In vivo imaging of growth cone and filopodial dynamics: Evidence for contact‐mediated retraction of filopodia leading to the tiling of sibling processes

Abstract

In the leech embryo, the peripheral comb cell (CC) sends out many nonoverlapping, growth cone-tipped processes that grow in parallel and serve as a scaffold for the migrating myocytes of the later-developing oblique muscle layer. To explore how the parallel arrangement is generated we first examined the arrangement of CC cytoskeletal components by expressing a tubulin-binding protein and actin, both tagged with fluorescent reporters. This revealed that the growth cones were compartmentalized into F-actin-rich filopodia and a microtubule-rich central region. Time-lapse analysis with a 2-photon laser scanning microscope revealed that the growth cones of the CC are highly dynamic, undergoing rapid filopodial extension and retraction. Measurements of filopodial lifespan and length revealed that most filopodia at the leading edge of the growth cone achieved significantly longer lifespans and length than lateral filopodia. Furthermore, for the short-lived lateral filopodia, apparent interaction with a neighboring process was found to be a significant predictor of their nearly immediate (within 2-4 minutes) retraction. When contact was experimentally prevented by ablating individual CCs, the filopodia from the growth cones of adjacent segmental neighbors were found to be significantly lengthened in the direction of the removed homolog. Treatment with low doses of cytochalasin D to disrupt F-actin assembly led to filopodial retraction and growth cone collapse and resulted in the bifurcation of many CC processes, numerous crossover errors, and the loss of parallelism. These findings indicate the existence of a contact-mediated repulsive interaction between processes of the CC.

Mechanical computation in neurons

“…Anatomical, biochemical and functional properties of growth cones and of their major components, lamellipodia and filopodia, have been thoroughly investigated (Aletta and Greene, 1988;Gomez and Letourneau, 1994;Baker and Macagno, 2007). The present work has two major aims: firstly, to examine in detail the kinetics and dynamics of filopodia and lamellipodia in an almost free environment and in a dense tissue; secondly, to provide a computational framework to rationalize the observed kinetics and dynamics.…”

“…The motion of filopodia and lamellipodia plays a major role in morphogenesis and neuronal differentiation: the exploratory motion of filopodia allows neurons to find the correct target and to establish the appropriate synaptic connections. Their motion has been analyzed and characterized to some extent by time lapse microscopy (Aletta and Greene, 1988;Gomez and Letourneau, 1994;Dent and Kalil, 2001;Baker et al, 2003;Baker and Macagno, 2007;Galbraith et al, 2007;Mongiu et al, 2007).…”

Neurite Outgrowth andIn VivoSensory Innervation Mediated by a CaV2.2–Laminin β2 Stop Signal

“…Neurite arbors efficiently innervate sensory surfaces while maintaining discrete spatial resolution in systems such as the mammalian retina (Wassle et al, 1981), the body wall of the leech and Drosophila (Grueber et al, 2003;Baker and Macagno, 2007), and zebrafish and Xenopus skin (Kitson and Roberts, 1983;Sagasti et al, 2005). In some cases, a tiled distribution is maintained by contact mediated repulsion (Emoto et al, 2004;Sagasti et al, 2005;Baker and Macagno, 2007;Hughes et al, 2007), whereas in other cases separate or additional mechanisms are involved (Grueber et al, 2003;Gallegos and Bargmann, 2004;Lin et al, 2004;Sagasti et al, 2005). These may include intrinsic limitation of neurite growth or instruction of neurite distribution by preexistent tiled patterns of extracellular matrix molecules within target tissue (Hari et al, 2004).…”

Self-avoidance and Tiling: Mechanisms of Dendrite and Axon Spacing

“…Comb cells are transient cells that may serve as a scaffold for muscle cells (Jellies and Kristan 1991). These cells possess numerous parallel extensions with regular spacing between them (Baker and Macagno 2007). A LAR homolog, HmLAR2, is expressed in these cells and knocking down its function with function-blocking antibodies, overexpression of the LAR ectodomain, or RNAi knockdown causes processes to adopt (Schmucker et al 2000;Yu et al 2009).…”