A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.

WIlsON: Web-based Interactive Omics VisualizatioN.

MOTIVATION: High throughput (HT) screens in the omics field are typically analyzed by automated pipelines that generate static visualizations and comprehensive spreadsheet data for scientists. However, exploratory and hypothesis driven data analysis are key aspects of the understanding of biological systems, both generating extensive need for customized and dynamic visualization. RESULTS: Here we describe WIlsON, an interactive workbench for analysis and visualization of multi-omics data. It is primarily intended to empower screening platforms to offer access to pre-calculated HT screen results to the non-computational scientist. Facilitated by an open file format, WIlsON supports all types of omics screens, serves results via a web-based dashboard, and enables end users to perform analyses and generate publication-ready plots. AVAILABILITY AND IMPLEMENTATION: We implemented WIlsON in R with a focus on extensibility using the modular Shiny and Plotly frameworks. A demo of the interactive workbench without limitations may be accessed at http://loosolab.mpi-bn.mpg.de. A standalone Docker container as well as the source code of WIlsON are freely available from our Docker hub https://hub.docker. com/r/loosolab/wilson, CRAN https://cran.r-project.org/web/packages/wilson/, and GitHub repository https://github.molgen.mpg.de/loosolab/wilson-apps, respectively.

Mono- and multi-nucleated ventricular cardiomyocytes constitute a transcriptionally homogenous cell population.

Individual adult ventricular cardiomyocytes are either mono- or multi-nucleated and undergo morphological changes during cardiac hypertrophy. However, corresponding transcriptional signatures, reflecting potentially different functions or the ability for cell-cycle entry, are not known. The aim of this study was to determine the transcriptional profile of mono- and multi-nucleated adult cardiomyocytes by single-cell RNA-sequencing (scRNA-seq) and to investigate heterogeneity among cardiomyocytes under baseline conditions and in pressure-induced cardiac hypertrophy. We developed an array-based approach for scRNA-seq of rod-shaped multi-nucleated cardiomyocytes from both healthy and hypertrophic hearts. Single-cell transcriptomes of mono- or multi-nucleated cardiomyocytes were highly similar, although a certain degree of variation was noted across both populations. Non-image-based quality control allowing inclusion of damaged cardiomyocytes generated artificial cell clusters demonstrating the need for strict exclusion criteria. In contrast, cardiomyocytes isolated from hypertrophic heart after transverse aortic constriction showed heterogeneous transcriptional signatures, characteristic for hypoxia-induced responses. Immunofluorescence analysis revealed an inverse correlation between HIF1α+ cells and CD31-stained vessels, suggesting that imbalanced vascular growth in the hypertrophied heart induces cellular heterogeneity. Our study demonstrates that individual mono- and multi-nucleated cardiomyocytes express nearly identical sets of genes. Homogeneity among cardiomyocytes was lost after induction of hypertrophy due to differential HIF1α-dependent responses most likely caused by none-homogenous vessel growth.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function.

BACKGROUND: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. METHODS: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. RESULTS: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. CONCLUSION: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

LimiTT: link miRNAs to targets.

BACKGROUND: MicroRNAs (miRNAs) impact various biological processes within animals and plants. They complementarily bind target mRNAs, effecting a post-transcriptional negative regulation on mRNA level. The investigation of miRNA target interactions (MTIs) by high throughput screenings is challenging, as frequently used in silico target prediction tools are prone to emit false positives. This issue is aggravated for niche model organisms, where validated miRNAs and MTIs both have to be transferred from well described model organisms. Even though DBs exist that contain experimentally validated MTIs, they are limited in their search options and they utilize different miRNA and target identifiers. RESULTS: The implemented pipeline LimiTT integrates four existing DBs containing experimentally validated MTIs. In contrast to other cumulative databases (DBs), LimiTT includes MTI data of 26 species. Additionally, the pipeline enables the identification and enrichment analysis of MTIs with and without species specificity based on dynamic quality criteria. Multiple tabular and graphical outputs are generated to permit the detailed assessment of results. CONCLUSION: Our freely available web-based pipeline LimiTT ( https://bioinformatics.mpi-bn.mpg.de/ ) is optimized to determine MTIs with and without species specification. It links miRNAs and/or putative targets with high granularity. The integrated mapping to homologous target identifiers enables the identification of MTIs not only for standard models, but for niche model organisms as well.

MIRPIPE: quantification of microRNAs in niche model organisms.

UNLABELLED: MicroRNAs (miRNAs) represent an important class of small non-coding RNAs regulating gene expression in eukaryotes. Present algorithms typically rely on genomic data to identify miRNAs and require extensive installation procedures. Niche model organisms lacking genomic sequences cannot be analyzed by such tools. Here we introduce the MIRPIPE application enabling rapid and simple browser-based miRNA homology detection and quantification. MIRPIPE features automatic trimming of raw RNA-Seq reads originating from various sequencing instruments, processing of isomiRs and quantification of detected miRNAs versus public- or user-uploaded reference databases. AVAILABILITY AND IMPLEMENTATION: The Web service is freely available at http://bioinformatics.mpi-bn.mpg.de. MIRPIPE was implemented in Perl and integrated into Galaxy. An offline version for local execution is also available from our Web site.

Proteomic and transcriptomic characterisation of FIA10, a novel murine leukemic cell line that metastasizes into the brain.

Brain metastasis leads to increased mortality and is a major site of relapse for several cancers, yet the molecular mechanisms of brain metastasis are not well understood. In this study, we established and characterized a new leukemic cell line, FIA10, that metastasizes into the central nervous system (CNS) following injection into the tail vein of syngeneic mice. Mice injected with FIA10 cells developed neurological symptoms such as loss of balance, tremor, ataxic gait and seizures, leading to death within 3 months. Histopathology coupled with PCR analysis clearly showed infiltration of leukemic FIA10 cells into the brain parenchyma of diseased mice, with little involvement of bone marrow, peripheral blood and other organs. To define pathways that contribute to CNS metastasis, global transcriptome and proteome analysis was performed on FIA10 cells and compared with that of the parental stem cell line FDCP-Mix and the related FIA18 cells, which give rise to myeloid leukemia without CNS involvement. 188 expressed genes (RNA level) and 189 proteins were upregulated (log2 ratio FIA10/FIA18 ≥ 1) and 120 mRNAs and 177 proteins were downregulated (log2 ratio FIA10/FIA18 ≤ 1) in FIA10 cells compared with FIA18 cells. Major upregulated pathways in FIA10 cells revealed by biofunctional analyses involved immune response components, adhesion molecules and enzymes implicated in extracellular matrix remodeling, opening and crossing the blood-brain barrier (BBB), molecules supporting migration within the brain parenchyma, alterations in metabolism necessary for growth within the brain microenvironment, and regulators for these functions. Downregulated RNA and protein included several tumor suppressors and DNA repair enzymes. In line with the function of FIA10 cells to specifically infiltrate the brain, FIA10 cells have acquired a phenotype that permits crossing the BBB and adapting to the brain microenvironment thereby escaping immune surveillance. These data and our model system FIA10 will be valuable resources to study the occurrence of brain metastases and may help in the development of potential therapies against brain invasion.

Antigen presentation plays positive roles in the regenerative response to cardiac injury in zebrafish.

In contrast to adult mammals, adult zebrafish can fully regenerate injured cardiac tissue, and this regeneration process requires an adequate and tightly controlled immune response. However, which components of the immune response are required during regeneration is unclear. Here, we report positive roles for the antigen presentation-adaptive immunity axis during zebrafish cardiac regeneration. We find that following the initial innate immune response, activated endocardial cells (EdCs), as well as immune cells, start expressing antigen presentation genes. We also observe that T helper cells, a.k.a. Cd4+ T cells, lie in close physical proximity to these antigen-presenting EdCs. We targeted Major Histocompatibility Complex (MHC) class II antigen presentation by generating cd74a; cd74b mutants, which display a defective immune response. In these mutants, Cd4+ T cells and activated EdCs fail to efficiently populate the injured tissue and EdC proliferation is significantly decreased. cd74a; cd74b mutants exhibit additional defects in cardiac regeneration including reduced cardiomyocyte dedifferentiation and proliferation. Notably, Cd74 also becomes activated in neonatal mouse EdCs following cardiac injury. Altogether, these findings point to positive roles for antigen presentation during cardiac regeneration, potentially involving interactions between activated EdCs, classical antigen-presenting cells, and Cd4+ T cells.

i2dash: Creation of Flexible, Interactive, and Web-based Dashboards for Visualization of Omics Data.

Data visualization and interactive data exploration are important aspects of illustrating complex concepts and results from analyses of omics data. A suitable visualization has to be intuitive and accessible. Web-based dashboards have become popular tools for the arrangement, consolidation, and display of such visualizations. However, the combination of automated data processing pipelines handling omics data and dynamically generated, interactive dashboards is poorly solved. Here, we present i2dash, an R package intended to encapsulate functionality for the programmatic creation of customized dashboards. It supports interactive and responsive (linked) visualizations across a set of predefined graphical layouts. i2dash addresses the needs of data analysts/software developers for a tool that is compatible and attachable to any R-based analysis pipeline, thereby fostering the separation of data visualization on one hand and data analysis tasks on the other hand. In addition, the generic design of i2dash enables the development of modular extensions for specific needs. As a proof of principle, we provide an extension of i2dash optimized for single-cell RNA sequencing analysis, supporting the creation of dashboards for the visualization needs of such experiments. Equipped with these features, i2dash is suitable for extensive use in large-scale sequencing/bioinformatics facilities. Along this line, we provide i2dash as a containerized solution, enabling a straightforward large-scale deployment and sharing of dashboards using cloud services. i2dash is freely available via the R package archive CRAN (https://CRAN.R-project.org/package=i2dash).

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex.

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

Interleukin-11 signaling promotes cellular reprogramming and limits fibrotic scarring during tissue regeneration.

Damage-induced fibrotic scarring limits tissue regeneration in mammals and is a leading cause of morbidity. In contrast, species like zebrafish can regenerate damaged tissues without excessive fibrosis. However, whether specific signaling pathways can both limit fibrosis and promote regeneration is unclear. Here, we show that interleukin-11 (Il-11)/Stat3 signaling has such a dual function. Zebrafish lacking Il-11 receptor function display severely compromised heart, fin, and scale regeneration. Deep phenotyping and transcriptional analysis of adult hearts and fins show that Il-11 signaling drives cellular reprogramming to orchestrate global and tissue-specific regenerative programs and broadly antagonizes hallmarks of adult mammalian scarring. Mechanistically, our data indicate that IL-11 signaling in endothelial cells antagonizes profibrotic transforming growth factor­ß signaling and endothelial-to-mesenchymal transition, limiting scarring and promoting cardiomyocyte repopulation, after injury. Overall, our findings position damage-induced Il-11/Stat3 signaling in a key role limiting fibrosis and promoting regeneration, revealing novel targets for regenerative therapies.

multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets.

Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set-specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.

ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation.

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.

Connect-four: genomic analyses of regenerating stem cells identifies zygotic Dux factors as tumor initiators.

How, if and in which cell types embryonic gene expression programs are elicited to induce tumor formation remains poorly understood. Through genomic analyses of regenerating, p53 deficient muscle stem cells we identified various oncogenomic amplifications, including but not limited to, the zygotic transcription factor Duxbl/DUXB to initiate tumorigenic transformation.

The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.

Histone3-lysine9 (H3K9) residues not only control gene expression, but also contribute to RNA splicing. Here, the H3K9 histone demethylase PHF8 was investigated in endothelial cells for its involvement in alternative splicing. An angiogenic sprouting assay shows the importance of PHF8 for endothelial cells. Immunoprecipitation reveals that PHF8 interacts with U1 spliceosomal proteins, such as SRPK1 and snRNP70. We identify the histocompatibility antigen HLA-G as a target of PHF8. The inclusion of HLA-G intron 4, with concomitant RNA Polymerase II accumulation at this intron is controlled by PHF8 and H3K9. Soluble HLA-G is generated after PHF8 knockdown, which leads to reduced T-cell proliferation. Collectively, PHF8 knockdown generates the immunosuppressive alternative splice product soluble HLA-G, which is secreted by endothelial cells to elicit a potential inhibitory effect on inflammation.

20-HETE promotes glucose-stimulated insulin secretion in an autocrine manner through FFAR1.

The long-chain fatty acid receptor FFAR1 is highly expressed in pancreatic ß-cells. Synthetic FFAR1 agonists can be used as antidiabetic drugs to promote glucose-stimulated insulin secretion (GSIS). However, the physiological role of FFAR1 in ß-cells remains poorly understood. Here we show that 20-HETE activates FFAR1 and promotes GSIS via FFAR1 with higher potency and efficacy than dietary fatty acids such as palmitic, linoleic, and α-linolenic acid. Murine and human ß-cells produce 20-HETE, and the ω-hydroxylase-mediated formation and release of 20-HETE is strongly stimulated by glucose. Pharmacological inhibition of 20-HETE formation and blockade of FFAR1 in islets inhibits GSIS. In islets from type-2 diabetic humans and mice, glucose-stimulated 20-HETE formation and 20-HETE-dependent stimulation of GSIS are strongly reduced. We show that 20-HETE is an FFAR1 agonist, which functions as an autocrine positive feed-forward regulator of GSIS, and that a reduced glucose-induced 20-HETE formation contributes to inefficient GSIS in type-2 diabetes.

Oncogenic Amplification of Zygotic Dux Factors in Regenerating p53-Deficient Muscle Stem Cells Defines a Molecular Cancer Subtype.

The identity of tumor-initiating cells in many cancer types is unknown. Tumors often express genes associated with embryonic development, although the contributions of zygotic programs to tumor initiation and formation are poorly understood. Here, we show that regeneration-induced loss of quiescence in p53-deficient muscle stem cells (MuSCs) results in rhabdomyosarcoma formation with 100% penetrance. Genomic analyses of purified tumor cells revealed spontaneous and discrete oncogenic amplifications in MuSCs that drive tumorigenesis, including, but not limited to, the amplification of the cleavage-stage Dux transcription factor (TF) Duxbl. We further found that Dux factors drive an early embryonic gene signature that defines a molecular subtype across a broad range of human cancers. Duxbl initiates tumorigenesis by enforcing a mesenchymal-to-epithelial transition, and targeted inactivation of Duxbl specifically in Duxbl-expressing tumor cells abolishes their expansion. These findings reveal how regeneration and genomic instability can interact to activate zygotic genes that drive tumor initiation and growth.

Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement.

Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.

The potassium channel KCNJ13 is essential for smooth muscle cytoskeletal organization during mouse tracheal tubulogenesis.

Tubulogenesis is essential for the formation and function of internal organs. One such organ is the trachea, which allows gas exchange between the external environment and the lungs. However, the cellular and molecular mechanisms underlying tracheal tube development remain poorly understood. Here, we show that the potassium channel KCNJ13 is a critical modulator of tracheal tubulogenesis. We identify Kcnj13 in an ethylnitrosourea forward genetic screen for regulators of mouse respiratory organ development. Kcnj13 mutants exhibit a shorter trachea as well as defective smooth muscle (SM) cell alignment and polarity. KCNJ13 is essential to maintain ion homeostasis in tracheal SM cells, which is required for actin polymerization. This process appears to be mediated, at least in part, through activation of the actin regulator AKT, as pharmacological increase of AKT phosphorylation ameliorates the Kcnj13-mutant trachea phenotypes. These results provide insight into the role of ion homeostasis in cytoskeletal organization during tubulogenesis.

Myh10 deficiency leads to defective extracellular matrix remodeling and pulmonary disease.

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.