Work Description

Title: Set of images for Figures 1 and 2 and 6 and Supplementary Figure 7 Open Access Deposited

http://creativecommons.org/licenses/by/4.0/
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Methodology
  • Zebrafish: Fish were maintained at approximately 28°C on a 14/10 h light/ dark cycle with standard husbandry procedures. Zebrafish lines, Tg(-5.5sws1: EGFP)kj9, Tg(-3.2sws2: mCherry)mi2007, Tg(trß2: tdTomato), Tg(-3.2sws2: EGFP), Tg(gnat2:H2A-CFP), and pigment mutant ruby carrying albino (slc45a)b4/b4 and roya9/a9 were used. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Michigan.

  • Histology: Retinal dissection, fixation and immunocytochemistry were performed as previously described. Briefly, the isolated retina was fixed in 4% paraformaldehyde with 5% sucrose in 0.1M phosphate buffer, pH 7.4, at 4°C overnight. After antigen retrieval with 10 mM sodium citrate in 0.05% tween 20 (pH 6.0), the retina was incubated in blocking buffer for 2 hours followed by primary antibody incubation, mouse anti-Zonula Occludens (ZO1-1A1, 1:200, ThermoFisher Scientific, Waltham, MA) and rabbit anti-GFP (1:200, ThermoFisher Scientific) at room temperature overnight. Incubation with secondary antibodies (Alexa Fluor 555 and 649, ThermoFisher) was performed at room temperature overnight, and the retina was flat-mounted on a glass slide. For retinal cross sections, affinity-purified rabbit polyclonal opsin antibodies, a gift from Dr. David R. Hyde, were used. Images were acquired with a Zeiss AxioImage ZI Epifluorescent Microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with an ApoTome attachment for optical sectioning structured illumination, Leica DM6000 Upright Microscope System (Leica Microsystems, Werzlarm Germany) and a Leica TCS SP5 confocal microscope equipped with Leica 40X HCX PL APO CS Oil Immersion lens.

  • CRISPR-Cas9 mediated mutation in the thrb gene: A genetic mutation targeting the type 2 isoform of the thrb gene (synonym, trß2) was generated by CRISPR-Cas9 gene editing methods. Briefly, pT7 gRNA vector (Addgene #46759) was used as a template to construct the thrb2 gRNA. PCR based method was performed using specific primers, 5’-GGGGTAATACGACTCACTATAGGCAACACAGCCAACCCTATGTTTTAGAGCTAGAAATAGCAAG-3’; 5’-AAAAAGCACCGACTCGGTGCC-3’. The MEGAscript T7 Kit (Ambion Inc., Austin, TX) was used to transcribe the gRNA. For the nlsCas9nl mRNA synthesis and purification, mMESSAGE mMACHINE T7 Transcription Kit (Ambion) and Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) were used. For genotyping of the thrb mutation, PCR fragments of the thrb gene, amplified using specific primer set, 5’-CATGGTGTAAGTGGCGGATATG -3’; 5’-TCCACTGCATCTGAGAGAAATCC-3’, were subjected to restriction with BstXI (New England Biolabs, Ipswich, MA).

  • Large tile scans of flat-mounted retinae: Large tile scans of entire flat-mounted retinae from adult Tg(sws1:EGFP) zebrafish immunostained for ZO1 were acquired with a Leica TCS SP8 LSCM (Leica Microsystems) equipped with Leica 20X PL APO Dry lens. The GFP signal was recovered by immunostaining with anti-GFP antibody. The White Light Laser was tuned to 555 nm for Alexa Fluor 555 and 649 nm for Alexa Fluor 649. The Leica HyD hybrid detectors were tuned to 600-641 nm for Alexa Fluor 555 and 701-751 nm for Alexa Fluor 649. Images were acquired at 700 Hz scan speed with a resolution of 2048 x 2048 pixels in the xy dimension with a 2.0 um interval between optical sections in the z-dimension.
Description
  • This dataset contains images of dissected and fixed retinae in which cones of specific subtypes are labeled either by transgenic expression of a fluorescent reporter or by antibody staining (Figures 1 and 2 and 6A and Supplementary Figure 7A). This dataset also contains images of dissected and fixed retinae in ZO1 is immunostained (Figure 6C-E and Supplementary Figure 7B). Please see the readme file for which files correspond to which figures.
Creator
Depositor
  • nunley@umich.edu
Contact information
Discipline
Funding agency
  • National Science Foundation (NSF)
  • National Institutes of Health (NIH)
Keyword
Citations to related material
  • Hayden Nunley, Mikiko Nagashima, Kamirah Martin, Alcides Lorenzo Gonzalez, Sachihiro C. Suzuki, Declan Norton, Rachel O. L. Wong, Pamela A. Raymond, David K. Lubensky. Defect patterns on the curved surface of fish retinae suggest mechanism of cone mosaic formation. bioRxiv 806679; doi: https://doi.org/10.1101/806679
  • Hayden Nunley, Mikiko Nagashima, Kamirah Martin, Alcides Lorenzo Gonzalez, Sachihiro C. Suzuki, Declan Norton, Rachel O. L. Wong, Pamela A. Raymond, David K. Lubensky. Defect patterns on the curved surface of fish retinae suggest mechanism of cone mosaic formation. PLoS Comput Biol. 2020 (under review)
Resource type
Last modified
  • 10/19/2020
Published
  • 10/19/2020
Language
DOI
  • https://doi.org/10.7302/1hp2-hc61
License
To Cite this Work:
Nunley, H., Nagashima, M., Martin, K., Lorenzo Gonzalez, A., Suzuki, S., Norton, D., Wong, R., Raymond, P., Lubensky, D. (2020). Set of images for Figures 1 and 2 and 6 and Supplementary Figure 7 [Data set]. University of Michigan - Deep Blue. https://doi.org/10.7302/1hp2-hc61

Files (Count: 12; Size: 705 MB)

Set of images for Figures 1 and 2 and 6 and Supplementary Figure 7

1. ‘120111 wm icc zpr1 s1s2 ctrl r2 40-1z.zvi’, ‘120111 wm icc zpr1 s1s2 ctrl r2
40-2z.zvi’, and ‘120111 wm icc zpr1 s1s2 ctrl r2 40-3z.zvi’ are examples of flat-mounted
retinae as shown in Fig. 2C
These are from an adult, double transgenic (Tg[sws1:GFP; sws2:mCherry]) line in which UV
and Blue cones express distinct fluorescent reporters under control of UV and Blue opsin
promoters, respectively. Antibody staining labels Red and Green cones.

2. ‘032813 FBY rh1 ZO1 r1 no gfp 63x-3 Nunley et al Fig 6E.zvi’ is the example image in
Fig. 6E
Apical plane of cones in retinal flat-mount from tbx2b mutant, which lacks UV cones. The
cone mosaic is disrupted in this mutant.

3. ‘070819 trb2 20x CMZ Nunley et al Fig. 6A.lif’ is the example image from Fig. 6A
Cross-section of wild-type retina in which immunostaining of Red cone opsin labels Red
cones.

4. ‘112012 citrate betacat 549 zo649 r4 63x-4 Nunley et al Fig 6C.zvi’ is the example
image from Fig. 6C
Apical plane of wild-type cone mosaic lattice in retinal flat-mount in which anti-ZO-1
stains cell profiles.

5. ‘122418 trb2-31bp ZO SP5 Nunley et al SI Fig 7B.lif’ is the example image from Fig.
S7B
Flat-mount retinal preparation of trβ2 mutant immunostained with ZO1

6. ‘122418 trb2-31bp ZP SP5 Nunley et al Fig. 6D.lif’ is the example image from Fig. 6D
Apical plane of cone mosaic in retinal flat-mount from trβ2 mutant, which lacks Red cones.

7. ‘122418 y-junction renamed Nunley et al Fig 1B.lif’ is the example image from Fig. 1B
Cone mosaic from flat-mount retinal preparation of an adult, triple transgenic fish,
Tg[sws2:GFP; trβ2:tdTomato; gnat2:CFP]. Blue cones express a fluorescent reporter under
control of the sws2 promoter, and Red cones express a fluorescent reporter under control
of the trβ2 promoter. All cones express an additional reporter under control of the gnat2
promoter. We distinguish between UV and Green cone subtypes based on morphology.

8. ‘triple_transgenic_no y-junction to y-junction comparison.tif’ shows examples of
triple transgenic fish (same line as in ‘122418 y-junction renamed Nunley et al
Fig 1B.lif’). One example image has no y-junction; the other does have a y-junction.

9. ‘triple_transgenic_y-junction summary.tif’ shows examples of triple transgenic fish
(same line as in ‘122418 y-junction renamed Nunley et al Fig 1B.lif’).

All examples have y-junctions.
10. ‘052215 R3 ZO UV.lif’ is an example of (raw) data for a tile scan of a flat-mounted
retina (similar to Fig. 3)

Nunley, H., Nagashima, M., Martin, K., Lorenzo Gonzalez, A., Suzuki, S., Norton, D.,
Wong, R., Raymond, P., Lubensky, D. Set of images for Figures 1 and 2 and 6 and
Supplementary Figure 7 [Data set]. University of Michigan - Deep Blue.
https://doi.org/10.7302/1hp2-hc61

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