Fig 2: Detection of the HA-tagged CLRN1 protein by immunoblotting. (A) Representative immunoblots showing the expression of the HA-tagged endogenous CLRN1 in retina homogenates from adult N-HA Clrn1 knock-in mice. Retinas from C57BL/6J mice (P40) were collected as controls. The last two lanes show a duplicate experiment representing retina homogenates from two different P28 N-HA Clrn1 knock-in mice. (B) Detection of CLRN1 in the HA-tagged Clrn1 mouse retina homogenates in the absence and presence of PNGase F enzyme. Note the shift in size towards a lower molecular mass in the presence of PNGase F. The C57BL/6J and Clrn1 KO mouse retina extracts were used as negative controls. (C) Detection of endogenous CLRN1 in adult human retina (human retina lysates in two different buffers are shown). Note the characteristic shift in size following PNGase F enzymatic treatment. (D) Detection of AAV-expressed N-terminal HA-tagged CLRN1 (main isoform) following subretinal delivery in C57BL/6J mice. Note: treatment with PNGase F resulted in a complete shift to a single lower-molecular-mass product, at the same size as endogenous HA-tagged CLRN1 shown in B. Due to the low level of endogenous HA-CLRN1, blots A, B, and C have a higher background and display nonspecific bands, which are also present in control C57BL/6J mice. In contrast, nonspecific bands are absent in blot D, due to the higher levels of recombinant AAV-expressed CLRN1.

Fig 3: Single-cell RNAseq analysis of Clrn1 expression in mouse, NHP, and human retina. (A) T-distributed stochastic neighbor embedding (t-SNE) was used to cluster cells. Each dot represents a single cell. The t-SNE plots place cells on the graph based on the expression of highly variable genes. This is a method that places similar cells in distinct clusters. Cells in each cluster were identified based on the expression of known cell markers listed in the Materials and methods section (a–c). Cells expressing Clrn1 are shown in red and non-expressing cells are grey (d–f). Clrn1-positive cells were found overlapping with Müller glia clusters in all three species. Cell populations are Müller glia (Muller), rods, cones, retinal ganglion cells (RGC), horizontal cells (HC), bipolar cells (BC), amacrine cells (AC), microglia (Mi), vascular endothelial (VE), retinal pigment epithelium (RPE), and astrocytes (AS). (B) Using Seurat, we extracted the expression of Clrn1 in all cell clusters. The scatter plots in B show cells in each cluster that are positive for Clrn1 in mouse (a), NHP (b), and human retinas (c). The violin plots show cell types with Clrn1-enriched expression. Shown here are cell populations including Müller glia, rods, cones, and bipolar cells (BC).

Fig 4: Clrn1 mRNA expression pattern in the mouse eye. (A) In situ hybridization using sections of mouse eye from postnatal day 8 (P8) probed with a Clrn1-specific probe. The signal appears as red punctate staining throughout the inner nuclear layer (INL). (B) The same section as in panel A, imaged by fluorescence microscopy (greyscale image). (C) P8 mouse eye section probed for dapB as a negative control. (D) P8 eye section showing the positive control polymerase II subunit mRNA expression (Polr2A probe). (E) Representative image (enlarged view) showing Clrn1 mRNA expression in the INL. (F) Polr2a positive control mRNA expression (enlarged view). Cell nuclei are stained with hematoxylin. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar = 50 µm.

Fig 5: Localization of CLRN1 transcripts in adult human retina by ISH. (A) Detection of CLRN1 mRNA with a human CLRN1-specific probe on a section of paraformaldehyde-fixed, paraffin-embedded human retina. Note the red punctate ISH signals within the INL and extending towards the NFL. (B) Expression of the human RNA polymerase II subunit A (POLR2A) as a positive control. RPE, retinal pigmented epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; NFL, nerve fiber layer. Scale bar = 50 µm.