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Metabolism and development are two important processes not often examined in the same context. The focus of the present study is the expression of specific peroxisomal genes, the subsequent biogenesis of peroxisomes, and their potential role in the metabolism associated with the development of Xenopus laevis embryos. The temporal and expression patterns of six peroxisomal genes (PEX5, ACO, PEX19, PMP70, PEX16, and catalase) were elucidated using RT-PCR. Functionally related peroxisomal genes exhibited similar expression patterns with their RNA levels elevated relatively late during embryogenesis. Using immunohistochemistry PMP70 and catalase protein was localized largely to dorsal-anterior structures. Peroxisomal function was assayed with peroxisomal targeted-GFP, which when microinjected, revealed peroxisomes in dorsal-anterior structures at stage 45. A requirement for peroxisomal function appears to be present only late in development as organogenesis is finishing, yolk stores are depleted, and ingestion commences.
Figure 1. Expression analysis of peroxisomal gene RNA levels during developmental stages of X. laevis. The results were obtained by quantifying band intensities of RT-PCR products for peroxisomal RNA as compared to EF1α on a 0.8% agarose gel. PEX5 expression is at its highest expression is a peak at stage 25, while other stages are low in relative expression (A). The expression of ACO exhibits a similar pattern to PEX5, however its early peak occurs at stage 20, and there is a steady increase at after the peak from stage 25 to 45 (B). PEX19 expression is highest as stage 45, however, stage 12 shows the second highest expression (C). PMP70 follows the same expression as PEX19 (D). The expression of PEX16 remains relatively low compared to its peak at Stage 45 (E). The expression of catalase is essentially the same as PEX16, having relatively low expression until stage 45 (F). Error bars are representative of three replications.Download figure to PowerPoint
Figure 2. Cross reactivity of mammalian peroxisomal antibodies with X. laevis protein. Western blot analysis for cross reactivity between X. laevis protein and antibodies for PMP70, Catalase. Each lane represents a independent protein sample. GAPDH was used as a control (A). Immunohistochemistry on X. laevis A6 cells using primary antibodies for PMP70 (B), catalase (C), and no primary antibody control (D). DAPI (blue), peroxisomal antibodies (red). Scale bar = 10μm.Download figure to PowerPoint
Figure 3. PMP70 [an antibody to abcd3] immunohistochemistry on sectioned X. laevis embryos. The stages present include 30 (A,C,E) and 45 (B,D,F). Tissue samples are of ectodermal and sub-ectodermal cells in the posteriorhead (A–B), ectodermal and sub-ectodermal cells in the anteriorhead (C–D), ventral ectodermal cells (E–F). DAPI (blue), actin filaments (green), peroxisomal signal (red). Scale bar = 10 μm.Download figure to PowerPoint
Figure 4. Catalase [antibody to cat.2] immunohistochemistry on sectioned X. laevis embryos. The stages present include 30 (A,C,E) and 45 (B,D,F). Tissue samples are of ectodermal and sub-ectodermal cells in the posteriorhead (A–B), ectodermal and sub-ectodermal cells in the anteriorhead (C–D), ventral ectodermal cells (E–F). DAPI (blue), actin filaments (green), peroxisomal signal (red). Scale bar = 10 μm.Download figure to PowerPoint
Figure 5. Microinjections of tagged and control GFP. Sections of X. laevis embryos microinjected with either GFP-KANL (A,C,E) or control GFP (B,D,F). Tissue samples are of sub-ectodermal cells in the posteriorhead (A–B),k sub-ectodermal cells in the anteriorhead (C–D), and ventral ectodermal cells (E–F). DAPI (blue), actin filaments (red), GFP (green). Scale bar = 10 μm.Download figure to PowerPoint
Figure 6. Stage 30 and 45 X. laevis embryos depicting location of histological sections used in Figures 3, 4 and 5. A lateral view of stage 30 (i) and stage 45 (ii) embryos demonstrating the approximate location and plane of histological section. Areas are cells of the ectoderm and sub-ectoderm in the posteriorhead (A), cells of the ectoderm and sub-ectoderm in the anteriorhead (B), and cells of the ventralectoderm (C). The approximate size of the embryos are approximately 5mm and 10mm for stage 30 and 45, respectively.Download figure to PowerPoint
