goat anti dynactin1  (Novus Biologicals)

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Part 1 : Example of an asymmetric division of multipotent progenitors, generating one Ath5+ and one Ath5- cell. Membranes of progenitors are labelled by hsp70:mkate2-ras (grey), nuclei by hsp70:H2B-RFP (grey). Ath5 expression is followed using ath5:GFP-CAAX (green). Time is shown in hours and minutes. Yellow and magenta dots label Ath5+ and Ath5- sister cells, respectively. Part 2 : Example of the division of an Ath5- sister cell. Cell nuclei are labelled by hsp70:H2B-RFP (grey) and Ath5 expression is followed using ath5:GFP-CAAX (green). Yellow dot labels Ath5+ sister cell. Magenta and cyan dots label Ath5- sister cell and its daughters.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Nuclei of progenitors are labelled by hsp70:H2B-RFP (grey) and Ath5 expression is followed using ath5:GFP-CAAX (green). Time is shown in hours and minutes. Yellow and magenta dot label sister cells.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Dynactin is labelled by hsp70:DNdynactin-mKate2 (grey), nuclei by hsp70:H2B-RFP (grey) and Ath5 expression is followed using ath5:GFP-CAAX (green). Time is shown in hours and minutes. Yellow and magenta dots label Ath5+ and Ath5- sister cell, respectively.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Part 1 : Tg(Tp1:H2B-mCherry),Tg(ath5:gap-GFP) double transgenic line is imaged at the onset of neurogenesis. Notch activity is shown by Tp1:H2BmCherry (grey), neurogenic cells by ath5:gap-GFP (green). Time is shown in hours and minutes. Part 2 : Basal translocation of a progenitor cell with Notch activity. Progenitor cells nuclei are labelled with hsp70:H2B-RFP (magenta), Notch activity is labelled by her4:EGFP (cyan). Blastomere transplantation of double transgenic Tg(her4:EGFP),Tg(hsp70:H2B-RFP) into wild type was used to achieve mosaic labelling. Time is shown in hours and minutes. White dot indicates a basal-translocating progenitor, cyan arrowheads point to her4 expression.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Stathmin is labelled by hsp70:Stathmin1-mKate2 (grey) and Ath5 expression is followed using ath5:GFP-CAAX (green). Time is shown in hours and minutes. Yellow and magenta dots label Ath5+ and Ath5- sister cell, respectively.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Membranes of progenitors are labelled by hsp70:mkate2-ras (grey), Sara endosomes by CFP-Sara (cyan) and Ath5 expression is followed using ath5:GFP-CAAX (green). Time is shown in hours and minutes, 1 min time resolution during division, 5 min time resolution afterwards to follow Ath5 expression onset. White arrow points to the asymmetrically distributed endosome. Yellow and magenta dots label Ath5+ and Ath5- sister cell, respectively.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques:

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Schematic of mosaic Ath5 expression in the retina (green) at 28 hpf. Injection of ath5:GFP-CAAX construct at 1 cell stage. ( B ) Example of an asymmetric multipotent progenitor division with regards to Ath5 expression onset. hsp70:H2B-RFP (nuclei, grey), hsp70:mkate2-ras (cell membrane, grey), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. ( C–C’ ) Schematics of multipotent progenitor cells dividing asymmetrically ( C ) or symmetrically ( C’ ) with regards to Ath5 expression. ( D ) Distribution of asymmetric vs symmetric divisions observed in live imaging experiments. N = number of embryos, n = number of divisions. ( E ) Ath5+ cells (grey) at 36 hpf in control (left) vs Notch inhibition (right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( F ) Ath5+ cells (grey) at 48 hpf in control (left) vs Notch inhibition (right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( G ) Schematic of retinal neuroepithelium measurements at 48 hpf, ( G’ ) retinal thickness control vs Notch inhibition, ( G’’ ) retinal diameter control vs Notch inhibition. Mann-Whitney test used for comparison. Vertical bars represent standard deviation. ( H ) Example of symmetric progenitor division upon Notch inhibition. hsp70:H2B-RFP (nuclei, grey), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. ( I ) Distribution of asymmetric vs symmetric divisions observed in live imaging experiments in Notch inhibition compared to controls. Figure 1—source data 1. Source data for panels G’,G’’.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Expressing, Injection, Construct, Membrane, Imaging, Control, Inhibition, MANN-WHITNEY, Comparison, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Notch inhibition affects progenitor division patterns. ( A ) Onset of Ath5 expression (green) in Ath5+ progenitors between divisions. ( B ) Example of Ath5- sister cell followed to next division. hsp70:H2B-RFP (nuclei, grey), ath5:GFP-CAAX (Ath5, green). Scale bar, 10 μm. ( C ) Schematics of lineage tree of Ath5- cell. ( D ) Ath5+ cells (grey) at 36 hpf in control (left) and upon Notch inhibition (right) with DAPT 50 μM. Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( E ) EdU+ cells (grey) at 42 hpf in control (left) and upon Notch inhibition (right) treated with 10 μM LY411575. Scale bar, 50 μm. The yellow lines delimit the apical and basal side of the retinal neuroepithelium. ( F ) Quantification of EdU-positive cells in the central portion of the retina in control (black) and upon Notch inhibition (blue). Unpaired t -test with Welch’s correction. Error bars represent standard deviation.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Inhibition, Expressing, Control, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Schematic of progenitor cell soma moving along the apicobasal axis between divisions. ( B ) Maximum basal position of sister cells. Paired t -test was used to compare sister cells. Lines connect sister cells. ( C ) Difference in maximum basal position between sister cells. Vertical error bars represent standard deviation. ( D ) Maximum basal position of sister cells in cases in which Ath5+ sister cells translocate more basally (27/35, 78%). ( E ) Maximum basal position of sister cells in cases in which Ath5- sister cells translocate more basally (8/35, 22%). ( F ) Difference in maximum basal position between sister cells, comparing Ath5+ and Ath5- cells at most basal positions. ( G ) Ath5+ progenitor trajectories between divisions. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( H ) Ath5- progenitor trajectories between divisions. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( I ) Mean + standard deviation of Ath5+ and Ath5- progenitor pooled tracks from panels G (Ath5+, green) and H (Ath5-, grey). ( J ) Asymmetric division upon DN-dynactin overexpression. hsp70:H2B-RFP (nuclei, grey), hsp70:DNdynactin-mKate2 (dynactin, grey) ath5:GFP-CAAX (Ath5, green). Scale bar, 10 μm. Dashed lines show apical and basal sides of the neuroepithelium. Magenta and yellow dots label sister cells. ( K ) Maximum basal position of sister cells upon dynactin inhibition. Paired t -test to compare sister cells. ( L ) Percentage of asymmetric and symmetric divisions observed upon disruption of dynactin function compared to control. Figure 2—source data 1. Source data for panels B, C, D, E, F, G, H and K. .

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Standard Deviation, Over Expression, Inhibition, Disruption, Control

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Asymmetric inheritance of basal process during progenitor division. hsp70:mKate2-ras labels cell membrane (grey). Arrowheads: basal process. Scale bar, 10 μm. Magenta and yellow dots label sister cells. Dashed lines show apical and basal side of retinal neuroepithelium. ( B ) Mean square displacement (MSD) for Ath5+ and Ath5- progenitors, calculated for basal translocation within the first 100 min after mitosis of the mother cell. Data from . ( C ) Maximum basal position of sister cells not inheriting the basal process (No BP) vs inheriting the basal process (BP) after division. ( D ) Proportion of Ath5+ and Ath5- progenitor cells inheriting the basal process. ( E ) Acetylated tubulin staining (magenta) of Tg(ath5:gap-GFP, cyan) with nuclei labelled by DAPI (grey) at 24 (upper left panel), 28 (upper right), 32 (bottom left) and 36 hpf (bottom right). Scale bar, 30 μm. Arrows mark acetylated tubulin staining in the primary cilium (upper left), retinal progenitors (upper right and bottom left) and retinal ganglion cells (bottom right). Dashed lines show apical and basal sides of the retinal neuroepithelium. ( F ) Example of basal translocation of progenitors upon Stathmin1 overexpression induced at 28 hpf. hsp70:Stathmin1-mKate2 (stathmin, grey), ath5:GFP-CAAX (Ath5, green). Scale bar, 5 μm. Magenta and yellow dots label sister cells. Dashed lines show apical and basal sides of the retinal neuroepithelium. ( G ) Percentage of asymmetric and symmetric divisions observed upon Stathmin1 overexpression compared to control. ( H ) Ath5- progenitor trajectories between divisions upon Stathmin1 overexpression. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( I ) Ath5+ progenitor trajectories between divisions upon Stathmin1 overexpression. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( J ) Mean and Standard Deviation of Ath5- progenitor trajectories in control (magenta) and Stathmin1 overexpression (grey). ( K ) Mean and Standard Deviation of Ath5+ progenitor trajectories in control (light green) and Stathmin1 overexpression (dark green). Figure 4—source data 1. Source data for panels B, C, H and I.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Membrane, Translocation Assay, Staining, Over Expression, Control, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Co-localisation of Sara-positive endosomes (RFP-Sara, magenta) and DeltaC staining (cyan) in 36 hpf embryos. Scale bar, 10 μm. Dashed line represents the apical side. ( A’ ) Close-up of co-localisation. Scale bar, 5 μm. ( B ) Co-localisation of Sara-positive endosomes (RFP-Sara, magenta) and Notch1b staining (cyan) in 36 hpf embryos. Scale bar, 10 μm. Dashed line represents the apical side. ( B’ ) Close-up of co-localisation. Scale bar, 5 μm. ( C ) Asymmetric distribution of Sara-positive endosomes in dividing progenitor cells in three different embryos fixed at three different developmental stages, 24, 28 and 36 hpf. Tg(actb1:HRAS-EGFP) (membrane, magenta), RFP-Sara (Sara-positive endosomes, cyan), DAPI (chromatin, grey). Scale bar, 10 μm. ( D ) Number of endosomes within dividing cells. n = 25 cells, 7 embryos. ( D’ ) Histogram of endosomal ratio between dividing cells. n = 25 cells, 7 embryos. ( E ) Asymmetric distribution of Sara-positive endosomes in dividing cells in live samples at 32 hpf. hsp70:mkate2-ras (membrane [grey], CFP-Sara [Sara-positive endosomes, cyan]). Scale bar, 10 μm. Arrowheads point to the endsosomes ( E’ ) YZ (left) and XZ (right) view of sister cells (grey) and Sara endosomes (cyan) at minute 3:00. Scale bar, 10 μm. Arrowheads point to the endsosomes. ( F ) Ath5+ cells (grey) in retinal neuroepithelium at 36 hpf in control (left) and Sara knockdown (Sara-MO, right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( F’ ) Tp1+ cells (grey) at 36 hpf in control (left) and Sara morpholino knockdown (right). Scale bar, 30 μm. The yellow line delimits the apical side of the retinal neuroepithelium and the lens. ( G ) Scheme recapitulating the main findings of this study presenting a working model for Sara-positive endosomes inheritance and its role in asymmetric divisions.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Staining, Membrane, Control, Knockdown

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Ath5+ cells (grey) in the retinal neuroepithelium at 36 hpf in embryos injected with control-MO (upper row) and 2.5 ng/embryo Sara-MO (lower row). Scale bar, 50 μm. The yellow lines delimit the apical and basal side of the retinal neuroepithelium. ( B ) pH3 staining (cyan) in 36 hpf Tg(ath5:gap-GFP) embryos (grey) injected with control-MO (upper row) and 2.5 ng/embryo Sara-MO (lower row). Scale bar, 50 μm. The yellow lines delimit the apical side of the retinal neuroepithelium. ( C ) EdU+ cells (grey) at 36 hpf in control (upper row) and upon Sara knockdown (lower row) in Tg(ath5:gap-GFP) embryos (green). Scale bar, 50 μm. ( D ) Quantification of EdU-positive cells in the central portion of the retina in control (black) and Sara knockdown (blue). Unpaired t -test with Welch’s correction. Error bars represent standard deviation. ( E ) Example of an asymmetric inheritance of Sara-positive endosomes (left) within asymmetric division of multipotent progenitors generating Ath5+ progenitors (right). hsp70:mkate2-ras (cells membrane, grey), CFP-Sara (Sara-positive endosomes, cyan), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. White arrow points to the Sara-positive endosome.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Injection, Control, Staining, Knockdown, Standard Deviation, Membrane

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: DNA constructs used in this study.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Construct, Concentration Assay

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Control, Software, Microscopy