The topographic projection from the eye to the tectum (amphibians and fish)/superior colliculus (birds and mammals) is a paradigm model system for studying mechanisms of neural wiring development. It has previously been proposed that retinal ganglion cell axons use distinct guidance strategies in fish vs. mammals, with direct guidance to the tectal target zone in the former and overshoot followed by biased branching toward the target zone in the latter. Here we visualized individual retinal ganglion cell axons as they grew over the tectum in zebrafish for periods of 10-21 hours and analyzed these results using an array of quantitative measures. We found that, although axons were generally guided directly toward their targets, this occurred without growth cone turning. Instead, axons branched dynamically and profusely throughout pathfinding, and successive branches oriented growth cone extension toward a target zone in a stepwise manner. These data suggest that the guidance strategies used between fish and mammals may be less distinct than previously thought.
The zebrafish retinotectal projection provides an attractive model system for studying many aspects of topographic map formation and maintenance. Visual connections initially start to form between 3 and 5 days postfertilization, and remain plastic throughout the life of the fish. Zebrafish are easily manipulated surgically, genetically, and chemically, and a variety of molecular tools exist to enable visualization and control of various aspects of map development. Here, we review zebrafish retinotectal map formation, focusing particularly on the detailed structure and dynamics of the connections, the molecules that are important in map creation, and how activity regulates the maintenance of the map.
BackgroundNormal brain function depends on the development of appropriate patterns of neural connections. A critical role in guiding axons to their targets during neural development is played by neuronal growth cones. These have a complex and rapidly changing morphology; however, a quantitative understanding of this morphology, its dynamics and how these are related to growth cone movement, is lacking.ResultsHere we use eigenshape analysis (principal components analysis in shape space) to uncover the set of five to six basic shape modes that capture the most variance in growth cone form. By analysing how the projections of growth cones onto these principal modes evolve in time, we found that growth cone shape oscillates with a mean period of 30 min. The variability of oscillation periods and strengths between different growth cones was correlated with their forward movement, such that growth cones with strong, fast shape oscillations tended to extend faster. A simple computational model of growth cone shape dynamics based on dynamic microtubule instability was able to reproduce quantitatively both the mean and variance of oscillation periods seen experimentally, suggesting that the principal driver of growth cone shape oscillations may be intrinsic periodicity in cytoskeletal rearrangements.ConclusionsIntrinsically driven shape oscillations are an important component of growth cone shape dynamics. More generally, eigenshape analysis has the potential to provide new quantitative information about differences in growth cone behaviour in different conditions.
The relative importance of neural activity versus activity-independent cues in shaping the initial wiring of the brain is still largely an open question. While activity is clearly critical for circuit rearrangements after initial connections have been made, whether it also plays a role in initial axon pathfinding remains to be determined. Here, we investigated this question using the guidance of zebrafish retinal ganglion cell axons to their targets in the tectum as a model. Recent results have implicated biased branching as a key feature of pathfinding in the zebrafish tectum. Using tetrodotoxin to silence neural activity globally, we found a decrease in the area covered by axon branches during pathfinding. After reaching the target, there were dynamic differences in axon length, area and the number of branches between conditions. However, other aspects of pathfinding were unaffected by silencing, including the ratio of branches directed toward the target, length, and number of branches, as well as turning angle, velocity, and number of growth cones per axon. These results challenge the hypothesis that neural connections develop in sequential stages of molecularly guided pathfinding and activity-based refinement. Despite a maintenance of overall guidance, axon pathfinding dynamics can nevertheless be altered by activity loss.
SUMMARYThe majority of neurons in the nervous system exhibit a polarized morphology, with multiple short dendrites and a single long axon. It is clear that multiple factors govern polarization in developing neurons, and the biased accumulation of intrinsic determinants to one side of the cell, coupled with responses to asymmetrically localized extrinsic factors, appears to be crucial. A number of intrinsic factors have been identified, but surprisingly little is known about the identity of the extrinsic signals. Here, we show in vivo that neuropilin-1 (Nrp1) and its co-receptor plexinA1 (Plxna1) are necessary to bias the extension of the dendrites of retinal ganglion cells to the apical side of the cell, and ectopically expressed class III semaphorins (Sema3s) disrupt this process. Importantly, the requirement for Nrp1 and Plxna1 in dendrite polarization occurs at a developmental time point after the cells have already extended their basally directed axon. Thus, we propose a novel mechanism whereby an extrinsic factor, probably a Sema3, acts through Nrp1 and Plxna1 to promote the asymmetric outgrowth of dendrites independently of axon polarization.
Branching is an important mechanism by which axons navigate to their targets during neural development. For instance, in the developing zebrafish retinotectal system, selective branching plays a critical role during both initial pathfinding and subsequent arborisation once the target zone has been reached. Here we show how quantitative methods can help extract new information from time-lapse imaging about the nature of the underlying branch dynamics. First, we introduce Dynamic Time Warping to this domain as a method for automatically matching branches between frames, replacing the effort required for manual matching. Second, we model branch dynamics as a birth-death process, i.e. a special case of a continuous-time Markov process. This reveals that the birth rate for branches from zebrafish retinotectal axons, as they navigate across the tectum, increased over time. We observed no significant change in the death rate for branches over this time period. However, blocking neuronal activity with TTX slightly increased the death rate, without a detectable change in the birth rate. Third, we show how the extraction of these rates allows computational simulations of branch dynamics whose statistics closely match the data. Together these results reveal new aspects of the biology of retinotectal pathfinding, and introduce computational techniques which are applicable to the study of axon branching more generally.
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.iii 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 molec...
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