Lefty misexpression suppresses patterning defects of <i>p18ahub</i> embryos.

<p>(A) Pie charts indicate fractions of embryos at 24 hpf with the indicated phenotypes. Number of embryos is at the center of each chart. Labels across indicate the amount of <i>lefty1 (lft1)</i> mRNA injected. Top row shows uninjected <i>p18ahub</i> control clutch for each experiment, middle row indicates <i>p18ahub</i> embryos injected with mRNA, and bottom row is WT embryos injected with mRNA. +1× <i>lft</i> mRNA corresponds to 1 pg mRNA. (B and C) <i>In situ</i> hybridization for <i>lft1</i> in a mid gastrula stage WT (B, n = 12) and <i>p18ahub</i> (C, n = 13) embryos; lateral views, dorsal facing.</p

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

<div><p>Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express <i>vox</i> and <i>ved</i>, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.</p></div

<i>p18ahub</i> mutants exhibit delayed progression of epiboly and severe dorsalization.

<p>Late blastula stage WT embryo (A), and age-matched <i>p18ahub</i> embryo (B). WT embryo at an early gastrula stage (C) with the shield (organizer) on the dorsal side (arrow). A <i>p18ahub</i> embryo at an early gastrula stage (D) displaying apparent radial hyperconvergence with no definitive dorsal side. (A,B) lateral views; (C,D) animal pole views. WT (E) and <i>p18ahub</i> (F) embryos at 24 hours post fertilization (hpf) (lateral views). (G) Pie chart depicting proportion of embryos with indicated phenotypes at 24 hpf sampled over a total of 2308 embryos. (H and I), <i>in situ</i> hybridization on 3–5-somite stage WT (H, n = 15) and age-matched <i>p18ahub</i> (I, n = 13) embryos for <i>six3</i> (<i>s</i>) expression in presumptive forebrain, <i>pax2.1</i> expression in the midbrain-hindbrain boundary (<i>p</i>) and pronephros (<i>p*</i>), <i>krox20</i> (<i>k</i>) in hindbrain rhombomeres 3 and 5, and <i>myod</i> (<i>m</i>) in paraxial mesoderm; dorsal view in (H), lateral view in (I), anterior to top. (J and K), 10-somite stage WT (J, n = 17, dorsal view), and age-matched <i>p18ahub</i> embryos (K, n = 18, anterior view) processed for <i>krox20 in situ</i> hybridization. (L–Q) <i>in situ</i> hybridization on mid gastrula stage WT and equivalent stage <i>p18ahub</i> embryos shown for: <i>otx2</i>, WT (L, n = 18) and <i>p18ahub</i> (M, n = 10); <i>cyp26a</i>, WT (N, n = 16) and <i>p18ahub</i> (O, n = 15); and <i>hoxb1b</i>, WT (P, n = 22) and <i>p18ahub</i> (Q, n = 21). Lateral views, anterior at top, dorsal to right.</p

<i>p18ahub</i> affects the Integrator Complex Subunit 6 (Ints6).

<p>(A) <i>p18ahub</i> was narrowed to an approximately 1.35 Mb interval between SSLP marker z7120 and an SSLP marker that we generated in BAC CR545476.14 (Zv9) (right marker) by examining meiotic recombination between markers flanking the mutation. The recombinants found in the number of meioses examined at each marker is shown and the positions of markers are approximate. (B) <i>p18ahub</i> is a T to A transition in an exon of <i>ints6</i> that converts valine 375 to an aspartate. An alignment of several Ints6 proteins was generated using ClustalW and reveals that V375 is nearly invariant among widely divergent species. (C and D) <i>In situ</i> hybridization for <i>ints6</i> transcripts on WT embryos at (C) 128-cell (n = 8) and (D) late blastula stage (n = 19); lateral views, animal to top. (E–G) Rescue experiments: pie charts show fractions of embryos evaluated at 1 dpf with indicated phenotypes. Numbers of embryos are shown in the center of each chart. (H) Uninjected <i>p18ahub</i> mutant embryos and (I) mutant embryos rescued by injection of 50 pg <i>ints6</i> mRNA, shown at 1 dpf.</p

Summary and model depicting Rbpms2 localizing domains, and consequences of <i>rbpms2</i> loss on Buc and Balbiani body architecture.

<p>(A) Chart depicting wild-type and mutant Rbpms2 proteins and the contribution of Rbpms2 functional domains to protein localization within subcellular aggregates of somatic and germline cell types. Y = yes, N = no, P = partial, ND = not determined. (B) In 293 (somatic) cells Rbpms2 (green) requires its C-terminus, but not the RNA binding domain for somatic cell granule localization; whereas, in somatic cells of the zebrafish blastula Rbpms2 accumulates in P-bodies (yellow and red dots) and Dcp2 negative granules (green dots) but not Tial-1 positive stress granules and requires an intact RNA binding domain. Similarly, in germline cells (PGCs and oocytes) the RNA binding domain, and presumably RNA binding is required for localization to germ granules and the Balbiani body, and the C-terminus contributes to the wild-type localization of the proteins. (C) Rbpms2 in wild-type primary oocytes promotes Balbiani body assembly or cohesiveness possibly through interaction with Bucky ball protein via the Rbpms2 C-terminus or via interaction with <i>buc</i> RNA. (D) In <i>rbpms2</i> double mutants, mitochondria are asymmetrically present within early oocytes and associate with nuage. Although Bucky ball protein is translated, no cohesive Buc structure forms in the Bb. Instead the Bucky ball domain is scattered and donut shaped, and large atypical electron dense bodies form in the cytoplasm, reflecting either failure to assemble the Balbiani body or its premature disassembly.</p

Reduced <i>bmp</i> expression but intact BMP signal transduction in <i>p18ahub</i> mutants.

<p>(A–H) <i>In situ</i> hybridization on early gastrula stage embryos or equivalent, except (C) and (D) are mid gastrula stage or equivalent. Embryos were stage-matched. Animal pole views, dorsal to the right for all. Expression of <i>bmp</i> ligand gene in WT embryos (A, n = 32, C, n = 43, and E, n = 21) and <i>p18ahub</i> embryos (B, n = 30, D, n = 18, and F, n = 16). Expression of the BMP antagonist <i>chordin</i> (<i>chd</i>) was confined to the dorsal side of WT embryos (G, n = 21) but was expanded around the entire margin of <i>p18ahub</i> embryos (H, n = 20) by early gastrulation. (I–N) Pie charts indicate fractions of embryos at 24 hpf that displayed the indicated phenotypes (categories described in the text). Number of embryos is shown at the center of each chart. <i>p18ahub</i> embryos (I, O, uninjected) that were injected with 20 pg <i>bmp2b</i> mRNA were rescued to WT or ventralized (K, P, +<i>bmp2b</i> mRNA) similarly to WT embryos (J, +<i>bmp2b</i> mRNA). <i>p18ahub</i> mutant embryos (L, uninjected) depleted of Chordin, Noggin1, and Follistatin-like 2b proteins by MO injection were similarly ventralized (N, +MO<i>cnf</i>) to WT embryos (M, +MO<i>cnf</i>).</p

Dorsal organizer gene expression is expanded in <i>p18ahub</i> mutants.

<p><i>In situ</i> hybridization using <i>goosecoid</i> (<i>gsc</i>) probe (A–D). Animal pole views, dorsal to right for (A) and (B); dorsal view, animal pole up in (C) and (D). WT embryos at early gastrula (A, n = 27) and mid gastrula stages (C, n = 41). <i>p18ahub</i> embryos (B, n = 33 and D, n = 10) displayed radially expanded <i>gsc</i> expression at the same stages. The embryo in (D) is tilted toward the viewer to show radial <i>gsc</i> expression. WT (E, n = 18) and <i>p18ahub</i> (F, n = 18) blastula stage embryos showed no obvious differences in the number or distribution of β-catenin immunopositive nuclei (arrows). (G–J) <i>In situ</i> hybridization for <i>bozozok (boz)</i>, lateral views, dorsal to right; insets show animal pole views. WT and <i>p18ahub</i> embryos showed normal <i>boz</i> expression at late blastula (G, n = 19 and H, n = 18) and early gastrula stages (I, n = 14 and J, n = 16, respectively). As described in the text, epiboly progression was delayed in <i>p18ahub</i> embryos. At 6 hpf WT embryos form a morphologically apparent organizer, indicated by a thickening of the blastoderm on the dorsal side of the embryo and <i>boz</i> expression within the hypoblast. In contrast, at 6 hpf <i>p18ahub</i> embryos still appeared to be in a late blastula stage, although their <i>boz</i> expression was similar to WT.</p

Quantification of fusion protein subcellular localization in zebrafish somatic cells.

<p>Quantification of fusion protein subcellular localization in zebrafish somatic cells.</p

Excessive axial mesoderm at the expense of ventrolateral mesoderm in <i>p18ahub</i> embryos.

<p>WT (A, n = 19) and stage-matched <i>p18ahub</i> (B, n = 20) embryos at a mid blastula stage, exhibited similar <i>nodal related 1 (ndr1)</i> expression. WT (C, n = 14) and age-matched <i>p18ahub</i> (D, n = 13) embryos at 6 hpf after synchronized matings displayed similar <i>lefty1</i> (<i>lft1</i>) expression, although blastoderm involution was not evident in the mutants. (A–D) are animal pole views, dorsal to right. (E–L) are mid gastrula stage, except (G and H) are 3–5 somite stage or equivalent. Dorsal views, animal to top, except (H–H″) are animal pole views. WT embryos (E, n = 6) displayed marginal and axial <i>ntl</i> expression. In contrast, some <i>p18ahub</i> embryos displayed only axial <i>ntl</i> expression distributed circumferentially (F, n = 16), while others displayed broadened axial and reduced ventrolateral <i>ntl</i> expression (F inset, n = 8). In WT embryos (G, n = 22) <i>ntl</i> was expressed in the notochord and tail bud mesenchyme. In equivalent stage <i>p18ahub</i> embryos, (H–H″, n = 21), <i>ntl</i> expression was observed in multiple notochords terminating in smaller tail bud-like domains. Consistent with ectopic axial <i>ntl</i> expression, the expression of <i>floating head</i> (<i>flh</i>), a marker of notochord precursors, confined dorsally in WT embryos (I, n = 20), was expressed around the entire embryonic margin in <i>p18ahub</i> embryos (J, n = 16). In WT (K, n = 18) <i>sox17</i> is expressed in endodermal precursor cells and in a single cluster of dorsal forerunner cells (arrow). In <i>p18ahub</i> embryos (L, n = 16) <i>sox17</i> expression indicates the presence of ectopic clusters of dorsal forerunner cells (arrows).</p

<i>rbpms2</i> mutant allele stability and localization activity in somatic cells.

<p>(A) RT-PCR to detect <i>rbpms2a</i> and <i>rbpms2b</i> maternal transcripts. Pink asterisks indicate likely nonsense mediated decay of mutant allele transcripts. (B-F) Wild-type GFP-Rbpms2a/b (B, D) and GFP-Rbpms2b^sa9329 (F) localize to granules in HEK 293 cells, while GFP-Rbpms2a^ae30 and GFP-Rbpms2b^ae32 are not localized. (G-K) Zebrafish blastula cells expressing GFP-Rbpms2 fusions. (G,H) Wild-type GFP-Rbpms2b localizes near the nucleus (H), is apparently associated with the centrosome/spindle in some cells, and (G) is in granules that are not positive for the stress granule marker Tial-1. (H) A subset of GFP-Rbpms2b positive granules are positive for the p-body marker Dcp2 (open arrowheads). (I) GFP-Rbpms2b^sa9329 localization to the centrosome/spindle but not granules. (J) GFP-Rbpms2b^ae32 and (K) GFP-Rbpms2a^ae30 localization. Insets show magnified views of the highlighted cells. Images are representative slices from Z-stacks of sphere stage embryos viewed from the animal pole.</p