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

Abstract

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.

Figure 1. Disheveled protein is highly enriched at the vegetal pole of eggs and early embryos.

S. purpuratus 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.

Figure 2. The Disheveled protein enriched at the vegetal pole is embedded in a vegetal cortical domain.

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.

Figure 3. The pool of Disheveled protein in the vegetal cortical domain is differentially post-translationally modified.

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 S. purpuratus 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.

Figure 4. Disheveled accumulation in the vegetal cortical domain begins during early oogenesis.

Immunolocalization of Dsh during oogenesis in Lytechinus pictus. 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.

Figure 5. The effect of cytoskeletal disruption on Disheveled localization and stability in the sea urchin egg.

(A-L) The effect of disrupting microfilaments and microtubules on the localization of Dsh to the vegetal cortical domain. S. purpuratus 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 S. purpuratus eggs following cytochalasin and colchicine treatment. (M) The effect of cytochalasin and colchicine treatment on the stability of Dsh in S. purpuratus 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.