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Dev Biol
2006 Feb 23;10751:20-5. doi: 10.1016/j.brainres.2005.12.105.
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Prion protein mRNA expression in Xenopus laevis: no induction during melanotrope cell activation.
van Rosmalen JW
,
Born JM
,
Martens GJ
.
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In mammals, the prion protein (PrP) is expressed in most tissues, but predominantly in neuronal tissues. Here, we investigated the temporal and spatial mRNA expression of PrP in the non-mammalian South African claw-toed frog Xenopus laevis. PrP transcripts were maternally expressed and detected throughout embryonic development, most strongly from neurulation onwards and including the tadpole stage. Microinjection of PrP mRNA into fertilized Xenopus eggs did not affect early embryonic development. In adult frogs, PrP mRNA expression was observed in all tissues examined, with high expression in brain, pituitary and testis. In Xenopus, the intermediate pituitarymelanotrope cells are involved in background adaptation of the animal and produce high levels of the prohormone proopiomelanocortin (POMC) when the melanotrope cells are active (i.e. when the animal is black-adapted). Remarkably and in contrast to most secretory pathway components, PrP was not upregulated in the melanotropes of black-adapted animals, arguing against a direct role of this protein in POMC biosynthesis.
Fig. 1.
Temporal expression pattern of PrP during Xenopus development. Total RNA was extracted from embryos at the indicated developmental stages and used for RT-PCR analysis (staging according to Nieuwkoop and Faber (1967)). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for the amount of input RNA. The generated PCR products were 225- and 230-bp in size for PrP and GAPDH, respectively. Negative controls without reverse transcriptase (not shown) or without cDNA for each primer pair used (H2O) showed no contamination of genomic DNA for all developmental stages.
Fig. 2.
PrP mRNA expression in various tissues of adult Xenopus. Total RNA was extracted from the indicated tissues including the neurointermediate lobe (NIL) and the anterior lobe (AL) of the pituitary of black-adapted (B) and white-adapted (W) animals and used for RT-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for the amount of input RNA. The generated PCR products were 225- and 230-bp in size for PrP and GAPDH, respectively. Negative controls without reverse transcriptase (not shown) or without cDNA for each primer pair used (H2O) showed no contamination of genomic DNA for all developmental stages.
Fig. 3.
Expression of GFP-PrPC and GFP-GPI fusion proteins in microinjected Xenopus embryos. In vitro synthesized GFP-PrP or GFP-GPI mRNA (1 ng) was microinjected into the animal pole of undivided fertilized Xenopus eggs. Expression of the GFP-PrPC (A–F) and GFP-GPI (G–L) fusion proteins was followed in time under a fluorescence microscope. Arrows indicate the sites of fusion protein expression. (A, G) Stage 9, (B, H) stage 12, (C, I) stage 14, (D, J) stage 22, (E, K) stage 26 and (F, L) stage 45 (staging according to Nieuwkoop and Faber (1967)). Scale bar equals 0.4 mm. (M) Western blot analysis of stage-9, -12 and -45 Xenopus embryos microinjected with 1 ng GFP-PrP mRNA, 1 ng GFP-GPI mRNA or 0.1× MR; an anti-GFP antibody (α-GFP) was used.
Fig. 4.
PrP mRNA expression in the neurointermediate and anterior pituitary of black- and white-adapted Xenopus. PrP (A) and POMC (B) mRNA expression levels in the neurointermediate lobe (NIL) and the anterior lobe (AL) of the pituitary of black-adapted (B; black bars) Xenopus compared with those of white-adapted (W; white bars) animals were determined by real-time quantitative RT-PCR. All values were normalized to GAPDH mRNA levels and are expressed as means ± SEM (n = 3). A significant difference is indicated by *** (P < 0.001).
