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

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

Additional file 1: Table S1. of Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

Tagging and coverage of MHC region markers. Table S2: Tagging and coverage of Tx-specific genes. Table S3: Untranslated regions (UTRs) considered in the TxArray design. Table S4: Loss-of-function variants included in the TxArray. Table S5: Copy number polymorphisms (CNPs) and variations (CNVs) included in the TxArray. (DOCX 54 kb