Requirement of histone deacetylase activity for the expression of critical photoreceptor genes

Background: Histone deacetylases (HDACs) play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins. Results: To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA), was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells. Conclusion: HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells

RCAS-RNAi: A loss-of-function method for the developing chick retina

BACKGROUND: The embryonic chick provides an excellent model system for studies of development. However, it has lacked an efficient loss-of-function method for studies of gene function. RESULTS: We show that avian retroviruses can deliver hairpins mediating RNA interference to the developing chick eye. These viruses 'knock down' specific genes in infected areas of the retina. The knock down persists as the retina matures and can be detected using in situ hybridization. Furthermore, the amount of retinal tissue affected can be controlled by manipulating the degree of infection. CONCLUSION: This technique provides a rapid and efficient loss-of-function option for studies in the developing chick retina

A Hybrid Photoreceptor Expressing Both Rod and Cone Genes in a Mouse Model of Enhanced S-Cone Syndrome

Rod and cone photoreceptors subserve vision under dim and bright light conditions, respectively. The differences in their function are thought to stem from their different gene expression patterns, morphologies, and synaptic connectivities. In this study, we have examined the photoreceptor cells of the retinal degeneration 7 (rd7) mutant mouse, a model for the human enhanced S-cone syndrome (ESCS). This mutant carries a spontaneous deletion in the mouse ortholog of NR2E3, an orphan nuclear receptor transcription factor mutated in ESCS. Employing microarray and in situ hybridization analysis we have found that the rd7 retina contains a modestly increased number of S-opsin–expressing cells that ultrastructurally appear to be normal cones. Strikingly, the majority of the photoreceptors in the rd7 retina represent a morphologically hybrid cell type that expresses both rod- and cone-specific genes. In addition, in situ hybridization screening of genes shown to be up-regulated in the rd7 mutant retina by microarray identified ten new cone-specific or cone-enriched genes with a wide range of biochemical functions, including two genes specifically involved in glucose/glycogen metabolism. We suggest that the abnormal electroretinograms, slow retinal degeneration, and retinal dysmorphology seen in humans with ESCS may, in part, be attributable to the aberrant function of a hybrid photoreceptor cell type similar to that identified in this study. The functional diversity of the novel cone-specific genes identified here indicates molecular differences between rods and cones extending far beyond those previously discovered

Crx, a Novel otx-like Homeobox Gene, Shows Photoreceptor-Specific Expression and Regulates Photoreceptor Differentiation

AbstractWe have isolated a novel otx-like homeobox gene, Crx, from the mouse retina. Crx expression is restricted to developing and mature photoreceptor cells. CRX bound and transactivated the sequence TAATCC/A, which is found upstream of several photoreceptor-specific genes, including the opsin genes from many species. Overexpression of Crx using a retroviral vector increased the frequency of clones containing exclusively rod photoreceptors and reduced the frequency of clones containing amacrine interneurons and Müller glial cells. In addition, presumptive photoreceptor cells expressing a dominant-negative form of CRX failed to form proper photoreceptor outer segments and terminals. Crx is a novel photoreceptor-specific transcription factor and plays a crucial role in the differentiation of photoreceptor cells

Gene expression changes within Müller glial cells in retinitis pigmentosa

Purpose: Retinitis pigmentosa (RP) is a progressive retinal degeneration in which the retina loses nearly all of its photoreceptor cells and undergoes major structural changes. Little is known regarding the role the resident glia, the Müller glia, play in the progression of the disease. In this article, we define gene expression changes in Müller glial cells (MGCs) from two different mouse models of RP, the retinal degeneration 1 (rd1) and rhodopsin knockout (Rhod-ko) models. The RNA repertoire of single MGCs was comprehensively profiled, and a comparison was made between MGCs from wild-type (WT) and mutant retinas. Two time points were chosen for analysis, one at the peak of rod photoreceptor death and one during the period of cone photoreceptor death. Methods: Retinas were dissociated, and single MGCs were chosen under a dissecting microscope using a micropipette. Single cell cDNAs were generated and genome-wide profiles were obtained by hybridization to Affymetrix arrays. A comparison was made among all samples to discover the changes in gene expression during the periods of rod and cone photoreceptor death. Results: MGCs respond to retinal degeneration by undergoing gliosis, a process marked by the upregulation of glial fibrillary acidic protein (Gfap). Many additional transcripts were found to change. These can be placed into functional clusters, such as retinal remodeling, stress response, and immune-related response. Conclusions: A high degree of heterogeneity among the individual cells was observed, possibly due to their different spatial proximities to dying cells and/or inherent heterogeneity among MGCs

Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice

BACKGROUND: In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. RESULTS: A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. CONCLUSIONS: This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA

Temporal order of bipolar cell genesis in the neural retina

NEURAL DEVELOPMENT www.neuraldevelopment.com Temporal order of bipolar cell genesis in the neural retina Eric M Morrow et al

Thyroid hormone components are expressed in three sequential waves during development of the chick retina

<p>Abstract</p> <p>Background</p> <p>Thyroid hormone (TH) is an important developmental regulator in many tissues, including the retina. TH is activated locally via deiodinase 2 (Dio2), and it is destroyed by deiodinase 3 (Dio3). The TH receptors, TRa and TRb, mediate TH activity through hormone and DNA binding, and interactions with transcription regulators.</p> <p>Results</p> <p>In the current work, the expression of these TH components was examined in the chick retina over time. Three waves of expression were characterized and found to be correlated with critical developmental events. The first wave occurred as progenitor cells began to make photoreceptors, the second as some cell types adopted a more mature location and differentiation state, and the third as Müller glia were generated. The cell types expressing the components, as well as the kinetics of expression within the cell cycle, were defined. TRb expression initiated during G2 in progenitor cells, concomitant with NeuroD and Otx2, which are expressed in early photoreceptor cells. TRb was expressed in photoreceptor cells for several days and then was reduced in expression level, as the expression of Crx, a later photoreceptor gene, became more evident. Dio3 was expressed throughout the cell cycle in progenitor cells. TRa was in most, if not all, retinal cells. Dio2 appeared transiently in a ventral (high) to dorsal gradient, likely in a subset of photoreceptor cells.</p> <p>Conclusion</p> <p>Multiple TH components were expressed in dynamic patterns in cycling progenitor cells and photoreceptors cells across the developing chick retina. These dynamic patterns suggest that TH is playing several roles in retinal development, both within the cycling progenitor cells and possibly with respect to the timing of differentiation of photoreceptor cells.</p

Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells

AbstractAn understanding of the number and types of progeny produced by progenitor cells during development provides a foundation for studies of when and where cell fate determination takes place. Lineal relationships can be revealed by the identification of descendents of cells that express a recombinase, such as Cre or Flp. This method provides data concerning gene expression history, but does not provide clonal resolution among the descendents. An alternative method employs retroviral labeling, which permits the identification of clones, but does not allow for the tracking of gene expression history. Here we report a combination of these methods to circumvent each method's limitations. By employing the specificity of Cre expression, and by selecting only a subset of cells with a Cre history for retroviral infection, clones with a gene expression history can be labeled. The method utilizes a conditional allele of the avian tumor virus receptor A (TVA), which allows infection of mouse cells following Cre activity, with mammalian retroviral vectors pseudotyped with the ASLV-A envelope glycoprotein (EnvA). We quantified the efficiency and specificity of this system in vivo and in vitro. We also generated a series of retroviral vectors encoding a variety of histochemical and fluorescent reporter genes that enable the tracking of mixtures of clones, thus enabling better resolution of clonal boundaries. This method and new vectors can be used to further our understanding of the gene expression patterns of progenitor cells that make particular daughter cells, as well as provide a platform for manipulating identified subsets of developing cells