- Email: [email protected]
[37] Genetic models to study guanylyl cyclase function
558
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[37]
[37] Genetic Models to Study Guanylyl Cyclase Function B y SUSAN W . ROBINSON a n d DAVID L. GARBERS
Introduction The process of vision is initiated by light activation of the visual pigment and a subsequent decrease in cGMP levels, leading to closure of cGMPgated ion channels and a decrease in intracellular calcium (reviewed in Refs. 1 and 2). It has been suggested that subsequent to this decrease in calcium concentration, an activation of retinal guanylyl cyclases by guanylyl cyclase-activating proteins (GCAP1 and GCAP2) 3'4 occurs, leading to, at least in part, a restoration of the cGMP-gated ion channel to its open or " d a r k " state. Two retina-specific guanylyl cyclases have been identified in mammals. In the human they are designated retGC1 and retGC25,6; in the rat the homologs are referred to as GC-E and GC-F. 7 These cyclases are members of the family of membrane guanylyl cyclase receptors. The members of this family all contain an extracellular, putative ligand-binding domain, a single membrane-spanning segment, and intracellular protein kinase-like and cyclase catalytic domains. 8 With respect to the putative ligand-binding domain, these cyclases remain orphan receptors. In the mouse, the gene for GC-E maps to chromosome 11 and the GC-F gene is localized on the X chromosome. 9 To date, no visual diseases have been mapped to Xq22, the region of the X chromosome where GC-F is located in humans. However, several retinal degenerations have been mapped near 17p13.1, the GC-E locus in humans. Missense and frameshift mutations in GC-E have been found in families with Leber 1 S. Yarfitz and J. B. Hurley, J. Biol. Chem. 269, 14329 (1994). 2 K.-W. Yau, Invest. Ophthalmol. Vis. Sci. 35, 9 (1994). 3 K. Palczewski, I. Subbaraya, W. A. Gorczyca, B. S. Helekar, C. C. Ruiz, H. Ohguro, J. Huang, X. Zhao, J. W. Crabb, R. S. Johnson, K. A. Walsh, M. P. Gray-Keller, P. B. Detwiler, and W. Baehr, Neuron 13, 395 (1994). 4 A. M. Dizhoor, E. V. Olshevskaya, W. J. Henzel, S. C. Wong, J. T. Stults, I. Ankoudinova, and J. B. Hurley, J. Biol. Chem. 270, 25200 (1995). 5 A. W. Shyjan, F. J. de Sauvage, N. A. Gillett, D. V. Goeddel, and D. G. Lowe, Neuron 9,
727 (1992). 6D. G. Lowe, A. M. Dizhoor, K. Liu, Q. Gu, M. Spencer, R. Laura, L. Lu, and J. B. Hurley, Proc. Natl. Acad. Sci. U.S.A. 92, 5535 (1995). 7 R.-B. Yang, D. C. Foster, D. L. Garbers, and H.-J. Ffille,Proc. Natl. Acad. Sci. U.S.A. 92, 602 (1995). 8B. J. Wedel and D. L. Garbers, Trends Endocrinol. Metab. 9, 213 (1998). 9R.-B. Yang, H.-J. FOlle,and D. L. Garbers, Genomics 31, 367 (1996).
METHODS IN ENZYMOLOGY,VOL.316
Copyright© 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00 $30.00
[37]
GENETIC ANALYSIS OF RETINAL GUANYLYL CYCLASES
559
congenital amaurosis, 1° an autosomal recessive disease characterized by blindness at birth, a normal fundus, and extinguished or markedly reduced electroretinogram (ERG). n In addition, two mutations in GC-E have been identified in families with CORD6, a form of autosomal dominant cone-rod dystrophy. 12A3 A classic approach to define the importance of a signaling pathway is to specifically block the pathway. However, specific inhibitors have not been available for most receptor guanylyl cyclases. In addition, in the retina the situation is further complicated by the presence of two closely related proteins that are expressed not only in the same tissue, but also within the same cell. 14 Thus, gene disruption appears to represent a particularly powerful method by which to define the functions of these guanylyl cyclases in vision and to delineate specific roles each cyclase might play. An advantage of this approach is the ability to identify possible developmental roles for the molecule, as well as defining its function in vivo. In addition, given the apparent involvement of GC-E, and perhaps also GC-F, in inherited retinal dystrophies these animals may represent useful models for the study and possible treatment of these diseases. Methods
Generation of GC-E-Deficient Mice The mouse GC-E gene has been cloned from a 129SV/J genomic library. 9 A neomycin resistance cassette has been introduced in exon 5. This disrupts the protein in the transmembrane domain and prevents expression of the catalytic domain, which is required for cGMP synthesis. The targeting vector has been introduced into mouse 129SV/J embryonic stem cells (SM1, from R. Hammer, University of Texas Southwestern Medical Center, Dallas, TX) and GC-E-deficient mice have been generated by standard techniques. 15 A similar targeting vector for the disruption of the GC-F gene has also been constructed (S. W. R. and D. L. G., unpublished data, 1999). 10 I. Perrault, J.-M. Rozet, P. Calvas, S. Gerber, A. Camuzat, H. Dollfus, S. Ch~telin, E. Souied, I. Ghazi, C. Leowski, M. Bonnemaison, D. Le Paslier, J. Fr6zal, J.-L. Duffer, S. Pittler, A. Munnich, and J. Kaplan, Nature Genet. 14, 461 (1996). u K. Mizuno, Y. Takei, M. L. Sears, W. S. Peterson, R. E. Carr, and L. M. Jampol, Am. J. Ophthalmol. 83, 32 (1977). 12 R. E. Kelsell, K. Gregory-Evans, A. M. Payne, I. Perrault, J. Kaplan, R.-B. Yang, D. L. Garbers, A. C. Bird, A. Y. Moore, and D. M. Hunt, Hum. Mol. Genet. 7, 1179 (1998). 13 I. Perrault, J.-M. Rozet, S. Gerber, R. E. Kelsell, E. Souied, A. Cabot, D. M. Hunt, A. Munnich, and J. Kaplan, Am. J. Hum. Gener 63, 651 (1998). 14 R.-B. Yang and D. L. Garbers, J. Biol. Chem. 272, 13738 (1997). 15 R. Ramirez-Solis, A. C. Davis, and A. Bradley, Methods Enzymol. 225, 855 (1993).
560
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[371
Histological Analysis Mice are sacrificed by carbon dioxide asphyxiation and the eyes are collected. Eyes are typically fixed overnight in 10% (v/v) formalin at 4 ° and embedded in paraffin. Sections (3 /zm thick) are cut through the optic nerve head. Sections are stained with hematoxylin and eosin to identify cell structure. Cone photoreceptors are identified on the basis of their nuclear morphology. Cone cell bodies characteristically have an oval shape with several clumps of chromatin and a large amount of lightly staining euchromatin, while rod nuclei are smaller and round with a single clump of chromatin and little euchromatin. 16
Electroretinogram Recordings Full-field corneal ERGs are recorded in a Ganzfeld dome from mice that are dark adapted for at least 12 hr. All manipulations are carried out under dim red light. Mice are anesthetized with ketamine (200 mg/kg) and xylazine (10 mg/kg) and pupils are dilated with cyclopentolate hydrochloride drops. A gold wire coil is placed on the cornea and referenced to a similar wire in the mouth; a needle electrode in the tail serves as ground. Signals are 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. Short-wavelength flashes [Wratten (Eastman Kodak, Rochester, NY) 47A: Amax 470 nm, halfbandwidth 55 nm] from -3.0 to -1.0 log scotopic troland-seconds (scot td. sec) in 0.3 log unit steps are produced by a Grass (Grass Instrument Company, Quincy, MA) photostimulator. High-intensity 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 are produced by a Novatron (Dallas, TX) flash unit. Double flashes are produced by two Novatron flash units, with a 1.5 log scot td-sec test flash followed by a 3.4 log scot td.sec probe flash.
Single-Cell Electrophysiology For recording from isolated rod photoreceptors, mice are dark adapted overnight and sacrificed under dim red light. All subsequent manipulations are carried out under infrared light. The retina is isolated as described by Sung et al. 17 Briefly, the retina is isolated in chilled oxygenated Leibovitz L-15 medium (GIBCO-BRL, Gaithersburg, MD) and placed on a glass capillary array (10-/~m-diameter capillaries; Galileo Electro-Optics, San a6 L. D. Carter-Dawson and M. M. LaVail, J. Comp. Neurol. 188, 245 (1979). 17 C.-H. Sung, C. Makino, D. Baylor, and J. Nathans, J. Neurosci. 14, 5818 (1994).
[371
G E N E T I C ANALYSIS OF R E T I N A L G U A N Y L Y L CYCLASES
561
Jose, CA) with the photoreceptors up. When needed, a piece of retina is chopped under L-15 medium containing DNase I (8 /xg/ml; Sigma, St. Louis, MO) and a suspension of fragments is transferred to the recording chamber. The outer segment of a single rod is drawn into a suction electrode connected to a current-to-voltage converter. The electrode is filled with a solution containing 134.5 mM Na +, 3.6 mM K +, 2.4 mM Mg 2÷, 1.2 mM Ca 2+, 136.3 mM CI-, 3 mM succinate, 3 mM L-glutamate, 10 mM glucose, 10 mM HEPES, 0.02 mM EDTA, Basal Media Eagle (BME) amino acid, and BME vitamin supplement. Membrane current is filtered with a low-pass eightpole Bessel filter at 30 Hz and digitized. Unpolarized 8-msec flashes at 500 nm (10-nm bandwidth) are used for stimulation.
Discussion Mice that contain a disrupted GC-E gene were obtained and the lack of GC-E expression in the retina was confirmed by Western blotting. TMThe development of the outer segment layer during the first 3 weeks postnatal appeared normal in GC-E null animals (data not shown). The overall retinal structure of these animals appears normal up to at least 18 months of age. In mice at 4 weeks of age, there is no difference in the number of cone photoreceptor nuclei between wild-type and knockout animals. TMHowever, at 5 weeks of age, the number of identifiable cones is sharply reduced and at subsequent ages few cone cell nuclei are present (Fig. 1). These data suggest that GC-E is not required for the development of cones, as similar numbers are present in null and wild-type mice at 4 weeks of age. However, the presence of GC-E appears to be required for the survival of these cells. This raises the possibility that cGMP is an important survival factor for photoreceptor cells, and that the survival of rods in GC-E null mice is due to the presence of GC-F in these cells. 14 ERGs were recorded from mice at various ages to determine how the absence of GC-E affects retinal function. The ERG is a reflection of the electrical activity of the retina and has two main components: the a-wave and the b-wave. The a-wave reflects the response of the photoreceptor cells to light and the b-wave is generated in the ceils of the inner nuclear layer. 19 In the GC-E null mice, the ERG is markedly reduced as early as 4 weeks of age. Figure 2A shows representative ERGs from 2-month-old mice. Both the a- and b-waves are reduced in the GC-E null animal; however, the 18R.-B. Yang, S. W. Robinson,W.-H.Xiong,K.-W.Yau, D. G. Birch, and D. L. Garbers,J. Neuroscience 19, 5889 (1999). 19E. L. Berson, in "Adler's Physiologyof the Eye" (W. M. Hart, ed.), p. 641. Mosby-Year Book, St. Louis,Missouri, 1992.
562
[37]
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
A
GC-E +1+
soo
200 "0
100
E
o -100
I
I
I
I
50
100
150
200
time (msec)
300 >
GC-E -/-
200.-I 100
,~
_
-100
I
0
50
100
150
200
time (msec) FIG. 2. ERGs from 2-month-old wild-type (GC-E + / + ) and null (GC-E - / - ) mice. (A) Responses to short-wavelength stimuli from - 3 to 1 log scot td" sec in 0.3 log unit steps. The a-wave is the initial small negative response; the positive b-wave is the main component. (B) ERGs showing the b-wave response from a 2-month-old GC-E + / + animal and from 1month-, 4-month-, and 6-month-old GC-E - / - mice. Left: Rod responses to a - 2 log scot t d . s e c flash. The response is reduced in 1-month-old null mice; however, rod responses continue to be detectable in older knockout mice and remain relatively stable. Right: Responses to a 1.44 log photopic td. sec flash in the presence of a rod-saturating (40 cd/m 2) background. This presumably represents only the cone response. The response is severely reduced in GCE - / - animals at 1 month and is undetectable at later ages.
maximal amplitude of the rod response did not decrease significantly with age in null animals (Fig. 2B). In addition, the response to white flashes in the presence of a rod-saturating background, which presumably reflects cone activity, was barely detectable at 4 weeks in GC-E - / - mice and was
[371
563
G E N E T I C ANALYSIS OF R E T I N A L G U A N Y L Y L CYCLASES
B
ROD
CONE
GC-E +/+ 2 month
GC-E -/- 1 month
GC-E 4- 4 month
~
GC-E -/- 6 month >
g 100 msec FIG. 2.
(continued)
undetectable at later ages (Fig. 2). Double-flash experiments indicated that the recovery of the photoresponse from a test flash was substantially faster in GC-E knockout mice (205 - 16 msec) than in wild-type animals (372 +__ 32 msec). The ERG data are in agreement with the histological data, in that they reflect the disappearance of the cone photoreceptors and are consistent with the maintenance of the overall retinal structure in the knockout mice. The effect of the absence of GC-E on rod function was examined by recording responses from isolated rods. The response to a saturating flash was comparable in wild-type and null animals. This is in contrast to the sharp reduction in the a-wave observed by ERG. Although the flash response rose with normal kinetics, the time-to-peak was increased, resulting in a higher sensitivity in GC-E knockout rods. In addition, the recovery of the flash response was faster in null than in wild-type animals, consistent with our observations in the double-flash ERG experiments. Thus, some discrepan-
564
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[37]
cies between E R G and single-cell recordings remain to be resolved in the GC-E null mice. The use of gene targeting to study the function of retinal guanylyl cyclases has both confirmed previous studies of the role of this molecule in vision, and has provided important new insights. As expected, GC-E plays an important role in the retinal response to light. In GC-E - / - animals, the ERG, which reflects the activity of the photoreceptor and inner retinal neurons, is reduced. The remaining responsiveness to light may be due to the expression of the second retinal guanylyl cyclase, GC-F, in rods. It will be possible to answer this question when mice lacking GC-F are available. Surprisingly, rather than a slowed recovery from a light flash, which might be expected in cells that are impaired in their ability to resynthesize cGMP, both E R G and single-rod recordings indicate that GC-E-deficient photoreceptors in fact have a speeded recovery process. This result suggests that rods in the GC-E null animals have undergone compensatory changes that have altered the kinetics of the photoresponse. Further studies will be important to pinpoint these changes and assess their significance. The identification of a form of cone-rod dystrophy (CORD6) associated with mutations in GC-E 12'13 further confirms the importance of GC-E for cone survival. The lack of rod cell death in the mouse model may be a reflection of the small number of cones in the mouse retina. Their loss may not cause as severe a disruption of retinal structure as the loss of cones in humans or other species with a more cone-dominated retina. Despite this difference, the GC-E null mice represent a unique animal model for testing treatments of cone dystrophies, including gene therapy approaches. Acknowledgments We thank our collaborators in the analysis of the GC-E knockout mice: Dr. David G. Birch, electroretinograms; and Drs. Wei-hong Xiong and King-Wai Yau, rod single-cell electrophysiology.
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[37]
[37] Genetic Models to Study Guanylyl Cyclase Function B y SUSAN W . ROBINSON a n d DAVID L. GARBERS
Introduction The process of vision is initiated by light activation of the visual pigment and a subsequent decrease in cGMP levels, leading to closure of cGMPgated ion channels and a decrease in intracellular calcium (reviewed in Refs. 1 and 2). It has been suggested that subsequent to this decrease in calcium concentration, an activation of retinal guanylyl cyclases by guanylyl cyclase-activating proteins (GCAP1 and GCAP2) 3'4 occurs, leading to, at least in part, a restoration of the cGMP-gated ion channel to its open or " d a r k " state. Two retina-specific guanylyl cyclases have been identified in mammals. In the human they are designated retGC1 and retGC25,6; in the rat the homologs are referred to as GC-E and GC-F. 7 These cyclases are members of the family of membrane guanylyl cyclase receptors. The members of this family all contain an extracellular, putative ligand-binding domain, a single membrane-spanning segment, and intracellular protein kinase-like and cyclase catalytic domains. 8 With respect to the putative ligand-binding domain, these cyclases remain orphan receptors. In the mouse, the gene for GC-E maps to chromosome 11 and the GC-F gene is localized on the X chromosome. 9 To date, no visual diseases have been mapped to Xq22, the region of the X chromosome where GC-F is located in humans. However, several retinal degenerations have been mapped near 17p13.1, the GC-E locus in humans. Missense and frameshift mutations in GC-E have been found in families with Leber 1 S. Yarfitz and J. B. Hurley, J. Biol. Chem. 269, 14329 (1994). 2 K.-W. Yau, Invest. Ophthalmol. Vis. Sci. 35, 9 (1994). 3 K. Palczewski, I. Subbaraya, W. A. Gorczyca, B. S. Helekar, C. C. Ruiz, H. Ohguro, J. Huang, X. Zhao, J. W. Crabb, R. S. Johnson, K. A. Walsh, M. P. Gray-Keller, P. B. Detwiler, and W. Baehr, Neuron 13, 395 (1994). 4 A. M. Dizhoor, E. V. Olshevskaya, W. J. Henzel, S. C. Wong, J. T. Stults, I. Ankoudinova, and J. B. Hurley, J. Biol. Chem. 270, 25200 (1995). 5 A. W. Shyjan, F. J. de Sauvage, N. A. Gillett, D. V. Goeddel, and D. G. Lowe, Neuron 9,
727 (1992). 6D. G. Lowe, A. M. Dizhoor, K. Liu, Q. Gu, M. Spencer, R. Laura, L. Lu, and J. B. Hurley, Proc. Natl. Acad. Sci. U.S.A. 92, 5535 (1995). 7 R.-B. Yang, D. C. Foster, D. L. Garbers, and H.-J. Ffille,Proc. Natl. Acad. Sci. U.S.A. 92, 602 (1995). 8B. J. Wedel and D. L. Garbers, Trends Endocrinol. Metab. 9, 213 (1998). 9R.-B. Yang, H.-J. FOlle,and D. L. Garbers, Genomics 31, 367 (1996).
METHODS IN ENZYMOLOGY,VOL.316
Copyright© 2000by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00 $30.00
[37]
GENETIC ANALYSIS OF RETINAL GUANYLYL CYCLASES
559
congenital amaurosis, 1° an autosomal recessive disease characterized by blindness at birth, a normal fundus, and extinguished or markedly reduced electroretinogram (ERG). n In addition, two mutations in GC-E have been identified in families with CORD6, a form of autosomal dominant cone-rod dystrophy. 12A3 A classic approach to define the importance of a signaling pathway is to specifically block the pathway. However, specific inhibitors have not been available for most receptor guanylyl cyclases. In addition, in the retina the situation is further complicated by the presence of two closely related proteins that are expressed not only in the same tissue, but also within the same cell. 14 Thus, gene disruption appears to represent a particularly powerful method by which to define the functions of these guanylyl cyclases in vision and to delineate specific roles each cyclase might play. An advantage of this approach is the ability to identify possible developmental roles for the molecule, as well as defining its function in vivo. In addition, given the apparent involvement of GC-E, and perhaps also GC-F, in inherited retinal dystrophies these animals may represent useful models for the study and possible treatment of these diseases. Methods
Generation of GC-E-Deficient Mice The mouse GC-E gene has been cloned from a 129SV/J genomic library. 9 A neomycin resistance cassette has been introduced in exon 5. This disrupts the protein in the transmembrane domain and prevents expression of the catalytic domain, which is required for cGMP synthesis. The targeting vector has been introduced into mouse 129SV/J embryonic stem cells (SM1, from R. Hammer, University of Texas Southwestern Medical Center, Dallas, TX) and GC-E-deficient mice have been generated by standard techniques. 15 A similar targeting vector for the disruption of the GC-F gene has also been constructed (S. W. R. and D. L. G., unpublished data, 1999). 10 I. Perrault, J.-M. Rozet, P. Calvas, S. Gerber, A. Camuzat, H. Dollfus, S. Ch~telin, E. Souied, I. Ghazi, C. Leowski, M. Bonnemaison, D. Le Paslier, J. Fr6zal, J.-L. Duffer, S. Pittler, A. Munnich, and J. Kaplan, Nature Genet. 14, 461 (1996). u K. Mizuno, Y. Takei, M. L. Sears, W. S. Peterson, R. E. Carr, and L. M. Jampol, Am. J. Ophthalmol. 83, 32 (1977). 12 R. E. Kelsell, K. Gregory-Evans, A. M. Payne, I. Perrault, J. Kaplan, R.-B. Yang, D. L. Garbers, A. C. Bird, A. Y. Moore, and D. M. Hunt, Hum. Mol. Genet. 7, 1179 (1998). 13 I. Perrault, J.-M. Rozet, S. Gerber, R. E. Kelsell, E. Souied, A. Cabot, D. M. Hunt, A. Munnich, and J. Kaplan, Am. J. Hum. Gener 63, 651 (1998). 14 R.-B. Yang and D. L. Garbers, J. Biol. Chem. 272, 13738 (1997). 15 R. Ramirez-Solis, A. C. Davis, and A. Bradley, Methods Enzymol. 225, 855 (1993).
560
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[371
Histological Analysis Mice are sacrificed by carbon dioxide asphyxiation and the eyes are collected. Eyes are typically fixed overnight in 10% (v/v) formalin at 4 ° and embedded in paraffin. Sections (3 /zm thick) are cut through the optic nerve head. Sections are stained with hematoxylin and eosin to identify cell structure. Cone photoreceptors are identified on the basis of their nuclear morphology. Cone cell bodies characteristically have an oval shape with several clumps of chromatin and a large amount of lightly staining euchromatin, while rod nuclei are smaller and round with a single clump of chromatin and little euchromatin. 16
Electroretinogram Recordings Full-field corneal ERGs are recorded in a Ganzfeld dome from mice that are dark adapted for at least 12 hr. All manipulations are carried out under dim red light. Mice are anesthetized with ketamine (200 mg/kg) and xylazine (10 mg/kg) and pupils are dilated with cyclopentolate hydrochloride drops. A gold wire coil is placed on the cornea and referenced to a similar wire in the mouth; a needle electrode in the tail serves as ground. Signals are 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. Short-wavelength flashes [Wratten (Eastman Kodak, Rochester, NY) 47A: Amax 470 nm, halfbandwidth 55 nm] from -3.0 to -1.0 log scotopic troland-seconds (scot td. sec) in 0.3 log unit steps are produced by a Grass (Grass Instrument Company, Quincy, MA) photostimulator. High-intensity 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 are produced by a Novatron (Dallas, TX) flash unit. Double flashes are produced by two Novatron flash units, with a 1.5 log scot td-sec test flash followed by a 3.4 log scot td.sec probe flash.
Single-Cell Electrophysiology For recording from isolated rod photoreceptors, mice are dark adapted overnight and sacrificed under dim red light. All subsequent manipulations are carried out under infrared light. The retina is isolated as described by Sung et al. 17 Briefly, the retina is isolated in chilled oxygenated Leibovitz L-15 medium (GIBCO-BRL, Gaithersburg, MD) and placed on a glass capillary array (10-/~m-diameter capillaries; Galileo Electro-Optics, San a6 L. D. Carter-Dawson and M. M. LaVail, J. Comp. Neurol. 188, 245 (1979). 17 C.-H. Sung, C. Makino, D. Baylor, and J. Nathans, J. Neurosci. 14, 5818 (1994).
[371
G E N E T I C ANALYSIS OF R E T I N A L G U A N Y L Y L CYCLASES
561
Jose, CA) with the photoreceptors up. When needed, a piece of retina is chopped under L-15 medium containing DNase I (8 /xg/ml; Sigma, St. Louis, MO) and a suspension of fragments is transferred to the recording chamber. The outer segment of a single rod is drawn into a suction electrode connected to a current-to-voltage converter. The electrode is filled with a solution containing 134.5 mM Na +, 3.6 mM K +, 2.4 mM Mg 2÷, 1.2 mM Ca 2+, 136.3 mM CI-, 3 mM succinate, 3 mM L-glutamate, 10 mM glucose, 10 mM HEPES, 0.02 mM EDTA, Basal Media Eagle (BME) amino acid, and BME vitamin supplement. Membrane current is filtered with a low-pass eightpole Bessel filter at 30 Hz and digitized. Unpolarized 8-msec flashes at 500 nm (10-nm bandwidth) are used for stimulation.
Discussion Mice that contain a disrupted GC-E gene were obtained and the lack of GC-E expression in the retina was confirmed by Western blotting. TMThe development of the outer segment layer during the first 3 weeks postnatal appeared normal in GC-E null animals (data not shown). The overall retinal structure of these animals appears normal up to at least 18 months of age. In mice at 4 weeks of age, there is no difference in the number of cone photoreceptor nuclei between wild-type and knockout animals. TMHowever, at 5 weeks of age, the number of identifiable cones is sharply reduced and at subsequent ages few cone cell nuclei are present (Fig. 1). These data suggest that GC-E is not required for the development of cones, as similar numbers are present in null and wild-type mice at 4 weeks of age. However, the presence of GC-E appears to be required for the survival of these cells. This raises the possibility that cGMP is an important survival factor for photoreceptor cells, and that the survival of rods in GC-E null mice is due to the presence of GC-F in these cells. 14 ERGs were recorded from mice at various ages to determine how the absence of GC-E affects retinal function. The ERG is a reflection of the electrical activity of the retina and has two main components: the a-wave and the b-wave. The a-wave reflects the response of the photoreceptor cells to light and the b-wave is generated in the ceils of the inner nuclear layer. 19 In the GC-E null mice, the ERG is markedly reduced as early as 4 weeks of age. Figure 2A shows representative ERGs from 2-month-old mice. Both the a- and b-waves are reduced in the GC-E null animal; however, the 18R.-B. Yang, S. W. Robinson,W.-H.Xiong,K.-W.Yau, D. G. Birch, and D. L. Garbers,J. Neuroscience 19, 5889 (1999). 19E. L. Berson, in "Adler's Physiologyof the Eye" (W. M. Hart, ed.), p. 641. Mosby-Year Book, St. Louis,Missouri, 1992.
562
[37]
ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
A
GC-E +1+
soo
200 "0
100
E
o -100
I
I
I
I
50
100
150
200
time (msec)
300 >
GC-E -/-
200.-I 100
,~
_
-100
I
0
50
100
150
200
time (msec) FIG. 2. ERGs from 2-month-old wild-type (GC-E + / + ) and null (GC-E - / - ) mice. (A) Responses to short-wavelength stimuli from - 3 to 1 log scot td" sec in 0.3 log unit steps. The a-wave is the initial small negative response; the positive b-wave is the main component. (B) ERGs showing the b-wave response from a 2-month-old GC-E + / + animal and from 1month-, 4-month-, and 6-month-old GC-E - / - mice. Left: Rod responses to a - 2 log scot t d . s e c flash. The response is reduced in 1-month-old null mice; however, rod responses continue to be detectable in older knockout mice and remain relatively stable. Right: Responses to a 1.44 log photopic td. sec flash in the presence of a rod-saturating (40 cd/m 2) background. This presumably represents only the cone response. The response is severely reduced in GCE - / - animals at 1 month and is undetectable at later ages.
maximal amplitude of the rod response did not decrease significantly with age in null animals (Fig. 2B). In addition, the response to white flashes in the presence of a rod-saturating background, which presumably reflects cone activity, was barely detectable at 4 weeks in GC-E - / - mice and was
[371
563
G E N E T I C ANALYSIS OF R E T I N A L G U A N Y L Y L CYCLASES
B
ROD
CONE
GC-E +/+ 2 month
GC-E -/- 1 month
GC-E 4- 4 month
~
GC-E -/- 6 month >
g 100 msec FIG. 2.
(continued)
undetectable at later ages (Fig. 2). Double-flash experiments indicated that the recovery of the photoresponse from a test flash was substantially faster in GC-E knockout mice (205 - 16 msec) than in wild-type animals (372 +__ 32 msec). The ERG data are in agreement with the histological data, in that they reflect the disappearance of the cone photoreceptors and are consistent with the maintenance of the overall retinal structure in the knockout mice. The effect of the absence of GC-E on rod function was examined by recording responses from isolated rods. The response to a saturating flash was comparable in wild-type and null animals. This is in contrast to the sharp reduction in the a-wave observed by ERG. Although the flash response rose with normal kinetics, the time-to-peak was increased, resulting in a higher sensitivity in GC-E knockout rods. In addition, the recovery of the flash response was faster in null than in wild-type animals, consistent with our observations in the double-flash ERG experiments. Thus, some discrepan-
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ANALYSIS OF ANIMAL MODELS OF RETINAL DISEASES
[37]
cies between E R G and single-cell recordings remain to be resolved in the GC-E null mice. The use of gene targeting to study the function of retinal guanylyl cyclases has both confirmed previous studies of the role of this molecule in vision, and has provided important new insights. As expected, GC-E plays an important role in the retinal response to light. In GC-E - / - animals, the ERG, which reflects the activity of the photoreceptor and inner retinal neurons, is reduced. The remaining responsiveness to light may be due to the expression of the second retinal guanylyl cyclase, GC-F, in rods. It will be possible to answer this question when mice lacking GC-F are available. Surprisingly, rather than a slowed recovery from a light flash, which might be expected in cells that are impaired in their ability to resynthesize cGMP, both E R G and single-rod recordings indicate that GC-E-deficient photoreceptors in fact have a speeded recovery process. This result suggests that rods in the GC-E null animals have undergone compensatory changes that have altered the kinetics of the photoresponse. Further studies will be important to pinpoint these changes and assess their significance. The identification of a form of cone-rod dystrophy (CORD6) associated with mutations in GC-E 12'13 further confirms the importance of GC-E for cone survival. The lack of rod cell death in the mouse model may be a reflection of the small number of cones in the mouse retina. Their loss may not cause as severe a disruption of retinal structure as the loss of cones in humans or other species with a more cone-dominated retina. Despite this difference, the GC-E null mice represent a unique animal model for testing treatments of cone dystrophies, including gene therapy approaches. Acknowledgments We thank our collaborators in the analysis of the GC-E knockout mice: Dr. David G. Birch, electroretinograms; and Drs. Wei-hong Xiong and King-Wai Yau, rod single-cell electrophysiology.