Jan Philipp Junker

Every cell is special: genome-wide studies add a new dimension to single-cell biology

Single-cell analyses have provided invaluable insights into studying heterogenity, signaling, and stochastic gene expression. Recent technological advances now open the door to genome-wide single-cell studies.

Genome-wide RNA Tomography in the Zebrafish Embryo

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Single-molecule mRNA detection and counting in mammalian tissue

We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis.

Identification of Molecular Compartments and Genetic Circuitry in the Developing Mammalian Kidney

Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.

Spatially Resolved Genome-wide Transcriptional Profiling Identifies BMP Signaling as Essential Regulator of Zebrafish Cardiomyocyte Regeneration

In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located near the wound border. To identify regulators of cardiomyocyte proliferation, we used spatially resolved RNA sequencing (tomo-seq) and generated a high-resolution genome-wide atlas of gene expression in the regenerating zebrafish heart. Interestingly, we identified two wound border zones with distinct expression profiles, including the re-expression of embryonic cardiac genes and targets of bone morphogenetic protein (BMP) signaling. Endogenous BMP signaling has been reported to be detrimental to mammalian cardiac repair. In contrast, we find that genetic or chemical inhibition of BMP signaling in zebrafish reduces cardiomyocyte dedifferentiation and proliferation, ultimately compromising myocardial regeneration, while bmp2b overexpression is sufficient to enhance it. Our results provide a resource for further studies on the molecular regulation of cardiac regeneration and reveal intriguing differential cellular responses of cardiomyocytes to a conserved signaling pathway in regenerative versus non-regenerative hearts.

Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars

A key goal of developmental biology is to understand how a single cell is transformed into a full-grown organism comprising many different cell types. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the developmental origin of cells remains challenging. Here we present LINNAEUS (lineage tracing by nuclease-activated editing of ubiquitous sequences)—a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes, generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae, and in heart, liver, pancreas, and telencephalon of adult fish. LINNAEUS provides a systematic approach for tracing the origin of novel cell types, or known cell types under different conditions.

Single-Cell Transcriptomics Enters the Age of Mass Production

Two publications in the current issue of Cell introduce novel methods for high-throughput single-cell transcriptomics by using droplet microfluidics and sophisticated barcoding schemes for transcriptional profiling of thousands of individual cells.

DAZL regulates Tet1 translation in murine embryonic stem cells

Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so‐called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA‐binding protein known to play a key role in germ‐cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5‐hydroxylation of methyl‐cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i‐mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1‐mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.

Tomo-Seq Identifies SOX9 as a Key Regulator of Cardiac Fibrosis During Ischemic Injury

Background: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. Methods: Although genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. Results: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, such as fibrosis, cardiomyocyte hypertrophy, and calcium handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the coregulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated that the majority of COL1-positive fibroblasts stem from a pool of SOX9-expressing cells, and in vivo loss of Sox9 blunted the cardiac fibrotic response on ischemic injury. The colocalization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. Conclusions: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq, we were able to unveil the unknown relevance of SOX9 as a key regulator of cardiac fibrosis, pointing to SOX9 as a potential therapeutic target for cardiac fibrosis.

Methods for lineage tracing on the organism-wide level

Determining the lineage origin of cell types is a major goal in developmental biology. Furthermore, lineage tracing is a powerful approach for understanding the origin of developmental defects as well as the origin of diseases such as cancer. There is now a variety of complementary approaches for identifying lineage relationships, ranging from direct observation of cell divisions by light microscopy to genetic labeling of cells using inducible recombinases and fluorescent reporters. A recent development, and the main topic of this review article, is the use of high-throughput sequencing data for lineage analysis. This emerging approach holds the promise of increased multiplexing capacity, allowing lineage analysis of large cell numbers up to the organism-wide level combined with simultaneous transcription profiling by single cell RNA sequencing.