FIGURE SUMMARY
Title

Uncoupling of neurogenesis and differentiation during retinal development

Authors
Engerer, P., Suzuki, S.C., Yoshimatsu, T., Chapouton, P., Obeng, N., Odermatt, B., Williams, P.R., Misgeld, T., Godinho, L.
Source
Full text @ EMBO J.

Vsx1+ progenitors undergo mitosis in different proliferative zones and match the expression of molecular markers of post‐mitotic BCs in their vicinity

A. Confocal images of a coronal cryostat section from a 2 dpf vsx1:GFP retina with immature, neuroepithelial (“unlaminated”, orange) and mature, laminated regions (“laminated”, cyan). Left panel, vsx1:GFP; right panel, vsx1:GFP shown in conjunction with pH3 antibody staining to label cells in late G2/M‐phase. Scale bar: 10 μm.

B, C High magnification images of (B) an apically dividing vsx1+ progenitor (orange arrowhead) in an unlaminated region where cells span the retina and express GFP weakly and (C) a non‐apically dividing vsx1+ progenitor (cyan arrowhead) in a laminated region where cells confine their processes to the OPL and IPL (dashed lines) and express high levels of GFP. Cellular membranes are labeled with BODIPY methyl ester. Scale bar: 10 μm.

D. Quantification of vsx1+ progenitor mitoses at apical (lightly shaded) and non‐apical (darkly shaded) locations in the unlaminated (“un”, orange) and laminated (“lam”, cyan) retina. Data are presented as mean ± SEM, 1,391 mitotic divisions, 80 sections from at least 14 fish.

E. Quantification of vsx1:GFP fluorescence intensity in dividing vsx1+ progenitors in the unlaminated (“un”, orange) and laminated (“lam”, cyan) retina. Data are presented as mean ± SEM, 86 progenitors, 43 sections from at least 13 eyes, Mann–Whitney U‐test, ***P ≤ 0.0001.

F. Correlation of vsx1:GFP fluorescence intensity in progenitors and their surrounding cells in the unlaminated (orange circles) and laminated (cyan circles) regions of the retina. Eighty‐six progenitors from 43 sections from at least 13 eyes, r2 = 0.78. Inset: Analysis for all pH3+ cells (n = 13) from a single section.

G. Confocal images of a cryostat section from a 2 dpf vsx1:GFP retina immunostained with antibodies against pH3 and Crx. Vsx1+ progenitors (GFP+ pH3+) are Crx negative (orange arrowheads) in the unlaminated retina (left panels, orange bars above panels) and are Crx positive (cyan arrowhead) in the laminated region (right panels, cyan bars above panels). Scale bar: 10 μm.

H. Correlation of Crx antibody staining intensity between mitotic BC progenitors and the vsx1+ cells in their vicinity. Thirty‐eight progenitors, 10 sections from at least five eyes, r2 = 0.87; orange circles, apical mitoses; cyan circles, non‐apical mitoses.

Morphological rearrangement of vsx1+ progenitors matches surrounding BCs

A. Confocal in vivo time‐lapse recording of a retina from the Q26 transgenic line (crossed to a UAS:memYFP reporter) showing a non‐apically dividing vsx1+ progenitor (pseudo‐colored cyan) with processes restricted to the IPL and OPL (dashed lines) during mitosis (0′). The last time point at which an apical process (open arrowhead) is detected is 89 min prior to mitosis. Scale bar: 10 μm.

B. Quantification of the distinct morphologies adopted by non‐apically dividing vsx1+ progenitors at M‐phase entry (122 progenitors, 17 fish). Open arrowheads indicate cytoplasmic processes extending beyond the synaptic layers (OPL and IPL, dashed lines).

C. Quantification of the time interval between retraction of the apical process (triangle) and mitosis (cyan circle) of non‐apically dividing vsx1+ progenitors shows a broad range from 18 min to more than 540 min. As only mitosis, but not process retraction was observed for the progenitor depicted with small cyan dots, the movie length of 540 min is an underestimate. 18 progenitors from 11 fish.

D. Schematic of apical process retraction (triangle) in a non‐apical vsx1+ progenitor (cyan soma) and the presence or absence of apical processes in the surrounding, post‐mitotic BCs.

E. Quantification of the percentage of surrounding post‐mitotic BCs without an apical process at the time when pre‐mitotic vsx1+ progenitors undergo apical process retraction (triangle, 60.3 ± 6.6%) and at the time when these progenitors undergo mitosis (cyan circle, 95.1 ± 1.7%). Data are presented as mean ± SEM, 17 progenitors, 10 fish.

Vsx1+ progenitors in the unlaminated and laminated region exhibit distinct cell biological behaviors

A. Concurrent visualization of nucleokinesis in a vsx1+ progenitor in the laminated retina (Q26; UAS:memYFP, upper panels) and the cell cycle stage (mOrange2‐PCNA mRNA, lower panels) by in vivo time‐lapse confocal imaging. Progenitor soma is pseudo‐colored cyan in YFP channel and outlined in cyan in mOrange channel. Scale bar: 10 μm.

B. Nuclear movement of vsx1+ progenitors in the unlaminated region (vsx1:GFP) of the retina prior to mitosis at the apical surface (orange circle). Seven cells, five fish. Black line represents the average position of all vsx1+ progenitors prior to mitosis in unlaminated regions.

C. Nuclear trajectories of vsx1+ progenitors in the laminated region that undergo mitosis at the INL/OPL interface (cyan circle, left panel) and post‐mitotic BCs in their vicinity (right panel). In the time interval during which trajectories were tracked, all 12 vsx1+ progenitors move to the INL/OPL interface prior to mitosis; 8 of 12 post‐mitotic surrounding BCs do so (five fish). Black lines represent the average position of all vsx1+ progenitors prior to mitosis in laminated regions (left panel) or of the post‐mitotic BCs (right panel).

D. Centrosomes (centrin4‐YFP mRNA, grayscale) of a vsx1+ progenitor (magenta mask, Q19) and post‐mitotic BCs in the laminated region concurrently translocate to the OPL and remain clustered there. The centrosome of the highlighted vsx1+ progenitor is pseudo‐colored green. Scale bar: 10 μm.

E. Continuation of the time‐lapse from (D) (centrosomes not depicted). The vsx1+ progenitor (magenta mask) shows transient lateral arborizations in the synaptic layers as wide as its soma (see brackets at −210′, −165′). A further example can be seen in (A) (see brackets at −67′).

The differentiation status of late‐born bipolar cells and earlier‐born bipolar cells in their vicinity are similar

A. Fluorescence intensity of a non‐apically dividing vsx1+ progenitor before it undergoes mitotic division (cyan trace), at the time of mitotic division (cyan circle) and of the two BC daughter cells (green ovals) over the course of 10 h (green traces).

B. Quantification of the fluorescence intensity of four pairs of BC daughters (green ovals) derived from non‐apically dividing vsx1+ progenitors (cyan circle) 10 h after division. The fluorescence intensity of each of the BC pairs was normalized to the mitotic cell from which they were derived. BC pairs depicted are derived from two fish. The different overall rates of GFP fluorescence increase can be explained by where along the differentiation gradient cells originate.

C. Comparison of the vsx1 expression levels of apical progenitors, non‐apical progenitors, and post‐mitotic BCs derived from non‐apically dividing progenitors 10 h following division. The fluorescence expression levels of cells surrounding each of the three cell categories are also quantified. Apically dividing vsx1+ progenitors (orange circle) and surrounding cells (dull orange bar, mean, and standard deviation, SD). Non‐apically dividing vsx1+ progenitors (cyan circle) and surrounding cells (dull cyan bar, mean, and SD). In total, 43 apically dividing progenitors and 524 cells in their surround, and 43 non‐apically dividing progenitors and 521 cells in their surround from at least 13 eyes were used for this analysis. Post‐mitotic BCs (green oval) and earlier‐born surrounding cells (dull green bar, mean, and standard deviation). Eleven post‐mitotic BCs derived from seven mitotic divisions and 105 cells in their surround from three fish were used for this analysis.

D. Confocal in vivo time‐lapse recording of the emergence of ribeye a puncta (pseudo‐colored magenta, ctbp2:mCherry‐ctbp2) in a BC derived from a non‐apically dividing vsx1+ progenitor (pseudo‐colored cyan, Q26; UAS:memYFP), 7 h following cell cycle exit. Insets of the magenta‐boxed region (at −118′ and 0′) of the axon terminal of the BC reveal the emergence of discernible ribeye a puncta at the 0′ time point (white arrowheads). Scale bars: 10 μm.

E. Quantification of the time interval between mitosis and clustering of ribeye puncta (triangle) in the axon terminals of the ensuing BC daughters. Eight BCs derived from seven non‐apically dividing progenitors (cyan circles) and eight BCs derived from apically dividing vsx1+ progenitors (orange circles) from three fish were used for this analysis. In seven of the eight BCs derived from apical progenitors (depicted as small orange dots), we observed clustering but not the mitotic division, thus the time interval in these cases is an underestimate.

F. Quantification of the axon terminal arbor width of BCs derived from non‐apical vsx1+ progenitors and the cells in their surround. At 10 h post‐division, mean arbor width for BCs was 4.34 ± 1.05 μm, SD; the mean arbor width for cells in their surround was 4.29 ± 1.24 μm, SD. In total, nine BCs derived from seven non‐apical divisions and 56 surround cells from six fish were used for analysis.

Neurogenesis and differentiation of vsx1+ progenitors are independent of each other

A. Schematic representation of expected outcomes if the immature retina (progenitors, P; neurons, N) is treated with HUA to delay the cell cycle. Upper panel: If there are multiple “fixed” progenitors, a block of cell division should stall progenitors at the differentiation state in which they normally would have undergone mitosis. The result would be a “salt‐and‐pepper” pattern of undifferentiated (light green) and differentiating progenitors (dark green). Lower panel: If cell cycle and differentiation are independent, all progenitors should homogenously differentiate. Open arrowheads indicate cytoplasmic processes not confined to the OPL and IPL, filled arrowheads indicate cytoplasmic processes confined to the synaptic layers.

B. Confocal images of a 2 dpf retina from a vsx1:GFP embryo injected with a p53 morpholino and mOrange2‐PCNA mRNA. A vsx1+ progenitor (dashed magenta outline) is shown before HUA treatment (left panel), at the time when it would have been “expected” to undergo mitosis (middle panel, orange diamond) and when it actually underwent mitosis (right panel, cyan circle). The retina and the vsx1+ progenitor continue to mature after the “expected” mitosis (retraction of cytoplasmic processes, mitosis at non‐apical location, and up‐regulation of GFP). Open arrowheads indicate cytoplasmic processes not confined to the OPL and IPL, filled arrowheads indicate cytoplasmic processes confined to the synaptic layers. Dotted lines indicate extent of vsx1+ cell somata across retinal thickness. Scale bar: 10 μm.

C. Progenitors that were expected to divide at the apical surface (exp), divided non‐apically (obs). 10 progenitors, four fish.

D. Quantification of vsx1:GFP fluorescence intensity of progenitors at the time when they were expected to undergo mitosis (exp, orange) and when they underwent mitotic division (obs, cyan). Data are presented as mean ± SEM, 10 progenitors, four fish. Mann–Whitney U‐test, ***P = 0.0002.

E. The fluorescence intensity of HUA‐treated vsx1:GFP progenitors at the time when they were expected to undergo mitosis (diamonds), and when they were observed to undergo mitosis (circles), plotted against the intensity of the surrounding cells (10 progenitors, four fish). One cell (cyan diamond) was expected to divide non‐apically. Green line indicates the fluorescence change of the lowest expressing progenitor for clarity.

In vivo time‐lapse recording of dividing BC progenitors in a vsx1:GFP retina

A.Confocal in vivo image of a 2 dpf vsx1:GFP retina. Expression of GFP, depicted using a “Fire” look‐up table (LUT), follows the retinal differentiation gradient, appearing first in the ventro‐nasal patch and subsequently in the nasal, dorsal, and temporal parts of the retina. Scale bar: 10 μm.

B.Time‐lapse images of the boxed region in (A). Left panels represent the raw levels of GFP fluorescence (Fire LUT). Right panels depict GFP fluorescence adjusted for better visualization (gray). Dashed circles mark mitotic events. Scale bar: 10 μm.

C. Individual mitotic events marked in (B) at progressively later times during development. GFP fluorescence levels of the mitotic progenitors and that of six cells in their surround. Orange circles, apical mitotic divisions; cyan circles, non‐apical mitotic divisions; gray squares, individual surrounding cells; black squares, average fluorescence of surrounding cells.

BC progenitors dividing in the laminated retina express Crx and ribeye a

A. Confocal image of a cryostat section of a 2 dpf crx:mCFP retina immunostained with antibodies against Crx and pH3. Co‐labeled cells are indicated by magenta dots. Scale bars: 10 μm.

B. In vivo image of a 2 dpf crx:mCFP retina. Expression of mCFP in the INL is largely limited to the “laminated” region (cyan bar above the figure panel) of the retina while only photoreceptors are labeled in the unlaminated parts of the retina (orange bar above panel). One crx:mCFP+ progenitor (cyan arrowhead) can be seen in an early stage of division, which judged by transgene expression levels, belongs to the most differentiated cells in this intermediate part of the INL. Scale bar: 10 μm.

C. In vivo time‐lapse images of a crx:mCFP+ progenitor undergoing mitotic division (pseudo‐colored cyan) in the INL of a 2 dpf embryo. Ninety‐seven such divisions were observed in four time‐lapse recordings of a total of 32.1 h. Scale bar: 10 μm.

D. Contrast inverted confocal images of an eye from a 2 dpf ctbp2:mEGFP transgenic fish immunostained to visualize GFP (left panel) and processed for fluorescence in situ hybridization to detect expression of a ribeye a specific exon (right panel). Scale bar: 10 μm.

E. High magnification of boxed area in (D). Expression of the ctbp2:mEGFP transgene and endogenous ribeye a mRNA is restricted to the INL in the laminated region of the retina (cyan bar over figure panel). Dashed line indicates onset of expression. Scale bar: 10 μm.

F. Confocal images of a 2 dpf ctbp2:mEGFP retina labeled to detect GFP, pH3, and ribeye a mRNA. A ribeye a+ pH3+ non‐apically dividing progenitor (magenta arrowheads, middle panel) can be seen in a laminated region of the retina (cyan bars above panels). Scale bar: 10 μm.

G. ctbp2:mEGFP+ progenitor cell undergoing mitotic division (pseudo‐colored cyan) in the INL during an in vivo time‐lapse recording of a 2 dpf retina. Eighty‐seven such divisions were observed in two time‐lapse recordings totaling 32.8 h. Scale bar: 10 μm.

Centrosomes of BCs are clustered at the OPL

A. In vivo confocal images of a 2 dpf retina in which centrosomes (centrin4 mRNA) and cellular membranes (BODIPY‐Texas Red) are labeled. The outer part of the INL is largely devoid of centrosomes. The emergence of the OPL (region to the right of the arrowhead) coincides with the clustering of centrosomes in this location. Solid gray line indicates the apical surface. Orange and cyan bars above the images represent unlaminated and laminated parts of the retina, respectively. Scale bar: 10 μm.

B. In vivo confocal images of a 2 dpf retina in which centrosomes (centrin4 mRNA) and a subset of BCs (Q19) are labeled. BC somata are devoid of centrosomes. The relocation of BC centrosomes from the apical surface (solid gray line) to the OPL coincides with the retraction of the apical process of BCs (arrowhead). Orange and cyan bars above the images represent unlaminated and laminated parts of the retina, respectively. Scale bar: 10 μm.

C. Confocal in vivo images of a 3 dpf retina with an isolated BC, in which the centrosome and cellular membranes are labeled with YFP and cerulean, respectively (plasmid encoding UAS:centrin4‐YFP/UAS:memCerulean was injected into Q26). The centrosome (green) is localized to the dendritic tuft at the OPL rather than in the soma. Scale bar: 5 μm.

D. Scheme illustrating distinct developmental steps (nucleokinesis, centrosome relocation, apical process retraction, transient arborizations of the apical process in the OPL and mitosis at the INL/OPL interface) for the vsx1+ progenitor from Fig 3D and E, observed by time‐lapse imaging. Nucleokinesis trajectories of the cell are color coded to indicate the local cytoarchitecture of the retina when these events took place (orange: unlaminated; cyan: laminated).

mOrange2‐PCNA allows for studying HUA‐induced delay of mitosis

A. Time‐lapse imaging of vsx1:GFP retinas expressing mOrange2‐PCNA was used to determine the time interval between the beginning of late S‐phase and the beginning of M‐phase for progenitors. In control fish the average time for this interval was 142 ± 48 min (mean ± 2 standard deviations, SD, 38 cells from six fish, orange circles represent single cells). HUA‐treated progenitors were significantly delayed compared to controls, with an average time interval of 534 ± 32 min (10 cells from four fish, P < 0.0001, Mann–Whitney U‐test, cyan circles). The delay with which each of the 10 HUA‐treated progenitors reached M‐phase was calculated by subtracting the average “expected” time interval between S‐ and M‐phase (obtained from control cells, orange diamond) from the observed time interval between S‐ and M‐phase for each HUA‐treated progenitor (cyan circles). An example of this calculation for the cell tracked in (B) is shown.

B. Time‐lapse confocal images of a HUA‐treated vsx1:GFP retina expressing mOrange2‐PCNA. Based on control experiments, the outlined cell (for which the delay in mitosis is depicted in A, cyan cell with a pink outline) would have been “expected” to undergo mitotic division around the 148 min time point (orange diamond; closest to the calculated mean “expected” time of 142 min), but entered M‐phase at 693 min (cyan circle). Scale bar: 10 μm.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ EMBO J.