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First published online November 21, 2008
doi: 10.1242/10.1242/dev.027912

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-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina

¹ Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA.
² Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

View larger version (109K): [in this window] [in a new window]   Fig. 1. Pcdh- genes are broadly expressed in the retina. (A-D) Retinas from Pcdh- fusg/fusg mice, expressing a Pcdh--GFP fusion protein, were stained with antibody to GFP (green in A) and DAPI (blue in A). Pcdh--GFP fusion proteins are concentrated in the process-rich outer plexiform layer (OPL), inner plexiform layer (IPL) and retinal fiber layer (arrow), as well as in outer segments (OS) of photoreceptors, outer and inner nuclear layers (ONL, INL) and the ganglion cell layer (GCL). (B-D) High-magnification images of areas labeled in A'. (E) In situ hybridization to P21 retina using a probe against the Pcdh- common intracellular domain. (F-K) Dissociated Pcdh- fusg/fusg retinal cells immunolabeled with cell-type-specific antibodies (red), anti-GFP (green) and DAPI (blue). Pcdh--GFP-positive cells are labeled with the photoreceptor (PR) marker recoverin, the horizontal cell (HZ) marker calbindin, the amacrine (AC) marker syntaxin, the RGC marker Brn3a, and the Müller Glia (MG) markers glutamine synthetase (H) and Sox9 (K). Scale bars: 50 µm in A,E; 10 µm in B-D.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Pcdh- proteins localize to retinal plexiform layers during postnatal development. (A-D) Immunolabeling of Pcdh- fusg/fusg retina at P0 (A), P3 (B), P7 (C) and P14 (D) with GFP antibody. At P0 (A) and P3 (B), Pcdh--GFP proteins are present in the IPL and retinal axon layer (arrow), and are distributed around cell somata in the neuroblast layer (NBL; other abbreviations as in Fig. 1). At P3, Pcdh- proteins are also present in presumptive horizontal cells (asterisk; inset in B). (C) By P7, Pcdh--GFPs are detected in the emerging OPL, as it develops between the ONL and INL. (D) By P14, the adult pattern of Pcdh--GFP localization (see Fig. 1) is attained. Scale bars: 20 µm in A; 10 µm in inset.

View larger version (122K): [in this window] [in a new window]   Fig. 3. Subcellular localization of Pcdh- proteins in the synaptic plexiform layers. (A-E) Confocal images of the OPL of Pcdh- fusg/fusg retinas double labeled with anti-GFP (green) and antibodies against proteins present in the synapses that photoreceptors form on horizontal and bipolar cells (red). (A) Bassoon, a component of synaptic ribbons in all photoreceptor nerve terminals. (B) PSD-95, a component of rod terminals (spherules). (C) Peanut agglutinin (PNA) labels cone terminals (pedicles). (D) PKC a component of rod bipolar dendrites (arrow). (E) Calbindin, a component of horizontal cell processes (open arrow) that terminate onto rod spherules as well as horizontal cell processes that stratify in the inner OPL (open arrowhead). Pcdh- proteins are present in both spherules and pedicles, and in bipolar and amacrine processes, but are not seen in horizontal cell varicosities (open arrow). (F) A single confocal plane of the IPL of Pcdh- fusg/fusg retinas labeled with anti-GFP (green) and antibodies to PSD-95 (red), which marks excitatory postsynaptic sites in this lamina. Fine Pcdh--GFP puncta are distributed throughout the IPL, and are present at but not restricted to synapses. Scale bars: 10 µm.

View larger version (104K): [in this window] [in a new window]   Fig. 4. Thinned INL and IPL in retinae lacking Pcdh- proteins. (A) Diagram of conditional Pcdh- inactivation alleles. Each Pcdh- protein is encoded by an mRNA, comprising one of 22 variable exons and the three constant `C' exons. In Pcdh- fdel, loxP sites flanking the entire Pcdh- locus result in deletion of all Pcdh- genes upon Cre-mediated recombination. In Pcdh- fcon3, loxP sites flank the final C3-GFP exon, resulting in truncated forms of Pcdh- proteins. In both alleles, GFP is fused to the C terminus of the C3 exon. (B-G) Sections from P18 control and Pcdh--deficient retinas, labeled with DAPI or anti-synaptophysin to highlight nuclear and synaptic layers, respectively. Retinal lamination is normal, and OPL and ONL are normal in thickness but IPL and INL are markedly thinned in mutants. (H) Quantification of nuclear and plexiform layer thickness in control (black), Chx10-Cre; Pcdh- fdel/fdel (white) and Chx10-Cre;Pcdh- fcon3/fcon3 (gray) retinas at P18. Bars show mean±s.e.m. from three or four animals of each genotype. **P<0.01, ANOVA and post-hoc Tukey test. (I) Quantification of retinal cell types in Pax6-Cre;Pcdh- +/fcon3 (black) and Pax6-Cre;Pcdh- fcon3/fcon3 (gray) P18 retinas. Bars show mean±s.e.m. from six to eight animals of each genotype. PR, photoreceptors by Po-pro1; HZ, calbindin+ horizontal cells; BP, Chx10+ bipolar cells; AC, Pax6+ amacrines; RGC, Brn3a+ retinal ganglion cells; MG, Sox9+ Müller Glia. Other abbreviations as in Fig. 1. Error bars indicate s.e.m. ***P<0.0001, Mann-Whitney non-parametric test. Scale bars: 50 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Thinning of Pcdh- mutant retina reflects increased apoptosis during a restricted postnatal period. (A-F) Nuclear labeling of Pcdh-del/del, Pcdh- fcon3/fcon3, and control retinas at E18.5 (A,B), P7 (C,D) and P15.0 (E,F). Mutant and control retinas are indistinguishable at E18.5 but INL and IPL are thinner in mutant retinas than in controls by P7. Although the INL becomes thinner in both mutants and controls over the subsequent 5 months, the difference between them is not progressive. (G,H) Thickness of NBL/INL (NBL at P0 and P3, INL at later stages) and IPL in control (black) and Chx10-Cre;Pcdh- fcon3/fcon3 mutant retina sections (red). The mutant NBL/INL and IPL develop normally through P3, then decline in thickness relative to controls over the next few weeks. Graphs show mean±s.e.m. from three or four animals. ***P<0.001, Student's t-test. (I) NBL/INL and IPL thickness in Pcdh- fcon3/fcon mutant retinas, expressed as percentage of control. (J,K) Increased numbers of apoptotic cells, marked by cleaved caspase 3 immunoreactivity (red), in Pcdh- fcon3/fcon3 mutant retinas at P7 compared with controls. (L,M) Quantification of cleaved caspase 3 immunopositive cells in control (black) and Pcdh- fcon3/fcon3 mutant (red) retinas. Differences between genotypes are significant in the NBL/IPL at P0 and P7, but in the ganglion cell layer only at P7. Results from three to six animals per stage. ***P<0.001; **P<0.01; *P<0.05; Student's t-test or Mann-Whitney test. Abbreviations as in Fig. 1. Scale bars: 20 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Cell autonomous and non-autonomous components of Pcdh--dependent cell survival. (A) Schematic of the recombination pattern in Pax6-Cre retina. In Pax6-Cre;Pcdh- fcon3/fcon3 mutants, all cells are Pcdh- negative in peripheral retina, but only Pax6-positive (+) amacrine cells are Pcdh- negative in the central sector. (B-E) Immunolabeling of central and peripheral regions of Pax6-Cre;Pcdh- +/fcon3 and Pax6-Cre;Pcdh- fcon3/fcon3 mutant retinas with GFP and Brn3a antibodies. Anti-GFP labels both Pax6-Cre-ires-GFP positive amacrine cells (asterisks) and Pcdh--GFP proteins (arrows). In unrecombined portions of the central Pax6-Cre;Pcdh- fcon3/+ and Pax6-Cre;Pcdh- fcon3/fcon3 retinas, Pcdh--GFP proteins are visible in the outer segments, OPL, and retinal axons (arrows). In peripheral regions, Pcdh--GFPs are absent. In both regions, mutant retinas have reduced numbers of Pax6-positive amacrine cells; Brn3a-positive RGCs are dramatically decreased in the mutant peripheral sector while slightly decreased in the central sector. (F) Quantification of retinal cell types in central regions of Pax6-Cre;Pcdh- fcon3/fcon3 retinas, expressed as a percentage of cells in Pax6-Cre;Pcdh- fcon3/+ littermates. Six to eight animals per genotype were analyzed; **P<0.05; **P<0.01; ***P<0.0001, by Student's t-test or Mann-Whitney test. Scale bar: 50 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Sublamina-specific targeting of amacrine and bipolar processes in the IPL in the absence of Pcdh- genes. (A,B) ChAT- (red) and vGluT3-positive amacrine subsets (green) in Chx10-Cre;Pcdh- fcon3/+ (control) and Chx10-Cre;Pcdh- fcon3/fcon3 retinas. ChAT-positive processes ramify in sublaminae (S) 2 and 4, and vGlut3-positive processes ramify in S3. (C,D) Synaptotagmin 2 (SytII) -positive OFF bipolar processes (red) and G13-positive ON bipolar processes (green) ramify in the outer and inner regions of the IPL, respectively. In all cases, laminar specificity is retained in mutants, but marker-laminae are reduced and disrupted. (E) Sketch of IPL sublaminae stained by the markers used in this study. Scale bars: 50 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 8. Laminar specificity and synapse formation by Pcdh--deficient neurons rescued from apoptosis. (A-D) Sections from retinas mutant for Bax (Chx10-Cre;Pcdh- fcon3/+ Bax-/-), Pcdh- (Chx10-Cre;Pcdh- fcon3/fcon3 Bax+/-), both (Chx10-Cre;Pcdh- fcon3/fcon3 Bax-/-) or neither (Chx10-Cre;Pcdh- +/fcon3 Bax+/-). Sections were stained with anti-bassoon (red) and Po-pro1 (blue). Thickness of IPL and INL are similar in Bax mutants and Bax, Pcdh- double mutants; both are thicker than those in Pcdh- mutants. (E-L) High power images of OPL (E,G,I,K) and IPL (F,H,J,L) from retinas in A-D. Density of synaptic puncta is similar in Bax mutants and Bax, Pcdh- double mutants (see Fig. S2 in the supplementary material for quantification). (M,N) Chx10-Cre;Pcdh- fcon3/fcon3 Bax-/- mutants immunostained for ChAT (red) and vGluT3 (green) (M) or synaptotagmin 2 (SytII, red), and G13 (green) (N). All processes make lamina-specific arbors (compare with Fig. 7) and disruptions seen in Pcdh- single mutants are absent in double mutants. Scale bars: 100 µm in A-D; 10 µm in E-L; 50 µM in M,N.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Visual processing in Pcdh- mutant retina. (A) Visual responses of RGCs in Pcdh- mutant retina to a flashing spot at photopic intensities. Background shading indicates periods of light On and Off. Each panel is a raster graph of firing from one neuron; each row is a repeat of the same stimulus; tick marks represent action potentials. Neurons vary greatly in whether they respond to light onset or offset, and whether firing is transient or sustained after the switch. (B) Direction-selective response of an RGC in Pcdh- mutant retina to a grating stimulus moving in eight different directions. Insets are histograms of spike times during one period of the grating (wavelength 664 µm, speed 664 µm/s). The polar plot reports the average firing rate as a function of direction. (C-H) Distribution of response parameters in mutant and control retinas. Each panel inspects a different characteristic of the visual response, and plots a cumulative histogram of that quantity for RGCs in mutant retinas (red curve, 115 cells, Chx10-Cre;Pcdh- fcon3/fcon3 or the peripheral region of the Pax6-Cre;Pcdh- fcon3/fcon3) and control retinas (black curve, 143 cells, genotype Pax6-Cre;Pcdh- +/fcon3 or Pcdh- fcon3/fcon3). The shaded range indicates 95% confidence interval. (C) Ratio of On and Off responses in mutant and control retinas. For each cell we computed an On-Off index from the experiment in A: (number of spikes fired during the on-phase of the spot)/(total number of spikes fired). (D) Size of the receptive field center, measured as the full width at half maximum of the receptive field profile b(x) (inset, see Eqn 2). (E) Speed of the response, measured as the time to peak of the temporal integration function a(t) (inset, see Eqn 2). (F) Average firing rate observed during stimulation with flickering gratings. (G,H) Threshold (G) and gain (H) of responses in a linear-nonlinear model (see Eqn 3).

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