Automated contour extraction for light-sheet microscopy images of zebrafish embryos based on object edge detection algorithm.

Embryo contour extraction is the initial step in the quantitative analysis of embryo morphology, and it is essential for understanding the developmental process. Recent developments in light-sheet microscopy have enabled the in toto time-lapse imaging of embryos, including zebrafish. However, embryo contour extraction from images generated via light-sheet microscopy is challenging owing to the large amount of data and the variable sizes, shapes, and textures of objects. In this report, we provide a workflow for extracting the contours of zebrafish blastula and gastrula without contour labeling of an embryo. This workflow is based on the edge detection method using a change point detection approach. We assessed the performance of the edge detection method and compared it with widely used edge detection and segmentation methods. The results showed that the edge detection accuracy of the proposed method was superior to those of the Sobel, Laplacian of Gaussian, adaptive threshold, Multi Otsu, and k-means clustering-based methods, and the noise robustness of the proposed method was superior to those of the Multi Otsu and k-means clustering-based methods. The proposed workflow was shown to be useful for automating small-scale contour extractions of zebrafish embryos that cannot be specifically labeled owing to constraints, such as the availability of microscopic channels. This workflow may offer an option for contour extraction when deep learning-based approaches or existing non-deep learning-based methods cannot be applied.

Structural basis for receptor-regulated SMAD recognition by MAN1.

Receptor-regulated SMAD (R-SMAD: SMAD1, SMAD2, SMAD3, SMAD5 and SMAD8) proteins are key transcription factors of the transforming growth factor-ß (TGF-ß) superfamily of cytokines. MAN1, an integral protein of the inner nuclear membrane, is a SMAD cofactor that terminates TGF-ß superfamily signals. Heterozygous loss-of-function mutations in MAN1 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. MAN1 interacts with MAD homology 2 (MH2) domains of R-SMAD proteins using its C-terminal U2AF homology motif (UHM) domain and UHM ligand motif (ULM) and facilitates R-SMAD dephosphorylation. Here, we report the structural basis for R-SMAD recognition by MAN1. The SMAD2-MAN1 and SMAD1-MAN1 complex structures show that an intramolecular UHM-ULM interaction of MAN1 forms a hydrophobic surface that interacts with a hydrophobic surface among the H2 helix, the strands ß8 and ß9, and the L3 loop of the MH2 domains of R-SMAD proteins. The complex structures also show the mechanism by which SMAD cofactors distinguish R-SMAD proteins that possess a highly conserved molecular surface.

Dullard deficiency causes hemorrhage in the adult ovarian follicles.

In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1-Cre-induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3-kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP-inducible inhibitory Smads, was up-regulated in the Dullard mutant ovaries. Tamoxifen-inducible Dullard deletion in the whole body using Rosa26-CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN-193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard-deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling.

Diabetes Impairs Wnt3 Protein-induced Neurogenesis in Olfactory Bulbs via Glutamate Transporter 1 Inhibition.

Diabetes is associated with impaired cognitive function. Streptozotocin (STZ)-induced diabetic rats exhibit a loss of neurogenesis and deficits in behavioral tasks involving spatial learning and memory; thus, impaired adult hippocampal neurogenesis may contribute to diabetes-associated cognitive deficits. Recent studies have demonstrated that adult neurogenesis generally occurs in the dentate gyrus of the hippocampus, the subventricular zone, and the olfactory bulbs (OB) and is defective in patients with diabetes. We hypothesized that OB neurogenesis and associated behaviors would be affected in diabetes. In this study, we show that inhibition of Wnt3-induced neurogenesis in the OB causes several behavioral deficits in STZ-induced diabetic rats, including impaired odor discrimination, cognitive dysfunction, and increased anxiety. Notably, the sodium- and chloride-dependent GABA transporters and excitatory amino acid transporters that localize to GABAergic and glutamatergic terminals decreased in the OB of diabetic rats. Moreover, GAT1 inhibitor administration also hindered Wnt3-induced neurogenesis in vitro Collectively, these data suggest that STZ-induced diabetes adversely affects OB neurogenesis via GABA and glutamate transporter systems, leading to functional impairments in olfactory performance.

The requirement of histone modification by PRDM12 and Kdm4a for the development of pre-placodal ectoderm and neural crest in Xenopus.

In vertebrates, pre-placodal ectoderm and neural crest development requires morphogen gradients and several transcriptional factors, while the involvement of histone modification remains unclear. Here, we report that histone-modifying factors play crucial roles in the development of pre-placodal ectoderm and neural crest in Xenopus. During the early neurula stage, PRDM12 was expressed in the lateral pre-placodal ectoderm and repressed the expression of neural crest specifier genes via methylation of histone H3K9. ChIP-qPCR analyses indicated that PRDM12 promoted the occupancy of the trimethylated histone H3K9 (H3K9me3) on the Foxd3, Slug, and Sox8 promoters. Injection of the PRDM12 MO inhibited the expression of presumptive trigeminal placode markers and decreased the occupancy of H3K9me3 on the Foxd3 promoter. Histone demethylase Kdm4a also inhibited the expression of presumptive trigeminal placode markers in a similar manner to PRDM12 MO and could compensate for the effects of PRDM12. ChIP-qPCR analyses revealed that promotion of the occupancy of H3K9me3 on the Foxd3, Slug, and Sox8 promoters was inhibited by Kdm4a overexpression. Taken together, these data indicate that histone modification was essential for pre-placodal ectoderm and neural crest development.

Wnt protein-mediated satellite cell conversion in adult and aged mice following voluntary wheel running.

Muscle represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle-derived stem cells, called satellite cells, play essential roles in regeneration after muscle injury in adult skeletal muscle. Although the molecular mechanism of muscle regeneration process after an injury has been extensively investigated, the regulation of satellite cells under steady state during the adult stage, including the reaction to exercise stimuli, is relatively unknown. Here, we show that voluntary wheel running exercise, which is a low stress exercise, converts satellite cells to the activated state due to accelerated Wnt signaling. Our analysis showed that up-regulated canonical Wnt/ß-catenin signaling directly modulated chromatin structures of both MyoD and Myf5 genes, resulting in increases in the mRNA expression of Myf5 and MyoD and the number of proliferative Pax7(+)Myf5(+) and Pax7(+) MyoD(+) cells in skeletal muscle. The effect of Wnt signaling on the activation of satellite cells, rather than Wnt-mediated fibrosis, was observed in both adult and aged mice. The association of ß-catenin, T-cell factor, and lymphoid enhancer transcription factors of multiple T-cell factor/lymphoid enhancer factor regulatory elements, conserved in mouse, rat, and human species, with the promoters of both the Myf5 and MyoD genes drives the de novo myogenesis in satellite cells even in aged muscle. These results indicate that exercise-stimulated extracellular Wnts play a critical role in the regulation of satellite cells in adult and aged skeletal muscle.

Establishment and culture optimization of a new type of pituitary immortalized cell line.

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms.

Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells.

Lipase member H is a novel secreted protein selectively upregulated in human lung adenocarcinomas and bronchioloalveolar carcinomas.

Lung cancer is one of the most frequent causes of cancer-related death worldwide. However, molecular markers for lung cancer have not been well established. To identify novel genes related to lung cancer development, we surveyed publicly available DNA microarray data on lung cancer tissues. We identified lipase member H (LIPH, also known as mPA-PLA1) as one of the significantly upregulated genes in lung adenocarcinoma. LIPH was expressed in several adenocarcinoma cell lines when they were analyzed by quantitative real-time polymerase chain reaction (qPCR), western blotting, and sandwich enzyme-linked immunosorbent assay (ELISA). Immunohistochemical analysis detected LIPH expression in most of the adenocarcinomas and bronchioloalveolar carcinomas tissue sections obtained from lung cancer patients. LIPH expression was also observed less frequently in the squamous lung cancer tissue samples. Furthermore, LIPH protein was upregulated in the serum of early- and late-phase lung cancer patients when they were analyzed by ELISA. Interestingly, high serum level of LIPH was correlated with better survival in early phase lung cancer patients after surgery. Thus, LIPH may be a novel molecular biomarker for lung cancer, especially for adenocarcinoma and bronchioloalveolar carcinoma.

Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices.

IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis.

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.

Structural and quantitative evidence for dynamic glycome shift on production of induced pluripotent stem cells.

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2-3 to α-2-6, and the expression of α-1-2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a "glycome shift" upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.

The Influence of Human Connections and Collaboration on Research Grant Success at Various Career Stages: Regression Analysis.

BACKGROUND: Documenting the grant acquisition characteristics of a highly selective group of researchers could provide insights into the research and faculty development of talented individuals, and the insights gained to foster such researchers will help university management strengthen their research capacity. OBJECTIVE: This study examines the role of human connections in the success of biomedical researchers in Japanese universities. METHODS: This study used grant data from the Grants-in-Aid for Scientific Research (GIA) program, the largest competitive research funding program in Japan, to collect information on projects and their implementation systems obtained throughout the participants' careers. Grant success was measured by the number and amounts of the awards obtained while participants occupied the role of principal investigator. Human connections were quantified by the number of projects in which the participants took part as members and were classified by their relationship with the project leader. Data were matched with information on career history, publication performance, and experience of the participants with government-funded programs apart from GIA and were analyzed using univariate and multivariate regression analyses. RESULTS: Early-career interpersonal relationships, as measured using the h-index value of the researchers who provided the participants with their initial experience as project members, had a positive effect on grant success. The experience of contributing to prestigious research programs led by top researchers dramatically increased the cumulative amount of GIA awards received by the participants over time. Univariate logistic regression analyses revealed that more interactions with upper-level researchers resulted in fewer acquisitions of large programs (odds ratio [OR] 0.67, 95% CI 0.50-0.89). Collaboration with peers increased the success rate of ≥2 research grants in large programs in situations in which both the participant and project leader were professors (OR 1.16, 95% CI 1.06-1.26). Tracking the process of research development, we found that collaboration during the periods of 10 to 14 years and 15 to 19 years after completing a doctorate degree determined the size of the project that the participant would obtain-interactions with peer researchers and subordinates during the 10- to 14-year postdegree period had positive effects on ≥2 large-program acquisitions (OR 1.51, 95% CI 1.09-2.09 and OR 1.31, 95% CI 1.10-1.57, respectively), whereas interactions with subordinates during the 15- to 19-year postdegree period also had positive effects (OR 1.25, 95% CI 1.25-1.07). Furthermore, relationships that remained narrowly focused resulted in limited grant success for small programs. CONCLUSIONS: Human networking is important for improving an individual's ability to obtain external funding. The results emphasize the importance of having a high-h-indexed collaborator to obtain quality information early in one's career; working with diverse, nonsupervisory personnel at the midcareer stage; and engaging in synergistic collaborations upon establishing a research area in which one can take more initiatives.

Spemann-Mangold organizer and mesoderm induction.

The discovery of the Spemann-Mangold organizer strongly influenced subsequent research on embryonic induction, with research aiming to elucidate the molecular characteristics of organizer activity being currently underway. Herein, we review the history of research on embryonic induction, and describe how the mechanisms of induction phenomena and developmental processes have been investigated. Classical experiments investigating the differentiation capacity and inductive activity of various embryonic regions were conducted by many researchers, and important theories of region-specific induction and the concept for chain of induction were proposed. The transition from experimental embryology to developmental biology has enabled us to understand the mechanisms of embryonic induction at the molecular level. Consequently, many inducing substances and molecules such as transcriptional factors and peptide growth factors involved in the organizer formation were identified. One of peptide growth factors, activin, acts as a mesoderm- and endoderm-inducing substance. Activin induces several tissues and organs from the undifferentiated cell mass of amphibian embryos in a concentration-dependent manner. We review the extent to which we can control in vitro organogenesis from undifferentiated cells, and discuss the application to stem cell-based regenerative medicine based on insights gained from animal experiments, such as in amphibians.

Correction: The Influence of Human Connections and Collaboration on Research Grant Success at Various Career Stages: Regression Analysis.

[This corrects the article DOI: 10.2196/49905.].

rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.

Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here, we demonstrate that a recombinant lectin probe, rBC2LCN, a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells, is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in culture medium containing fluorescent dye-labeled rBC2LCN. Fluorescent dye-labeled rBC2LCN was also successfully used to separate live pluripotent stem cells from a mixed cell population by flow cytometry.

N-Cadherin is a prospective cell surface marker of human mesenchymal stem cells that have high ability for cardiomyocyte differentiation.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Activin A regulates growth of gastro-intestinal epithelial cells by mediating epithelial-mesenchymal interaction.

The importance of epithelial-mesenchymal interaction on the development of gastro-intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin-3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region-specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial-mesenchymal interaction in the developing GI tract.

The thioredoxin-related redox-regulating protein nucleoredoxin inhibits Wnt-beta-catenin signalling through dishevelled.

Dishevelled (Dvl) transduces signals from the Wnt receptor, Frizzled, to downstream components, leading to the stabilization of beta-catenin and subsequent activation of the transcription factor T cell factor (TCF) and/or lymphoid enchancer factor (LEF). However, the mechanism of Dvl action remains unclear. Here, we report that nucleoredoxin (NRX), a thioredoxin (TRX) family protein, interacts with Dvl. Overexpression of NRX selectively suppresses the Wnt-beta-catenin pathway and ablation of NRX by RNA-interference (RNAi) results in activation of TCF, accelerated cell proliferation and enhancement of oncogenicity through cooperation with mitogen-activated extracellular signal regulated kinase kinase (MEK) or Ras. We find that cells respond to H(2)O(2) stimulation by activating TCF. Redox-dependent activation of the Wnt-beta-catenin pathway occurs independently of extracellular Wnts and is impaired by RNAi of NRX . In addition, association between Dvl and NRX is inhibited by H(2)O(2) treatment. These data suggest a relationship between the Wnt-beta-catenin pathway and redox signalling through redox-sensitive association of NRX with Dvl.

Xenopus furry contributes to release of microRNA gene silencing.

A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.