ER and mitochondria are absent from the periphery of stage II <i>mgn</i> mutant oocytes.

<p>Transmission electron micrographs of stage II wild-type (A–A″) and <i>mgn</i> mutant (B–B″) oocytes. ER and mitochondria are distributed throughout wild-type stage II oocytes (A–A″) but are absent from the periphery of stage II <i>mgn</i> mutant oocytes (B–B″). Arrowheads indicate oocyte nuclei. Positions of regions shown at high magnification (A′, A″, B′ and B″) are indicated by the respective colored asterisks in A and B. Dashed blue line outlines the electron dense region in which most of the ER and mitochondria are located. Scale bars = 20 microns (A, B) and 1 microns (A′, A″, B′ and B″).</p

Localization of stable microtubules but not actin is disrupted in <i>mgn</i> mutant oocytes.

<p>Stage I oocytes stained with an antibody to acetylated tubulin to label stable microtubules (A,B) or rhodamine phalloidin to label actin cytoskeleton (C,D). In wild-type stage I oocytes, acetylated microtubules are uniformly distributed throughout the oocyte (A,A′), whereas in <i>mgn</i> mutant oocytes, acetylated microtubules are largely absent from peripheral regions of the oocyte (B,B′). DAPI staining around oocytes labels nuclei of surrounding somatic follicle cells. Green = acetyated tubulin; blue = DAPI. In both wild-type (C) and <i>mgn</i> mutant oocytes (D), actin is localized to the nucleus and at the cortex. Arrowheads indicate oocyte nuclei. Red = phalloidin. Scale bars = 25 microns. All images are single optical sections.</p

<i>mgn</i> is required for animal-vegetal polarity of the egg.

<p>Nomarski images of eggs from a wild-type (heterozygous sibling) female (A) and a <i>mgn</i> mutant female (B) one hour post activation. Eggs from <i>mgn</i> mutants exhibit cytoplasm surrounding the yolk (arrowheads) rather than restricted to the blastodisc at the animal pole as in wild-type (lateral view, animal pole up). In addition, <i>mgn</i> eggs are frequently smaller and display variable elevation of the chorion (arrow). Scale bar = 250 microns.</p

<i>mgn</i> mutant oocytes exhibit an enlarged Balbiani body and absence of mitochondria and ER from the periphery of the oocyte.

<p>Oocytes stained with DiOC₆ to label ER and mitochondria (A–E). In a wild-type mid stage I oocyte, the nucleus is in the middle of the oocyte (arrowhead) and the Balbiani body is at the future vegetal side of the oocyte (A, arrow). (B,C) Mutant oocytes exhibit an enlarged Balbiani body (arrows), and an absence of ER, mitochondria and the Balbiani body from the periphery. Images are single optical sections. In stage II wild-type oocytes (D), ER and mitochondria are localized throughout the oocyte, whereas in <i>mgn</i> mutant oocytes (E), ER and mitochondria are concentrated in the middle of the oocyte and are absent from peripheral regions. Images are single optical sections of 0.5 micron oocyte sections. (F) Bar graph depicting size of Balbiani body (Bb) during stage I of oogenesis. 50–70 micron oocytes, n = 10 wild-type and 10 mutant oocytes; 70–90 micron oocytes, n = 15 wild-type and 15 mutant oocytes; 90–110 micron oocytes, n = 15 wild-type and 15 mutant oocytes. Arrowheads indicate nuclei. (A–C) scale bars = 25 microns. (D,E) scale bars = 100 microns.</p

The <i>mgn</i> mutation causes defects during oogenesis.

<p>Sections of wild-type and mutant oocytes stained with hematoxylin (purple) and eosin (pink). The cytoplasm of stage I zebrafish oocytes is strongly basophilic, resulting in strong purple staining, while the mitochondria-rich Balbiani body is slightly acidophilic and stains pale pink with eosin. In wild-type mid stage I oocytes (A), the nucleus is localized at the center of the oocyte and the Balbiani body is near the future vegetal cortex. In <i>mgn</i> mutant stage I oocytes (B), the nucleus is asymmetrically localized and the Balbiani body, which frequently remains close to the nucleus, is surrounded by a region that is lightly stained with eosin. Wild-type stage II oocytes (C) have a central nucleus surrounded by cortical granules (CG). In stage II <i>mgn</i> mutant oocytes (D), the nucleus is mislocalized and CG accumulate opposite to the nucleus, in and around a faint eosin-stained area (arrow). During stage III, wild-type oocytes (E) accumulate yolk in the center of the oocyte and CG localize uniformly around the cortex, whereas stage III <i>mgn</i> mutant oocytes (F) display an uneven distribution of CG at the cortex. Also note stage II oocyte at right in (F). Arrowheads indicate oocyte nuclei; arrows indicate Balbiani body in (A,B); asterisks indicate CG. Scale bars = 50 microns (A,B), 100 microns (C,D), and 200 microns (E,F).</p

Sequences for additional oligos used for 613.9 kb capture.

<p>Sequences for additional oligos used for 613.9 kb capture.</p

Amplification of <i>hecate/grip2a</i>-dependent symmetry breaking event by a general animal-directed long-range transport system.

<p>A) Cortical shifts of various vegetally localized components, including <i>wnt8a</i> mRNA, Sybu protein and <i>grip2a</i> mRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Lu1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Tran1" target="_blank">[8]</a>; this report) are short-range and dependent on microtubule bundling and alignment, itself dependent on <i>hec</i> function. In wild-type embryos, such a short-range shift generates a symmetry breaking event that is subsequently amplified by long-range, animally-directed transport mechanism independent of <i>hec</i> function and not restricted to the prospective dorsal axis. B) In <i>hec</i> mutant embryos, neither reorganization of vegetal microtubules into aligned bundles nor a short-range shift occur, so that, even though long-range transport remains intact, vegetal determinant transport to the animal pole is affected. The mechanistic basis for the long-range transport, occurring in the region of a loosely organized mediolateral microtubule cortical network remains to be determined (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#s3" target="_blank">Discussion</a>).</p

Defects in the vegetal localization of <i>wnt8a</i> mRNA and Sybu protein.

<p>A–D) Off-center shift of <i>wnt8a</i> mRNA is affected in <i>hec</i> mutants. Whole mount in situ hybridization of wild-type embryos (A,C) and <i>hec</i> mutant embryos (B,D) at the 1- (A,B, 30 mpf) and 4- (C,D, 60 mpf) cell stages. Images show representative embryos. A majority of wild-type embryos showed a clear off-center shift (85%, n = 27 at 30 mpf and 74%, n = 47 at 60 mpf). A majority of <i>hec</i> mutant embryos showed vegetal localization without a shift at 30 mpf (79%, n = 33, remaining embryos show no localization) and absence of localization at 60 mpf (89%, n = 38, remaining embryos show reduced vegetal localization without a shift). The apparent label at the base of the blastodisc is observed in a majority of mutant embryos (71%, n = 38) but not in wild-type (C) or control embryos labeled with other probes (not shown) and may reflect remaining <i>wnt8a</i> mRNA that has lost anchoring at the vegetal pole and has moved animally through the action of axial streamers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Fuentes1" target="_blank">[83]</a>. E–I) Localization of Sybu protein is affected in <i>hec</i> mutants. Whole mount immunofluorescence to detect Sybu protein of untreated wild-type (E,G) and <i>hec</i> mutant (F,H,) embryos and nocodazole-treated wild-type embryos (I) at the indicated stages. In wild-type embryos, an off-center shift in Sybu protein can be observed starting at 30 mpf (G). In <i>hec</i> mutants, Sybu protein becomes undetectable levels by this same time point (H). Patterns of localization of Sybu protein at 10 mpf and 20 mpf time points (combined n: 32 WT, 19 mutant for 10–20 mpf), and 30 mpf and 40 mpf time points were similar and have been combined. 59% (n = 32) of wild-type and 63% (n = 19) of <i>hec</i> mutant embryos showed centered vegetal localization during 10–20 mpf. At 30–40 mpf, the percent of embryos that showed vegetal localization, now with an off-center shift, was reduced to 25% (n = 28) in wild-type, and 0% (n = 25) of <i>hec</i> mutants showed any localization at these time points. Treatment of wild-type embryos with nocodazole inhibits the shift but does not result in delocalization from the vegetal cortex (I, embryo at 40 mpf), as previously shown <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>. Magnification bars in (D) and (I) correspond to 100 µm for panels sets (A–D) and (E–I), respectively.</p

Long-range animally-directed transport is not affected in <i>hecate</i> mutants.

<p>A–C) Paths of injected 0.2 um fluorescent beads after injection into the vegetal pole with a single (A,B) or double (C) injection in wild-type (A,C) or <i>hec</i> mutant (B) embryos. (A′–C′) show merged imaged including the fluorescent channel (shown in A–C) and corresponding DIC optics at low intensity. The extent and frequency of bead transport appeared similar in wild-type and mutant embryos (A,B, see text). Injections into two opposite sides of the vegetal pole results in multiple animally-directed paths, indicating that the entirety of the mediolateral cortex is competent for bead movement. Arrowheads and arrows in (A–C) indicate site of injection in the vegetal region and animally-directed paths along mediolateral regions, respectively. Magnification bar in (C′) corresponds to 100 µm for all panels.</p

Molecular identification of the <i>hecate</i> locus.

<p>A) Linkage map of the <i>hec</i> locus. The number of recombinants over the total number of analyzed meiosis is indicated. <i>hec</i> linkage was initially identified between SSLP markers z59658 and z24511 on chromosome 8. Fine mapping analysis with newly identified RFLP markers further narrowed the region between the gene <i>gpd1a-1</i> and the RFLP zC150E8y. B) Contig of five BAC clones covering the <i>hec</i> critical region. CH73-233M11, CH73-272M14, CH73-250D21, DKEY-43H14 and CH211-150E8 are five sequenced and overlapping BAC clones in this interval. C) Exon-intron structure of the <i>hec/grip2a</i> gene, which contains 16 exons. The <i>hec^p06ucal</i>, <i>hec^t2800</i> and <i>hec^p08ajug</i> alleles each cause a premature stop-codon in exon 4, exon 10 and exon 12, respectively. D) Sequence traces of the cDNA products from wild-type and the three mutant <i>hec</i> alleles. Nucleotide substitutions are indicated by the red box. Mutant cDNAs show a C-A transversion in codon 118 (<i>hec^p06ucal</i>), a C-T transversion in codon 414 (<i>hec^t2800</i>), or a C-T transversion in codon 499 (<i>hec^p08ajug</i>), all creating premature STOP codons. E) Schematic diagram showing the protein domain structures of Grip2a in the wild-type and mutant alleles. Red boxes represent conserved PDZ domains.</p