The developmental sequence of gene expression within the rod photoreceptor lineage in embryonic zebrafish.

In postembryonic zebrafish, rod photoreceptors are continuously generated from progenitors in the inner nuclear layer, which are derived from radial Müller glia that express the transcription factor pax6. We used BrdU incorporation, in combination with in situ hybridization for cell-specific transcription factors, to establish the patterns of gene expression during rod lineage maturation in the embryonic zebrafish. Downregulation of pax6 expression was accompanied by sporadic upregulation of expression of the transcription factors NeuroD/nrd, rx1, crx, and Nr2e3/pnr. As cells of the rod lineage entered the outer nuclear layer, they became homogeneous, coordinately expressing NeuroD, rx1, crx, and Nr2e3. Postmitotic, maturing rods also expressed nrl, rod opsin, and rod transducin/gnat1. The presence of rx1 within the rod lineage and in maturing rods indicates that rx1 is not cone-specific, as previously reported, and suggests a high degree of molecular similarity between rod and cone progenitor populations in the zebrafish.

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina.

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.

The Xenopus ortholog of the nuclear hormone receptor Nr2e3 is primarily expressed in developing photoreceptors.

Nr2e3 is a nuclear hormone receptor that is involved in rod photoreceptor differentiation. The Nr2e3 gene was previously identified in humans, mice, zebrafish and chicken. In all species, Nr2e3 expression is restricted to the retina and is believed to have a role in rod photoreceptor specification and maintenance. Here we report the identification and characterization of the Xenopus Nr2e3. We found that Nr2e3 is primarily expressed in developing rod photoreceptors. In contrast to other species, Nr2e3 is also expressed in the notochord and pineal gland during Xenopus laevis development.

Phototransduction in mouse rods and cones.

Phototransduction is the process by which light triggers an electrical signal in a photoreceptor cell. Image-forming vision in vertebrates is mediated by two types of photoreceptors: the rods and the cones. In this review, we provide a summary of the success in which the mouse has served as a vertebrate model for studying rod phototransduction, with respect to both the activation and termination steps. Cones are still not as well-understood as rods partly because it is difficult to work with mouse cones due to their scarcity and fragility. The situation may change, however.

Mosaic Eyes is a novel component of the Crumbs complex and negatively regulates photoreceptor apical size.

Establishment of apical-basal cell polarity has emerged as an important process during development, and the Crumbs complex is a major component of this process in Drosophila. By comparison, little is known about the role of Crumbs (Crb) proteins in vertebrate development. We show that the FERM protein Mosaic Eyes (Moe) is a novel regulatory component of the Crumbs complex. Moe coimmunoprecipitates with Ome/Crb2a and Nok (Pals1) from adult eye and in vitro interaction experiments suggest these interactions are direct. Morpholino knockdown of ome/crb2a phenocopies the moe mutations. Moe and Crumbs proteins colocalize apically and this apical localization requires reciprocal protein function. By performing genetic mosaic analyses, we show that moe- rod photoreceptors have greatly expanded apical structures, suggesting that Moe is a negative regulator of Crumbs protein function in photoreceptors. We propose that Moe is a crucial regulator of Crumbs protein cell-surface abundance and localization in embryos.

The CNTF/LIF signaling pathway regulates developmental programmed cell death and differentiation of rod precursor cells in the mouse retina in vivo.

Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.

Morphogenesis of the different types of photoreceptors of the chicken (Gallus domesticus) retina and the effect of amblyopia in neonatal chicken.

Despite the great variety in chicken photoreceptors, existing morphogenetic studies only deal with two types: rods and cones. We have therefore examined by scanning electron microscopy the first appearance and maturation of different retinal photoreceptors in 36 chicken embryos (Gallus domesticus), aged 5-19 days prehatching. On day 5 of incubation, chicken retinae were only composed of proliferating ventricular cells devoid of photoreceptors. On day 8, outer mitotic cells were separated from inner differentiating photoreceptors, by the transient layer of Chievitz. Ball-like protrusions appeared at the ventricular surface, representing the first signs of photoreceptor inner segment formation. From day 10 onward, double cones, single cones, and rods could be clearly distinguished, and occasional cilia were detected at their tip. On day 12, inner segments had increased in length and diameter, and frequently carried a cilium representing the beginning of outer segment formation. On day 14, most photoreceptors displayed a distinct outer segment. On day 19, photoreceptors had essentially assumed adult morphology. Based on the shape of their outer segments, two subtypes of cones and three subtypes of double cones could be distinguished. Throughout development, we observed microvilli close to maturing photoreceptors, either originating from their lateral sides, from their tip, or from Müller cells. Microvillus density peaked between day 12 and 14, indicating an important role in photoreceptor morphogenesis. Unilateral occlusion of the eyes of posthatching chicken reduced the proportion of double cones to single cones in the retina, indicating dependence of retinal morphogenesis upon functional activity of visual cells.

Transgenic mice expressing Cre-recombinase specifically in retinal rod bipolar neurons.

PURPOSE: To establish a transgenic mouse line that expresses Cre-recombinase in retinal rod bipolar cells for the generation of rod bipolar cell-specific knockout mutants. METHODS: The IRES-Cre-cDNA fragment was inserted into a 173-kb bacterial artificial chromosome (BAC) carrying the intact Pcp2 gene, by using red-mediated recombineering. Transgenic mice were generated with the modified BAC and identified. The Cre-transgenic mice were crossed with ROSA26 and Z/EG reporter mice to detect Cre-recombinase activity. RESULTS: X-gal staining showed that strong Cre-recombinase activities were present in retinal inner nuclear layers and cerebellar Purkinje cells. Double staining with an anti-GFP antibody and an anti-PKCalpha antibody (specific for retinal rod bipolar cells) revealed that Cre-recombinase activity localized exclusively to the rod bipolar cells in the retina. CONCLUSIONS: A mouse BAC-Pcp2-IRES-Cre transgenic line that expresses Cre-recombinase in retinal rod bipolar neurons has been established. Because mutations in some ubiquitously expressed genes may result in retinal degenerative diseases, the mouse strain BAC-Pcp2-IRES-Cre will be a useful new tool for investigating the effects of retinal rod bipolar cell-specific gene inactivation.

GDF11 controls the timing of progenitor cell competence in developing retina.

The orderly generation of cell types in the developing retina is thought to be regulated by changes in the competence of multipotent progenitors. Here, we show that a secreted factor, growth and differentiation factor 11 (GDF11), controls the numbers of retinal ganglion cells (RGCs), as well as amacrine and photoreceptor cells, that form during development. GDF11 does not affect proliferation of progenitors-a major mode of GDF11 action in other tissues-but instead controls duration of expression of Math5, a gene that confers competence for RGC genesis, in progenitor cells. Thus, GDF11 governs the temporal windows during which multipotent progenitors retain competence to produce distinct neural progeny.

The rod photoreceptor pattern is set at the optic vesicle stage and requires spatially restricted cVax expression.

How and when positional identities in the neural retina are established have been addressed primarily with respect to the topographic projections of retinal ganglion cells onto their targets in the brain. Although retinotectal map formation is a prominent manifestation of retinal patterning, it is not the only one. Photoreceptor subtypes are arranged in distinct, species-specific patterns. The mechanisms used to establish photoreceptor patterns have been relatively unexplored at the mechanistic level. We performed ablations of the eye anlage in chickens and found that removal of the anterior or dorsal optic vesicle caused loss of the area centralis, which is a rod-free central area of the retina, and severely disorganized other aspects of the rod pattern. These observations indicate that the anteroposterior and dorsoventral distribution of rods is determined by the optic vesicle stage. To investigate the molecular mechanisms involved, the rod distribution was analyzed after viral misexpression of several patterning genes that were previously shown to be important in positional specification of retinal ganglion cells. Ectopic expression of FoxG1, SOHo1,or GH6 transcription factors expressed in the anterior optic vesicle and/or optic cup, respectively, did not affect the rod pattern. This pattern therefore appears to be specified by an activity acting before, or in parallel with, these factors. In contrast, misexpression of the ventrally restricted transcription factor, cVax, severely disturbed the rod pattern.

Notch-Delta signaling is required for spatial patterning and Müller glia differentiation in the zebrafish retina.

Notch-Delta signaling has been implicated in several alternative modes of function in the vertebrate retina. To further investigate these functions, we examined retinas from zebrafish embryos in which bidirectional Notch-Delta signaling was inactivated either by the mind bomb (mib) mutation, which disrupts E3 ubiquitin ligase activity, or by treatment with gamma-secretase inhibitors, which prevent intramembrane proteolysis of Notch and Delta. We found that inactivating Notch-Delta signaling did not prevent differentiation of retinal neurons, but it did disrupt spatial patterning in both the apical-basal and planar dimensions of the retinal epithelium. Retinal neurons differentiated, but their laminar arrangement was disrupted. Photoreceptor differentiation was initiated normally, but its progression was slowed. Although confined to the apical retinal surface as in normal retinas, the planar organization of cone photoreceptors was disrupted: cones of the same spectral subtype were clumped rather than regularly spaced. In contrast to neurons, Müller glia failed to differentiate suggesting an instructive role for Notch-Delta signaling in gliogenesis.

Developmental regulation of calcium-dependent feedback in Xenopus rods.

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.

The transcription factor Bhlhb4 is required for rod bipolar cell maturation.

Retinal bipolar cells are essential to the transmission of light information. Although bipolar cell dysfunction can result in blindness, little is known about the factors required for bipolar cell development and functional maturation. The basic helix-loop-helix (bHLH) transcription factor Bhlhb4 was found to be expressed in rod bipolar cells (RB). Electroretinograms (ERGs) in the adult Bhlhb4 knockout (Bhlhb4(-/-)) showed that the loss of Bhlhb4 resulted in disrupted rod signaling and profound retinal dysfunction resembling human congenital stationary night blindness (CSNB), characterized by the loss of the scotopic ERG b-wave. A depletion of inner nuclear layer (INL) cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development. Other retinal cell populations including photoreceptors were unaltered. The timing of RB cell depletion in the Bhlhb4(-/-) mouse suggests that Bhlhb4 is essential for RB cell maturation.

The expression of the Leber congenital amaurosis protein AIPL1 coincides with rod and cone photoreceptor development.

PURPOSE: The Leber congenital amaurosis (LCA) protein AIPL1 is present only in the rod photoreceptors of the adult human retina and is excluded from the cone photoreceptors. LCA, however, is characterized by an absence of both rod and cone function at birth or shortly thereafter. Therefore, this study was conducted to determine whether AIPL1 is present in the rod and cone photoreceptors of the developing human retina. In addition, the expression of NUB1, a putative AIPL1-interacting partner, was examined. METHODS: A comprehensive spatiotemporal examination of AIPL1 distribution during development was performed by immunohistochemistry, using a previously characterized AIPL1 anti-serum. Immunofluorescence confocal microscopy was used to examine the coexpression of AIPL1 with the long/medium (L/M) and short (S) wavelength-sensitive cone photoreceptors in the developing human retina. The spatiotemporal distribution of NUB1 was also examined by immunohistochemistry, using a newly developed anti-serum to the C terminus of NUB1. RESULTS: AIPL1 protein was detected by 11.8 fetal weeks in the central fetal human retina. With continued development, AIPL1 expression spread gradually toward peripheral retina. AIPL1 was expressed in the L/M and S cone photoreceptors in addition to the rods of the developing human retina. NUB1 was localized in cell nuclei throughout the human fetal and adult eye at all time points. CONCLUSIONS: The pattern of AIPL1 expression closely follows the centroperipheral gradient in photoreceptor development. The data suggest that AIPL1 is essential for the normal development of both rod and cone photoreceptor cells and that mutations in the AIPL1 gene cause the death or dysfunction of photoreceptors early in development resulting in blindness or severely impaired vision at birth.

Investigation of the migration path for new rod photoreceptors in the adult cichlid fish retina.

In the retina of adult teleost, precursor cells divide in the outer nuclear layer and give rise to new rod photoreceptors. These new rods migrate from the outer limiting membrane to the inner edge of the outer nuclear layer (ONL) before differentiating. In order to understand which cues these cells use during migration and insertion at the appropriate location we combined cell-specific stains in the retina of the cichlid fish Haplochromis burtoni, viewed with confocal laserscan microscopy: Dividing cells were labeled with bromodeoxyuridine (BrdU), Müller glial cells, cone photoreceptors, and horizontal cells were detected by specific antibodies. During the migration phase (24 to 48 h after BrdU uptake) up to 46% of BrdU-labeled cells were spindle shaped and radially oriented. Most of them were in direct proximity to Müller cell processes. Four days after BrdU-uptake, most labeled cells (91%) were found in the inner portion of the ONL and displayed a spherical shape. This marks the end of the movement of the new rods. At this stage, the labeled cells showed no preference to lie near glial fibers but were often found close to the pedicles of double cones. The leading edge of migrating cells reached into the outer plexiform layer (OPL) but not further than processes of horizontal cells. This is beyond the location of mature rods. We hypothesize that the cells are repelled in the OPL and insert back in the ONL to differentiate as rods.

GDNF regulates chicken rod photoreceptor development and survival in reaggregated histotypic retinal spheres.

PURPOSE: To investigate the role of glial-cell-line-derived neurotrophic factor (GDNF) on proliferation, differentiation, and apoptosis of different retinal cell types--in particular, photoreceptor cells. METHODS: Reaggregated histotypic spheres, derived from retinal cells of the E6 chicken embryo were used. Under rotation, so-called rosetted spheroids arose by aggregation of dissociated retinal cells, followed by the proliferation, migration, differentiation and programmed cell death of particular cell types. Rosetted spheroids were cultured under serum-reduced conditions, either in the absence or presence of 50 ng/mL GDNF. At appropriate stages, rosetted spheroids were analyzed by using conventional staining and immunolabeling with antibodies against different retinal cell types. RESULTS: At early stages of culture, the application of GDNF to rosetted spheroids significantly increased and sustained the rate of proliferation. In particular, a de novo production of rod photoreceptors was observed, whereas cone photoreceptors and amacrine, horizontal, ganglion, and Müller cells were not affected. In addition, in GDNF-treated cultures, rod photoreceptors differentiated earlier than in nontreated cultures. In older rosetted spheroids raised in absence of GDNF, rod but not cone photoreceptors underwent apoptosis. By supplementation with GDNF, the percentage of dying rod photoreceptors was dramatically reduced (31%-6% at 8 days in culture, 71%-3% at 10 days in culture). Both the mitogenic and survival promoting effect of GDNF were dose dependent. CONCLUSIONS: The results strongly suggest that GDNF, at least in vitro, affects rod photoreceptors. Depending on the developmental stage, GDNF regulates their proliferation, differentiation, and survival.

Visualization of rod photoreceptor development using GFP-transgenic zebrafish.

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.

Photoreceptor plasticity in reaggregates of embryonic chick retina: rods depend on proximal cones and on tissue organization.

Plasticity of photoreceptors and their integration into epithelial structures homologous to an outer nuclear layer (ONL), was investigated in embryonic chick retinal cell reaggregates by immunohistochemistry using an antibody specific for red plus green cones (RG-cones) and an antibody for rods. If reaggregates are raised in the presence of pigmented epithelium (RPE), completely reconstructed, stratified retinal spheres are produced, where all rods and cones are integrated into an outer laminar ONL, similar to a normal retina. In the absence of RPE, 'rosetted' spheres form which contain internal rosettes homologous to an ONL. Only a minor fraction of cones and rods of 'rosetted' spheres are located within rosettes, while a larger fraction is diffusely displaced in nonorganized areas, thus, not contributing to an ONL-like epithelium. In both types of spheres, the total percentage of RG-cones was similar to the in vivo retina, indicating that expression of cones is autonomous. Following cones, after about one day, rods developed only within already existing RG-cone clusters. Thereby, the ratio of rods to RG-cones increases as the tissue organization decreases: for stratified spheres this ratio is, 0.50 (1 rod/2 cones; similar to mature retina); for rosettes, 0.74 (3 rods/4 cones) and for nonorganized areas, 1.09 (1 rod/1 cone) -- a higher ratio under our conditions has never been detected. Thus, rod expression depends strictly on the presence of nearby cones; their relative numbers are distinctively adjusted according to the cytoarchitecture of the tissue environment. The biomedical implications of these findings are briefly discussed.

Fra-1 replaces c-Fos-dependent functions in mice.

Structure-function analysis as well as studies with knock-out and transgenic mice have assigned distinct functions to c-Fos and Fra-1, two components of the transcription factor AP-1 (activator protein-1). To test whether Fra-1 could substitute for c-Fos, we generated knock-in mice that express Fra-1 in place of c-Fos. Fra-1 rescues c-Fos-dependent functions such as bone development and light-induced photoreceptor apoptosis. Importantly, rescue of bone cell differentiation, but not photoreceptor apoptosis, is gene-dosage dependent. Moreover, Fra-1 fails to substitute for c-Fos in inducing expression of target genes in fibroblasts. These results show that c-Fos and Fra-1 have maintained functional equivalence during vertebrate evolution.