Routes of Choroidal Venous Flow in High Myopia

A few reports in the ophthalmic literature have described choroidal blood outflow through posterior routes. Most of these patients were highly myopic; therefore, a correlation between such posterior routes and high myopia has been suspected. We examined highly myopic eyes using indocyanine green (ICG) videoangiography to investigate the prevalence and clinical significance of posterior routes. We examined 255 highly myopic eyes (146 patients) using ICG videoangiography. All had refractive errors greater than -8.25 D. We also examined a control group consisting of 42 eyes (26 patients) that had refractive errors within ±3D. Of the 255 highly myopic eyes, 61 (23.9%) had choroidal blood outflow through posterior routes. These routes were classified by type of vein according to its penetration site. One was the choriovaginal vein, which drains into the margin of the optic nervehead. The other was the macular vortex vein, which penetrates the sclera near the macula. On the other hand, only one of the 42 eyes (2.4%) in the control group showed choroidal outflow by a posterior route. The prevalence of posterior routes was significantly higher in the highly myopic eyes than in the control eyes (P < 0.05). As posterior routes for choroidal blood outflow were observed in nearly 25% of highly myopic eyes, these vessels would appear to be one of the major routes of posterior choroidal outflow in these eyes.