Filopodial dynamics and growth cone stabilization in Drosophila visual circuit development

Abstract

Filopodial dynamics are thought to control growth cone guidance, but the types and roles of growth cone dynamics underlying neural circuit assembly in a living brain are largely unknown. To address this issue, we have developed long-term, continuous, fast and high-resolution imaging of growth cone dynamics from axon growth to synapse formation in cultured Drosophila brains. Using R7 photoreceptor neurons as a model we show that >90% of the growth cone filopodia exhibit fast, stochastic dynamics that persist despite ongoing stepwise layer formation. Correspondingly, R7 growth cones stabilize early and change their final position by passive dislocation. N-Cadherin controls both fast filopodial dynamics and growth cone stabilization. Surprisingly, loss of N-Cadherin causes no primary targeting defects, but destabilizes R7 growth cones to jump between correct and incorrect layers. Hence, growth cone dynamics can influence wiring specificity without a direct role in target recognition and implement simple rules during circuit assembly.DOI: http://dx.doi.org/10.7554/eLife.10721.001

Frazzled promotes growth cone attachment at the source of a Netrin gradient in the Drosophila visual system

“…This system is similar to one developed by Hiesinger and colleagues to study the cellular mechanism of neural superposition, the choreographed re-distribution of R1-6 growth cones from ommaditial bundles to lamina cartridges (Langen et al, 2015). In the medulla neuropil, Hiesinger and colleagues used ex vivo live imaging to study R7 targeting, specifically characterizing the role of the Ca2+-dependent cell adhesion molecule, N-cadherin, in mediating adhesive interaction between growth cones and the developing neuropil (Özel et al, 2015).

Video 1.Medulla Registration.The developmental roll of the medulla is corrected in preparation for growth cone segmentation and tracking. DOI: http://dx.doi.org/10.7554/eLife.20762.007

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Modality-specific circuits for skylight orientation in the fly visual system

“…We, therefore wondered how DRA.R7 and DRA.R8 cells could reliably choose between different post-synaptic partners when terminating in such close proximity within their respective M6 sublayers. In order to get some insight into this process, we decided to characterize the time series of DRA.R8 photoreceptor axons growing into the medulla, using 2-photon live imaging 42 (see material and methods) ( Figure 7A, Supplemental Movie 3). Imaging sparsely labeled R7 and/or R8 cells (within the DRA and outside of it) from an ex vivo brain explant over the course of 18 hours revealed the temporal dynamics of their individual outgrowth behavior during layer targeting ( Figure 7B).…”

“…To achieve this DRA-specific connectivity, polarizationsensitive DRA.R8 cells differ from their color-sensitive counterparts both in layer targeting and the distribution of their presynaptic sites. Finally, using live imaging of ingrowing photoreceptor axons 42 , we visualize the temporal sequence of R7 and R8 axons targeting to the M6 layer, thereby temporally separating synaptic partner choices. Our work reveals an instance for how repetitive microcircuits, organized in highly stereotypical medulla columns, can become reorganized locally in order to adapt them to their modality-specific needs.…”

Wiring visual systems: common and divergent mechanisms and principles

“…3B). R7 growth cones never actively extend thereafter, forming early contacts with their main postsynaptic partners, Dm8; new layers form through intercalations between these early R8 and R7 layers [32,43] (Fig. 3B).…”

“…The role of N-cadherin is often interpreted as ‘broadly adhesive’ and functioning redundantly with other factors, in part because mutant neurons often target correctly during early development [1,9,39]. However, R7 live imaging revealed that cadN mutant fly R7 axons actively jump between correct and incorrect medulla layers, arguing against a strict role for CadN in preferential adhesion within the correct layer [43]. Subsequent to its own targeting, L3 produces the secreted attractant netrin, which influences R8 growth cones as they actively extend to this same layer [48].…”