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 crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates.

© 2017 Wiley Periodicals, Inc. & M Most animal eggs have a primordial polarity, termed the animalvegetal (AV) axis, that frequently predicts the anterior-posterior (AP) axis of the embryo and adult. Transformation of the AV polarity of the egg to the AP polarity of the embryo in many species is mediated by the localized activation of canonical Wnt (cWnt) signaling in vegetal blastomeres by maternal determinants. In the sea urchin egg, cWnt signal transduction is facilitated by localized “activation” of the Disheveled protein, a central regulator of Wnt signaling. While Disheveled protein is broadly distributed in the oocyte and early embryo, it is highly enriched at the eggs’ vegetal pole (top left, inset; diameter is ~72 μm). In isolated egg cortices, Disheveled is tightly associated with the cortex (background image), and forms 0.1-0.2 μm diameter puncta that appear to be embedded between short actin fi laments (bottom left). This pool of Disheveled is differentially posttranslationally modifi ed, and is later inherited by the blastomeres that activate cWnt signaling to specify the endomesoderm. The mechanisms that tether Disheveled to the vegetal cortical domain and subsequently activate it locally are not known, but elucidation of these processes will be critical for understanding how the AV axis is initially specifi ed and how this polarity leads to early patterning of animal embryos.

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.

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

INTRODUCTION The discovery that -catenin is an early activator of endomesoderm specification provided a key observation that led to the assembly of a model endomesoderm gene regulatory network (GRN) in the sea urchin (Wikramanayake et al., 1998; Logan et al., 1999; Davidson et al., 2002; Oliveri et al., 2002). Many experiments expanded that early network model by adding a number of transcription factors and signals. Cis-regulatory analyses plus new mechanistic insights continue to update causal information on how embryonic sea urchin cells are specified and diversify (Davidson, 2006; Oliveri et al., 2006; Smith et al., 2007; Croce and McClay, 2010). Those early network models also stimulated interest in networks more broadly in other systems (Sandmann et al., 2007; Zeitlinger et al., 2007; Owraghi et al., 2009). A remaining gap in the sea urchin network is the issue of how the developmental program is initiated in the first place. Boveri (Boveri, 1901) was among the first to observe that the endomesoderm originates from the vegetal half of the embryo, and studies with egg fragments experimentally confirmed that observation (Horstadius, 1927; Horstadius, 1928). More recent studies in the starfish showed that a relatively small region of the vegetal hemisphere contained the material necessary for endoderm specification (Kuraishi and Osanai, 1994). If that small vegetal region was removed, an archenteron failed to appear (Kiyomoto and Shirai, 1993). Those studies, and others like them, extending to vertebrates, strongly suggested that information near the vegetal pole was important for activating endomesoderm specification, but the identity of the active principle in the vegetal region remained unknown. Experiments with -catenin first suggested that the canonical Wnt pathway is involved in activation of the endomesoderm GRN (Emily-Fenouil et al., 1998; Wikramanayake et al., 1998; Logan et al., 1999). Exploring this further, Weitzel et al. (Weitzel et al., 2004) showed that expressed -catenin-GFP protein initially was found throughout the zygote, but over a short time, beginning at the 60-cell stage, was degraded in animal blastomeres and stabilized in vegetal blastomeres. The stabilization required another member of the Wnt pathway, the cytoplasmic effector Dishevelled (Dsh) (Weitzel et al., 2004). Though Dsh mRNA was ubiquitously distributed during cleavage, it was observed that an expressed Dsh-GFP protein reporter accumulated at the vegetal cortex of the egg, and further studies showed that a specific peptide in the Dsh molecule was necessary for the polarized protein accumulation (Leonard and Ettensohn, 2007). These data suggested a possible mechanism for localized activation of the endomesoderm gene regulatory network involving localized components of the Wnt pathway in the vegetal cortex of the egg. The present study was undertaken to establish the role of the vegetal cortex in activating the endomesoderm GRN and to identify the maternal determinants necessary for this process. Here, we show that the vegetal cortex, possibly exclusively through the canonical Wnt pathway, provides a localized center for endomesoderm specification, and that maternal Wnt6 is essential to trigger endoderm specification. Development 138, 3297-3306 (2011) doi:10.1242/dev.058792 © 2011. Published by The Company of Biologists Ltd