Blocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis, but not secondary mesoderm formation.

Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca(2+) signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis.

Detecting expression patterns of Wnt pathway components in sea urchin embryos.

The animal-vegetal (A-V) axis is a maternally established asymmetry that is present in most animal eggs, and it plays an important role in germ-layer segregation. Recent work has shown that the canonical Wnt signaling pathway plays an evolutionarily conserved role in specifying and patterning this axis. However, the precise mechanisms by which this pathway is activated in the early embryo to pattern the A-V axis are not known in most animals. The availability of the Strongylocentrotus purpuratus genome sequence, the ability to experimentally manipulate eggs and early embryos using embryological and molecular tools, and the superior optical clarity of sea urchin embryos makes them an important model for investigating the role of the canonical Wnt pathway in specifying and patterning the A-V axis. Here, we provide detailed protocols for determining the expression and localization of mRNA and proteins in early sea urchin embryos, which can be used in studies examining the regulation of Wnt signaling along the A-V axis.

Functional analysis of Wnt signaling in the early sea urchin embryo using mRNA microinjection.

The Wnt pathway is a highly conserved signal transduction pathway that plays many critical roles in early animal development. Recent studies have shown that this pathway plays a conserved role in the specification and patterning of the animal-vegetal (A-V) axis in sea urchins and sea anemones. These observations have suggested that the common ancestor to cnidarians and bilaterians used the Wnt signaling pathway for specifying and patterning this maternally established axis. Because the A-V axis plays a critical role in germ layer segregation, a better understanding of how the Wnt pathway is regulated along the A-V axis will provide key insight into the molecular mechanisms regulating germ layer segregation and germ layer evolution in animal embryos. Here, we provide a detailed protocol for using mRNA microinjection that can be used to analyze Wnt signaling in early sea urchin embryos. This protocol can also be adapted to introduce morpholino anti-sense oligonucleotides into sea urchin embryos.

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages.

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.

An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.