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

Activation of Wnt/ß-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-3ß-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.

A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning.

Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method.

Reconstruction of a starfish embryo provides unique morphogenesis during the developmental process that is not observed in normal development. Here, we established a novel method for reconstruction from single embryos/larvae. By using this method, we investigated the morphogenetic capabilities in critical steps during the reconstruction process as showed by the reconstructed embryos generated from embryos/larvae at the six developmental stages, or from segregated ectodermal and/or endomesodermal cells. Additionally, the novel method addressed several problems found in prior methods related to reproducibly generating reconstructed embryos. In the reconstructions from the various stage embryos/larvae, the morphogenetic capabilities were substantively reduced in the reconstructed embryos generated from 3-day bipinnaria (3dBp). The combination experiments using ectodermal or endomesodermal cells segregated from 2dBp or 3dBp showed a reduction of the morphogenetic capabilities in both cells types in 3dBp. The reconstructed embryos generated from ectodermal or endomesodermal cells segregated from 2dBp possessed partial morphological features, such as formation of the epithelium or blastopore, but all failed to develop into bipinnariae. These results indicate two limitations of the morphogenetic capabilities during the reconstruction process. Firstly, the morphogenetic capabilities to reconstruct an embryo are considerably reduced between 2dBp and 3dBp. Secondly, cells specified as ectoderm or endomesoderm possess limited morphogenetic capabilities to reconstruct bipinnaria. Furthermore, our results demonstrate that the interaction between these specified cell types is required for reconstruction.

Spiralian gastrulation: germ layer formation, morphogenesis, and fate of the blastopore in the slipper snail Crepidula fornicata.

BACKGROUND: Gastrulation is a critical step in bilaterian development, directly linked to the segregation of germ layers, establishment of axes, and emergence of the through-gut. Theories about the evolution of gastrulation often concern the fate of the blastopore (site of endomesoderm internalization), which varies widely in a major branch of bilaterians, the Spiralia. In this group, the blastopore has been said to become the mouth, the anus, both, or neither. Different developmental explanations for this variation exist, yet no modern lineage tracing study has ever correlated the position of cells surrounding the blastopore with their contribution to tissues of the mouth, foregut, and anus in a spiralian. This is the first study to do so, using the gastropod Crepidula fornicata. RESULTS: Crepidula gastrulation occurs by epiboly: the first through third quartet micromeres form an epithelial animal cap that expands to cover vegetal endomesodermal precursors. Initially, descendants of the second and third quartet micromeres (2a-2d, 3a-3d) occupy a portion of the blastopore lip. As the blastopore narrows, the micromeres' progeny exhibit lineage-specific behaviors that result in certain sublineages leaving the lip's edge. Anteriorly, cells derived from 3a(2) and 3b(2) undergo a unique epithelial-to-mesenchymal transition involving proliferation and a collective movement of cells into the archenteron. These cells make a novel spiralian germ layer, the ectomesoderm. Posteriorly, cells derived from 3c(2) and 3d(2) undergo a form of convergence and extension that involves zippering of cells and their intercalation across the ventral midline. During this process, several of these cells, as well as the 2d clone, become displaced posteriorly, away from the blastopore. Progeny of 2a-2c and 3a-3d make the mouth and foregut, and the blastopore becomes the opening to the mouth. The anus forms days later, as a secondary opening within the 2d(2) clone, and not from the classically described "anal cells", which we identify as the 3c(221) and 3d(221) cells. CONCLUSIONS: Our analysis of Crepidula gastrulation constitutes the first description of blastopore lip morphogenesis and fates using lineage tracing and live imaging. These data have profound implications for hypotheses about the evolution of the bilaterian gut and help explain observed variation in blastopore morphogenesis among spiralians.

E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis.

Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. Caenorhabditis elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two Cullin-RING ubiquitin ligase (CRL) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/ß-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we propose that ubiquitin-mediated protein degradation, like many transcription factors, function broadly as regulators driving developmental progression during embryogenesis in C. elegans.

Dose-dependent nuclear ß-catenin response segregates endomesoderm along the sea star primary axis.

In many invertebrates, the nuclearization of ß-catenin at one pole of the embryo initiates endomesoderm specification. An intriguing possibility is that a gradient of nuclear ß-catenin (nß-catenin), similar to that operating in vertebrate neural tube patterning, functions to distinguish cell fates in invertebrates. To test this hypothesis, we determined the function of nß-catenin during the early development of the sea star, which undergoes a basal deuterostomal mode of embryogenesis. We show that low levels of nß-catenin activity initiate bra, which is expressed in the future posterior endoderm-fated territory; intermediate levels are required for expression of foxa and gata4/5/6, which are later restricted to the endoderm; and activation of ets1 and erg in the mesoderm-fated territory requires the highest nß-catenin activity. Transcription factors acting downstream of high nß-catenin segregate the endoderm/mesoderm boundary, which is further reinforced by Delta/Notch signaling. Significantly, therefore, in sea stars, endomesoderm segregation arises through transcriptional responses to levels of nß-catenin activity. Here, we describe the first empirical evidence of a dose-dependent response to a dynamic spatiotemporal nß-catenin activity that patterns cell fates along the primary axis in an invertebrate.