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

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

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

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

Localization of <i>grip2a</i> mRNA in wild-type and mutant oocytes.

<p>A–C) Whole mount in situ hybridization of dissected ovaries from wild-type (A), <i>bucky ball (buc</i>, B) and <i>magellan (mgn</i>, C) mutant females. In (A–C) oocytes at stage III of development are indicated. Smaller oocytes are at stages I and II, which are difficult to differentiate in whole mounts at this magnification. The <i>grip2a</i> mRNA localization domain is observed in an asymmetric cortical position in wild-type oocytes (A) but is unlocalized and diffuse in <i>buc</i> oocytes (B) and internally-located in <i>mgn</i> mutants (C). D–J) Sections of wild-type and mutant oocytes at the indicated stages after labeling to detect <i>grip2a</i> mRNA. Stages are as indicated in the panel and were determined by size and oocyte morphology according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Selman1" target="_blank">[101]</a>. D–F) Wild-type oocytes showing localization to the presumptive Balbiani Body (D) and subsequent localization to a cortical domain of the oocyte corresponding to the presumptive vegetal pole (E–F). G,H) <i>buc</i> mutant oocytes lack the Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Bontems1" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Marlow1" target="_blank">[43]</a> and the <i>grip2a</i> mRNA subcellular localization domain in stage I and II oocytes. I) <i>mgn</i> mutant stage I oocytes exhibit an enlarged Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a> and displayed an enlarged <i>grip2a</i> mRNA localization domain. J) Stage II mgn mutant oocytes fail to localize transcripts to the vegetal pole which instead persist in an internal domain <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a>, as observed also for <i>grip2a</i> mRNA. Number of oocytes examined were as follows: wild-type: early stage I: 13, stage II: 12; <i>buc</i>: early stage I: 35, stage II: 26; <i>mgn</i>: early stage I: 23, stage II: 18. Magnification bar in (C) corresponds to 250 µm for panels (A–C), and in (J) to 50 µm for panels (D–J).</p

Microtubule reorganization at the vegetal cortex is affected in <i>hecate</i> mutants.

<p>A–F) Cortical microtubule network at the vegetal pole in wild-type (A) and <i>hec</i> mutant (B–F) embryos at 20 mpf. Microtubules appear oriented in the same direction and bundled in wild-type embryos (A). The extent of co-orientation and bundling is greatly reduced in <i>hec</i> mutant embryos (B), where microtubules form multiple aster-like structures which can have a well focused-center (C,D) or can exhibit a central microtubule-free zone (E,F) and often overlap (F) or interdigitate (D). The relatively unbundled microtubule arrangement shown in (B) also corresponds to a sector of a large aster-like structure emanating from a not shown central core. Up to 6 aster-like structures were observed in the vegetal cortex of a single embryo. G,H) Cortex in mediolateral regions shows a loose and apparently random network of microtubules which appears similar in both wild-type (G) and <i>hec</i> mutant (H) embryos (n = 8 for wild-type and mutants). All images are z-axis projections of confocal image stages. The phenotype was fully (100%) penetrant according to the two main categories (wild-type, aligned and bundled microtubules; mutant, radialy oriented and unbundled microtubules, with 10 wt and 25 <i>hec</i> mutant embryos imaged. Magnification bar in (H) corresponds to 40 µm for all panels.</p

Axis induction defects in embryos from mothers homozygous for three different <i>hecate</i> mutant alleles.

<p>A–F) Side views (except as indicated in (F)) of live embryos at 24 hpf showing a range of phenotypes from most severe (A) to normal (E) and double-axis embryos (F). A) Class V4 embryo exhibiting complete radial symmetry and lacking all anterior (e.g. head structures) or dorsalmost (e.g. notochord) structures. B) Class V3 embryo exhibiting a rudimentary axis but lacking all anterior and dorsalmost structures. C) Class V2 embryo lacking anteriormost structures such as the eyes, but showing formation of more posterior head structures such as the otic vesicles (black arrowhead, also indicated in D). D) Class V1 embryo with a relatively complete axis but reduced anterior structures, such as the eyes (white arrowhead, also labeled in E), and lacking a properly formed notochord. E) Normal embryo derived from <i>hec</i> mutant females indistinguishable from wild-type. Note somites in (E) are chevron-shaped, while they are blocky in (B–D) indicative of defects in notochord formation, or encircling the entire embryo in (A). F) A double-axis embryo found in <i>hec</i> mutant clutches exhibiting weak expressivity. Insert in the lower left shows the left axis indicated by the dashed rectangle, out of focus in the main image. Insert in the upper left panel shows a dorsal view, showing the bifurcated axis. Note the lack of defined anterior structures in both axes, as well as the lack of a notochord along the trunk, also reflected by block-shaped somites in this region. Images in A–F are side views, except for upper left insert in (F). G–I) Animal views of live wild-type embryos (G) and embryos from females homozygous for each of the three <i>hec</i> mutant alleles. The dorsal thickening or shield (arrow) is absent in mutant embryos. K–V) In situ hybridization analysis to detect expression of dorsally-expressed genes (<i>gsc</i>, <i>chd</i>) and ventrally-expressed genes (<i>eve 1</i>). The expression domains of <i>gsc</i> and <i>cho</i> is reduced in <i>hec</i> mutant embryos, while the expression domain of <i>eve</i> is expanded in these embryos. (K–V) are animal view of embryos, dorsal to the right when identifiable, at the shield stage (6 hpf). Magnification bars in (F) and (V) correspond to 100 µm for panels sets (A–F) and (G–V), respectively. Dorsal view insert in panel (F, upper left) has been reduced to 75% size.</p

Whole mount in situ hybridization analysis of the expression of zebrafish <i>grip2a</i> mRNA in early embryos.

<p>A–E) <i>grip2a</i> mRNA is localized in a slightly off-center position at the vegetal pole of early embryos, and remains in that position until the sphere stage when it becomes undetectable. F) Control sense probe. G) The domain of <i>grip2a</i> mRNA localization in <i>hec</i> mutants is reduced in intensity and appears to lack an off-center shift. H,I) Representative control (DMSO)- and nocodazole-treated embryos showing the lack of off-center shift in nocodazole-treated embryos (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422.s004" target="_blank">Figure S4</a> for details). All panels are side views (except for (B), which is a vegetal view of the embryo in (A)) at the following stages: A,B: 1-cell (20 mpf), C,F,G: 2-cell (45 mpf), D: 64-cell (2 hpf), E: sphere (4 hpf). (H,I) are at 40 mpf, which approximately corresponds to the 2-cell stage (C) in untreated embryos. Magnification bar in (I) corresponds to 100 µm for all panels.</p

Germ plasm recruitment and PCG determination appears unaffected in <i>hecate</i> mutants.

<p>A–F) Whole mount in situ hybridization of 4-cell stage (60 mpf) embryos to label the germ line-specific genes <i>dazl1</i> (A,B,D,E), and <i>vasa</i> (C,F) in wild-type (A–C) and <i>hec</i> mutant (D–F) embryos. Early localization of <i>dazl</i> mRNA to the vegetal pole (A,D, side views), and <i>dazl</i> mRNA and <i>vasa</i> mRNA to the germ plasm as it becomes recruited to the furrows of the first and second cleavage cycles (B,C,E,F, animal views). Localization of dazl domains of recruitment at the furrow is not detected in the side views as the levels of mRNA in these domains are relatively low and the focal plane is not optimal for their visualization. G,H) PGC determination, as determined by <i>vasa</i> expression in 24-hour embryos (G), appears unaffected in <i>hec</i> mutants (H), although the PGCs in mutant embryos do not become clustered as in wild-type due to the aberrant cell specification in these mutants. Quantification of the number of <i>vasa</i>-expressing cells is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422.s009" target="_blank">Figure S9</a>. Magnification bar in (H) corresponds to 100 µm for all panels.</p