Pan Y , Martinez-De Luna RI , Lou CH , Nekkalapudi S , Kelly LE , Sater AK , El-Hodiri HM .

EY015480 NEI NIH HHS , R01 EY015480-05 NEI NIH HHS , R01 EY015480 NEI NIH HHS

	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.

Andreazzoli, Role of Xrx1 in Xenopus eye and anterior brain development. 1999, Pubmed, Xenbase

Andreazzoli, Role of Xrx1 in Xenopus eye and anterior brain development. 1999, Pubmed , Xenbase
Bailey, Regulation of vertebrate eye development by Rx genes. 2004, Pubmed , Xenbase
Batni, Characterization of the Xenopus rhodopsin gene. 1996, Pubmed , Xenbase
Boatright, A major cis activator of the IRBP gene contains CRX-binding and Ret-1/PCE-I elements. 1997, Pubmed
Boyer, Core transcriptional regulatory circuitry in human embryonic stem cells. 2005, Pubmed
Casarosa, Xrx1, a novel Xenopus homeobox gene expressed during eye and pineal gland development. 1997, Pubmed , Xenbase
Casarosa, Xrx1 controls proliferation and multipotency of retinal progenitors. 2003, Pubmed , Xenbase
Chen, The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation. 2002, Pubmed
Chen, Co-expression of Argonaute2 Enhances Short Hairpin RNA-induced RNA Interference in Xenopus CNS Neurons In Vivo. 2009, Pubmed , Xenbase
Chen, Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specific genes. 1997, Pubmed
Cheng, Photoreceptor-specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. 2004, Pubmed
Chuang, Zebrafish genes rx1 and rx2 help define the region of forebrain that gives rise to retina. 2001, Pubmed
Chuang, Expression of three Rx homeobox genes in embryonic and adult zebrafish. 1999, Pubmed
Danno, Molecular links among the causative genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression. 2008, Pubmed , Xenbase
Decembrini, Dicer inactivation causes heterochronic retinogenesis in Xenopus laevis. 2008, Pubmed , Xenbase
Deschet, Expression of the medaka (Oryzias latipes) Ol-Rx3 paired-like gene in two diencephalic derivatives, the eye and the hypothalamus. 1999, Pubmed , Xenbase
Diederichs, Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. 2008, Pubmed
Dorval, CHX10 targets a subset of photoreceptor genes. 2006, Pubmed
El-Hodiri, xnf7 functions in dorsal-ventral patterning of the Xenopus embryo. 1997, Pubmed , Xenbase
Faehnle, Argonautes confront new small RNAs. 2007, Pubmed
Furukawa, rax, a novel paired-type homeobox gene, shows expression in the anterior neural fold and developing retina. 1997, Pubmed
Furukawa, Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation. 1997, Pubmed
Furukawa, rax, Hes1, and notch1 promote the formation of Müller glia by postnatal retinal progenitor cells. 2000, Pubmed
Ghai, Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina. 2009, Pubmed
Huang, Metamorphosis is inhibited in transgenic Xenopus laevis tadpoles that overexpress type III deiodinase. 1999, Pubmed , Xenbase
Hutvagner, Argonaute proteins: key players in RNA silencing. 2008, Pubmed
Höck, The Argonaute protein family. 2008, Pubmed
Ishibashi, Distinct roles of maf genes during Xenopus lens development. 2001, Pubmed , Xenbase
Kelly, Pbx1 and Meis1 regulate activity of the Xenopus laevis Zic3 promoter through a highly conserved region. 2006, Pubmed , Xenbase
Kikuchi, The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site. 1993, Pubmed
Kimura, Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor-specific gene expression. 2000, Pubmed
Lerner, Sp4 is expressed in retinal neurons, activates transcription of photoreceptor-specific genes, and synergizes with Crx. 2005, Pubmed
Li, Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis. 2006, Pubmed , Xenbase
Liu, Expression of the Xvax2 gene demarcates presumptive ventral telencephalon and specific visual structures in Xenopus laevis. 2001, Pubmed , Xenbase
Loosli, Loss of eyes in zebrafish caused by mutation of chokh/rx3. 2003, Pubmed
Loosli, Medaka eyeless is the key factor linking retinal determination and eye growth. 2001, Pubmed
Ma, Retina-specific cis-elements and binding nuclear proteins of carp rhodopsin gene. 2001, Pubmed
Mani, Xenopus rhodopsin promoter. Identification of immediate upstream sequences necessary for high level, rod-specific transcription. 2001, Pubmed , Xenbase
Martinez, Erx, a novel retina-specific homeodomain transcription factor, can interact with Ret 1/PCEI sites. 1998, Pubmed
Mathers, Regulation of eye formation by the Rx and pax6 homeobox genes. 2000, Pubmed
Mathers, The Rx homeobox gene is essential for vertebrate eye development. 1997, Pubmed , Xenbase
Mears, Nrl is required for rod photoreceptor development. 2001, Pubmed
Mitton, The leucine zipper of NRL interacts with the CRX homeodomain. A possible mechanism of transcriptional synergy in rhodopsin regulation. 2000, Pubmed
Moritz, Xenopus laevis red cone opsin and Prph2 promoters allow transgene expression in amphibian cones, or both rods and cones. 2002, Pubmed , Xenbase
Moriya, Preference for background color of the Xenopus laevis tadpole. 1996, Pubmed , Xenbase
Nakazawa, Arrestin gene mutations in autosomal recessive retinitis pigmentosa. 1998, Pubmed
Nelson, The developmental sequence of gene expression within the rod photoreceptor lineage in embryonic zebrafish. 2008, Pubmed
Nelson, Retinal homeobox 1 is required for retinal neurogenesis and photoreceptor differentiation in embryonic zebrafish. 2009, Pubmed
Ohuchi, Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development. 1999, Pubmed , Xenbase
Otteson, Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-1 and G-rich repressor elements. 2005, Pubmed
Otteson, Kruppel-like factor 15, a zinc-finger transcriptional regulator, represses the rhodopsin and interphotoreceptor retinoid-binding protein promoters. 2004, Pubmed
Paddison, RNA interference: the new somatic cell genetics? 2002, Pubmed
Pan, The Rx-like homeobox gene (Rx-L) is necessary for normal photoreceptor development. 2006, Pubmed , Xenbase
Perron, The genetic sequence of retinal development in the ciliary margin of the Xenopus eye. 1998, Pubmed , Xenbase
Seufert, Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain development. 2005, Pubmed , Xenbase
Sparrow, A simplified method of generating transgenic Xenopus. 2000, Pubmed , Xenbase
Swaroop, A conserved retina-specific gene encodes a basic motif/leucine zipper domain. 1992, Pubmed
Turner, Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. 1994, Pubmed , Xenbase
Valverde, Analysis of the IRBP gene as a cause of RP in 45 ARRP Spanish families. Autosomal recessive retinitis pigmentosa. Interstitial retinol binding protein. Spanish Multicentric and Multidisciplinary Group for Research into Retinitis Pigmentosa. 1998, Pubmed
Viczian, XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina. 2003, Pubmed , Xenbase
Voronina, Mutations in the human RAX homeobox gene in a patient with anophthalmia and sclerocornea. 2004, Pubmed
Wang, QRX, a novel homeobox gene, modulates photoreceptor gene expression. 2004, Pubmed
Wells, The identification of E2F1-specific target genes. 2002, Pubmed
Whitaker, Conserved transcriptional activators of the Xenopus rhodopsin gene. 2004, Pubmed , Xenbase
Wilson, The nature of dominant mutations of rhodopsin and implications for gene therapy. 2003, Pubmed
Wu, The role of Xenopus Rx-L in photoreceptor cell determination. 2009, Pubmed , Xenbase
Zhang, Targeted expression of the dominant-negative FGFR4a in the eye using Xrx1A regulatory sequences interferes with normal retinal development. 2003, Pubmed , Xenbase
den Hollander, A homozygous missense mutation in the IRBP gene (RBP3) associated with autosomal recessive retinitis pigmentosa. 2009, Pubmed , Xenbase