Determination of the membrane topology of PORCN, an O-acyl transferase that modifies Wnt signalling proteins.

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos.

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.

The Use of Chick Embryos to Study Wnt Activity Gradients.

The chick spinal cord provides a valuable model for assessing Wnt signaling activity. Loss or gain of function constructs that are transfected by electroporation can be directed to a single side of the spinal cord, thus leaving the contralateral side as an internal control. Here, we describe a method for measuring Wnt signaling via the use of BAT-Gal, a ß-catenin dependent Wnt reporter.

Divergent effects of Porcupine and Wntless on WNT1 trafficking, secretion, and signaling.

Loss-of-function studies have identified Porcupine (PORCN) and Wntless (WLS) as essential mediators of Wnt secretion and signaling. Whereas PORCN is thought to palmitoylate Wnt proteins, WLS is believed to transport palmitoylated Wnt proteins to the cell surface. However, little is known about how these two proteins cooperate to regulate Wnt palmitoylation, trafficking, secretion, and signaling. We first investigated possible interactions between PORCN, WLS, and WNT1, by carrying out co-immunoprecipitation studies. These studies demonstrate the existence of a complex containing PORCN and WLS. They further show that PORCN and WLS compete for binding to WNT1. Then, we used gain-of-function studies to investigate the cooperation between PORCN and WLS as well as possible biochemical interactions between PORCN, WLS, and WNT1. Consistent with the proposed roles for PORCN and WLS, we show that overexpression of PORCN promotes palmitoylation of WNT1 while overexpression of WLS does not. Overexpression of PORCN enhances the ability of WLS to promote WNT1 trafficking to the cell surface as well as secretion, but decreases the ability of WLS to activate WNT1 signaling in target cell. These observations suggest that the levels of WNT1 on the cell surface and in the media are not the sole determinants of the activation of Wnt signaling in target cells.

Measuring empathy for human and robot hand pain using electroencephalography.

This study provides the first physiological evidence of humans' ability to empathize with robot pain and highlights the difference in empathy for humans and robots. We performed electroencephalography in 15 healthy adults who observed either human- or robot-hand pictures in painful or non-painful situations such as a finger cut by a knife. We found that the descending phase of the P3 component was larger for the painful stimuli than the non-painful stimuli, regardless of whether the hand belonged to a human or robot. In contrast, the ascending phase of the P3 component at the frontal-central electrodes was increased by painful human stimuli but not painful robot stimuli, though the interaction of ANOVA was not significant, but marginal. These results suggest that we empathize with humanoid robots in late top-down processing similarly to human others. However, the beginning of the top-down process of empathy is weaker for robots than for humans.

Beautiful friendship: Social sharing of emotions improves subjective feelings and activates the neural reward circuitry.

Humans have a strong tendency to affiliate with other people, especially in emotional situations. Here, we suggest that a critical mechanism underlying this tendency is that socially sharing emotional experiences is in itself perceived as hedonically positive and thereby contributes to the regulation of individual emotions. We investigated the effect of social sharing of emotions on subjective feelings and neural activity by having pairs of friends view emotional (negative and positive) and neutral pictures either alone or with the friend. While the two friends remained physically separated throughout the experiment-with one undergoing functional magnetic resonance imaging and the other performing the task in an adjacent room-they were made aware on a trial-by-trial basis whether they were seeing pictures simultaneously with their friend (shared) or alone (unshared). Ratings of subjective feelings were improved significantly when participants viewed emotional pictures together than alone, an effect that was accompanied by activity increase in ventral striatum and medial orbitofrontal cortex, two important components of the reward circuitry. Because these effects occurred without any communication or interaction between the friends, they point to an important proximate explanation for the basic human motivation to affiliate with others, particularly in emotional situations.

Concentration-dependent effects of WNTLESS on WNT1/3A signaling.

BACKGROUND: WNTLESS (WLS) is a multi-transmembrane protein that transports Wnt ligands from the Golgi to the cell surface. Although WLS loss-of-function experiments in the developing central nervous system reveal phenotypes consistent with defects in WNT1 and WNT3A signaling, data from complementary gain-of-function experiments have not yet been reported. Here, we report the phenotypic consequences of WLS overexpression in cultured cells and in the developing chick spinal cord. RESULTS: Overexpression of small amounts of WLS along with either WNT1 or WNT3A promotes the Wnt/ß-catenin pathway in HEK293T cells, while overexpression of higher levels of WLS inhibits the Wnt/ß-catenin pathway in these cells. Similarly, overexpressed WLS inhibits the Wnt/ß-catenin pathway in the developing spinal cord, as assessed by cell proliferation and specification. These effects appear to be Wnt-specific as overexpression of WLS inhibits the expression of FZD10, a target of ß-catenin-dependent transcription. CONCLUSIONS: Our results show that overexpression of WLS inhibits Wnt/ß-catenin signaling in the spinal cord. As the activation of the Wnt/ß-catenin pathway in the spinal cord requires WNT1 or WNT3A, our results are consistent with a model in which the relative concentration of WLS to Wnt regulates WNT1/3A signaling in the developing spinal cord.

Frizzled10 mediates WNT1 and WNT3A signaling in the dorsal spinal cord of the developing chick embryo.

BACKGROUND: WNT1 and WNT3A drive a dorsal to ventral gradient of ß-catenin-dependent Wnt signaling in the developing spinal cord. However, the identity of the receptors mediating downstream functions remains poorly understood. RESULTS: In this report, we show that the spatiotemporal expression patterns of FZD10 and WNT1/WNT3A are highly correlated. We further show that in the presence of LRP6, FZD10 promotes WNT1 and WNT3A signaling using an 8xSuperTopFlash reporter assay. Consistent with a functional role for FZD10, we demonstrate that FZD10 is required for proliferation in the spinal cord. Finally, by using an in situ proximity ligation assay, we observe an interaction between FZD10 and WNT1 and WNT3A proteins. CONCLUSIONS: Together, our results identify FZD10 as a receptor for WNT1 and WNT3A in the developing chick spinal cord.

Differential palmit(e)oylation of Wnt1 on C93 and S224 residues has overlapping and distinct consequences.

Though the mechanisms by which cytosolic/intracellular proteins are regulated by the post-translational addition of palmitate adducts is well understood, little is known about how this lipid modification affects secreted ligands, such as Wnts. Here we use mutational analysis to show that differential modification of the two known palmit(e)oylated residues of Wnt1, C93 and S224, has both overlapping and distinct consequences. Though the relative roles of each residue are similar with respect to stability and secretion, two distinct biological assays in L cells show that modification of C93 primarily modulates signaling via a ß-catenin independent pathway while S224 is crucial for ß-catenin dependent signaling. In addition, pharmacological inhibition of Porcupine (Porcn), an upstream regulator of Wnt, by IWP1, specifically inhibited ß-catenin dependent signaling. Consistent with these observations, mapping of amino acids in peptide domains containing C93 and S224 demonstrate that acylation of C93 is likely to be Porcn-independent while that of S224 is Porcn-dependent. Cumulatively, our data strongly suggest that C93 and S224 are modified by distinct enzymes and that the differential modification of these sites has the potential to influence Wnt signaling pathway choice.

Identification and characterization of subpopulations of Pax3 and Pax7 expressing cells in developing chick somites and limb buds.

Pax3 and Pax7 are closely related paired-boxed family transcription factors that are known to play important roles in embryonic and adult myogenesis. Previous reports describing the expression of Pax3 and Pax7 transcripts reveal expression in many overlapping domains. In this manuscript, we extend these studies by examining the protein expression profiles for Pax3 and Pax7 in developing chick somites and limbs with cellular resolution. Our studies show the existence of distinct subpopulations of cells in the somite and developing limb that are defined by the relative expression levels of Pax3 and Pax7. We also show that Pax3 and Pax7 negatively regulate each other's expression in the dermomyotome, thus providing a possible mechanism for the maintenance of observed expression patterns in the dermomyotome. Further characterization of Pax3- and/or Pax7-positive cells in the dermomyotome and myotome with respect to proliferation and differentiation reveals subpopulations of cells with distinct properties.

Porcupine-mediated lipid-modification regulates the activity and distribution of Wnt proteins in the chick neural tube.

A long-term goal of developmental biology is to understand how morphogens establish gradients that promote proper tissue patterning. A number of reports describe the formation of the Wg (Wnt1) gradient in Drosophila and have shown that Porcupine, a predicted membrane-bound O-acyl transferase, is required for the correct distribution of Wg protein. The discovery that Wnts are palmitoylated on a conserved cysteine residue suggests that porcupine activity and Wnt palmitoylation are important for the generation of Wnt gradients. To establish the role of porcupine in Wnt gradient formation in vertebrates, we tested the role of porcupine/Wnt palmitoylation in human embryonic kidney 293T cells and in the chick neural tube. Our results lead us to conclude that: (1) vertebrate Wnt1 and Wnt3a possess at least one additional site for porcupine-mediated lipid-modification; (2) porcupine-mediated lipid-modification of Wnt proteins promotes their activity in 293T cells and in the chick neural tube; and (3) porcupine-mediated lipid-modification reduces the range of activity of Wnt1 and Wnt3a in the chick neural tube. These findings highlight the importance of porcupine-mediated lipid modifications in the formation of vertebrate Wnt activity gradients.

Differential inhibition of Wnt-3a by Sfrp-1, Sfrp-2, and Sfrp-3.

Secreted frizzled related proteins (Sfrps) are extracellular attenuators of Wnt signaling that play important roles in both embryogenesis and oncogenesis. Although Sfrps are generally thought to bind and sequester Wnts away from active receptor complexes, very little is known about the specificity of Sfrp family members for various Wnts. In the developing chick neural tube, sfrp-1, 2, and 3 transcripts are expressed in and adjacent to the dorsal neural tube, where Wnt-1 and Wnt-3a are expressed. To better define the possible roles of Sfrp-1, 2, and 3 in the neural tube, we first tested the ability of purified Sfrps to inhibit Wnt-3a-induced accumulation of beta-catenin in L cells. We find that both Sfrp-1 and Sfrp-2 can inhibit Wnt-3a activity while Sfrp-3 cannot. To determine where Sfrp-1 and Sfrp-2 impinge on the Wnt signaling pathway, we tested the ability of these Sfrps to inhibit Wnt signaling induced by the addition of LiCl, an inhibitor of GSK-3. Sfrp-1 and Sfrp-2 are unable to inhibit the accumulation of beta-catenin in LiCl-treated cells, suggesting that the ability of Sfrps to inhibit the accumulation of beta-catenin is GSK-3 dependent. We have further shown that Sfrp-2 inhibits the ability of ectopic Wnt-3a to stimulate proliferation in the developing chick neural tube. These results provide the framework for understanding how Sfrps function to regulate Wnt-3a activity in developing embryos and in cancer.

SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas.

Epithelioid sarcoma is a rare soft tissue neoplasm of uncertain lineage that usually arises in the distal extremities of adults, presents a high rate of recurrences and metastases and frequently poses diagnostic dilemmas. The recently reported large-cell "proximal-type" variant is characterized by increased aggressiveness, deep location, preferential occurrence in proximal/axial regions of older patients, and rhabdoid features. Previous cytogenetic studies indicated that the most frequent alterations associated with this tumor entity affect chromosome 22. In this study, combined spectral karyotyping, fluorescence in situ hybridization, and array-based comparative genomic hybridization analyses of two proximal-type cases harboring a rearrangement involving 10q26 and 22q11 revealed that the 22q11 breakpoints were located in a 150-kb region containing the SMARCB1/INI1 gene, and that homozygous deletion of the gene was present in the tumor tissue. The SMARCB1/INI1 gene encodes for an invariant subunit of SWI/SNF chromatin remodeling complex and has been previously reported to act as a tumor suppressor gene frequently inactivated in infantile malignant rhabdoid tumors. We analyzed SMARCB1/INI1 gene status in nine additional epithelioid sarcoma cases (four proximal types and five conventional types) and altogether we identified deletions of SMARCB1/INI1 gene in 5 of 11 cases, all proximal types. We confirmed and further extended the number of cases with SMARCB1/INI1 inactivation to 6 of 11 cases, by real-time quantitative PCR analysis of mRNA expression and by SMARCB1/INI1 immunohistochemistry. Overall, these results point to SMARCB1/INI1 gene involvement in the genesis and/or progression of epithelioid sarcomas. Analysis of larger series of epithelioid sarcomas will be necessary to highlight putative clinically relevant features related to SMARCB1/INI1 inactivation.

A proliferative role for Wnt-3a in chick somites.

The proper patterning of somites to give rise to sclerotome, dermomyotome, and myotome involves the coordination of many different cellular processes, including lineage specification, cell proliferation, cell death, and differentiation, by intercellular signals. One such family of secreted signaling proteins known to influence somite patterning is the Wnt family. Although the participation of Wnt-3a in the patterning of dorsal structures in the somite is well established, no clear consensus has emerged about the cellular processes that are governed by Wnt-3a in the somite. The recent demonstration that Wnt-3a has a proliferative role in the neural tube [Development 129 (2002) 2087] suggested that Wnt-3a might also act to regulate proliferation in somites. To test this hypothesis, we first analyzed the effects of Wnt-3a on segmental plate and somite explants (from Hamburger and Hamilton stage 10 chick embryos) grown in culture. These studies indicate that Wnt-3a is capable of maintaining and/or inducing expression of both Pax-3 and Pax-7, transcription factors that have been implicated in proliferation. To directly test for a role in proliferation, explants were immunostained with antibodies against phospho-histone H3. Explants treated with Wnt-3a show an increase in the percentage of cells expressing phospho-histone H3 as compared to controls. To test the proliferative effect of Wnt-3a in vivo, we ectopically expressed Wnt-3a in chick neural tubes via electroporation. Consistent with previous studies, ectopic expression of Wnt-3a in vivo results in a mediolateral expansion of the dermomyotome and myotome. We now show that proliferation of dorsal/dermomyotomal cells is significantly enhanced by ectopic Wnt-3a. Collectively, our explant and in vivo studies indicate that an increase in proliferation plays an important role in the expansion of the dermomyotome and myotome in Wnt-3a-treated embryos. Furthermore, our results demonstrate that small changes in proliferation can dramatically influence patterning and morphogenesis.