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

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 types 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 retina and early 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.This dataset contains the data, code, and scripts to reproduce the results in the manuscript, "Lineage motifs: developmental modules for control of cell type proportions." Within this resource, data and scripts are organized by figure. All code is written in Python, with analysis scripts provided as Jupyter notebooks

Lineage motifs: developmental modules for control of cell type proportions (post-revision)

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 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 or extrinsic interactions. Applying LMA to published datasets reveals spatial and temporal organization of cell fate commitment in retina and early 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.This dataset contains the data, code, and scripts to reproduce the results in the manuscript, "Lineage motifs: developmental modules for control of cell type proportions." Within this resource, data and scripts are organized by figure. All code is written in Python, with analysis scripts provided as Jupyter notebooks

Modeling of the RAG Reaction Mechanism

SummaryIn 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

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

Data for "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription "

This dataset provides the raw and analyzed data as well as the code to recreate results reported in the paper "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription". The dataset is organized based on the figures and figure panels of the paper, with the codes provided as Matlab and R scripts. Please contact the corresponding author for any questions or comments regarding the data, the code, the methods, or the reagents used here.Related Publication:</p> In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription</p> Askary, Amjad Caltech</p> Nature Biotechnology</p> https://doi.org/10.1038/s41587-019-0299-4</p>en

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

<div><p>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 <i>barx1</i>, as well as <i>in vivo</i> 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 <i>barx1</i> expression in the dorsal arches, with mutation of <i>barx1</i> rescuing some aspects of dorsal skeletal patterning in <i>jag1b</i> mutants. We also identified <i>prrx1a</i> and <i>prrx1b</i> as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal <i>barx1</i>+ pre-cartilage condensations. Simultaneous loss of <i>jag1b</i> and <i>prrx1a/b</i> better rescued lower facial defects of <i>edn1</i> 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 <i>edn1</i> 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.</p></div

Accelerated cartilage differentiation in ventral-intermediate arch NCCs.

<p>(A) Schematic of pharyngeal arch patterning in zebrafish. At early patterning stages (~28 hpf), the first two pharyngeal arches (pa1, pa2) are divided into distinct dorsal (blue) and ventral/intermediate (green stripe) domains, with the latter resolving into intermediate (light green) and ventral (dark green) domains by 36 hpf. Notch activity governs the dorsal domain, Edn1 the intermediate domain, and Bmp signaling the ventral domain. The anterior maxillary domain (grey) is not significantly influenced by any of these pathways. The facial cartilages of the larval skeleton (5 dpf) are color-coded based on their arch origins. Hm, hyomandibula; Pq, palatoquadrate; M, Meckel’s; Sy, symplectic; Ch, ceratohyal. (B) <i>barx1</i> (green) is upregulated ventrally (≤ 26 hpf, white open arrowhead) well before dorsal second arch expression can be detected (~32 hpf, white arrowhead). NCCs express the <i>sox10</i>:<i>GFP</i> transgene (blue). Shown are maximum intensity projections of confocal z-stacks of single-color <i>in situs</i> co-stained with a GFP antibody. The orientation of the dorsal (D)-ventral (V) axis is indicated. (C) Stills from a time-lapse movie (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005967#pgen.1005967.s016" target="_blank">S1 Movie</a>) show the emergence of facial cartilages (<i>sox10</i>:<i>DsRed</i>+, magenta) from <i>fli1a</i>:<i>EGFP</i>+ ectomesenchyme (green). <i>sox10</i>:<i>DsRed</i>+ chondrocytes appear in a stereotyped sequence within the facial cartilages, with cells of the intermediate Sy and Pq cartilages detectable first at 56 hpf, followed by the ventral M and Ch cartilages at 60 hpf and the dorsal Hm at 65 hpf. (D) The same sequence of cartilage differentiation is seen slightly earlier in stills from a time-lapse movie of <i>col2a1a</i>_<i>BAC</i>:GFP fish (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005967#pgen.1005967.s017" target="_blank">S2 Movie</a>). The time-lapses in B and C were performed with a 20x objective using 0.5x digital magnification. Et, ethmoid cartilage. (E) Color-coded schematic of the sequence of chondrocyte differentiation in the facial skeleton. The orientations of the D-V and anterior (A)-posterior (P) axes are indicated. Scale bar in B = 20 μm; scale bars in C, D = 100 μm.</p

Partially overlapping functions of Prrx1a/b and Jagged-Notch signaling in dorsal cartilage development.

<p>(A-F) The expression of <i>prrx1a</i> and <i>prrx1b</i> (magenta) is largely normal in <i>jag1b</i> mutants (B, D) but is upregulated in ventral arch NCCs (white arrows) and reduced in dorsal NCCs upon forced Notch activation in <i>hsp70I</i>:<i>Gal4</i>; <i>UAS</i>:<i>NICD</i> embryos subjected to a 20–24 hpf heat-shock treatment. <i>dlx2a</i> expression (green) marks all arch NCCs. (G-I) <i>edn1</i> mutants display a loss of ventral <i>barx1</i> expression (green) and gain of <i>prrx1b</i> (magenta) (white arrowhead) (H). In <i>jag1b</i>; <i>edn1</i> mutants, there is partial recovery of ventral <i>barx1</i> expression in the second arch (white open arrowhead), which corresponds to regions where the ectopic expression of <i>prrx1b</i> is restored to control levels. (J, K) In 7/10 <i>prrx1a</i>; <i>prrx1b</i> mutants, <i>jag1b</i> expression is partially reduced in the dorsal second arch (yellow arrow). (L-O) Dissections of facial cartilage and bone derived from the first two arches show additive phenotypes in <i>jag1b</i>; <i>prrx1a</i>; <i>prrx1b</i> triple mutants. Similar to <i>prrx1a</i>; <i>prrx1b</i> double mutants, triple mutants display ectopic cartilage connecting Pq to the otic cartilage (black arrows). However, similar to <i>jag1b</i> single mutants, <i>jag1b</i>; <i>prrx1a</i>; <i>prrx1b</i> triple mutants also display irregularities in the main body of Pq (black arrowheads). Scale bar in K = 20 μm; scale bar in O = 100 μm.</p