Biomedical subjects

Ruben Adler

Publications and source records attributed to Ruben Adler.

11 recordsLinked to original sources

PubMed2007

Nonredundant role of Akt2 for neuroprotection of rod photoreceptor cells from light-induced cell death.

The Akt kinases mediate cell survival through phosphorylation and inactivation of apoptotic machinery components. Akt signaling provides a trophic signal for transformed retinal neurons in culture, but the in vivo role of Akt activity is unknown. In this study, we found that all three Akt isoforms were expressed in rod photoreceptor cells. We investigated the functional roles of Akt1 and Akt2, two of the isoforms of Akt, and their biological significance in light-induced retinal degeneration. Consistent with the hypothesis that Akt activity is important to circumvent stress-induced apoptosis, herein we report the novel finding that rod photoreceptor cells in Akt2 knock-out mice exhibited a significantly greater sensitivity to stress-induced cell death than rods in heterozygous or wild-type mice. Under similar conditions, Akt1 deletion had no effect on the retina. The presence of three Akt isoforms in the retina is suggestive of a functional redundancy; however, our studies clearly demonstrate that, under stress, Akt1 and Akt3 cannot complement the specific survival signals driven by Akt2. Furthermore, we show that Akt2 is specially activated is response to light stress. The results presented in this study provide the first direct evidence that Akt2 has a nonredundant neuroprotective role in photoreceptor survival and maintenance.

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PubMed2006

Effects of follistatin overexpression on cell differentiation in the chick embryo retina.

Although activin is expressed in the embryonic central nervous system (CNS), its possible functions in the regulation of CNS neuronal differentiation remain largely unknown. We have investigated this question in the retina, a well-characterized CNS structure previously shown to respond to activin in vitro, and to express activin subunits and receptors in vivo. RCAS retroviruses were used to overexpress in the chick retina in ovo either follistatin (FS), an activin-binding protein and inhibitor, or alkaline phosphatase (AP), as control. FS-treated retinas appeared normal until ED 8, when they showed a reduction of the inner plexiform layer, accompanied by a marked decrease in the frequency of amacrine cells. The territory lacking amacrine cells showed downregulation of transcription factors necessary for amacrine cell differentiation, such as Pax6 and AP2alpha, accompanied by ectopic expression of transcription factors associated with the development of horizontal or bipolar neurons, such as Prox1, Chx10 and NeuroM. Increases in cell death were also observed in FS-treated retinas. Taken together with previous in vitro studies, our results suggest that activin is a powerful regulator of neuronal differentiation in the central nervous system.

Activins↗

PubMed2006

Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window.

Pax6 mutations cause complex ocular malformations, but it is uncertain whether early eye development normally requires Pax6 function in both the optic vesicle (OV) and the lens epithelium, or only in the latter. To investigate this question, we electroporated the OV with anti-Pax6 or control morpholinos before the onset of lens placode formation. Pax6 downregulation was already detectable in the OV 10 h after anti-Pax6 treatment, and was accompanied by a significant increase in the death of OV cells. A small eye-like phenotype developed thereafter, whose severity was developmental stage-dependent. When treatment was applied at Hamburger Hamilton (HH) stage 10, there was no optic cup formation, and lens development was abortive despite normal Pax6 expression in the lens epithelium. Treatment at HH stage 11 resulted in structurally normal lens and optic cup, although the latter showed abnormal expression domains for several transcription factors. Early eye development therefore requires cell-autonomous Pax6 function not only in the lens but also in the optic vesicle, where it plays a hitherto unknown role in cell survival. The results, moreover, indicate that there is a critical stage during which Pax6 expression in the OV is necessary for normal lens development.

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PubMed2006

Bone morphogenetic protein 7 increases chick photoreceptor outer segment initiation.

PURPOSE: The purpose of this study was to investigate the regulation of photoreceptor differentiation and outer segment elongation by the growth factor BMP7. METHODS: Dissociated low-density embryonic day 6 (E6) chick retinal cultures were grown for 6 days in the presence of BMP7, other members of the TGF-beta family of growth factors, or control vehicle. Cultured cells were characterized using microscopy, immunocytochemistry, and RT-PCR. Antibodies against visinin and GABA were used to distinguish photoreceptors from nonphotoreceptor cells, and monoclonal antibodies rhodopsin (rho) 4D2, OS-2, and COS-1 were used to distinguish subpopulations of cones and rods. RT-PCR was used to investigate mRNAs encoding visual pigments. RESULTS: Photoreceptors treated with BMP7 initiated outer segment elongation more frequently than photoreceptors in control cultures. The effect on outer segment initiation was confined to rods and to green opsin-expressing cones and appeared not to involve an increase in outer segment length. BMP7 did not appear to affect the survival, proliferation, or differentiation of progenitors or the fate of photoreceptors or amacrine cells in vitro. BMP5 and GDF5 showed weaker stimulatory effects than BMP7 on outer segment formation, whereas activin, BMP2, and BMP4 inhibited visual pigment expression and outer segment formation, and BMP6 had no detectable effects. CONCLUSIONS: BMP7 must be added to the list of candidate molecules capable of stimulating outer segment formation.

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PubMed2005

Roles of cell-intrinsic and microenvironmental factors in photoreceptor cell differentiation.

Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific "marker" as a proxy to identify photoreceptor cell fate is problematic.

Activins↗

PubMed2005

Challenges in the study of neuronal differentiation: a view from the embryonic eye.

Progress in the study of the molecular mechanisms that regulate neuronal differentiation has been quite impressive in recent years, and promises to continue to an equally fast pace. This should not lead us into a sense of complacency, however, because there are still significant barriers that cannot be overcome by simply conducting the same type of experiments that we have been performing thus far. This article will describe some of these challenges, while highlighting the conceptual and methodological breakthroughs that will be necessary to overcome them.

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PubMed2004

A method for analysis of gene expression in isolated mouse photoreceptor and Müller cells.

PURPOSE: Molecular analysis of complex phenomena, such as selective death of photoreceptors and their rescue by neuro-protective agents, has been hindered by limitations of techniques for investigating gene expression in individual cells within a heterogeneous tissue such as the retina. The purpose of this study was to develop methods to assess gene expression in single retinal cells. METHODS: Individual cells from papain-dissociated mouse retinae were captured with micropipettes and identified by morphology and by immunocytochemistry. Single cell cDNA libraries were generated by poly-d(T)-primed reverse transcription, poly-d(A) tailing of first strand cDNA, and en masse PCR-amplification using a custom made oligo-d(T). PCR was used to investigate gene expression in cDNAs from individual cells. RESULTS: Dissociated rod and Müller glia cells maintained their morphology, which correlated with their immunocytochemical properties. RPE cells were recognized by their pigmentation. With the exception of bipolar cells, non-photoreceptor neurons were only identifiable by immunocytochemistry. Abundant cDNA could be synthesized from each individual cell. Cell-specific "markers" were detected by PCR almost exclusively in the predicted cell types. The expression of neurotrophic factor receptors was consistent with previous biological studies. CONCLUSIONS: These studies establish a method to compare, investigate, and analyze gene expression in individual cells of the retina.

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PubMed2003

Gene discovery in the embryonic chick retina.

PURPOSE: The chick embryo is a powerful model system for the study of retinal development. However, analysis of gene expression in the chick retina has lagged behind biological studies. The purpose of this study was to identity and characterize genes expressed in the chick embryo retina as candidate molecules involved in the development and function of photoreceptors and other retinal cell types. METHODS: RNA from embryonic day (ED) 18 White Leghorn chick embryo retinae was used to generate an oligo dT-primed cDNA library. Bacterial colonies representing five thousand individual clones were arrayed onto nylon membranes using a microarray robot. Replicate membranes were hybridized with cDNA probes synthesized from ED 18 retina, brain and liver. Clones that appeared preferentially expressed in retina were identified by homology searches, and their spatial and temporal expression patterns were analyzed by in situ hybridization. RESULTS: Two hundred and seventy-two clones were identified. Approximately forty percent of the clones represented potential novel genes, including ESTs, hypothetical proteins and clones with no assigned identities. Furthermore, many genes were identified that are the putative chick orthologues of genes cloned from other species. We determined the expression pattern of several clones for which sequence homologies suggested possible roles in transcriptional regulation, apoptosis or intercellular signaling. Their corresponding mRNAs were expressed in the embryonic retina in topographically specific, developmentally regulated patterns. CONCLUSIONS: We identified and characterized genes in the chick embryo retina using a combination of microarray analysis and in situ hybridization. Analysis of the expression patterns suggests involvement of several of these genes in key events during embryogenesis.

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PubMed2002

The role of bone morphogenetic proteins in the differentiation of the ventral optic cup.

The ventral region of the chick embryo optic cup undergoes a complex process of differentiation leading to the formation of four different structures: the neural retina, the retinal pigment epithelium (RPE), the optic disk/optic stalk, and the pecten oculi. Signaling molecules such as retinoic acid and sonic hedgehog have been implicated in the regulation of these phenomena. We have now investigated whether the bone morphogenetic proteins (BMPs) also regulate ventral optic cup development. Loss-of-function experiments were carried out in chick embryos in ovo, by intraocular overexpression of noggin, a protein that binds several BMPs and prevents their interactions with their cognate cell surface receptors. At optic vesicle stages of development, this treatment resulted in microphthalmia with concomitant disruption of the developing neural retina, RPE and lens. At optic cup stages, however, noggin overexpression caused colobomas, pecten agenesis, replacement of the ventral RPE by neuroepithelium-like tissue, and ectopic expression of optic stalk markers in the region of the ventral retina and RPE. This was frequently accompanied by abnormal growth of ganglion cell axons, which failed to enter the optic nerve. The data suggest that endogenous BMPs have significant effects on the development of ventral optic cup structures.

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PubMed2002

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation.

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.

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PubMed2002

Effects of homeobox genes on the differentiation of photoreceptor and nonphotoreceptor neurons.

PURPOSE: The homeobox genes Pax6 and Chx10 are diffusely expressed in proliferating, undifferentiated retina neuroepithelial cells. Distinct, topographically specific expression patterns emerge, however, as postmitotic cells become organized into layers. The hypothesis that the product of each gene may be necessary for the differentiation of particular nonphotoreceptor neuron subsets and that their absence may be required for progenitor cells to differentiate as photoreceptors was tested in this study. METHODS: Neural retinas from 5-day-old chick embryos were dissociated, cultured at low density, and cotransfected with a plasmid expressing the green fluorescent protein (GFP) reporter gene, and a plasmid expressing Pax6, Chx10, Optx2, or the control gene lacZ. After further culture, the cells were fixed and processed for the detection of cell-specific markers. RESULTS: Nonphotoreceptor neurons increased threefold with Chx10 and almost sixfold with Pax6, compared with cells transfected with lacZ. The frequency of GFP(+) cells immunoreactive with the ganglion cell-specific antibody RA4 was unchanged by Chx10, but was increased twofold by Pax6. Conversely, Chx10 and Pax6 expression diminished the photoreceptor population to approximately 35% and 15% of control values, as determined by morphologic analysis, visinin immunocytochemistry, and peanut lectin binding. Optx2 had some inhibitory effects on photoreceptor differentiation, which were accompanied by marked increases in the frequency of morphologically undifferentiated cells. CONCLUSIONS: These results are consistent with the hypothesis that Chx10 and Pax6 promote the differentiation of nonphotoreceptor neurons while inhibiting the differentiation of photoreceptor cells.

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