Heterogeneity in mitotic position and behaviour of HCprs. (A) Scheme of zebrafish retina. Neurons: photoreceptors (PRs, magenta); horizontal cells (HCs, green); bipolar cells (BCs, yellow); amacrine cells (ACs, blue) and retinal ganglion cells (RGCs, magenta). Layers: outer nuclear layer (ONL), inner nuclear layer (INL), ganglion cell layer (GCL), outer plexiform layer (OPL) and inner plexiform layer (IPL). (B) Montage of HC lamination 36 hpf-76 hpf. lhx-1:eGFP(HC and PR, green), ptf1a:dsRed (AC and HC, magenta). Scale bar: 10 µm. (C) Montage representing a typical example of HCpr mitosis length. Time is relative to mitosis onset. Red arrowhead indicates pre-mitotic protrusive cell; yellow dot indicates mitotic cell; red dots indicates sister cells after division; and dotted white line indicates IPL. Scale bar: 5 µm. (D) Position and angle of HCpr mitosis is heterogenous. Yellow arrowheads indicate mitotic HCprs; white dots indicate sister HCs after division; white dotted line indicates OPL (top) and IPL (bottom). Scale bar: 10 µm. (D′) Percentage distribution of HCpr position of division. (E) Position (µm), (F) duration (min) and (G) angle of HCpr mitosis. (H,I) Frequency of mitotic (H) position and (I) duration. (J) Plot of mitotic duration versus position shows no strong correlation. Regression analysis was carried out using Pearson's correlation (r).

HCprs undergo long heterogenous cell cycles. (A) Montage of the HCpr cell cycle. Trβ2:tdTomato (HCpr, green) and PCNA-GFP (nuclei, magenta). White arrowheads indicate the tracked cell. The OPL is indicated by a dotted white line. Scale bar: 10 µm. Bottom panels show Trβ2:tdTomato and PCNA-GFP individually. Scale bar: 5 µm. (B) HCpr cell cycle phases occur at different positions in the INL. Trβ2:tdTomato (HCpr, green) and PCNA-GFP (nuclei, magenta) are shown. White arrowheads indicate the cell that was followed. The OPL is indicated by a dotted white line. Scale bar: 10 µm. Bottom panels show Trβ2:tdTomato and PCNA-GFP individually. Scale bar: 5 µm. (C) Distribution of HCpr cell cycle length. Line indicates the mean. (D) Scatter plot of HCpr cell cycle length versus HCpr position shows a lack of strong dependence. Regression analysis was carried out using Pearson correlation (r). (E) Distribution of HCpr cell cycle phase lengths: S, G2 and M. Lines indicate the means.

Bi-directional HC migration is heterogenous but HCprs always stopover within the amacrine cell layer and heterogeneity in HC depth depends on IPL formation. (A) Montage of HC migration from HCpr birth to final positioning. lhx-1:eGFP (HC and PR, green) and Utrophin-mCherry (cell outline, magenta). Red dot indicates tracked cell; white arrowhead indicates apical process before detachment; red and cyan dots indicate sister HCs after terminal division. Time is relative to HC birth. White lines indicate apical surface (top) and basal membrane (bottom). White dotted lines indicate OPL (top) and IPL (bottom). Scale bar: 5 µm. (B) Scatter plot of HC birth to max apical position. Data are mean±s.d. (C) Single cell trajectories of HC bidirectional migration from birth to final positioning as a percentage of the total thickness of the retina. The orange and green lines provide examples of a shallow and a deep track, respectively. The shadow indicates the initial stage of migration. (D) Mean square displacements±s.d. of HC for different phases; from birth to apical process retraction (solid line) from retraction to final positioning (dashed line). (E) Representative examples of HC migration along the apical-basal and nasal-temporal retinal axis. (F) Distribution of HC maximum migration depth. Data are mean±s.d. with individual data points indicated. (G) Percentage of HC maximum migration depth relative to the retinal thickness, before (black circles) and after (magenta circles) IPL (black line) formation. (H) Examples of HC migration before and after IPL formation. lhx-1:eGFP (HC and PRs, green) and ptf1a:dsRed (ACs and HCs, magenta). Solid and dashed white lines indicate apical surface and IPL, respectively. White arrowheads indicate HC position. Scale bar: 10 µm.

(A) HCs subtypes and markers in the zebrafish retina. B-C) Typical examples of HCprs labelled with (B) Connexin55.5:rasGFP (Cx55.5) and (C) Trb2:tdTomato. Red arrowheads point to HCs within the HC layer. Dashed line represents the OPL. Scale bar 10μm. Insets of the red-boxed region shows magnified HC. Scale bar 5μm. (D) Schematic showing measurement of HC distance from the OPL at rounding (left) (analyzed data can be found in Figure 1E and divided by marker the same data is seen in Supplementary Figure 1E) and HC angle of division (right) (analyzed data can be found in Figure 1G). Scale Bar, 5 μm. (E) Position of mitotic HCprs for different HC marker populations. (The pooled version of this analysis can be found in Figure 1E this is the same data divided by marker) (F) Schematic showing depth of migration measurements. Relative thickness of the INL was quantified by drawing a line (D) from the OPL to the IPL using Fiji Line tool. To quantify HC position, a line (d’) was drawn from the center of HC to the OPL. Relative percentage of HC depth was defined by dividing d’/D. For earlier developmental stages before the IPL formation, thickness of the INL was measured once the IPL was formed within the same region (analyzed data can be found in Figure 3 F and G).

Heterogeneity in mitotic position and behaviour of committed HCpr subtypes. (A) Rare example of HCpr dividing in the RGC layer. ptf1a:GAL4-VP16, UAS:gap-YFP (green, HCpr and AC); ath5:RFP (PR and RGC). White arrow: mitotic HCpr. White dashed line: OPL (top), IPL (bottom). Scale Bar, 10 μm. (B) Spread of metaphase to anaphase duration in mitotic HCprs. (C) Example of HCpr spindle rocking before mitosis. lhx-1:eGFP (HCpr, green); H2Bmcherry (nuclei, magenta), white arrows: position of metaphase plate. White dashed line: the OPL (top), the IPL (bottom). Scale Bar, 5 μm. (D) Distribution of HCpr mitotic angle versus position does not show a strong correlation. (E) Distribution of HCpr angle versus length of mitosis does not show a strong correlation.

Acknowledgments
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