Automatic Detection of Medial and Lateral Compartments from Histological Sections of Mouse Knee Joints Using the Single-Shot Multibox Detector Algorithm.

OBJECTIVE: Although mouse osteoarthritis (OA) models are widely used, their histological analysis may be susceptible to arbitrariness and inter-examiner variability in conventional methods. Therefore, a method for the unbiased scoring of OA histology is needed. In this study, as the first step for establishing this system, we developed a computer-vision algorithm that automatically detects the medial and lateral compartments of mouse knee sections in a rigorous and unbiased manner. DESIGN: A total of 706 images of coronal sections of mouse knee joints stained by hematoxylin and eosin, safranin O, or toluidine blue were randomly divided into training and validation images at a ratio of 80:20. A model to detect both compartments automatically was built by machine learning using a single-shot multibox detector (SSD) algorithm with training images. The model was tested to determine whether it could accurately detect both compartments by analyzing the validation images and 52 images of sections stained with Picrosirius red, a method not used for the training images. RESULTS: The trained model accurately detected both medial and lateral compartments of all 140 validation images regardless of the staining method employed, severity of articular cartilage defects, and the anatomical positions and conditions of the sections. Our model also correctly detected both compartments of 50 of 52 Picrosirius red-stained images. CONCLUSIONS: By applying deep learning based on the SSD algorithm, we successfully developed a model that detects the locations of the medial and lateral compartments of tissue sections of mouse knee joints with high accuracy.

Cdc42 is required for male germline niche development in mice.

Spermatogonial stem cells (SSCs) are maintained in a special microenvironment called a niche. However, much is unknown about components that constitute the niche. Here, we report that Cdc42 is essential for germline niche development. Sertoli cell-specific Cdc42-deficient mice showed normal premeiotic spermatogenesis. However, germ cells gradually disappeared during haploid cell formation and few germ cells remained in the mature testes. Spermatogonial transplantation experiments revealed a significant loss of SSCs in Cdc42-deficient testes. Moreover, Cdc42 deficiency in Sertoli cells downregulated GDNF, a critical factor for SSC maintenance. Cdc42-deficient Sertoli cells also exhibited lower nuclear MAPK1/3 staining. Inhibition of MAP2K1 or depletion of Pea15a scaffold protein downregulated GDNF expression. A screen of transcription factors revealed that Cdc42-deficient Sertoli cells downregulate DMRT1 and SOX9, both of which are critical for Sertoli cell development. These results indicate that Cdc42 is essential for niche function via MAPK1/3-dependent GDNF secretion.

OGG1 protects mouse spermatogonial stem cells from reactive oxygen species in culture†.

Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage.

Covalent Hyperbranched Polymer Self-Assemblies of Three-Way Junction DNA for Single-Molecule Devices.

A hyperbranched polymer (HBP) made of three-way junction (TWJ) DNAs is reported. Three types of 26-mer DNAs with 5'-ends modified with psoralen (PSN) were synthesized. All had self-complementary sequences starting from the 5'-end to the sixth base (AAGCTT), allowing intermolecular hybridization. The base sequences of the remaining 20-mer sites were designed so that upon hybridization, three strands had a TWJ structure with a mass of 25,000 that could be further grown by forming HBPs. PSN photochemically reacts to form interstrand cross-links that increase the polymer stability. Aggregates [(380 ± 44) nm and (65 ± 6) nm] detected with dynamic light scattering for TWJ-DNA solutions were also imaged by electron microscopy and atomic force microscopy, providing evidence of hyperbranched polymerization. The TWJ unit also polymerized on solid substrates such as Au and glass and formed self-assembled monolayers (SAMs). The HBP SAMs were integrated into commercial Pt-interdigitated electrode arrays. The DNA devices had current-voltage curves typical of metal-insulator-metal Schottky diodes; the effective barrier heights and the ideality factors were 0.52 ± 0.002 eV and 21 ± 3.2, respectively. The series resistances were (26 ± 3.3) × 106 Ω, which may provide insights into DNA electron transport. The DNA HBP enables stable electrical connections with probe electrodes and will be an important single-molecule platform.

A lipidome atlas in MS-DIAL 4.

We present Mass Spectrometry-Data Independent Analysis software version 4 (MS-DIAL 4), a comprehensive lipidome atlas with retention time, collision cross-section and tandem mass spectrometry information. We formulated mass spectral fragmentations of lipids across 117 lipid subclasses and included ion mobility tandem mass spectrometry. Using human, murine, algal and plant biological samples, we annotated and semiquantified 8,051 lipids using MS-DIAL 4 with a 1-2% estimated false discovery rate. MS-DIAL 4 helps standardize lipidomics data and discover lipid pathways.

Wnt/ß-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone.

BACKGROUND: Both loss- and gain-of-function of Wnt/ß-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/ß-catenin signaling in the superficial zone (SFZ) of articular cartilage. METHODS: Wnt/ß-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/ß-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/ß-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/ß-catenin signaling in SFZ cells. RESULTS: Wnt/ß-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific ß-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific ß-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by ß-catenin knockout, while it was upregulated by ß-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/ß-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. CONCLUSIONS: We demonstrated that Wnt/ß-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

Correction to: Adamts17 is involved in skeletogenesis through modulation of BMP­Smad1/5/8 pathway.

In the published article, the Fig. 4a was published incorrectly.

Adamts17 is involved in skeletogenesis through modulation of BMP-Smad1/5/8 pathway.

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.

ROS amplification drives mouse spermatogonial stem cell self-renewal.

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.

Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF-κB pathway.

Exposure of articular cartilage to excessive mechanical loading is deeply involved in the pathogenesis of osteoarthritis. Here, we identify gremlin-1 as a mechanical loading-inducible factor in chondrocytes, detected at high levels in middle and deep layers of cartilage after cyclic strain or hydrostatic pressure loading. Gremlin-1 activates nuclear factor-κB signalling, leading to subsequent induction of catabolic enzymes. In mice intra-articular administration of gremlin-1 antibody or chondrocyte-specific deletion of Gremlin-1 decelerates osteoarthritis development, while intra-articular administration of recombinant gremlin-1 exacerbates this process. Furthermore, ras-related C3 botulinum toxin substrate 1 activation induced by mechanical loading enhances reactive oxygen species (ROS) production. Amongst ROS-activating transcription factors, RelA/p65 induces Gremlin-1 transcription, which antagonizes induction of anabolic genes such as Sox9, Col2a1, and Acan by bone morphogenetic proteins. Thus, gremlin-1 plays essential roles in cartilage degeneration by excessive mechanical loading.

Biphasic regulation of chondrocytes by Rela through induction of anti-apoptotic and catabolic target genes.

In vitro studies have shown that Rela/p65, a key subunit mediating NF-κB signalling, is involved in chondrogenic differentiation, cell survival and catabolic enzyme production. Here, we analyse in vivo functions of Rela in embryonic limbs and adult articular cartilage, and find that Rela protects chondrocytes from apoptosis through induction of anti-apoptotic genes including Pik3r1. During skeletal development, homozygous knockout of Rela leads to impaired growth through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela does not alter growth. In articular cartilage, homozygous knockout of Rela at 7 weeks leads to marked acceleration of osteoarthritis through enhanced chondrocyte apoptosis, whereas heterozygous knockout of Rela results in suppression of osteoarthritis development through inhibition of catabolic gene expression. Haploinsufficiency or a low dose of an IKK inhibitor suppresses catabolic gene expression, but does not alter anti-apoptotic gene expression. The biphasic regulation of chondrocytes by Rela contributes to understanding the pathophysiology of osteoarthritis.

Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2.

Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1(fl/fl) mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-Cre(ERT);Hes1(fl/fl) mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.

S100A1 and S100B are dispensable for endochondral ossification during skeletal development.

S100A1 and S100B are induced by the SOX trio transcription factors (SOX9, SOX5, and SOX6) in chondrocytes, and inhibit their hypertrophic differentiation in culture. However, functional roles of S100A1 and S100B during in vivo skeletal development are yet to be determined. Here we show that mice deficient of both the S100a1 and S100b genes displayed normal skeletal growth from embryonic stage to adulthood. Although no compensatory upregulation of other S100 family members was observed in S100a1/S100b double mutants, the related S100a2, S100a4, S100a10, and S100a11 were expressed at similarly high levels to S100a1 and S100b in mouse primary chondrocytes. Furthermore, overexpression of these other S100 members suppressed the hypertrophic differentiation of chondrocytes in vitro as efficiently as S100A1 and S100B. Taken together, the present study demonstrates that S100A1 and S100B are dispensable for endochondral ossification during skeletal development, most likely because their deficiency may be masked by other S100 proteins which have similar functions to those of S100A1 and S100B.

Determination of differential gene expression profiles in superficial and deeper zones of mature rat articular cartilage using RNA sequencing of laser microdissected tissue specimens.

Superficial zone (SFZ) cells, which are morphologically and functionally distinct from chondrocytes in deeper zones, play important roles in the maintenance of articular cartilage. Here, we established an easy and reliable method for performance of laser microdissection (LMD) on cryosections of mature rat articular cartilage using an adhesive membrane. We further examined gene expression profiles in the SFZ and the deeper zones of articular cartilage by performing RNA sequencing (RNA-seq). We validated sample collection methods, RNA amplification and the RNA-seq data using real-time RT-PCR. The combined data provide comprehensive information regarding genes specifically expressed in the SFZ or deeper zones, as well as a useful protocol for expression analysis of microsamples of hard tissues.

Identification of fibroblast growth factor-18 as a molecule to protect adult articular cartilage by gene expression profiling.

To identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions, we initially compared four types of chondrocytes: articular (AA) versus growth plate (AG) cartilage chondrocytes in adult rats, and superficial layer (IS) versus deep layer (ID) chondrocytes of epiphyseal cartilage in infant rats. Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The results were validated by real-time RT-PCR analysis. Among them, Hoxd1, Fgf18, and Esm1 were expressed more strongly in AA than in IS. Fgf18 was the extracellular and secreted factor that decreased glycosaminoglycan release and depletion from the cartilage, and enhanced proliferation of articular chondrocytes. Fgf18 was strongly expressed in the articular cartilage chondrocytes of adult rats. In a surgical rat osteoarthritis model, a once-weekly injection of recombinant human FGF18 (rhFGF18) given 3 weeks after surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with significant effect at 10 µg/week of rhFGF18. As the underlying mechanism, rhFGF18 strongly up-regulated Timp1 expression in the cell and organ cultures, and inhibition of aggrecan release by rhFGF18 was restored by addition of an antibody to Timp1. In conclusion, we have identified Fgf18 as a molecule that protects articular cartilage by gene expression profiling, and the anticatabolic effects may at least partially be mediated by the Timp1 expression.

Enrichment of mouse spermatogonial stem cells based on aldehyde dehydrogenase activity.

Spermatogonial stem cells (SSCs) comprise a small population of germ cells that have self-renewal potential. However, studies on SSCs are hampered by the lack of SSC-specific markers. Although the cryptorchid operation is often used to obtain an enriched SSC population by destroying differentiating germ cells using high body temperature, SSCs in cryptorchid testes have different biological characteristics than those in a normal environment. Therefore, it is necessary to develop new methods for SSC selection. In this study, we report a method of isolating SSCs from wild-type mouse testes based on their functional characteristics using aldehyde dehydrogenase (ALDH) activity levels, which have been successfully used for stem cell isolation from many self-renewing tissues. Testis cells selected using CD9 or CDH1, both of which are expressed by SSCs, exhibit ALDH activity in flow cytometric analyses. However, spermatogonial transplantation revealed that SSCs do not show ALDH activity, whereas somatic stem cells have high ALDH activity levels. Nevertheless, SSCs could be enriched 183.7-fold based on CDH1 preselection and transplantation of cells that lacked ALDH activity. In contrast, we failed to enrich SSCs from cultured spermatogonia, which exhibited ALDH upregulation in vitro. These results suggest that SSCs are unique among tissue-specific stem cells in their regulation of ALDH activity. Development of a new technique for SSC isolation from wild-type testes based on their functional properties will facilitate investigation of SSCs in a normal testicular environment.

Insufficient ability of Rac1b to perturb cystogenesis.

Rac1b is frequently expressed in a number of human cancer cells. It is still unclear, however, whether Rac1b causes morphological abnormalities in epithelial tissues. To investigate whether Rac1b induces morphological changes in 3-dimensional epithelial structures, we utilized an auxin-dependent protein expression system, which enabled us to rapidly induce and evaluate Rac1b function in MDCK (Madin-Darby Canine Kidney) cysts, a model for polarized epithelial structure. Cells carrying the wild-type Rac1, Rac1b and constitutively active Rac1V12 gene were morphologically indistinguishable from normal, when their coding proteins were not expressed. However, upon protein induction, Rac1V12, but not the wild-type Rac1 or Rac1b, significantly induced the luminal cell accumulation. Live cell imaging with cell cycle indicators showed that expression of Rac1V12, but not the wild-type Rac1 or Rac1b, promoted cell cycle progression. From these results, we concluded that the expression of Rac1b per se cannot induce cell proliferation. Rather, it is considered that Rac1b expression may participate in progression of malignancy.