(A–D) Crb1 (C, red) localizes primarily internally in photoreceptors of nok mutant retinas at 4.5 dpf. The arrows indicate the subcellular enrichment of Crb1. The inset in D shows a magnified photoreceptor. G/R are visualized in blue and actin is shown in green. D is the merged image of A–C.
(E–H) Crb2 (red) localizes primarily internally in photoreceptors of nok mutant retinas at 4.5 dpf. The arrows indicate the subcellular enrichment of Crb2. The inset in H shows a magnified photoreceptor. G/R are visualized in blue and actin is shown in green. H is the merged image of E–G.
The apical end of the retinas in panels A–L is oriented on top. Scale bars indicate 10 μm.

(A) A schematic drawing of Nok’s position in the inner segment and its spatial relationship with the OLM.
(B) The two mechanisms by which Nok participates in maintaining the integrity of the ONL: through establishing/maintaining the OLM, and likely through establishing a potential novel type of cell-cell adhesion complex between photoreceptors. The former mechanism likely involves the participation of Muller cells in wildtype retinas; whereas the latter may concern only photoreceptors and likely involves Crumbs proteins.

(A–D) Crb1 (red) localizes to the cell membrane regions in the inner segments of photoreceptors in the adult retina at 12 mpf. The arrowheads point out the Crb1 staining in the cell membrane regions between the members of G/R (blue). Green staining indicates the localization of actin. D is the merged image of A–C.
(E–H) Crb1 (red) localizes apically to the OLM, which is visualized by ZO1 staining (blue) and actin staining (I, green, arrowheads) in the adult retina. H is the merged image of E–G.
(I–L) Crb2 (red) localizes to the cell membrane regions in the inner segments of photoreceptors in the adult retina at 12 mpf. The arrowheads point out the Crb2 staining in the cell-cell junctional regions between the members of G/R (blue). Green staining indicates the localization of actin and the OLM is indicated with arrowheads (M). L is the merged image of I–K.
(M–P) Crb1 (red, arrows) localizes to the interior regions of the photoreceptor rosettes in N-cad mutant retinas at 4.5 dpf. G/R are visualized in blue and actin is shown in green. P is the merged image of M–O.
(Q–T) Crb2 (red, arrow) localizes to the interior regions of the photoreceptor rosettes in N-cad mutant retinas at 4.5 dpf. G/R are visualized in blue and actin is shown in green. T is the merged image of Q–S.
Sections A–L are oriented with apical side of the retina on the top. Scale bars indicate 10 μm.

(A–D) In wildtype retinal neuroepithelium, ZO-1(blue) colocalizes to the adherens junction-associated actin bundles (green) at 35 hpf (arrows). Nok (red) localizes immediately apical to the ZO-1 staining. D is the merged image of A–C. The asterisk indicates the lens. Apical side of the retina is oriented on top.
(E–H) In N-cad mutant retinas at 35 hpf, ZO-1 (blue) and actin bundle staining (green) indicate that retinal epithelial cells adhere to each other to form a ring-like structure (arrows) at 35 hpf. Nok (red) localizes apical to the ZO-1 staining in a more interior region within the cluster of cells. H is the merged image of E–G. The asterisk indicates the lens.
(I–L) In nok mutant retinas at 35 hpf, ZO-1(blue) and adherens junction-associated actin bundles (green) localize ectopically to the interior of the retinal neuroepithelium (arrows). Mutant Nok (red) does not concentrate to the adherens junctions (K). L is the merged image of I–K.
Scale bars indicate 10 μm.

(A–D) Muller glial cells extend processes basally into the inner limiting membrane (arrowhead) and apically into the OLM (arrow) in wildtype embryo retinas at 4.5 dpf. The Muller glial processes in the OLM (B, arrows, red) colocalize with actin staining (C, green, arrows) in the OLM (D, arrows). A merged image of B–C and the image of G/R (blue) is shown in D. The apical side of the neural retina is on top.
(E–H) Loss of N-cad function leads to disruption of retinal layers in N-cad mutant retinas at 4.5 dpf. However, the photoreceptor cells adhere to each other and form rosettes (E, bracket). The rosettes are visualized by staining for G/R (blue). Dashed lines indicate presumable photoreceptor cells other than G/R in a rosette (E). Strong actin staining (green) is found at the apical ends of the photoreceptors in the interior of rosettes (H, arrow). Processes of Muller glial cells (red) are not detectable between photoreceptors in the interior of the rosettes (F and H). A merged image of E–G is shown in H.
Scale bars indicate 10 μm.

(A–D) In wildtype retinas at 4.5 dpf, ZO-1 (blue) localizes to the OLM, basal to the Nok staining (red). The OLM is indicated by actin staining (A, arrow). A merged image of A–C is shown in D. Apical side of the retina is oriented on top.
(E–H) ZO-1 staining (blue) reveals the existence and location of the OLM-like structures in the interior regions of the rosettes in N-cad mutant retinas at 4.5 dpf (white arrows). The inset shows the magnification of a portion of the interior region, which is indicated by a white arrow in H. The Nok staining (red) inside the rosettes is concentrated more apically (black arrow) than that of ZO-1 (black arrowhead) (H, inset). A merged image of E–G is shown in H.
(I–L) ZO-1 forms rudimentary assemblies in nok mutant retinas at 4.5 dpf. Unlike Nok, subcellular accumulation of ZO-1 in photoreceptors is not affected by the loss of Nok function, as evidenced by the punctate ZO-1 staining in nok mutant retinas (J, red, arrows). These ZO-1 positive foci are also enriched with actin, as in the wildtype OLM (I and K, arrows), and they localize to membrane regions of the retinal cells that are presumably photoreceptors (L, arrows, asterisks). K is the merged image of I and J. L shows a magnified region of K.
Scale bars indicate 10 μm.

(A–D) Nok staining (C, arrowheads, red) localizes to cell-cell junctional regions in the inner segments at 6 dpf. Green/red double cones (G/R) are visualized by zpr1 antibody (blue). The OLM (A, arrowheads) is positioned basal to the Nok staining. The regions where Nok localizes are also enriched with actin (arrows, A). Asterisks indicate the localization of ellipsoids, which are the most apical regions in the inner segments and are enriched with mitochondria but negative for zpr1 staining. A merged image of A–C is shown in D. G/R is the abbreviation for green/red double cones.
(E and F) Tangential imaging of the inner segments reveals that Nok (red) is associated with the cell membrane of photoreceptors. Nok’s expression pattern resembles that of the OLM (green), which is immediately basal to Nok.
(G–J) Nok localizes in the inner segments of the adult retina at 12 mpf. Counter staining for G/R reveals that Nok localizes to the junctional interface between photoreceptors in the inner segments. The arrowheads indicate that the Nok staining is in the junctional regions between the pairing members of G/R, which extend into the distal region of the ONL. Asterisks indicate the localization of ellipsoids. Merged image of G–I is shown in J.
Sections, except for E and F, are oriented with apical side of the retina on top. Scale bars indicate 10 μm.

(A) The movement of wildtype ROP (rod opsin promoter)::GFP cells in wildtype retinas was observed live under a confocal microscope between 50 hpf and 51 hpf. The images of GFP-positive cells at the start time are shown in green and those taken one hour later in red. The images of the two time points are merged, which reveals that a majority of wildtype ROP::GFP cells are spatially stationary in the retina, even though some photoreceptors show tilting at certain angles (arrows). Apical side of the retina is oriented on top.
(B) The majority of ROP::GFP cells in nok mutant retinas between 50 hpf and 51 hpf move significantly during a period of one hour.
(C) The mobility of the GFP-positive cells in wild type, mutant, or mosaic retinas are surveyed and cells are cataloged into seven groups based on their mobility. The survey shows that over 70% of mutant ROP::GFP cells move significantly in the mutant retina, in the range of 2–6 um/hr; whereas over 70% of wildtype ROP::GFP cells fall into the category of 0–1 um/hr. Furthermore, wildtype ROP::GFP cells in nok mutant retinas show increased mobility compared to those in wildtype retinas but decreased mobility compared to those in the pure mutant retina. The number of cells surveyed is indicated in parentheses.
Scale bars indicate 10 μm. Numbers of cells examined are indicated in parentheses.

(A–D) In the N-cad mutant retina at 4.5 dpf, Nok (C, red, arrows) is only found in the interior regions of photoreceptor rosettes, where actin is also enriched. Blue indicates G/R. Dashed lines in D indicate the regions presumably occupied by other types of photoreceptors. A merged image of A–C is shown in D.
(E–H) In the nok mutant retina at 4.5 dpf, photoreceptors do not form rosettes but rather scatter randomly at 5 dpf. G/R are visualized in blue. Nok staining (red) in retinal cells is diffuse. Some Nok positive cells are zpr1-positive as well, indicating that these are G/R (arrows); other Nok-positive cells are zpr1-negative (arrowheads), indicating that they are other types of photoreceptor cells. H is the merged image of E–G.
(I). Western Blot analysis of protein extract of about one fifth of an adult eye (lane 1) and the extract of 190, 000 purified rods (lane 2) demonstrates that rods express Nok. Molecular weight markers are indicated on the right. The arrow indicates the position of full length Nok protein. Two weaker bands are noticeable in lane 2 between 50 and 37 kDa markers; they likely derive from the degradation of Nok during the process of rod isolation.
Scale bar indicates 10 μm.