Journal: eLife

Article Title: Dynamics of primitive streak regression controls the fate of neuromesodermal progenitors in the chicken embryo

doi: 10.7554/eLife.64819

Figure Lengend Snippet: ( A ) Schematic diagram showing the strategy used to decipher if the neuromesodermal progenitor (NMP) territory is a mix of monopotent cells (left) or composed of bipotent cells (right). Schemes show an example of a cell that has been marked by retroviral barcoding or genetic color coding and its expected outcome in the different cases (arrows). The color indicates the neural (red, N), the mesodermal (green, M), and the neuromesodermal (gold, NM) identities. ( B ) Experimental procedure showing the infected or electroporated region of the epiblast at stage 5HH (left, green) and the stage at which embryos were harvested for analysis (n = 3). ( C ) (left) Diagram showing the neural tube (red) and paraxial mesoderm (green) in the anterior (light) and posterior (dark) regions of the embryo. (Right) Pie graphs showing the distribution of the neural (red) and mesodermal (green) cells anterior (light) or posterior (dark) to the 27th somite in the seven clones identified by retrovirus labeling analyzed (n = 110 cells in three embryos). ( D ) Confocal z-section showing the region of a stage 17HH embryo shown in ( H ) and acquired using three separated laser paths to retrieve the color codes genetically encoded as described in . ( E ) Triplot diagrams showing the distribution of descendants of cells labeled with different Nucbow combinations in the anterior (top) and posterior (bottom) regions of seven clones in a representative stage 17HH embryo. Each symbol represents a cell identified based on the percentage of red, blue, and yellow expressed. The symbols are colored based on their clonal identity. Squares: neural cells; stars: mesodermal cells. ( F ) (left) Region analyzed showing the different axial levels. (Right) Axial distribution of the clones in three-stage 17HH embryos. Red bars: neural cells; green bars: mesodermal cells. ( G ) Quantification of the different clones: mesodermal (M, green), neural (N, red), and bipotent neuromesodermal clones (NM, gold) at stage 17HH (left) and stage 20HH (right) (n = 16 clones, 271 cells in three embryos) and (n = 40 clones, 519 cells in three embryos), respectively. ( H ) Experimental procedure showing the electroporated region of the epiblast at stage 5HH (left, green) and the stage at which embryos were harvested for analysis (n = 3). ( I ) Confocal z-section using three-color imaging corresponding to the posterior region of a stage 20HH embryo shown in ( H ). ( J ) Triplots showing the distribution of 10 representative clones in the anterior (left) and posterior (right) regions of a stage 20HH embryo electroporated at stage 5HH. Squares: neural cells; stars: mesodermal cells. ( K ) (left) Region analyzed showing the different axial levels. (Right) Axial distribution of the clones in three embryos. Green bars: mesodermal cells; red bars: neural cells, double line: anteroposterior axis. M: mesoderm; N: neural; NM: neuromesodermal; S: somite; HL: hindlimb; D: dorsal views. Anterior to the top. Scale bar: 100 µm. Figure 2—source data 1. Retrovirus and Brainbow labeling of chicken embryo. Figure 2—source data 2. Matlab code for clone identification.

Article Snippet: Software, algorithm , K mean Brainbow clustering , , MATLAB , .

Techniques: Retroviral, Infection, Clone Assay, Labeling, Imaging

Journal: eLife

Article Title: Dynamics of primitive streak regression controls the fate of neuromesodermal progenitors in the chicken embryo

doi: 10.7554/eLife.64819

Figure Lengend Snippet:

Article Snippet: Software, algorithm , K mean Brainbow clustering , , MATLAB , .

Techniques: Northern Blot, Transgenic Assay, Expressing, Recombinant, Software, Retroviral

Journal: Journal of neuroscience methods

Article Title: Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow

doi: 10.1016/j.jneumeth.2014.11.009

Figure Lengend Snippet: The Brainbow design, and recombination events that can support expression of each FP. (A) The Brainbow transcription unit, as constructed in E. coli, before Cre-mediated recombination; the initial order of the FP genes. The Brainbow transcription unit contains the VGLUT1 promoter (black segment with arrow), loxP sites (black triangles), and four FP genes (EmGFP (Tsien, 1998), green; mOrange2 (Shaner et al., 2008), yellow; LSSmKate2 (Piatkevich et al., 2010), red; or EBFP2 (Ai et al., 2007), blue); the orientation of each gene is indicated by a white arrow; and each gene is followed by a unique polyadenylation site (A1+ to A4+). Cre-mediated recombination between loxP sites in an inverted orientation leads to inversion, which is reversible; Cre-mediated recombination between loxP sites in the same orientation leads to deletion, which is irreversible. (B) Inversions can support expression of each FP gene. The first line shows the product of an inversion between the first and second loxP sites. The second line shows the product of an inversion between the first and fourth loxP sites; and the third line shows the product of a subsequent inversion between the first and second loxP sites. (C) A deletion followed by an inversion can support expression of each FP gene. The first line shows the product of a deletion between the second and fourth loxP sites in the initial orientation; the second line shows the product of a subsequent inversion. The third line shows the product of a deletion between the first and third loxP sites in the initial orientation; the fourth line shows the product of a subsequent inversion.

Article Snippet: The Brainbow cassette was synthesized (Genscript) as four fragments, each in pUC57, and then assembled.

Techniques: Expressing, Construct

Journal: Journal of neuroscience methods

Article Title: Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow

doi: 10.1016/j.jneumeth.2014.11.009

Figure Lengend Snippet: The structure of pVGLUT1brainbow and the production of Brainbow arrays by rolling circle DNA replication. (A) A schematic diagram of pVGLUT1brainbow. The VGLUT1 promoter (wavy segment with arrow) supports expression of Brainbow (clear segment). The vector backbone contains an HSV-1 origin of DNA replication (oriS, black circle with white interior in the short line segment) and an HSV-1 a sequence (cross hatched segment), which contains the packaging site; these elements support replication and packaging of the vector, respectively. A cassette of three polyadenylation sites (tri-poly A, black segment) was placed 5′ to the VGLUT1 promoter to reduce any effects on expression from the HSV-1 immediate early 4/5 promoter (contained in short line segment). Sequences from pBR322 (diagonal line segment) support growth in E. coli. (B) Concatamers of the vector are produced during the second, rolling circle phase, of HSV-1 DNA replication (Roizman and Sears, 1993). A HSV-1 genome-sized concatamer of the vector is packaged into a HSV-1 particle (Fraefel et al., 1996; Roizman and Sears, 1993); the HSV-1 genome is ~152 kb, and pVGLUT1brainbow is ~20 kb; thus, an array of 7 or 8 pVGLUT1brainbow is packaged into a HSV-1 particle.

Article Snippet: The Brainbow cassette was synthesized (Genscript) as four fragments, each in pUC57, and then assembled.

Techniques: Expressing, Plasmid Preparation, Sequencing, Produced

Journal: Journal of neuroscience methods

Article Title: Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow

doi: 10.1016/j.jneumeth.2014.11.009

Figure Lengend Snippet: pVGLUT1brainbow supports labeling of POR cortex neurons and axons, and distant axons in PER cortex, with different hues. Rats were sacrificed at 4 days (A–E) or 8 days (F) after gene transfer, brains were sectioned, and confocal stacks were analyzed. (A) A brightfield, low power view of POR cortex in a section proximal to the injection site. The arrow indicated the rhinal sulcus. (B) A low power view of Brainbow labeled neurons in the same section as in (A). (C) A high power view of the boxed area from (B). Neurons and proximal axons that contain different hues are shown. Arrows, cell bodies; arrowheads, axons. (D and E) High power views of neurons and proximal axons, in POR cortex, that contain different hues, from different sections. Some of the labeled cell bodies contain proximal processes, particularly in (D). Also, some axons that are not connected to a cell body are visible. (F) A high power view of PER cortex that shows one axon for ~50 um in length (arrow) and other axons in cross section (arrowheads). Scale bars: (A and B) 250 μm, (C–E) 50 μm, (F) 50 μm.

Article Snippet: The Brainbow cassette was synthesized (Genscript) as four fragments, each in pUC57, and then assembled.

Techniques: Labeling, Injection

Journal: Journal of neuroscience methods

Article Title: Neurons can be labeled with unique hues by helper virus-free HSV-1 vectors expressing Brainbow

doi: 10.1016/j.jneumeth.2014.11.009

Figure Lengend Snippet: pVGLUT1brainbow can label neurons and proximal axons in the hippocampal dentate gyrus with different hues. Rats were sacrificed at 4 days after gene transfer, and confocal stacks were collected. (A) A brightfield, low power view of the hippocampus and adjacent areas in a section proximal to the injection site. (B) A low power view of Brainbow labeled neurons in the same section as in (A). (C) A medium power view of the boxed area from (B) showing Brainbow labeled cells. Arrows, cell bodies. (D) A high power view of the boxed area from (C) showing Brainbow labeled neurons and proximal axons. Arrows, cell bodies; arrowheads, axons. (E and F) High power views of Brainbow labeled neurons and proximal axons from different sections that contain the dentate gyrus. Some of the labeled cell bodies contain proximal processes, particularly a blue neuron on the left side of (E) contains an axon that extends for ~50 μm. Also, some axons that are not connected to a cell body are visible. Scale bars: (A) 500 μm, (B) 250 μm, (C) 100 μm, (D–F) 50 μm.

Article Snippet: The Brainbow cassette was synthesized (Genscript) as four fragments, each in pUC57, and then assembled.

Techniques: Injection, Labeling

Journal: Frontiers in Neuroscience

Article Title: An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

doi: 10.3389/fnins.2023.1241919

Figure Lengend Snippet: Epifluorescence images of PhiYFP-immunoreactivity in (A) the somatosensory cortex, (B) the hippocampus, and (C) the dorsal raphe (DR) of a mouse with the Brainbow 3.2 transgene. The PhiYFP-signal is absent from the DR. I and VI, cortical layers I and VI; Aq, aqueduct; CA1, field CA1 of the hippocampus; DG, dentate gyrus; DRD, dorsal DR; DRV, ventral DR; mlf, medial longitudinal fasciculus. Scale bar = 100 μm.

Article Snippet: The Brainbow adeno-associated viruses (AAVs) (AAV-EF1a-BbTagBY [Addgene #45185-AAV9] and AAV-EF1a-BbChT [Addgene #45186-AAV9]) ( ) were stored at −75°C.

Techniques:

Journal: Frontiers in Neuroscience

Article Title: An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

doi: 10.3389/fnins.2023.1241919

Figure Lengend Snippet: Confocal images of the immunoreactivity of the three Brainbow fluorophores (with the red, green, and blue channels merged) in the dorsal raphe (DR) of a Tph2 - iCreER mouse (male) that received an intracranial injection of the Brainbow -AAVs into the DR. This mouse was allowed to survive for around 3 months after the tamoxifen treatment and was around 1 year old at the time of the tissue collection. (A) A low-power image of the DR. (B) A high-power image of the lateral DR. (C) A high-power image of the ventral DR. Aq, aqueduct; DRD, dorsal DR; DRL, lateral DR; DRV, ventral DR; mlf, medial longitudinal fasciculus. Scale bars = 150 μm (A) , 30 μm (B,C) .

Article Snippet: The Brainbow adeno-associated viruses (AAVs) (AAV-EF1a-BbTagBY [Addgene #45185-AAV9] and AAV-EF1a-BbChT [Addgene #45186-AAV9]) ( ) were stored at −75°C.

Techniques: Injection

Journal: Frontiers in Neuroscience

Article Title: An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

doi: 10.3389/fnins.2023.1241919

Figure Lengend Snippet: Confocal images of fibers immunoreactive for the three Brainbow fluorophores (with the red, green, and blue channels merged) in the diencephalon and telencephalon of a Tph2 - iCreER mouse (male) that received an intracranial injection of Brainbow -AAVs in the dorsal raphe (DR). This mouse was allowed to survive for around 3 months after the tamoxifen treatment and was around 1 year old at the time of the tissue collection. (A,B) The left and right habenulas in the same section. In this geometrically constrained space, individual fibers appear to produce local high-density islands (pink and green in the images). (C) The basolateral amygdala. (D–G) Four enlarged parts of (C) that show highly tortuous but readily separable trajectories (D,E) and unambiguous branching points (F,G) . Scale bars = 20 μm (A–C) and 5 μm (D–G) .

Article Snippet: The Brainbow adeno-associated viruses (AAVs) (AAV-EF1a-BbTagBY [Addgene #45185-AAV9] and AAV-EF1a-BbChT [Addgene #45186-AAV9]) ( ) were stored at −75°C.

Techniques: Injection

Journal: Frontiers in Neuroscience

Article Title: An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

doi: 10.3389/fnins.2023.1241919

Figure Lengend Snippet: Simulated fibers produced by a step-wise random walk, in which the direction of each step was drawn from the von Mises-Fisher probability distribution with the concentration parameter κ. In (A–C) , the diameter of the fibers is 1 μm and each step is 1.5 μm in length. The tortuosity index ( t ) of each fiber, calculated as the fiber length divided by the Euclidean distance between the fiber ends ( ; ), is also shown. (A) Six realizations of a walk with κ = 20 and 30 steps (the fiber length of 45 μm), approximately corresponding to the mean segment length in a 40 μm-thick section . Each fiber is shown in three non-perpendicular orientations that demonstrate caveats of two-dimensional interpretations (the same fiber may appear very different, depending on its orientation with respect to the imaging plane). (B) Six realizations of a walk with κ = 20 and 300 steps (the fiber length of 450 μm). The tortuosity index can be suboptimal in that it can strongly vary in fibers produced by the same stochastic process. (C) Single realizations of walks with six κ values (from 2 to 64) and 300 steps (the fiber length of 450 μm). (D) A simulation of Brainbow-labeled fibers. A set of 300 fibers, each with κ = 15 and 300 steps of 1.5 μm, was computed in a volume matching the z-stack in (185 μm × 185 μm × 21 μm). Each fiber was seeded randomly in the volume (using the uniform distribution in the three dimensions). The segments of the fibers that escaped the volume were considered to be outside the “section” and are not shown. Each fiber was assigned a random color. The fiber radius at each step was independently drawn from the normal distribution with the mean of 0.20 μm and the standard deviation of 0.15 μm. (E) A magnified part of (D) .

Article Snippet: The Brainbow adeno-associated viruses (AAVs) (AAV-EF1a-BbTagBY [Addgene #45185-AAV9] and AAV-EF1a-BbChT [Addgene #45186-AAV9]) ( ) were stored at −75°C.

Techniques: Produced, Concentration Assay, Imaging, Labeling, Standard Deviation

Journal: Frontiers in Neuroscience

Article Title: An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

doi: 10.3389/fnins.2023.1241919

Figure Lengend Snippet: An assessment of the accuracy of the tracing algorithm. (A) The maximum-intensity projection of a subset volume of a z-stack of the basolateral amygdala containing Brainbow-labeled fibers . Scale bar = 10 μm. (B) Twenty automatically traced fibers (with the step of 0.75 μm and no “color” information). Nine traces required no manual correction of any of the points, six traces required one point to be corrected, three traces required two points to be corrected, and two traces required three points to be corrected. In total, 18 of 637 points (2.8%) had to be corrected. Many of the corrections were due to two fibers crossing in the same optical section (in grayscale images). Single-point errors are often easily detectable in maximum-intensity projections (e.g., the trace may jump to another fiber) and can be corrected before the dataset is used in stochastic analyses. (C) The traces shown in isolation. The mean of the estimated κ values was 26 (at the step of 0.75 μm). (D–F) The maximum-intensity projections of the “red,” “green,” and “blue” channels, respectively. The asterisks (one, two, and three) show three “color-isolated” fibers and their corresponding traces.

Article Snippet: The Brainbow adeno-associated viruses (AAVs) (AAV-EF1a-BbTagBY [Addgene #45185-AAV9] and AAV-EF1a-BbChT [Addgene #45186-AAV9]) ( ) were stored at −75°C.

Techniques: Labeling, Isolation

Journal: The EMBO Journal

Article Title: Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

doi: 10.1038/s44318-024-00136-y

Figure Lengend Snippet: ( A ) The palmuscle-Multi and myofiber:iCre#1 transgenic constructs. ( B ) Schematic drawing of individual myofibers before and after Cre activation. Addition of tamoxifen (Tam) activates Cre recombinase, which acts on Brainbow-based cassettes to convert label-free myofibers into color-barcoded myofibers. ( C ) Whole-animal view of a live palmuscle-Multi zebrafish larva at 10 dpf. ( D ) Magnified view of the pectoral fin, craniofacial, and trunk myofibers. ( E ) Representative cross-sectional view of a myotome (left). Schematic outlines of color-barcoded myofibers are shown on the right. ( F ) Color space analysis of 2057 individual myofibers from 32 myotomes captured from a single palmuscle-Multi . About 50 distinct hues were detected in live animals upon Cre activation. ( G ) The palmuscle-Multi and myofiber:iCre#2 transgenic constructs. ( H ) Timeline of the treatment and tracking scheme. ( I ) Schematic drawing of the Tg(palmuscle-Multi; myofiber:iCre#2) larva before and after Cre activation. ( J ) Whole-animal view of the Tg(palmuscle-Multi; myofiber:iCre#2) larva without Dox and Tam treatment. ( K ) The pectoral fin, craniofacial and trunk region displayed multicolor myofibers upon treatment with Dox and Tam. n = number of animals ( J ). Stitched image ( C , D , J ). Scale bars, 200 µm ( C , J ); 100 µm ( D , K ). dpf, days post-fertilization. .

Article Snippet: A 2.2-kb promoter sequence upstream of the mylpfa gene (Ju et al, ) was used to generate following transgenic lines: Tg(mylpfa:palm-mTurquoise2)as69 , Tg(mylpfa:H2A-mCherry)as70 , and Tg(mylpfa:Brainbow1.0L)as71 , and Tg(mylpfa:H2A-mCherry-2A-GC3AI)as72 , and Tg(mylpfa:LifeAct-mScarlet)as76 , the Brainbow reporter sequence was a gift from Joshua Sanes (Addgene plasmid # 18721) (Livet et al, ).

Techniques: Transgenic Assay, Construct, Activation Assay

Journal: The EMBO Journal

Article Title: Whole-body replacement of larval myofibers generates permanent adult myofibers in zebrafish

doi: 10.1038/s44318-024-00136-y

Figure Lengend Snippet: ( A ) Timeline of the tracking scheme. ( B – D ) Long-term time-lapse imaging of the same myofibers in different anatomical regions, including pectoral fin myofibers ( B ), craniofacial myofibers ( C ), and trunk myofibers ( D ). White arrows highlight myofibers that “disappear” from later time points. ( E , F ) Quantitative changes in the tagged myofiber number. Of note, the analyses include only larvae with standard length more than 9 mm at 28 dpf. ( G ) Schematic drawing of zebrafish at larval, juvenile and adult stages, reflecting their relative size difference. ( H ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 14, 42, 70, and 180 dpf. White arrows point to remaining myofibers. White dashed line encircles the area containing fast muscle fibers (42, 70, and 180 dpf). F310 Ab stains fast myofibers. ( I , J ) Quantitative analysis of tagged myofiber number ( I ), and percent area occupied by the tagged myofibers ( J ). The tagged myofiber numbers from two to three consecutive cross-sections were counted and averaged for each individual. ( K ) Timeline of the tracking scheme. ( L ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 29, 42, and 70 dpf. White arrows point to remaining myofibers at 42 dpf. White dashed line encircles the area containing fast muscle fibers. F310 Ab stains fast myofibers. ( M , N ) Quantitative analysis of tagged myofiber number ( M ), and percent area occupied by the tagged myofibers ( N ). The tagged myofiber numbers from two to four consecutive cross-sections were counted and averaged for each individual. ( O ) Timeline of the treatment and tracking scheme. ( P , Q ) Histological examinations of the tagged myofibers in the middle-trunk region of the animals at 29, 43 dpf, and 10 mpf showed no leaky Cre activity ( P ). A short pulse of Dox and Tam labeled most of the trunk fast myofibers ( Q ). White dashed line encircles the area containing fast muscle fibers. Number of animals examined—8, 6, 4 (29 dpf, 43 dpf, 10 mpf, P ); 8, 5, 3 (29 dpf, 43 dpf, 10 mpf, Q ). F310 Ab stains fast myofibers. ( R ) Schematic drawing of the palmuscle-Multi cassettes before and after Cre activation. qPCR primers were designed to detect transcripts from the recombined Brainbow cassettes, targeting the common sequences of mCherry, mYFP, and mCerulean. ( S ) Timeline of the sampling scheme. ( T , T’ ) RT-qPCR analysis of the whole-animal myofiber loss at 14, 28, 42 and 70 dpf ( T ), and an enlarged view of the 70 dpf data ( T’ ). Of note, three separate body compartments—Anterior (A), Middle (M), and Posterior (P)—were collected at 70 dpf, owing to the substantial size of the fish at this stage. Data from biological replicates are shown as mean ± standard deviation ( I , J , M , N ) or mean ± standard error ( T, T’ ). m = myofibers ( E , F ). n = number of animals ( E , F , I , J , M , N ) or biological repeats ( T , T’ ). Stitched image ( H , L , P , Q ). Scale bar, 100 µm ( B – D ); 100 µm ( H , 14 dpf) and ( L , P , Q , 29 dpf); 300 µm ( H , L , 42 dpf; P , Q , 43 dpf); 500 µm ( H , L , 70 and 180 dpf; P , Q , 10 mpf). dpf, days post-fertilization. mpf, months post-fertilization. .

Article Snippet: A 2.2-kb promoter sequence upstream of the mylpfa gene (Ju et al, ) was used to generate following transgenic lines: Tg(mylpfa:palm-mTurquoise2)as69 , Tg(mylpfa:H2A-mCherry)as70 , and Tg(mylpfa:Brainbow1.0L)as71 , and Tg(mylpfa:H2A-mCherry-2A-GC3AI)as72 , and Tg(mylpfa:LifeAct-mScarlet)as76 , the Brainbow reporter sequence was a gift from Joshua Sanes (Addgene plasmid # 18721) (Livet et al, ).

Techniques: Imaging, Activity Assay, Labeling, Activation Assay, Sampling, Quantitative RT-PCR, Standard Deviation