Search

The spatial organization of cells within a given tissue plays a crucial role in determining their overall function. Driven by the importance of understanding this organization, spatial transcriptomics is quickly coming to the forefront of new Next Generation Sequencing (NGS) applications with the ability to map cellular gene expression back to a specific location within the tissue. To address the growing interest in this area of research, embryonic chick trunk tissue was used to conduct a comparison of two spatial transcriptomic solutions as we aim to determine the capabilities of each and how they might benefit the research at our Institute. The first method of this evaluation, Slide-seq, was published in Science by Macosko et al. This approach uses a 'puck' coated with barcoded beads in known positions at 10um resolution to capture the spatial information of mRNA from a given tissue section placed on the puck. The second method in the comparison is a commercialized kit from 10X Genomics called Visium. In this approach, the spatial information of mRNA from a given tissue section is obtained using 55um barcoded spots in known positions within a designated 'capture area' on a special microscope slide. The Visium solution also uses H&E staining and brightfield imaging of the tissue to inform the 10X Genomics Space Ranger analysis software tool following sequencing. Both methods require fresh frozen tissue sections at 10um thickness, use poly(dT) to capture mRNA transcripts for cDNA synthesis, and construct NGS libraries that utilize Illumina sequencing platforms to deliver unbiased spatial analysis. A 10X Genomics 3' Single Cell RNA-seq dataset was also generated from the embryonic chick trunk tissue to inform and assist the analysis of both methods. Here we present our assessment of the two spatial methods and the challenges we faced in this pilot.

Comprehensive transcriptional characterization of mRNA and small RNA in early Drosophila germline stem cells reveals novel piRNA clusters, transposon dynamics, and alternative splicing events., Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3′ UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

Anterior segment dysgenesis is often associated with cornea diseases, cataracts and glaucoma. The ciliary body (CB) is a part of the anterior segment that secretes aqueous and vitreous humor that nourishes the eye. Although defective Notch signaling in the CB disrupts CB morphogenesis and causes eye degeneration, the underlying mechanisms remain missing. Here, this study shows that NOTCH signaling controls CB morphogenesis by directly regulating Nectin1 expression, and maintains the vitreous and eye structures by regulating the expression of vitreous proteins and gap junction protein Cx43. Genetic inactivation of Rbpj or both Notch2 and Notch3 in the CB not only disrupts CB morphogenesis but also causes CB epithelial detachment and vitreous shrinkage in the developing eye, and eye degeneration in adults. Molecularly, NOTCH signaling transcriptionally controls the expression of adhesion protein Nectin1 known to be essential for CB morphogenesis, and controls eye pressure and the vitreous through stabilizing gap junction protein Cx43 known to be important for CB secretion. Mechanistically, Nectin1 directly binds and stabilizes Cx43. Finally, NOTCH signaling in the CB also directly controls the expression of vitreous proteins. Therefore, this study has provided important mechanistic insight into how NOTCH signaling controls CB morphogenesis and secretion, and also further suggests the potential involvement of CB secretion in various eye diseases, including retinal degeneration.

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in theDrosophilaovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3′ UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001, eLife digest An animal or plant has many different types of cells that have specific roles in the life of the organism. These cells are organized into tissues. In most tissues in adult animals, small groups of cells called stem cells are responsible for replacing the other cells that have been lost due to disease, injury, or as part of normal body maintenance. The ‘germ line’ stem cells of female fruit flies—which produce female sex cells (or eggs)—are an effective system for studying how stem cells are regulated. These cells live in an area of the ovary called a stem cell niche. Each time a stem cell divides, it produces one stem cell and one other daughter cell. This daughter cell then moves into another niche called the ‘differentiation’ niche and undergoes a series of divisions that produce the egg cells. The differentiation niche is formed by escort cells and is crucial for producing the egg cells, but it is not clear how the escort cells promote this process, or how the niche is maintained. Wang et al. have now studied the differentiation niche in more detail. The experiments show that a cell communication system called Wnt signaling maintains the differentiation niche by controlling the ability of the escort cells to grow and divide. If Wnt signaling is defective, the differentiation niche is lost, which disrupts the formation of egg cells. Further experiments show that two proteins called Wnt2 and Wnt4 in the differentiation niche—which activate Wnt signaling—act as signals to regulate the niche, mainly by controlling the expression of four particular genes. These four genes encode enzymes that remove ‘reactive oxygen species’ from cells. Wang et al.'s findings have revealed an important role for Wnt signaling in maintaining the differentiation niche. The next step is to figure out the details of how this works. DOI: http://dx.doi.org/10.7554/eLife.08174.002

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. Here we report that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.

Chromatin immunoprecipitation followed by sequencing the precipitated DNA (ChIP-Seq) is the state-of-the-art method to study protein-DNA interactions. ChIP-Seq allows identification of binding sites of proteins across the entire genome in an unbiased manner. One of the major limitations of currently available ChIP-Seq protocols is the necessity to isolate sufficient amounts of immune precipitated DNA for subsequent sequencing. The NARG 2013/14 study evaluated library preparation alternatives starting from one and two orders of magnitude DNA less than standard protocols. Library preparation kits from seven different commercial providers were utilized in this project. Some of these kits were intended for low input while other kits were used outside of specifications of the manufacturers. Aliquots of the same preparation of ChIPed DNA were processed using the standard protocol for 10 ng of input DNA and the evaluated library preparation alternatives for 1 ng and 100 pg of input DNA. Each library type was prepared at two different ABRF Member labs and sequencing was done on a single Illumina HiSeq flow cell.