Fig. 4

Clarin-1 localizes in mechanosensory hair cell bodies and at the outer limiting membrane and lateral contacts between photoreceptors in the larval retina. (A-D) Cross sections showing Clarin-1 enrichment in apical regions of cell bodies (brackets) and at synapses (arrowheads) of hair cells of the ear (anterior macula shown in A, C) and neuromasts (B). Clarin-1 does not colocalize with actin filaments in the hair cell stereocilia (white arrows in C and D). Kinocilia are labeled with Acetylated Tubulin antibody (AT) and stereocilia are labeled with Actin antibody. (E-H) Cross sections through the retina of 3 dpf (E and F) and 7 dpf (G and H) larvae labeled with DrClrn1 and Glutamine synthetase (GS, panel E) or Actin (panel G) antibodies. Clarin-1 is enriched at synapses (OPL indicated with white closed arrow in F), and in the inner nuclear layer (white open arrow in F) at 3 dpf. Blue box in ventral region of E and F indicates region magnified in panels I and J. A full retina view at 7 dpf shows Clarin-1 label in the photoreceptors (ONL), Inner nuclear layer (INL) and ganglion cell layer (GCL). (I-L) High magnification views of 3 dpf (I and J), and 5 dpf (K and L) retinas. Clarin-1 is enriched at the outer limiting membrane (OLM) and in the outer plexiform layer (OPL). Glial cell processes are seen passing through the layer of the photoreceptor nuclei (ONL). At 3 dpf, the apical structures of photoreceptors, the inner and outer segments, are rudimentary and the retinal pigmented epithelium (RPE) lies close to the OLM. By 5 dpf, cone apices have extended such that space between the OLM and the RPE has increased, and fine, filamentous enrichments of Clarin-1 can be observed between cone inner segments (asterisks in K and L; high magnification image shown in L). (M and N) Transverse section through the 7 dpf retina to visualize the photoreceptor layer from the top down. The meshwork of cell junctions that make up the OLM derive from Müller cell processes labeled by Glutamine synthetase (GS). Clarin-1 localization partially overlaps, and is also enriched at the lateral interfaces extending from the periphery of the transverse cut (white arrows in I). (O and P) Clarin-1 is seen in close proximity to actin at the lateral contacts between photoreceptors in the 7 dpf retina. Scale bars: E, F: 50 µm; A, C, D, G-P: 10 µm. B and L: 5 µm. Schematics show the plane of sectioning for retinal tissue.