A Notch-Gli2 axis sustains Hedgehog responsiveness of neural progenitors and Müller glia.

Neurogenesis is regulated by the dynamic and coordinated activity of several extracellular signalling pathways, but the basis for crosstalk between these pathways remains poorly understood. Here we investigated regulatory interactions between two pathways that are each required for neural progenitor cell maintenance in the postnatal retina; Hedgehog (Hh) and Notch signalling. Both pathways are activated in progenitor cells in the postnatal retina based on the co-expression of fluorescent pathway reporter transgenes at the single cell level. Disrupting Notch signalling, genetically or pharmacologically, induces a rapid downregulation of all three Gli proteins and inhibits Hh-induced proliferation. Ectopic Notch activation, while not sufficient to promote Hh signalling or proliferation, increases Gli2 protein. We show that Notch regulation of Gli2 in Müller glia renders these cells competent to proliferate in response to Hh. These data suggest that Notch signalling converges on Gli2 to prime postnatal retinal progenitor cells and Müller glia to proliferate in response to Hh.

A novel mutation in two Hmong families broadens the range of STRA6-related malformations to include contractures and camptodactyly.

PDAC (also termed Matthew Wood) syndrome is a rare, autosomal recessive disorder characterized by pulmonary hypoplasia/aplasia, diaphragmatic defects, bilateral anophthalmia, and cardiac malformations. The disorder is caused by mutations in STRA6, an important regulator of vitamin A and retinoic acid metabolism. We describe six cases from four families of Hmong ancestry, seen over a 30 years period in California. These include: (i) consanguineous siblings with a combination of bilateral anophthalmia, diaphragmatic abnormalities, truncus arteriosus, and/or pulmonary agenesis/hypoplasia; (ii) a singleton fetus with bilateral anophthalmia, pulmonary agenesis, cardiac malformation, and renal hypoplasia; (iii) a sibling pair with a combination of antenatal contractures, camptodactyly, fused palpebral fissures, pulmonary agenesis, and/or truncus arteriosus; (iv) a fetus with bilateral anophthalmia, bushy eyebrows, pulmonary agenesis, heart malformation, and abnormal hand positioning. The phenotypic spectrum of PDAC syndrome has until now not included contractures or camptodactyly. Sequencing of STRA6 in unrelated members of families three and four identified a novel, shared homozygous splice site alteration (c.113 + 3_4delAA) that is predicted to be pathogenic. We hypothesize this may represent a unique disease allele in the Hmong. We also provide a focused review of all published PDAC syndrome cases with confirmed or inferred STRA6 mutations, illustrating the phenotypic and molecular variability that characterizes this disorder.

Identification of novel retinoic acid target genes.

Retinoic acid is required for diverse ontogenic processes and as such identification of the genes and pathways affected by retinoic acid is critical to understanding these pleiotropic effects. The presomitic mesoderm of the E8.5 mouse embryo is composed of undifferentiated cells that are depleted of retinoic acid, yet are competent to respond to the retinoid signal. We have exploited these properties to use this tissue to identify novel retinoic acid-responsive genes, including candidate target genes, by treating E8.5 embryos with retinoic acid and assessing changes in gene expression in the presomitic mesoderm by microarray analysis. This exercise yielded a cohort of genes that were differentially expressed in response to exogenous retinoic acid exposure. Among these were a number of previously characterized retinoic acid targets, validating this approach. In addition, we recovered a number of novel candidate target genes which were confirmed as retinoic acid-responsive by independent analysis. Chromatin immunoprecipitation assays revealed retinoic acid receptor occupancy of the promoters of certain of these genes. We further confirmed direct retinoic acid regulation of the F11r gene, a new RA target, using tissue culture models. Our results reveal a significant number of potential RA targets implicated in embryonic development and offer a novel in vivo system for better understanding of retinoid-dependent transcription.

Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity.

INTRODUCTION: Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer. METHODS: Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis. RESULTS: Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn-null animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn-null environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15). CONCLUSIONS: Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

Hedgehog regulates Norrie disease protein to drive neural progenitor self-renewal.

Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.

Mutations in the enzyme glutathione peroxidase 4 cause Sedaghatian-type spondylometaphyseal dysplasia.

BACKGROUND: Sedaghatian-type spondylometaphyseal dysplasia (SSMD) is a neonatal lethal form of spondylometaphyseal dysplasia characterised by severe metaphyseal chondrodysplasia with mild limb shortening, platyspondyly, cardiac conduction defects, and central nervous system abnormalities. As part of the FORGE Canada Consortium we studied two unrelated families to identify the genetic aetiology of this rare disease. METHODS AND RESULTS: Whole exome sequencing of a child affected with SSMD and her unaffected parents identified two rare variants in GPX4. The first (c.587+5G>A) was inherited from the mother, and the second (c.588-8_588-4del) was de novo (NM_001039848.1); both were predicted to impact splicing of GPX4. In vitro studies confirmed the mutations spliced out part of exon 4 and skipped exon 5, respectively, with both resulting in a frameshift and premature truncation of GPX4. Subsequently, a second child with SSMD was identified; although DNA from the child was not available, the two unaffected parents were found by Sanger sequencing to each carry the same heterozygous stop mutation in exon 3 of GPX4, c.381C>A, p.Tyr127* (NM_001039848.1). CONCLUSIONS: Our identification of truncating mutations in GPX4 in two families affected with SSMD supports the pathogenic role of mutated GPX4 in this very rare disease. GPX4 is a member of the glutathione peroxidase family of antioxidant defence enzymes and protects cells against membrane lipid peroxidation. GPX4 is essential for early embryo development, regulating anti-oxidative and anti-apoptotic activities. Our findings highlight the importance of this enzyme in development of the cardiac, nervous, and skeletal systems.

Cdx regulates Dll1 in multiple lineages.

Vertebrate Cdx genes encode homeodomain transcription factors related to caudal in Drosophila. The murine Cdx homologues Cdx1, Cdx2 and Cdx4 play important roles in anterior-posterior patterning of the embryonic axis and the intestine, as well as axial elongation. While our understanding of the ontogenic programs requiring Cdx function has advanced considerably, the molecular bases underlying these functions are less well understood. In this regard, Cdx1-Cdx2 conditional mutants exhibit abnormal somite formation, while loss of Cdx1-Cdx2 in the intestinal epithelium results in a shift in differentiation toward the Goblet cell lineage. The aim of the present study was to identify the Cdx-dependent mechanisms impacting on these events. Consistent with prior work implicating Notch signaling in these pathways, we found that expression of the Notch ligand Dll1 was reduced in Cdx mutants in both the intestinal epithelium and paraxial mesoderm. Cdx members occupied the Dll1 promoter both in vivo and in vitro, while genetic analysis indicated interaction between Cdx and Dll1 pathways in both somitogenesis and Goblet cell differentiation. These findings suggest that Cdx members operate upstream of Dll1 to convey different functions in two distinct lineages.

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development.

BACKGROUND: The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. RESULTS: Using a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking. CONCLUSIONS: We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

Identification of Wnt/ß-catenin modulated genes in the developing retina.

PURPOSE: During mammalian eye development, the restriction of Wnt/ß-catenin signaling at the junction of the neural retina and the retinal pigment epithelium in the peripheral eyecup is required for the development of the ciliary margin, a non-neural region of the eyecup that is the precursor of the ciliary body and iris of the adult eye. METHODS: To identify genes that are modulated by ß-catenin activity in the embryonic retina, we performed gene expression profiling in Li(+)-treated retinal explants, a pharmacological model of ß-catenin activation. The Li(+)-modulated gene data set was searched for ß-catenin/T-cell specific transcription factor binding sites. RESULTS: Functional annotations of this data set revealed significant enrichments for genes involved in chromatin organization, neurogenesis, and cell motion/migration. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the modulation of 12 genes in Li(+)-treated explants and retinas of mice with Cre-mediated induction of constitutively active ß-catenin (ß-cat(act)). In situ hybridization revealed ß-catenin-specific upregulation of cyclin-dependent kinase inhibitor 1A (P21) [Cdkn1a] and tumor necrosis factor receptor superfamily, member 19 (Tnfrsf19) in the developing retina consistent with the antineurogenic and proliferation changes associated with ectopic Wnt/ß-catenin signaling in the eyecup. CONCLUSIONS: This data set of Li(+)-modulated genes provides a valuable resource for characterizing the Wnt/ ß-catenin regulated gene network in eyecup patterning.

Retinoic acid receptor-related orphan receptor alpha regulates a subset of cone genes during mouse retinal development.

Color vision is supported by retinal cone photoreceptors that, in most mammals, express two photopigments sensitive to short (S-opsin) or middle (M-opsin) wavelengths. Expression of the Opn1sw and Opn1mw genes, encoding S-opsin and M-opsin, respectively, is under the control of nuclear receptors, including thyroid hormone receptor beta2 (TRbeta2), retinoid X receptor gamma (RXRgamma), and RORbeta, a member of the retinoic acid receptor-related orphan receptor (ROR) family. We now demonstrate that RORalpha, another member of the ROR family, regulates Opn1sw, Opn1mw, as well as Arr3 (cone arrestin) in the mouse retina. RORalpha expression is detected in cones by postnatal day 3 and maintained through adulthood. The retinas of staggerer mice, carrying a null mutation of RORalpha, show significant down-regulation of Opn1sw, Opn1mw, and Arr3. RORalpha acts in synergy with cone-rod homeobox transcription factor (Crx), to activate the Opn1sw promoter in vitro. Chromatin immunoprecipitation assays reveal that RORalpha directly binds to the Opn1sw promoter, Opn1mw locus control region, and the Arr3 promoter in vivo. Our data suggest that RORalpha plays a crucial role in cone development by directly regulating multiple cone genes.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin(+) cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP(+) embryonic cones and postnatal Nrl(-/-) 'cods' into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Norrin/Frizzled4 signalling in the preneoplastic niche blocks medulloblastoma initiation.

The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/- mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.

Recruitment of the rod pathway by cones in the absence of rods.

In mammalian retinas, rods and cones connect to distinct sets of bipolar cells. In the retina of Nrl (neural retina leucine zipper) knock-out mice, in which rods fail to form and all photoreceptors are cones, rod bipolar cells have normal morphology, pattern of staining, and lamination, but they form synaptic connections with cones. Hence, retinal interneurons are not entirely committed to the choice of their synaptic partners; also, their morphology and pattern of distribution of their processes in the synaptic layers of the retina are not instructed by the establishment of synaptic connections with cognate photoreceptor(s). These findings are of considerable relevance for our understanding of mechanisms responsible for cell recognition during development and in the context of using transplanted tissue to restore vision in retinal degeneration.

GRK1-dependent phosphorylation of S and M opsins and their binding to cone arrestin during cone phototransduction in the mouse retina.

The shutoff mechanisms of the rod visual transduction cascade involve G-protein-coupled receptor (GPCR) kinase 1 (GRK1) phosphorylation of light-activated rhodopsin (R*) followed by rod arrestin binding. Deactivation of the cone phototransduction cascade in the mammalian retina is delineated poorly. In this study we sought to explore the potential mechanisms underlying the quenching of the phototransduction cascade in cone photoreceptors by using mouse models lacking rods and/or GRK1. Using the "pure-cone" retinas of the neural retina leucine zipper (Nrl) knock-out (KO, -/-) mice (Mears et al., 2001), we have demonstrated the light-dependent, multi-site phosphorylation of both S and M cone opsins by in situ phosphorylation and isoelectric focusing. Immunoprecipitation with affinity-purified polyclonal antibodies against either mouse cone arrestin (mCAR) or mouse S and M cone opsins revealed specific binding of mCAR to light-activated, phosphorylated cone opsins. To elucidate the potential role of GRK1 in cone opsin phosphorylation, we created Nrl and Grk1 double knock-out (Nrl-/-Grk1-/-) mice by crossing the Nrl-/- mice with Grk1-/- mice (Chen et al., 1999). We found that, in the retina of these mice, the light-activated cone opsins were neither phosphorylated nor bound with mCAR. Our results demonstrate, for the first time in a mammalian species, that cone opsins are phosphorylated and that CAR binds to phosphorylated cone opsins after light activation.

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.

PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.

Characterization of new transcripts enriched in the mouse retina and identification of candidate retinal disease genes.

PURPOSE: Most retinal disease genes are preferentially expressed in photoreceptors, the light-sensitive cells involved in phototransduction. In addition, some of the genes linked to retinal diseases are essential for normal retinal development. The goal of this study was to identify new transcripts enriched in photoreceptors involved in retinal development or diseases. METHODS: To isolate uncharacterized retinal transcripts, the bioinformatic method Digital Differential Display (DDD) was used. RNA in situ hybridization was used to characterize gene-expression patterns. RESULTS: Twenty-seven mouse ESTs highly represented in retinal libraries were identified. Eight ESTs were predominantly expressed in photoreceptors and/or in the retinal pigment epithelium (RPE), whereas transcripts for other ESTs were detected more ubiquitously in the retinal cells or abundantly in ganglion cells and/or the inner nuclear layer. Mapping of the corresponding human orthologues of the photoreceptor/RPE-enriched genes revealed that two of them are candidate disease genes for retinitis pigmentosa, loci RP22 and RP28. Both of these are predominantly expressed in rod photoreceptors. The candidate RP22 gene codes for a putative transmembrane protein showing homology to Cln8 (ceroid lipofuscinosis, neuronal 8), in which gene mutations are associated with photoreceptors degeneration in mice. Also identified were two genes expressed in photoreceptors that are candidate disease genes for recessive Bardet-Biedl syndrome type 3 (BBS3) and recessive ataxia with RP (AXPC1). CONCLUSIONS: This study demonstrates how bioinformatic analysis can be used to identify novel tissue-specific genes relevant to development and diseases.

Gene expression profile of native human retinal pigment epithelium.

PURPOSE: To generate a profile of genes expressed in the native human retinal pigment epithelium and identify candidate genes for retinal and macular diseases. METHODS: Two cDNA libraries (one amplified, the other unamplified) were constructed using RNA isolated from native human RPE sheets. The sequence from the 5' end was obtained for randomly selected clones from the two libraries. Of these, more than 2000 expressed sequence tags (ESTs) were analyzed for similarity to sequences and gene clusters in public databases. RESULTS: EST analysis revealed several known RPE-expressed genes and more than 500 genes that have been characterized previously but were not known to be expressed in the RPE. Transthyretin and 90-kDa heat shock protein represent the most abundant transcripts identified in these RPE libraries. More than 200 novel ESTs and putative proteins were identified. An additional 344 sequences matched only the human genomic sequence. CONCLUSIONS: High-complexity cDNA libraries were generated from native human RPE. Analysis of ESTs generated from these libraries has yielded a profile of genes expressed in the native RPE. Several of the identified genes are known to play a significant role in the RPE. Novel ESTs, putative proteins, and genomic hits may represent as yet unidentified RPE-expressed genes and many of these, mapping in the region of retinal disease loci, may serve as candidate genes. In addition, the nonredundant set of more than 1100 genes and ESTs described herein will be a valuable resource for generating gene microarrays, which can assist in delineating RPE expression profiles during human disease pathogenesis.

Connexin 36 in photoreceptor cells: studies on transgenic rod-less and cone-less mouse retinas.

PURPOSE: Rod-cone gap junctions permit transmittal of rod visual information to the cone pathway. A recent report has shown that this transfer does not occur in mice in which the gap junction protein connexin 36 is knocked out indicating that rod-cone gap junctions are assembled from this protein. It remains unresolved, however, whether rods, cones or both express connexin 36. We have tried to address this question with the use of transgenic rod-less and cone-less mice. METHODS: Deletion of Nrl, a transcription factor, results in a complete loss of rods with a concomitant increase in S-cones. We used this as the rod-less (cone-only) model. Cone-less (rod-only) retinas were from mice expressing an attenuated diphtheria toxin gene under the control of a promoter selective for cones. Nearly all long wavelength cones and 95% of short wavelength cones are missing in this model. Fixed retinal sections from these two models and age matched controls were used to detect connexin 36 gap junctions by immunofluorescence. RESULTS: Punctate immunofluorescence, indicating the presence of gap junctions, was observed in the inner and outer plexiform layers of both wild type and cone-less and rod-less retinas. Our assumption was that immunofluorescence due to photoreceptor gap junctions would be observed in the outer plexiform layer. In all the animals, most of the immunofluorescence was in the inner plexiform layer, with only a marginal reaction in the outer plexiform layer. In cone-only (rod-less) retina, immunofluorescence in the outer plexiform layer increased by more than 20 fold compared to wild type. In rod-only (cone-less) retina, the outer plexiform layer showed about a 30% decrease in immunofluorescence. In both rod-less and cone-less retinas, immunofluorescence in the inner plexiform layer was higher than in the wild type by 25-50%. CONCLUSIONS: Cones constitute only about 3% of photoreceptors in the wild type retina while they make up 100% of the photoreceptors in cone-only retina. This increase in their numbers coincided with a 20 fold increase in immunofluorescence in the outer plexiform layer, strongly suggesting that cones express connexin 36. Conversely, when the cone numbers went down from 3% to near zero in cone-less retina, immunofluorescence decreased by about 30% in the outer plexiform layer, suggesting again that cones express the connexin and that they contribute to its presence disproportionately more than their numbers indicate. The results from both rod-less and cone-less animals are strongly indicative of cones expressing connexin 36, but are not sufficient to conclude whether rods express the protein. An unexpected observation from our experiments is that immunofluorescence increases slightly in the inner plexiform layer in both rod-less and cone-less retina for reasons that need further investigation.

Mutation screening of patients with Leber Congenital Amaurosis or the enhanced S-Cone Syndrome reveals a lack of sequence variations in the NRL gene.

PURPOSE: To determine if mutations in the retinal transcription factor gene NRL are associated with retinopathies other than autosomal dominant retinitis pigmentosa (adRP). METHODS: Genomic DNA was isolated from blood samples obtained from 50 patients with Leber Congenital Amaurosis (LCA), 17 patients with the Enhanced S-Cone Syndrome (ESCS), and a patient with an atypical retinal degeneration that causes photoreceptor rosettes with blue cone opsin. The 5' upstream region (putative promoter), untranslated exon 1, coding exons 2 and 3, and exon-intron boundaries of the NRL gene were analyzed by direct sequencing of the PCR-amplified products. RESULTS: Complete sequencing of the NRL gene in DNA samples from this cohort of patients revealed only one nucleotide change. The C->G transversion at nucleotide 711 of NRL exon 3 was detected in one LCA patient; however, this change did not alter the amino acid (L237L). CONCLUSIONS: No potential disease causing mutation was identified in the NRL gene in patients with LCA, ESCS, or the atypical retinal degeneration. Together with previous studies, our results demonstrate that mutations in the NRL gene are not a major cause of retinopathy. To date, only missense changes have been reported in adRP patients, and sequence variations are rare. It is possible that the loss of NRL function in humans is associated with a more complex clinical phenotype due to its expression in pineal gland in addition to rod photoreceptors.