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Fig. 1. Xpat protein is present throughout oogenesis and early development. Oocytes (A), eggs and embryos (B) were extracted either in 0.125 M KCl medium and the supernatant analysed by SDS gel electrophoresis and Western blotting, or in 0.5 M KCl and the pellet analysed. In the former extraction, the bulky yolk proteins are insoluble and in the latter they are soluble. A small sample from oocytes injected with 1 ng Xpat mRNA was run in parallel to confirm co-migration with the endogenous band detected (A).
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Fig. 2. Xpat protein (red) is a constituent of germ plasm. (A) Anti-Xpat heavily stains particles concentrated in the mitochondrial cloud (mc) of a stage I oocyte, and pre-clouds (pc) around the nucleus (n). (B, C) At stage II, Xpat is still focused in the mitochondrial cloud, along with endogenous Xpat mRNA, detected by injecting fluorescent antisense Xpat (green, overlay, Câ²). This oocyte is viewed looking in from the point where the mitochondrial cloud touches the cortex. (D) Germ plasm islands in the vegetal cortex of a stage VI oocyte, detected by anti-Xpat (D), mitochondrial anti-VDAC (Dâ², overlay in Dʺ). (E) Field of germ plasm islands at the vegetal pole (see inset of the unfertilised egg). (F) Islands at the 2-cell stage, shown in transverse optical section. (G) A PGC deep in the endoderm of a stage 28 embryo, also stained with DAPI (left, blue). (H) A PGC, immunostained for Xpat, and the cellular outline drawn in grey, demonstrates similarity to a conventionally stained PGC in the tailbud embryo (I), from Kamimura et al. (1980). (J) Nuclei in epidermal cells of tailbud embryos also contain Xpat. Scale bars, 20 μm.
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Fig. 3. Exogenously expressed Xpat organises germ plasm-like structures. (A) In situ hybridisation to Xpat mRNA in the unfertilised egg. (BâF) Fluorescence after equatorial injection of Xpat-GFP mRNA. At 18 h, the Xpat-GFP was around the injection point (B, fluorescence, Bâ², white light) and localised to the cortex (white arrowhead in bisected oocyte, C) as well as the nucleus, n (D). At the cortex, it was particulate. By 55 h, the fluorescent particles moved to the vegetal pole and formed a field of germ plasm-like aggregates (E) in the cortex (F). (G) Control GFP was uniformly distributed in the oocyte. (H) In the absence of vitellogenin, a similar field of Xpat-GFP structures formed in the pigmented animal pole. (I) An Xpat-GFP vegetal aggregate is similar in structure to endogenous Xpat germ islands revealed by anti-Xpat antibody (J). (K, L) In stage I oocytes, 24 h after injection, Xpat-GFP forms particles, accumulating around the mitochondrial cloud (mc) and pre-clouds (pc). (M) Xpat-GFP granules in an epidermal cell of a neurula.
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Fig. 4. The vegetal field of Xpat-GFP aggregates contracts during maturation and fertilisation, like endogenous germ plasm. (A) A field of Xpat-GFP aggregates at the vegetal pole of a stage VI oocyte before maturation, seen with a combination of fluorescence and white light, or (B) with fluorescence alone. (C) The same oocyte after maturation, then pricked to simulate fertilisation and left for 7 min. (D) The decrease in size of the field of large granules through maturation, pricking at a time equivalent to the first cell cycle. Maturation took 6 h after progesterone addition and the oocyte was pricked at 6.5 h.
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Fig. 5. Xpat-GFP recruits mitochondria. Xpat-GFP is shown in green and mitochondria by staining with TMRE (red) after 2 days incubation. (A, B) Xpat-GFP aggregates in the vegetal hemisphere drew in mitochondria from deeper layers, shown in two optical sections of living oocytes 1.6 μm apart. (C) Anti-VDAC staining (red) of fixed Xpat-GFP-expressing oocytes confirmed this result. (D) In some cases, mitochondria (Dâ²) became integrated into the Xpat-GFP aggregates (D; overlay Dʺ). (E) The same is seen in aggregates formed in the animal hemisphere, after animal mRNA injection and omitting vitellogenin. Scale bars, 20 μm, AâC; 10 μm, DâE.
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Fig. 6. Microtubule-based transport of Xpat. (A) In stage I oocytes, Xpat particles (Anti-Xpat, red) are closely associated with microtubules (green), here shown by immunofluorescence. (B–B′ and Bʺ) Xpat germ plasm islands in stage VI oocytes (red) are in dense areas of microtubules (green, overlay B′ and Bʺ), as are Xpat-GFP aggregates (C, Xpat-GFP green, microtubules in red). (D–K) Effects of inhibitors on Xpat-GFP. In control oocytes, Xpat-GFP forms cortical particles at the equator by 18 h after mRNA injection (see Fig. 3D). (D, E) These Xpat-GFP particles have formed aggregates at the vegetal cortex by 55 h. (F, G) Nocodazole interferes with vegetal localisation and aggregation, particularly to the cortex. (H, I) Taxol, which bundles microtubules, produces chains of particles all through the oocyte, largely in the cortex. (J, K) Microfilament disruption with cytochalasin D allows some cortical localisation, but the cortex appears to fragment and the particles form only small, disorganised aggregates, largely at the vegetal pole. (L–Lʺ) Dynein, stained with an intermediate chain antibody (green) is present in intense local foci in the cortex and also as fine particles in germ plasm islands (arrows), identified with Xpat antibody (red). (Lʺ) Overlay. (M–Mʺ) Xpat-GFP (green) co-localises with immunostained Dynein (blue). Overlay (Mʺ). 20 μm scale bars in confocal sections (A–C), 4 μm (L), 10 μm (M). 50 μm scale bars in surface views of oocytes (D–J). Panels E, G, I and K are bisected oocytes, fixed at 80°C for 30 s; the control section is Fig. 3F.
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