Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination - PubMed (original) (raw)

. 2017 Mar 15;144(6):1097-1106.

doi: 10.1242/dev.142760. Epub 2017 Feb 7.

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Self-organising aggregates of zebrafish retinal cells for investigating mechanisms of neural lamination

Megan K Eldred et al. Development. 2017.

Abstract

To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48 h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.

Keywords: Cell sorting; Layer formation; Müller cells; Organoid; Reaggregation; SoFa.

© 2017. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.

Fig. 1.

Dissociation, culture and re-aggregation of zebrafish retinal cells. (A,B) Schematic representing retinas dissected from 24 hpf zebrafish (A), collected into glass dishes and dissociated into single cells (B). (C) Agarose microwell dish cast from the 3D Petri Dish PDMS Mould (adapted, with permission, from

http://www.microtissues.com

). (D) Schematic representing the seeding chamber of the 3D Petri dish. After seeding, cells settle into individual wells. (E-J) Time-lapse images of a single well from the 3D Petri dish showing 24 hpf cells re-aggregating. (H) Cells are almost fully reaggregated 3 h after seeding. (J) Cells have undergone compaction 15 h after seeding. Time is in minutes and hours after seeding. Scale bar: 100 μm.

Fig. 2.

Fig. 2.

A self-organising retina: identification of zebrafish retinal cells and characterisation of organisation. The main cell types of the retina can be identified in the SoFa1 transgenic line (Almeida et al., 2014) using a combination of genetically tagged cell fate markers: Atoh7:gapRFP labels RGC, AC/HC and PR cell membranes; Ptf1a:cytGFP labels AC/HC cytoplasm; and Crx:gapCFP labels BP and PR membranes. (A) Central sagittal section of a region of the SoFa1 retina. Scale bar: 20 μm. (B) Dissociated cells of the SoFa1 line. Scale bar: 20 μm. (C-F) Individual cells are identified based on their spectral expression: (C) RGCs express membrane RFP; (D) AC/HCs express cytoplasmic GFP and membrane RFP; (E) BPs express membrane CFP; and (F) PRs express membrane CFP and RFP. Scale bar: 5 μm. (G-L) Central sagittal section of a retinal aggregate cultured using the SoFa1 line. (G) Crx:gapCFP-expressing cells are found in the centre of the aggregate. (H) Ptf1a:cytGFP-expressing cells are found in a distinct ring around the Crx:gapCFP population. (I) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (J) Merge of channels represented in G-I. (K) DAPI. (L) Bright-field image. Scale bar: 10 μm. (M-P) Generation of analysis of cellular organisation using custom-made Matlab scripts. (M) A mask is fitted to the aggregate using the DAPI channel. (N) Successive isocontours are fitted from the periphery to the centre of the aggregate. (O) Fluorescence is measured along each contour and plotted as a relative fluorescence intensity (_y_-axis) against radial position (in pixels) (_x_-axis). (P) Fluorescence profiles for each channel are plotted as an empirical cumulative distribution function (ECDF) (_y_-axis) against radial position [radial units (ru)] (_x_-axis). The dotted diagonal line represents a theoretically perfect even distribution of fluorescence from centre to periphery.

Fig. 3.

Fig. 3.

Retinal pigment epithelium is not required for zebrafish retinal self-organisation. Fluorescence profiles are generated for SoFa1 aggregates cultured either with or without RPE cells. (A-F) Central sagittal section of a SoFa1 aggregate with RPE. (A) Crx:gapCFP-expressing cells are found in the centre of the aggregate. (B) Ptf1a:cytGFP-expressing cells are found in a ring around the edge of the Crx:gapCFP population. (C) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (D) Merge of channels represented in (A-C). (E) DAPI. (F) Bright-field image. Pigment-expressing RPE cells can be seen near the centre of the aggregate (arrows). Scale bar: 10 μm. (G) Fluorescence profiles for the aggregate represented in A-F. (H) ECDF plot for the aggregate represented in A-F. (I-N) Central sagittal section of a SoFa1 aggregate without RPE. (I) Crx:gapCFP-expressing cells are found in the centre of the aggregate. (J) Ptf1a:cytGFP-expressing cells are found in a ring around the edge of the Crx:gapCFP population. (K) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (L) Merge of channels represented in I-K. (M) DAPI. (N) Bright-field image. No pigment-expressing RPE cells can be seen. Scale bar: 10 μm. (O) Fluorescence profiles for the aggregate represented in I-N. (P) ECDF plot for the aggregate represented in I-N. (Q) Average fluorescence profiles with shaded error for aggregates with RPE (_n_=15, three experimental repeats). (R) Average fluorescence profiles with shaded error for aggregates without RPE (_n_=15, three experimental repeats). (S) Average ECDF plots for aggregates with RPE. (T) Average ECDF plots for aggregates without RPE. (U) Area (in arbitrary units) is calculated between the ECDF for the Crx:gapCFP population and the ECDF for the Ptf1a:cytGFP population of cells, and compared between aggregates with RPE (+RPE) and without RPE (–RPE) (_n_=15 for each condition, Mann–Whitney two-tailed _t_-test, _P_>0.05).

Fig. 4.

Fig. 4.

Müller glia are important in zebrafish retinal self-organisation. Fluorescence profiles are generated for SoFa1 aggregates treated either with 25 μM DAPT to prevent the differentiation of Müller glia or with DMSO as a control. (A-F) Central sagittal section of a SoFa1 aggregate treated with DMSO. (A) Crx:gapCFP-expressing cells are found in the centre of the aggregate. (B) Ptf1a:cytGFP-expressing cells are found in a ring around the edge of the Crx:gapCFP population. (C) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (D) Merge of channels represented in A-C. (E) DAPI. (F) Bright-field image. Scale bar: 10 μm. (G) Fluorescence profiles for the aggregate represented in A-F. (H) ECDF plot for the aggregate represented in A-F. (I-N) Central sagittal section of a SoFa1 aggregate treated with 25 μM DAPT. (I) Some Crx:gapCFP-expressing cells are found in the centre of the aggregate, and some are found nearer the edge. (J) Ptf1a:cytGFP-expressing cells are found throughout the aggregate. (K) Atoh7:gapRFP-expressing cells are found throughout the aggregate. (L) Merge of channels presented in I-K. (M) DAPI. (N) Bright-field image. Scale bar: 10 μm. (O) Fluorescence profiles for the aggregate represented in I-N. (P) ECDF plot for the aggregate represented in I-N. (Q) Average fluorescence profiles with shaded error for aggregates treated with DMSO (_n_=15, three experimental repeats). (R) Average fluorescence profiles with shaded error for aggregates treated with 25 μM DAPT (_n_=15, three experimental repeats). (S) Average ECDF plots for aggregates treated with DMSO. (T) Average ECDF plots for aggregates treated with 25 μM DAPT. (U) Area (in arbitrary units) is calculated between the ECDF for the Crx:gapCFP population and the ECDF for the Ptf1a:cytGFP population of cells, and compared between aggregates treated with DMSO and aggregates treated with 25 μM DAPT (_n_=15 for each condition, Mann–Whitney two-tailed _t_-test, P<0.0001).

Fig. 5.

Fig. 5.

Late application of DAPT allows Müller glia to be generated and aggregates to self-organise. Aggregates are cultured in the presence of DAPT applied at 45-48 hpf onwards to block the differentiation of Müller glia or at 63 hpf onwards to allow the differentiation of some Müller glia compared with DMSO control. (A,B) Aggregates cultured in the presence of DMSO show several GFAP-positive cells (indicated by arrows). (C,D) Aggregates cultured in the presence of DAPT from 45-48 hpf onwards have little or no GFAP-positive cells. (E,F) Aggregates cultured in the presence of DAPT from 63 hpf onwards have several GFAP-positive cells (indicated by arrows). Scale bar: 10μm. (G) Average ECDF plots for aggregates treated with DMSO. (H) Average ECDF plots for aggregates treated with DAPT from 45-48 hpf onwards. (I) Average ECDF plots for aggregates treated with DAPT from 63 hpf onwards. (J) Area (in arbitrary units) is calculated between the ECDF for the Crx:gapCFP population and the ECDF for the Ptf1a:cytGFP population of cells, and compared between aggregates treated with DMSO and aggregates treated with DAPT from 45-48 hpf onwards (_n_=32 for DMSO, _n_=20 for DAPT at 45-48 hpf, Mann–Whitney two-tailed _t_-test, P<0.0001). (K) Area (arbitrary units) compared between the ECDF plots of aggregates treated with DAPT from 45-48 hpf onwards and aggregates treated with DAPT from 63 hpf onwards (_n_=20 for DAPT at 45-48 hpf, _n_=26 for DAPT at 63 hpf, Mann–Whitney two-tailed _t_-test, _P_<0.0001). (L) Area (arbitrary units) compared between the ECDF plots of aggregates treated with DMSO and aggregates treated with DAPT from 63 hpf onwards (_n_=32 for DMSO, _n_=26 for DAPT at 63 hpf, Mann–Whitney two-tailed _t_-test, _P_>0.05). (M) Areas of data from all conditions.

References

    1. Almeida A. D., Boije H., Chow R. W., He J., Tham J., Suzuki S. C. and Harris W. A. (2014). Spectrum of Fates: a new approach to the study of the developing zebrafish retina. Development 141, 1971-1980. 10.1242/dev.104760 -DOI -PMC -PubMed
    1. Ben-Shaul Y. B., Hausman R. E. and Moscona A. A. (1980). Age-dependent differences in cognin regeneration on embryonic retina cells: immunolabeling and SEM studies. Dev. Neurosci. 3, 66-74. 10.1159/000112378 -DOI -PubMed
    1. Bernardos R. L. and Raymond P. A. (2006). GFAP transgenic zebrafish. Gene Expr. Patterns 6, 1007-1013. 10.1016/j.modgep.2006.04.006 -DOI -PubMed
    1. Boije H., Rulands S., Dudczig S., Simons B. D. and Harris W. A. (2015). The independent probabilistic firing of transcription factors: a paradigm for clonal variability in the zebrafish retina. Dev. Cell 34, 532-543. 10.1016/j.devcel.2015.08.011 -DOI -PMC -PubMed
    1. Caviness V. S. and Sidman R. L. (1973). Time of origin of corresponding cell classes in the cerebral cortex of normal and reeler mutant mice: An autoradiographic analysis. J. Comp. Neurol. 148, 141-151. 10.1002/cne.901480202 -DOI -PubMed

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