The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic ...target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.
Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA ...staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.
Maternal gene products drive early development when the newly formed embryo is transcriptionally inactive. During the maternal-zygotic transition, embryonic transcription is initiated and many ...maternal RNAs are degraded. Multiple mechanisms regulate the birth of zygotic RNAs and the death of maternal RNAs. Genome activation appears to rely in part on the sequestration of transcriptional repressors by the exponentially increasing amount of DNA during cleavage divisions. Maternal RNA degradation is induced by the binding of proteins and microRNAs to the 3' untranslated region of target RNAs.
Morphogens are long-range signaling molecules that pattern developing tissues in a concentration-dependent manner. The graded activity of morphogens within tissues exposes cells to different signal ...levels and leads to region-specific transcriptional responses and cell fates. In its simplest incarnation, a morphogen signal forms a gradient by diffusion from a local source and clearance in surrounding tissues. Responding cells often transduce morphogen levels in a linear fashion, which results in the graded activation of transcriptional effectors. The concentration-dependent expression of morphogen target genes is achieved by their different binding affinities for transcriptional effectors as well as inputs from other transcriptional regulators. Morphogen distribution and interpretation are the result of complex interactions between the morphogen and responding tissues. The response to a morphogen is dependent not simply on morphogen concentration but also on the duration of morphogen exposure and the state of the target cells. In this review, we describe the morphogen concept and discuss the mechanisms that underlie the generation, modulation, and interpretation of morphogen gradients.
During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 ...cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branch points contained cells that express genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework with which to reconstruct complex developmental trees from single-cell transcriptomes.
The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell ...types. We combined single-cell RNA-seq (scRNA-seq) with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ∼13,000 habenular cells with 4× cellular coverage identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a reference atlas created a resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions.
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•Establishment of a single-cell RNA-seq protocol for neurons in zebrafish•Identification of 18 distinct habenular types•Retention of neuronal types between larva and adult•RNA-FISH and image registration to build a gene expression atlas
Pandey et al. use scRNA-seq to define more than a dozen different neuronal types in the zebrafish habenula. Cell types are retained between larva and adult.
Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ...ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternal-zygotic transition, the maintenance of pluripotency, the patterning of the body axes, the specification and differentiation of cell types and the morphogenesis of organs. Drawing from several animal model systems, we describe two emerging themes for ncRNA function: promoting developmental transitions and maintaining developmental states. These examples also highlight the roles of ncRNAs in ensuring a robust commitment to one of two possible cell fates.
Multicellular systems develop from single cells through distinct lineages. However, current lineage-tracing approaches scale poorly to whole, complex organisms. Here, we use genome editing to ...progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease.
Nodal morphogens Schier, Alexander F
Cold Spring Harbor perspectives in biology,
11/2009, Volume:
1, Issue:
5
Journal Article
Peer reviewed
Open access
Nodal signals belong to the TGF-beta superfamily and are essential for the induction of mesoderm and endoderm and the determination of the left-right axis. Nodal signals can act as morphogens-they ...have concentration-dependent effects and can act at a distance from their source of production. Nodal and its feedback inhibitor Lefty form an activator/inhibitor pair that behaves similarly to postulated reaction-diffusion models of tissue patterning. Nodal morphogen activity is also regulated by microRNAs, convertases, TGF-beta signals, coreceptors, and trafficking factors. This article describes how Nodal morphogens pattern embryonic fields and discusses how Nodal morphogen signaling is modulated.
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