Wilk K et al. (2005), Delivery of germinal granules and localized RNA...

XB-ART-2235

Int J Dev Biol 2005 Jan 01;491:17-21. doi: 10.1387/ijdb.041906kw.

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Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud.

Wilk K , Bilinski S , Dougherty MT , Kloc M .

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During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex. Thus the fragmentation of the cloud that occurs later in oogenesis is irrelevant to the movement of METRO-localized RNAs and germinal granules. On the basis of these findings, we propose here a revised model of germinal granule and localized RNAs delivery to the oocyte vegetal cortex via the METRO pathway.

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Species referenced: Xenopus
Genes referenced: cat2 nanos1 pgat

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	Fig. 1. Electron micrographs of cross/oblique sections of the mitochondrial cloud in Xenopus oocytes. (A) In stage I oocyte, the mitochondrial cloud is a spherical, compact organelle composed of thousands of mitochondria (gray specs) and hundreds of germinal granules. Germinal granules (in red) are mainly concentrated at the vegetal tip of the cloud. (B) Stage II oocyte shows many interconnected islands containing mitochondria (grey specs) and germinal granules (in red). Germinal granules were artificially colored. (C) METRO region of mitochondrial cloud from stage I oocyte showing germinal granules (red arrows) between mitochondria. Bars, 10 μm.
	Fig. 2. Sections of oocytes hybridized with a digoxigenin-labeled antisense Xcat2 RNA probe and stained blue with NBT/BCIP alkaline phosphatase substrate. (A) In an early stage I oocyte the mitochondrial cloud (mc) is located at the oocyte nucleus (on) and the germinal granules (dark blue dots) are distributed more or less randomly within the whole cloud. (B) In early stage II oocyte the mitochondrial cloud extends between the nucleus and vegetal cortex and germinal granules are concentrated as a layer at its vegetal tip (the METRO region). Several �delayed� germinal granules (arrowheads) are visible in the animal (nuclear) region of the cloud. In a stage II oocyte (C) and an early stage III oocyte (D) the mitochondrial cloud is visible as an unfragmented, single entity with a layer of germinal granules at the vegetal cortex. (E) A Late stage III oocyte with a single layer of germinal granules (arrows) anchored at the vegetal cortex. Abbreviations: mc, mitochondrial cloud; on, oocyte nucleus. Bars, 50 μm.
	Fig.3. Semithin sections through oocytes hybridized with Xcat2 (A,C) and Xpat (B) antisense RNA probes, labeled with nanogold and silver enhanced. (A). In a stage II oocyte, the unfragmented mitochondrial cloud (mc) extends between the layer of homogenous cytoplasm (stars) surrounding the nucleus (on) and vegetal cortex. The germinal granules containing localized Xcat2 mRNA (artificially colored in red) are visible as a layer at the vegetal cortex in the METRO region. (B) Fragment of stage II oocyte showing the unfragmented mitochondrial cloud with a layer of Xpat mRNA (outlined artificially in red) at the vegetal cortex in the METRO region. (C) In a late stage III oocyte, germ plasm islands (fragments of mitochondrial cloud) are at the vegetal cortex. Arrows indicate the position of several of germ plasm islands, which are barely visible at such a low magnification. Bars in A,B, 25 μm; bar in C, 50 μm.
	Fig. 4. Three-dimensional reconstructions of stage II oocytes hybridized with Xcat2 (A,B) or Xpat (C,D) mRNA probes, labeled with nanogold and silver enhanced. The mitochondrial cloud (light blue) is visible as an unfragmented entity extending between the nucleus (yellow) and vegetal cortex. The surface of the cloud is uneven and convoluted. Groups of mitochondria (secondary clouds, dark blue) are also visible around the nucleus. (A, B) Germinal granules (red dots) are located at the vegetal tip of the cloud at the METRO region of the oocyte. (C,D) Xpat mRNA (red) is concentrated in the METRO region of the cloud.
	Fig. 5. Revised model of delivery of germinal granules and METROlocalized RNAs to the vegetal cortex of Xenopus oocytes. Steps 1 and 2 indicate stage I of oogenesis, in which the spherical mitochondrial cloud (light purple) is located near the nucleus (blue) but is separated from it by a thin layer of homogenous cytoplasm (dark yellow). The germinal granules (dark purple) and METRO-localized RNAs are initially distributed at random within the cloud and subsequently become aggregated at its vegetal tip (the METRO region, step 2). In the old model (shown to the left), from stage II of oogenesis onward, the mitochondrial cloud breaks down and the germinal granules and METRO-localized RNAs move within the mitochondrial cloud fragments (steps 3a and 4a) and become anchored at the vegetal cortex between stages III and VI of oogenesis (step 5). In the revised model (shown to the right), in stage II of oogenesis, the unbroken mitochondrial cloud expands to span between the nucleus and vegetal cortex, pushing the germinal granules and METRO-localized RNAs toward the vegetal cortex (steps 3b and 4b) where they become anchored. In stage II of oogenesis the mitochondrial cloud become uneven and convoluted, which on the sections can give the impression of separated islands (step 4b). Subsequently, between stage III and VI of oogenesis, the mitochondrial cloud breaks down (step 4b�). The occasional �delayed� germinal granules are rarely visible in the fragments of the cloud (step 4b�).