XB-ART-35563Dev Dyn July 1, 2007; 236 (7): 1970-9.
Transformation of undifferentiated progenitors into specific cell types is largely dependent on temporal and spatial expression of a complex network of transcription factors. Here, we examined whether neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3 transcription factors contribute to cell fate determination. We cloned the Xenopus Nr2e3 gene and showed that its temporal and spatial expression is similar to its mammalian ortholog. We tested its in vivo function by misexpressing these transcription factors in Xenopus eye primordia, demonstrating that either human Nr2e3 or Nrl directed photoreceptor precursors to become rods at the expense of cones. Furthermore, overexpression of Xenopus Nrl dramatically increased the number of lens fibers, whereas human Nrl did not, suggesting evolutionary divergence of function of the Nrl gene family. Misexpression of Nrl and Nr2e3 together were more effective than either transcription factor alone in directing precursors to the rod fate.
PubMed ID: 17377979
Article link: Dev Dyn
Species referenced: Xenopus
Genes referenced: calb1 crx nr2e1 nr2e3 nrl otx2 rho rpe
Antibodies: Calb1 Ab3 Rho Ab1
Article Images: [+] show captions
|Figure 1. Xenopus Nr2e3 gene is highly conserved with other vertebrates. A: Comparison of human Nr2e3 with Xenopus Nr2e3. The sequence identities of DNA binding domains (DBD) and ligand binding domains (LBD) were revealed by using DNAStar (Stratagene). Numbers in the bars show the percentage of amino acid (AA) sequence identity to hNr2e3. The numbers above the bar indicate the boundaries of different regions based on AA residue position. Xenopus AA residue positions are relative due to the fact that exon 1 is missing. The dashed line represents the unidentified sequence. B: Phylogenetic relationship of Nr2e3 and Nr2e1 family members. Maximum parsimony algorithm with bootstraping was used to determine relationships. Species abbreviations are as follows: hs, Homo sapiens; mm, Mus musculus; xl, Xenopus laevis; gg, Gallus gallus. C: Reverse transcriptase-polymerase chain reaction (RT-PCR) of adult Xenopus heads (stages 20, 24, 36), eyes (stage 42). and retina (adult) RNA using xOtx2, xOtx5b, xL-Nrl, xNr2e3, xRho, and H4 (loading control) primers. D: Timing of Xenopus retinogenesis. E: In situ hybridizations of Xenopus laevis retina at different stages with xNr2e3 probe. Staining can be seen in the outer nuclear layer of the photoreceptors only when antisense probe is used. The negative control (using sense probe) does not produce any staining. Scale bar is 50 mu m. F,G: Embryos at stage 17-18 were lipofected in eye anlagen with either CMV-empty vector (F) or CMV-hNr2e3 together with CMV-GFP (G) and immunostained with cone antibody (anti-calbindin) and DAPI. Cones appear red (untransfected) or yellow (transfected), while rods always appear green (F,G lower panels). The fates of lipofected cells were determined and quantified as the average percentage of each cell type. H: Student's t-test was used to determine significance. Asterisks denote P < 0.05. GC, ganglion; Am&BP, amacrine and bipolar, H, horizontal; R, rod; C, cone; Mu, Müller glia; RPE, retina pigment epithelia; OS, outer segment; ONL, outer nuclear layer; INL inner nuclear layer; IPL inner plexiform layer; GCL, ganglion cell layer.|
|Figure 2. A,B: Cells overexpressing hNrl or xL-Nrl differentiate into rods. Representative retinas lipofected with xL-Nrl (A) and hNrl (B) immunostained with anti-calbindin (cones) and 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI). C: The fates of lipofected cells were determined and quantified as the average percentage of each cell type. Analysis of variance (analysis of variance [ANOVA]), single-factor, was used to determine significance. Asterisks denote P <0.05. GC, ganglion; Am&BP, amacrine and bipolar, H, horizontal; R, rod; C, cone; Mu, Müller glia. D,E: Many of the xL-Nrl lipofected tadpoles exhibited increased numbers of GFP-positive lens fiber cells compared with lipofections with empty vector or hNrl. Quantitative analysis (ANOVA, single-factor) revealed that the increase of GFP-positive lens fiber cells in xL-Nrl lipofected retinas is statistically significant (P <0.01). G,H: While anti-rhodopsin antibody stained exclusively rods in empty vector lipofected animals (G), many of the xL-Nrl lipofected retinas exhibited rhodopsin expression in nonphotoreceptor cells (H, arrows).Download figure to PowerPoint|
|Figure 3. Overexpressing multiple transcription factors within the same cell. A–D: Representative retinas lipofected with Otx5b (A), Otx5b+xL-Nrl (B), hNrl+hNr2e3 (C), Otx5b+xL-Nrl+hNr2e3 (D) immunostained with anti-calbindin (cones) and 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI). E: The fates of lipofected cells were determined and quantified as the average percentage of each cell type. F: Summary of Tukey post hoc multiple comparison test. Statistically significant differences (P <0.05) in cell numbers are shown in green (increase) or red (decrease). White blocks appear when there is no statistical difference.Download figure to PowerPoint|