Neurons must develop characteristic and often complex morphologies to form functional circuits of the nervous system. Development of proper neuronal structure requires precise regulation of multiple cellular processes, including axon formation, outgrowth and branching [1–4]. Axon formation creates a distinct molecular and functional compartment of the neuron and the position of axon initiation is often critical for establishing neuronal polarity . Axons initiate and extend led by highly protrusive growth cones that sense environmental guidance cues. As growth cones advance, the axon shaft undergoes consolidation, a process that suppresses protrusive activity behind the growth cone and maintains axon polarity . Axon branching and/or the extension of multiple axons allow neurons to innervate multiple targets [2, 6]. The formation of axon branches in specific locations and prevention of branching at inappropriate sites requires local regulation of signals that promote or suppress protrusions. These processes are critical for neuronal development, yet the mechanisms that regulate axon formation, consolidation and branching in the complex in vivo environment remain poorly understood.
Precise regulation of filamentous actin (F-actin) dynamics is essential for the cell motility processes underlying neuronal morphogenesis [1, 4, 7, 8]. A necessary first step in axon formation is accumulation of F-actin at the initiation site, which was first shown in situ during axonogenesis in grasshopper sensory neurons . In addition, the organization of F-actin into filopodia is a key prerequisite for neurite formation in cultured mouse cortical neurons . In Caenorhabditis elegans, the specific site of F-actin accumulation and axon formation is determined by extracellular UNC-6/netrin, which locally activates a signaling pathway that controls the actin regulatory protein MIG10/lamellipodin [11, 12]. In mammalian in vitro models of neuronal polarization, actin regulation is also a critical convergence point for polarity signals (reviewed in [13, 14]). Nonetheless, our understanding of the mechanisms controlling the process of initial axon emergence from the cell body and the underlying actin rearrangements in vivo is far from complete.
During outgrowth, the polarized structure of axons must be established and maintained by consolidation along the axon shaft and restriction of F-actin-based protrusions to the growth cone [1, 4]. In vitro, consolidation is mediated in part by Rho-kinase inhibition of F-actin at the growth cone neck . Along the axon shaft, calpain proteolytic activity is required to inhibit protrusive activity by reducing levels of cortactin, an actin regulator . Thus, active suppression of F-actin protrusive activity along the axon shaft is required for proper axon extension and to prevent ectopic branching. To date, the process of axon consolidation in vivo has not been investigated.
The formation of axon branches also relies on dynamic rearrangements of the actin cytoskeleton [4, 6, 17–19]. Axon branching can occur by at least two distinct modes: growth cone bifurcation or interstitial branching off the axon shaft [2, 6, 17]. Although bifurcation is necessary for sensory axon target innervation in vivo[20, 21], the cytoskeletal rearrangements involved in this process are largely unknown. Interstitial branching appears to be the predominant branching mode in most neuron types studied thus far [2, 6, 22–24]. During interstitial branching in cultured cortical neurons, F-actin accumulates at branch initiation points and actin polymerization is required for branch formation [25, 26]. Branch promoting factors have been shown to act through derepression of calpain-mediated consolidation, thereby releasing the inhibition of F-actin polymerization . Still, little is known about the dynamics of axon branching or the underlying F-actin rearrangements in vivo. Studies using live imaging of F-actin in vitro have been instrumental to our understanding of axon formation, outgrowth and branching [1, 4, 15, 25, 26]. Here, we image F-actin during neuronal morphogenesis in intact embryos, where axons must integrate multiple signals that influence their motility.
Recent studies have brought to light the importance of intrinsic transcription factor regulation in neuronal morphogenesis . Transcription factor expression not only defines neuronal identity, but also can dictate axon trajectory choices downstream of identity decisions. For example, specific combinations of LIM homeodomain (LIM-HD) and homeobox domain (Hox) transcription factors are expressed in subsets of spinal motor neurons, and these determine the motor axon trajectories and innervation of specific muscle targets [28–31]. In retinal ganglion cells, cell-specific expression of Zic2, a zinc-finger transcription factor, and Isl2, a LIM-HD transcription factor, regulate the midline crossing choice of retinal ganglion cell axons at the optic chiasm [32, 33]. These transcription factors mediate axon pathway choices at least in part by regulating expression of Eph family axon guidance receptors [30, 34, 35]. In general, however, the mechanisms by which transcription controls axon motility remain largely unknown.
The zebrafish Rohon-Beard (RB) spinal sensory neurons provide an ideal model to investigate mechanisms controlling neuronal development in vivo because of their unique and stereotyped morphology [36, 37]. RB neurons extend two types of axons, centrals and peripherals, with distinct trajectories and behaviors . Each neuron extends ascending and descending central axons that grow longitudinally within the spinal cord to form the dorsal longitudinal fasciculus, and a single peripheral axon that exits the spinal cord and branches in the skin. The differential pathways of central and peripheral RB axons are determined in part by differing responses to extracellular signals . Furthermore, LIM-HD transcription factors of the Islet family and their cofactors, CLIMs, are essential intrinsic regulators of RB neuron morphology [39, 40]. Ubiquitous expression of a dominant negative CLIM (DN-CLIM) protein disrupts LIM-HD transcriptional activity and results in the reduction or elimination of RB peripheral axons without an apparent effect on the central axons or RB cell fate [39, 40]. Thus, these transcription factors are selectively required for peripheral axon formation and/or guidance. In this study, we analyze how LIM-HD transcription factors control cell motility processes and the F-actin cytoskeleton during neuronal morphogenesis in vivo.
To investigate the mechanisms controlling RB morphology and to understand how two axons from one cell are guided differently, we used live imaging of axon behaviors and F-actin distribution in vivo. We were able to visualize changes in F-actin distribution during dynamic processes such as axon consolidation and branching. We show that extensive F-actin protrusive activity precedes formation of the peripheral axon, and that F-actin concentrates at the site of peripheral axon initiation. In addition, we found that DN-CLIM differentially affects the motility of peripheral and central axons. In most cases, peripheral axons failed to initiate in DN-CLIM-expressing embryos; however, those that formed had significantly reduced growth rates and branching. In contrast, central axon growth rates were faster in DN-CLIM-expressing embryos. Finally, we found that disruption of LIM-HD activity did not affect F-actin-based protrusive activity or the ability of F-actin to accumulate at peripheral axon initiation sites, indicating that F-actin dynamics were unperturbed by DN-CLIM. However, DN-CLIM did influence the location of F-actin accumulation and axon initiation, suggesting LIM-HD activity may function in axon positioning.