mok^s309 Mutants Have a Complex Retina Phenotype

(A and B) Whole-mount lateral views of live 4.5 dpf zebrafish larvae. In mok^s309 mutants (B), the eyes are smaller than in the wild-type (A), but external morphology is otherwise indistinguishable.

(C and D) Horizontal sections of 5 dpf retinas stained with DAPI (blue) and Zpr1 antibody (green). mok^s309 retinas (D) have an expanded GCL (dashed white lines), compared to wild-type (C), and no photoreceptors, as revealed by Zpr1 staining.

(E and F) Coronal sections of 3 dpf retinas stained by TUNEL assay. mok^s309 retinas (F) have increased apoptosis, particularly in the photoreceptor layer (asterisks), compared to wild-type (E).

(G and H) Horizontal sections of 48 hpf retinas expressing GFP driven by the atoh7 promoter (green) and stained with DAPI (blue) and HuC/D antibody (red). mok^s309 retinas (H) have an increased number of GFP-expressing RGCs compared to wild-type (G).

Scale bars, 500 μm (A and B) and 100 μm (C–H).

mok^s309 Retinas Have Increased RGCs and Decreased Bipolar and Müller Glia Cells Due to Premature Neurogenesis

(A–H) Horizontal 5 dpf retina sections. GFP expression under the control of atoh7 (A and E) and Brn3c (Pou4f3) (B and F) promoters reveals an increase in RGCs in mok^s309 (E and F) as compared to the wild-type (A and B), with some RGCs ectopically located outside the GCL (yellow arrowheads in [E] and [F]). The Müller glia marker GS (C and G) and the bipolar cell marker PKCα (D and H) show fewer immunoreactive cell bodies (yellow arrowheads) in mok^s309 (G and H) as compared to wild-type (C and D).

(I–P) Horizontal 50 hpf retina sections stained for IdU (green, injected at 26 hpf), BrdU (red, injected at 38 hpf), and DAPI (blue). mok^s309 retinas (M–P) show more IdU-positive and BrdU-negative cells than wild-type, indicating that a larger number of progenitors had exited the cell cycle between 26 and 38 hpf.

Scale bars, 100 μm.

Cell Transplantation Analysis Reveals that mok Acts Cell Autonomously

(A–D) Representative sections of 5 dpf chimeric retinas. The transplanted cells are clearly identified by the expression of H2A-GFP marker (green) in their nuclei. Cell transplantation shows that mok^s309 mutant clones in wild-type host retinas (B) have a higher propensity to generate neurons located in the GCL compared to control (A). Conversely, wild-type clones in mok^s309 host retinas preferentially generate INL neurons (C). mok^s309 mutant clones in mok^s309 host retinas are shown for comparison (D). Dashed lines indicate the outer limit of the GCL. Scale bars, 100 μm.

(E) Quantification of the transplantation results showing the distribution of clones in the three retinal nuclear layers. ^***p < 0.001; ^*p < 0.01. Error bar indicates SEM.

Expression of Dnct1 and Its Function in INM

(A) Western blotting of extracts from 4 dpf embryos shows that Dnct1 is undetectable in mok^s309.

(B) Time course analysis of Dnct1 expression by western blot in wild-type zebrafish (numbers on top indicate hours after fertilization).

(C and D) Whole-mount in situ hybridization shows dnct1 enriched in the head and eye region (C) and in the notochord (D). Scale bars, 100 μm.

(E and F) Coronal sections of 2 dpf retinas stained for the mitotic marker PH3. In mok^s309 (F), a number of mitotic cells are sparsely located throughout the retina, while, in the wild-type (E), they are confined within 2–3 cell diameters from the ventricular surface. Dashed lines demarcate the apical (right) and basal (left) domains. Asterisks indicate mitotic cells residing in the developing lens.

(G) Scatter plot of wild-type (gray triangles) and mok^s309 mutant (black squares) circles, showing the maximum basal distance of nuclei during INM from 30–48 hpf.

(H) Histogram showing that these populations are statistically different (p = 0.001, Wilcoxon two-sample test). Error bar indicates SEM.

A Gradient of Notch Signaling along the Apical-Basal Axis of the Developing Retina

(A–C) Coronal sections of 26 hpf retinas showing mRNA expression levels of components of the Notch/Delta signaling pathway. In situ hybridization shows higher levels of notch1a close to the apical surface of the retina (A) and deltaB and deltaC close to the basal surface (B and C).

(D) Optical section of a 33 hpf retina expressing her4:dRFP and H2A-GFP transgenes in a mosaic manner.

(E and F) Coronal sections of 26 hpf retinas stained with anti-DeltaC antibody, showing punctate cytoplasmic staining distributed in the basal half of the tissue both in wild-type and mok^s309 retinas.

(G and H) Coronal sections of 26 hpf retinas stained with anti-Dnct1 antibody, showing an enrichment at the apical surface in wild-type retinas (G), which is virtually absent in mutants (H).

(I) Coronal section of 26 hpf retina; α-tubulin staining reveals the parallel orientation of microtubules to the apical-basal axis.

(J–L) Sections of mouse retina. Activated Notch1 antibody labels a subset of nuclei at the apical surface (J). Anti-Dnct1 and anti-BBS4 antibodies show a cytoplasmic, punctated staining enriched at the apical surface. γ-tubulin staining (G, H, K, L) reveals the apical localization of the centrioles in retinal progenitors. In (D)–(L), DAPI (blue) stains the nuclei. In all panels, apical surface is on the left.

Scale bars, 25 μm (A–I) and 50 μm (J–L).

Atoh7 Loss of Function and Notch Activation Can Rescue Late Cell Fates in mok^s309

(A–D) Compared to wild-type (A), mok mutants (B) have an excess of RGCs and few bipolar cells, labeled by PKCα immunoreactivity. lak (atoh7) mutants fail to generate RGCs and overproduce bipolar neurons (C). lak/mok double mutants have rescued bipolar cell production.

(E–H) Heat-shocked induction of an activated form of Notch receptor NICD at 32 hpf drives progenitors toward a Müller glia fate (GS positive) in both wild-type (G) and mok^s309 retinas (H). The presence of the transgenes (hsp:Gal4, UAS:NICD) has no effect on Müller glia differentiation in the absence of heat shock (E and F).

Scale bars, 100 μm.

Disruption of the Nuclear Anchor to Dynactin Phenocopies the mok^s309 Mutation

(A and B) Expression of the Müller glia marker GS is reduced in the retina of larvae injected with a syne2a-MO (B) compared to control MO-injected larvae (A). Scale bars, 100 μm.

(C and D) Representative examples of sections of 5 dpf retinas overexpressing a control vector (C) or a dominant-negative Syne2a (KASH, [D]) under the control of a heat-shock promoter. The cells that express the constructs are identified by the expression of GFP marker (green). Clones of cells that express the syne2a dominant-negative construct preferentially generate GCL neurons. Scale bars, 50 μm.

(E) Quantification of the KASH overexpression results, showing the distribution of clones in the three retinal nuclear layers. ^*p < 0.01, ^**p < 0.005. Error bar indicates SEM.

(F) A model of the mechanism that couples INM with graded Notch activation.

moks³⁰⁹ Embryos Have Increased Apoptosis Localized in the Eye/Head Region
(A, B) Whole-mount TUNEL staining of 2.5 dpf embryos. mok mutants (B) show a dramatic increase of apoptotic cells in the eye and in the head, compared to the wildtype
(A). Scale bars: 500mm.

atoh7 Expression Is Prolonged in moks³⁰⁹ Retinas
(A-F) Lateral eye view showing atoh7 expression, detected by in situ hybridization. atoh7 expression is normal during early retinogenesis in moks³⁰⁹ mutants (26 and 36 hpf, compare [A, B] with [D, E]). Later, at 42 hpf, when atoh7 expression in wildtype is restricted to the ciliary marginal zone at the periphery of the retina (C), moks³⁰⁹ retinas show significantly higher expression (F). Scale bars: 50mm.

moks³⁰⁹ Retinas Have Increased RGCs and Decreased Bipolar Cells
(A, C) In situ analysis in 5 dpf retinas shows that mok^s309 mutants have ectopic cells expressing the RGC marker brn3b outside the GCL (arrows in [C]).
(B, D) In situ analysis of the bipolar neuron marker vsx1 shows that its expression is undetectable in mok^s309 5 dpf retina sections.

moks³⁰⁹ Retinas Have Normal Number of Amacrine and Horizontal Cells
(A, B) 5 dpf retina section showing pax6:GFP expression (green) and stained with antiparvalbumin antibody (red). Both markers identify a subset of amacrine cells. These cells are specified in normal number in mok^s309 mutants although their nuclei are sometimes positioned at a greater distance from the inner plexiform layer (IPL) (compare white arrowheads in [A] versus [B]).
(C, D) 5 dpf retina section stained with anti-GAD65/67 antibody. This marker labels both amacrine cells (yellow arrowhead) and horizontal cells (white arrowheads). Their specification is unaffected in mok^s309 mutants, but amacrine cell nuclei are often mislocalized (compare yellow arrowheads in [C] versus [D]).

Premature Cell-Cycle Exit in moks³⁰⁹
(A-D) 40 hpf retina section stained for BrdU (red, 12 hours after injection), PCNA (green, labelling proliferating cells), and DAPI (blue). mok^s309 mutant retinas (E-H) show increased BrdU positive and PCNA negative cells indicating increased cell cycle exit in the time between the BrdU injection and the fixation. Scale bars: 100mm.

Phenocopy of moks³⁰⁹ Phenotype by Morpholino Knockdown of Dynactin-1 Function
(A-C) Retina sections of 5 dpf embryos injected with splicing morpholino against dynactin-1 gene (dnct1Mo) or control morpholino (ctrlMo). Dnct1Mo injection resulted in a mild (B) or severe phenotyope (C) overall resembling the mok^s309 phenotype in thickening of the GCL (B,C) and disruption of ONL (C).
(D, E) Absence of opsin-GFP localization in dnct1Mo injected embryos was observed in the transgenic line expressing a GFP fused to the C-terminal 44 amino acids of Xenopus rhodopsin, in a subset of photoreceptors (Perkins et al., 2002). When GFP signal was detected in the injected embryos it was mislocalized around the nucleus as well as in the photoreceptor outer segment (E′) as reported in mok (Doerre and Malicki, 2001).

moks³⁰⁹ Mutants Have Normal Apical-Basal Marker Distribution
(A-D) 28 hpf retina sections stained with an antibody recognizing the basal lamina marker Laminin (green in [A, B]) and the apically distributed markers aPKCegreen in [C, D]) and the cell junction component ZO-1 (red in [C, D]). No obvious difference is detected between mutant (B, D) and wildtype (A, C) retinas.
(E,F) 28 hpf retina section stained with phalloidin (red) to detect a-actin and expressing Pard3-GFP (green). The majority of the Pard3-GFP protein is localized at the apical surface where it overlaps with the actin rich adherens-junctions. Nuclei of cells expressing the Pard3-GFP construct are marked by white stars. Nuclei counterstained with DAPI in blue. Scale bars 25 μm.

Retinal Neuroepithelia Show Changes in Notch Activity with Nuclear Migration
A. Montage of selected confocal planes showing decrease of the fluorescence signal in her4:dRFP pixel intensity with basal-directed nuclear migration.
B. Montage of the same cell later in development showing increased pixel intensity with apical-directed migration. In 11/15 cells moving nuclei apical to basal dRFP intensity decreased, while in 13/15 cells moving nuclei from basal to apical dRFP intensity increased. Time (hr:min) from initial pixel measurements is indicated in upper right. Integrated pixel intensity from the 543nm channel is indicated next to the region of interest. Apical surface is located in the upper right of each panel, while basal surface is to the lower left.

Decreased BBS4 Localization in moks³⁰⁹ Mutants
(A, B) 24 hpf retina sections stained for BBS4 (green) and g-tubulin (red). in wildtype embryos BSS4 protein localizes in aggregates near the apical surface of the retina (A). This localization is greatly reduced in mok^s309 mutants (B). Nuclei counterstained with DAPI in blue. Scale bars 25 μm.

Intact Localization of the Endocytic Compartment in dnct1Mo- Injected Embryos
(A-D) Optical sections of 24 hpf live embryo retinas, transiently expressing the endocyting compartment markers Numb and Rab11 GFP fusion constructs (EGFP-Nb1 and EGFP-rab11, respectively). No differences were detected in the localization of both proteins in dnct1Mo or ctrlMo injeceted embryos.