The Journal of Neuroscience, November 2, 2005, ():
Function of Atypical Protein Kinase C 
in Differentiating Photoreceptors Is Required for Proper Lamination of Mouse Retina
J. Neurosci. Koike et al.
25: 10290
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Supplemental figure 1. X-gal staining of the embryonic retina
X-gal staining of the retina from Crx-Cre/CAG-CAT-Z mouse (A, B) and CAG-CAT-Z mouse (C, D) at E12.5 (A, C) and E16.5 (B, D). Scare bar, 100 μm. We could detect the X-gal staining at the outermost layer of the retina where photoreceptor committed cells are located (A, B). The X-gal signal in the inner layer (A, B) is considered to be background because we could detect the signal also in the inner layer of the control retinas (C, D) when treated with X -gal.
- supplemental material
-
Supplemental figure 2. Progenitors in Crx-Cre retina do not express Cre recombinase.
Immunostaining of Crx-cre retinas at E11.5 (A, D, G), E12.5 (B, E, H) and E15.5 (C, F, I).
(A-C) Progenitors were immunostained with anti-Ki-67 antibody (green). Ki-67-positive cells were distributed throughout the layer except in the innermost layer and also in the part of the outermost layer.
(D-F) Cre recombinase expressing cells were immunostained with anti-Cre recombinase antibody (red). At E11.5, Cre-expressing cells were not detected (D). At E12.5 and E15.5, Cre-expressing cells were localized at the outermost layer where photoreceptor-committed cells are normally localized (E, F).
(G-I) Merge image of Ki-67-positive and Cre-expressing cells. Note that the anti-Ki-67 and anti-Cre signals do not overlap each other. Scale bar, 50μm.
- supplemental material
-
Supplemental figure 3. Immunostaining by anti-beta galactosidase antibody and Ki-67 antibody
The E12.5 retinas of Crx-Cre/CAG-CAT-Z (A), and CAG-CAT-Z (B) mice were double stained with rabbit anti-beta-galactosidase antibody (red, Cappel, Aurora, OH) and anti-Ki-67 antibody (green). Inner limiting membrane (ILM) is indicated by white dotted line. Scale bar shows 25μm. Arrows indicate beta-galactosidase positive cells located at the outermost layer; arrowheads indicate cells located at inner layer. (C) Expression pattern of Crx mRNA at E14.5. Some of the Crx-expressing cells are localized at the inner layer (arrowheads). We consider that beta-gal positive cells located in the inner layer (A, arrowheads) are migrating photoreceptor-committed cells such as detected in C (arrowheads).
- supplemental material
-
Supplemental figure 4. Phalloidin staining and immunostaining by anti-Brn3b antibody
The layer structure was detected by staining with phalloidin (green) in the control retinas (A, C) and aPKCλ CKO retina (B, D) at E11.5 (A, B) and E15.5 (C, D). At E15.5, laminar structure was largely disorganized in the CKO retina (D), but the innermost layer (indicated by star) was not affected and showed organized laminar structure. (E) Ganglion cells were detected by immunostaining with an anti-Brn3b antibody (E15.5). Double star indicates the inner layer. (B’, D’ and E’) The same pictures of B, D and E, and outer limiting membrane (OLM) and ILM are indicated by white dotted line. Scale bar, 50μm. Immunostaining results show that some ganglion cells were dispersed in the E15.5 retina, but others are properly located in the innermost layer (designated by double star), which are considered to be early-differentiated ganglion cells.
- supplemental material
-
Supplemental figure 5. The expression of aPKCλ, Par3 and Par6 is disrupted in aPKCλ CKO retina.
(A-F) Immunostaining of aPKCλ (A, B), Par3 (C, D) and Par6 (E, F) in control (A, C, E) and aPKCλ CKO retina (B, D, F) at E14.5. Arrows indicate apical edge. Scale bar, 10μm. (G) Western blot analysis of aPKCλ, Par3, Par6 and aPKCζ in control and aPKCλ CKO retina at P6. Reduction of aPKCζ protein in CKO retina is considered to reflect the reduction of aPKCλ because of the cross-reactivity of the anti-aPKC ζ antibody to aPKCλ.
- supplemental material
-
Supplemental figure 6. N-cadherin, β-catenin and afadin are colocalized with aPKCλ.
(A-C) Immunostaining of aPKCλ (red) and β-catenin (A, green), N-cadherin (B, green) and afadin (C, green) in wild type retina at E15.5.
Nuclei were counterstained with TOTO-3 (blue). Scale bar, 10μm. Arrows indicate apical edge.
- supplemental material
-
Supplemental figure 7. S-phase retinal progenitors were dislocated in the aPKC lambda CKO retina.
Freshly dissected E15.5 control retina (A) and aPKCλ CKO retina (B) were incubated with BrdU and S-phase retinal progenitors detected by anti-BrdU antibody. Outer limiting membrane (OLM) is indicated by a white dotted line. Scale bar, 50μm.
- supplemental material
-
Supplemental figure 8. aPKCλ is essential for polarity formation in photoreceptor and complete retinal lamination
(A) Scheme of embryogenesis, neurogenesis and gliogenesis in neural retina and correlation with lamination defect in aPKCλ CKO retina. Neurogenesis and gliogenesis begin around at E11.5 and E17, respectively, in neural retina. Cre expression begins around E12.5 in postmitotic photoreceptors under the control of the Crx promoter. Note that the lamination defect in aPKCλ CKO retina is observed before gliogenesis begins.
(B) Model illustration explains how aPKCλ is essential for polarity formation of photoreceptor and complete retinal lamination. In a normal retina, aPKCλ first contributes to AJC formation between differentiating photoreceptor cells or between differentiating photoreceptor cells and progenitors by establishing adherens junction. Subsequently, differentiating post-mitotic cells, such as ganglion cells, migrate to their predicted layer. Photoreceptor-committed cells polarize and become mature photoreceptors. In aPKCλ CKO retina, photoreceptor-committed cells fail to form AJC. Progenitors then fail to anchor at the apical edge due to lack of AJC formation with photoreceptor-committed cells. Progenitors differentiate in abnormal positions and fail to migrate into the predicted layer. Photoreceptor-committed cells fail to polarize and scatter.
- supplemental material
-
Supplemental figure 9. N-cadherin and β-catenin are colocalized with aPKCλ.
(A, B) Immunostaining of aPKCλ (red) & N-cadherin (A, green), and β-catenin (B, green) in wild type retina at E11.5. Nuclei were counterstained with TOTO-3 (blue). Scale bar, 10μm. Note that adherens junctions are formed before photoreceptors are generated.
