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Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR.

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi-mediated knock-down of Nup153 alters the expression of approximately 5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation-an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR-binding is used for chromosomal organization that enables gene expression control.

Reproduction is highly dependent on environmental and physiological factors including nutrition, mating stimuli and microbes. Among these factors, microbes facilitate vital functions for host animals such as nutritional intake, metabolic regulation, and enhancing fertility under poor nutrition conditions. However, detailed molecular mechanisms by which microbes control germline maturation, leading to reproduction, remain largely unknown. In this study, we show that environmental microbes exert a beneficial effect on Drosophila oogenesis by promoting germline stem cell (GSC) proliferation and subsequent egg maturation via acceleration of ovarian cell division and suppression of apoptosis. Moreover, insulin-related signaling is not required; rather, the ecdysone pathway is necessary for microbe-induced increase of GSCs and promotion of egg maturation, while juvenile hormone contributes only to increasing GSC numbers, suggesting that hormonal pathways are activated at different stages of oogenesis. Our findings reveal that environmental microbes can enhance host reproductivity by modulating host hormone release and promoting oogenesis. Environmental microbes enhance Drosophila oogenesis by increasing the number of germline stem cells, suppressing apoptosis and accelerating ovarian cell division through hormonal pathways.

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi-mediated knock-down of Nup153 alters the expression of ~5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation--an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR-binding is used for chromosomal organization that enables gene expression control.

Piwi-interacting RNAs (piRNAs), a class of 23- to 29-nt gonad-specific small RNAs, function to combat transposons in gonads. piRNAs are thought to be processed and amplified in membrane-less granules called nuage in germline cells. In Drosophila, two PIWI family proteins, several Tudor-domain containing (Tdrd) proteins and RNA helicases are assembled at perinuclear region of germline cells, forming nuage to process into piRNAs. Among those, Tejas (Tej), a fly homolog of mouse Tdrd5, has been known as a robust nuage component required for piRNA biogenesis in germline cells, yet its molecular functions remained elusive. To understand its molecular basis on nuage assembly and functions for piRNA biogenesis, we investigated subcellular localization of fluorescent-tagged nuage proteins including Tej and monitored the behavior of piRNA precursors. Tej functions as a core component for assembly of Vasa and Spindle-E to nuage granules through distinct motifs, respectively. The loss of Tej function resulted in malformation of nuage and accumulation of piRNA precursors en route in processing, perturbing further piRNA biogenesis in germline cells. Our study also revealed that the low complexity region of Tej regulates the mobility of nuage by phase separation. Collectively, we propose that Tej plays a pivotal role in processing of piRNA precursors by assembling RNA helicases, Vasa and Spindle-E, to nuage, by controlling the dynamics of nuage components.

HemK2 is a highly conserved methyltransferase spanning from yeast to humans. Despite its conservation, the identification of its genuine substrates has been controversial, and its biological importance in higher organisms remains unclear. In this study, we elucidate the role of HemK2 in the methylation of eukaryotic Release Factor 1 (eRF1), a process essential for female germline development in Drosophila melanogaster. Knockdown of hemK2 in the germline cells (hemK2-GLKD) induces apoptosis in these cells, accompanied by a pronounced decrease in both eRF1 methylation and protein synthesis. The overexpression of a methylation-deficient eRF1 variant recapitulates the defects observed in hemK2-GLKD, suggesting that eRF1 is a primary methylation target of HemK2. Furthermore, hemK2-GLKD leads to significant reduction mRNA levels in germline cell. We demonstrate that these defects in oogenesis and protein synthesis can be partially restored by inhibiting the No-Go Decay pathway. In addition, hemK2 knockdown is associated with increased disome formation, suggesting that disruptions in eRF1 methylation may provoke ribosomal stalling, which subsequently activates translation-coupled mRNA surveillance mechanisms that degrade actively-translated mRNAs. We propose that HemK2-mediated methylation of eRF1 is critical for ensuring efficient protein production and mRNA stability, which are vital for the generation of high-quality eggs.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Fig6F and Fig7E were added; Fig7D revised; Supplementary FigS7B moved to FigS7C; New FigS7B added; Supplementary Table S2 revised.

Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR.

Gene expression studies have typically focused on finding differentially expressed genes or pathways between two or more conditions. More recently, single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as the study of gene expression variation within cell types from cell lines or from relatively similar cells in tissues, organs or tumors. However, although single-cell RNA-seq provides quantitative and comprehensive expression data in a developing embryo, it is not yet clear whether this can replace conventional in situ screens for finding developmentally important genes; moreover, current single-cell data analysis approaches typically cluster cells into types based on a common set of genes or identify more variable or differentially expressed genes using predefined groups of cells, limiting their use for finding genes with novel expression patterns. Here we present a method that comprehensively finds cell-specific patterns of developmentally important regulators directly from single-cell gene expression data of the Ciona embryo, a marine chordate. We recover many of the known expression patterns directly from our single-cell RNA-seq data and despite extensive previous screens, we succeed in finding new cell-specific patterns and genes, which we validate by in situ and single-cell qPCR.

Katakori is a symptom name that is unique to Japan, and refers to myofascial pain syndrome-like clinical signs in the shoulder girdle. Various methods of pain relief for katakori have been reported, but in the present study, we examined the clinical effects of multi-acupuncture point injections (MAPI) in the acupuncture points with which we empirically achieved an effect, as well as the anatomical sites affected by liquid medicine. The subjects were idiopathic katakori patients (n = 9), and three cadavers for anatomical investigation. BL-10, GB-21, LI-16, SI-14, and BL-38 as the WHO notation were selected as the acupuncture point. Injections of 1 mL of 1% w/v mepivacaine were introduced at the same time into each of these points in the patients. Assessment items were the Pain Relief Score and the therapeutic effect period. Dissections were centered at the puncture sites of cadavers. India ink was similarly injected into each point, and each site that was darkly-stained with India ink was evaluated. Katakori pain in the present study was significantly reduced by MAPI. Regardless of the presence or absence of trigger points, pain was significantly reduced in these cases. Dark staining with India ink at each of the points in the anatomical analysis was as follows: BL-10: over the rectus capitis posterior minor muscle and rectus capitis posterior major muscle fascia; GB-21: over the supraspinatus muscle fascia; LI-16: over the supraspinatus muscle fascia; SI-14: over the rhomboid muscle fascia; and BL-38: over the rhomboid muscle fascia. The anatomical study suggested that the drug effect was exerted on the muscles above and below the muscle fascia, as well as the peripheral nerves because the points of action in acupuncture were darkly-stained in the spaces between the muscle and the muscle fascia.