Role of PKN1 in Retinal Cell Type Formation

Abstract

We recently identified PKN1 as a developmentally active gatekeeper of the transcription factor neuronal differentiation-2 (NeuroD2) in several brain areas. Since NeuroD2 plays an important role in amacrine cell (AC) and retinal ganglion cell (RGC) type formation, we aimed to study the expression of NeuroD2 in the postnatal retina of WT and Pkn1^-/- animals, with a particular focus on these two cell types. We show that PKN1 is broadly expressed in the retina and that the gross retinal structure is not different between both genotypes. Postnatal retinal NeuroD2 levels were elevated upon Pkn1 knockout, with Pkn1^-/- retinae showing more NeuroD2⁺ cells in the lower portion of the inner nuclear layer. Accordingly, immunohistochemical analysis revealed an increased amount of AC in postnatal and adult Pkn1^-/- retinae. There were no differences in horizontal cell, bipolar cell, glial cell and RGC numbers, nor defective axon guidance to the optic chiasm or tract upon Pkn1 knockout. Interestingly, we did, however, see a specific reduction in SMI-32⁺ α-RGC in Pkn1^-/- retinae. These results suggest that PKN1 is important for retinal cell type formation and validate PKN1 for future studies focusing on AC and α-RGC specification and development.

Keywords: NeuroD2; amacrine cells; protein kinase N1; retinal development; retinal ganglion cells.

Figure 4

Pkn1^−/− retinae show a reduced number of α-RGC. (a) Retinal sections prepared from P10 old littermates were stained for calbindin and SMI-32. Scale bar refers to 20 µm. SMI-32⁺ cells in the RGC layer per 500 µm were analyzed in a blinded manner (data are presented as individual n-values, referring to separate animals, with mean ± S.E.M., one-way ANOVA with Tukey’s multiple comparison test, (*) p < 0.05, (**) p < 0.01, ns not significant). (b) Adult retinal flat mounts were stained for SMI-32 and the pan-RGC marker RBPMS. Upper scale bar refers to 100 and lower scale bar refers to 50 µm. (c) The total amount of SMI-32⁺ cells (137 µm × 137 µm; data are presented as individual n-values, referring to separate animals, with mean ± S.E.M., (***) p < 0.001, unpaired t-test), and (d) the relative amount of SMI-32⁺ cells as a percentage of RBPMS⁺ cells per field was analyzed (137 µm × 137 µm; data are presented as individual n-values, referring to separate animals with mean ± S.E.M., (**) p < 0.01, unpaired t-test). (e) Optic nerve fibers were stained for SMI-32. Pictures are representative of 3 animals per genotype. Scale bar refers to 50 µm. (f) Optic tracts were stained for SMI-32. Pictures are representative of two animals per genotype. Scale bar refers to 50 µm.

Figure 1

Pkn1 knockout results in elevated developmental retinal NeuroD2 levels. (a) Retinal sections from P10 old WT animals were probed for PKN1 expression by RNAscope in situ hybridization. (b) There were no differences in retinal layer thickness between P10 old littermates (data are presented as individual n-values, referring to separate animals, with mean ± S.E.M., p > 0.05, one-way ANOVA). Representative image stained with Hoechst to assess layer thickness. Analysis was performed by an experimenter blinded to the genotype. (c) Representative image of adult WT and Pkn1^−/− retinae, stained with Hoechst, to assess retinal layer thickness. (d) Retinal layer thickness of adult WT and Pkn1^−/− retinae was not different. Analysis was performed by an experimenter blinded to the genotype (data are presented as individual n-values, referring to separate animals, with mean ± S.E.M., p > 0.05, unpaired t-test). (e) The number of ONL rows was not affected by Pkn1 knockout (data are presented as individual n-values, referring to separate animals, with mean ± S.E.M., p > 0.05, unpaired t-test). (f) Retinal protein extracts prepared from P10 old littermates were tested for NeuroD2 expression by Western blotting. NeuroD2 levels were related to the loading control GAPDH (data are shown as individual n-values, referring to separate animals, with mean ± S.E.M., one-way ANOVA with Dunnett’s multiple comparison test, (*) p < 0.05, ns not significant). (g) Expression of NeuroD2 was analyzed in retinal sections prepared from P10 old littermates. Layers are labelled in the Hoechst channel and magnified pictures of the INL (dashed rectangles) are shown with NeuroD2 and Hoechst combined. The analysis of the number of NeuroD2⁺ cells in the INL was performed by an experimenter blinded to the genotype (data are shown as individual n-values, referring to separate animals with mean ± S.E.M., one-way ANOVA with Dunnett’s multiple comparison test, (*) p < 0.05, ns not significant). All scale bars refer to 50 µm. INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; OPL: outer plexiform layer; RGC: retinal ganglion cell layer.

Figure 2

Pkn1 knockout affects retinal amacrine cells. (a) Retinal sections prepared from P10 old littermates and adult animals were stained for Dab1. The number of Dab1⁺ cells in the INL was analyzed (data are presented as individual n-values, referring to separate animals with mean ± S.E.M. (*) p < 0.05, (**) p < 0.01, unpaired t-test). (b) High-magnification images of the INL of P10 retinae stained for NeuroD2 (magenta), Dab1 (green) and Hoechst. Images are representative of 3 animals/genotype. (c) Retinal sections prepared from P10 old littermates and from adult animals were stained for calretinin. Images are representative of 3–8 separate animals per genotype per age. (d) Retinal sections prepared from P10 old littermates and from adult animals were stained for calbindin. Images are representative of 3–5 separate animals per genotype per age. Arrowhead refers to horizontal cells. All scale bars refer to 50 µm. INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC retinal ganglion cell layer. All analyses are presented in Table 1 and Table 2.

Figure 3

Pkn1 knockout does not affect total RGC numbers or chiasm formation. (a) Total amount of RGCs, stained with the pan-RGC marker RBPMS, was not different in retinal flat mounts of WT and Pkn1^−/− animals (field refers to a 137 µm × 137 µm square; data are presented as individual n-values, referring to separate animals, with mean ± S.E.M. p > 0.05, unpaired t-test). Scale bar refers to 50 µm. ONH: optic nerve head. (b) Chiasm formation is not affected by Pkn1 knockout. The neuronal tracers DiI and DiD were applied to retinae of E15.5 embryos. Chiasm width, length, optic nerve (ON) and optic tract (OT) diameter were analyzed by an experimenter blinded to the genotype (data are presented as individual n-values, referring to separate animals, with mean ± S.E.M. p > 0.05, unpaired t-test). Scale bar refers to 100 µm.