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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 ...

Lewis & Clark College

The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease.

Mapping the development of cerebellar Purkinje cells in zebrafish.

Brainbow is a genetic cell-labeling technique where hundreds of different hues can be generated by stochastic and combinatorial expression of a few spectrally distinct fluorescent proteins. Unique color profiles can be used as cellular identification tags for multiple applications such as tracing axons through the nervous system, following individual cells during development, or analyzing cell lineage. In recent years, Brainbow and other combinatorial expression strategies have expanded from the mouse nervous system to other model organisms and a wide variety of tissues. Particularly exciting is the application of Brainbow in lineage tracing, where this technique has been instrumental in parsing out complex cellular relationships during organogenesis. Here we review recent findings, new technical improvements, and exciting potential genetic and genomic applications for harnessing this colorful technique in anatomical, developmental, and genetic studies.

Brainbow: new resources and emerging biological applications for multicolor genetic labeling and analysis.

The lateral line sensory system in fish detects movements in the water and allows fish to respond to predators, prey, and other stimuli. As the lateral line forms in the first two days of zebrafish development, axons extend caudally along the lateral surface of the fish, eventually forming synapses with hair cells of neuromasts. Growing lateral line axons are located superficially under the skin and can be labeled in living zebrafish using fluorescent protein expression. This system provides a relatively straightforward approach for in vivo time-lapse imaging of neuronal development in an undergraduate setting. Here we describe an upper-level neurobiology laboratory module in which students investigate aspects of axonal development in the zebrafish lateral line system. Students learn to handle and image living fish, collect time-lapse videos of moving mitochondria, and quantitatively measure mitochondrial dynamics by generating and analyzing kymographs of their movements. Energy demands may differ between axons with extending growth cones versus axons that have already reached their targets and are forming synapses. Since relatively little is known about this process in developing lateral line axons, students generate and test their own hypotheses regarding how mitochondrial dynamics may differ at two different time points in axonal development. Students also learn to incorporate into their analysis a powerful yet accessible quantitative tool, the kymograph, which is used to graph movement over time. After students measure and quantify dynamics in living fish at 1 and 2 days post fertilization, this module extends into independent projects, in which students can expand their studies in a number of different, inquiry-driven directions. The project can also be pared down for courses that wish to focus solely on the quantitative analysis (without fish handling), or vice versa. This research module provides a useful approach for the design of open-ended laboratory research projects that integrate the scientific process into undergraduate Biology courses, as encouraged by the AAAS and NSF Vision and Change Initiative. 

In Vivo Time-Lapse Imaging in the Zebrafish Lateral Line: A Flexible, Open-Ended Research Project for an Undergraduate Neurobiology Laboratory Course.

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.

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

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.

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

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Tamily A. Weissman

Biology Department

Lewis & Clark College

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

Tamily A. Weissman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Biology Department

Lewis & Clark College

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

Biology Department