, 1723 (2007);
et al.Gerald H. Jacobs,
Engineered to Express a Human Cone
Emergence of Novel Color Vision in Mice
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on March 27, 2007
Emergence of Novel Color
Vision in Mice Engineered to Express
a Human Cone Photopigment
Gerald H. Jacobs,1* Gary A. Williams,1Hugh Cahill,2,3,4Jeremy Nathans2,3,4,5
Changes in the genes encoding sensory receptor proteins are an essential step in the evolution
of new sensory capacities. In primates, trichromatic color vision evolved after changes in
X chromosome–linked photopigment genes. To model this process, we studied knock-in mice
that expressed a human long-wavelength–sensitive (L) cone photopigment in the form of an
X-linked polymorphism. Behavioral tests demonstrated that heterozygous females, whose retinas
contained both native mouse pigments and human L pigment, showed enhanced long-wavelength
sensitivity and acquired a new capacity for chromatic discrimination. An inherent
plasticity in the mammalian visual system thus permits the emergence of a new dimension of
sensory experience based solely on gene-driven changes in receptor organization.
cone pigment most sensitive to short (S) wave-
lengths is encoded on an autosome, and a second
cone pigment most sensitive to middle (M) or
long (L) wavelengths is encoded on the X chro-
evolution of trichromacy (2). In Old World pri-
of the ancestral X-chromosomal cone pigment
gene (3). By contrast, in most New World mon-
keys, cone pigment diversity derives from poly-
morphic variation in a single X-linked gene (4, 5).
tion produces a mosaic of spectrally distinct cone
types, which provides the foundation for trichro-
macy. The polymorphic X-linked pigment genes
in New World monkeys may represent the an-
cestral arrangement for primates, as judged by the
observation that the same three amino acid sub-
and L pigments in all present-day primates (3).
and appropriate neural wiring, and it has been
argued that the organization of the primate retina,
and in particular the low-convergence midget bi-
polar and ganglion cell system, is such that the
addition of a new class of cone photoreceptors
may be all that is required for comparing M
versus L cone signals (6–8).
Our experiments used genetically engineered
mice to model the first step in the evolution of
primate trichromacy. We asked whether the sud-
he retinas of most nonprimate mammals
den acquisition of an additional and spectrally
distinct pigment and its production in a subset of
cones suffice to permit a new dimension of chro-
matic discrimination that would imply that (i) the
mammalian brain is sufficiently plastic that it can
extract and compare a new dimension of sensory
first inherited an additional X-chromosome allele
would have immediately enjoyed a selective ad-
vantage with respect to chromatic discrimina-
tion. We produced a line of mice in which most
of the coding sequences of the normal mouse
X-chromosomal M pigment gene [specifying a
pigment with wavelength of maximum absorp-
tion lmax~510 nm, hereafter “M”] was replaced
with a human L pigment cDNA (specifying a
“L”) (9). A similar knock-in mouse has recently
been produced (10). As in New World monkeys,
breeding yields mice with three distinct com-
plements of M/L pigments: Males and homozy-
gous females have either M or L pigments, and
heterozygous females have a mixture of the two
pigments. Earlier we observed that cones con-
taining the L pigment could transduce light
signals with an efficiency roughly equal to that
gram (ERG), and that the responses of individual
retinal ganglion cells (RGCs) in M/L hetero-
in chromatic sensitivity despite the large number
of a typical mouse RGC. This functional hetero-
geneity likely reflects the spatial graininess of M
inactivation, which is consistent with the ~50-mm
patches in the retina (9).
We first performed an analysis of cone-based
spectral sensitivity using ERG flicker photometry
(11) [see also supporting online material (SOM)].
in the ultraviolet range (12, 13); we minimized its
activation by using lights of relatively long wave-
length. Spectral sensitivity functions, obtained
from mice whose retinas contained either the M
pigment (n = 12 mice) or the L pigment (n = 17
mice), were fitted with photopigment absorption
functions (Fig. 1, A and B). The spectral maxima
Spectral sensitivity functions, similarly obtained
by linearly summing the spectra from mice with
either M or L pigments. On average, there is a
twofold larger contribution from the M pigment
than from the L pigment (presumably reflecting a
individual variability in this relative weighting
(Fig. 1D). The latter likely reflects stochastic vari-
ation in X-chromosome inactivation (14).
Although heterozygotes have, on average,
greater M than L sensitivity, this is not because
the L pigment compromises cone viability or
M/L opsin demonstrated nearly identical densities
only one or the other of these pigments [mean M
cone densities ±SD = 8129 ± 995 per mm2and
mean L cone densities ±SD = 8288 ± 952 per
mm2(n = 2 mice for each genotype)]. Second,
ERG voltage versus intensity (V-log I) functions
when the intensity of the stimulating light is
specified according to its calculated effectiveness
for each pigment (Fig. 1E), implying that signals
the same efficiency.
To examine whether vision is altered by the
added photopigment, we tested mice in a be-
havioral three-alternative forced-choice discrimi-
identify which one of the three test panels was
location of the correct choice varying randomly
between trials (15). The L pigment has greater
sensitivity to long wavelengths than does the M
sensitivity to such lights in mice whose retinas
contain the L pigment. Increment thresholds were
determined for 11 mice (four having only M
pigment and seven M/L heterozygotes) by briefly
adding either of two monochromatic test lights
(500 and 600 nm) to one of the three stimulus
between the 500- and 600-nm test lights were
significantly smaller for M/L heterozygotes (Fig.
1F), indicating that these mice can extract visual
information from L cones.
variations in spectral composition irrespective of
variations in intensity. To assess this possibility,
we derived brightness matches between a mono-
chromatic test light and a series of standard lights
of different wavelengths (see SOM) and then
asked whether mice could discriminate between a
of 600 nm. At each trial, the location of the test
its intensity varied randomly in steps of 0.1 log
1Neuroscience Research Institute and Department of Psychol-
ogy, University of California, Santa Barbara, CA 93106, USA.
2Department of Neuroscience, Johns Hopkins Medical School,
Baltimore, MD 21205, USA.3Department of Ophthalmology,
Johns Hopkins Medical School, Baltimore, MD 21205, USA.
4Howard Hughes Medical Institute, Johns Hopkins Medical
School, Baltimore, MD 21205, USA.
lecular Biology and Genetics, Johns Hopkins Medical School,
Baltimore, MD 21205, USA.
*To whom correspondence should be addressed. E-mail:
5Department of Mo-
VOL 315 23 MARCH 2007
on March 27, 2007
units to encompass a range of ±0.5 log units
tested a mouse whose retina contained M but not
L pigment. As expected, despite extensive testing
(totaling ~15,600 trials), no significant discrim-
ination was achieved (open circle in Fig. 2A).
With the use of the same procedures, two M/L
heterozygotes also failed to show evidence of
color discrimination (open square and triangle in
Fig. 2A; ~16,000 trials per subject).
The two M/L heterozygotes that failed in the
wavelength discrimination had relatively skewed
M:L ratios (78:22 and 65:35). Unbalanced rep-
resentation of M and L cones has little impact
on human color vision (16, 17), but, with smaller
numbers of cones, we thought that this might not
trials), an M/L heterozygote with a more ba-
lanced M:L ratio (44:56) successfully discrimi-
nated 500-nm from 600-nm lights. The averaged
asymptotic performance across the full range of
brightness variation at this test wavelength is in-
dicated by the vertical bar and arrow in Fig. 2A.
The test wavelength was then progressively dis-
placed in steps of 5 or 10 nm toward 600 nm; the
mouse succeeded at each of these discriminations
until the test and standard lights were separated by
This process was repeated for six other standard
lights covering the range from 570 to 630 nm,
yielding in each case a qualitatively similar result.
which the test and standard lights were set to the
cumulative results of this control procedure (Fig.
2B) show that (i) discrimination failed when both
chromatic and brightness cues were eliminated
predict the points of discrimination failure. Two
additional M/L heterozygotes also succeeded at
the color discrimination task (Fig. 2A).
For the M/L heterozygotes, wavelength dis-
crimination varied across the range tested,
being most acute at 590 to 600 nm (Fig. 2C).
The U-shaped wavelength discrimination func-
tion is qualitatively like those for other dichro-
matic visual systems (1); in particular, it is similar
tritanopes (18), a dichromatic subtype in which
color vision is based only on M and L pigments
withpeaks at~530and~560nm,respectively. At
present, we cannot fully explain the variation
among M/L heterozyotes in the color discrimina-
tion task, but it presumably reflects some com-
vagaries in the spatial distribution of cone types
vis-à-vis RGC receptive fields, or central factors
related to memory, intelligence, or motivation.
To assess dichromatic color matches, we used
a test light that was composed of an additive
mixture of two monochromatic lights. Using the
same brightness control measures as before, we
to determine what proportions could be discrimi-
nated from two monochromatic standard lights
(600 and 580 nm, in successive experiments).
Results obtained from the M/L heterozygote with
summarized in Fig.2,D and E. Inboth cases,this
animal successfully discriminated the combina-
the 530-nm component. However, as more of the
620-nm component was added, discrimination
became more difficult, eventually dropping to
chance performance, thereby defining a color
match. Dichromatic color matches can be pre-
dicted by calculating the proportions of each test
pigments is equal to the absorption from the stan-
dard light. These predicted matches (downward-
pointing blue arrows in Fig. 2, D and E) are close
to the actual location of discrimination failure, as
is also seen for a human equivalently tested (red
square and red arrow in Fig. 2D). These results
imply that color vision in this M/L heterozygous
between the M and L pigments.
In primates, midget bipolar and ganglion cells
mediate the chromatically opponent M versus L
color vision pathway (19, 20). Like many non-
primate mammals, the mouse lacks a midget
in M/L heterozygotes must be subserved by other
means. Most mouse RGCs have a receptive field
center with an antagonistic surround (22), albeit a
weak one, and chromatic information could be
to these two regions. In a variation on this idea,
chromatic information could also be extracted
simply based on variation among RGCs in the
total M versus L weightings.
In M/L heterozygous mice and in heterozy-
gous New World monkeys, the stochastic process
Fig. 1. (A) ERG spectral sensitivity for mice expressing the M pigment. Data points are mean values for
12 animals. The curve is that for the best-fitting photopigment absorption function. (B) Mean spectral
sensitivity function for 18 mice expressing the human L pigment. (C) Spectral sensitivity for 82
heterozygous mice. The curve is the best-fit linear summation of curves derived from those in (A) and (B).
(D) Distribution of the L:M cone weightings required to best fit each of the heterozygous mice
represented in (C). (E) Mean V-log I functions obtained from activation of either mouse M or human
L pigments. Light intensity has been specified according to its calculated effectiveness on each of
these pigments. sec, seconds; sr, steradians. For derivation of the fitted functions, see SOM. (F)
Increment-threshold measurements. The inset schematizes the discrimination context in which, on each
trial, a monochromatic test light was added to any one of the three panels, all of which were steadily
illuminated with identical achromatic light. The colored bars depict the difference in the thresholds
obtained for 500- and 600-nm test lights for mice whose retinas contained either mouse M (n = 4 mice)
or both M and L (n = 7 mice) pigments. Error bars in [(A) to (C)] and [(E) and (F)] indicate 2 SDs.
23 MARCH 2007 VOL 315
on March 27, 2007
(X-chromosome inactivation) by which M versus Download full-text
L cone territories are generated and the presumed
lack of any molecular distinction between M and
L cones other than pigment content have parallels
in a recent model for M versus L cone develop-
ment in Old World primates, in which M versus L
pigment gene expression is hypothesized to occur
in a stochastic and independent manner in each
cell, unaccompanied by any other differences in
inactivation lends indirect support to this Old
World primate model by demonstrating the pos-
sibility of behavioral trichromacy in the context
These results have general implications for the
evolution of sensory systems. In the visual, ol-
factory, and gustatory systems, genetic manipu-
the existing complement of receptors or replaced
an endogenous receptor protein with a new one
(24–27). In each case, the behavioral or electro-
physiological responses show the predicted ex-
pansion or modification of sensitivity. However,
these earlier experiments were not designed to
create an additional class of sensory neurons or to
facilitate the emergence of neural circuitry for
the present experiments, X-chromosome inacti-
vation has provided the means to both express a
non-native receptor protein and to localize it to a
distinct class of primary sensory neurons. Our
observation that the mouse brain can use this in-
that alterations in receptor genes might be of
immediate selective value not only because they
expand the range or types of stimuli that can be
detected but also because they permit a plastic
nervous system to discriminate between new and
existing stimuli. Additional genetic changes that
refine the downstream neural circuitry to more
efficiently extract sensory information could then
follow over many generations.
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28. We thank J. Fenwick for help with animal testing. This
work was supported by grant EY002052 from the
National Eye Institute (G.H.J.), the Howard Hughes
Medical Institute (J.N.), and the Visual Neurosciences
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Supporting Online Material
Materials and Methods
14 December 2006; accepted 21 February 2007
Fig. 2. Tests of color vision in mice. (A) Wavelength discrimination. The inset symbolizes the context in
which the mouse was required to detect which of three stimulus panels was distinctively illuminated; its
luminance and position relative to that of thestandardwavelength (600nm) wererandomizedfrom trial
to trial. Lower left symbols show asymptotic performance levels (for the final 100 trials ± 1 SD) for three
animals at the calculated brightness match (see SOM) and forvalues ± 0.1 log units from that point (the
with M:L ratios of 78:22 and 65:35, respectively). The solid circles connected by lines are the asymptotic
performance levels achieved at the calculated brightness match by the M/L heterozygote with an M:L
ratio of 44:56.Averaged performance over the full range of luminance variation for a test wavelength of
solid squares show results from two other heterozygous animals (M:L ratios of 46:54 and 53:47,
respectively). For the latter animal, the standard wavelength was 610 nm. (B) Results from the
embedded brightness control test. The circles are asymptotic performance levels for cases where the test
and comparison lights were set to the same wavelength and the test light was systematically varied in
luminance relative (re) to the value required for a brightness match. The results were cumulated across
seven standard wavelengths. The horizontal dashed line indicates chance performance. (C) Wavelength
between the test and standard wavelengths that is required for successful discrimination (at the 95%
confidence level). Symbols for individual animals are the same as those in (A). The dashed line
connecting standard wavelengths of 580 and 570 nm for one female indicates that it failed to
must exceed 60 nm. (D) Color-matching data. Plotted are asymptotic levels of performance achieved by
an M:L heterozygous mouse (44:56 M:L ratio) at the brightness matchwhere the test light was a variable
mixture of 530- and 620-nm lights, and the standard light was 600 nm. The location of the predicted
match based on the M and L photopigments is indicated by the blue arrow. The matchmade by a human
trichromat (red square) is shown along with the predicted human match (red arrow). (E) Color-matching
data. Details are the same as for (D) except that the standard light was 580 nm. The shaded area in [(A)
and (B)] and [(D) and (E)] indicates chance performance (at the 95% confidence level).
VOL 315 23 MARCH 2007
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