Journal: Nature Communications

Article Title: Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq

doi: 10.1038/s41467-024-47290-9

Figure Lengend Snippet: A A UMAP projection of 8226 single cells with five cell lineages (colors as indicated). Each cell is colored by its lineage assignments. B Histograms (y-axis; number of genes) of trajectory-specific maternal (red, top) and zygotic (blue, bottom) significantly differentially regulated genes, per trajectory (x-axis, color-coded as in ( A )). Each set of trajectory-specific genes is divided into up-regulated (right) and down-regulated (left) genes. Some genes are differentially regulated in more than one trajectory. C – E Trajectory-specific model fits (solid lines) to interpolated zygotic (right) trajectory (light blue dots) or non-trajectory (dark blue dots) and maternal (left) trajectory (light red dots) or non-trajectory (dark red dots) expression levels (y-axis, log 2 scale) across 11 pseudotime bins (x-axis) for genes with trajectory specific regulation. Expression levels were interpolated separately for cells that are assigned to a trajectory or those not assigned to it across 11 pseudotime bins. Gene name and trajectory are indicated on top. Gray lines represent fits that match both trajectory and non-trajectory data, and therefore retain the null hypothesis of similar regulation within and outside a trajectory. F Left: hybridization chain reaction (HCR) in situs against dendra mRNA with “neutral“ UTRs, mOxBFP mRNA with “neutral UTRs”, and krt8 that marks enveloping layer (EVL) cells. Right: diagrams of injected reporter mRNAs. G Quantification of ratio of mOxBFP to dendra HCR fluorescence in the enveloping layer (EVL) and deep layer (rest of the blastoderm) in “neutral” UTR reporter injections from n = 6 independent embryos. Box plots show median (line), 1st and 3rd quartiles (hinges), and 1.5 inter-quartile ranges (whiskers). Significance was calculated using a two-sided Student’s t test. Each sample was measured twice in the two different regions. H , I Similar to ( F , G ), except using mOxBFP mRNA with epcam UTRs in n = 12 independent embryos.

Article Snippet: Embryos were then pre-hybridized in HCR probe hybridization buffer (Molecular Instruments) for 2 h at 37 °C with shaking at 300 rpm in a ThermoMixer C. To prepare probe working solution, 1 μL of each 1 μM HCR probe was diluted in 500 μL of probe hybridization buffer at 37 °C.

Techniques: Expressing, Hybridization, Injection, Fluorescence

Journal: PLoS Genetics

Article Title: CREB mediates the C . elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-β expression

doi: 10.1371/journal.pgen.1009678

Figure Lengend Snippet: (A) Representative images of L1 larval wild-type or crh-1 mutant animals expressing daf-7 p:: gfp transgene in the presence (+) or absence (-) of ascr#5. Shown is GFP expression or daf-7 smFISH signal in the ASI neurons. All images for wild-type versus crh-1 mutant animals were taken with identical camera settings, including the same exposure time. Anterior is to the left. Scale bar: 50 μm. (B) The relative level of daf-7 p:: gfp fluorescence in the ASI neurons of L1 larval animals of the indicated genotypes in the absence (-) of ascr#5. (C) Scatter plot of fluorescence intensity of daf-7 smFISH signal in the ASI neurons of wild-type or crh-1 mutant animals in the presence (+) or absence (-) of ascr#5. The median is indicated by a horizontal line. Each dot is the fluorescence intensity of a single neuron; n ≥ 30 neurons total each, at least two independent experiments. (D) Representative images of L1 larval wild-type or crh-1 mutant animals expressing str-3 p:: gfp in the ASI neurons. Scale bar: 10 μm. (E) The relative level of str-3 p:: gfp fluorescence in the ASI neurons of L1 larval animals of the indicated genotypes in the absence (-) of ascr#5. n ≥ 30 for each. Error bars indicate SEM. ***, ††† , or ### indicates different from wild-type, absence of ascr#5, or crh-1 mutants at p < 0.001, respectively (student t-test). Arrowheads indicate the ASI neurons (A and D) .

Article Snippet: Then, a hybridization buffer containing the daf-7 probe (125 nM, Biosearch) was added and incubated overnight at 30°C.

Techniques: Mutagenesis, Expressing, Fluorescence

Journal: PLoS Genetics

Article Title: CREB mediates the C . elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-β expression

doi: 10.1371/journal.pgen.1009678

Figure Lengend Snippet: (A) Percent of L2d formed by animals of the indicated genotypes when grown in the presence of live OP50 food and ascr#5 pheromone. N ≥ 4 for each. (B) Representative images of wild-type (left column), crh-1 mutant (middle columns), or daf-7 mutant animals expressing of flp-8 p:: gfp in a subset of touch receptor neurons in the presence of live OP50 food and ascr#5 pheromone. The color-matched boxed regions of the second column each are shown at higher magnification. hAH, hour after hatching. Scale bars: 50 μm. Images of wild-type and crh-1 mutants are originated from . (C) Percent of flp-8 p:: gfp expression in the AVM neurons by animals of the indicated genotypes when grown in the presence of live OP50 food and ascr#5 pheromone. n ≥ 30 for each. *** indicates different from wild-type at p < 0.001(student t-test). (C-E) Percent of L2d formed by animals of the indicated genotypes when grown in the presence of live OP50 food and ascr#5 pheromone. N ≥ 8 (C) , 6 (D) , 4 (E) for each. Error bars represent the SEM. ### or ††† indicates different from crh-1 or daf-7 mutants at p < 0.001, respectively (student t-test).

Article Snippet: Then, a hybridization buffer containing the daf-7 probe (125 nM, Biosearch) was added and incubated overnight at 30°C.

Techniques: Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: CREB mediates the C . elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-β expression

doi: 10.1371/journal.pgen.1009678

Figure Lengend Snippet: (A) Shown are the conserved mammalian CRE motif and the newly identified CRE motif in the daf-7 promoter. (B) The percentage of transgenic animals expressing daf-7 p:: gfp reporter construct in the ASI neurons is shown. GFP fluorescence was observed either in wild-type or crh-1 mutant animals. The strength of GFP expression is indicated by the number of + symbols. Wild-type nucleotides are indicated in red, mutated nucleotides in blue. An arrowhead indicates daf-7 p-mutated CRE promoter. At least two independent extrachromosomal lines for each construct were examined. n ≥ 30 for each. (C) Representative images of L1 larval wild-type or crh-1 mutant animals expressing daf-7 p:: gfp or daf-7 p-mutated CRE:: gfp in the ASI neurons. Scale bar: 50 μm. (D) Scatter plot of fluorescence intensity of daf-7 p:: gfp fluorescence in the ASI neurons of adult wild-type or crh-1 mutant animals with (+) or without (-) of CRE site mutation. The median is indicated by a horizontal line. Each dot is the fluorescence intensity of a single neuron; n ≥ 30 neurons total each, at least two independent experiments. Error bars represent the SEM. $ $ $ indicates different at p < 0.001, respectively (student t-test). (E) Nuclear extracts isolated from control (2 nd lane) or GFP- crh-1 (3 rd , 4 th , 5 th lane) transfected cells were analyzed by EMSA using biotinylated daf-7 p-CRE probes (2 nd , 3 rd , 4 th , 5 th lane) with non-biotinylated daf-7 p-WT-CRE (4 th lane) and daf-7 p-Mut-CRE (5 th lane) competitors as indicated. An arrowhead indicates a complex of CRE probes and CRH-1.

Article Snippet: Then, a hybridization buffer containing the daf-7 probe (125 nM, Biosearch) was added and incubated overnight at 30°C.

Techniques: Transgenic Assay, Expressing, Construct, Fluorescence, Mutagenesis, Isolation, Transfection

Journal: PLoS Genetics

Article Title: CREB mediates the C . elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-β expression

doi: 10.1371/journal.pgen.1009678

Figure Lengend Snippet: CRH-1 positively regulates dauer-inhibiting daf-7 TGF-β expression in the ascr#5-sensing ASI neurons. In crh-1 mutants, the decreased daf-7 TGF-β expression provides a sensitized genetic background for further ascr#5-mediated daf-7 TGF-β repression via srg-36/37 GPCRs which leads to transient L2d formation and flp-8 expression of AAP-TRN.

Article Snippet: Then, a hybridization buffer containing the daf-7 probe (125 nM, Biosearch) was added and incubated overnight at 30°C.

Techniques: Expressing