Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

Abstract

Purpose: The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site.

Methods: X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos.

Results: We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities.

Conclusions: vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system.

Figure 1

Identification of Xenopus laevis Vsx1 and Vsx2. A: Predicted protein sequences of X. laevis Vsx1 and Vsx2. Sequences were aligned using ClustalW. The black and gray backgrounds indicate amino acid identity and similarity, respectively. The highly conserved octapeptide motif (OP; box), homeodomain (dashed line), and CVC domain (double underline) are indicated. The orthopedia-aristaless-Rx (OAR; Vsx2) and Rinx-Vsx1 (RV; Vsx1) domains are also indicated. The triple underline indicates the region of Vsx2 lacking from the short form. B: Phylogenetic representation of ClustalW alignment of the Vsx1 and Vsx2 amino acid sequences from X. laevis, zebrafish, chicken, and mouse, Caenorhabditis elegans Ceh-10, and X. laevis Rx-L.

Figure 2

vsx2 undergoes alternative splicing of exon 5. A: A graphic depiction of the long (vsx2-l) and short (vsx2-s) forms of vsx2 showing alternative 3′ splice site use in intron 4. The locations and sequences of the splice sites (arrow; red text) are indicated by their position relative to the exons (gray boxes) and intron (black lines). B: The splice site matrix scores of the splicing signals found in intron 4 according to the Neural Network (NN) and Alternative Splice Site Predictor (ASSP) bioinformatics programs. Both programs recognize vsx2-l as a strong 3′ splice site (high homology to consensus splicing signals) of intron 4 and vsx2-s as a weaker 3′ acceptor splice site (intermediate homology to splicing signals) for intron 4. ss, splice site.

Figure 3

vsx1 and vsx2 are expressed in the INL of the maturing Xenopus laevis retina. A–H: Expression of vsx1 (A–D) and vsx2 (E–H) at st 38, 41, 45, and 50 (as indicated). Brackets indicate the ciliary marginal zone (CMZ). I–L: Overlapping expression of vsx1 and vsx2 decreases as the retina matures. Double in situ hybridization was performed on sections prepared from paraffin-embedded tadpoles using differently labeled probes for vsx1 (red) and vsx2 (blue). I, inner nuclear layer; G, ganglion cell layer; L, lens. Scale bars = 1 µM. Scale bar in A applies to B, C, E, F, and G; scale bar in D applies to H.

Figure 4

DNA binding activity of Vsx1 and 2 and transcriptional transactivation activity of Vsx2 isoforms. A: Vsx1 and Vsx2 can bind the photoreceptor conserved element-1 (PCE-1) site in vitro. Electrophoretic mobility shift assay (EMSA) performed using a radiolabeled PCE-1 probe, in vitro translated Vsx1 or Vsx2 proteins, and unlabeled wild-type (W) or mutant (M) PCE-1, BAT (B), or Ret4 (R) oligonucleotide competitors. Arrows indicate specific DNA–protein complexes. B: Vsx2_L, but not Vsx2_S, inhibits activation of XOP-Luc by otx5b and XLmaf. The luciferase assay was performed using lysates from embryos coinjected with the XOP-Luc reporter plasmid and otx5b + XLmaf and/or Vsx2_L or Vsx2_S RNAs as shown. Values are presented as fold mean normalized luciferase activity (relative to the reporter only, no effector control); error bars denote standard deviation from the mean. P, protein; C, competitor. * p<0.03; ** p<0.01.