Panel A shows a gene schematic comparing Piccolo and Piccolino exon organization and transcript structures with numbered exons. Panel B shows a gene recombination diagram illustrating Cre mediated deletion between loxP sites generating modified allele. Panel C shows fluorescence microscopy images of wild type retina displaying Piclo and CAR localization with merged views. Panel D shows fluorescence microscopy images of knockout retina showing reduced Piclo signal and CAR localization comparison. Panel E shows a western blot detecting anti Piclo protein bands in wild type and knockout retinal samples. Panel F shows super resolution microscopy images of synaptic ribbon protein RIBEYE organization with enlarged structural reconstruction. Panel G shows super resolution microscopy images of active zone protein Bassoon with enlarged structural reconstruction views. Panel H shows merged super resolution image comparing synaptic ribbon and active zone alignment in wild type retina. Panel I shows a bar graph comparing synaptic ribbon length between wild type and knockout retinas. Panel J and J prime show electron microscopy images comparing synaptic ribbon morphology in wild type and knockout photoreceptor terminals. Panel J double prime shows a bar graph comparing synaptic ribbon height between wild type and knockout retinas. Panel K shows a bar graph comparing active zone length between wild type and knockout retinas.
Generation and characterization of a rod photoreceptor–specific Pclo knockout mouse line (Pclo cKO ). (A) Exon-intron structure of the Pclo gene indicating splicing of full-length Piccolo (top) and Piccolino (bottom). (B) Schematic representation of the genetic strategy used for generating PclocKO ms. (C and D) Fluorescence micrographs of vertical cryostat sections through the retinae of PcloWT (C) and PclocKO ms (D) double-labeled for Pclo (cyan) and cone arrestin (CAR, red). Arrowheads (➤) in D point to Pclo-positive cone photoreceptor terminals. (E) Western blot of whole retinal lysates of PcloWT and Pclo cKO synaptosomal fractions (P2) labeled with Pclo4 antibodies. (F and F′) CLSM images of vertical sections (left), whole-mount retina (middle), and a single SR (right) labeled against RIBEYE to measure SR length in PcloWT (F) and PclocKO ms (F′). (G and G′) CLSM images of vertical sections (left), whole-mount retina (middle), and a single SR (right) labeled against Bassoon (Bsn) to measure AZ length in PcloWT (G) and PclocKO ms (G′). (H and H′) Merge of PcloWT and PclocKO SR and AZ stainings shown in F and G and F′ and G′, respectively. (I) Quantification of SR length in PcloWT and PclocKO rod photoreceptors. Data are mean ± SD, **P = 0.0029, unpaired t test, n = 100 ribbons per animal, three animals. (J and J′) Representative EM micrographs of PcloWT (J) and PclocKO (J′) rod photoreceptor SRs used for measuring SR height (dashed line). (J″) Quantification of rod photoreceptor SR height in PcloWT and PclocKO retinae. Data are mean ± SEM, ****P < 0.0001, unpaired t test, 18–33 ribbons per animal, three animals. (K) Quantification of AZ length in PcloWT and PclocKO rod photoreceptors. Data are mean ± SD, **P = 0.0051, unpaired t test, n = 100 ribbons per animal, three animals. Scale bar = 20 µm (overview) and 2 µm (high-power view) in D for C and D, 20 µm in F–G′; 1 µm in H and H′ for single-ribbon views in F, F', G, G′, H, and H′ and 0.5 µm in J′ for J and J″. loxP, locus of X-over P1; Cre, Cre-recombinase; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Source data are available for this figure: SourceData F1.