osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face.

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways - Jagged-Notch, Endothelin1 (Edn1), and Bmp - regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face.

Spatial motifs reveal patterns in cellular architecture of complex tissues.

Spatial organization of cells is crucial to both proper physiological function of tissues and pathological conditions like cancer. Recent advances in spatial transcriptomics have enabled joint profiling of gene expression and spatial context of the cells. The outcome is an information rich map of the tissue where individual cells, or small regions, can be labeled based on their gene expression state. While spatial transcriptomics excels in its capacity to profile numerous genes within the same sample, most existing methods for analysis of spatial data only examine distribution of one or two labels at a time. These approaches overlook the potential for identifying higher-order associations between cell types - associations that can play a pivotal role in understanding development and function of complex tissues. In this context, we introduce a novel method for detecting motifs in spatial neighborhood graphs. Each motif represents a spatial arrangement of cell types that occurs in the tissue more frequently than expected by chance. To identify spatial motifs, we developed an algorithm for uniform sampling of paths from neighborhood graphs and combined it with a motif finding algorithm on graphs inspired by previous methods for finding motifs in DNA sequences. Using synthetic data with known ground truth, we show that our method can identify spatial motifs with high accuracy and sensitivity. Applied to spatial maps of mouse retinal bipolar cells and hypothalamic preoptic region, our method reveals previously unrecognized patterns in cell type arrangements. In some cases, cells within these spatial patterns differ in their gene expression from other cells of the same type, providing insights into the functional significance of the spatial motifs. These results suggest that our method can illuminate the substantial complexity of neural tissues, provide novel insight even in well studied models, and generate experimentally testable hypotheses.

Lineage motifs as developmental modules for control of cell type proportions.

In multicellular organisms, cell types must be produced and maintained in appropriate proportions. One way this is achieved is through committed progenitor cells or extrinsic interactions that produce specific patterns of descendant cell types on lineage trees. However, cell fate commitment is probabilistic in most contexts, making it difficult to infer these dynamics and understand how they establish overall cell type proportions. Here, we introduce Lineage Motif Analysis (LMA), a method that recursively identifies statistically overrepresented patterns of cell fates on lineage trees as potential signatures of committed progenitor states or extrinsic interactions. Applying LMA to published datasets reveals spatial and temporal organization of cell fate commitment in zebrafish and rat retina and early mouse embryonic development. Comparative analysis of vertebrate species suggests that lineage motifs facilitate adaptive evolutionary variation of retinal cell type proportions. LMA thus provides insight into complex developmental processes by decomposing them into simpler underlying modules.

Lineage motifs: developmental modules for control of cell type proportions.

In multicellular organisms, cell types must be produced and maintained in appropriate proportions. One way this is achieved is through committed progenitor cells that produce specific sets of descendant cell types. However, cell fate commitment is probabilistic in most contexts, making it difficult to infer progenitor states and understand how they establish overall cell type proportions. Here, we introduce Lineage Motif Analysis (LMA), a method that recursively identifies statistically overrepresented patterns of cell fates on lineage trees as potential signatures of committed progenitor states. Applying LMA to published datasets reveals spatial and temporal organization of cell fate commitment in zebrafish and rat retina and early mouse embryo development. Comparative analysis of vertebrate species suggests that lineage motifs facilitate adaptive evolutionary variation of retinal cell type proportions. LMA thus provides insight into complex developmental processes by decomposing them into simpler underlying modules.

Publisher Correction: In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription.

Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.

N4: a precise and highly sensitive promoter predictor using neural network fed by nearest neighbors.

Promoters, the genomic regions proximal to the transcriptional start sites (TSSs) play pivotal roles in determining the rate of transcription initiation by serving as direct docking platforms for the RNA polymerase II complex. In the post-genomic era, correct gene prediction has become one of the biggest challenges in genome annotation. Species-independent promoter prediction tools could also be useful in meta-genomics, since transcription data will not be available for micro-organisms which are not cultivated. Promoter prediction in prokaryotic genomes presents unique challenges owing to their organizational properties. Several methods have been developed to predict the promoter regions of genomes in prokaryotes, including algorithms for recognition of sequence motifs, artificial neural networks, and algorithms based on genome's structure. However, none of them satisfies both criteria of sensitivity and precision. In this work, we present a modified artificial neural network fed by nearest neighbors based on DNA duplex stability, named N4, which can predict the transcription start sites of Escherichia coli with sensitivity and precision both above 94%, better than most of the existed algorithms.

Building and maintaining joints by exquisite local control of cell fate.

We owe the flexibility of our bodies to sophisticated articulations between bones. Establishment of these joints requires the integration of multiple tissue types: permanent cartilage that cushions the articulating bones, synovial membranes that enclose a lubricating fluid-filled cavity, and a fibrous capsule and ligaments that provide structural support. Positioning the prospective joint region involves establishment of an "interzone" region of joint progenitor cells within a nascent cartilage condensation, which is achieved through the interplay of activators and inhibitors of multiple developmental signaling pathways. Within the interzone, tight regulation of BMP and TGFß signaling prevents the hypertrophic maturation of joint chondrocytes, in part through downstream transcriptional repressors and epigenetic modulators. Synovial cells then acquire further specializations through expression of genes that promote lubrication, as well as the formation of complex structures such as cavities and entheses. Whereas genetic investigations in mice and humans have uncovered a number of regulators of joint development and homeostasis, recent work in zebrafish offers a complementary reductionist approach toward understanding joint positioning and the regulation of chondrocyte fate at joints. The complexity of building and maintaining joints may help explain why there are still few treatments for osteoarthritis, one of the most common diseases in the human population. A major challenge will be to understand how developmental abnormalities in joint structure, as well as postnatal roles for developmental genes in joint homeostasis, contribute to birth defects and degenerative diseases of joints. WIREs Dev Biol 2017, 6:e245. doi: 10.1002/wdev.245 For further resources related to this article, please visit the WIREs website.

Ancient origin of lubricated joints in bony vertebrates.

Synovial joints are the lubricated connections between the bones of our body that are commonly affected in arthritis. It is assumed that synovial joints first evolved as vertebrates came to land, with ray-finned fishes lacking lubricated joints. Here, we examine the expression and function of a critical lubricating protein of mammalian synovial joints, Prg4/Lubricin, in diverse ray-finned fishes. We find that Prg4 homologs are specifically enriched at the jaw and pectoral fin joints of zebrafish, stickleback, and gar, with genetic deletion of the zebrafish prg4b gene resulting in the same age-related degeneration of joints as seen in lubricin-deficient mice and humans. Our data support lubricated synovial joints evolving much earlier than currently accepted, at least in the common ancestor of all bony vertebrates. Establishment of the first arthritis model in the highly regenerative zebrafish will offer unique opportunities to understand the aetiology and possible treatment of synovial joint disease.

Iroquois Proteins Promote Skeletal Joint Formation by Maintaining Chondrocytes in an Immature State.

An early event in skeletal joint development is the specification of articular chondrocytes at the joint surface. Articular chondrocytes are distinct in producing lower levels of cartilage matrix and not being replaced by bone, yet how they acquire these properties remains poorly understood. Here, we show that two members of the Iroquois transcriptional repressor family, Irx7 and Irx5a, function to block chondrocyte maturation at the developing hyoid joint of zebrafish. These Irx factors suppress the production of cartilage matrix at the joint in part by preventing the activation of a col2a1a enhancer by Sox9a. Further, both zebrafish Irx7 and mouse IRX1 are able to repress cartilage matrix production in a murine chondrogenic cell line. Iroquois proteins may therefore have a conserved role in keeping chondrocytes in an immature state, with the lower levels of cartilage matrix produced by these immature cells contributing to joint flexibility.

Modeling of the RAG reaction mechanism.

In vertebrate V(D)J recombination, it remains unclear how the RAG complex coordinates its catalytic steps with binding to two distant recombination sites. Here, we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation. The reaction schemes with the best fitting capability (1) strongly favor a RAG tetrameric complex over a RAG octameric complex; (2) indicate that once a RAG complex brings two recombination signal sequence (RSS) sites into synapsis, the synaptic complex rarely disassembles; (3) predict that the binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (4) show that the RAG binding properties permit strong distinction between RSS sites within active chromatin versus nonspecific DNA or RSS sites within inactive chromatin. The results provide general insights for synapsis by nuclear proteins as well as more specific testable predictions for the RAG proteins.

Mechanistic basis for RAG discrimination between recombination sites and the off-target sites of human lymphomas.

During V(D)J recombination, RAG targeting to correct sites versus off-target sites relies on both DNA sequence features and on chromatin marks. Kinetic analysis using the first highly active full-length purified RAG1/RAG2 complexes has now allowed us to define the important catalytic features of this complex. We found that the overall rate of nicking, but not hairpinning, is critical for the discrimination between correct (optimal) versus off-target (suboptimal) sites used in human T-cell lymphomas, and we show that the C-terminal portion of RAG2 is required for this. This type of kinetic analysis permits us to analyze only the catalytically active RAG complex, in contrast to all other methods, which are unavoidably confounded by mixture with inactive RAG complexes. Moreover, we can distinguish the two major features of any enzymatic catalysis: the binding constant (K(D)) and the catalytic turnover rate, k(cat). Beyond a minimal essential threshold of heptamer quality, further suboptimal heptamer deviations primarily reduce the catalytic rate constant k(cat) for nicking. Suboptimal nonamers reduce not only the binding of the RAG complex to the recombination site (K(D)) but also the catalytic rate constant, consistent with a tight interaction between the RAG complex and substrate during catalysis. These features explain many aspects of RAG physiology and pathophysiology.