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.

Distinct Frizzled receptors independently mediate endomesoderm specification and primary archenteron invagination during gastrulation in Nematostella.

Endomesodermal cell fate specification and archenteron formation during gastrulation are tightly linked developmental processes in most metazoans. However, studies have shown that in the anthozoan cnidarian Nematostella vectensis, Wnt/ß-catenin (cWnt) signalling-mediated endomesodermal cell fate specification can be experimentally uncoupled from Wnt/Planar Cell Polarity (PCP) signalling-mediated primary archenteron invagination. The upstream signalling mechanisms regulating cWnt signalling-dependent endomesoderm cell fate specification and Wnt/PCP signalling-mediated primary archenteron invagination in Nematostella embryos are not well understood. By screening for potential upstream mediators of cWnt and Wnt/PCP signalling, we identified two Nematostella Frizzled homologs that are expressed early in development. NvFzd1 is expressed maternally and in a broad pattern during early development while NvFzd10 is zygotically expressed at the animal pole in blastula stage embryos and is restricted to the invaginating cells of the presumptive endomesoderm. Molecular and morphological characterization of NvFzd1 and NvFzd10 knock-down phenotypes provide evidence for distinct regulatory roles for the two receptors in endomesoderm cell fate specification and primary archenteron invagination. These results provide further experimental evidence for the independent regulation of endomesodermal cell fate specification and primary archenteron invagination during gastrulation in Nematostella. Moreover, these results provide additional support for the previously proposed two-step model for the independent evolution of cWnt-mediated cell fate specification and Wnt/PCP-mediated primary archenteron invagination.

Carbon Nitride Dots: A Selective Bioimaging Nanomaterial.

In contrast to the recent immense attention in carbon nitride quantum dots (CNQDs) as a heteroatom-doped carbon quantum dot (CQD), their biomedical applications have not been thoroughly investigated. Targeted cancer therapy is a prominently researched area in the biomedical field. Here, the ability of CNQDs as a selective bioimaging nanomaterial was investigated to assist targeted cancer therapy. CNQDs were first synthesized using four different precursor sets involving urea derivatives, and the characteristics were compared to select the best candidate material for bioapplications. Characterization techniques such as UV-vis, luminescence, X-ray photoelectron spectroscopy, nuclear magnetic resonance spectroscopy, and transmission electron microscopy were used. These CNQDs were analyzed in in vitro studies of bioimaging and labeling using pediatric glioma cells (SJGBM2) for possible selective biolabeling and nanodistribution inside the cell membrane. The in vitro cellular studies were conducted under long-wavelength emission without the interference of blue autofluorescence. Thus, excitation-dependent emission of CNQDs was proved to be advantageous. Importantly, CNQDs selectively entered SJGBM2 tumor cells, while it did not disperse into normal human embryonic kidney cells (HEK293). The distribution studies in the cell cytoplasm indicated that CNQDs dispersed into lysosomes within approximately 6 h after the incubation. The CNQDs exhibited great potential as a possible nanomaterial in selective bioimaging and drug delivery for targeted cancer therapy.

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo.

Canonical Wnt/ß-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with ß-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

Polarized Dishevelled dissolution and reassembly drives embryonic axis specification in sea star oocytes.

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes.1 However, the mechanisms that generate cellular asymmetries within the oocyte and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled (Dvl) is a cytoplasmic Wnt pathway effector required for axis development in diverse species,2-4 but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dvl localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially regulated Dvl dynamics.

Metformin derived carbon dots: Highly biocompatible fluorescent nanomaterials as mitochondrial targeting and blood-brain barrier penetrating biomarkers.

Carbon dots (CDs) have been intensively studied since their discovery in 2004 because of their unique properties such as low toxicity, excellent biocompatibility, high photoluminescence (PL) and good water dispersibility. In this study metformin derived carbon dots (Met-CDs) were synthesized using a microwave assisted method. Met-CDs were meticulously characterized using ultra-violet spectroscopy (UV-vis), photoluminescence (PL), Fourier Transform Infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force (AFM) and transmission electron (TEM) microscopies. According to results of cytotoxicity studies, Met-CDs possess low-toxicity and excellent biocompatibility towards both non-tumor and tumor cell lines indicating that Met-CDs are outstanding candidates for living cell bioimaging studies. Furthermore, bioimaging studies have displayed that Met-CDs can penetrate the cell membrane and disperse throughout the cell structure including the nucleus and mitochondria. More specifically, Met-CDs tend to start localizing selectively inside the mitochondria of cancer cells, but not of non-tumor cells after 1 h of incubation. Finally, a zebrafish study confirmed that Met-CDs cross the blood-brain barrier (BBB) without the need of any other ligands. In summary, this study presents synthesis of Met-CDs which feature abilities such as mitochondrial and nucleus localizations along with BBB penetration.

Gel-like carbon dots: A high-performance future photocatalyst.

To protect water resources, halt waterborne diseases, and prevent future water crises, photocatalytic degradation of water pollutants arouse worldwide interest. However, considering the low degradation efficiency and risk of secondary pollution displayed by most metal-based photocatalysts, highly efficient and environmentally friendly photocatalysts with appropriate band gap, such as carbon dots (CDs), are in urgent demand. In this study, the photocatalytic activity of gel-like CDs (G-CDs) was studied using diverse water pollution models for photocatalytic degradation. The degradation rate constants demonstrated a remarkably enhanced photocatalytic activity of G-CDs compared with most known CD species and comparability to graphitic carbon nitride (g-C3N4). In addition, the rate constant was further improved by 1.4 times through the embedment of g-C3N4 in G-CDs to obtain CD-C3N4. Significantly, the rate constant was also higher than that of g-C3N4 alone, revealing a synergistic effect. Moreover, the use of diverse radical scavengers suggested that the main contributors to the photocatalytic degradation with G-CDs alone were superoxide radicals (O2-) and holes that were, however, substituted by O2- and hydroxyl radicals (OH) due to the addition of g-C3N4. Furthermore, the photocatalytic stabilities of G-CDs and CD-C3N4 turned out to be excellent after four cycles of dye degradation were performed continuously. Eventually, the nontoxicity and environmental friendliness of G-CDs and CD-C3N4 were displayed with sea urchin cytotoxicity tests. Hence, through various characterizations, photocatalytic degradation and cytotoxicity tests, G-CDs proved to be an environmentally friendly and highly efficient future photocatalyst.

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.

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/ß-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/ß-catenin pathway.

Detecting expression patterns of Wnt pathway components in Nematostella vectensis embryos.

The anthozoan cnidarian Nematostella vectensis has emerged as a key model system for evolutionary developmental biology studies, and this animal' usefulness will grow with the recent sequencing of its genome. In particular, work done in Nematostella is providing insight into the role of the Wnt pathway in the evolution of pattern formation. This chapter describes methods to maintain and spawn these animals, and detailed protocols to detect expression patterns of Wnt pathway components in Nematostella eggs and embryos.

Wnt signaling in the early sea urchin embryo.

Wnt signaling regulates a remarkably diverse array of cellular and developmental events during animal embryogenesis and homeostasis. The crucial role that Wnt signaling plays in regulating axial patterning in early embryos has been particularly striking. Recent work has highlighted the conserved role that canonical Wnt signaling plays in patterning the animal-vegetal (A-V) axis in sea urchin and sea anemone embryos. In sea urchin embryos, the canonical Wnt signaling pathway is selectively turned on in vegetal cells as early as the 16-cell stage embryo, and signaling through this pathway is required for activation of the endomesodermal gene regulatory network. Loss of nuclear beta-catenin signaling animalizes the sea urchin embryo and blocks pattern formation along the entire A-V axis. Nuclear entry of beta-catenin into vegetal cells is regulated cell autonomously by maternal information that is present at the vegetal pole of the unfertilized egg. Analysis of Dishevelled (Dsh) regulation along the A-V axis has revealed the presence of a cytoarchitectural domain at the vegetal pole of the unfertilized sea urchin egg. This vegetal cortical domain appears to be crucial for the localized activation of Dsh at the vegetal pole, but the precise mechanisms are unknown. The elucidation of how Dsh is selectively activated at the vegetal cortical domain is likely to provide important insight into how this enigmatic protein is regulated during canonical Wnt signaling. Additionally, this information will shed light on the origins of embryonic polarity during animal evolution. This chapter examines the roles played by the canonical Wnt signaling pathway in the specification and patterning of the A-V axis in the sea urchin. These studies have led to the identification of a novel role for canonical Wnt signaling in regulating protein stability, and continued studies of Wnt signaling in this model system are likely to reveal additional roles for this pathway in regulating early patterning events in embryos.

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.

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.

Two Extracellular Matrices From Oocytes of the Marine Shrimp Sicyonia ingentis that Independently Mediate Only Primary or Secondary Sperm Binding: (sperm/fertilization/shrimp/gamete binding/extracellular matrix).

During spawning, female Sicyonia ingentis simultaneously release ova and stored nonmotile sperm and mix them externally to initiate gamete interaction. Sperm bind to a thin vitelline envelope (VE) via their anterior appendage and within seconds are induced to undergo acrosomal exocytosis. The sperm penetrate the VE and become secondarily bound to the surface coat (SC), a glycocalyx on the oocyte surface. In this study, both extracellular matrices were isolated from S. ingentis oocytes. Isolated VEs mediated only primary sperm binding (i.e., before the acrosome reaction), while the isolated SCs mediated only secondary sperm binding (i.e., after acrosomal exocytosis). Isolated S. ingentis VEs were used to characterize primary sperm binding activity. The two extracellular matrices differ morphologically and possess different polypeptide profiles. Soluble fractions of isolated VEs inhibited primary sperm binding in a concentration dependent manner, and immunolocalization of VE components demonstrated highly localized VE binding sites at the tip of the sperm anterior appendage by which sperm bind eggs. Extensive Pronase digestion of VE components did not affect sperm binding activity of solubilized VE components, while complete deglycosylation with trifluoromethanesulfonic acid destroyed sperm binding activity. However, neither alkaline treatment nor enzyme digestion using glycosidases specific for asparagine and serine/ threonine linked oligosaccharides affected sperm binding activity.

Sperm of the Shrimp Sicyonia ingentis Undergo a Bi-Phasic Capacitation Accompanied by Morphological Changes.

Sperm removed from seminal receptacles of female Sicyonia ingentis can be induced to undergo a bi-phasic acrosome reaction (AR), acrosomal exocytosis followed by filament formation, using egg water (EW). Sperm removed from males will not undergo any phase of the AR when incubated with EW, indicating that these sperm undergo a capacitation process after insemination. Freshly molted females (functional virgins) were placed in aquaria with males and monitored for copulation. Mated females were isolated and allowed to carry sperm for specific periods of time. At these time points, sperm were removed and assayed for the ability to undergo the AR using EW. The results indicate that sperm are competent to undergo acrosomal exocytosis after approximately 25 hr, while competency to form acrosomal filaments is not achieved until around 145 hr post-insemination. Morphological examination of sperm removed from males and sperm removed from females revealed dramatic differences. Microscopic evidence indicates that some of the morphological changes seen during capacitation are necessary for the successful completion of the AR.

Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a beta-catenin dependent pathway.

Sea urchin (Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of beta-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC(50) of creosote for complete exogastrulation being 1.57 ppm, while the EC(50)s of the PAHs ranged from 0.41 ppm (2.0 microM) to 4.33 ppm (33.5 microM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against beta-catenin, a multi-functional protein known to be involved in cell-cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of beta-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since beta-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types.

Nuclearization of ß-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 ß-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 ß-catenin (actß-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 actß-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 actß-cat mRNA-injected mesomeres were tracked, we found that injected mesomeres formed mesenchyme-like and pigment cells, but endoderm was induced. Higher concentrations of actß-cat mRNA were less likely to induce endoderm or pigment cells, but had similar mesenchyme-like cell production to 0.5 pg/pL actß-cat mesomere/AH chimeras. CONCLUSIONS: Our results show that nuclear ß-catenin is sufficient to endow naïve cells with the ability to act as an organizing center and that ß-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.

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.

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 ß-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.