Developmental dynamics of cone photoreceptors in the eel

Background: Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?Results: The Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their lambda(max) values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction).Conclusions: Larger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells

Dominant Cone-Rod Dystrophy: A Mouse Model Generated by Gene Targeting of the GCAP1/Guca1a Gene

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions

Photopigment expression in ‘<i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>Representative cryosections from five month-old mice stained with DAPI and (<b>a</b>) a combined anti-L/M- and S-cone opsin antibody (red) and PNA (green), and (<b>b</b>) an anti-rhodopsin (rod opsin) antibody (red). For both, upper panels show fluorescent light micrographs, and lower panels show single slices from confocal microscopy. The expression of both cone and rod opsin appears similar in both <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice compared with wild-type littermates, although the rod opsin staining indicates a shortening of rod outer segments. PNA staining (green) in (<b>a</b>) indicates a reduced number of cones in the mutant retinae which is more pronounced in homozygous than in heterozygous mutant mice. INL inner nuclear layer, ONL outer nuclear layer, IS/OS inner segment/outer segment.</p

Electroretinography of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>(<b>a</b>) Representative ERG traces recorded at five months of age. Light-adapted, cone-mediated ERG (upper trace, right eye, lower trace, left eye) is reduced in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice compared to wild-type littermates, as is the cone-mediated flicker response to 10- and 15-Hz stimuli (* and # respectively). (<b>b</b>) Representative ERG traces recorded at 12 months of age. There is a greater reduction in cone function in mutant mice, with light-adapted flash responses further attenuated and flicker responses almost extinguished. (<b>c</b>) Averaged photopic, cone-mediated b-wave amplitudes from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice recorded over a twelve month period from birth. There is a progressive loss of cone function over time in both heterozygous and homozygous mutant mice compared to wild-type littermates (<i>n = 8</i> per genotype). (<b>d</b>) ERG b-wave amplitudes from <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice plotted as a percentage of wild-type amplitudes over a twelve month period from birth.</p

cGMP levels in retinae of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>cGMP levels were measured in six week-old mice, before the onset of degeneration as evidenced by retinal structure and function. Levels of cGMP were corrected for total protein content of each sample.</p

Reduced rod function in <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p>(<b>a</b>) Representative ERG traces taken from dark-adapted 5 month-old wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice, showing a reduction in a- and b-wave amplitude in mutant mice in the rod-dominated ERG in response to a series of increasing stimulus intensities. (<b>b</b>) Representative ERG traces taken from dark-adapted 12 month-old wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice, showing that rod function is relatively spared when compared with cone function. (<b>c</b>) Averaged scotopic, rod-dominated b-wave amplitudes from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice recorded over a twelve month period from birth. Mean values were calculated from b-wave amplitudes from 100 mcds/m² flash intensity, which corresponds to a rod-only response. Note the age-related decline in wild-type mice. (<b>d</b>) Rod b-wave amplitudes in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice plotted as a percentage of wild-type amplitudes over a twelve month period from birth.</p

E155G mutation in GCAP1 causes photoreceptor degeneration.

<p>Photomicrographs of resin-embedded sections from (<b>a</b>) wild-type (<b>b</b>) <i>Guca1a</i>^+/COD3, and (<b>c</b>) <i>Guca1a</i>^COD3/COD3 mice at five months of age. There is a marked thinning of the outer nuclear layer (ONL) in mutant eyes as shown by the loss of photoreceptor nuclei. There is also an apparent shortening of outer segment length, particularly in the <i>Guca1a</i>^COD3/COD3 homozygous mice. Photoreceptor loss in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice was quantified by counting photoreceptor nuclei in photomicrographs of sections from paraffin-embedded eyes taken at fixed positions around the optic nerve. At five months of age (<b>d</b>), there is 30% loss in <i>Guca1a</i>^+/COD3 and a 46% loss in <i>Guca1a</i>^COD3/COD3 mice. At 12 months of age (<b>e</b>), this has progressed to a 32% loss in <i>Guca1a</i>^+/COD3 and a 49% loss in <i>Guca1a</i>^COD3/COD3 mice. * indicates statistical significance at the 1% probability level. Note that in the lower panels of (a), PNA shows non-cone staining above the ONL and INL. INL inner nuclear layer, ONL outer nuclear layer, IS/OS inner segment/outer segment.</p

Mutant GCAP1 leads to progressive loss of cone photoreceptors.

<p>(<b>a</b>) Representative retinal flat mounts taken from 5 month-old mice stained with PNA and anti-cone opsin antibody. There are fewer PNA-positive cone cells present in mutant compared to wild-type retinae, as well as a fewer opsin-expressing cells in mutant versus wild-type retinae. Quantitative analysis shows that there are significantly fewer cone opsin-expressing cells in mutant retinae than PNA-positive cells, although the labelling of both markers is significantly reduced in <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice as compared to wild-type littermates. (<b>b</b>) Representative retinal flatmounts taken from 12 month-old mice confirms progression of cone degeneration; in mutant mice, there are fewer opsin-positive cells at 12 months compared to wild-type littermates.</p

Impaired recovery from bright flash of <i>Guca1a</i>^COD3 ‘knock-in’ mice.

<p><b>T</b>he a-wave amplitude from a bright test flash, presented at varying times after a conditioning flash, is plotted as a proportion of the amplitude elicited by the conditioning flash. A value of 0 indicates the amplitude from the second flash is the same as that from the first; full recovery from a bright conditioning flash occurs within two seconds in wild-type mice, and the second flash elicits an amplitude half that of the first when the inter-stimulus interval is approximately 1000 ms. This recovery time is extended to 1600 ms in both heterozygous and homozygous knock-in mice, whilst the amplitude from the second flash fails to reach that elicited by the first flash during the time period of recording.</p

Generation of <i>Guca1a</i>^COD3 ‘knock-in’ mice with E155G mutation in GCAP1.

<p>(<b>a</b>) A vector targeting the endogenous <i>Guca1a</i> locus was constructed to include an A-to-G transversion at nucleotide position 19 in exon 4 of <i>Guca1a</i> (red circle), as well as a <i>loxP</i>-flanked (blue arrow heads) neomycin resistance gene within intron 3. Following <i>Cre</i>-mediated excision of the <i>Neo</i> selectable marker, the resulting locus contained the A-to-G change in exon 4 plus a residual 34 bp <i>loxP</i> sequence in intron 3. This was used to distinguish between mutant and native alleles by PCR – the position of the primers used is indicated by the small blue arrows on the <i>Cre</i>-deleted <i>Guca1a</i> locus. (<b>b</b>) PCR amplicons from wild-type, <i>Guca1a</i>^+/COD3 and <i>Guca1a</i>^COD3/COD3 mice (lanes 3, 4 and 5 respectively), with 1 kb DNA ladder (lane 1) and no-DNA control (lane 2). The wild type allele generates a band at 734 bp whereas the mutant allele generates a 768 bp band which includes the residual intronic <i>loxP</i> sequence. Wild-type mice have therefore a single band at 734 bp, homozygous <i>Guca1a</i>^COD3/COD3 mice have a single band at 768 bp, and heterozygous <i>Guca1a</i>^+/COD3 mice have both bands. (<b>c</b>) Sequence of wild type and targeted <i>Guca1a</i> allele showing A-to-G transversion at nucleotide position 19 of exon 4.</p