Semaphorin Receptors Antagonize Wnt Signaling Through Beta-Catenin Degradation.

Precise control of morphogen signaling levels is essential for proper development. An outstanding question is: what mechanisms ensure proper morphogen activity and correct cellular responses? Previous work has identified Semaphorin (SEMA) receptors, Neuropilins (NRPs) and Plexins (PLXNs), as positive regulators of the Hedgehog (HH) signaling pathway. Here, we provide evidence that NRPs and PLXNs antagonize Wnt signaling in both fibroblasts and epithelial cells. Further, Nrp1/2 deletion in fibroblasts results in elevated baseline Wnt pathway activity and increased maximal responses to Wnt stimulation. Notably, and in contrast to HH signaling, SEMA receptor-mediated Wnt antagonism is independent of primary cilia. Mechanistically, PLXNs and NRPs act downstream of Dishevelled (DVL) to destabilize ß-catenin (CTNNB1) in a proteosome-dependent manner. Further, NRPs, but not PLXNs, act in a GSK3ß/CK1-dependent fashion to antagonize Wnt signaling, suggesting distinct repressive mechanisms for these SEMA receptors. Overall, this study identifies SEMA receptors as novel Wnt pathway antagonists that may also play larger roles integrating signals from multiple inputs.

Influence of CUREs on STEM retention depends on demographic identities.

Research has shown that undergraduate research experiences can have substantive effects on retaining students in science, technology, engineering and mathematics (STEM). However, it is impossible to provide individual research experiences for every undergraduate student, especially at large universities. Course-based undergraduate research experiences (CUREs) have become a common approach to introduce large numbers of students to research. We investigated whether a one-semester CURE that replaced a traditional introductory biology laboratory course could increase retention in STEM as well as intention to remain in STEM, if the results differed according to demography, and investigated the possible motivational factors that might mediate such an effect. Under the umbrella of the Authentic Research Connection (ARC) program, we used institutional and survey data from nine semesters and compared ARC participants to non-participants, who applied to ARC but either were not randomly selected or were selected but chose not to enroll in an ARC section. We found that ARC had significant effects on demographic groups historically less likely to be retained in STEM: ARC participation resulted in narrowing the gaps in graduation rates in STEM (first vs continuing-generation college students) and in intention to major in STEM [females vs males, Persons Excluded because of Ethnicity or Race (PEERs) vs non-PEERs]. These disproportionate boosts in intending STEM majors among ARC students coincide with their reporting a greater sense of student cohesiveness, retaining more interest in biology, and commenting more frequently that the course provided a useful/valuable learning experience. Our results indicate that CUREs can be a valuable tool for eliminating inequities in STEM participation, and we make several recommendations for further research.

Preventing erosion of X-chromosome inactivation in human embryonic stem cells.

X-chromosome inactivation is a paradigm of epigenetic transcriptional regulation. Female human embryonic stem cells (hESCs) often undergo erosion of X-inactivation upon prolonged culture. Here, we investigate the sources of X-inactivation instability by deriving new primed pluripotent hESC lines. We find that culture media composition dramatically influenced the expression of XIST lncRNA, a key regulator of X-inactivation. hESCs cultured in a defined xenofree medium stably maintained XIST RNA expression and coating, whereas hESCs cultured in the widely used mTeSR1 medium lost XIST RNA expression. We pinpointed lithium chloride in mTeSR1 as a cause of XIST RNA loss. The addition of lithium chloride or inhibitors of GSK-3 proteins that are targeted by lithium to the defined hESC culture medium impeded XIST RNA expression. GSK-3 inhibition in differentiating female mouse embryonic stem cells and epiblast stem cells also resulted in a loss of XIST RNA expression. Together, these data may reconcile observed variations in X-inactivation in hESCs and inform the faithful culture of pluripotent stem cells.

gammaCOP is required for apical protein secretion and epithelial morphogenesis in Drosophila melanogaster.

BACKGROUND: There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the gammaCOP gene, which encodes a subunit of the COPI coatomer complex. PRINCIPAL FINDINGS: We found that gammaCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic gammaCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in gammaCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, gammaCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in gammaCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis. CONCLUSIONS/SIGNIFICANCE: These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of gammaCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the gammaCOP phenotype to the effect on a specific key target protein.

Developmental roles of the Mi-2/NURD-associated protein p66 in Drosophila.

The NURD and Sin3 histone deacetylase complexes are involved in transcriptional repression through global deacetylation of chromatin. Both complexes contain many different components that may control how histone deacetylase complexes are regulated and interact with other transcription factors. In a genetic screen for modifiers of wingless signaling in the Drosophila eye, we isolated mutations in the Drosophila homolog of p66, a protein previously purified as part of the Xenopus NURD/Mi-2 complex. p66 encodes a highly conserved nuclear zinc-finger protein that is required for development and we propose that the p66 protein acts as a regulatory component of the NURD complex. Animals homozygous mutant for p66 display defects during metamorphosis possibly caused by misregulation of ecdysone-regulated expression. Although heterozygosity for p66 enhances a wingless phenotype in the eye, loss-of-function clones in the wing and the eye discs do not have any detectable phenotype, possibly due to redundancy with the Sin3 complex. Overexpression of p66, on the other hand, can repress wingless-dependent phenotypes. Furthermore, p66 expression can repress multiple reporters in a cell culture assay, including a Wnt-responsive TCF reporter construct, implicating the NURD complex in repression of Wnt target genes. By co-immunoprecipitation, p66 associates with dMi-2, a known NURD complex member.

Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless.

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.

Regulating morphogen gradients in the Drosophila wing.

During development, diffusible ligands, known as morphogens, are thought to move across fields of cells, regulating gene expression in a concentration dependent manner. The case for morphogens has been convincingly made for the Decapentapleigic (Dpp), Wingless (Wg) and Hedgehog (Hh) proteins in the Drosophila wing. In each case, the concentration of the morphogen's receptor plays an important role in shaping the morphogen gradient, through influencing ligand transport and/or stability. However, the relationships between each ligand/receptor pair are different. The role of heparan sulfated proteoglycans, endocytosis and novel exovesicles called argosomes in regulating morphogen distribution will also be discussed.

Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila.

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate target gene expression. Wg signaling causes nuclear translocation of Armadillo, the fly beta-catenin, which then complexes with the DNA-binding protein TCF, enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. This work describes a ubiquitously expressed protein, called Pygopus, which is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. Our data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF.