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The Journal of Neuroscience, July 15, 1999, 19(14):5889-5897
Disruption of a Retinal Guanylyl Cyclase Gene Leads to
Cone-Specific Dystrophy and Paradoxical Rod Behavior
Ruey-Bing
Yang2,
Susan
W.
Robinson1,
Wei-Hong
Xiong5,
King-Wai
Yau5,
David G.
Birch3, 4, and
David L.
Garbers1, 2
1 Howard Hughes Medical Institute and Departments of
2 Pharmacology and 3 Ophthalmology, University
of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, 4 Retina Foundation of the Southwest, Dallas, Texas 75231, and 5 Howard Hughes Medical Institute and Department of
Neuroscience, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205
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ABSTRACT |
One of two orphan photoreceptor guanylyl cyclases that are highly
conserved from fish to mammals, GC-E (or retGC1) was eliminated by gene
disruption. Expression of the second retinal cyclase (GC-F) as well as
the numbers and morphology of rods remained unchanged in GC-E null
mice. However, rods isolated from such mice, despite having a normal
dark current, recovered from a light flash markedly faster.
Unexpectedly, the a- and b-waves of electroretinograms (ERG) from
dark-adapted null mice were suppressed markedly. Cones, initially present in normal numbers in the retina, disappeared by 5 weeks, based on ERG and histology. Thus, the GC-E-deficient mouse
defines a model for cone dystrophy, but it also demonstrates that
morphologically normal rods display paradoxical behavior in their
responses to light.
Key words:
guanylyl cyclases; retina; photoreceptors; gene
disruption; cone dystrophy; mice; cyclic GMP; guanylyl cyclase-E
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INTRODUCTION |
Vision begins in retinal
photoreceptors, where the photoisomerization of the visual pigment
leads to a G-protein-mediated activation of a phosphodiesterase
hydrolyzing cGMP. As a result, the intracellular cGMP level decreases,
and cGMP-gated ion channels close to produce a membrane
hyperpolarization (for review, see Lagnado and Baylor, 1992 ; Yarfitz
and Hurley, 1994; Yau, 1994). The closure of these channels results in
a decrease in the intracellular Ca2+ concentration,
which is proposed to act via guanylyl cyclase-activating proteins
(GCAP1 and GCAP2) to increase guanylyl cyclase activity (Palczewski et
al., 1994; Dizhoor et al., 1995) (for review, see Polans et al., 1996).
The increase in cyclase activity then would represent an important
mechanism for photoreceptor adaptation and recovery.
Two eye-specific guanylyl cyclases have been identified in the mammal;
these are designated RetGC-1 and RetGC-2 in human (Shyjan et al., 1992;
Lowe et al., 1995) and guanylyl cyclase-E (GC-E) and GC-F in rat (Yang
et al., 1995). They are members of the family of membrane receptor
guanylyl cyclases, all of which contain an extracellular putative
ligand-binding domain, an apparent single transmembrane segment, and
intracellular protein kinase homology and cyclase catalytic domains
(Garbers and Lowe, 1994). With respect to the putative ligand-binding
domain, the retinal cyclases remain orphan receptors. That there are
two eye-specific guanylyl cyclases, both containing apparent
ligand-binding domains conserved across human, rat, cow, and fish
(Seimiya et al., 1997), implies significant pressure to retain the
nature of the domain, possibly for the purpose of ligand recognition.
The retinal guanylyl cyclases appear predominantly, but not
exclusively, in the photoreceptor outer segment layer. In the rod-dominant rat retina, GC-E appears uniformly distributed in the
outer segment layer (Cooper et al., 1995). In cat, monkey, and human
retinas, which have a significant number of cones, the GC-E-like
immunoreactivity appears greater in cone than in rod outer segments
(Dizhoor et al., 1994; Liu et al., 1994; Cooper et al., 1995). In the
cone-rich retina of chicken, GC-E immunostaining is strong and uniform
across the outer segment layer (Cooper et al., 1995). The relative
distribution of GC-F in the outer segment layer is not well documented.
Based on double-label immunogold electron microscopy, GC-F appears to
be coexpressed with GC-E in the same rod outer segment (Yang and
Garbers, 1997). In monkey retina, in situ hybridization
experiments indicate a uniform distribution of the message for GC-F
across the outer segment layer, but it has not been established whether
this cyclase is also present in cones (Lowe et al., 1995).
In this paper we report the disruption of the GC-E gene. We have found
that mice lacking the gene have morphologically normal rods at least up
to 1 year of age, but the cones rapidly degenerate. The rods have a
normal dark current despite the absence of functional GC-E and no
compensatory increase in the expression of GC-F.
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MATERIALS AND METHODS |
Generation of GC-E-deficient mice. The GC-E gene
(Gucy2e) from the mouse strain 129/SvJ was used to construct
a targeting vector. A neomycin-resistance gene cassette replaced a
portion of exon 5, which codes for the transmembrane region, leaving a coding sequence that could yield only a truncated protein (see Fig.
1a). SM1 embryonic stem (ES) cells (from Dr. Robert Hammer, University of Texas Southwestern Medical Center, Dallas, TX) derived from the inbred 129/SvJ mouse line were cultured on an irradiated STO-LIF fibroblast feeder layer (Ramirez-Solis et al., 1993). ES cells
(1.0 × 107) were electroporated at 0.23 kV,
500 µFD with 25 µg of linearized targeting vector DNA. G418 (180 µg/ml) (Life Technologies, Gaithersburg, MD) and gancyclovir
(1 µM) were added to the medium for selection for 4-5 d,
until ES cell colonies formed. To identify clones with targeted
disruption of the GC-E gene, we expanded the ES cells and
prepared genomic DNA for Southern blot analysis (see Fig. 1b). Chimeric mice were generated by injecting the
GC-E-disrupted ES cells into C57BL/6J blastocysts that subsequently
were implanted into the uteri of pseudopregnant foster females. Seven
chimeras were born and mated with C57BL/6J mice to test for germline
competency of the targeted clone; one of these mice transmitted the
mutant allele. ES cell-derived progeny were screened for the presence of the targeted allele by Southern blot (see Fig. 1b) and/or
PCR analysis. Heterozygous animals were inbred to generate homozygous mice lacking a functional GC-E gene, verified by Southern blot analysis. The targeted allele has been maintained in both 129/SvJ and
C57BL/6J backgrounds.
Western blot and histological analyses. For Western blotting
the posterior portion of the eyecup was collected and homogenized in
1× Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 0.005% bromophenol blue, and 5% 2-mercaptoethanol). Total
proteins were separated by SDS-polyacrylamide gel electrophoresis,
transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-P,
Millipore, Bedford, MA), and probed with antisera against GC-E, GC-F,
or rds/peripherin (1:1000 dilution) as described (Travis et
al., 1991; Yang and Garbers, 1997). For histology the eyes were
removed, fixed overnight at 4°C with a solution of 10% formalin, and
embedded in paraffin. Sections of 3 or 4 µm were cut through the
optic nerve head and stained with hematoxylin and eosin. Cone cells were identified by their nuclear structure (Carter-Dawson and LaVail,
1979) or by labeling with peanut agglutinin (PNA) as described by
Blanks and Johnson (1984). For PNA labeling, the sections were deparaffinized with xylene and rehydrated through an ethanol series. After being blocked with 500 µg/ml bovine serum albumin in
PBS, the sections were incubated with 100 µg/ml
biotin-conjugated PNA (Vector Laboratories, Burlingame, CA) for 20 min
at room temperature. Nonspecific binding was determined by incubation
with biotin-PNA plus 50 mM D-galactose (Sigma,
St. Louis, MO). Labeling was detected with the Vectastain ABC
peroxidase kit (Vector Laboratories), followed by diaminobenzidine
substrate. Sections were counterstained with methyl green.
Electroretinogram (ERG) recordings. Full-field corneal ERGs
were obtained in a Ganzfeld dome from mice of indicated genotypes and
ages. After at least 12 hr of dark adaptation, the mice were anesthetized with ketamine (200 mg/kg) and xylazine (10 mg/kg). Pupils
were dilated with local cyclopentolate hydrochloride drops before
study. A gold wire coil placed on one cornea was referenced to a
similar gold wire in the mouth. A needle electrode in the tail served
as a ground. Signals were amplified 10,000-fold with a Tektronix
(Beaverton, OR) AM502 differential amplifier (3 dB down at 2 and 10,000 Hz), digitized (sampling rate 1.25-5 kHz), and averaged on a personal
computer. Two different flash stimulators were used. A Grass
photostimulator provided 10 msec, short-wavelength flashes (Wratten
47A:  max = 470 nm; half-bandwidth = 55 nm)
from  3.0 to 1.0 log scotopic troland-seconds (scot td-sec) in 0.3 log unit steps. A high-intensity flash unit (Novatron, Dallas, TX)
produced 1.3 msec, short-wavelength flashes (Wratten W47B: max = 449 nm; half-bandwidth = 47 nm) from 1.0 to 3.4 log scot td-sec in 0.3 log unit steps.
The leading edge of the rod a-wave was fit by the Lamb and Pugh (1992)
model for the activation phase of phototransduction, which gives:
![P3(i,t)≅[1−<UP>exp</UP>(<UP>−</UP>i · S(t−t<SUB><UP>d</UP></SUB>)<SUP>2</SUP>)]RmP3 <UP>for</UP> t>t<SUB><UP>d</UP></SUB>](/content/vol19/issue14/fulltext/5889/img001.gif)
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(1)
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where P3 is the sum of the responses of individual
rods. The amplitude of P3 is a function of flash intensity
(i) and time (t) after flash onset. S
is a sensitivity parameter that scales i. RmP3 is
the maximum response, and td is a brief delay.
In the double-flash experiments two Novatron flash units were used
within the full-field dome. A 1.5 log scot td-sec test flash was
followed by a 3.4 log scot td-sec probe flash. The degree of recovery
of the a-wave at various times t after the test flash was
calculated from the size of the a-wave evoked by the probe flash
divided by that evoked by the probe flash in the absence of the
test flash. The recovery time course was fit by an exponential decline
function:
![<UP>exp</UP>[−(t−T)/&tgr;],](/content/vol19/issue14/fulltext/5889/img002.gif)
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(2)
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where T is the critical delay before any response
decline begins and  is the decline time constant.
Single-cell electrophysiology. The procedures were primarily
according to Sung et al. (1994). GC-E null or wild-type mice aged 8-10
weeks were dark-adapted overnight. Animals were killed by
CO2 asphyxiation under dim red light. All subsequent
procedures were performed under infrared light. The retina was isolated
from the enucleated eye in chilled, oxygenated Leibovitz's L-15 medium (Life Technologies) and placed photoreceptor-side up on a glass capillary array (10-µm-diameter capillaries; Galileo Electro-Optics, Sturbridge, MA) on which the retina was held by suction, allowing the
vitreous humor to be removed by moving a razor blade between the retina
and the array. The retinal pieces were stored in L-15 medium on ice
until use. When needed, a piece of retina was chopped under L-15 medium
containing 8 µg/ml deoxyribonuclease (Sigma) with a razor blade
mounted on a lever arm, and a suspension of small retinal fragments was
transferred into the recording chamber. The chamber temperature was
held at 36-38°C by perfusing it continuously with heated solution
buffered with bicarbonate and bubbled with 95%
O2/5% CO2, pH 7.4. The outer
segment of an isolated rod or a rod projecting from a small fragment of
retina was drawn into a suction electrode connected to a
current-to-voltage converter. The recorded membrane current was
filtered with a low-pass, eight-pole Bessel filter at 30 Hz and digitized.
The suction electrode was filled with a solution containing (in
mM): 134.5 Na+, 3.6 K+, 2.4 Mg2+, 1.2 Ca2+, 136.3 Cl , 3 succinate, 3 L-glutamate, 10 glucose, 10 HEPES, and 0.02 EDTA plus basal
medium Eagle (BME) amino acid supplement and BME vitamin supplement (Life Technologies). The perfusion medium was the same except that 20 mM NaHCO3 replaced an equimolar
amount of NaCl. The optical bench design was as previously described
(Baylor et al., 1979a). Unpolarized 8 msec flashes at 500 nm (10 nm
bandwidth) were used for stimulation throughout.
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RESULTS |
Generation of GC-E-deficient animals
The mouse GC-E gene (Gucy2e) was disrupted by replacing
a portion of exon 5, which codes for the transmembrane region, with a
neomycin-resistance gene cassette (Fig.
1a; see Materials and Methods). The targeted allele was maintained in both 129/SvJ and C57BL/6J backgrounds. The homozygous null animals (Fig. 1b)
were fully viable and fertile, indicating that GC-E is not required for
normal development. Moreover, there were no noticeable abnormalities in
the appearance or behavior of the knock-out animals when they were
compared with their wild-type and heterozygous littermates in the
normal animal-housing environment.
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Figure 1.
Targeted disruption of the GC-E gene in mice.
a, Maps of the wild-type GC-E locus, the targeting
vector, and the mutated locus. The coding exons of the GC-E gene are
shown as open boxes. The map of the targeting vector
shows the replacement of a portion of exon 5 coding for the
transmembrane region with the neomycin-resistance gene
(Neor). Two copies of the
thymidine kinase (TK) gene were placed at the
5'-end of the targeting vector for negative selection. The expected
sizes of BglII-generated fragments from the wild-type
and disrupted GC-E genes detected with a 5'-flanking probe (bold
bar) are shown. b, Southern blot analysis of DNA
from ES cell clones and tail DNA from littermates. The appearance of a
7 kb band from the mutant allele is indicated. c,
Western blot analysis of retinal extracts from 10-week-old wild-type
(+/+) and homozygous null (/) mice. Anti-GC-E, GC-F (Yang and
Garbers, 1997), and rds/peripherin antisera (Travis et
al., 1991) were used for immunodetection.
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Western-blot analysis was performed to confirm the absence of GC-E
expression in the knock-out animals. The posterior portion of the
eyecup was homogenized in Laemmli buffer, and total proteins were
separated by SDS-PAGE. Proteins were transferred to PVDF membranes and
blotted with an antiserum specific to the C-terminal portion of GC-E
(Yang and Garbers, 1997). This confirmed that the mutant allele
eliminates the expression of GC-E (Fig. 1c). The expression
level of GC-F was unchanged in 10-week-old homozygous null mice, as was
the level of rds/peripherin, an integral membrane glycoprotein located in photoreceptor outer segment disks (Connell et
al., 1991; Travis et al., 1991) (Fig. 1c). Even at 6 months of age the levels of GC-F, rds/peripherin, and rhodopsin
were comparable in wild-type and null animals (data not shown),
suggesting that the rod outer segments remained mainly intact in the
null animals.
Retinal structure of GC-E-deficient mice
Under light microscopy the development of the outer segment layer
in animals 1-3 weeks of age appeared normal in GC-E null animals. The
overall retinal structure continued to appear normal up to 1 year, with
unaltered thickness of the various retinal layers (Fig.
2). The number of photoreceptor nuclei
was counted from retinal sections from two wild-type and two null
animals. At 12 months of age the wild-type retina contained 151 ± 12 photoreceptor nuclei/2000 µm2, whereas the GC-E
/ retina contained 140 ± 12 photoreceptor nuclei/2000
µm2 (mean ± SEM). The retinas of
heterozygous animals likewise appeared morphologically normal at all
ages (data not shown).
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Figure 2.
Retinal morphology of wild-type (a,
c) and GC-E knock-out mice (b, d) at 10 weeks
(a, b) and 12 months (c, d) of age. Shown
are light micrographs of 4 µm sections cut through the optic nerve
head stained with hematoxylin and eosin. Magnification, 100×.
OS, Outer segment; IS, inner segment;
ONL, outer nuclear layer; OPL, outer
plexiform layer; INL, inner nuclear layer;
IPL, inner plexiform layer; GCL, ganglion
cell layer.
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Although with light microscopy the outer segments of cone cells cannot
be identified in the rod-dominant mouse retina, these cells can be
distinguished by their nuclear morphology. Cone cell bodies can be
recognized on the basis of their oval shape containing one to three
clumps of chromatin and a large amount of lightly staining euchromatin;
rods, on the other hand, have a round nucleus containing a single clump
of chromatin with little euchromatin (Carter-Dawson and LaVail, 1979).
At 4 weeks of age, the earliest age to identify reliably the cone cell
nuclei by this method, the number of cone cell bodies is similar in
wild-type and null animals. By 5 weeks of age, however, the number of
identifiable cones has been reduced dramatically in the null mice, with
only an occasional cone cell body recognizable in the outer nuclear layer (Fig. 3a-d). At later
ages the number of cone cells appears to remain stable, suggesting the
continued survival of the cones that were present at 5 weeks of age.
Thus, although the lack of GC-E does not seem to affect the
differentiation of cones, these cells degenerate rapidly at 4-5 weeks
of age, with a few cones surviving at late ages. The fact that the
overall thickness of the outer nuclear layer does not noticeably change
despite cone degeneration again suggests that the rods remain
relatively intact.
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Figure 3.
Cone photoreceptors are lost between
4 and 5 weeks of age in GC-E knock-out mice. Left
column, wild-type animals (+/+); right
column, GC-E null animals (/) . Shown are light
micrographs of 4-µm-thick hematoxylin- and eosin-stained sections
from animals of 4 weeks (a, b) and 5 weeks (c,
d) of age. Few cone cell nuclei are detectable in / mice at
5 weeks of age. However, cone cell nuclei are clearly evident in
wild-type littermates (arrows). Magnification, 250×.
Also shown are peanut agglutinin labeling of retinas from 4-week-old
(e, f) and 5-week-old (g,
h) mice. Cone photoreceptors (arrowheads) are
present in equal numbers in 4-week-old wild-type and null animals and
5-week-old wild-type mice, but they are not present in 5-week-old GC-E
/ mice. Magnification, 100×.
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The lectin PNA has been shown to label specifically the cone cell outer
and inner segments in several species, including mouse (Blanks and
Johnson, 1984). We used this lectin to label cone cells in wild-type
and GC-E knock-out retinas to confirm further the time course of cone
cell loss in the knock-out mice. At the age of 4 weeks the labeling of
scattered photoreceptor outer and inner segments, which are presumably
cones, was observed in both wild-type and null animals (Fig.
3e,f). Cone inner segments were stained the most
strongly. Equivalent labeling of wild-type and null animals also was
observed in 3-week-old animals (data not shown). The specificity of PNA
binding was confirmed by competitive inhibition of binding by 50 mM D-galactose (data not shown). No PNA label
was present in the retina of 5-week-old GC-E null mice, although
labeling was unchanged in wild-type animals (Fig. 3g,h). This confirmed the disappearance of cone cell nuclei that we had observed in sections stained with hematoxylin and eosin.
ERG measurements
To examine whether the loss of GC-E affects retinal function, we
measured full-field ERGs in wild-type, heterozygous, and null animals.
The ERGs of GC-E null animals showed alterations as early as 1 month of
age (Fig. 4a, right column),
with the rod a-wave (top panel) and b-wave
(middle panel) both markedly reduced. Cone responses
to white flashes in the presence of a rod-saturating background
(bottom panel) were barely detectable. Interestingly, this reduction in ERG response was evident before the apparent disappearance of cone cells, which occurred ~5 weeks of age. At 5 months of age (Fig. 4b) there was no further decrease in rod ERG amplitude, but the cone ERG was nondetectable and, in fact, was
absent as early as 2 months of age (data not shown). Values for the
sensitivity parameter (S) and the maximal amplitude
(RmP3) of the rod response were calculated for each animal
by fitting Equation 1 (see Materials and Methods) to the leading edge
of the rod a-wave (dashed curves in Fig. 4a,b,
top panel). The values for S, which
reflects the amplification of the activation phase of
phototransduction, were comparable between knock-out and wild-type animals at all ages. However, RmP3 was consistently lower
for GC-E / mice up to 1 year of age (Fig. 4c). By
comparison, the ERG responses of heterozygous mice were normal up to 1 year of age (data not shown). The reduced a-wave suggests that rod
function is compromised in the null animals despite their apparently
normal morphology. The reduction of the b-wave, which is approximately proportional to the a-wave reduction, is not surprising because it
arises primarily from the electrical activity of secondary neurons
(Ogden, 1994) and therefore is influenced by the a-wave. Finally, the
disappearance of the cone response is consistent with the morphological
finding that the number of cone cells is reduced sharply in animals
that are older than 5 weeks.
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Figure 4.
Full-field ERG responses from wild-type and
GC-E-deficient mice. a, b,
Top, Responses to short-wavelength flashes from 1.8 to
3.4 log scot td-sec in 0.3 log unit steps. Dashed curves
are fits of Equation 1 to the leading edge of the responses.
Middle, Responses to short-wavelength stimuli ranging
from 3 to 1 log scot td-sec (0.3 log unit steps). The b-wave is the
main component. Bottom, Cone responses to white flashes
from 0.24 to 1.44 log photopic troland-seconds (0.3 log unit steps) in
the presence of a rod-saturating (40 cd/m2)
background. No cone response is detectable in the / animal at the
age of 5 months. c, Plot of maximal amplitude of ERG
a-wave (RmP3) with increasing age. Data are averaged
(mean ± SEM) from 20 wild-type and 17 / animals.
d, Decline of RmP3 as a function of the
interflash interval in a double-flash experiment (see Materials and
Methods) from representative animals. The best-fit curves are
exponential declines. The critical period T is 208 msec
for the GC-E null mouse and 467 msec for the wild-type
mouse.
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Double-flash experiments were performed to determine whether recovery
from activation was altered in GC-E null animals. In this protocol the
recovery of the underlying photoresponse to a test flash was probed
with a second intense flash that followed the test flash at various
times (interflash intervals). As shown in Figure 4d, the
derived photoresponse of a representative GC-E / animal begins to
decline from saturation sooner than that of a wild-type animal. The
mean period of saturation of the a-wave (parameter T, Eq. 2
in Materials and Methods) at the intensity used for the first flash was
significantly shorter (p < 0.001; t
test) in the null animals (205 ± 16 msec; mean ± SEM;
n = 6) than in wild-type animals (372 ± 32 msec;
n = 8).
Rod photoreceptor electrophysiology
The reduction in the ERG a-wave in GC-E null animals prompted us
to examine the impact of the absence of GC-E on rod function. Responses
of single dark-adapted rods, either isolated or projecting from
fragments of mechanically dissociated retina, were recorded with a
suction pipette (Baylor et al., 1979a) (see Materials and Methods). In
contrast to the reduction in the ERG a-wave amplitude, the saturated
response elicited by a bright flash from rods of null animals was
normal in amplitude (rmax, Table
1), suggesting that the dark current
through the cGMP-activated channels was approximately unchanged. There
were changes in other respects, however. Although the flash response of
a rod from knock-out animals rose with normal kinetics, the response
exhibited an increased time-to-peak (tp,
Table 1) before decaying more rapidly than normal (compare Fig.
5a with b). The
rods from null mice also show oscillations during and after recovery
from a light flash; the reason for this remains unclear. Such
oscillations have been demonstrated previously in calculations under
conditions in which Ca2+ buffering is increased
(Nikonov et al., 1998). Additionally, after the light flash the
wild-type mice demonstrate multiple apparent phases of recovery (Fig.
5a), whereas the null mice show predominantly one phase
(Fig. 5b). The longer time-to-peak with the same trajectory
led to a higher sensitivity of the GC-E null rods, which can be seen
from the flash intensity-response relations in Figure 5c
(also Io, Table 1). This increase in the
flash sensitivity would not be detected by the ERG measurements because
the b-wave intrudes and truncates the a-wave (see Fig. 4a, top
panel). The higher sensitivity of rods from mutant mice
also was indicated by the larger amplitude of the single-photon
response (a, Table 1), calculated from the variance/mean
ratio of the response amplitudes of the cell to repetitive, identical
dim flashes (see Baylor et al., 1979b). These results indicate that
there is a disparity between ERG and single-rod recordings with respect
to the rod response amplitude; however, with both techniques a faster
recovery of rods from light is apparent.
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Figure 5.
Suction pipette recordings from single
rods of wild-type and GC-E null mice. a,
b, Normalized responses of a rod from a wild-type mouse
(a) and a GC-E / mouse
(b) to 500 nm flashes of increasing strength.
Each trace is the averaged response from multiple flash
trials. The records were low-pass-filtered at 30 Hz. Flash monitor
output is shown by the bottom trace in each
panel. The maximal response for a was
14.3 pA and for b was 10.7 pA. c,
Relation between the peak amplitude of flash response and the flash
intensity for the two rods shown in a and
b. Wild-type, squares; GC-E /,
circles. Curves are fit with the exponential saturation
function, r/rmax = 1 exp(ki), where k is a constant
inversely proportional to the sensitivity of the cell and
i is the flash strength. Half-maximal responses occurred
at 53 photons/µm2 for the wild-type rod and 20 photons/µm2 for the GC-E / rod.
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DISCUSSION |
A severe reduction in cones and a vanishing cone response to light
as a function of age represent the dominant phenotype of GC-E null
mice. However, such mice also display a decreased rod response to
light, based on ERG recordings. The marked decrease in the ERG response
occurs despite the morphologically normal appearance of the rods. The
likeliest consequence of the absence of GC-E is a reduction of the cGMP
level in cones in both dark and light conditions. This decrease itself
could facilitate degeneration, or alternatively, the cessation of the
dark current could be the primary effector of cone loss. Chronic light
exposure can cause degeneration as well, possibly via mechanisms
similar to that induced by the elimination of GC-E (Fain and Lisman,
1993). We do not know if null mice kept in the dark would retain
cones in the absence of GC-E.
The a-wave of the dark-adapted ERG is much smaller in amplitude than
normal for the null animals, suggesting a substantial loss of rod
function, in part concomitant with cone degeneration. However,
single-rod recordings suggest that the saturated response of individual
rods is close to normal. Thus, the reason for the reduction in the
a-wave is unclear. Possibly, because of the disappearance of cones and
associated changes in the extracellular matrix, the transretinal
resistance decreases. As a result, the same current response generated
in rods would produce a smaller ERG a-wave. Even before the
disappearance of cone cells at ~5 weeks of age, the functioning of
these cells may be compromised, leading to alterations in retinal
function despite apparently normal morphology as assessed with light
microscopy. A similar disparity between the ERG a-wave and single-rod
recordings has been reported previously for mice lacking the
phospholipase C  4 enzyme normally present in rods (Jiang et al.,
1996; Peng et al., 1997).
Despite a normal dark current the flash response of rods from null mice
is aberrant. The response rises with kinetics similar to control but
then has a longer time-to-peak, thus reaching higher amplitude for a
given flash intensity (i.e., a larger single-photon response). These
characteristics often are associated with negative-feedback control on
phototransduction. An interruption of the intracellular Ca2+ decline, for example, could result in a light
response rising unchecked via negative feedback (Yau, 1994). In this
scenario, however, the flash response is expected to decline more
slowly (Yau, 1994), opposite to the accelerated recovery kinetics
observed in both ERG and single-rod recordings. Based on modeling, the peculiar response characteristics demonstrated by the GC-E null rods in
principle can be explained by particular conditions with intracellular
Ca2+ buffering (Nikonov et al., 1998). However, no
experiments to measure the effects of a lack of GC-E on photoreceptor
Ca2+ have been performed yet.
What is the implication of an unchanged rod dark current in GC-E /
mice? The simplest interpretation would be that GC-E is present
insignificantly, if at all, in rods and therefore contributes little to
cGMP synthesis, with this function being performed by GC-F.
Alternatively, GC-E may be important for rod cGMP synthesis, but the
dark current remains constant in null animals because there is a
compensatory increase in the number of cGMP-activated channels on the
plasma membrane or an increase in the affinity of the channels for
cGMP. However, there is no evidence for such compensatory changes
because the length of the rod outer segment of null animals appears
normal, as do the activation properties of the channels and their
density on the plasma membrane (data not shown). Another potential
compensation for the lack of GC-E is an increase in the activity of
GC-F. Although GC-F expression is unchanged (see Results), its
catalytic activity could increase if the inhibition by
Ca2+ is changed, possibly because of a decrease in
the free Ca2+ concentration in the mutant rods in
darkness. However, this is not expected, given that the free
Ca2+ concentration in the steady state should depend
only on the dark current and the Ca2+ efflux through
the
Na+/Ca2+,K+
exchanger (Yau, 1994). In other words, with the dark current near
normal and the properties of the exchanger presumably unchanged, the
free Ca2+ concentration should stay constant, unless
a minor difference in the dark current is able to change the feedback
control on GC-F dramatically.
The gene for GC-F is located in region Xq22 of the human X chromosome
(Yang et al., 1996), to which no retinal diseases have been mapped. The
GC-E gene is located in mouse chromosome 11, syntenic with human
chromosome 17p13.1 (Oliveira et al., 1994; Yang et al., 1996), which
has been linked with several retinopathies (Joshi et al., 1997).
Recently, mutations in GC-E have been identified in human retinal
diseases. Missense and frame-shift mutations in the human GC-E gene
that are expected to render the protein nonfunctional have been
associated with Leber congenital amaurosis, characterized by
early-onset and widespread rod/cone degeneration (Perrault et al.,
1996). Like Leber's disease, the mouse GC-E null phenotype is
recessive; unlike the disease, however, the mouse phenotype consists
primarily of cone degeneration. One might argue that, unlike the
rod-dominant mouse retina, the human retina has substantially more
cones so that cone degeneration would disrupt the integrity of the
human retina more severely and consequently cause concomitant rod
degeneration. Other missense mutations of the human GC-E gene lead to a
form of cone/rod dystrophy (CORD6), a disease that initially affects
cones, followed by rod degeneration later (Kelsell et al., 1998;
Perrault et al., 1998). Although CORD6 resembles the mouse GC-E null
phenotype, the human disease is hereditarily dominant. Furthermore, the
GC-E missense mutations associated with CORD6 are thought to affect
dimerization of the enzyme rather than to cause its complete
elimination, as in the knock-out. Finally, in the chicken a naturally
occurring null mutation of the orthologous GC-E gene has been found to
cause autosomal recessive retinal degeneration (Ulshafer et al., 1984; Semple-Rowland et al., 1998). Although the degeneration in the rd chicken apparently differs from that in the GC-E null
mouse by affecting both rods and cones, the difference in this case again could be explained by the chicken retina being cone-rich (having
a cone/rod ratio much higher than in the mouse retina).
Another complexity that should be considered in interpreting our
results and the disease phenotypes has to do with GCAP1 and GCAP2, the
Ca2+-binding proteins that regulate GC-E and GC-F
function. There appears to be uniform agreement, based on
immunocytochemistry, that GCAP1 is present in both rod and cone outer
segments (Palczewski et al., 1994; Frins et al., 1996; Cuenca et al.,
1998; Howes et al., 1998). The localization of GCAP2 is more
controversial and perhaps species-dependent. It may be expressed in
both rod and cone outer segments (Otto-Bruc et al., 1997; Cuenca et
al., 1998; Howes et al., 1998), although biochemical experiments
suggest that GCAP1 and not GCAP2 is the major stimulator of guanylyl
cyclase in rod outer segments (Otto-Bruc et al., 1997). It was
demonstrated recently that a missense mutation in the human GCAP1 gene
leads to autosomal dominant cone dystrophy (Payne et al., 1998),
apparently because of constitutive activation of guanylyl cyclase
(Dizhoor et al., 1998; Sokal et al., 1998). The degeneration of cones
and the sparing of rods suggest that GCAP1 is functionally important in
cones but perhaps not as critical in rods. The observation of a similar
phenotype, i.e., cone degeneration, when GC-E or GCAP1 is mutated
suggests that tight regulation of guanylyl cyclase activity in these
cells may be crucial for their survival.
The GC-E-deficient mouse demonstrates that this protein is essential
for normal retinal structure and function. It appears that GC-E is
required for the survival of cone photoreceptors at an apparently
critical period at the age of ~5 weeks, whereas rod photoreceptors
appear to remain normal even at advanced ages. Although the kinetics of
activation of phototransduction is unchanged in rods, the maximum ERG
response is reduced markedly, and recovery after a light flash is
faster in mice lacking GC-E. These results suggest an important role
for this guanylyl cyclase in both the survival of a subclass of retinal
cells and in the delicate balance of cellular processes that lead to vision.
| |
FOOTNOTES |
Received Dec. 9, 1998; revised April 27, 1999; accepted May 4, 1999.
This research was supported in part by a grant-in-aid of research from
the National Academy of Sciences (D.L.G.), Sigma Xi, The Scientific
Research Society (R.B.Y.), and the National Eye Institute (K.-W.Y and
D.G.B., Grant EY05235). This work was supported by the Howard Hughes
Medical Institute. We thank Dr. Gabriel Travis for providing
rds/peripherin and rhodopsin antibodies, Valerie Mach
and Kristen Rossi for assistance in maintaining the mouse colony, and
Dr. Clint Makino for valuable help and advice on the recordings from
single mouse rods.
Correspondence should be addressed to Dr. David L. Garbers, Howard
Hughes Medical Institute, 5323 Harry Hines Boulevard, Dallas, TX
75235-9050.
Dr. Yang's present address: Department of Molecular Biology, Genentech
Incorporated, 1 DNA Way, South San Francisco, CA 94080.
| |
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26218 - 26229.
[Abstract]
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A. Mendez, M. E. Burns, I. Sokal, A. M. Dizhoor, W. Baehr, K. Palczewski, D. A. Baylor, and J. Chen
Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors
PNAS,
August 14, 2001;
98(17):
9948 - 9953.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION
