Adaptive expansion of ERVK solo-LTRs is associated with Passeriformes speciation events.

Endogenous retroviruses (ERVs) are ancient retroviral remnants integrated in host genomes, and commonly deleted through unequal homologous recombination, leaving solitary long terminal repeats (solo-LTRs). This study, analysing the genomes of 362 bird species and their reptilian and mammalian outgroups, reveals an unusually higher level of solo-LTRs formation in birds, indicating evolutionary forces might have purged ERVs during evolution. Strikingly in the order Passeriformes, and especially the parvorder Passerida, endogenous retrovirus K (ERVK) solo-LTRs showed bursts of formation and recurrent accumulations coinciding with speciation events over past 22 million years. Moreover, our results indicate that the ongoing expansion of ERVK solo-LTRs in these bird species, marked by high transcriptional activity of ERVK retroviral genes in reproductive organs, caused variation of solo-LTRs between individual zebra finches. We experimentally demonstrated that cis-regulatory activity of recently evolved ERVK solo-LTRs may significantly increase the expression level of ITGA2 in the brain of zebra finches compared to chickens. These findings suggest that ERVK solo-LTRs expansion may introduce novel genomic sequences acting as cis-regulatory elements and contribute to adaptive evolution. Overall, our results underscore that the residual sequences of ancient retroviruses could influence the adaptive diversification of species by regulating host gene expression.

Spatial analysis toolkits for RNA in situ sequencing.

Spatial transcriptomics (ST) is featured by high-throughput gene expression profiling within their native cell and tissue context, offering a means to investigate gene regulatory networks in tissue microenvironment. In situ sequencing (ISS) is an imaging-based ST technology that simultaneously detects hundreds to thousands of genes at subcellular resolution. As a highly reproducible and robust technique, ISS has been widely adapted and undergone a series of technical iterations. As the interest in ISS-based spatial transcriptomic analysis grows, scalable and integrated data analysis workflows are needed to facilitate the applications of ISS in different research fields. This review presents the state-of-the-art bioinformatic toolkits for ISS data analysis, which covers the upstream and downstream analysis workflows, including image analysis, cell segmentation, clustering, functional enrichment, detection of spatially variable genes and cell clusters, spatial cell-cell interactions, and trajectory inference. To assist the community in choosing the right tools for their research, the application of each tool and its compatibility with ISS data are reviewed in detailed. Finally, future perspectives and challenges concerning how to integrate heterogeneous tools into a user-friendly analysis pipeline are discussed. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico.

Characterization of the Nucleus Pulposus Progenitor Cells via Spatial Transcriptomics.

Loss of refreshment in nucleus pulposus (NP) cellularity leads to intervertebral disc (IVD) degeneration. Nevertheless, the cellular sequence of NP cell differentiation remains unclear, although an increasing body of literature has identified markers of NP progenitor cells (NPPCs). Notably, due to their fragility, the physical enrichment of NP-derived cells has limited conventional transcriptomic approaches in multiple studies. To overcome this limitation, a spatially resolved transcriptional atlas of the mouse IVD is generated via the 10x Genomics Visium platform dividing NP spots into two clusters. Based on this, most reported NPPC-markers, including Cathepsin K (Ctsk), are rare and predominantly located within the NP-outer subset. Cell lineage tracing further evidence that a small number of Ctsk-expressing cells generate the entire adult NP tissue. In contrast, Tie2, which has long suggested labeling NPPCs, is actually neither expressed in NP subsets nor labels NPPCs and their descendants in mouse models; consistent with this, an in situ sequencing (ISS) analysis validated the absence of Tie2 in NP tissue. Similarly, no Tie2-cre-mediated labeling of NPPCs is observed in an IVD degenerative mouse model. Altogether, in this study, the first spatial transcriptomic map of the IVD is established, thereby providing a public resource for bone biology.

Combined amplification-based single-molecule fluorescence in situ hybridization with immunofluorescence for simultaneous in situ detection of RNAs and proteins.

We present a combined amplification-based single-molecule fluorescence in situ hybridization and immunofluorescence (asmFISH-IF) method for the detection of multiple RNAs and proteins simultaneously in cells and formaldehyde-fixed and paraffin-embedded tissue sections. We showed that performing asmFISH before immunofluorescence gives a better IF signal than the opposite. Our asmFISH-IF method could help study the interplay of RNA and protein, helping to understand their functions.

Lymphatic endothelial transcription factor Tbx1 promotes an immunosuppressive microenvironment to facilitate post-myocardial infarction repair.

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge syndrome disease gene that encodes a T-box transcription factor (TF). We found induced profound lymphangiogenic and immunomodulatory gene expression changes in lymphatic endothelial cells (LECs) after myocardial infarction (MI). The activated LECs penetrated the infarcted area and functioned as intramyocardial immune hubs to increase the numbers of tolerogenic dendritic cells (tDCs) and regulatory T (Treg) cells through the chemokine Ccl21 and integrin Icam1, thereby inhibiting the expansion of autoreactive CD8+ T cells and promoting reparative macrophage expansion to facilitate post-MI repair. Mimicking its timing and implementation may be an additional approach to treating autoimmunity-mediated cardiac diseases.

Chromogenic Visualization of Single RNA Molecules In Situ with Duplex Capability by Rolling Circle Amplification with Alkaline Phosphatase.

Visualizing spatial patterns of gene expression by optical microscopy at single-molecule resolution represents a long-standing challenge for imaging and molecular engineering technologies. In this study, we developed a method for visualizing mRNA with duplex capability by optical microscopy using rolling circle amplification with streptavidin-modified alkaline phosphatase (SA-ALP) to provide highly selective and sensitive RNA detection. ALP-based RNA detection provides comparable sensitivity and specificity to fluorescence-based in situ assays and similar performance to the current RNAscope technique for single-molecule RNA detection, but with improved ease of operation. This versatile and relatively user-friendly method of single-molecule RNA visualization can also overcome common problems of background interference. Our findings show that the red spots generated by the Fast Red staining in situ are readily quantified by image analysis, even in samples with heavy melanin deposition, supporting the clinical translation of this assay to improve diagnostic assays for recalcitrant tissues. This system was adaptable for duplex assays with multiple probes and multiple stains, which is ALP with horseradish peroxidase to produce red and brown signals to simultaneously visualize two different RNA targets. The duplex assay can be successfully applied to quantify mRNA expression from two genes in situ within single cells and multiple cell types. With the advantages of high sensitivity and low hardware requirements, this versatile and user-friendly method of RNA visualization may enable low-resource institutions to conduct previously inaccessible diagnostic or research questions about the in situ expression and distribution of RNAs at single-molecule resolution.

Improved in situ sequencing for high-resolution targeted spatial transcriptomic analysis in tissue sections.

Spatial transcriptomics enables the study of localization-indexed gene expression activity in tissues, providing the transcriptional landscape that in turn indicates the potential regulatory networks of gene expression. In situ sequencing (ISS) is a targeted spatial transcriptomic technique, based on padlock probe and rolling circle amplification combined with next-generation sequencing chemistry, for highly multiplexed in situ gene expression profiling. Here, we present improved in situ sequencing (IISS) that exploits a new probing and barcoding approach, combined with advanced image analysis pipelines for high-resolution targeted spatial gene expression profiling. We develop an improved combinatorial probe anchor ligation chemistry using a 2-base encoding strategy for barcode interrogation. The new encoding strategy results in higher signal intensity as well as improved specificity for in situ sequencing, while maintaining a streamlined analysis pipeline for targeted spatial transcriptomics. We show that IISS can be applied to both fresh frozen tissue and formalin-fixed paraffin-embedded tissue sections for single-cell level spatial gene expression analysis, based on which the developmental trajectory and cell-cell communication networks can also be constructed.

Single-molecule RNA in situ detection in clinical FFPE tissue sections by vsmCISH.

Although RNA plays a vital role in gene expression, it is less used as an in situ biomarker for clinical diagnostics than DNA and protein. This is mainly due to technical challenges caused by the low expression level and easy degradation of RNA molecules. To tackle this issue, methods that are sensitive and specific are needed. Here, we present an RNA single-molecule chromogenic in situ hybridization assay based on DNA probe proximity ligation and rolling circle amplification. When the DNA probes hybridize into close proximity to the RNA molecules, they form a V-shape structure and mediate the circularization of circle probes. Thus, our method was termed vsmCISH. We successfully applied our method to assess HER2 mRNA expression status in invasive breast cancer tissue and investigated the utility of albumin mRNA ISH for differentiating primary from metastatic liver cancer. The promising results on clinical samples indicate that our method has great potential for application in diagnosing diseases using RNA biomarkers.

Novel In Situ Hybridization Assay for Chromogenic Single-Molecule Detection of Human Papillomavirus E6/E7 mRNA.

RNA plays a vital role in the physiological and pathological processes of cells and tissues. However, RNA in situ hybridization applications in clinical diagnostics are still limited to a few examples. In this study, we developed a novel in situ hybridization assay for human papillomavirus (HPV) E6/E7 mRNA by taking advantage of specific padlock probing and rolling circle amplification, combined with chromogenic readout. We designed padlock probes for 14 types of high-risk HPV and demonstrated that E6/E7 mRNA could be visualized in situ as discrete dot-like signals using bright-field microscopy. Overall, the results are consistent with the clinical diagnostics lab's hematoxylin and eosin (H&E) staining and p16 immunohistochemistry test results. Our work thus shows the potential applications of RNA in situ hybridization for clinical diagnostics using chromogenic single-molecule detection, offering an alternative technical option to the current commercially available kit based on branched DNA technology. IMPORTANCE In situ detection of viral mRNA expression in tissue samples is of great value for pathological diagnosis to access viral infection status. Unfortunately, conventional RNA in situ hybridization assays lack sensitivity and specificity for clinical diagnostic purposes. Currently, the commercially available branched DNA technology-based single-molecule RNA in situ detection method offers satisfactory results. Here, we present our padlock probe- and rolling circle amplification-based RNA in situ hybridization assay for detecting HPV E6/E7 mRNA expression in formalin-fixed paraffin-embedded tissue sections, providing an alternative yet robust method for viral RNA in situ visualization that is also applicable to different types of diseases.

Two modified density gradient centrifugation methods facilitate the isolation of mouse Leydig cells.

Preparation of sufficient mouse Leydig cells (LCs) with high purity is a prerequisite for investigations of the biological/pathological functions of LCs in mouse models. Density gradient centrifugation based on discontinuous Percoll gradients is an effective method (defined as regular method) for LC isolation. In this study, we developed two modified methods for LC isolation and compared their performance with that of the regular method. Modified method 1 integrated the crude LCs into the 50% Percoll solution before centrifugation. Modified method 2 sequentially used 50 and 60% Percoll solutions to isolate LCs. The purity of LCs was approximately 88.4, 91.3, and 79.7% derived from the regular, modified 1, and modified 2 methods, respectively. The yields of LCs in the same respective order were approximately 1.7 × 105, 3.9 × 105, and 11.9 × 105 cells per 108 interstitial cells input. Modified method 1 attained higher purity and yields than those of the regular method. Although the purity of LCs was relatively low for modified method 2, it could be used before further purification by, for example, fluorescence-activated or magnetic-activated cell sorting, owing to its simplicity and high yields. Therefore, our study provided alternative methods to facilitate LC isolation in mice.

Catalytic DNA-Assisted Mass Production of Arbitrary Single-Stranded DNA.

Synthetic single-stranded (ss) DNA is a cornerstone for life and materials science, yet the purity, quantity, length, and customizability of synthetic DNA are still limiting in various applications. Here, we present PECAN, paired-end cutting assisted by DNAzymes (DNA enzymes or deoxyribozymes), which enables mass production of ssDNA of arbitrary sequence (up to 7000 nucleotides, or nt) with single-base precision. At the core of PECAN technique are two newly identified classes of DNAzymes, each robustly self-hydrolyzing with minimal sequence requirement up- or down-stream of its cleavage site. Flanking the target ssDNA with a pair of such DNAzymes generates a precursor ssDNA amplifiable by pseudogene-recombinant bacteriophage, which subsequently releases the target ssDNA in large quantities after efficient auto-processing. PECAN produces ssDNA of virtually any terminal bases and compositions with >98.5 % purity at the milligram-to-gram scale. We demonstrate the feasibility of using PECAN ssDNA for RNA in situ detection, homology-directed genome editing, and DNA-based data storage.

Spatial Transcriptomics for Tumor Heterogeneity Analysis.

The molecular heterogeneity of cancer is one of the major causes of drug resistance that leads to treatment failure. Thus, better understanding the heterogeneity of cancer will contribute to more precise diagnosis and improved patient outcomes. Although single-cell sequencing has become an important tool for investigating tumor heterogeneity recently, it lacks the spatial information of analyzed cells. In this regard, spatial transcriptomics holds great promise in deciphering the complex heterogeneity of cancer by providing localization-indexed gene expression information. This study reviews the applications of spatial transcriptomics in the study of tumor heterogeneity, discovery of novel spatial-dependent mechanisms, tumor immune microenvironment, and matrix microenvironment, as well as the pathological classification and prognosis of cancer. Finally, future challenges and opportunities for spatial transcriptomics technology's applications in cancer are also discussed.

Polymerase chain reaction-based assays facilitate the breeding and study of mouse models of Klinefelter syndrome.

Klinefelter syndrome (KS) is one of the most frequent genetic abnormalities and the leading genetic cause of nonobstructive azoospermia. The breeding and study of KS mouse models are essential to advancing our knowledge of the underlying pathological mechanism. Karyotyping and fluorescence in situ hybridization are reliable methods for identifying chromosomal contents. However, technical issues associated with these methods can decrease the efficiency of breeding KS mouse models and limit studies that require rapid identification of target mice. To overcome these limitations, we developed three polymerase chain reaction-based assays to measure specific genetic information, including presence or absence of the sex determining region of chromosome Y (Sry), copy number of amelogenin, X-linked (Amelx), and inactive X specific transcripts (Xist) levels. Through a combined analysis of the assay results, we can infer the karyotype of target mice. We confirmed the utility of our assays with the successful generation of KS mouse models. Our assays are rapid, inexpensive, high capacity, easy to perform, and only require small sample amounts. Therefore, they facilitate the breeding and study of KS mouse models and help advance our knowledge of the pathological mechanism underlying KS.

Interneuron origin and molecular diversity in the human fetal brain.

Precise generation of excitatory neurons and inhibitory interneurons is crucial for proper formation and function of neural circuits in the mammalian brain. Because of the size and complexity of the human brain, it is a challenge to reveal the rich diversity of interneurons. To decipher origin and diversity of interneurons in the human fetal subpallium, here we show molecular features of diverse subtypes of interneuron progenitors and precursors by conducting single-cell RNA sequencing and in situ sequencing. Interneuron precursors in the medial and lateral ganglionic eminence simultaneously procure temporal and spatial identity through expressing a combination of specific sets of RNA transcripts. Acquisition of various interneuron subtypes in adult human brains occurs even at fetal stages. Our study uncovers complex molecular signatures of interneuron progenitors and precursors in the human fetal subpallium and highlights the logic and programs in the origin and lineage specification of various interneurons.

Glycogen accumulation and phase separation drives liver tumor initiation.

Glucose consumption is generally increased in tumor cells to support tumor growth. Interestingly, we report that glycogen accumulation is a key initiating oncogenic event during liver malignant transformation. We found that glucose-6-phosphatase (G6PC) catalyzing the last step of glycogenolysis is frequently downregulated to augment glucose storage in pre-malignant cells. Accumulated glycogen undergoes liquid-liquid phase separation, which results in the assembly of the Laforin-Mst1/2 complex and consequently sequesters Hippo kinases Mst1/2 in glycogen liquid droplets to relieve their inhibition on Yap. Moreover, G6PC or another glycogenolysis enzyme-liver glycogen phosphorylase (PYGL) deficiency in both human and mice results in glycogen storage disease along with liver enlargement and tumorigenesis in a Yap-dependent manner. Consistently, elimination of glycogen accumulation abrogates liver growth and cancer incidence, whereas increasing glycogen storage accelerates tumorigenesis. Thus, we concluded that cancer-initiating cells adapt a glycogen storing mode, which blocks Hippo signaling through glycogen phase separation to augment tumor incidence.

Liquid Biopsy, ctDNA Diagnosis through NGS.

Liquid biopsy with circulating tumor DNA (ctDNA) profiling by next-generation sequencing holds great promise to revolutionize clinical oncology. It relies on the basis that ctDNA represents the real-time status of the tumor genome which contains information of genetic alterations. Compared to tissue biopsy, liquid biopsy possesses great advantages such as a less demanding procedure, minimal invasion, ease of frequent sampling, and less sampling bias. Next-generation sequencing (NGS) methods have come to a point that both the cost and performance are suitable for clinical diagnosis. Thus, profiling ctDNA by NGS technologies is becoming more and more popular since it can be applied in the whole process of cancer diagnosis and management. Further developments of liquid biopsy ctDNA testing will be beneficial for cancer patients, paving the way for precision medicine. In conclusion, profiling ctDNA with NGS for cancer diagnosis is both biologically sound and technically convenient.

CENPL, ISG20L2, LSM4, MRPL3 are four novel hub genes and may serve as diagnostic and prognostic markers in breast cancer.

As a highly prevalent disease among women worldwide, breast cancer remains in urgent need of further elucidation its molecular mechanisms to improve the patient outcomes. Identifying hub genes involved in the pathogenesis and progression of breast cancer can potentially help to unveil mechanism and also provide novel diagnostic and prognostic markers. In this study, we integrated multiple bioinformatic methods and RNA in situ detection technology to identify and validate hub genes. EZH2 was recognized as a key gene by PPI network analysis. CENPL, ISG20L2, LSM4, MRPL3 were identified as four novel hub genes through the WGCNA analysis and literate search. Among these, many studies on EZH2 in breast cancer have been reported, but no studies are related to the roles of CENPL, ISG20L2, MRPL3 and LSM4 in breast cancer. These four novel hub genes were up-regulated in tumor tissues and associated with cancer progression. The receiver operating characteristic analysis and Kaplan-Meier survival analysis indicated that these four hub genes are promising candidate genes that can serve as diagnostic and prognostic biomarkers for breast cancer. Moreover, these four newly identified hub genes as aberrant molecules in the maintenance of breast cancer development, their exact functional mechanisms deserve further in-depth study.

Imaging of individual transcripts by amplification-based single-molecule fluorescence in situ hybridization.

An amplification-based single-molecule fluorescence in situ hybridization (asmFISH) assay is introduced that exploits improved probe design for highly specific imaging of individual transcripts in fixed cells and tissues. In this method, a pair of DNA ligation probes are ligated on RNA templates upon specific hybridization, followed by probe circularization based on enzymatic DNA ligation and rolling circle amplification for signal boosting. The method is more efficient and specific than the padlock probe assay for detection of the same RNA molecules and discrimination of single nucleotide polymorphisms. Moreover, asmFISH is a versatile method which can be applied not only to cultured cells, but also to fresh frozen and formalin-fixed, paraffin-embedded tissue sections.

Single Cell RNA Expression Analysis Using Flow Cytometry Based on Specific Probe Ligation and Rolling Circle Amplification.

Conventional flow cytometry has been widely used for high-throughput single-cell gene expression analysis using specific antibody staining. However, this is limited by the availability of high-quality antibodies. We developed a novel flow cytometry RNA detection technique termed RCA-Flow for single-cell RNA expression analysis. We showed that it is able to analyze not only mRNAs but also microRNAs and circular RNAs that are otherwise difficult to analyze by other flow cytometry techniques. The versatility for high-throughput analysis of different types of RNA molecules makes our method possess great potential for both biomedical and clinical applications.

Visualization of individual microRNA molecules in fixed cells and tissues using target-primed padlock probe assay.

MicroRNAs (miRNAs) are key regulators of gene expression at the posttranscriptional level. Precisely profiling of miRNA expression will help us to better understand their roles in normal and diseased cells and tissues. Here we describe in situ miRNA detection by padlock probing and miRNA target-primed rolling circle amplification. We optimized our protocol and showed it can be applied to both fixed cells and tissue sections. The method can be used in basic research and potentially in clinical diagnostics in the future.