XB-ART-40937Dev Biol March 15, 2010; 339 (2): 494-506.
Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product.
The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type.
PubMed ID: 20060393
PMC ID: PMC2830336
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: ago2 ces3.7 crx myc nrl opn1lw pmel rax rho rpe
Antibodies: Isl1/2 Ab1 Peanut Agglutinin Rho Ab2
Article Images: [+] show captions
|Fig. 1. Rhodopsin and red cone opsin (RCO) are Rx targets. (A–C) Rx is co-expressed with rhodopsin and RCO in photoreceptors. In situ hybridization on sections from paraffin-embedded st 41 tadpoles using probes for Rx (A), rhodopsin (B) or RCO (C). (D, E) Chromatin immunoprecipitation (ChIP) results indicating that myc-tagged Rx (MT-Rx) can bind to the rhodopsin (D) and RCO (E) promoters in vivo. Results are presented as the CT of each sample normalized to the CT of a “no antibody” control. p < 0.003 (XOP), p < 0.002 (RCO).|
|Fig. 3. Generation of Rx knockdown embryos. (A) A portion of X. laevis Rx1A was selected as a target for development of an shRNA. Alignment of the shRNA target sequence with corresponding regions of X. laevis Rx2A and mouse Rx. (B) Tadpoles transgenic for the Rx shRNA plasmid appeared normal. Bright light or UV-light views of transgenic tadpoles at st 41. (C) Retinas from Rx shRNA transgenic tadpoles appeared histologically normal. Hematoxylin and eosin stained sections of paraffin-embedded wild type (left panel) or Rx shRNA transgenic tadpole. (D) Rx shRNA transgenic tadpoles have reduced levels of both Rx1A and Rx2A as determined by quantitative RT-PCR (qRT-PCR) performed using total RNA purified from isolated tadpole heads (st 41). (E) Rx1A is expression is reduced by in situ hybridization performed using 8 μM sections of paraffin-embedded tadpoles at st 38, 41, and 45 (from left to right). The reduction in Rx expression appears to increase as development progresses.|
|Fig. 4. Exogenous Argonaute2 (Ago2) exacerbates the effects of shRNA-mediated Rx knockdown. (A) Exogenous Ago2 exacerbates the Rx shRNA knockdown phenotype. Embryos were generated by intra-cytosolic sperm injection (ICSI) with Rx shRNA transgene and injected with RNA encoding X. laevis Ago2 RNA. Embryos were co-injected with dsRed Express RNA as a lineage tracer. Embryos were photographed under white light (i, ii, iii), red fluorescence to visualize dsRed lineage tracer (iâ ², iiâ ², iiiâ ²), or blue fluorescence to visualize Rx shRNA transgene (iâ ³, iiâ ³, iiiâ ³). Embryos receiving only the Rx shRNA (panels iâ iii) or Ago2 RNA (panels iâ ³â iiiâ ³) have apparently normal eyes while embryos transgenic for the Rx shRNA and receiving Ago2 RNA in the developing eye exhibited abnormally developed eyes (panels iâ ²â iiiâ ²). (B) Exogenous Ago2 exacerbates the effects of Rx shRNA on Rx expression. Wholemount in situ hybridization using an Rx antisense riboprobe and embryos receiving either the Rx shRNA transgene (ii), Ago2 RNA (iii), or both (iv). Control embryos (i) received neither. These results are presented in graph form in (C).|
|Fig. 6. Knockdown of photopigment gene expression can be rescued by expression of mouse Rx. (A) Top: Schematic diagram of the X. tropicalis Rx genomic locus. Shown are: two of the three ultra-conserved elements (UCEs) (UCE2 and UCE3, purple), 3 kb promoter (yellow), first coding exon (blue). Bottom: Schematic diagram of Rx regulatory region construct UCE2 + tRx3000, containing the 3 kb Rx promoter and UCE2. (B) Expression of tRx3000/GFP transgene in tailbud embryos. (i) white light image of st 20 neural tube stage embryo; (ii) fluorescent image of the same embryo shown in (i); (iii) wholemount in situ hybridization of tailbud stage (st 28) tRx3000/GFP transgenic embryo using an antisense riboprobe to GFP. (C) In situ hybridization of sections of paraffin-embedded st 41 tadpoles using an antisense riboprobe to GFP. (i) The tRx3000/GFP transgene is expressed in the photoreceptor layer, in the INL, and the CMZ. It is not expressed in the distal portion of the CMZ where retinal stem cells are found. (ii) Addition of UCE2 to the tRx3000/GFP transgene drives expression throughout the CMZ. (D) Schematic diagram of rescue construct. The construct includes the mRx coding region driven by UCE2 + tRx3000 and a dsRed expression cassette driven by the CMV promoter for selection of transgenic embryos. (E) Expression of photopigment genes rhodopsin and red cone opsin is not reduced in embryos transgenic for both Rx shRNA and the mRx by qRT-PCR.|
|Fig. 7. Specific degeneration of photoreceptors in visually impaired Rx shRNA tadpoles. (A–C) Hematoxylin and eosin staining of sections prepared from paraffin-embedded st 50 tadpoles. Black arrows indicate nuclei in the outer nuclear layer (ONL). Red arrow indicates a gap in the ONL. (D–F) Immunohistochemical staining of sections from paraffin-embedded tadpoles using an antibody raised against rhodopsin (RetP1). Black arrows indicate RetP1-positive cells in the photoreceptor layer. Red arrow indicates a gap in RetP1 staining in the photoreceptor layer. (G–I) Staining of sections prepared from paraffin-embedded tadpoles using peanut agglutinin (PNA). Black arrows indicate PNA-positive cells in the photoreceptor layer. Red arrow indicates a gap in PNA staining in the photoreceptor layer. Wild type (A, D, G), Rx shRNA transgenic (B, E, H), or Rx shRNA + mRx rescue construct cotransgenic (C, F, I) tadpoles were raised to st 50 and tested for visual function (Table 1). Rx shRNA transgenic tadpoles with impaired visual function exhibited abnormal photoreceptor histology, including missing nuclei (red arrow) compared to a nontransgenic control (A, B). Photoreceptor histology was normal in a tadpole transgenic for both Rx shRNA and mRx (C). Rod and cone photoreceptors of Rx shRNA tadpoles exhibited reduced staining with rhodopsin and PNA (red arrows in E and H) compared to nontransgenic controls (D, E, G, H). Rhodopsin and PNA staining appeared normal in tadpoles transgenic for both the Rx shRNA and mRx (F, I).|
|pmel (premelanosome protein) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.|
|Supplementary Figure S1. The silver [pmel] gene is specifically expressed in the developing eye at tailbud stages. A. Wholemount in situ hybridization of late tailbud stage embryo (st 34) using silver antisense riboprobe. Silver is expressed in the eye. B. The embryos shown in (A) was embedded in paraffin and sectioned. The in situ hybridization signal is localized to the outer layer of the eye, the retinal pigmented epithelium (RPE). C. In situ hybridization performed using sections of a paraffin-embedded st 41 tadpole. Expression of the silver gene (blue color) is restricted to the RPE (brown color)[in the] lens.|
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