A conserved RNA switch for acetylcholine receptor clustering at neuromuscular junctions in chordates.

In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "mini-gene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream AChR clustering. We present evidence that, although the overall pathway is well conserved, there are some surprising differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.

Secondary production of the central rangeland region of the United States.

Rangelands are the dominant land use across a broad swath of central North America where they span a wide gradient, from <350 to >900 mm, in mean annual precipitation. Substantial efforts have examined temporal and spatial variation in aboveground net primary production (ANPP) to precipitation (PPT) across this gradient. In contrast, net secondary productivity (NSP, e.g., primary consumer production) has not been evaluated analogously. However, livestock production, which is a form of NSP or primary consumer production supported by primary production, is the dominant non-cultivated land use and an integral economic driver in these regions. Here, we used long-term (mean length = 19 years) ANPP and NSP data from six research sites across the Central Great Plains with a history of a conservative stocking to determine resource (i.e., PPT)-productivity relationships, NSP sensitivities to dry-year precipitation, and regional trophic efficiencies (e.g., NSP:ANPP ratio). PPT-ANPP relationships were linear for both temporal (site-based) and spatial (among site) gradients. The spatial PPT-NSP model revealed that PPT mediated a saturating relationship for NSP as sites became more mesic, a finding that contrasts with many plant-based PPT-ANPP relationships. A saturating response to high growing-season precipitation suggests biogeochemical rather than vegetation growth constraints may govern NSP (i.e., large herbivore production). Differential sensitivity in NSP to dry years demonstrated that the primary consumer production response heightened as sites became more xeric. Although sensitivity generally decreased with increasing precipitation as predicted from known PPT-ANPP relationships, evidence suggests that the dominant species' identity and traits influenced secondary production efficiency. Non-native northern mixed-grass prairie was outperformed by native Central Great Plains rangeland in sensitivity to dry years and efficiency in converting ANPP to NSP. A more comprehensive understanding of the mechanisms leading to differences in producer and consumer responses will require multisite experiments to assess biotic and abiotic determinants of multi-trophic level efficiency and sensitivity.

Molecular Subtypes of Head and Neck Cancer in Patients of African Ancestry.

PURPOSE: The purpose of this study was to better understand the complex molecular biomarkers and signatures of head and neck cancer (HNC) among Black patients and identify possible molecular changes associated with HNC disparities. EXPERIMENTAL DESIGN: Molecular subtypes and genomic changes in HNC samples from patients of African and European ancestry in The Cancer Genome Atlas, Memorial Sloan Kettering Cancer Center, Broad Institute, MD Anderson Cancer Center, and John Hopkins University were identified. Molecular features (genomic, proteomic, transcriptomic) associated with race and genomic alterations associated with clinical outcomes were determined. An independent cohort of HNC tumor specimens was used to validate the primary findings using IHC. RESULTS: Black patients were found to have a younger age at diagnosis, more aggressive tumor types, higher rates of metastasis, and worse survival compared with White patients. Black patients had fewer human papillomavirus-positive tumor types and higher frequencies of laryngeal subtype tumors. Higher frequencies of TP53, MYO18B, KMT2D, and UNC13C mutations and a lower frequency of PIK3CA mutations were observed in Black patients. Tumors of Black patients showed significant enrichment of c-MYC and RET-tyrosine signaling and amplifications. A significant increase in tumor expression of c-MYC in Black patients was observed and was associated with poor survival outcomes in the independent cohort. CONCLUSIONS: Novel genomic modifications and molecular signatures may be related to environmental, social, and behavioral factors associated with racial disparities in HNC. Unique tumor mutations and biological pathways have potential clinical utility in providing more targeted and individualized screening, diagnostic, and treatment modalities to improve health outcomes.

Asymmetric Fitness of Second-Generation Interspecific Hybrids Between Ciona robusta and Ciona intestinalis.

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species of tunicates in the same ascidian genus, Ciona robusta and Ciona intestinalis, can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to characterize the genetic basis of simple traits, and further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids suggested that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. We observed asymmetric fitness, whereby the C. intestinalis maternal lines fared more poorly in our system, pointing to maternal origins of species-specific sensitivity. We discuss the possibility that asymmetrical second generation inviability and infertility emerge from interspecific incompatibilities between the nuclear and mitochondrial genomes, or other maternal effect genes. This work paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.

Initial characterization of Wnt-Tcf functions during Ciona heart development.

In vertebrate embryos, the cardiopharyngeal mesoderm gives rise to both cardiac and branchiomeric head muscles. The canonical Wnt signaling pathway regulates many aspects of cardiomyocyte specification, and modulates a balance between skeletal and cardiac myogenesis during vertebrate head muscle development. However, the role of Wnt signaling during ascidian cardiopharyngeal development remains elusive. Here, we documented the expression of Wnt pathway components during cardiopharyngeal development in Ciona, and generated tools to investigate potential roles for Wnt signaling, and its transcriptional effector Tcf, on heart vs. pharyngeal muscle fate specification. Neither focused functional analyses nor lineage-specific transcriptome profiling uncovered a significant role for Tcf during early cardiac vs. pharyngeal muscle fate choice. By contrast, Wnt gene expression patterns of Frizzled4 and Lrp4/8 and CRISPR/Cas9-mediated Tcf knock-down suggested a later requirement for Wnt signaling during heart morphogenesis and/or cardiomyocyte differentiation. This study provides a provisional set of reagents to study Wnt signaling function in Ciona, and promising insights for future analyses of Wnt functions during heart organogenesis.

An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time.

In embryos, multipotent progenitors divide to produce distinct progeny and express their full potential. In vertebrates, multipotent cardiopharyngeal progenitors produce second-heart-field-derived cardiomyocytes, and branchiomeric skeletal head muscles. However, the mechanisms underlying these early fate choices remain largely elusive. The tunicate Ciona emerged as an attractive model to study early cardiopharyngeal development at high resolution: through two asymmetric and oriented divisions, defined cardiopharyngeal progenitors produce distinct first and second heart precursors, and pharyngeal muscle (aka atrial siphon muscle, ASM) precursors. Here, we demonstrate that differential FGF-MAPK signaling distinguishes between heart and ASM precursors. We characterize a feed-forward circuit that promotes the successive activations of essential ASM determinants, Hand-related, Tbx1/10 and Ebf. Finally, we show that coupling FGF-MAPK restriction and cardiopharyngeal network deployment with cell divisions defines the timing of gene expression and permits the emergence of diverse cell types from multipotent progenitors.

CitSci.org: A New Model for Managing, Documenting, and Sharing Citizen Science Data.

Citizen science projects have the potential to advance science by increasing the volume and variety of data, as well as innovation. Yet this potential has not been fully realized, in part because citizen science data are typically not widely shared and reused. To address this and related challenges, we built CitSci.org (see www.citsci.org), a customizable platform that allows users to collect and generate diverse datasets. We hope that CitSci.org will ultimately increase discoverability and confidence in citizen science observations, encouraging scientists to use such data in their own scientific research.

Regulation and evolution of cardiopharyngeal cell identity and behavior: insights from simple chordates.

The vertebrate heart arises from distinct first and second heart fields. The latter also share a common origin with branchiomeric muscles in the pharyngeal mesoderm and transcription regulators, such as Nkx2-5, Tbx1 and Islet1. Despite significant progress, the complexity of vertebrate embryos has hindered the identification of multipotent cardiopharyngeal progenitors. Here, we summarize recent insights in cardiopharyngeal development gained from ascidian models, among the closest relatives to vertebrates. In a simplified cellular context, progressive fate specification of the ascidian cardiopharyngeal precursors presents striking similarities with their vertebrate counterparts. Multipotent cardiopharyngeal progenitors are primed to activate both the early cardiac and pharyngeal muscles programs, which segregate following asymmetric cells divisions as a result of regulatory cross-antagonisms involving Tbx1 and Nkx2-5 homologs. Activation of Ebf in pharyngeal muscle founder cells triggers both Myogenic Regulatory Factor-associated differentiation and Notch-mediated maintenance of an undifferentiated state in distinct precursors. Cross-species comparisons revealed the deep conservation of the cardiopharyngeal developmental sequence in spite of extreme genome sequence divergence, gene network rewiring and specific morphogenetic differences. Finally, analyses are beginning to uncover the influence of surrounding tissues in determining cardiopharyngeal cell identity and behavior. Thus, ascidian embryos offer a unique opportunity to study gene regulation and cell behaviors at the cellular level throughout cardiopharyngeal morphogenesis and evolution.

Surrounding tissues canalize motile cardiopharyngeal progenitors towards collective polarity and directed migration.

Collectively migrating cells maintain group polarity and interpret external cues to reach their destination. The cardiogenic progenitors (also known as trunk ventral cells, TVCs) of the ascidian Ciona intestinalis provide a simple chordate model with which to study collective migration. Bilateral pairs of associated TVCs undergo a stereotyped polarized migration away from the tail towards the ventral trunk, arguably constituting the simplest possible example of directed collective migration. To identify tissues contributing to TVC polarity and migration, we quantified the contact between TVCs and surrounding tissues, and blocked the secretory pathway in a tissue-specific manner. Even though TVCs normally migrate as an invariably determined leader-trailer polarized pair of adherent cells, they are capable of migrating individually, albeit a shorter distance and with altered morphology. The mesenchyme contacts newborn TVCs and contributes to robust specification of the trailer but appears to have only minor effects on directed migration. The notochord does not contact the TVCs but contributes to the onset of migration. The trunk endoderm first contacts the leader TVC, then 'encases' both migrating cells and provides the inputs maintaining leader-trailer polarity. Migrating TVCs adhere to the epidermis and need this contact for their cohesion. These phenomenological studies reveal that inherently motile cardiopharyngeal progenitors are channeled into stereotyped behaviors by interactions with surrounding tissues.

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling.

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.

Complex interactions between GSK3 and aPKC in Drosophila embryonic epithelial morphogenesis.

Generally, epithelial cells must organize in three dimensions to form functional tissue sheets. Here we investigate one such sheet, the Drosophila embryonic epidermis, and the morphogenetic processes organizing cells within it. We report that epidermal morphogenesis requires the proper distribution of the apical polarity determinant aPKC. Specifically, we find roles for the kinases GSK3 and aPKC in cellular alignment, asymmetric protein distribution, and adhesion during the development of this polarized tissue. Finally, we propose a model explaining how regulation of aPKC protein levels can reorganize both adhesion and the cytoskeleton.

Spatially defined Dsh-Lgl interaction contributes to directional tissue morphogenesis.

The process of epithelial morphogenesis defines the structure of epidermal tissue sheets. One such sheet, the ventral epidermis of the Drosophila embryo, shows both intricate segmental patterning and complex cell organization. Within a segment, cells produce hair-like denticles in a stereotypical and highly organized pattern over the surface of the tissue. To understand the cell biological basis of this process, we examined cell shapes and alignments, and looked for molecules that showed an asymmetric distribution in this tissue. We found that apical polarity determinants and adherens junctions were enriched at the dorsal and ventral borders of cells, whereas basolateral determinants were enriched at the anterior and posterior borders. We report that the basolateral determinant Lgl has a novel function in the planar organization of the embryonic epidermis, and this function depends on Dsh and myosin. We conclude that apical-basal proteins, used to establish polarity within a cell, can be independently co-opted to function in epithelial morphogenesis.

GSK3beta affects apical-basal polarity and cell-cell adhesion by regulating aPKC levels.

The dynamic rearrangement of cell-cell contacts is required for the establishment of functional epithelial cell sheets. However, the signaling pathways and cellular mechanisms that initiate and maintain this polarity are not well understood. We show that loss of the Wnt signaling component GSK3 beta results in increased levels of aPKC and leads to defects in apical-basal polarity. We find that GSK3 beta directly phosphorylates aPKC, which likely promotes its ubiquitin-mediated proteosomal degradation. aPKC increases the levels of Armadillo and stabilizes adherens junctions. These results suggest that the Wnt pathway component GSK3 beta regulates the polarity determinant aPKC, which in turn affects cell-cell contacts during the development of polarized tissues.

Epithelial polarity: interactions between junctions and apical-basal machinery.

Epithelial polarity is established and maintained by competition between determinants that define the apical and basolateral domains. Cell-cell adhesion complexes, or adherens junctions, form at the interface of these regions. Mutations in adhesion components as well as apical determinants normally lead to an expansion of the basolateral domain. Here we investigate the genetic relationship between the polarity determinants and adhesion and show that the levels of the adhesion protein Armadillo affect competition. We find that in arm mutants, even a modest reduction in the basolateral component lgl leads to a full apical domain expansion or lgl phenotype. By using an allelic series of Armadillo mutations, we show that there is a threshold at which basolateral expansion can be reversed. Further, in embryos lacking the Wingless signaling component zw3, the same full apical expansion occurs again with only a reduction in lgl. We propose a model where zw3 regulates protein levels of apical and adhesion components and suggest that a reciprocal interaction between junctions and polarity modules functions to maintain stable apical and basolateral domains.