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Neuronal migration prevents spatial competition in retinal morphogenesis

Nature volume 620, pages 615–624 (2023)Cite this article

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Abstract

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1,2,3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.

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Fig. 1: Emerging PRs undergo bidirectional translocation in the zebrafish retina with direction-dependent kinetics.
Fig. 2: Bidirectional PR translocation is conserved in human tissue and human organoids.
Fig. 3: Stable microtubules are required for basal PR migration but dispensable for apical migration.
Fig. 4: Actomyosin is the main driver of the apical migration of PRs.
Fig. 5: PR migration prevents congestion of the mitotic zone.

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Data availability

Raw RNA-seq data are accessible through the NCBI GEO series accession number GSE194158. Source data are provided with this paper.

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Acknowledgements

We thank the laboratory of C.N. for project discussions; E. Barriga, M. Cayouette, A. Grapin-Botton, W. Harris and C. Modes for comments on the manuscript; S. Kaufmann, H. Hollak, T. Ferreira and J. Coelho for experimental support; the Computer Department and the Light Microscopy, FACS, Scientific Computing, RNA-seq and Fish facilities at the Instituto Gulbenkian de Ciência (IGC) and the MPI-CBG for technical support and help with statistical analyses; T. Namba and A. Swaroop for sharing DNA constructs; M. Heide for support with the human organoid culture; R. Mateus for hosting experiments; and T. Paixao for help with statistical analyses. E.N. and J.I. were associated with the IMPRS-CellDevoSys PhD program and E.N. was also a member of the IBB–Integrative Biology and Biomedicine PhD program. This work was supported by the MPI-CBG, the Fundação Calouste Gulbenkian-IGC, the German Research Foundation (NO 1069/5-1) and an ERC consolidator grant (H2020 ERC-2018-CoG-81904).

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  1. Martin Weigert

    Present address: Institute of Bioengineering, School of Life Sciences École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

Authors and Affiliations

  1. Instituto Gulbenkian de Ciência, Oeiras, Portugal

    Mauricio Rocha-Martins, Elisa Nerli, Jenny Kretzschmar & Caren Norden

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Mauricio Rocha-Martins, Elisa Nerli, Jenny Kretzschmar, Martin Weigert, Jaroslav Icha, Eugene W. Myers & Caren Norden

  3. Center for Systems Biology Dresden (CSBD), Dresden, Germany

    Mauricio Rocha-Martins, Elisa Nerli, Martin Weigert & Eugene W. Myers

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Contributions

M.R.-M.: conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing (original draft and revised draft) and supervision. E.N.: formal analysis, investigation, methodology, validation and writing (revised draft). J.K.: formal analysis, investigation, methodology and validation. M.W.: software, investigation and methodology. J.I.: investigation and methodology. E.W.M.: resources, supervision and software. C.N.: conceptualization, funding acquisition, project administration, resources, writing (original draft and revised draft) and supervision.

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Correspondence to Mauricio Rocha-Martins or Caren Norden.

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Extended data figures and tables

Extended Data Fig. 1 Identity and migratory behaviour of emerging PRs.

a, Example of denoising of the Tg(crx:gap-CFP) signal using the deep-learning-based algorithm CARE. Dashed white box indicates areas shown in a’. Lookup table shows min and max signal values. b, Bidirectional migration of PR (PR). ath5:GFP-CAAX (grey) labels early neurogenic progenitors; Time is displayed in hours:minutes. Yellow dots mark PR; Magenta dots mark RGC. c, Bidirectional migration of PR (PR). trβ2:tdTomato (grey) labels L-cone PRs; Time is displayed in hours:minutes. White dot marks apical birth; yellow dots mark PR. d, Genes involved in PR differentiation (crx), subtype specification and opsin expression (thrb, neurod1, six7, rxrga) as well as PR function (rcvrn2, rcvrna, cnga3a) that are upregulated in the migrating Crx+ cells at 42 hpf. RNA-seq was performed as described in the Methods. Fold change calculated between emerging PRs (Crx+, Ath5+, Ptf1a−) and progenitors (Crx−, Ath5−, Ptf1a−). P value calculated using Wald test (two-sided) corrected for multiple comparisons with the Benjamini–Hochberg correction. Scale bars: 10 μm (b,c), 25 μm (a).

Source Data

Extended Data Fig. 2 Kinetics of PR migration.

a, Trajectories of PR bidirectional migration from birth to final positioning relative to the apical surface (0 μm) shown as individual trajectories (n = 49 cells, N = 17 embryos) with superimposed mean trajectory ± s.d. b, Directionality ratio of PRs’ basal and apical migration shown as mean of all the tracks ± s.e.m. (n = 33 cells, N = 15 embryos). c,d, Basal (c) and apical (d) velocities (absolute mean velocities) of progenitors and PRs calculated from the trajectories shown in Fig. 1g–h. P value for comparison between cell types using Mann–Whitney test (two-sided): 0.0076 (c) and 0.0065 (d). The lower and upper hinges of the box indicate the 25th and 75th percentile, respectively; middle line indicates the median, whiskers indicate minimum and maximum. e,f, Density plot of absolute mean velocities (basal, e; apical, f) resampled with replacement 105 times for bootstrap estimation of differences (progenitor – PR): mean difference of −0.086 µm min−1 (e) and 0.17 µm min−1 (f).

Source Data

Extended Data Fig. 3 PR migration in human tissue and human organoids.

a, Spatial distribution of OTX2+ PRs along the apical–basal axis of the neuroepithelium of 11-PCW human retina from peripheral to central regions. OTX2 (yellow) labels OTX2+ PRs; DAPI (blue) labels nuclei. b, Spatial distribution of recoverin+ cells along the apical–basal axis of the neuroepithelium of human retinal organoids at an early stage of lamination (D78). Recoverin (yellow) labels recoverin+ PRs; HuC/HuD (magenta) labels RGC and amacrine cells; DAPI (blue) labels nuclei. c, Identity of cells expressing GFP under the control of the human CRX promoter at 16 days after electroporation (12 OTX2+ of 13 GFP+ cells analysed, N = 5 organoids). Endogenous OTX2 (cyan); hcrx:GFP (yellow); caggs:Lyn-tdTomato (magenta). d, Partial trajectories of the migration of PRs positive for hcrx-driven GFP relative to the apical surface (0 μm) and the start of apical migration (0 min). Scale bars: 20 μm (c), 50 μm (a,b).

Source Data

Extended Data Fig. 4 Role of stable microtubules in PR migration.

a, Correlation between PR and sister RGC positions relative to the apical surface (0 μm). Each dot represents an individual time point from 20 pairs of PR–RGC cells (450 time points, N = 7 embryos). b, Bidirectional migration of sister PRs after Ath5 morpholino-mediated knockdown. Tg(ath5:gap-RFP) (grey) labels early neurogenic progenitors. Yellow and blue dots mark sister PRs. c, Distribution of stable microtubules (MTs) in apically migrating PRs at 42 hpf. Tg(crx:gap-CFP) (grey) labels PRs; acetylated tubulin labels stable MTs, lookup table shows minimum and maximum signal values. Dashed white box indicates areas shown in c’ and c”. Arrowheads mark basal processes of PRs. d, Effect of colcemid-induced MT depolymerization on PR basal migration. Embryos were treated with 100 µM of colcemid (DMSO for control) from 30 hpf for 8 h. Tg(crx:gap-CFP) labels PRs, lookup table shows minimum and maximum signal values; phalloidin (grey) labels F-actin. Arrowheads mark PRs. e,f, Effect of mosaic overexpression of stathmin on PR migration. e, ath5:GFP-CAAX (magenta) labels early neurogenic progenitors; hsp70:Stathmin1-mKate2 (yellow). White dots mark PR. f, Trajectories of PRs upon stathmin overexpression (n = 8 cells, N = 3 embryos) relative to the apical surface (0 μm) and typically behaving wild-type (WT) cell from Extended Data Fig. 2a for comparison. g,h, Effect of tissue-wide stathmin-induced MT depolymerization on PR apical migration. Heat shock induction of stathmin expression at 42 hpf. Tg(crx:gap-CFP) (grey) labels PRs. For prevalence of phenotype, see Extended Data Fig. 5e. Scale bars: 5 μm (e), 10 μm (b), 20 μm (c,d,g,h). In b,e,g–h, time is displayed in hours:minutes.

Source Data

Extended Data Fig. 5 Role of actomyosin in PR apical migration.

a, Actin distribution during PR translocation. ath5:Utrophin-GFP labels actin in early neurogenic progenitors, lookup table shows minimum and maximum signal values. Time is displayed in hours:minutes. Yellow dots mark PR; arrowheads mark actin enrichment. b, Expression levels of rock and mylk genes in PRs at 42 hpf shown as mean ± s.d. (N = 5 embryos). c,d, Time series of retinas treated with blebbistatin (25 μM; d) or DMSO (control; c). Tg(crx:gap-CFP) (grey) labels PRs. Time is displayed in hours:minutes. e, Live assessment of chemical and genetic interferences on PR migration. P values were calculated using Fisher’s exact test (two-sided). Scale bars: 5 μm (a), 20 μm (c,d).

Source Data

Extended Data Fig. 6 PR migration behaviour and apical occupancy.

a, Correlation between maximum basal position and duration of PR migration. Dots represent individual cells (n = 49 cells, N = 17 embryos). b, Proliferative status of the retina during PR migration at 42 hpf. Tg(crx:gap-CFP) (magenta) labels PRs; EdU (yellow) labels cells in S-phase. c, Scheme of FACS sorting of PRs and bipolar cells. d, Box plots of the percentages of PRs and bipolar cells in the cell population negative for ptf1a:Gal4/UAS:gap-YFP measured by FACS (experiments = 6, retinas per sample = 25, minimum live cells sorted per experiment = 10,000, total live events sorted = 107,361). The lower and upper hinges of the box indicate the 25th and 75th percentile, respectively; middle line indicates the median, whiskers indicate minimum and maximum. Dots represent individual measurements. e, Co-expression of crx and ath5 promoter-driven reporters at 42 hpf. Tg(crx:gap-CFP) (magenta) labels PRs; Tg(ath5:gap-RFP) (cyan) labels RGCs, PRs and neurogenic progenitors. Dashed white boxes indicate areas shown in e’. Yellow dots mark PRs. f, Measurement of apical surface area of retinal tissue at 42 hpf using the Fiji plug-in Volume Manager. Tg(crx:gap-CFP) (grey) labels PRs. Yellow grid marks the apical surface. g, Measurement of the cross-sectional area of apical PRs at 42 hpf. Tg(crx:gap-CFP) (grey) labels PRs. Yellow line in the XY view outlines the plane shown in XZ view (g’). h, Abundance of basal PRs at 42 hpf. Tg(crx:gap-CFP) labels PRs, colour represents z depth, lookup table shows minimum and maximum z depths. i, Progenitor movements and apical mitosis after colcemid treatment in an area in which PRs had not yet emerged. Embryos were treated with 25 μM colcemid. Tg(crx:gap-CFP) in magenta labels PRs; hsp70:H2B-RFP in yellow labels apically migrating nuclei. Time is displayed as hours:minutes. Blue and white dots mark progenitor cells; arrowheads mark apical mitoses. Scale bars: 10 μm (g,i), 15 μm (b), 20 μm (f,e,h).

Source Data

Extended Data Fig. 7 Blocking the basal migration of PRs causes apical congestion.

a,b, Effect of stathmin-induced blockage of basal PR migration on PR apical occupancy at 42 hpf. Heat shock induction of stathmin overexpression at 30 hpf. Blue line outlines the plane shown in the XZ view (a’,b’) which corresponds to the resliced view of the apical mitotic zone. a,b, Tg(crx:gap-CFP) labels PRs, colour represents z depth, lookup table shows minimum and maximum z depths. a’,b’, Tg(crx:gap-CFP) (grey) labels PRs. a”,b”, Binary mask of the Crx signal generated using the Ilastik pixel classification tool. White and black areas show Crx signal and background, respectively. c,d, Violin plots of local occupancy levels (1,000 bootstrapped ROIs per embryo) of retinas shown in a,b (c) and pooled measurements (d). The lower and upper dotted lines indicate the 25th and 75th percentile, respectively; the middle line indicates the median. d’, Relative frequency of occupancy levels calculated from pooled measurements shown in d. P value for comparison between CTR (without heat shock; N = 7 embryos) and stathmin OVE (N = 14 embryos) using Mann–Whitney test (two-sided): > 0.0001. Scale bars: 10 μm (a’,b’), 20 μm (a,b).

Source Data

Extended Data Fig. 8 Position of mitotic cells during apical congestion by PRs.

a,b, Effect of stathmin-induced blockage of basal PR migration on the position of mitotic cells at 42 hpf, related to Fig. 5f–h. Heat shock induction of stathmin overexpression at 30 hpf. Dashed blue line marks the apical zone shown in XZ view (a’,b’). Dashed white line marks the ROI rendered in 3D (normal shading mode) using Imaris (a”,b”). Tg(crx:gap-CFP) (magenta) labels PRs; pH3 (yellow) labels mitotic cells; DAPI (blue) labels nuclei. c, Effect of stathmin-induced blockage of PR migration on the position of mitotic cells, related to Supplementary Video 10. Single Z plane of non-apical division upon apical congestion. Heat shock induction of stathmin expression at 30 hpf. Tg(crx:gap-CFP) (magenta) labels PRs; H2B-GFP (yellow) labels nuclei. Blue dots mark dividing cell. Time is displayed in hours:minutes. Scale bars: 5 μm (c), 10 μm (a’,a”,b’, b”), 20 μm (a,b).

Extended Data Fig. 9 Effects of mosaic and tissue-wide overexpression of stathmin on the position of mitotic cells and basal neurons.

a, Mosaic overexpression of stathmin does not impair neurogenic divisions per se. Time series of neurogenic progenitors negative (CTR) or positive for stathmin (stathmin OVE). ath5:GFP-CAAX (magenta) labels early neurogenic progenitors; hsp70:Stathmin1-mKate2 (yellow) labels stathmin. Blue dots mark neurogenic progenitors. Arrowheads mark mitoses. White dashed lines delineate the apical surface of the neuroepithelium. Time is displayed in hours:minutes. b, Positioning of neurogenic divisions shown as mean ± s.d. (CTR (Ath5+/Stathmin−) = 10 cells; stathmin OVE (Ath5+/Stathmin+) = 11 cells; N = 3 embryos from independent experiments). P value for comparison between CTR and stathmin OVE using Mann–Whitney test (two-sided): 0,8633. c–f, Effect of stathmin-induced blockage of PR migration on the position of progenitor divisions and RGCs at 56 hpf. Heat shock induction of stathmin overexpression at 30 hpf. c,d, Tg(hsp70:Stathmin1-mKate2) (blue) labels stathmin; pH3 (yellow) labels mitotic cells; DAPI (grey) labels nuclei. Arrowheads mark ectopic mitoses. e,f, Tg(crx:gap-CFP) (magenta) labels PRs; Zn-5 (cyan) labels RGCs. Arrowheads mark ectopic PRs. Scale bars: 5 μm (a), 40 μm (e,f), 50 μm (c,d).

Source Data

Extended Data Fig. 10 Emergence and translocation of delaminated PRs during apical congestion by PRs.

a, Time series of the appearance of delaminated PRs upon stathmin-induced apical congestion. b, Image of the whole eye after live imaging. Dashed white box indicates the area shown in b’. c, Time series of the translocation of delaminated PRs upon stathmin-induced apical congestion. a–c, Heat shock induction of stathmin overexpression at 30 hpf. Tg(crx:gap-CFP) labels PRs, lookup table shows minimum and maximum signal values. White arrowheads mark delaminated PRs. In a,c, time is displayed as hours:minutes. d, Effect of stathmin-induced blockage of PR migration on the position of PRs at 72 hpf. Heat shock induction of stathmin overexpression at 30 hpf. Zpr1 (cyan) and Tg(crx:gap-CFP) (magenta) label PRs; DAPI (grey) labels nuclei. Arrowheads mark ectopic PRs in the basal neuronal layer. Scale bars: 20 μm (a,c), 50 μm (b,d).

Extended Data Fig. 11 Schematic summary of mechanisms that drive PR migration and its relevance for the coordination of tissue growth and organization.

a, PRs emerge at the apical surface of the retinal neuroepithelium and undergo bidirectional somal translocation that entails fast and directed movement towards the basal lamina followed by a basal pause and saltatory movements towards the apical surface. Basal and apical migration display different kinetics owing to the involvement of distinct cytoskeleton machineries. Although basal migration depends on stable microtubules, they are dispensable for apical translocation, which depends on actomyosin contractions at the cell rear. b, PR bidirectional migration is concurrent with cell proliferation and tissue growth. c, Bidirectional migration of PRs delays lamination, helping to coordinate tissue growth and organization by securing space at the apical surface for incoming divisions of progenitor cells. This bidirectional mode of somal translocation is crucial to maintain tissue integrity and function, as blockage of PR movements leads to congestion of the mitotic zone that causes progenitor delamination and secondary neuronal lamination defects.

Supplementary information

Supplementary Fig. 1

Gating strategy for FACS Sorting of Spectrum of Fates line for transcriptomics analysis and quantification of retinal cell types.

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Supplementary Video 1

Bidirectional migration of photoreceptors in the zebrafish retina, related to Fig. 1. (Part 1) Tg(ath5:gap-RFP) (grey) labels early neurogenic progenitors; Tg(crx:gap-CFP) labels PRs, lookup table shows min and max signal values. (Part 2) trβ2:tdTomato (grey) labels L-cone PRs. Time is displayed in hours:minutes. Yellow dot marks PR.

Supplementary Video 2

Photoreceptor layer formation in the zebrafish retina, related to Fig. 1. Tg(ath5:gap-RFP) (grey) labels early neurogenic progenitors; Tg(crx:gap-CFP) labels PRs, lookup table shows min and max signal values. Time is displayed in hours:minutes.

Supplementary Video 3

Bidirectional migration of photoreceptors in human retinal organoids, related to Fig. 2. hcrx:GFP (yellow) labels PR; caggs:Lyn-tdTomato (magenta) labels transfected cells. Blue dot marks PR. Time is displayed in hours:minutes.

Supplementary Video 4

Effect of mosaic stathmin overexpression on photoreceptor migration, related to Fig. 3. ath5:GFP-CAAX (magenta) labels early neurogenic progenitors; hsp70:Stathmin1-mKate2 (yellow). Blue dots mark PRs. Part 1 shows complete stalling of PR translocation. Part 2 shows loss of directionality phenotype. Time is displayed in hours:minutes.

Supplementary Video 5

Effect of tissue-wide interference with microtubules or myosin on photoreceptor migration, related to Figs. 3, 4 and 5. Effect of stathmin-induced microtubule depolymerization on PR basal migration (Part 1) and apical migration (Part 2). Heat shock induction of stathmin expression at 36 hpf (Part 1) and 42 hpf (Part 2). Tg(crx:gap-CFP) (grey) labels PRs, Tg(hsp70:Stathmin1-mKate2) in blue. (Part 3) Effect of myosin inhibitor blebbistatin on PR apical migration. Time series of retinas treated with blebbistatin (25 μM) or DMSO (control). Tg(crx:gap-CFP) (grey) labels PRs. (Part 4) Effect of actomyosin activity inhibitor Rockout on PR apical migration. Time series of retinas treated with Rockout (125 μM) or DMSO (control). Tg(crx:gap-CFP) (grey) labels PRs. Time is displayed in hours:minutes.

Supplementary Video 6

Myosin distribution during photoreceptor migration, related to Fig. 4. ath5:GFP-CAAX (magenta) labels early neurogenic progenitors; ath5:MRLC2-mKate2 labels myosin, lookup table shows min and max signal values. Yellow dot marks PR; arrowheads mark myosin enrichments. Time is displayed in hours:minutes.

Supplementary Video 7

Effect of colcemid-induced blockage of PR migration on progenitor divisions, related to Fig. 5. (Part 1) Subapical mitoses upon colcemid-induced blockage of basal PR migration. (Part 2) Progenitor movements and apical mitosis upon colcemid treatment in an area in which PRs did not yet emerge. Embryos were treated with 25 μM of colcemid. Tg(crx:gap-CFP) in magenta labels PRs; hsp70:H2B-RFP in yellow labels apically migrating progenitor nuclei. Blue and white dots mark progenitor cells. Time is displayed in hours:minutes.

Supplementary Video 8

3D rendering of cells entering mitosis at subapical positions upon colcemid-induced blockage of basal PR migration, related to Fig. 5 and Supplementary Video 7 (Part 1). Embryos were treated with 25 μM of colcemid. Tg(crx:gap-CFP) in magenta labels PRs; hsp70:H2B-RFP in yellow labels apically migrating progenitor nuclei. Volume rendering (Maximum Intensity Projection Mode) using Imaris. Time is displayed in hours:minutes:seconds.

Supplementary Video 9

Effect of stathmin-induced blockage of PR migration on position of mitotic cells, related to Fig 5. (Part 1-3) Examples of non-apical divisions upon apical congestion. Heat shock induction of stathmin expression at 30 hpf. Tg(crx:gap-CFP) in magenta labels PRs; H2B-GFP in yellow labels nuclei. Blue dots mark dividing cells. Time is displayed in hours:minutes.

Supplementary Video 10

3D rendering of non-apical divisions upon stathmin-induced blockage of PR migration, related to Fig 5. (Part 1-2) Examples shown in 2D in Supplementary Video 9. Heat shock induction of stathmin expression at 30 hpf. Tg(crx:gap-CFP) in magenta labels PRs; H2B-GFP in yellow labels nuclei. Volume rendering (Maximum Intensity Projection Mode) using Imaris. Manual segmentation of mitotic cells and automatic segmentation of Crx signal in yellow and magenta, respectively. Time is displayed in hours:minutes.

Supplementary Video 11

Time series of the appearance of delaminated photoreceptors upon stathmin-induced apical congestion, related to Fig.5. Heat shock induction of stathmin expression at 30 hpf. Tg(crx:gap-CFP) labels PRs, lookup table shows min and max signal values. White arrowheads mark delaminated PRs. Time is displayed in hours:minutes.

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Rocha-Martins, M., Nerli, E., Kretzschmar, J. et al. Neuronal migration prevents spatial competition in retinal morphogenesis. Nature 620, 615–624 (2023). https://doi.org/10.1038/s41586-023-06392-y

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