Differences in activation of intracellular signaling in primary human retinal endothelial cells between isoforms of VEGFA 165.

Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR. Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA 165 a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA 165 b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA 165 b was only half of the maximum activation from VEGFA 165 a. At a lower intermediate dose, where VEGFA 165 a activated intracellular signaling stronger than VEGFA 165 b, the changes in VEGFA target gene expression were generally greater with VEGFA 165 a. Conclusions: In primary HRMECs, VEGFA 165 a could maximally activate MAPK and AKT at lower concentrations where VEGFA 165 b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA 165 a and VEGFA 165 b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.

Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia.

Treatment of a mouse model of oxygen-induced retinopathy (OIR) with recombinant human Norrin (Norrie Disease Protein, gene: NDP) accelerates regrowth of the microvasculature into central ischemic regions of the neural retina, which are generated after treatment with 75% oxygen. While this reduces the average duration and severity of ischemia overall, we do not know if this accelerated recovery of the microvasculature results in any significant survival of retinal ganglion cells (RGCs). The purpose of this study was to investigate ganglion cell survival with and without the intravitreal injection of Norrin in the murine model of oxygen induced retinopathy (OIR), using two strains of mice: C57BL/6J and Thy1-YFP mice. Intravitreal injections of Norrin or vehicle were done after five days of exposure to 75% oxygen from ages P7 to P12. The C57BL/J mice were followed by Spectral-Domain Optical Coherence Tomography (SD-OCT), and the average nerve fiber layer (NFL) and inner-plexiform layer (IPL) thicknesses were measured at twenty-four locations per retina at P42. Additionally, some C57BL/J retinas were flat mounted and immunostained for the RGC marker, Brn3a, to compare the population density of surviving retinal ganglion cells. Using homozygous Thy1-YFP mice, single intrinsically fluorescent RGCs were imaged in live animals with a Micron-III imaging system at ages P21, 28 and P42. The relative percentage of YFP-fluorescent RGCs with dendritic arbors were compared. At age P42, the NFL was thicker in Norrin-injected OIR eyes, 14.4 µm, compared to Vehicle-injected OIR eyes, 13.3 µm (p = 0.01). In the superior retina, the average thickness of the IPL was greater in Norrin-injected OIR eyes, 37.7 µm, compared to Vehicle-injected OIR eyes, 34.6 µm (p = 0.04). Retinas from Norrin injected OIR mice had significantly more surviving RGCs (p = 0.03) than vehicle-injected mice. Based upon NFL thickness and counts of RGCs, we conclude that Norrin treatment, early in the ischemic phase, increased the relative population density of surviving RGCs in the central retinas of OIR mice.

Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice.

PURPOSE: The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA's adoption as an anticonvulsant. This generated speculation for VPA's potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA's potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA's effects on the rate of photoreceptor loss in two strains of mice, Pde6b(rd1/rd1) and Pde6b(rd10/rd10) . METHODS: The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6b(rd1/rd1) mice. Pde6b(rd1/rd1) mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6b(rd10/rd10) mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6b(rd10/rd10) mice to measure retinal function. RESULTS: In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9-P21) dramatically slowed the loss of rod photoreceptors in Pde6b(rd1/rd1) mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6b(rd10/rd10) mice (P17-P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6b(rd10/rd10) mice earlier (P9-P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9-P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals. CONCLUSIONS: A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6b(rd1/rd1) mice (P9-P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA's efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17-P28) started when the central retina was mature.

Mechanisms Underlying Rare Inherited Pediatric Retinal Vascular Diseases: FEVR, Norrie Disease, Persistent Fetal Vascular Syndrome.

Familial Exudative Vitreoretinopathy (FEVR), Norrie disease, and persistent fetal vascular syndrome (PFVS) are extremely rare retinopathies that are clinically distinct but are unified by abnormal retinal endothelial cell function, and subsequent irregular retinal vascular development and/or aberrant inner blood-retinal-barrier (iBRB) function. The early angiogenesis of the retina and its iBRB is a delicate process that is mediated by the canonical Norrin Wnt-signaling pathway in retinal endothelial cells. Pathogenic variants in genes that play key roles within this pathway, such as NDP, FZD4, TSPAN12, and LRP5, have been associated with the incidence of these retinal diseases. Recent efforts to further elucidate the etiology of these conditions have not only highlighted their multigenic nature but have also resulted in the discovery of pathological variants in additional genes such as CTNNB1, KIF11, and ZNF408, some of which operate outside of the Norrin Wnt-signaling pathway. Recent discoveries of FEVR-linked variants in two other Catenin genes (CTNND1, CTNNA1) and the Endoplasmic Reticulum Membrane Complex Subunit-1 gene (EMC1) suggest that we will continue to find additional genes that impact the neural retinal vasculature, especially in multi-syndromic conditions. The goal of this review is to briefly highlight the current understanding of the roles of their encoded proteins in retinal endothelial cells to understand the essential functional mechanisms that can be altered to cause these very rare pediatric retinal vascular diseases.

The transcription factor neural retina leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter.

Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors in the mammalian retina. In the Nrl(-/-) mouse retina, photoreceptor precursors fail to produce rods and generate functional cone photoreceptors that predominantly express S-opsin. Previous global expression analysis using microarrays revealed dramatically reduced expression of myocyte enhancer factor Mef2c in the adult Nrl(-/-) retina. We undertook this study to examine the biological relevance of Mef2c expression in retinal rod photoreceptors. Bioinformatics analysis, rapid analysis of cDNA ends (5'-RACE), and reverse transcription coupled with qPCR using splice site-specific oligonucleotides suggested that Mef2c is expressed in the mature retina from an alternative promoter. Chromatin immunoprecipitation (ChIP) studies showed the association of active RNA polymerase II and acetylated histone H3 just upstream of Mef2c exon 4, providing additional evidence for the utilization of an alternative promoter in the retina. In concordance, we observed the binding of NRL to a putative NRL-response element (NRE) at this location by ChIP-seq and electrophoretic mobility shift assays. NRL also activated the Mef2c alternative promoter in vitro and in vivo. Notably, MEF2C could support Rhodopsin promoter activity in rod photoreceptors. We conclude that Mef2c expression from an alternative promoter in the retina is regulated by NRL. Our studies also implicate MEF2C as a transcriptional regulator of homeostasis in rod photoreceptor cells.

A Survey of Multigenic Protein-Altering Variant Frequency in Familial Exudative Vitreo-Retinopathy (FEVR) Patients by Targeted Sequencing of Seven FEVR-Linked Genes.

While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were: NDP (ChrX), CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution: LRP5 13/33 (40%), FZD4 9/33 (27%), ZNF408 6/33 (18%), (KIF11 3/33 (9%), NDP 1/33 (3%), CTNNB1 1/33 (3%). Most protein-altering variants, 85%, were found in three genes: FZD4, LRP5, and ZNF408. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46, n = 30) compared to subjects confirmed unaffected by FEVR (0.95, n = 20), (p = 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.

Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.

PURPOSE: During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites. METHODS: High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10-12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT-PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d). RESULTS: ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region. CONCLUSIONS: High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene's promoter complex.

FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo.

BACKGROUND: FIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes. RESULTS: FIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina. CONCLUSION: FIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank.

PURPOSE: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags. METHODS: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). RESULTS: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. CONCLUSIONS: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

Ocular coherence tomography image data of the retinal laminar structure in a mouse model of oxygen-induced retinopathy.

The data presented in this article are related to the research paper entitled "Norrin treatment improves ganglion cell survival in an oxygen-induced model of retinal ischemia" (Dailey et al., 2017) [1] This article describes treatment with the human Norrin protein, an atypical Wnt-protein, to improve the survival of retinal ganglion cells in a murine model of Oxygen-Induced Retinopathy (OIR). That study utilized Optical coherence tomography (OCT) to visualize retinal layers at high resolution in vivo, and to quantify changes to nerve fiber layer thickness. Organization of the laminar structure of other retinal layers in this model in vivo, were not known because of uncertainties regarding potential artifacts during the processing of tissue for traditional histology. The OCT image data provided here shows researchers the retinal laminar structural features that exist in vivo in this popular mouse OIR model. Traditional H&E stained retinal tissue sections are also provided here for comparison.

Effects of anti-VEGF treatment on the recovery of the developing retina following oxygen-induced retinopathy.

PURPOSE: Inhibition of VEGF is widely used in patients to control neovascularization and decrease vascular permeability. To date, the effect of VEGF inhibition has not been evaluated in the developing retina such as that seen in premature infants. The goal of this study was to address the effect of anti-VEGF treatment on retinal development of a mouse model of retinopathy. METHODS: C57BL/6J mice were evaluated using a model of oxygen-induced retinopathy. Test animals were treated at postnatal day (P) 14 with intravitreal injections of the VEGF inhibitor aflibercept (2.5 or 10 µg) in one eye. Control animals were treated with injection of PBS in one eye. The noninjected fellow eyes were used as internal controls. Areas of avascular retina and neovascular tufts in injected (treated) eyes and noninjected fellow eyes were determined at P17, and the difference related to these characteristics was obtained among them. To evaluate the effect of VEGF inhibition on neurogenesis, focal ERG was performed at P21 and P42. Histologic evaluation of the retinal structure was also evaluated at P42. RESULTS: Aflibercept treatment reduced the amount of neovascular tufts but significantly increased the area of avascular retina (low dose and high dose) at P17. The delayed vascular growth corresponded to decreased ERG amplitudes (at P21 and P42) and structural changes in the retinal layers that persisted (at P42), despite vascular recovery. CONCLUSIONS: Inhibition of VEGF in developing eyes has the short-term effect of delayed vascular growth and the long-term effects of decreased function with persistent changes in the neuroretinal structures.

FIZ1 is expressed during photoreceptor maturation, and synergizes with NRL and CRX at rod-specific promoters in vitro.

FIZ1 (Flt-3 Interacting Zinc-finger) interacts and co-purifies with the rod-specific transcription factor NRL (Neural Retina Leucine zipper). We hypothesize that FIZ1 is part of an interface between cell-specific factors, like NRL, and more ubiquitous regulatory networks that vary the absolute expression levels of some rod-specific genes (i.e. Rhodopsin). As part of an ongoing exploration of FIZ1's role in neural retina, in vivo, we have taken the first look at FIZ1 expression in the developing mouse retina during the retinal maturation period. Using the normal C57B6 mouse as a model, multiple approaches were used including: immunoblotting, immunohistochemistry, and quantitative real-time PCR. Functional implications of FIZ1/NRL interaction, on NRL- and CRX-mediated activation of the Rhodopsin (Rho) and cGMP-phosphodiesterase beta-subunit gene (PDE6B) promoters, were examined by co-transfection assays. Immunoblot analysis revealed that FIZ1 protein levels were lowest in immature mouse neural retina (P0). FIZ1 concentration increased at least ten-fold as the neural retina matured to the adult state (P21 and later). Immunohistochemical comparison of immature post-natal and mature adult retina revealed increasing FIZ1 protein in photoreceptors, the inner plexiform layer, and the ganglion cell layer. Total retinal Fiz1 mRNA content increased as the neural retina matured. The expected increase in Rho mRNA level was also monitored as a genetic marker of photoreceptor maturation. In transient co-transfection assays of CV1 cells, FIZ1 synergized with NRL to activate transcription from the Rho and PDE6B gene promoters with some differences. In the case of the Rho promoter, FIZ1 synergized when both NRL and CRX were present. With the PDE6B promoter, FIZ1 synergized with NRL alone, and the inclusion of CRX decreased this synergy. These findings support previous evidence that FIZ1 is present in rod-photoreceptors (co-immunoprecipitation from nuclear-protein extracts with rod-specific NRL). FIZ1 expression increases in the neural retina during the retinal maturation period. Additionally, in vitro experiments demonstrate that FIZ1 has the potential to significantly increase the NRL-mediated activation of photoreceptor-specific promoters. While CRX is not a strong activator of the PDE6B promoter, alone or with NRL, CRX decreased the synergy of NRL with FIZ1.

Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor.

NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase beta-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.

Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors.

NR2E3, a photoreceptor-specific orphan nuclear receptor, is believed to play a pivotal role in the differentiation of photoreceptors. Mutations in the human NR2E3 gene and its mouse ortholog are associated with enhanced S-cones and retinal degeneration. In order to gain insights into the NR2E3 function, we performed temporal and spatial expression analysis, yeast two-hybrid screening, promoter activity assays and co-immunoprecipitation studies. The Nr2e3 expression was localized preferentially to the rod, and not to the cone, photoreceptor nuclei in rodent retina. The yeast two-hybrid screening of a retinal cDNA library, using NR2E3 as the bait, identified another orphan nuclear receptor NR1D1 (Rev-erbalpha). The interaction of NR2E3 with NR1D1 was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. In transient transfection studies using HEK 293 cells, both NR2E3 and NR1D1 activated the promoters of rod phototransduction genes synergistically with neural retina leucine zipper (NRL) and cone-rod homeobox (CRX). All four proteins, NR2E3, NR1D1, NRL and CRX, could be co-immunoprecipitated from the bovine retinal nuclear extract, suggesting their existence in a multi-protein transcriptional regulatory complex in vivo. Our results demonstrate that NR2E3 is involved in regulating the expression of rod photoreceptor-specific genes and support its proposed role in transcriptional regulatory network(s) during rod differentiation.

The minimal transactivation domain of the basic motif-leucine zipper transcription factor NRL interacts with TATA-binding protein.

The basic motif-leucine zipper (bZIP) transcription factor NRL controls the expression of rhodopsin and other phototransduction genes and is a key mediator of photoreceptor differentiation. To delineate the molecular mechanisms underlying transcriptional initiation of rod-specific genes, we characterized different regions of the NRL protein using yeast-based autoactivation assays. We identified 35 amino acid residues in the proline- and serine-rich N-terminal region (called minimal transactivation domain, MTD), which, when combined with LexA or Gal4 DNA binding domains, exhibited activation of target promoters. Because this domain is conserved in all proteins of the large Maf family, we hypothesized that NRL-MTD played an important role in assembling the transcription initiation complex. Our studies showed that the NRL protein, including the MTD, interacted with full-length or the C-terminal domain of TATA-binding protein (TBP) in vitro. NRL and TBP could be co-immunoprecipitated from bovine retinal nuclear extract. TBP was also part of c-Maf and MafA (two other large Maf proteins)-containing complex(es) in vivo. Our data suggest that the function of NRL-MTD is to activate transcription by recruiting or stabilizing TBP (and consequently other components of the general transcription complex) at the promoter of target genes, and a similar function may be attributed to other bZIP proteins of the large Maf family.