Co-operation between the mutant nanos1-3′Δ6 and the wild-type 3′UTR in germ plasm RNP localisation.

<p>Stage VI oocytes injected with Cy3-<i>nanos1 3′Δ6</i> RNA display a very weak localisation pattern after 48 h in culture. Localization of this RNA was greatly improved when co-injected with Cy5-labelled RNA consisting of the full length <i>nanos1</i> 3′UTR.</p

Summary of experiments testing the localisation of Xvelo proteins and mutants.

*<p>eGFP-XveloFLΔ3 localisation was always very weak, and only discernable at very high gain settings during confocal microscopy. If localisation ability was strictly based on the criteria used for the other full length proteins or mutants (ie constant lower gain) they would probably all be scored as negative.</p><p>N is the number of experiments using different batches of oocytes The number of oocytes displaying germ plasm localisation are scored with respect to the total number injected. For eGFP-XveloFL and eGFP-XveloSV the numbers shown are exclusively for experiments in which the mutants were also tested. Similar data were obtained for these proteins in a much larger number of experiments.</p

Effect of RNA depletion on the size of germ plasm islands and the localisation of YFP-Hermes.

<p>Stage VI oocytes were injected with RNA encoding YFP-Hermes plus 11 ng of each antisense oligo. After culturing for 48 hours in OCM, mitochondria were stained with TMRE and the germ plasm region examined by confocal microscopy. Overlays of YFP-Hermes (green) and mitochondria (red) are shown, and the AS oligos used indicated on each panel.</p

Localisation of ‘early pathway’ mRNAs and Hermes protein into the germ plasm of stage VI oocytes.

<p>A. The procedure for examining the cortical distribution of labelled RNAs. After injection and incubation of oocytes in OCM (with or without vitellogenin-containing serum) for 24 to 72 h, oocytes were held in an inverted position between a slide and a coverslip, in a chamber made with a latex spacer. They were then examined by confocal microscopy using a 40× oil-immersion lens. B. Full-length Cy5-labelled <i>nanos1</i> and <i>Xpat</i> RNAs localise in islands of particles at the vegetal pole 48 h after injection. C. Low power stereo microscope view from the side of the vegetal pole of a whole stage VI oocyte, 24 h after injection with mRNA encoding YFP-Hermes. YFP-Hermes protein is clearly localised to a field of fluorescent islands at the vegetal pole (white arrows), typical of germ plasm markers. In the stereo microscope the depth of focus is large so that a much larger area than that occupied by the germ plasm is in focus. D,E. In these islands Cy5-labelled <i>nanos-1</i> and <i>Xpat</i> RNA’s co-localise with YFP-Hermes protein, following co-injection of mRNA encoding YFP-Hermes. All the above oocytes were visualized live, as they are in later figures unless otherwise stated. F. Internal distribution of Cy5-<i>nanos1</i> RNA between the nucleus and the vegetal cortex of a fixed stage VI oocyte. Following injection of RNA, oocytes were cultured for 48 h in OCM, fixed and hemisected with a scalpel prior to visualization by confocal microscopy using a Z-stack.</p

Summary of localisation patterns displayed by injected RNA.

<p>In each table cell the number of oocytes showing a given localisation with respect to the total number of injected oocytes is shown. N = the number of experiments using oocytes from different females. Cy5 or Cy3-labelled RNAs were routinely injected, with or without RNA encoding YFP-Hermes. The YFP-Hermes alone denotes localisation by the translated protein following injection of the RNA. FL; full length.</p

Sub-cellular localisation of GFP fusion proteins and of the sites of where they interact.

<p>Stage VI oocytes were injected with mRNA encoding the constructs indicated, and cultured in OCM for 48/1% formaldehyde at -20°C overnight, rehydrated and sectioned in an animal/vegetal plane by hand. Low power fluorescent stereo microscope images are shown.</p

FRAP experiment showing that Hermes protein in germ plasm exchanges with a cytoplasmic pool YFP-Hermes.

<p>A. Confocal images, before during and after the bleaching step. B. Quantification of the bleached area and a small unbleached control region. C. Low power image to show that injected RNA diffuses more slowly than protein. Oocytes were co-injected at the equator with Cy5-<i>nanos1</i> and <i>YFP-Hermes</i> RNAs (The latter does not have its own UTRs, so would not localise). After 48 h the vegetal pole was visualised and it is seen that the Cy5-<i>nanos1</i> RNA has diffused more slowly from the injection point, at the top left, than Hermes protein.</p

YFP-Hermes localises into particles in islands containing concentrated mitochondria.

<p>A. Stage VI oocytes were injected with RNA encoding YFP-Hermes and after 18 h mitochondria were stained with TMRE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061847#pone.0061847-Machado1" target="_blank">[22]</a>, prior to visualisation of the vegetal cortex, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061847#pone-0061847-g001" target="_blank">Figure 1A</a>. B Stage IV oocytes were injected with Cy5-<i>nanos1</i> RNA and after 24 h the oocytes were stained with TMRE and analysed as in A.</p

The behaviour of endogenous and exogenous germ plasm molecules during oocyte maturation.

<p>A–D. Fixed oocytes stained with antisera against Hermes (green) and Xpat (red). E–H. Live oocytes expressing YFP-Hermes (green) and injected Cy5-<i>nanos1</i> (red). A. The wide field of large islands in a control oocyte; the overlay of Hermes and Xpat is shown. B. A similar view of newly fertilised eggs showing the dramatic reduction in fluorescent islands. C. Detail of islands in a control oocyte. D Detail of a fertilised egg. Note that Hermes and Xpat are in distinct particles. E. The wider field of YFP-Hermes coincident with Cy5-<i>nanos1</i> is similar to the endogenous molecules in A. F. When these oocytes were matured with progesterone the field was reduced, as in B, but less so; this is expected, since fertilised eggs have progressed further. G,H. Details of the fields in E and F respectively.</p

Distribution of GFP-tagged Hermes interacting candidates in the germ plasm region of stage VI oocytes.

<p>The vegetal pole of oocytes was injected with Cy5-<i>nanos1</i> and mRNAs encoding GFP-tagged proteins as indicated. After culturing in OCM for 48 hours the cortex of live oocytes was examined by confocal microscopy. Overlays are shown on the right. Xvelo proteins localise strongly to germ plasm RNPs, but Rbm42b and particularly Rbm24b localise more diffusely in the general vicinity of the germ plasm. Control RNA encoding GFP protein is shown in F.</p