In vivo aggregation of presynaptic alpha-synuclein is not influenced by its phosphorylation at serine-129.

Abnormal aggregation of the α-synuclein protein is a key molecular feature of Parkinson's disease and other neurodegenerative diseases. The precise mechanisms that trigger α-synuclein aggregation are unclear, and it is not known what role aggregation plays in disease pathogenesis. Here we use an in vivo zebrafish model to express several different forms of human α-synuclein and measure its aggregation in presynaptic terminals. We show that human α-synuclein tagged with GFP can be expressed in zebrafish neurons, localizing normally to presynaptic terminals and undergoing phosphorylation at serine-129, as in mammalian neurons. The visual advantages of the zebrafish system allow for dynamic in vivo imaging to study α-synuclein, including the use of fluorescence recovery after photobleaching (FRAP) techniques to probe protein mobility. These experiments reveal three distinct terminal pools of α-synuclein with varying mobility, likely representing different subpopulations of aggregated and non-aggregated protein. Human α-synuclein is phosphorylated by an endogenous zebrafish Polo-like kinase activity, and there is a heterogeneous population of neurons containing either very little or extensive phosphorylation throughout the axonal arbor. Both pharmacological and genetic manipulations of serine-129 show that phosphorylation of α-synuclein at this site does not significantly affect its mobility. This suggests that serine-129 phosphorylation alone does not promote α-synuclein aggregation. Together our results show that human α-synuclein can be expressed and measured quantitatively in zebrafish, and that disease-relevant post-translational modifications occur within neurons. The zebrafish model provides a powerful in vivo system for measuring and manipulating α-synuclein function and aggregation, and for developing new treatments for neurodegenerative disease.

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

Multicolor lineage tracing using in vivo time-lapse imaging reveals coordinated death of clonally related cells in the developing vertebrate brain.

The global mechanisms that regulate and potentially coordinate cell proliferation & death in developing neural regions are not well understood. In particular, it is not clear how or whether clonal relationships between neural progenitor cells and their progeny influence the growing brain. We have developed an approach using Brainbow in the developing zebrafish to visualize and follow multiple clones of related cells in vivo over time. This allows for clear visualization of many dividing clones of cells, deep in proliferating brain regions. As expected, in addition to undergoing interkinetic nuclear migration and cell division, cells also periodically undergo apoptosis. Interestingly, cell death occurs in a non-random manner: clonally related cells are more likely to die in a progressive fashion than cells from different clones. Multiple members of an individual clone die while neighboring clones appear healthy and continue to divide. Our results suggest that clonal relationships can influence cellular fitness and survival in the developing nervous system, perhaps through a competitive mechanism whereby clones of cells are competing with other clones. Clonal cell competition may help regulate neuronal proliferation in the vertebrate brain.

Combining near-infrared fluorescence with Brainbow to visualize expression of specific genes within a multicolor context.

Fluorescent proteins are a powerful experimental tool, allowing the visualization of gene expression and cellular behaviors in a variety of systems. Multicolor combinations of fluorescent proteins, such as Brainbow, have expanded the range of possible research questions and are useful for distinguishing and tracking cells. The addition of a separately driven color, however, would allow researchers to report expression of a manipulated gene within the multicolor context to investigate mechanistic effects. A far-red or near-infrared protein could be particularly suitable in this context, as these can be distinguished spectrally from Brainbow. We investigated five far-red/near-infrared proteins in zebrafish: TagRFP657, mCardinal, miRFP670, iRFP670, and mIFP. Our results show that both mCardinal and iRFP670 are useful fluorescent proteins for zebrafish expression. We also introduce a new transgenic zebrafish line that expresses Brainbow under the control of the neuroD promoter. We demonstrate that mCardinal can be used to track the expression of a manipulated bone morphogenetic protein receptor within the Brainbow context. The overlay of near-infrared fluorescence onto a Brainbow background defines a clear strategy for future research questions that aim to manipulate or track the effects of specific genes within a population of cells that are delineated using multicolor approaches.

Management-driven evolution in a domesticated ecosystem.

Millennia of human land-use have resulted in the widespread occurrence of what have been coined 'domesticated ecosystems'. The anthropogenic imprints on diversity, composition, structure and functioning of such systems are well documented. However, evolutionary consequences of human activities in these ecosystems are enigmatic. Calluna vulgaris (L.) is a keystone species of coastal heathlands in northwest Europe, an ancient semi-natural landscape of considerable conservation interest. Like many species from naturally fire-prone ecosystems, Calluna shows smoke-adapted germination, but it is unclear whether this trait arose prior to the development of these semi-natural landscapes or is an evolutionary response to the anthropogenic fire regime. We show that smoke-induced germination in Calluna is found in populations from traditionally burnt coastal heathlands but is lacking in naturally occurring populations from other habitats with infrequent natural fires. Our study thus demonstrates evolutionary imprints of human land-use in semi-natural ecosystems. Evolutionary consequences of historic anthropogenic impacts on wildlife have been understudied, but understanding these consequences is necessary for informed conservation and ecosystem management.

Conditional cold avoidance drives between-population variation in germination behaviour in Calluna vulgaris.

BACKGROUND AND AIMS: Across their range, widely distributed species are exposed to a variety of climatic and other environmental conditions, and accordingly may display variation in life history strategies. For seed germination in cold climates, two contrasting responses to variation in winter temperature have been documented: first, an increased ability to germinate at low temperatures (cold tolerance) as winter temperatures decrease, and secondly a reduced ability to germinate at low temperatures (cold avoidance) that concentrates germination towards the warmer parts of the season. METHODS: Germination responses were tested for Calluna vulgaris, the dominant species of European heathlands, from ten populations collected along broad-scale bioclimatic gradients (latitude, altitude) in Norway, covering a substantial fraction of the species' climatic range. Incubation treatments varied from 10 to 25 °C, and germination performance across populations was analysed in relation to temperature conditions at the seed collection locations. KEY RESULTS: Seeds from all populations germinated rapidly and to high final percentages under the warmer incubation temperatures. Under low incubation temperatures, cold-climate populations had significantly lower germination rates and percentages than warm-climate populations. While germination rates and percentages also increased with seed mass, seed mass did not vary along the climatic gradients, and therefore did not explain the variation in germination responses. CONCLUSIONS: Variation in germination responses among Calluna populations was consistent with increased temperature requirements for germination towards colder climates, indicating a cold-avoidance germination strategy conditional on the temperature at the seeds' origin. Along a gradient of increasing temperatures this suggests a shift in selection pressures on germination from climatic adversity (i.e. low temperatures and potential frost risk in early or late season) to competitive performance and better exploitation of the entire growing season.

Exploration versus exploitation in polydomous ant colonies.

In socially foraging species resource information can be shared between individuals, increasing foraging success. In ant colonies, nestmate recruitment allows high exploitation rates at known resources however, to maximise foraging efficiency this must be balanced with searching for new resources. Many ant species form colonies inhabiting two or more spatially separated but socially connected nests: this type of organisation is known as polydomy. Polydomous colonies may benefit from increased foraging efficiency by carrying out dispersed-central place foraging. However, decentralisation of the colony may affect recruitment success by limiting interaction between ants based in separate nests. We use an agent-based model which compares the foraging success of monodomous and polydomous colonies in different food environments, incorporating recruitment through pheromone trails and group foraging. In contrast to previous results we show that polydomy is beneficial in some but not all cases. Polydomous colonies discover resources at a higher rate, making them more successful when food is highly dispersed, but their relative success can be lowered by limitations on recruitment success. Monodomous colonies can have higher foraging efficiency than polydomous colonies by exploiting food more rapidly. The results show the importance of interactions between recruitment strategy, colony size, and colony organisation.

Diet will help reduce cholesterol levels.

A cardio-protective diet, rich in omega-3 oils and fresh fruit and vegetables, will help lower cholesterol levels. In this article, a dietitian provides answers to frequently asked questions and advice on good dietary practice.