Generation of X. laevis Lines With Cell Type–Specific Expression of TRAP Transgenes
An optimal construct for carrying out TRAP studies in the X. laevis retina was determined to be a direct fusion of enhanced green fluorescent protein (EGFP) coding sequence, a linker sequence [2× SGGGG], and the full coding sequence of X. laevis L10a gene (see Experimental Procedures section). This EGFP-rpl10a cDNA was placed behind three upstream regulatory control sequences: 17.5 kb from the zebrafish islet2b gene (isl2b) (Pittman et al., 2008), 1.3 kb from the X. laevis rhodopsin gene (Zhang et al., 2008) (here referred to as rho), or 1.7 kb from the X. tropicalis fatty acid binding protein 7 gene (fabp7, also named basic lipid binding protein or blbp), to drive expression in X. laevis RGCs, rods, and Müller cells, respectively. The resulting transgenic F0 tadpoles were screened for EGFP expression using an epifluorescence stereomicroscope and grown to sexual maturity. These transgenic frogs were then mated to wildtype frogs. The resulting transgenic F1 progeny, as selected by EGFP fluorescence, were grown to the tadpole stage 57 (Nieuwkoop and Faber, 1994), and their retinas were examined for EGFP fluorescence together with antibody markers for RGCs, rods, and Müller cells. In retina sections from Tg(isl2b:EGFP-rpl10a) embryos, EGFP fluorescence was confined to the ganglion cell layer (GCL) with an occasional cell in the inner nuclear layer (INL), presumed to be a displaced RGC (arrowhead, Fig. 1; Tg(isl2b:EGFP-rpl10a)). Labeling with an antibody to γ-synuclein (sncg), a gene expressed in most or all RGCs of multiple mammals (Surgucheva et al., 2002; Mu et al., 2004; Soto et al., 2010) confirmed EGFP-Rpl10a to be expressed within RGC somata. Two of three Tg(isl2b:EGFP-rpl10a) lines showed nearly identical expression. A third line with very high expression of the transgene in RGCs also had low-level expression in the outer nuclear layer (ONL) and was discarded. In retinas from tadpoles expressing the EGFP-rpl10a transgene under the control of the rho upstream sequences, the EGFP-Rpl10a fusion protein localized in the outer nuclear layer (ONL), the location of photoreceptor cells (Fig. 1, Tg(rho:EGFP-rpl10a)). Labeling with a rhodopsin antibody showed that the transgene was expressed only in ONL cells that are rods and was not expressed in cone photoreceptors. Three different lines with the Tg(rho:EGFP-rpl10a) transgene showed rod-specific expression, though their expression differed in intensity. The expression of the EGFP-rpl10a transgene under regulatory control of the fabp7 upstream sequences was expected to occur exclusively in Müller cells. However, by comparison to immunostaining using a Müller cell–specific anti-Fabp7 antibody, the Tg(fabp7:EGFP-rpl10a) transgene expression was highest in Müller cells but also occurred in other retinal cells in all of six different Tg(fabp7:EGFP-rpl10a) frog lines tested. Thus, the Tg(fabp7:EGFP-rpl10a) frog lines were used only as a reference for the two other lines, Tg(isl2b:EGFP-rpl10a) and Tg(rho:EGFP-rpl10a), which did have cell type–specific expression.
Figure 1. X. laevis lines have specific expression of EGFP-Rpl10a in rods or RGCs. Representative images show GFP fluorescence of the EGFP-rpl10a transgene in retinal sections of stage 57 F1 progeny (green; left column) from lines expressing the EGFP-rpl10a cDNA under the control of zebrafish islet2b (isl2b), X. laevis rhodopsin (rho), or X. tropicalis fatty acid binding protein 7 (fabp7) upstream regulatory sequences. Immunostaining using cell type–specific antibodies (red; second column) specific for RGCs (Sncg), rods (Rho), and Müller cells (Fabp7) confirms cell type-specificity of the Tg(rho:EGFP-rpl10a) and Tg(isl2b:EGFP-rpl10a) lines but not Tg(fabp7:EGFP-rpl10a) line. DAPI shows the nuclear cell layers (blue; 3rd column) while a composite image of the GFP fluorescence, antibody, DAPI and brightfield identifies any overlap between endogenous GFP fluorescence and immunostaining (Merge + Phase; 4th column). Arrowheads point to a presumed displaced RGC. Arrows point to Müller cell processes. RPE, retinal pigment epithelium; ONL/INL, outer and inner nuclear layers; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 20 μm.
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To determine whether the transgenic animals expressed the expected protein products, Western blotting of whole eye lysates from F1 embryos expressing the EGFP-Rpl10a fusion protein and from F1 embryos expressing a cytoplasmic GFP transgene (GFP-cyto), both under regulatory control of the RGC-specific isl2b promoter, were analyzed. Antibodies against the GFP variants confirmed that the Tg(isl2b:GFP) and Tg(isl2b:EGFP-rpl10a) transgenes each produced only one major protein product of the expected size, 27 and 56 kD, respectively (Fig. 2A). To confirm the ribosomal localization of the transgenic EGFP-Rpl10a protein, confocal microscopy was used to determine the distribution of the EGFP-Rpl10a protein within rods and RGCs (Fig. 2B,C). In rods, the EGFP-Rpl10a protein localized primarily to the photoreceptor inner segment, the main site of protein translation, but also to nucleoli, site of ribosome assembly (arrowhead, Fig. 2B). Similarly, in RGCs the EGFP-Rpl10a protein localized to the cytoplasm, where most translation occurs, as well as to the nucleoli (Fig. 2C).
Figure 2. EGFP-Rpl10a protein localizes properly and can be used to selectively enrich for RNAs. A: Western analysis of whole eye lysates from transgenic tadpoles expressing cytoplasmic GFP (GFP-cyto) and EGFP-Rpl10a, both under control of the same islet2b regulatory sequence, and from wildtype control tadpoles (Wt) show transgene protein products of the expected size. B: Representative image shows a 2D projection of a series of optical sections (Z-stack) showing nucleolar and inner segment localization of the EGFP-Rpl10a protein in rods. C: Representative image shows a 2D projection of a Z-stack showing nucleolar and cytoplasmic localization of the transgenic EGFP-Rpl10a protein in RGCs. Arrowheads in B and C point to nucleoli. D: RNA recovery from unconjugated beads (−) or EGFP-coated beads (+) using lysates of Tg(isl2b:EGFP-Rpl10a) (L10a), Tg(isl2b:GFP) (GFP-cyto), and from non-transgenic wildtype (Wt) retinas, show significant RNA recovery only in Tg(isl2b:EGFP-Rpl10a) lysates in the presence of the EGFP antibody. Grey horizontal line shows the 0.5-ng limit of detection of the bioanalyzer. E: The residual unbound RNA fraction from the samples shown in D was comparable in all samples. Data in D and E represent the mean RNA levels from 12 retinas averaged for three replicates, and statistical significance between individual samples was established by pair-wise ANOVA comparisons (***P < 0.001). F: Representative superimposed electropherogram traces from TRAP EGFP-rpl10a transgenic samples affinity purified with EGFP conjugated beads (red trace) and unconjugated beads (black trace) show high abundance 18S and 28S ribosomal RNAs and lower abundance mRNAs only in EGFP-rpl10a samples extracted using EGFP- conjugated beads. Right insets show corresponding RNA gels for each of the traces. Scale bars = 2 μm.
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To determine whether the tagged ribosomes could be used to selectively enrich for translating mRNAs, retina extracts from tadpoles expressing the EGFP-rpl10a or GFP cDNAs under the control of the isl2b upstream sequences and from wildtype control embryos were subjected to affinity purification using magnetic beads coated with anti-EGFP antibodies. Following the affinity purification, RNA was purified from both the bead-bound IP fraction and the unbound total fraction (Fig. 2D). Control unconjugated beads failed to pull down any significant amount of RNA. Amongst the EGFP-conjugated bead-bound fractions, only retinas from EGFP-rpl10a embryos yielded significant amounts of mRNA (one-way ANOVA; P < 0.001). As expected, the RNA extracted from the residual unbound total fractions were variable and exceeded the immunoprecipitated amounts, but showed no significant difference between the groups (one way-ANOVA; P > 0.5) (Fig. 2E). Confirming a selective pull-down of mRNAs associated with transgenic ribosomes, sharp ribosomal 28S and 18S RNA peaks and lower molecular weight mRNAs were only detected in the case where EGFP-conjugated beads were used on lysates from Tg(isl2b:EGFP-rpl10a) retinas (Fig. 2F). No TRAPed RNAs were detectable in the control samples (Fig. 2F).
TRAP mRNAs Accurately Represent Expression Profiles of Individual Cell Types
Four genes known or presumed to be expressed at low, middle, or high levels were selected to represent each of three cell types (RGCs, rods, and Müller cells): sncg, rbpms (a.k.a. hermes), brn3d (a.k.a. pou4f1.2), and barhl2 (a.k.a. xbh1) for RGCs (Gerber et al., 1999; Patterson et al., 2000; Hutcheson and Vetter, 2001; Agathocleous et al., 2009), rho, gnat1, pde6a, and crx for rods (Batni et al., 1996; Knox et al., 1998; Calvert et al., 2000; Viczian et al., 2003; Muradov et al., 2010), and rlbp1, vim, fabp7, and notch1 for Müller cells (Dent et al., 1989; Coffman et al., 1990; Dorsky et al., 1995). Most of these genes are expected to be expressed in only one cell type, whereas others such as barhl2 and crx are highest in one cell type but also expressed in other cell types (Patterson et al., 2000; Viczian et al., 2003; see Supp. Fig. S3, which is available online). In addition, a set of three genes (gapdh, rpl6, rpl10a) presumed to have broad expression in all retinal cell types and typically used as reference genes (Kusner et al., 2004; Chen et al., 2008; Roesch et al., 2008) were also selected. To confirm the expression levels and the cell type-specificity of the genes, all 15 genes were analyzed by in situ hybridization (ISH) using retina sections from wildtype stage 57 tadpoles. Representative images of the mRNA expression pattern detected by ISH for three genes (rbpms, gnat1, and rlpb1) exemplify the cell type specificity in RGCs, rods, and Müller cells, respectively (Fig. 3). All 12 genes chosen for their cell type–specific expression were confirmed as specific or highly enriched in the expected cell type by ISH or immunostaining (Figs. 1 and 3; Supp. Fig. S3). Surprisingly, the genes presumed to be ubiquitous were broadly expressed but revealed differences in expression levels between different cell types. For instance, gapdh was expressed more highly in a subset of cells in the INL and GCL, presumed to be horizontal cells and a subset of RGCs, respectively, based on cell size and morphology, and was not observed in pigment epithelial cells (Fig. 3). The two other genes presumed to be ubiquitous (rpl6 and rpl10a) were expressed at comparable levels in differentiated retinal cells, but were expressed at higher levels in the undifferentiated cells of the ciliary marginal zone (Supp. Fig. S4).
Figure 3. In situ hybridization shows expression patterns of a subset of the genes used to assess cell-type enrichment by TRAP. Representative images of retina sections from wildtype stage 57 tadpoles show mRNA expression patterns for genes selectively expressed in RGCs, rods, and Müller cells (rbpms, gnat1, rlpb1, respectively) and a gene expressed widely in multiple cell types (gapdh). A composite image of the in situs (shown in top row), DAPI and brightfield is used to identify cell layers (Merge DAPI + Phase; 2nd row). RPE, retinal pigment epithelium; ONL/INL, outer and inner nuclear layers; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 20 μm.
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To determine whether the TRAP method provided effective enrichment of mRNAs from the targeted cell types, qPCR was used to quantify the expression of the described 15 genes in the mRNA eluted from the beads (TRAPed mRNA) obtained from the retinas of tadpoles expressing EGFP-rpl10a under control of the three regulatory sequences. Additionally, EGFP primers were used to assess transgene expression levels in the various transgenic lines. Changes in gene expression are typically assessed by measuring the genes of interest relative to a ubiquitously expressed control gene such as gapdh (relative qRT-PCR methods) (Pfaffl, 2001). However, since ISH revealed differences in expression between different cell types of all tested control genes, absolute qPCR methods were used to measure mRNA copy number based on standard curves generated for each of the 16 genes of interest (Supp. Fig. S2).
To determine whether the TRAP method could enrich for mRNAs from different cell types, the TRAPed mRNAs in each of the three transgenic genotypes (Tg(isl2b:EGFP-rpl10a), Tg(rho:EGFP-rpl10a), and Tg(fabp7:EGFP-rpl10a)) were compared to mRNA harvested from unbound fraction obtained from whole eye lysates (no transgene), and are here presented in four graphs according to whether the genes were expected to be enriched in RGCs (Fig. 4A), rods (Fig. 4B), Müller cells (Fig. 4C), or were expected to be ubiquitous (Fig. 4D). These data represent averages from a minimum of three independent biological replicate experiments. For the chosen RGC-specific genes (Fig. 4A), there were highly significant differences among genotypes (P < 0.001). In pair–wise comparisons between individual genotypes, Tg(isl2b:EGFP-rpl10a) and Tg(rho:EGFP-rpl10a) were significantly different for all four RGC genes (Tukey test, P < 0.005). The degree of enrichment of genes between the Tg(isl2b:EGFP-rpl10a) and Tg(rho:EGFP-rpl10a) lines varied, with levels of RGC-specific genes expressed at high (sncg), moderate (brn3d; rbpms), and low (barhl2) levels showing enrichments of 350-, 500-, 87-, and 87-fold, respectively (Fig. 4A). There was also a significant enrichment of these genes when comparing TRAPed mRNA from the Tg(isl2b:EGFP-rpl10a) lines to the two control samples expected to contain mRNA from all retina cell types, the TRAPed mRNAs from the Tg(fabp7:EGFP-rpl10a) lines and the unbound mRNA from non transgenic (wildtype) whole eyes (Fig. 4A; Tukey test, P < 0.05). The average enrichment in the Tg(isl2b:EGFP-rpl10a) samples for brn3d, sncg, and rbpms was 73-, 15-, and 3-fold relative to the Tg(fabp7:EGFP-rpl10a) samples, and 58-, 10-, and 4-fold relative to whole eye mRNA, respectively (Tukey test, P < 0.05). Barhl2, a gene with inherently low expression, showed the least enrichment between the Tg(isl2b:EGFP-rpl10a) and both control genes, 3- and 2-fold over Tg(fabp7:EGFP-rpl10a) TRAPed and whole eye mRNAs, respectively (Fig. 4A).
Figure 4. Lines expressing EGFP-rpl10a in rods and RGCs enable efficient enrichment of cell type–specific mRNAs. Graphs show absolute expression levels of mRNA for affinity-purified TRAPed mRNAs from transgenic lines (blue, Tg(rho:EGFP-Rpl10a); red, Tg(isl2b:EGFP-Rpl10a); black, Tg(fabp7:EGFP-Rpl10a)) and from the unbound fraction of wildtype control eyes (grey, Wildtype). To determine the gene expression copy number, a standard curve was generated for each of the genes tested by qPCR (Supp. Fig. S2). Due to large differences in absolute gene expression values, genes expressed at low levels require a different y-axis scale (see inset bar graphs for barhl2 [A], pde6a and crx [B], and notch1 [C]). Asterisks reflect the level of significance for individual pair-wise comparisons between the marked genotype and either one other genotype (bracket) or all other genotypes (Tukey test; **P < 0.005; ***P < 0.001).
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For genes selected for their expression in rods (Fig. 4B), overall, there were significant differences among genotypes (P < 0.005). As expected, enrichment was largest in comparing the Tg(rho:EGFP-rpl10a) lines relative to the Tg(isl2b:EGFP-rpl10a) lines. In pair-wise comparisons among genotypes of genes expressed at high (rho) or moderate (gnat1) levels, there was 100- and 95-fold enrichment, respectively (Tukey test, P < 0.05). For genes expressed at low levels (pde6a and crx), the magnitude of the enrichment was less, 48- and 41-fold (Fig. 4B). While enrichment was also observed in rod-specific genes when comparing Tg(rho:EGFP-rpl10a) TRAPed mRNA to Tg(fabp7:EGFP-rpl10a) TRAPed mRNA and unbound whole retina mRNA, the magnitude of this enrichment was considerably less and did not reach statistical significance.
Overall, significant differences among genotypes were also seen in Müller cell–specific genes (P < 0.005) (Fig. 4C). Pair-wise comparison of the Müller cell–specific gene expressed at highest levels (rlbp1) was enriched in the Tg(fabp7:EGFP-rpl10a) TRAPed mRNAs relative to that of unbound whole eye mRNA, confirming that the Tg(fabp7:EGFP-rpl10a) transgene is expressed at higher levels in Müller cells relative to other retinal cells (Tukey test, P < 0.05). With the additional exception of fabp7 expression in RGCs (pair-wise comparison of Tg(fabp7:EGFP-rpl10a) and Tg(isl2b:EGFP-rpl10a); Tukey test, P < 0.05), Müller cell genes were selectively underrepresented in the TRAPed mRNAs from rho- and isl2b-driven EGFP-rpl10a lines (Fig. 4C). As expected, control genes were found in both the Tg(rho:EGFP-rpl10a) and Tg(isl2b:EGFP-rpl10a) TRAPed mRNAs at comparable levels (Fig. 4D; P > 0.78). Since among the mRNAs translated in the transgenic animals were the EGFP-rpl10a transgenes themselves, their level of expression was also assessed. While a trend indicating higher EGFP-rpl10a mRNA expression levels in rod-specific lines was observed, the level of expression was generally comparable in the different EGFP-rpl10a lines (Fig. 4D).
As a means of assessing the efficiency of TRAP in the X. laevis retina, two independent metrics previously used to asses TRAP efficiency in mice (Dougherty et al., 2010) were calculated. First, the ratio of affinity-purified TRAPed RNAs (IP) relative to the unbound fraction of RNA present in the whole eyes from wildtype samples (Totalwt) was determined for each genotype (Fig. 5A,B). Plots showing the IP/Totalwt ratio confirmed that both the Tg(isl2b:EGFP-rpl10a) (Fig. 5A) and Tg(rho:EGFP-rpl10a) (Fig. 5B) lines show cell type specificity in RGC genes and rod genes, respectively. Second, a specificity index (SI), which rank-orders IP ratios between genotypes, was also calculated. In this analysis, 50 sets of permuted non-rank ordered SI data were compared to the normalized and rank-ordered data (according to http://www.bactrap.org/downloads/Specificity.r). The distributions of these data were then compared to determine the probability that a particular gene was present in the population above chance. As evidenced by the clustering of SIs in RGC and rod genes (Fig. 5C), this second statistical measure also identified these genes as being cell type–specific. In addition, the Müller cell gene rlbp1 was also shown to be enriched in the Tg(fabp7:EGFP-rpl10a) TRAPed mRNA (Fig. 5C).
Figure 5. The IP/Total ratio and Specificity Index (SI) provide independent measures to identify genes enriched above background levels in different cell types. A,B: Data for each of three biological replicates are plotted as the ratio of the absolute expression level for the IP for Tg(isl2b:EGFP-Rpl10a) (A) and Tg(rho:EGFP-Rpl10a) (B) TRAPed mRNAs over the absolute expression level for RNAs purified from total unbound fraction from a reference wildtype sample (Total) for each gene. Standard error of the mean (error bars) was calculated based on the standard deviation for a ratio as described in the Experimental Procedures section. C: Scatter plot of the SI for all cell types plotted against individual genes shows a clustering of cell type–specific enrichment for RGCs (red highlighted cluster) and rod phototoreceptor cells (blue highlighted cluster). Genotypes on graph are as indicated isl2b = Tg(isl2b:EGFP-rpl10a); rho = Tg(rho:EGFP-rpl10a); fabp7 = Tg(fabp7:EGFP-rpl10a). P values are significant at an SI less than 4 (dashed line, P < 0.1).
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To determine the line-to-line reproducibility of the TRAP method in the X. laevis retina, two additional lines expressing the Tg(rho:EGFP-rpl10a) transgene and one additional line expressing each of the Tg(isl2b:EGFP-rpl10a) and Tg(fabp7:EGFP-rpl10a) transgenes were also examined by qPCR. These data, comprising multiple replicates of individual lines and multiple lines of the same genotype, were examined using a principal components analysis (PCA). In plotting the compound data (Fig. 6A), the first principal component plotted along the X-axis (PC1) showed that the highest variation through the data was largely attributable to genotype. On one end of PC1, all Tg(isl2b:EGFP-rpl10a) samples, including data from two different transgenic lines, formed a distinct cluster (Fig. 6A; red inverted triangles), while all the Tg(rho:EGFP-rpl10a) samples, generated from three different transgenic lines, formed a second cluster at the opposite end of the axis (Fig. 6A; blue triangles). Both the whole eye mRNAs (Fig. 6A; grey diamonds) and the Tg(fabp7:EGFP-rpl10a) TRAPed mRNAs (Fig. 6A; black squares) were virtually contiguous and showed little variation along the PC1 axis. The second principal component plotted along the Y-axis (PC2) showed significant variation among different biological replicates, including within the Tg(isl2b:EGFP-rpl10a) and Tg(rho:EGFP-rpl10a) lines. A scree plot showed that the third component contributed significantly to the variance (Supp. Fig. S5). Analysis of the PCA plot along the second and third dimensions (Supp. Fig. S5) also showed separation of the two cell type–specific groups (RGCs and rods) and an overlap between the third group (Tg(fabp7;EGFP-rpl10a)) and the wildtype samples.
Figure 6. TRAP provides cell-type enrichment for genes of different absolute expression levels and reproducibility across different transgenic lines in the X. laevis retina. A: PCA of individual samples showing qPCR-based expression levels from TRAPed replicates from the same GFP-rpl10a frog lines as well as from different lines. B: PCA of the contribution of each of the 16 analyzed genes on the overall variance. The placement and length of the vectors show the contribution to the variance for each gene. The gene expression data have been standardized to a value of 1, demarked by the tan circle (see Experimental Procedures section).
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To assess the relative contribution of each gene to the variability in the data, individual gene data for all samples were analyzed in a second PCA plot, where vector length approximates the standard deviation and angles between the vectors approximate the degree of correlation between the genes (Greenacre, 2010). This analysis showed cell type–specific genes to be tightly clustered along PC1 with more variability along PC2, and that genes expressed at both high and low levels contributed nearly equally to the separation of different genotypes along PC1. Overall, PCA analysis demonstrated that TRAP in the X. laevis retina shows low variability both within individual replicates for a given line and between different lines.