Genetically engineered mice with an additional class of cone photoreceptors: implications for the evolution of color vision - PubMed (original) (raw)
Genetically engineered mice with an additional class of cone photoreceptors: implications for the evolution of color vision
Philip M Smallwood et al. Proc Natl Acad Sci U S A. 2003.
Abstract
Among eutherian mammals, only primates possess trichromatic color vision. In Old World primates, trichromacy was made possible by a visual pigment gene duplication. In most New World primates, trichromacy is based on polymorphic variation in a single X-linked gene that produces, by random X inactivation, a patchy mosaic of spectrally distinct cone photoreceptors in heterozygous females. In the present work, we have modeled the latter strategy in a nonprimate by replacing the X-linked mouse green pigment gene with one encoding the human red pigment. In the mouse retina, the human red pigment seems to function normally, and heterozygous female mice express the human red and mouse green pigments at levels that vary between animals. Multielectrode array recordings from heterozygous female retinas reveal significant variation in the chromatic sensitivities of retinal ganglion cells. The data are consistent with a model in which these retinal ganglion cells draw their inputs indiscriminately from a coarse-grained mosaic of red and green cones. These observations support the ideas that (i) chromatic signals could arise from stochastic variation in inputs drawn nonselectively from red and green cones and (ii) tissue mosaicism due to X chromosome inactivation could be one mechanism for driving the evolution of CNS diversity.
Figures
Fig. 4.
Relative weightings of red and green cone inputs in the ganglion cell population. Results shown are from five different animals. Each data point represents the fraction of red cone input, r, for an individual ganglion cell. (A) Results from an R/Y hemizygote, three G/R heterozygotes, and a G/Y hemizygote. Blue error bars below the data points represent the spread of the data points (mean ± SD). Black error bars above the data points denote the experimental uncertainty (2 × SD of the noise) in each measurement, averaged over all cells in the experiment. n, number of ganglion cells. The skew of the R/Y data points from the expected value of 1.0 likely reflects a combination of uncertainties in the red pigment spectral sensitivity curve and the spectral output of the LCD panel. (B) Scatter plots of two independent measurements of r; “even” and “uneven” refer, respectively, to data obtained during the interleaved even and odd minutes of the recording session (see Materials and Methods). Each data point represents one cell. (Left) Three G/R retinas. (Right) G/Y and R/Y retinas. The dotted line represents identical values. In G/R retinas, the cell-to-cell variation in r exceeds the variation from measurement error.
Fig. 6.
The measured spectral variation among retinal ganglion cells can be explained by the spatial heterogeneity of X inactivation. The SD of the red cone fraction, r, within a ganglion cell population is plotted against the average fraction. Squares, measured values (mean ± SEM) for ganglion cells in three G/R retinas (Fig. 4). Triangles, calculated values from the four patchy cone mosaics in Fig. 5, assuming that each ganglion cell pools input from the overlying cones with a Gaussian weighting function (SD 65 μm). The curve, the expected dependence if the cone mosaic consists of 50-μm2 tiles, was assigned randomly to red and green cones with probability r and 1 – r, respectively.
Fig. 5.
X-inactivation mosaicism in four β-galactosidase transgenic retinas. Retinas were obtained from adult female mice heterozygous for the hydroxymethylglutaryl CoA (HMGcoA) reductase promoter-lacZ transgene as described in ref. . (Left) Original images of X-gal-stained retinal flatmounts sectioned in the plane of the retina at the level of the outer nuclear layer. Blue, X-gal; red, neutral red staining of nuclei. (Right) The same images processed with an intensity threshold to obtain binary maps of the X-gal-stained regions.
Fig. 1.
Generation of the human red knock-in allele. (A) Restriction map of the mouse green pigment gene showing exons 1–6 (black bars) and the 0.7-kb _Eco_RI–_Bam_HI probe used for embryonic stem cell screening and mouse genotyping (Top). The human red pigment knock-in construct in which the 5′ half of mouse exon 2 is joined in-frame to the corresponding region of human red pigment cDNA hs7 (Middle; ref. 6), and the final floxed human red knock-in allele from which the phosphoglycerate kinase-neo selection cassette has been removed (Bottom). Brackets indicate the region deleted. (B) Southern blot genotyping of mouse tail DNA using the 0.7-kb probe. Samples are from male mice hemizygous for the mouse green pigment gene (G/Y) or the human red pigment knock-in allele (R/Y), and heterozygous female mice (G/R). Y, Y chromosome. (C) Schematic of the transmembrane disposition of the human red pigment showing the junction used to create the mouse green/human red hybrid pigment in the center of transmembrane helix 1 (horizontal line). Amino acids that differ between the mouse green and human red pigments are shown as filled circles. The five amino acid dimorphisms that determine the different wavelengths of maximal absorption among members of the long wavelength visual pigment subfamily are labeled. Positions 197 and 308 account for the different absorption maxima of the mouse green and human red pigments (21).
Fig. 2.
Normal numbers of longer wavelength cones in the R/Y retina. Tenmicrometer sections of dorsal retina were immunostained with anti-human red/green carboxyl-terminal antibodies and Texas red secondary antibody (red). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). The number and lamination of nuclei and the density of immunoreactive cone outer segments are indistinguishable between WT and R/Y retinas.
Fig. 3.
ERG spectral sensitivity functions from red cone knock-in mice. (A) The data points are mean values for 3 G/G and G/Y mice, 10 R/Y and R/R mice, and 10 G/R mice. The data from mice expressing only a single longer wave pigment are fit to the photopigment templates of Govardovskii et al. (27). The best curve fit to the data from the G/R mice is a linear summation of the green (57%) and red (43%) curves. For the R/Y and R/R group and the G/R group, in which 10 mice each were analyzed over the same 25 wavelengths, the average SDs were 0.054 log units (R/Y and R/R) and 0.089 log units (G/R). The larger SD in the G/R group presumably derives from interanimal variability in the ratio of red:green cones. (B) Results from an ERG test for spectral response univariance. Plotted are the differences (in log units) for the 500- and 600-nm photometric equations obtained in the alternate presence of 500- and 600-nm adapting lights. Values near zero indicate spectral univariance; positive values imply the presence of two independently adaptable spectral mechanisms.
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