Mechanistically distinct mouse models for CRX-associated retinopathy.

Cone-rod homeobox (CRX) protein is a "paired-like" homeodomain transcription factor that is essential for regulating rod and cone photoreceptor transcription. Mutations in human CRX are associated with the dominant retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congenital Amaurosis (LCA), with variable severity. Heterozygous Crx Knock-Out (KO) mice ("+/-") have normal vision as adults and fail to model the dominant human disease. To investigate how different mutant CRX proteins produce distinct disease pathologies, we generated two Crx Knock-IN (K-IN) mouse models: Crx(E168d2) ("E168d2") and Crx(R90W) ("R90W"). E168d2 mice carry a frameshift mutation in the CRX activation domain, Glu168del2, which is associated with severe dominant CoRD or LCA in humans. R90W mice carry a substitution mutation in the CRX homeodomain, Arg90Trp, which is associated with dominant mild late-onset CoRD and recessive LCA. As seen in human patients, heterozygous E168d2 ("E168d2/+") but not R90W ("R90W/+") mice show severely impaired retinal function, while mice homozygous for either mutation are blind and undergo rapid photoreceptor degeneration. E168d2/+ mice also display abnormal rod/cone morphology, greater impairment of CRX target gene expression than R90W/+ or +/- mice, and undergo progressive photoreceptor degeneration. Surprisingly, E168d2/+ mice express more mutant CRX protein than wild-type CRX. E168d2neo/+, a subline of E168d2 with reduced mutant allele expression, displays a much milder retinal phenotype, demonstrating the impact of Crx expression level on disease severity. Both CRX([E168d2]) and CRX([R90W]) proteins fail to activate transcription in vitro, but CRX([E168d2]) interferes more strongly with the function of wild type (WT) CRX, supporting an antimorphic mechanism. E168d2 and R90W are mechanistically distinct mouse models for CRX-associated disease that will allow the elucidation of molecular mechanisms and testing of novel therapeutic approaches for different forms of CRX-associated disease.

Feedback activation of neurofibromin terminates growth factor-induced Ras activation.

BACKGROUND: Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown. RESULTS: Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels. CONCLUSIONS: Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.

Ras activation revisited: role of GEF and GAP systems.

Ras is a prototypical small G-protein and a central regulator of growth, proliferation and differentiation processes in virtually every nucleated cell. As such, Ras becomes engaged and activated by multiple growth factors, mitogens, cytokines or adhesion receptors. Ras activation comes about by changes in the steady-state equilibrium between the inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states of Ras, resulting in the mostly transient accumulation of Ras-GTP. Three decades of intense Ras research have disclosed various families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as the two principal regulatory elements of the Ras-GDP/GTP loading status. However, with the possible exception of the GEF Sos, we still have only a rudimentary knowledge of the precise role played by many GEF and GAP members in the signalling network upstream of Ras. As for GAPs, we even lack the fundamental understanding of whether they function as genuine signal transducers in the context of growth factor-elicited Ras activation or rather act as passive modulators of the Ras-GDP/GTP cycle. Here we sift through the large body of Ras literature and review the relevant data for understanding the participation and precise role played by GEFs and GAPs in the process of Ras activation.

Ras palmitoylation is necessary for N-Ras activation and signal propagation in growth factor signalling.

Ras GTPases undergo post-translational modifications that govern their subcellular trafficking and localization. In particular, palmitoylation of the Golgi tags N-Ras and H-Ras for exocytotic transport and residency at the PM (plasma membrane). Following depalmitoylation, PM-Ras redistributes to all subcellular membranes causing an accumulation of palmitate-free Ras at endomembranes, including the Golgi and endoplasmic reticulum. Palmitoylation is unanimously regarded as a critical modification at the crossroads of Ras activity and trafficking control, but its precise relevance to native wild-type Ras function in growth factor signalling is unknown. We show in the present study by use of palmitoylation-deficient N-Ras mutants and via the analysis of palmitate content of agonist-activated GTP-loaded N-Ras that only palmitoylated N-Ras becomes activated by agonists. In line with an essential role of palmitoylation in Ras activation, dominant-negative RasS17N loses its blocking potency if rendered devoid of palmitoylation. Live-cell Ras-GTP imaging shows that N-Ras activation proceeds only at the PM, consistent with activated N-Ras-GTP being palmitoylated. Finally, palmitoylation-deficient N-Ras does not sustain EGF (epidermal growth factor) or serum-elicited mitogenic signalling, confirming that palmitoylation is essential for signal transduction by N-Ras. These findings document that N-Ras activation proceeds at the PM and suggest that depalmitoylation, by removing Ras from the PM, may contribute to the shutdown of Ras signalling.

G-protein betagamma-complex is crucial for efficient signal amplification in vision.

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary ßγ-complex in this process remains a mystery. Here, we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin ßγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.

Performance of a six-methylation-marker assay on self-collected cervical samples - A feasibility study.

OBJECTIVES: In high-income countries, a high proportion of cervical cancers is diagnosed in screening non-attendees. One approach to improve screening coverage is to offer self-sampling for human papillomavirus (HPV) testing. However, especially young women are often HPV positive without having a precancerous lesion in need of treatment. To improve the rather low specificity of HPV testing additional markers such as DNA-methylation can be used. The aim of this feasibility study was to examine the performance of the methylation marker assay GynTect®, comprising six methylation markers, on dry self-collected cervico-vaginal samples compared to physician-taken samples. METHODS: We recruited 89 patients from our colposcopy clinic of whom 87 qualified for the study. The women took a self-sample with the Evalyn-Brush. Afterwards the planned colposcopy was performed and smears for cytology and reference HPV testing were taken as well as a biopsy in cases of abnormalities. Physician-taken and self-collected samples were tested for HPV DNA and were analyzed with GynTect®. RESULTS: We obtained 95.5 % valid results for the self-collected samples which was very close to the physician-taken samples. Only about half of the self-collected samples were GynTect® positive in comparison to the physician-taken samples. GynTect® scores were significantly lower for self-collected than for physician-taken samples (p = 0.001, paired t-test). The overall concordance for GynTect® results was moderate (kappa 0.394; p < 0.001). For HPV testing we obtained a good concordance (kappa 0.586; p < 0.001). The GynTect® results for the self-collected samples showed a sensitivity for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+) of 26.1 % (95 %-CI: 0.13-0.46) and a specificity of 95.6 % (95 %-CI: 0.85-0.99), in comparison to a sensitivity of 45.5 % (95 %-CI: 0.27-0.65) and a specificity of 78.3 % (95 %-CI: 0.64-0.88) for the physician-taken samples. CONCLUSIONS: GynTect® methylation marker testing has a satisfactory amount of valid results on self-collected samples. However, the results of the self-collected samples differed clearly in comparison to the reference samples. To justify an application in screening, a larger study with more cases of high-grade cervical dysplasia and HPV positive patients will be needed.

Laccase-Enzyme Treated Flax Fibre for Use in Natural Fibre Epoxy Composites.

Natural fibres have a high potential as reinforcement of polymer matrices, as they combine a high specific strength and modulus with sustainable production and reasonable prices. Modifying the fibre surface is a common method to increase the adhesion and thereby enhance the mechanical properties of composites. In this study, a novel sustainable surface treatment is presented: the fungal enzyme laccase was utilised with the aim of covalently binding the coupling agent dopamine to flax fibre surfaces. The goal is to improve the interfacial strength towards an epoxy matrix. SEM and AFM micrographs showed that the modification changes the surface morphology, indicating a deposition of dopamine on the surface. Fibre tensile tests, which were performed to check whether the fibre structure was damaged during the treatment, showed that no decrease in tensile strength or modulus occurred. Single fibre pullout tests showed a 30% increase in interfacial shear strength (IFSS) due to the laccase-mediated bonding of the coupling agent dopamine. These results demonstrate that a laccase + dopamine treatment modifies flax fibres sustainably and increases the interfacial strength towards epoxy.

Regulation of photoreceptor gene expression by Crx-associated transcription factor network.

Rod and cone photoreceptors in the mammalian retina are special types of neurons that are responsible for phototransduction, the first step of vision. Development and maintenance of photoreceptors require precisely regulated gene expression. This regulation is mediated by a network of photoreceptor transcription factors centered on Crx, an Otx-like homeodomain transcription factor. The cell type (subtype) specificity of this network is governed by factors that are preferentially expressed by rods or cones or both, including the rod-determining factors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; and cone-determining factors, mostly nuclear receptor family members. The best-documented of these include thyroid hormone receptor beta2 (Tr beta2), retinoid related orphan receptor Ror beta, and retinoid X receptor Rxr gamma. The appropriate function of this network also depends on general transcription factors and cofactors that are ubiquitously expressed, such as the Sp zinc finger transcription factors and STAGA co-activator complexes. These cell type-specific and general transcription regulators form complex interactomes; mutations that interfere with any of the interactions can cause photoreceptor development defects or degeneration. In this manuscript, we review recent progress on the roles of various photoreceptor transcription factors and interactions in photoreceptor subtype development. We also provide evidence of auto-, para-, and feedback regulation among these factors at the transcriptional level. These protein-protein and protein-promoter interactions provide precision and specificity in controlling photoreceptor subtype-specific gene expression, development, and survival. Understanding these interactions may provide insights to more effective therapeutic interventions for photoreceptor diseases.

MLL1 is essential for retinal neurogenesis and horizontal inner neuron integrity.

Development of retinal structure and function is controlled by cell type-specific transcription factors and widely expressed co-regulators. The latter includes the mixed-lineage leukemia (MLL) family of histone methyltransferases that catalyze histone H3 lysine 4 di- and tri-methylation associated with gene activation. One such member, MLL1, is widely expressed in the central nervous system including the retina. However, its role in retinal development is unknown. To address this question, we knocked out Mll1 in mouse retinal progenitors, and discovered that MLL1 plays multiple roles in retinal development by regulating progenitor cell proliferation, cell type composition and neuron-glia balance, maintenance of horizontal neurons, and formation of functional synapses between neuronal layers required for visual signal transmission and processing. Altogether, our results suggest that MLL1 is indispensable for retinal neurogenesis and function development, providing a new paradigm for cell type-specific roles of known histone modifying enzymes during CNS tissue development.

Crx-L253X Mutation Produces Dominant Photoreceptor Defects in TVRM65 Mice.

Purpose: The cone-rod homeobox (CRX) transcription factor is essential for photoreceptor gene expression, differentiation, and survival. Human CRX mutations can cause dominant retinopathies of varying onset and phenotype severity. In animal models, dominant frameshift Crx mutations introduce a premature termination codon (PTC), producing inactive truncated proteins that interfere with normal CRX function. Previously, a mutant mouse, TVRM65, was reported to carry a recessive late PTC mutation, Crx-L253X. More detailed phenotype analysis of the pathogenicity of Crx-L253X sheds new light on the variability of CRX-linked diseases. Methods: Homozygous (L253X/X); heterozygous (L253X/+); Crx-/- and control C57BL/6J (WT) mice were analyzed at various ages for changes in retinal function (ERG), morphology (histology) and photoreceptor gene expression (qRT-PCR). Results: At 1 month, L253X/X mice lack visual function, show greater reductions in retinal thickness, and distinct gene expression changes relative to Crx-/-, suggesting that the phenotype of L253X/X is more severe than Crx-/-. L253X/+ mice have reduced rod/cone function, but normal retinal morphology at all ages tested. qRT-PCR assays described a complex phenotype in which both developing and mature photoreceptors are unable to maintain proper gene expression. L253X mRNA/protein is overexpressed relative to normal Crx, suggesting a pathogenic mechanism similar to early PTC mutations. However, the overexpression is less pronounced, correlating with a relatively mild dominant phenotype. Conclusions: The L253X mouse provides a valuable model for CRX-associated retinopathy. The pathogenicity of CRX frameshift mutations depends on the position of the PTC, which in turn determines the degree of mutant mRNA/protein overproduction.

Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice.

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.

Water consumption and biomass production of protoplast fusion lines of poplar hybrids under drought stress.

Woody crops such as poplars (Populus) can contribute to meet the increasing energy demand of a growing human population and can therefore enhance the security of energy supply. Using energy from biomass increases ecological sustainability as biomass is considered to play a pivotal role in abating climate change. Because areas for establishing poplar plantations are often confined to marginal sites drought tolerance is one important trait for poplar genotypes cultivated in short rotation coppice. We tested 9-month-old plants of four tetraploid Populus tremula (L.) × P. tremuloides (Michx.) lines that were generated by protoplast fusion and their diploid counterpart for water consumption and drought stress responses in a greenhouse experiment. The fusion lines showed equivalent or decreased height growth, stem biomass and total leaf area compared to the diploid line. The relative height increment of the fusion lines was not reduced compared to the diploid line when the plants were exposed to drought. The fusion lines were distinguished from the diploid counterpart by stomatal characteristics such as increased size and lower density. The changes in the stomatal apparatus did not affect the stomatal conductance. When exposed to drought the carbohydrate concentrations increased more strongly in the fusion lines than in the diploid line. Two fusion lines consumed significantly less water with regard to height growth, producing equivalent or increased relative stem biomass under drought compared to their diploid relative. Therefore, these tetraploid fusion lines are interesting candidates for short rotation biomass plantation on dry sites.

Inner ear pathology in the mucopolysaccharidosis VII mouse.

Mucopolysaccharidosis type VII (MPS VII, Sly syndrome) is caused by dysfunction of the acid hydrolase beta-D-glucuronidase. The defect results in the accumulation of incompletely degraded glycosaminoglycans within lysosomes of a wide array of cell types. MPS VII is associated with mixed (conductive and sensorineural) hearing loss, vision defects, shortened stature, mental retardation and decreased lifespan. Whether the sensorineural component of hearing loss in MPS VII involves degeneration of cochlear sensory cells is not yet clear. The MPS VII mouse resembles its human counterpart in all major aspects, and has been the focus of extensive research seeking to correct MPS VII and other lysosomal storage diseases. The value of potential treatments for this hearing loss can be determined only if cochlear pathology in this model is well characterized. We examined threshold sensitivity, frequency tuning, hair cell density and the appearance of the cochlea and vestibular organs in MPS VII mice ranging from 1.0 to 7.5 months of age. At all ages, lysosomal storage is pronounced within cells of spiral limbus, spiral prominence, spiral ligament and glial cells, but not within organ of Corti, stria vascularis, or neurons. Within the vestibular maculae and cristae, both hair cells and supporting cells also show lysosomal storage. Although hearing thresholds are never normal, reduction in the sharpness of frequency tuning is not apparent until 2.5 months of age, suggesting that the sensorineural component of hearing loss begins in adulthood. No evidence was found for cell loss within the organ of Corti, or any other structure, however. Our results suggest that sensorineural hearing loss in the MPS VII mouse is not caused by degeneration, but may arise from alterations in mass and stiffness of cochlear structures or impaired sensory cell function. They also indicate a possible vestibular component in MPS VII.

Transcription coactivators p300 and CBP are necessary for photoreceptor-specific chromatin organization and gene expression.

Rod and cone photoreceptor neurons in the mammalian retina possess specialized cellular architecture and functional features for converting light to a neuronal signal. Establishing and maintaining these characteristics requires appropriate expression of a specific set of genes, which is tightly regulated by a network of photoreceptor transcription factors centered on the cone-rod homeobox protein CRX. CRX recruits transcription coactivators p300 and CBP to acetylate promoter-bound histones and activate transcription of target genes. To further elucidate the role of these two coactivators, we conditionally knocked out Ep300 and/or CrebBP in differentiating rods or cones, using opsin-driven Cre recombinase. Knockout of either factor alone exerted minimal effects, but loss of both factors severely disrupted target cell morphology and function: the unique nuclear chromatin organization seen in mouse rods was reversed, accompanied by redistribution of nuclear territories associated with repressive and active histone marks. Transcription of many genes including CRX targets was severely impaired, correlating with reduced histone H3/H4 acetylation (the products of p300/CBP) on target gene promoters. Interestingly, the presence of a single wild-type allele of either coactivator prevented many of these defects, with Ep300 more effective than Cbp. These results suggest that p300 and CBP play essential roles in maintaining photoreceptor-specific structure, function and gene expression.

Epigenetic regulation of retinal development and disease.

Epigenetic regulation, including DNA methylation, histone modifications, and chromosomal organization, is emerging as a new layer of transcriptional regulation in retinal development and maintenance. Guided by intrinsic transcription factors and extrinsic signaling molecules, epigenetic regulation can activate and/or repress the expression of specific sets of genes, therefore playing an important role in retinal cell fate specification and terminal differentiation during development as well as maintaining cell function and survival in adults. Here, we review the major findings that have linked these mechanisms to the development and maintenance of retinal structure and function, with a focus on ganglion cells and photoreceptors. The mechanisms of epigenetic regulation are highly complex and vary among different cell types. Understanding the basic principles of these mechanisms and their regulatory pathways may provide new insight into the pathogenesis of retinal diseases associated with transcription dysregulation, and new therapeutic strategies for treatment.

Improvements in mucopolysaccharidosis I mice after adult retroviral vector-mediated gene therapy with immunomodulation.

Mucopolysaccharidosis I (MPS I) is caused by deficient alpha-L-iduronidase (IDUA) activity and results in the accumulation of glycosaminoglycans and multisystemic disease. Gene therapy could program cells to secrete mannose 6-phosphate-modified IDUA, and enzyme in blood could be taken up by other cells. Neonatal retroviral vector (RV)-mediated gene therapy has been shown to reduce the manifestations of murine MPS I; however, intravenous injection of RV into adults was ineffective owing to a cytotoxic T lymphocyte (CTL) response against transduced cells. In this study, prolonged inhibition of CD28 signaling with CTLA4-Ig, or transient administration of CTLA4-Ig with an anti-CD40 ligand antibody or with an anti-CD4 antibody, resulted in stable expression in most mice that received RV as adults. Mice with stable expression had 81 +/- 41U/ml IDUA activity in serum. This resulted in reductions in bone disease, improvements in hearing and vision, and reductions in biochemical and pathological evidence of lysosomal storage in most organs. Improvements in brain were likely due to diffusion of enzyme from blood. However, aortic disease was refractory to treatment. This demonstrates that most manifestations of MPS I can be prevented using adult gene therapy if an immune response is blocked.

Development of sensory, motor and behavioral deficits in the murine model of Sanfilippo syndrome type B.

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B) is caused by a deficiency in the lysosomal enzyme N-acetyl-glucosaminidase (Naglu). Children with MPS IIIB develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death. The murine model of MPS IIIB demonstrates lysosomal distention in multiple tissues, a shortened life span, and behavioral changes. PRINCIPAL FINDINGS: To more thoroughly assess MPS IIIB in mice, alterations in circadian rhythm, activity level, motor function, vision, and hearing were tested. The suprachiasmatic nucleus (SCN) developed pathologic changes and locomotor analysis showed that MPS IIIB mice start their daily activity later and have a lower proportion of activity during the night than wild-type controls. Rotarod assessment of motor function revealed a progressive inability to coordinate movement in a rocking paradigm. Purkinje cell counts were significantly reduced in the MPS IIIB animals compared to age matched controls. By electroretinography (ERG), MPS IIIB mice had a progressive decrease in the amplitude of the dark-adapted b-wave response. Corresponding pathology revealed shortening of the outer segments, thinning of the outer nuclear layer, and inclusions in the retinal pigmented epithelium. Auditory-evoked brainstem responses (ABR) demonstrated progressive hearing deficits consistent with the observed loss of hair cells in the inner ear and histologic abnormalities in the middle ear. CONCLUSIONS/SIGNIFICANCE: The mouse model of MPS IIIB has several quantifiable phenotypic alterations and is similar to the human disease. These physiologic and histologic changes provide insights into the progression of this disease and will serve as important parameters when evaluating various therapies.

Carbonic anhydrase XIV deficiency produces a functional defect in the retinal light response.

Members of the carbonic anhydrase (CA) family play an important role in the regulation of pH, CO(2), ion, and water transport. CA IV and CA XIV are membrane-bound isozymes expressed in the eye. CA IV immunostaining is limited to the choriocapillaris overlying the retina, whereas CA XIV is expressed within the retina in Müller glial cells and retinal pigment epithelium. Here, we have characterized the physiological and morphological phenotype of the CA IV-null, CA XIV-null, and CA IV/CA XIV-double-null mouse retinas. Flash electroretinograms performed at 2, 7, and 10 months of age showed that the rod/cone a-wave, b-wave, and cone b-wave were significantly reduced (26-45%) in the CA XIV-null mice compared with wild-type littermates. Reductions in the dark-adapted response were not progressive between 2 and 10 months, and no differences in retinal morphology were observed between wild-type and CA XIV-null mice. Müller cells and rod bipolar cells had a normal appearance. Retinas of CA IV-null mice showed no functional or morphological differences compared with normal littermates. However, CA IV/CA XIV double mutants showed a greater deficit in light response than the CA XIV-null retina. Our results indicate that CA XIV, which regulates extracellular pH and pCO(2), plays an important part in producing a normal retinal light response. A larger functional deficit in the CA IV/CA XIV double mutants suggests that CA IV can also contribute to pH regulation, at least in the absence of CA XIV.