In vivo imaging of the zebrafish retinotectal map

Kita, Elizabeth M. (2015). In vivo imaging of the zebrafish retinotectal map PhD Thesis, Queensland Brain Institute, The University of Queensland. doi:10.14264/uql.2015.685

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Author Kita, Elizabeth M.
Thesis Title In vivo imaging of the zebrafish retinotectal map
School, Centre or Institute Queensland Brain Institute
Institution The University of Queensland
DOI 10.14264/uql.2015.685
Publication date 2015-06-05
Thesis type PhD Thesis
Open Access Status Other
Supervisor Geoffrey Goodhill
Ethan Scott
Total pages 226
Language eng
Subjects 0608 Zoology
Formatted abstract
The precise neural connections that form during development support the creation of a functioning nervous system. An accessible experimental model for this process is the retinotectal system in zebrafish, which develops rapidly and can be observed in vivo throughout its establishment using confocal time-lapse imaging. The retinotectal connection is topographic, such that the spatial relationships of the retinal ganglion cell (RGC) somas in the retina are maintained by the arborization locations of their axons on the tectum. However, how axons find their correct arborization location on this topographic map is still debated. While initial studies suggested that zebrafish RGCs were guided directly by a growth cone to their target on the map, recent observations challenge that claim. My work characterizes a novel guidance pattern for RGCs pathfinding on the tectum. Once pathfinding through the tectal neuropil, zebrafish RGCs continually add and remove branches. The directionality of the branches is biased, in that more are pointing towards the eventual arborization zone than away from it. Growth cones, which tip some of the branches, extend mostly in straight lines rather than progressing through turns towards the target. The distance to the target is decreased by the axon branching in many directions, and selecting a branch that decreases the distance to support further rounds of branching. The mechanism of biased branching could be based on known types of input that guide connectivity during development and we focused on the contributions of activity and molecular guidance cue gradients.

We hypothesized that silencing neural activity using TTX could alter the navigation methods used, but many of the characteristics of axon pathfinding were similar despite global silencing. The area covered by the branches during pathfinding decreases when the retinotectal system is electrically silent, but the biased branch ratio remains. Several measures of the arbors after the target is reached show differing patterns without activity, supporting previous evidence that activity is an important regulation of arborization dynamics.

Molecularly, the retinotectal map is established through gradients of guidance cues that guide axons to their target location, cause them to stop advancing, and elaborate a terminal arbor. Through morpholino knock down we observe the different effects that two of these guidance proteins, ephrin-A5b and ephrin-A2, have on individual axons as they navigate. Overall, knockdown of ephrin-A5b tends to increase length, area and number of branches, while the knockdown of ephrin-A2 has the opposite effect on these measures. Additionally, a subset of axons show phenotypes that are masked by the grouped data, where several form loops instead of travelling in rather straight trajectories, or alternatively, grow long and remain relatively branchless as they travel towards the caudal extent of the tectum.

The following thesis gives quantitative, detailed insight into some of the ways that activity and molecular cues sculpt the formation of the retinotectal connection at the level of the individual axons.
Keyword Axon guidance
Zebrafish (Danio rerio)
Optic Tectum
Developmental neuroscience
Neural Activity
Topographic map
Axon branching
Timelapse imaging

Document type: Thesis
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Created: Tue, 02 Jun 2015, 10:01:02 EST by Elizabeth Kita on behalf of Queensland Brain Institute