Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

<div><p>Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved cytoarchitectural domain that specifies the AV axis in metazoan ova. </p> </div

Disheveled accumulation in the vegetal cortical domain begins during early oogenesis.

<p>Immunolocalization of Dsh during oogenesis in <i>Lytechinus </i><i>pictus</i>. Sections of the ovary were dissected and oocytes at different stages were released by gentle shaking of the tissue in seawater. (A-E) No Dsh protein is detected in primary oocytes. Cortical Dsh localization is detected in the mature egg on the right of the primary oocyte (white arrows in A and D). (E) Bright field view. (F-J) Midsize oocytes showing the first detectable accumulation of Dsh in the vegetal cortical domain (white arrow). Note the MTOC at the opposite end of the Dsh staining (white asterisk). (J) Bright field view. (K-N) Large oocytes show strong Dsh labeling in the vegetal cortical domain (white arrow). Note that strong Dsh staining is also seen in the MTOC (white asterisk) at this stage. Expression of Dsh protein is shown in red, filamentous actin is visualized with fluorescein phalloidin (green) and nuclei are visualized with DAPI staining (blue). Arrows indicate Dsh staining and the asterisks indicate the MTOC. (BF) Bright field view.</p

Disheveled protein is highly enriched at the vegetal pole of eggs and early embryos.

<p><i>S</i>. <i>purpuratus</i> eggs and embryos were processed for immunofluorescence and analyzed using scanning confocal microscopy. (A-F) Developmental stages from an unfertilized egg to a 60-cell stage embryo. Dsh was immunolocalized using an anti-Dsh antibody (red), filamentous actin was visualized using fluorescein phalloidin (green), and nuclei were visualized using DAPI (blue). Top panels show Dsh staining and the corresponding bottom panels show an overlay of Dsh with phalloidin and DAPI. All images are oriented with the animal pole towards the top and vegetal pole towards the bottom. (A) An unfertilized egg showing the asymmetric enrichment of Dsh at one pole. (B) Zygote stage. After fertilization the Dsh pattern becomes more punctate. (C) 8-cell stage embryo. Punctate Dsh staining is seen in the four cells. (D) 16-cell stage embryo. Punctate Dsh staining is observed in the micromeres and the macromeres indicating that the Dsh accumulation at earlier stages is at the vegetal pole. (E) 32-cell stage embryo. Punctate Dsh staining is seen in the macromeres and the vegetal tier cells. (F) 60-cell stage embryo. Dsh puncta are seen predominantly in the micromeres and the veg2 tier.</p

The pool of Disheveled protein in the vegetal cortical domain is differentially post-translationally modified.

<p>Approximately 40 mg of total protein from (A) eggs, (B) isolated cortical fragments, (C) 16-cell stage embryos, (D) 16-cell stage micromeres, were collected from <i>S</i>. <i>purpuratus</i> and then subjected to 2D gel electrophoresis, separating the proteins first by charge (on a pH 4 to pH 7 IPG strip) then by size, and then to Western blot analysis using anti-Dsh antibodies. Dsh (red) isoforms in isolated cortex samples and micromere samples are enriched near the acidic (left) side of the gel, while whole embryo samples show the presence of Dsh protein species that are not detected in the isolated cortex and 16-cell stage micromeres. Tubulin (green) serves as an internal control. </p

The Disheveled protein enriched at the vegetal pole is embedded in a vegetal cortical domain.

<p>Cortices were collected from S. purpuratus eggs and zygotes, prepared for anti-Dsh immunofluorescence and viewed using scanning confocal microscopy. (A-C) Wide field view of cortices isolated from unfertilized eggs shows the accumulation of Dsh in the vegetal cortical domain (—, 50µm). (D-F) A high magnification view of a single cortex labeled by anti-Dsh antibodies and phalloidin shows a uniform F-actin distribution and concentrated Dsh in one domain, (—, 10µm). Note the punctate appearance of the Dsh staining. (G-I) A cortex isolated from a zygote labeled with anti-Dsh antibodies and phalloidin shows that Dsh remains anchored in the vegetal cortical domain following fertilization (—, 10µm). (J-L) A higher magnification view of the Dsh puncta at the vegetal cortex shows that Dsh is embedded between short actin filaments, (—, 2.5µm). Insets on the left and top of each panel from D-L shows a 90˚ rotation of the 3D confocal dataset shown in D-L (x-y) and allows the same image to be viewed in cross sections, x-z and z-y, showing Dsh is embedded between short actin filaments.</p

The effect of cytoskeletal disruption on Disheveled localization and stability in the sea urchin egg.

<p>(A-L) The effect of disrupting microfilaments and microtubules on the localization of Dsh to the vegetal cortical domain. <i>S</i>. <i>purpuratus</i> eggs were treated with DMSO (A, E, I), cytochalasin B (B, F, J), cytochalasin D (C, G, K) or colchicine (D, H, L) for 20 minutes, washed and then incubated for a total time period of two hours. Following the incubation the eggs were processed for Dsh immunofluorescence (A-D) and for F-actin staining using fluorescein phalloidin (E-H). (I-L) are bright field views of the corresponding fluorescent images. (A) Control DMSO treated eggs show Dsh localization at one pole of the egg. (B) and (C) Dsh localization was abolished in eggs treated with 10 mg/ml cytochalasin B or D for 20 minutes, washed and incubated in seawater for a total time of 2 hours. (D) Eggs treated with 100 mM colchicine for 2 hours showed no effect on Dsh localization to the vegetal cortical domain. (M-O) Western blot analysis of Dsh protein in <i>S</i>. <i>purpuratus</i> eggs following cytochalasin and colchicine treatment. (M) The effect of cytochalasin and colchicine treatment on the stability of Dsh in <i>S</i>. <i>purpuratus</i> eggs. In some cases (lanes 2, 3, 5) the eggs were exposed to DMSO or the cytoskeletal disrupting drugs for 20 minutes, washed and then incubated for a total time of 2 hours prior to processing them for Western blot analysis. In other cases (lanes 4, 6, 7) the eggs were left in the drugs for the 2 hour incubation period prior to processing for Western blot analysis. Untreated control eggs (lane 1) , DMSO- (lane 2), and colchicine-treated (lane 7) eggs express the Dsh protein, while there is no Dsh detected in the cytochalasin-treated eggs (lanes 3-6). (N, O) Eggs were briefly exposed to either cytochalasin B or D for 5 minutes and then incubated in seawater for approximately 2 hours. Samples were collected for Western blot analysis at the times indicated in the figure. Loss of Dsh protein reactivity on the Western blots can be observed starting at 1:45 hr in cytochalasin B treated eggs, and at around 1:30 hours in cytochalasin D treated eggs. The same batch of eggs was used for immunofluorescence and Western blot analyses.</p

An early global role for Axin is required for correct patterning of the anterior-posterior axis in the sea urchin embryo

Activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3β-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a critical conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates

A dual role for Axin in establishing the anterior-posterior axis in the early sea urchin embryo

Abstract The activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres—in cleavage stage embryos—to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical negative regulator of cWnt signaling, during early sea urchin embryogenesis. Sea urchin Axin is ubiquitously expressed in early embryos and by the blastula stage the expression of the gene becomes restricted to the posterior end of the embryo. Strikingly, knockdown of Axin protein levels using antisense Axin morpholinos (MO) led to ectopic nuclearization of β-catenin and activation of endomesoderm gene expression in anterior blastomeres in early embryos. These embryos developed a severely posteriorized phenotype that could be fully rescued by co-injection of Axin MO with wild-type Axin mRNA. Axin is known to negatively regulate cWnt by its role in mediating β-catenin stability within the destruction complex. Consistent with this function overexpression of Axin by mRNA injection led to the downregulation of nuclear β-catenin, inhibition of endomesoderm specification and a strong anteriorization of embryos. Axin has several well-defined domains that regulate its interaction with β-catenin and the key regulators of the destruction complex, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase 3β(GSK-3β), and Dishevelled (Dvl). Using Axin constructs with single deletions of the binding sites for these proteins we showed that only the GSK-3βbinding site on Axin is required for its inhibition of cWnt in the sea urchin embryo. Strikingly, overexpression of the GSK-3β-binding domain alone led to embryos with elevated levels of endomesoderm gene expression and a strongly posteriorized phenotype. These results indicated that Axin has a critical global role in inhibiting cWnt signaling in the early sea urchin embryo, and moreover, that the interaction of Axin with GSK-3βis critical for this inhibition. These results also add to the growing body of evidence that Axin plays a global function in suppressing cWnt signaling in early embryos and indicates that modulation of Axin function may be a critical early step during patterning of the AP axis during bilaterian developmen