Strabismus-mediated primary archenteron invagination is uncoupled from Wnt/β-catenin-dependent endoderm cell fate specification in Nematostella vectensis (Anthozoa, Cnidaria): Implications for the evolution of gastrulation

<p>Abstract</p> <p>Background</p> <p>Gastrulation is a uniquely metazoan character, and its genesis was arguably the key step that enabled the remarkable diversification within this clade. The process of gastrulation involves two tightly coupled events during embryogenesis of most metazoans. Morphogenesis produces a distinct internal epithelial layer in the embryo, and this epithelium becomes segregated as an endoderm/endomesodermal germ layer through the activation of a specific gene regulatory program. The developmental mechanisms that induced archenteron formation and led to the segregation of germ layers during metazoan evolution are unknown. But an increased understanding of development in early diverging taxa at the base of the metazoan tree may provide insights into the origins of these developmental mechanisms.</p> <p>Results</p> <p>In the anthozoan cnidarian <it>Nematostella vectensis</it>, initial archenteron formation begins with bottle cell-induced buckling of the blastula epithelium at the animal pole. Here, we show that bottle cell formation and initial gut invagination in <it>Nematostella </it>requires NvStrabismus (NvStbm), a maternally-expressed core component of the Wnt/Planar Cell Polarity (PCP) pathway. The NvStbm protein is localized to the animal pole of the zygote, remains asymmetrically expressed through the cleavage stages, and becomes restricted to the apical side of invaginating bottle cells at the blastopore. Antisense morpholino-mediated NvStbm-knockdown blocks bottle cell formation and initial archenteron invagination, but it has no effect on Wnt/ß-catenin signaling-mediated endoderm cell fate specification. Conversely, selectively blocking Wnt/ß-catenin signaling inhibits endoderm cell fate specification but does not affect bottle cell formation and initial archenteron invagination.</p> <p>Conclusions</p> <p>Our results demonstrate that Wnt/PCP-mediated initial archenteron invagination can be uncoupled from Wnt/ß-catenin-mediated endoderm cell fate specification in <it>Nematostella</it>, and provides evidence that these two processes could have evolved independently during metazoan evolution. We propose a two-step model for the evolution of an archenteron and the evolution of endodermal germ layer segregation. Asymmetric accumulation and activation of Wnt/PCP components at the animal pole of the last common ancestor to the eumetazoa may have induced the cell shape changes that led to the initial formation of an archenteron. Activation of Wnt/ß-catenin signaling at the animal pole may have led to the activation of a gene regulatory network that specified an endodermal cell fate in the archenteron.</p

Autonomous Cell Specification: Overview

Autonomous cell specification is a form of specification in which the developing cell is able to differentiate (express proteins typical of the mature cell) without receiving external signals. This is possible when cytoplasmic determinants (the cytoplasmic regulatory factors necessary for specification) are partitioned into the cell during cleavage.Autonomous cell specification is a form of specification in which the developing cell is able to differentiate (express proteins typical of the mature cell) without receiving external signals. This is possible when cytoplasmic determinants (the cytoplasmic regulatory factors necessary for specification) are partitioned into the cell during cleavage

3 - Otx, β-Catenin, and the Specification of Ectodermal Cell Fates in the Sea Urchin Embryo

This chapter reviews the current status of the specification events leading to the differentiation of ectodermal cell types in sea urchin embryo. In sea urchins, the fates of all the major cell types are specified very early in embryogenesis, and it is the establishment of these individual cell types, rather than reiterative patterning along a body axis, which is critical for embryonic development. This chapter describes the embryological and molecular features that make ectodermal cell fate specification an attractive experimental system. The prevailing model that the activities of uniformly distributed transcription factors are regionally modulated by external signaling pathways is evaluated in light of new experiments. A comparison is made of the relevance of recent findings on sea urchin development to other organisms and how evolutionary mechanisms influence cell fate-specification processes is considered. The chapter also presents the current evidence linking extracellular signaling events with ectoderm-specific gene expression

Visualizing egg and embryonic polarity

During development metazoan embryos have to establish the molecular coordinates for elaboration of the embryonic body plan. Typically, bilaterian (bilaterally symmetric animals) embryos establish anterior-posterior (AP) and dorsal-ventral (DV) axes, and in most cases the AP axis is established first. For over a century it has been known that formation of the AP axis is strongly influenced by the primary axis of the egg, the animal-vegetal (AV) axis. The molecular basis for how the AV axis influences AP polarity remains poorly understood, but sea urchins have proven to be important for elucidating the molecular basis for this process. In fact, it is the first model system where a critical role for Wnt signaling in specification and patterning the AV and AP axis was first established. One current area of research is focused on identifying the maternal factors that regulate localized activation of Wnt/β-catenin signaling at the vegetal pole during development. An essential tool for this work is the means to identify the AV polarity in oocytes and eggs. This permits investigation into how polarity is established and allows development of experimental strategies to identify maternal factors that contribute to and control axial polarity. This chapter provides protocols to accomplish this in sea urchin eggs and early embryos. We describe simple methods to visualize polarity including direct observation of eggs and oocytes, using a microscope for overt morphological signs of polarity, and more extensive methods involving localization of known factors indicative of inherent embryonic polarity, such as the upstream regulators of the Wnt/β-catenin pathway

Nuclearization of β-catenin in ectodermal precursors confers organizer-like ability to induce endomesoderm and pattern a pluteus larva

BACKGROUND: In many bilaterians, asymmetric activation of canonical Wnt (cWnt) signaling at the posterior pole is critical for anterior-posterior (AP) body axis formation. In 16-cell stage sea urchins, nuclearization of β-catenin in micromeres activates a gene regulatory network that defines body axes and induces endomesoderm. Transplanting micromeres to the animal pole of a host embryo induces ectopic endomesoderm in the mesomeres (ectoderm precursors) whereas inhibiting cWnt signaling blocks their endomesoderm-inducing activity and the micromeres become ectoderm-like. We have tested whether ectopic activation of cWnt signaling in mesomeres is sufficient to impart the cells with organizer-like abilities, allowing them to pattern normal embryonic body axes when recombined with a field of mesomeres. RESULTS: Fertilized eggs were microinjected with constitutively active Xenopus β-catenin (actβ-cat) mRNA and allowed to develop until the 16-cell stage. Two mesomeres from injected embryos were then recombined with isolated animal halves (AH) from uninjected 16-cell stage embryos. Control chimeras produced animalized phenotypes (hollow balls of ectoderm) and rarely formed skeletogenic mesoderm (SM)-derived spicules, endoderm or pigment cells, a type of non-skeletogenic mesoderm (NSM). In contrast, over half of the 0.5 pg/pL actβ-cat mesomere/AH chimeras formed a partial or complete gut (exhibiting AP polarity), contained mesenchyme-like cells similar to SM, and produced pigment cells. At three days, chimeras formed plutei with normal embryonic body axes. When fates of the actβ-cat mRNA-injected mesomeres were tracked, we found that injected mesomeres formed mesenchyme-like and pigment cells, but endoderm was induced. Higher concentrations of actβ-cat mRNA were less likely to induce endoderm or pigment cells, but had similar mesenchyme-like cell production to 0.5 pg/pL actβ-cat mesomere/AH chimeras. CONCLUSIONS: Our results show that nuclear β-catenin is sufficient to endow naïve cells with the ability to act as an organizing center and that β-catenin has both cell-autonomous and non-autonomous effects on cell fate specification in a concentration-dependent manner. These results are consistent with the hypothesis that a shift in the site of early cWnt signaling in cleaving embryos could have modified polarity of the main body axes during metazoan evolution

Requirement of SpOtx in Cell Fate Decisions in the Sea Urchin Embryo and Possible Role as a Mediator of β-Catenin Signaling

We show here that the homeodomain transcription factor SpOtx is required for endoderm and aboral ectoderm formation during sea urchin embryogenesis. SpOtx target genes were repressed by fusing the SpOtx homeodomain to an active repression domain of Drosophila Engrailed. The Engrailed–SpOtx fusion protein reduced the expression of endoderm- and aboral ectoderm-specific genes and inhibited the formation of endoderm and aboral ectoderm cell types. Coexpressing activated β-catenin with Engrailed–SpOtx did not overcome the inhibition of endoderm and aboral ectoderm formation, suggesting that SpOtx functioned either downstream of or parallel to nuclear β-catenin. Embryos expressing C-cadherin, which blocks nuclear translocation of β-catenin, have defects in endoderm and aboral ectoderm formation. Coexpressing SpOtx with C-cadherin restored aboral ectoderm-specific gene expression and aboral ectoderm morphology, but with C-cadherin present, SpOtx was not sufficient for endoderm formation. Our results show that SpOtx plays a key role in the activation of aboral ectoderm- and endoderm-specific gene expression and, in addition, suggest that SpOtx mediates some of β-catenin's functions in endoderm and aboral ectoderm formation

β-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal–vegetal axis in sea urchin embryos are largely unknown. Nuclear β-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that β-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that β-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal–vegetal axis. Our results also reveal similarities between the sea urchin animal–vegetal axis and the vertebrate dorsal–ventral axis, suggesting that these axes share a common evolutionary origin