(A) Effect of protein synthesis inhibition on the downregulation of Nrl and Crx by TSA

<p><b>Copyright information:</b></p><p>Taken from "Requirement of histone deacetylase activity for the expression of critical photoreceptor genes"</p><p>http://www.biomedcentral.com/1471-213X/7/78</p><p>BMC Developmental Biology 2007;7():78-78.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1914050.</p><p></p> P2 retinal explant cultures were treated with TSA, TSA plus cycloheximide, or DMSO for 3 hours, followed by Northern blot analysis. (B) Effect of HDAC inhibition on histone acetylation by western blot analysis. (C) Effect of HDAC inhibition on the acetylation of non-histone proteins by western blot analysis. The blot was sequentially probed with a pan acetyl-lysine antibodyn (top blot), an acetyl-tubulin antibody (middle blot), and an α-tubulin antibody (bottom blot)

(A) Northern blot analysis of the time course of changes in retinal gene expression when HDAC activity was inhibited in P2 mouse retinal explants

<p><b>Copyright information:</b></p><p>Taken from "Requirement of histone deacetylase activity for the expression of critical photoreceptor genes"</p><p>http://www.biomedcentral.com/1471-213X/7/78</p><p>BMC Developmental Biology 2007;7():78-78.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1914050.</p><p></p> (B) Effect of HDAC inhibition on Nrl and Crx expression in P21 mouse retinal explants by Northern blot analysis

Northern blot analysis of the expression of Otx2, Nrl, and Crx when P2 mouse retinal explants were treated with 1 μM TSA or DMSO for 3 hours

<p><b>Copyright information:</b></p><p>Taken from "Requirement of histone deacetylase activity for the expression of critical photoreceptor genes"</p><p>http://www.biomedcentral.com/1471-213X/7/78</p><p>BMC Developmental Biology 2007;7():78-78.</p><p>Published online 29 Jun 2007</p><p>PMCID:PMC1914050.</p><p></p

A Partial View of the Rod Photoreceptor Transcriptional Regulatory Network

<p>Note that green lines indicate activation, and yellow and red lines indicate weak and strong repression, respectively. The dotted lines associated with a question mark indicate that it is not known whether Nrl directly represses the target genes in question or whether its repression is mediated by a downstream transcription factor (“X”). Note that <i>Nr2e3</i> appears to negatively regulate its own transcription. The regulatory linkages depicted in this diagram are not necessarily direct. The weak activation of some rod-specific genes by <i>Nr2e3</i> is omitted from this diagram for clarity. Also not shown is the role of other photoreceptor transcription factors, such as <i>Crx</i>.</p

The Onset of <i>rhodopsin</i> Expression Is Delayed in the <i>rd7</i> Mutant Retina

<p>Note the nearly undetectable staining for <i>rhodopsin</i> in this P6 mutant retina (top right). The majority of rod-specific genes did not show this delay in expression onset, as indicated by the normal amount of staining for <i>Pde6a</i> in the mutant at P6 (bottom images).</p

Cone and Rod Gene Expression in the <i>rd7</i> Mutant at P14

<p>The upper sets of photomicrographs demonstrate examples of type I and type II cone gene derepression in the <i>rd7</i> mutant retina as explained in the main text. The bottom left images show several rod-specific genes that are essentially unchanged in the <i>rd7</i> background at P14. The bottom right images show the expression pattern of three photoreceptor transcription factors in the <i>rd7</i> mutant. Abbreviations in the lower left hand corner of each pair of panels represent the gene symbols summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010011#pgen-0010011-g001" target="_blank">Figure 1</a>.</p

Expression Patterns of Several Novel Cone Genes Up-Regulated in <i>rd7</i>

<p>In the wild-type images (wt), note the scattered, weakly positive cells at the scleral edge of the outer nuclear layer in a cone distribution. All of the genes show marked up-regulation in the <i>rd7</i> mutant. <i>Bub1b</i> and <i>Tcta</i> show transcript localization predominantly to the inner segment of the photoreceptors. Retinas are oriented such that the scleral edge is up.</p

Cone-Specific and Cone-Enriched Genes Evaluated in the <i>rd7</i> Mutant by Microarray and In Situ Hybridization

<div><p>The color coding of text in the column “Gene Name” is as follows: light blue (G1–G15), genes previously reported in the literature to have cone-specific or cone-enriched patterns of expression; yellow (G16–G25), novel cone genes identified in an unrelated study (unpublished data); dark green (G26–G36), novel cone genes identified in the present study that were up-regulated in <i>rd7;</i> light green (G37–G46), additional genes found to be up-regulated in <i>rd7</i> by microarray in the present study but that had either weak or inapparent cone-specific signal on in situ hybridization; white (G47–G53), additional genes up-regulated by microarray at two different timepoints but with either unusual expression patterns or nonconfirmatory in situ hybridizations. The column “ID” contains identifiers used in the present paper to refer to specific genes. “GenBank ID” contains the GenBank accession number of the clone used to make the probe for in situ hybridization. Within this column, “lab clone” indicates that the probe used for in situ hybridization derived from a clone in our laboratory. The region of the gene to which it corresponds is indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010011#pgen-0010011-st001" target="_blank">Table S1</a>. Columns “P0” through “P21” contain the results of microarray experiments at the given postnatal dates. P0, P6, and P14 time points represent analyses on cDNA microarrays; the P21 time point represents data from an Affymetrix microarray (mouse genome 432 2.0). A red cell with a single up arrow indicates that the gene in question was up-regulated in three out of three microarrays at that time point (as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010011#s4" target="_blank">Materials and Methods</a>). Those cells labeled orange with a single up arrow and asterisk indicate that the gene in question was up-regulated in two out of three microarrays at that time point. The column “In Situ” lists the type of derepression seen for the gene in question in the <i>rd7</i> mutant retina (type I and type II are described in the main text). Genes designated “unclassified” represent patterns of derepression that were difficult to classify as either type I or type II (see main text for more details). “Wild type” in this column indicates that the in situ hybridization pattern in the <i>rd7</i> mutant retina was not different from the wild-type pattern; and “special” indicates a special pattern of expression discussed more fully in the main text. The column “Expression Pattern” contains a concise description of the wild-type expression pattern of the gene in question. In the case of genes for which no signal was obtained on in situ hybridization in the present study, the specified expression pattern derives from reports in the literature. Within this column, “cone > rod” indicates that the gene is expressed in all photoreceptors, but at higher levels in cones than rods; “cone?” indicates very weak staining in a cone-like distribution.</p><p>BP, bipolar cells; EP, early photoreceptor expression pattern; IS, inner core segment localization; MG, Müller glia; N/A, not available on the microarray; NS, no signal detected on in situ hybridization; RPE, retinal pigment epithelium.</p></div

The <i>rd7</i> Mutant Retina Contains a Morphologically Hybrid Photoreceptor Cell Type in Addition to Supernumerary S-Opsin–Positive Cones

<div><p>(A and B) Toluidine blue-stained semi-thin sections of the outer nuclear layer (scleral edge oriented up).</p><p>(C and D) Hand-drawn diagrams of the cells in (A) and (B), respectively. Cells with the nuclear features of cones are highlighted in blue. Note that the number of such cells is greater in the mutant, and their cell bodies are scattered throughout the outer nuclear layer. In addition, the overall columnar architecture of the outer nuclear layer seen in the wild type is disrupted in this portion of the mutant retina. Other portions of the mutant retina with fewer supernumerary cone cells, however, retain the normal columnar appearance (unpublished data).</p><p>(E and F) Images of the outer nuclear layer (scleral edge up) stained by in situ hybridization for S-opsin. Note the typical pattern of staining at the scleral edge of the outer nuclear layer in the wild type. The <i>rd7</i> mutant retina shows supernumerary S-opsin–positive cells scattered throughout the outer nuclear layer in a distribution very similar to the supernumerary cone cells seen in (B). Since images (E) and (F) derive from different retinas than those depicted in (A) and (B), the location of the individual cells do not correspond.</p><p>(G and H) Electron micrographs of the outer nuclear layer (10,000× magnification). Note the uniform distribution of rod cell bodies in the wild type (G). The cell bodies are nearly round and consist almost exclusively of a nucleus with a single, dense mass of heterochromatin. In the <i>rd7</i> mutant (H), two types of cell are shown. The ovoid one with a lesser quantity of heterochromatin, paler euchromatin, and two juxtanuclear mitochondria (yellow arrow) represents a typical cone cell body. The adjacent cell with a more “rod-like” mass of heterochromatin and a single juxtanuclear mitochondrion (red arrow) represents one of the hybrid photoreceptors discussed more fully in the main text.</p></div

Some of the Genes Up-Regulated in <i>rd7</i> Show an Early Photoreceptor Pattern of Expression

<p><i>Gnb3</i> and <i>Thrb2</i> are both previously characterized cone genes that show staining at the scleral edge of the embryonic mouse retina in cells that will differentiate into photoreceptors. <i>Ece1</i> and <i>Otop3</i> are two novel cone genes identified in this study that were up-regulated in <i>rd7</i> and also showed an early photoreceptor pattern of expression. <i>Prdm1</i> and RIKEN cDNA 1300018I05 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010011#pgen-0010011-g001" target="_blank">Figure 1</a>, genes G48 and G49, respectively) are two other genes that had either undetectable signal <i>(Prdm1)</i> or no apparent change in expression pattern in adult <i>rd7</i> mutants (RIKEN cDNA 1300018I05), but which also showed staining in the embryonic retina in a presumptive photoreceptor pattern. All images are from E17.5 retina except <i>Gnb3,</i> which was from E16.</p