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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.042531

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Knockout of GARPs and the β-subunit of the rod cGMP-gated channel disrupts disk morphogenesis and rod outer segment structural integrity

¹ Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA
² Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
³ Department of Physiological Science, Jules Stein Eye Institute, UCLA School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA
⁴ Department of Ophthalmology, Jules Stein Eye Institute, UCLA School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. Gene targeting of the murine Cngb1 locus. (A) Map of β-subunit and GARP exons and corresponding transcripts. Representations of transcripts encoding Cngb1 gene products are shown below an exon map of the locus. The mGARP2 transcript is composed of exons 1-12 and a unique exon designated 12a. Transcript mGARP1 consists of exons 1-18 and alternate exon 16a. The 3' exon of mGARP1 contains all of exon 18 found in the β-subunit transcript and an additional predicted 364 bp of sequence downstream of the end of exon 18 (designated 18L). The β-subunit transcript (Cngb1) consists of exons 1-33 excluding the two alternate exons (12a and 16a). Exons 1 and 2 and the predicted promoter region are within the region targeted for deletion. (B) Schematic of genome deletion strategy. The targeting plasmid pCNGB1-KO contains a PGK-Neo cassette flanked by a 1.4 kb short arm and a 7 kb long arm of Cngb1 excluding 3.5 kb of genomic sequence that contains a predicted promoter and exons 1 and 2. The first ATG of the protein coding region for all three protein products is within exon 2 (ATG). Some of the relevant restriction sites for cloning are shown (see Materials and Methods). (C) Genotype analysis. A 1.8 kb PCR product was generated with primer pair a/c specific for the WT allele, and a 1.6 kb PCR product was produced with primer pair a/b specific for the deleted allele. Multiplex PCR with all three primers a/b/c yielded both products using heterozygous mouse genomic DNA as a template. In the absence of template DNA, no PCR products were observed (not shown). (D) RT-PCR of the targeted region. A primer pair that amplifies a 254 bp product containing all of exon 2 was used to amplify cDNA generated from each genotype. No product was generated in the absence of cDNA (lane –) or in the KO sample (lane –/–). All samples could be amplified with control primer pairs (not shown). (E) Northern blot analysis of Cngb1 KO and WT littermate total RNA. Using a cDNA probe spanning exons 4-9, a strong band of about 1.6 kb and a weaker 6.2 kb band are observed, consistent with transcripts encoding GARP2 and the β-subunit, respectively. Distinct GARP1 transcript is not apparent; however, there are faint, somewhat diffuse, bands that might represent the low-abundance GARP1. There is no signal in homozygous KO mice, indicating that Cngb1-related transcripts are either absent or of very low abundance. Reprobing of the same blot with a G3PDH DNA probe demonstrated comparable loading of RNA in each lane (not shown).

View larger version (132K): [in this window] [in a new window]   Fig. 2. Morphology of PN30 WT and Cngb1 KO mouse retina. (A) In the WT mouse retina (+/+) the normal stratification into layers of appropriate thickness is observed. In contrast to the knockout retina (–/–), rods appear uniformly cylindrical and have thicker RIS and ROS layers. (B,C) In the knockout retina both ROS and RIS are shorter and nonuniform in appearance, and disoriented. All other layers appear normal in thickness. Orig. mag. x400 (B). Scale bar: 50 µm (C). RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Expression of photoreceptor proteins in the Cngb1 knockout mouse. (A) Cngb1-related gene products are undetectable in mouse ROS. In normal and heterozygous mice, β-subunit and GARP2 proteins are readily detected and a weaker GARP1 band is observed. In homozygous –/– KO mice no β-subunit or GARP proteins are detected consistent with the Cngb1 KO being a true null. (B) The cGMP-gated cation channel -subunit is significantly reduced in knockout ROS homogenates. Monoclonal antibody PMc 2G11 against the channel -subunit (CNGA1) detected the 63 kDa channel subunit in WT and heterozygous mice but a weak signal with apparent degradation product was seen in homozygous Cngb1 KO mice. (C) The level of expression of several outer segment proteins is reduced in the homozygous Cngb1 KO mice. (See Table 1 for quantitative analysis.) β-Actin was used as a loading control.

View larger version (87K): [in this window] [in a new window]   Fig. 4. Immunolocalization of ROS proteins. Frozen retina sections from PN30 WT littermates, heterozygotes [GARP (+/–)], and homozygous Cngb1 KO [GARP (–/–)] were labeled (green) with antibodies against the channel β-subunit and GARP (upper panels), the channel -subunit (CNGA1, middle panels), and peripherin-2 (Per/rds, lower panels). Nuclei are counterstained with DAPI (blue). Scale bar: 50 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 5. Ectopic disk morphogenesis in Cngb1 KO mice. (A) Ectopic disk membranes initiate at the connecting cilia. Overgrown disc membranes extend from the connecting cilia (arrowhead) of a rod photoreceptor cell in a PN 20 KO retina. (B) Low power TEM view of an ROS-RIS interface in PN 60 KO retina. Ectopic membranes are apparent on most ROS in the field (arrowheads). Only a portion of each cell is observed due to disorientation of the rod cells. Orig. mag. x6500 (A); x1100 (B). Scale bars: 500 nm (A); 2000 nm (B).

View larger version (211K): [in this window] [in a new window]   Fig. 6. Ultrastructure of PN 20 WT and KO retina. (A) PN 20 WT mouse. WT mice show well developed, nicely aligned, tightly packed outer segments. (B-D) PN 20 Cngb-X1 KO mice. In the KO mice, tightly packed but misaligned, misshapen outer segments are apparent. Abnormal elongation of disks is clearly observed in the region demarcated by arrowheads. Orig. mag. x2100 (A,B); x1650 (C,D). Scale bars: 500 µm.

View larger version (166K): [in this window] [in a new window]   Fig. 7. Ultrastructure of PN60 WT and KO retina. To further define the Cngb1-X1 KO phenotype and examine the basis for the apparent structural abnormalities of the photoreceptor, retinas from PN60 mice were analyzed by TEM. (A) PN 60 WT Mouse. ROS appear as uniform stacks of disks surrounded by a thin plasma membrane. (B-D) PN 60 Cngb1-X1 KO mice. By contrast, in addition to loss of cells, ROS are shorter and have a unique abnormal morphology defined by extra-mebranous disk-like material (arrowheads). Swirls of disk membrane are occasionally seen as marked by an arrow in D. Orig. mag. x2100 (A,D); x1650 (B,C). Scale bars: 500 µm.

View larger version (109K): [in this window] [in a new window]   Fig. 8. Scanning electron microscopy analysis of WT and KO retina. Shown are scanning micrographs of PN 35 Cngb1-X1 and WT midperipheral retina. RPE is at the top of the images. (A,C) WT retina. Uniform cylindrical tightly packed outer segments are apparent. (B,D) Cngb1-X1 KO mouse retina. The KO mouse retina outer segments appear misshapen with abnormal strictures and increased girth consistent with the overgrown disks and ROS appearance observed by TEM. Scale bars: 15 µm (A,B); 7.5 µm (C,D).

View larger version (17K): [in this window] [in a new window]   Fig. 9. Single rod cell response analyses in WT and KO mice. Typical responses for young adult (PN 18 – PN 24) WT (A) and KO (B) mice are shown. Cells were given flashes of increasing light intensities ranging from 16 to 6200 photon/µm2 (1.2 to 3.7 log photon/µm2) for the WT mouse rod and from 370 to 52,000 photon/µm2 (2.4 to 4.7 log photon/µm2) for the KO mouse rod. (C) Intensity-response data for WT and Cngb1 KO rods. The intensity response graph plots the amplitude of the light response vs. the stimulus strength that generated the response. Flash responses for each rod were normalized to the maximum dark current for each rod and then averaged. The left ordinate is for the WT rods and the right ordinate, which is tenfold expanded, is for the KO rods.

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