Transgenic Overexpression of LARGE Induces alpha-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle

Background: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of alpha-dystroglycan. Overexpression of LARGE induces hyperglycosylation of alpha-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.Methodology/Principal Findings: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with alpha-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.Conclusions/Significance: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and alpha-dystroglycan hyperglycosylation in muscle should be safe

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGF(delta/delta) mouse model of amyotrophic lateral sclerosis

Background: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGF(delta/delta) mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGF(delta/delta) mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGF(delta/delta) mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGF(delta/delta) mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGF(delta/delta) mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy

MycoRed: Betalain pigments enable in vivo real-time visualisation of arbuscular mycorrhizal colonisation.

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis

The land plant-specific MIXTA-MYB lineage is implicated in the early evolution of the plant cuticle and the colonization of land.

The evolution of a lipid-based cuticle on aerial plant surfaces that protects against dehydration is considered a fundamental innovation in the colonization of the land by the green plants. However, key evolutionary steps in the early regulation of cuticle synthesis are still poorly understood, owing to limited studies in early-diverging land plant lineages. Here, we characterize a land plant specific subgroup 9 R2R3 MYB transcription factor MpSBG9, in the early-diverging land plant model Marchantia polymorpha, that is homologous to MIXTA proteins in vascular plants. The MpSBG9 functions as a key regulator of cuticle biosynthesis by preferentially regulating expression of orthologous genes for cutin formation, but not wax biosynthesis genes. The MpSBG9 also promotes the formation of papillate cells on the adaxial surface of M. polymorpha, which is consisitent with its canonical role in vascular plants. Our observations imply conserved MYB transcriptional regulation in the control of the cutin biosynthesis pathway as a core genetic network in the common ancestor of all land plants, implicating the land plant-specific MIXTA MYB lineage in the early origin and evolution of the cuticle

Towards a synthesized critique of neoliberal biodiversity conservation

During the last three decades, the arena of biodiversity conservation has largely aligned itself with the globally dominant political ideology of neoliberalism and associated governmentalities. Schemes such as payments for ecological services are promoted to reach the multiple ‘wins’ so desired: improved biodiversity conservation, economic development, (international) cooperation and poverty alleviation, amongst others. While critical scholarship with respect to understanding the linkages between neoliberalism, capitalism and the environment has a long tradition, a synthesized critique of neoliberal conservation - the ideology (and related practices) that the salvation of nature requires capitalist expansion - remains lacking. This paper aims to provide such a critique. We commence with the assertion that there has been a conflation between ‘economics’ and neoliberal ideology in conservation thinking and implementation. As a result, we argue, it becomes easier to distinguish the main problems that neoliberal win-win models pose for biodiversity conservation. These are framed around three points: the stimulation of contradictions; appropriation and misrepresentation and the disciplining of dissent. Inspired by Bruno Latour’s recent ‘compositionist manifesto’, the conclusion outlines some ideas for moving beyond critique

Next generation sequencing to investigate genomic diversity in Caryophyllales

Pucker B, Feng T, Brockington SF. Next generation sequencing to investigate genomic diversity in Caryophyllales. bioRxiv. 2019.AbstractCaryophyllales are a highly diverse and large order of plants with a global distribution. While some species are important crops like Beta vulgaris, many others can survive under extreme conditions. This order is well known for the complex pigment evolution, because the red pigments anthocyanin and betalain occur with mutual exclusion in species of the Caryophyllales. Here we report about genome assemblies of Kewa caespitosa (Kewaceae), Macarthuria australis (Macarthuriaceae), and Pharnaceum exiguum (Molluginaceae) which are representing different taxonomic groups in the Caryophyllales. The availability of these assemblies enhances molecular investigation of these species e.g. with respect to certain genes of interest.</jats:p

TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation.

The Arabidopsis genome contains three genes encoding proteins of the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily. TTG1 is a known regulator of epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2 are known regulators of the circadian clock. Here, we discovered a new central role for TTG1 WDR proteins as regulators of the circadian system, as evidenced by the lack of detectable circadian rhythms in a triple lwd1 lwd2 ttg1 mutant. This shows that there has been subfunctionalization via protein changes within the angiosperms, with some TTG1 WDR proteins developing a stronger role in circadian clock regulation while losing the protein characteristics essential for pigment production and epidermal cell specification, and others weakening their ability to drive circadian clock regulation. Our work shows that even where proteins are very conserved, small changes can drive big functional differences.CAA acknowledges support from the Cambridge University Botanic Garden Research Fund. TJH was supported by BBSRC UK grant BB/M006212/1 awarded to AARW

An efficient field and laboratory workflow for plant phylotranscriptomic projects

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141349/1/aps31600128.pd