A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Normal light response, photoreceptor integrity, and rhodopsin dephosphorylation in mice lacking both protein phosphatases with EF hands (PPEF-1 and PPEF-2).

Rhodopsin dephosphorylation in Drosophila is a calcium-dependent process that appears to be catalyzed by the protein product of the rdgC gene. Two vertebrate rdgC homologs, PPEF-1 and PPEF-2, have been identified. PPEF-1 transcripts are present at low levels in the retina, while PPEF-2 transcripts and PPEF-2 protein are abundant in photoreceptors. To determine if PPEF-2 alone or in combination with PPEF-1 plays a role in rhodopsin dephosphorylation and to determine if retinal degeneration accompanies mutation of PPEF-1 and/or PPEF-2, we have produced mice carrying targeted disruptions in the PPEF-1 and PPEF-2 genes. Loss of either or both PPEFs has little or no effect on rod function, as mice lacking both PPEF-1 and PPEF-2 show little or no changes in the electroretinogram and PPEF-2-/- mice show normal single-cell responses to light in suction pipette recordings. Light-dependent rhodopsin phosphorylation and dephosphorylation are also normal or nearly normal as determined by (i) immunostaining of PPEF-2-/- retinas with the phosphorhodopsin-specific antibody RT-97 and (ii) mass spectrometry of C-terminal rhodopsin peptides from mice lacking both PPEF-1 and PPEF-2. Finally, PPEF-2-/- retinas show normal histology at 1 year of age, and retinas from mice lacking both PPEF-1 and PPEF-2 show normal histology at 3 months of age, the latest time examined. These data indicate that, in contrast to loss of rdgC function in Drosophila, elimination of PPEF function does not cause retinal degeneration in vertebrates.

Four novel mutations in the RPE65 gene in patients with Leber congenital amaurosis.

Leber congenital amaurosis (LCArpar; is a heterogeneous disorder representing the congenital forms of retinitis pigmentosa accounting for about 5% of all retinal dystrophies. The RPE65 gene product is required for regeneration of the visual pigment for phototransduction. Defects in the RPE65 gene have so far been shown to account for approximately 10 % of known cases of LCA. Here we describe four additional novel mutations in the RPE65 gene (c.889delA, c.131G>A, c.1249G>C, c.430T>G) and several novel polymorphisms in a large series of LCA patients. Hum Mutat 18:164, 2001.

Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins-termed secreted Frizzled-related proteins-that function as antagonists of Wnt signalling. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway.

Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene.

Wnt signaling has been implicated in the control of cell proliferation and in synapse formation during neural development, and these actions are presumed to be mediated by frizzled receptors. In this paper we report the phenotype of mice carrying a targeted deletion of the frizzled-4 (fz4) gene. fz4(-/-) mice exhibit three distinct defects: (1) progressive cerebellar degeneration associated with severe ataxia, (2) absence of a skeletal muscle sheath around the lower esophagus associated with progressive esophageal distension and dysfunction, and (3) progressive deafness caused by a defect in the peripheral auditory system unaccompanied by loss of hair cells or other auditory neurons. As assayed using a lacZ knock-in reporter, fz4 is widely expressed within the CNS. In particular, fz4 is expressed in cerebellar Purkinje cells, esophageal skeletal muscle, and cochlear inner hair cells, and the absence of Fz4 in these cells is presumed to account for the fz4(-/-) phenotype. In contrast to the early cell proliferation and patterning effects classically ascribed to Wnts, the auditory and cerebellar phenotypes of fz4(-/-) mice implicate Frizzled signaling in maintaining the viability and integrity of the nervous system in later life.

Cellular and subcellular localization, N-terminal acylation, and calcium binding of Caenorhabditis elegans protein phosphatase with EF-hands.

The RdgC/PPEF family of serine/threonine protein phosphatases is distinguished by the presence of C-terminal EF-hands and neuron-specific expression, including frequent expression in primary sensory neurons. Here we report that the sole Caenorhabditis elegans PPEF (CePPEF) homolog is also highly expressed in primary sensory neurons and is not found outside the nervous system. Neurons expressing CePPEF include the ciliary chemosensory neurons AWB and AWC; and within these neurons, CePPEF is highly enriched in the sensory cilia. In transgenic C. elegans and in transfected 293 cells, CePPEF is membrane-associated, and the N terminus of CePPEF is necessary and sufficient for this membrane association. [(3)H]Myristate and [(3)H]palmitate labeling studies in 293 cells demonstrated that this association was mediated by myristoylation at Gly(2) and palmitoylation at Cys(3). Introducing the G2A or C3S mutation into CePPEF greatly reduced membrane association in 293 cells and in transgenic nematodes. A recombinant C-terminal fragment of CePPEF containing two putative EF-hands bound between one and two Ca(2+) ions/protein, and mutation of residues presumed to ligand calcium in the two putative EF-hands led to diminished calcium binding. These results establish the first direct evidence for fatty acylation and calcium binding of a PPEF family member and demonstrate a remarkable conservation of sensory neuron expression among the members of this distinctive family of protein phosphatases.

ABCR, the ATP-binding cassette transporter responsible for Stargardt macular dystrophy, is an efficient target of all-trans-retinal-mediated photooxidative damage in vitro. Implications for retinal disease.

A large body of experimental and clinical data have documented the damaging effects of light exposure on photoreceptor cells although the identities of the biologically relevant molecular targets of photodamage are still uncertain. Several lines of evidence point to retinoids or retinoid derivatives as chromophores that can mediate light damage. We report here that ABCR, a photoreceptor-specific transporter involved in the recycling of all-trans-retinal, is unusually sensitive to photooxidation damage mediated by all-trans-retinal in vitro. Partial loss of ABCR function is responsible for Stargardt macular dystrophy, which is associated with accumulation of A2E, a diretinoid adduct within the retinal pigment epithelium. Photodamage to ABCR causes it to aggregate in SDS gels and results in the loss of retinal-stimulated ATPase activity. Peripherin/RDS and ROM-1, two structural proteins that colocalize with ABCR at the outer segment disc rim, are also significantly more susceptible to all-trans-retinal-mediated photodamage than are the major proteins from the rod outer segment. These observations imply that there may be specific protein targets of photodamage within the outer segment, and they may be especially relevant to assessing the risk of light exposure in those individuals who already have diminished ABCR activity due to mutation in one or both copies of the ABCR gene.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 during craniofacial morphogenesis.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during chicken craniofacial development. cFHF-4 is expressed in the mesenchyme of the frontonasal process, and in the mesenchyme and ectoderm of the mandibular processes. The expression of cFHF-4 and other genes implicated in facial patterning have been analyzed in talpid(2) embryos or in the presence of exogenous retinoic acid. Talpid(2) mutants show abnormal patterns of gene expression, including up-regulation of cFHF-4 in the developing face, which correlate with defects in cartilage formation. By contrast, expression of cFHF-4 in the developing face is strongly downregulated by teratogenic doses of all-trans retinoic acid in a dose-dependent manner. Low levels of retinoic acid that produce distal upper beak truncations do not affect cShh, c-Patched-1, or c-Bmp-2 expression in the face, but downregulate cFHF-4 in the frontonasal process.

Mechanistic studies of ABCR, the ABC transporter in photoreceptor outer segments responsible for autosomal recessive Stargardt disease.

ABCR is an ABC transporter that is found exclusively in vertebrate photoreceptor outer segments. Mutations in the human ABCR gene are responsible for autosomal recessive Stargardt disease, the most common cause of early onset macular degeneration. In this paper we review our recent work with purified and reconstituted ABCR derived from bovine retina and from cultured cells expressing wild type or site-directed mutants of human ABCR. These experiments implicate all-trans-retinal (or Schiff base adducts between all-trans-retinal and phosphatidylethanolamine) as the transport substrate, and they reveal asymmetric roles for the two nucleotide binding domains in the transport reaction. A model for the retinal transport reaction is presented which accounts for these experimental observations.

Biochemical defects in ABCR protein variants associated with human retinopathies.

Mutations in the gene encoding ABCR (ABCA4), a photoreceptor-specific ATP-binding cassette (ABC) transporter, are responsible for autosomal recessive Stargardt disease (STGD), an early onset macular degeneration, and some forms of autosomal recessive cone-rod dystrophy and autosomal recessive retinitis pigmentosa. Heterozygosity for ABCA4 mutations may also represent a risk factor for age-related macular degeneration (AMD), although this idea is controversial. An ongoing challenge in the analysis of ABCA4-based retinopathies arises from the observation that most of the ABCA4 sequence variants identified so far are missense mutations that are rare in both patient and control populations. With the current sample size of most sequence variants, one cannot determine statistically whether a particular sequence variant is pathogenic or neutral. A related challenge is to determine the degree to which each pathogenic variant impairs ABCR function, as genotype-phenotype analyses indicate that age of onset and disease severity correlate with different ABCA4 alleles. To address these questions, we performed a functional analysis of human ABCR and its variants. These experiments reveal a wide spectrum of biochemical defects in these variants and provide insight into the transport mechanism of ABCR.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.

Isoform diversity among fibroblast growth factor homologous factors is generated by alternative promoter usage and differential splicing.

Fibroblast growth factor (FGF) homologous factors-1, -2, -3, and -4 (FHFs 1-4; also referred to as FGFs 11-14) comprise a separate branch of the FGF family and have been implicated in the development of the nervous system and limbs. We report here the characterization of multiple isoforms of FHF-1, -2, -3, and -4 which are generated through the use of alternative start sites of transcription and splicing of one or more of a series of alternative 5'-exons. Several isoforms show different subcellular distributions when expressed in transfected tissue culture cells, and the corresponding differentially spliced transcripts show distinct expression patterns in developing and adult mouse tissues. Together with the evolutionary conservation of the FHF isoforms among human, mouse, and chicken, these data indicate that alternative promoter use and differential splicing are important regulatory processes in controlling the activities of this subfamily of FGFs.

Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.

Retinol dehydrogenase (RDH), the enzyme that catalyzes the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment, was the first visual cycle enzymatic activity to be identified. Previous work has shown that this enzyme utilizes NADPH, shows a marked preference for all-trans-retinal over 11-cis-retinal, and is tightly associated with the outer segment membrane. This paper reports the identification of a novel member of the short chain dehydrogenase/reductase family, photoreceptor RDH (prRDH), using subtraction and normalization of retina cDNA, high throughput sequencing, and data base homology searches to detect retina-specific genes. Bovine and human prRDH are highly homologous and are most closely related to 17-beta-hydroxysteroid dehydrogenase 1. The enzymatic properties of recombinant bovine prRDH closely match those previously reported for RDH activity in crude bovine rod outer segment preparations. In situ hybridization and RNA blotting show that the PRRDH gene is expressed specifically in photoreceptor cells, and protein blotting and immunocytochemistry show that prRDH localizes exclusively to both rod and cone outer segments and that prRDH is tightly associated with outer segment membranes. Taken together, these data indicate that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment.

What can we learn about age-related macular degeneration from other retinal diseases?

Age-related macular degeneration (AMD) is increasingly recognized as a complex genetic disorder in which one or more genes contribute to an individual's susceptibility for developing the condition. Twin and family studies as well as population-based genetic epidemiologic methods have convincingly demonstrated the importance of genetics in AMD, though the extent of heritability, the number of genes involved, and the phenotypic and genetic heterogeneity of the condition remain unresolved. The extent to which other hereditary macular dystrophies such as Stargardts disease, familial radial drusen (malattia leventinese), Best's disease, and peripherin/RDS-related dystrophy are related to AMD remains unclear. Alzheimer's disease, another late onset, heterogeneous degenerative disorder of the central nervous system, offers a valuable model for identifying the issues that confront AMD genetics.

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.

Mutually exclusive expression of human red and green visual pigment-reporter transgenes occurs at high frequency in murine cone photoreceptors.

This study examines the mechanism of mutually exclusive expression of the human X-linked red and green visual pigment genes in their respective cone photoreceptors by asking whether this expression pattern can be produced in a mammal that normally carries only a single X-linked visual pigment gene. To address this question, we generated transgenic mice that carry a single copy of a minimal human X chromosome visual pigment gene array in which the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and beta-galactosidase reporters. As determined by histochemical staining, the reporters are expressed exclusively in cone photoreceptor cells. In 20 transgenic mice carrying any one of three independent transgene insertion events, an average of 63% of expressing cones have alkaline phosphatase activity, 10% have beta-galactosidase activity, and 27% have activity for both reporters. Thus, mutually exclusive expression of red and green pigment transgenes can be achieved in a large fraction of cones in a dichromat mammal, suggesting a facile evolutionary path for the development of trichromacy after visual pigment gene duplication. These observations are consistent with a model of visual pigment expression in which stochastic pairing occurs between a locus control region and either the red or the green pigment gene promotor.