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Common Mechanisms Underlying Growth Cone Guidance and Axon Branching Katherine Kalil,1,2 Gyorgyi Szebenyi,2* and Erik W. Dent1 1 2 Neuroscience Training Program and Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706 Received 20 April 2000; accepted 25 April 2000 ABSTRACT: During development, growth cones direct growing axons into appropriate targets. However, in some cortical pathways target innervation occurs through the development of collateral branches that extend interstitially from the axon shaft. How do such branches form? Direct observations of living cortical brain slices revealed that growth cones of callosal axons pause for many hours beneath their cortical targets prior to the development of interstitial branches. High resolution imaging of dissociated living cortical neurons for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing behaviors and enlargement of the growth cone. After a new growth cone forms and resumes forward advance, filopodial and lamellipodial remnants of the large paused growth cone are left behind on the axon shaft from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch The development of appropriate connections is essential for the correct functioning of the nervous system. Thus, intense interest has focused on the nerve growth cone at the axon tip that guides the growing axon along appropriate pathways and into targets. However, it has been known for many years that axons can *Present address: Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75235-9111. Correspondence to: K. Kalil ([email protected]). Contract grant sponsor: National Institutes of Health; contract grant number: NS14428 and NS34270 (KK). Contract grant sponsor: National Institutes of Health predoctoral training grant award; contract grant number: GM07507 (EWD). © 2000 John Wiley & Sons, Inc. points, we fluorescently labeled microtubules in living cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the growth cone. In both regions microtubules reorganize into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly developing branches with anterograde and retrograde movements. Although axon branching of dissociated cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to axon branching and suggest that common mechanisms underlie directed axon growth from the terminal growth cone and the axon shaft. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 145–158, 2000 Keywords: axon branching; growth cone behaviors; microtubules; fibroblast growth factor; time-lapse imaging also establish connections by extending collateral branches from the shaft of the primary axon into the target. Although growth cones are able to bifurcate, careful observations by O’Leary and colleagues (reviewed in O’Leary et al., 1990) showed that in vivo layer 5 cerebral cortical axons innervate their targets by developing interstitial branches from the axon shaft and not through bifurcation of the terminal growth cone. It was also shown that collateral branches form by a process of delayed budding from the primary axon many millimeters behind the growth cone. These findings led to the view that for efferent cortical axons the role of the growth cone at the tip of the primary axon is limited to guidance and elongation along appropriate pathways. Accordingly, growth cones of layer 5 cortical axons were thought to bring 145 146 Kalil et al. the axons into the vicinity of their targets but fail to respond to target cues and thus have no overt role in target selection (O’Leary and Koester, 1993). However, recent studies from our laboratory and others have shown that growth cone behaviors may serve to demarcate axon branch points (Halloran and Kalil, 1994; Yamamoto et al., 1997; Szebenyi et al., 1998; Davenport et al., 1999), that new growth from growth cones and axon branches may involve similar reorganization of the cytoskeleton (Dent et al., 1999a,b), and that multifunctional cues can influence axon guidance as well as collateral branching (Wang et al., 1999). Taken together these studies are beginning to identify common mechanisms that directly link growth cone guidance and axon branching. ROLE OF INTERSTITIAL AXON BRANCHING IN TARGET INNERVATION Studies in vivo have shown that interstitial collateral branches form at right angles from the axon shaft at considerable distances behind the growth cone and up to several days after the growth cone has advanced beyond the target innervated by the branch (Kuang and Kalil, 1994; O’Leary and Terashima, 1988). These delays in branching are thought to account for waiting periods between the arrival of axons in their target regions and development of innervation. Delayed interstitial axon branching has been demonstrated in two major efferent cortical pathways: the corpus callosum (Hogan and Berman, 1990; Norris and Kalil, 1991), which interconnects the two cerebral hemispheres, and the corticospinal tract, which arises from layer 5 of the sensorimotor cortex and innervates the pontine nuclei (O’Leary and Terashima, 1988; O’Leary et al., 1990) and the spinal cord (Kuang and Kalil, 1994). It is known that cortical axons make transient errors in their caudal trajectories, such that visual cortical axons extend past their targets in the pontine nuclei and grow inappropriately into the pyramidal tract in the spinal cord (O’Leary et al., 1990). Axons from forelimb areas of the sensorimotor cortex also make projection errors, often growing past appropriate targets in the cervical spinal cord and extending into the lumbar cord. Despite these projection errors, corticospinal connections are topographically appropriate from the earliest stages of development (Kuang and Kalil, 1994). These connections form by interstitial axon collaterals that branch into spinal targets where they develop elaborate terminal arbors. Similarly, retinal growth cones also overshoot their tectal targets but develop topographic tectal connections by extension of interstitial branches at appropri- ate target sites (Simon and O’Leary, 1992a,b). These results demonstrate a relative lack of precision in axon tracts versus specificity of target innervation by axon collaterals and suggest that in such systems the primary growth cone selects the appropriate pathway but grows past and ignores target regions. Subsequently, according to this view, the axon shaft responds to secreted target-derived factors and extends an interstitial branch into a specific target. Thus, pathfinding by growth cones and target selection by axon collaterals had been regarded by some authors as separate phenomena (O’Leary and Koester, 1993; Kennedy and Tessier-Lavigne, 1995; Bolz and Castellani, 1997; Joosten, 1997). GROWTH CONE BEHAVIORS IN TARGET REGIONS A series of studies on cocultures of explanted cerebral cortex and their targets in three-dimensional collagen gels have supported in vivo models of delayed interstitial branching by showing that collateral branches can be induced to extend toward targets by the release of a target-derived, diffusible chemoattractant. Observations of cocultured explants from the cortex and basilar pons, for example, showed that a diffusible activity from the pons specifically attracts layer 5 cortical axons at a distance. Importantly, the use of retrograde dye labeling also revealed that many of the axons attracted to the pontine target are actually collateral branches (Heffner et al., 1990), consistent with the mode of establishment of corticopontine connections in vivo (O’Leary et al., 1990). Time-lapse imaging of these cocultures confirmed that the collateral branches stimulated by the pons-derived activity form interstitially from the axon shaft and not by growth cone bifurcation (Sato et al., 1994). Although most of the branches are transient, more branches become stabilized in the presence of the pontine explant, suggesting that the target-derived activity promotes the initiation as well as stabilization of interstitial collaterals. In living brain slices of the corticopontine pathway (Bastmeyer and O’Leary, 1996) similar axon activity was described in the form of dynamic varicosities and filopodia-like extensions that sometimes become branches. Observations of developing retinotectal arbors in living tadpoles (Harris et al., 1987; O’Rourke et al., 1994; Witte et al., 1996) and zebrafish (Kaethner and Stuermer, 1992, 1994) also illustrated the importance of interstitial branching in the formation of axon arbors. Although none of these studies of interstitial branching addressed the role of the growth cone in Mechanisms of Axon Branch Formation target selection, other observations suggested that changes in growth cone morphology and behavior may be manifested at decision regions related to target recognition or branch points. Growth cones in fixed tissue were shown to exhibit dramatic differences in their morphologies, depending on their locations. In tracts and pathways simple forms predominate, but growth cones have more complex morphologies in decision regions where they change direction and approach or enter targets (Tosney and Landmesser, 1985; Caudy and Bentley, 1986; Bovolenta and Mason, 1987; Holt, 1989; Norris and Kalil, 1991, 1992). Consistent with these morphological observations, video microscopy of growth cone behaviors in semiintact preparations of the vertebrate brain have correlated elongated streamlined forms with growth cone advance and large complex forms with growth cone pausing, particularly at decision regions where growth cones need to recognize targets or make decisions about crossing the CNS midline (Harris et al., 1987; Kaethner and Stuermer, 1992; Sretavan and Reichhardt, 1993; Godement et al., 1994; Halloran and Kalil, 1994; Mason and Wang, 1997). We have used time-lapse video microscopy in slice preparations of early postnatal cortex to show that over many hours of development, growth cones in different regions of the callosal pathway have strikingly different behaviors (Halloran and Kalil, 1994). In the callosal tract growth cones advance rapidly and steadily, displaying continual shape changes. These primary growth cones do not make turns into cortical targets but extend well beyond them. Subsequently, axon branches tipped by small growth cones develop interstitially from the axon shaft and grow dorsally toward the overlying sensorimotor cortex. Growth cones of these axon collateral branches have uniformly small compact shapes and extend relatively slowly within the cortex in straight radial trajectories, in keeping with earlier electron microscopic evidence that callosal axons are guided into cortical targets by extension along radial glial processes (Norris and Kalil, 1992). The most dramatic behaviors were observed in regions of the callosum beneath cortical targets, where growth cones have elaborate morphologies and complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. These behaviors are likely to reflect recognition of cortical target signals by the growth cone. Importantly, interstitial branches to cortical targets develop at those points where complex growth cone pausing behaviors previously occurred, suggesting a link between growth cone pausing and axon branching. A timelapse study of behaviors of growing thalamic axons in 147 organotypic cocultures of the lateral geniculate nucleus (LGN) and visual cortex (Yamamoto et al., 1997) supported this view by demonstrating that regions of growth cone pausing are well correlated with axon branching. When axons from the LGN encounter layer 4 of the visual cortex, the normal target of LGN axons, they stop growing for several hours and often retract, with concomitant collapse of the growth cone. The emergence of a branch behind the growth cone several hours later suggested that target-derived signals may induce growth cone pausing and elicit growth of interstitial axon branches (Yamamoto et al., 1997). ROLE OF THE PRIMARY GROWTH CONE IN AXON BRANCHING Studies in brain slices and organotypic cultures have the advantage of preserving in situ the cellular and molecular cues present during CNS development. However, typically only a small region of the axon can be imaged at any given time and for periods of only a few hours. Moreover, the resolution of individual axons in these preparations is not sufficient to demonstrate precisely how growth cone behaviors are related to axon branching. Therefore, to study in detail the development of interstitial axon branches in relation to growth cone behaviors, we have used highresolution imaging of dissociated neurons from the cerebral cortex (Szebenyi et al., 1998). We chose for analysis pyramidal neurons from early postnatal sensorimotor cortex because in vivo layer 5 pyramidal neurons give rise to efferent axons that branch interstitially to cortical and subcortical targets (O’Leary and Terashima, 1988; Norris and Kalil, 1992; Kuang and Kalil, 1994). A disadvantage of this approach is that behaviors of dissociated neurons occur in the absence of normal target cues. However, an advantage of studying neurons in relative isolation is the ability to carry out imaging of single neurons at high resolution without the confounding influence of cell– cell interactions. By continuously imaging cortical neurons at frequent intervals for periods up to 5 days, we were able to observe the entire length of growing axons and follow the development of interstitial branches from the initial behaviors of the primary growth cone through the elongation of stable branches. Thus, lengthy periods of observation encompassed the history of the primary growth cone in relation to subsequent development of branching along the axon shaft. We found that cultured cortical neurons develop axon branches in a manner similar to cortical neurons 148 Kalil et al. in vivo (Szebenyi et al., 1998). Cortical neurons initially extend numerous minor processes from the cell body that are approximately equal in length and tipped by growth cones. One of the processes then continues to elongate and becomes the single axon. This scenario is also consistent with development of hippocampal neurons in culture (Dotti et al., 1988). About 20 – 40 h after plating, branches begin to extend interstitially from the axon shaft (Fig. 1) and form growth cones at their tips. In contrast to numerous transient short filopodia, branches persist for several days and grow to lengths averaging ⬎130 m. The numbers (4 –5), clustering (2–5), positions (up to 1 mm behind the primary growth cone), and delays (several hours to several days) in extension of axon branches correspond to features of delayed interstitial branches observed on developing cortical axons in vivo (O’Leary and Terashima, 1988; Kuang and Kalil, 1994) and in situ (Bastmeyer and O’Leary, 1996). Interestingly, as happens in vivo (O’Leary et al., 1990) the primary axon distal to the interstitial branch often ceases to grow or degenerates. Importantly, we found that pausing behaviors by the primary axonal growth cone are highly correlated with interstitial axon branching (Szebenyi et al., 1998), as we had previously observed in situ in target regions of the corpus callosum (Halloran and Kalil, 1994). Growth cone pausing behaviors are characterized by repeated cycles of collapse, retraction, and extension without net forward advance. During pausing behaviors, which could last from 1 to 30 h in dissociated cultures, growth cones become on average six times larger than those that are advancing and have large spread out lamellipodia. The greatly expanded lamellipodium then reorganizes by forming a new growth cone at its tip (Fig. 2). By the end of the pausing period, this new primary growth cone emerges from the tip of the lamellipodium to lead the growing axon, whereas the rest of the large paused growth cone remains behind on the axon in the form of filopodial and lamellar activity. After delays of several hours to several days after the primary growth cone resumes forward advance, interstitial axon branches tipped by growth cones subsequently emerge from filopodial and lamellar protrusions on these active regions of the axon. These results are consistent with recent findings in cultures of retinal ganglion cell axons encountering repellent cues. When retinal growth cones are induced to collapse by contact with inhibitory target cells from the tectum, with cells transfected with repulsive ephrin molecules or by mechanical manipulations, lateral extensions in the form of filopodia and lamellipodia develop rapidly from the axon shaft behind the growth cone (Daven- port et al., 1999). Lateral extensions from the axon of the collapsed growth cone (or even from trailing fasciculated axons) can then continue growing to form branches tipped by growth cones extending in new orthogonal directions. Our results (Szebenyi et al., 1998) suggest a novel mechanism whereby the primary growth cone demarcates future branch points by pausing, reorganizing and leaving behind active remnants on the axon from which interstitial branches later emerge. This result essentially eliminates the distinction often made between delayed interstitial branching and growth cone bifurcation because the division of the original primary growth cone into a new growth cone and an active remnant on the axon shaft is actually a bifurcation. Although this work was carried out on isolated neurons in the absence of targets, the results are consistent with the notion that in vivo it is the growth cone that recognizes targets that will be innervated by delayed interstitial branches. According to this model (Fig. 3) interstitial branching results directly from target recognition by the growth cone, suggesting that guidance of the primary growth cone and target selection by axon collaterals are not separate events but closely related phenomena. CYTOSKELETAL REORGANIZATION UNDERLYING AXON BRANCHING The actin and microtubule cytoskeleton of neuronal growth cones plays a central role in shape changes and reorienting behaviors underlying axon guidance. Microtubules (MTs) in growth cones provide structural support and act as tracks for transport of vesicles as they do in the axon. During growth and navigation of the axon, the MT array within the growth cone reorganizes and reorients toward the future direction of axon outgrowth (Sabry et al., 1991; Tanaka and Kirschner, 1991; Lin and Forscher, 1993; Tanaka et al., 1995; Tanaka and Sabry, 1995; Suter et al., 1998). Filamentous actin (F-actin), which occupies peripheral regions of the growth cone (Forscher and Smith, 1988; Bridgman and Dailey, 1989; Lewis and Bridgman, 1992), drives motility of the veil-like lamellipodia and occupies filopodia, the finger-like microspikes that protrude from the leading edge of the growth cone. A major unanswered question is how the actin and microtubule cytoskeleton interact within the growth cone during guidance behaviors (O’Connor and Bentley, 1993; Letourneau, 1996; Suter and Forscher, 1998). For example, local accumulation of Factin could result in tension from actin–microtubule interactions that would pull the central microtubule Mechanisms of Axon Branch Formation 149 Figure 2 Enlargement and reorganization of a growth cone during prolonged pausing. During the 18-h pausing period, the lamella of the growth cone gradually enlarges. At 6 h a prominent microtubule loop in the growth cone is apparent under phase optics. At 12 h the new primary growth cone is emerging from the tip of the reorganized growth cone. By 18 h the primary growth cone resumes elongation, and a large lamellar expansion remains behind on the axon shaft. Etchings on the coverslips serve as landmarks. Scale bar ⫽ 30 m. Reprinted from Szebenyi et al. (1998) with permission. domain toward a target site. On the other hand, depletion of F-actin, through the attenuation of retrograde flow, could result in microtubule advance to- Figure 1 Extension of interstitial branches from regions of the axon where prolonged growth cone pausing occurs. Representative images were taken from a series of images acquired at 3-min intervals during the 3-day observation period. Between 5 and 20 h, the growth cones on the branched axon pause in the region indicated by the arrows. Later, between 35 and 53 h, a cluster of axon branches extends interstitially from the axon in this pausing region. Scale bar ⫽ 100 m. Reprinted from Szebenyi et al. (1998) with permission. 150 Kalil et al. Figure 3 Schematic representation of different stages in axon branching. (A) A growth cone of an efferent cortical axon is advancing toward its target, indicated by the circle. (B) The primary growth cone pauses for extended time periods in the vicinity of the target and enlarges. (C) After the primary growth cone resumes forward advance, remnants of the reorganized growth cone are left behind as filopodial or lamellar activity along the axon shaft. (D) After a time delay, an interstitial axon branch emerges from a region of lamellar activity at some distance behind the primary growth cone and extends toward the target. Reprinted from Szebenyi et al. (1998) with permission. ward positive extracellular cues (reviewed in Letourneau, 1996; Suter and Forscher, 1998). Local regulation of axonal morphogenesis by neurotrophins (Berninger and Poo, 1996) implies that they induce cytoskeletal rearrangements (Gallo et al., 1997), leading to directed extension of the growth cone. The role that MTs play in axon growth is not limited to their continuous rearrangement at the terminal growth cone. Studies on cultured hippocampal neurons suggest that MTs fragment within the region of the axon where interstitial branches form (Yu et al., 1994). Moreover, in a recent study demonstrating induction of filopodia on regions of axons in contact with nerve growth factor (NGF)-coupled beads, it was found that local debundling of microtubules occurs in the axon shaft along with actin accumulation (Gallo and Letourneau, 1998). To begin to understand the cytoskeletal mechanisms underlying directed axon growth, we investigated specific changes that occur in the MT array within the terminal growth cone and at sites of interstitial axon branch formation. Some authors have argued that the reorganization of the MT array is based solely on the assembly and disassembly of MTs and not on their movement through the cytoplasm (reviewed in Hirokawa et al., 1997), whereas other authors have argued that individual MTs can interact with motor proteins that actively transport them to new locations (reviewed in Baas, 1997; 1999; Baas and Brown, 1997). To date, this issue remains controversial, in large part because of results based on relatively low-resolution fluorescence analyses. Nevertheless, there is compelling evidence from indirect studies that individual MTs are capable of movement (Yu et al., 1996; Slaughter et al., 1997; Gallo and Letourneau, 1999). We took a direct approach by visualizing the movements of individual MTs with high-resolution time-lapse fluorescence digital imaging of dissociated cortical neurons microinjected with fluorescently labeled tubulin (Dent et al., 1999b). We hypothesized that in order for new growth to occur at the growth cone and from interstitial axon branches, MTs must rearrange from a bundled array to a more plastic configuration. We therefore investigated the reorganization of the bundled MT array during transitions from quiescent to growth states and determined whether individual MTs in these regions are capable of independent movement. During pausing, growth cones are typically large and flat (Szebenyi et al., 1998) and in their central regions MTs form prominent loops (Fig. 4) characteristic of slowly growing axons (Tsui et al., 1984; Lankford and Klein, 1990; Sabry et al., 1991; Tanaka and Kirschner, 1991). We found that during transitions of the growth cones from quiescent to growth states, MTs undergo a dramatic reorganization that involves an initial breaking away of short MTs from the MT loop, followed by a splaying apart of the looped MTs and their entry into branches forming from the growth cone (Fig. 5). MTs explore growth cone lamellipodia with forward, backward, and lateral movements, and individual MTs are able to move independently of one another. To observe MT reorganization and movements at axon Mechanisms of Axon Branch Formation 151 Figure 4 Cortical neurons fixed and stained with fluorescent phalloidin, which binds actin filaments and antibodies to ␣-tubulin, which label microtubules. (A) MTs (pseudocolored green) in the axon splay apart at branch points, as shown in the inset. Actin filaments (pseudocolored red) are concentrated in distal regions of the growth cone and developing axon branches. (B) MTs form a prominent loop in the central region of the growth cone and are closely apposed to actin filaments in the distal region of the growth cone, as shown in the inset. In both images regions of overlap between MTs and actin filaments appear yellow. Scale bar ⫽ 10 m; inset ⫽ 5 m. branch points, we focused on the expanded regions of the axon shaft that resemble flat lamellipodia from which axon branches develop (Szebenyi et al., 1998). Observations at early stages of branch formation revealed disruptions in the bundled MT array where MTs splay apart (Figs. 4 and 6). MTs explore filopodial processes extending from the axon [Fig. 6(A)] and continue to invade elongating branches [Fig. 6(B)]. Only those filopodia that contain MTs develop into branches, whereas those lacking MTs either disappear or remain as filopodia. However, even longer branches tipped by growth cones and heavily invested with MTs are capable of regressing, suggesting that although MT invasion is necessary for development of branches, the presence of MTs does not guarantee the survival of a branch. In cases where filopodia develop along the axon shaft but MTs within the axon remain bundled, branches never form, even though MTs frequently penetrate into these transient filopodia. These results suggest that development of branches from the axon shaft involves local splaying apart of the MT bundle accompanied by breakdown of longer MTs and invasion of shorter MTs into nascent branches. This reorganization of MTs is similar to that observed during formation of branches from paused growth cones. MTs invade developing branches from the axon shaft and move forward and backward within them, sometimes growing and shrinking at the same time. Even longer MTs were seen to retreat from growing axon branches. Imaging of branch formation along the axon shaft over extended time periods revealed that MTs undergo continual redistribution during the simultaneous extension of some processes, accompa- 152 Kalil et al. nied by invasion of MTs and the regression of others concomitant with a loss of MTs. This suggests that anterograde and retrograde MT transport, in addition to MT polymerization and depolymerization, play a role in directing MTs toward branches favored for growth and away from processes that retract. The retrograde movement of MTs was unexpected, because MT movement in intact cells has thus far only been documented in the anterograde direction, when MTs move parallel to their own long axis (Terasaki et al., 1995; Keating et al., 1997). Our overall impression was that shorter MTs have more complex and rapid movements. Individual MTs sometimes move at constant rates but can exhibit saltatory movements, changing speed and direction within seconds and accelerating rapidly to velocities up to 30 m/min. MTs longer than 10 m tend to move at rates significantly slower than those shorter than 10 m. The inverse relationship of MT length and transport rates may be due to more drag (Willard and Simon, 1983) on longer MTs. If this were correct, increasing the length of a MT by polymerization and further stabilizing it through interactions with MTassociated proteins (Desai and Mitchison, 1997) would result in slower MT movements. In contrast, in regions of new growth, short MTs undergoing more active movements may be required for rapid exploration of growth cones and developing branches. As MTs in these processes invade regions favored for growth, some of the MTs could then become stabilized in preferred directions by elongating and slowing down. Recent studies indicate that cytoplasmic dynein is a key motor protein that drives MT movement in cellular extracts (Heald et al., 1996) and in neurons (Ahmad et al., 1998). Similarities in the rates of anterograde and retrograde movement observed in the present study suggest that the same motor might Figure 5 Movement and fragmentation of individual MTs in a paused growth cone. (A) A large paused growth cone has a prominent MT loop in the central region. MT movements shown in image sequences (B) and (C) occur in regions indicated by the boxes. In sequence (B) a MT elongates while moving rapidly into the peripheral lamellipodium and then shortens while moving laterally (40 – 60 s). In sequence (C) a MT elongates (0 –30 s) and then fragments into two shorter MTs (40 s). The shorter MT segment remains stationary without elongating or shortening (50 –70 s), whereas the longer MT segment grows slightly (50 s) and then fragments a second time (60 –70 s). Matching images in (B⬘) and (C⬘) highlight in white the MTs shown in (B) and (C). Scale bar ⫽ 5 m. Reprinted from Dent et al. (1999) with permission. Mechanisms of Axon Branch Formation 153 Figure 6 Splaying apart of the MT array within the axon shaft during interstitial branching. The sequence of black and white with matching pseudocolor images shows changes in the MT array before (A, A⬘) and during (B, B⬘) development of an interstitial branch. Pseudocolor images indicate fluorescence intensity from low to high as shown in the scale. Arrows in the first image in the sequence in (A) point to two regions where MTs are splayed apart in contrast to the bundled array in the nonbranching region of the axon shaft (arrowhead). P and D refer to proximal and distal segments of the axon, respectively. Six minutes later the MTs in the upper region (arrow) have formed a bundle, whereas those in the lower region (arrow ) remain splayed. At 28 min MTs have invade a filopodial process (arrow). In (B) the same region of the axon is shown 5 h later when MTs are invading an interstitial branch developing in the position of the filopodium shown at 28 min in (A). Arrows in (B) indicate distal ends of the MTs. Times are shown in hours and minutes. Scale bar ⫽ 5 m. Reprinted from Dent et al. (1999) with permission. be responsible for both types of movement. In theory, the forces generated by cytoplasmic dynein could result in the transport of MTs with their plus (Ahmad et al., 1998) or minus (Heald et al., 1996) ends leading (reviewed in Baas, 1999). Previous ultrastructural studies on cultured hippocampal neurons suggested that the presence of short MTs at branch points result from the fragmentation of longer microtubules (Yu et al., 1994). Our direct observations confirmed that indeed MTs do undergo fragmentation (Fig. 5). During transitions from quiescent to growth states, fragmentation of longer MTs would result in a higher number of shorter MTs ideally suited for rapid exploratory movements within growth cones and developing branches. After invasion into appropriate regions, these short MTs could then elongate and become stabilized, allowing for further growth of the axon to occur. Fragmentation of MTs in neurons may be locally regulated. For example, focal application of a calcium ionophore to axons of Aplysia neurons was shown to elicit new growth cones that develop into branched neuritic processes (Ziv and Spira, 1997). In these branching regions the MT array appears to be discontinuous, suggesting that locally induced MT fragmentation is correlated with growth of new axonal processes. What mechanisms might account for MT fragmentation in the axon? Several recent studies suggest that the protein katanin, 154 Kalil et al. known to have MT-severing properties in vitro (McNally and Vale, 1993; Hartman et al., 1998), is present in a variety of cell types including neurons (McNally and Thomas, 1998; Ahmad et al., 1999). One interesting possibility is that MT fragmentation may be regulated by intrinsic and/or extrinsic factors that locally activate katanin. Our results demonstrate a similar reorganization of the microtubule cytoskeleton at the terminal growth cone and at sites of branch formation (Fig. 7). This should perhaps not be surprising, given that interstitial branches form from regions of activity on the axon shaft at sites where the terminal growth cone has previously paused (Szebenyi et al., 1998). The similar MT behaviors in these two regions are probably important for enabling individual MTs to move more effectively into the lamellipodia and filopodia of growth cones and developing interstitial branches. It has long been recognized in vivo that after a developing cortical axon extends an interstitial branch toward a callosal or spinal target the region of the cortical axon distal to the branch degenerates (O’Leary et al., 1990). This process is probably accompanied by a redistribution of MTs. At present the mechanisms that regulate the long-term redistribution of MTs are unknown, but it is compelling to speculate that the kinds of anterograde and retrograde MT movements that we observed might be a key factor in determining whether an axon branch degenerates or continues to grow and stabilize. Thus far we have considered only the reorganization and movement of MTs during new axonal growth. Although actin is known to play an important role in regulating changes in the distribution of MTs at the growth cone, the precise nature of actin–microtubule interactions in the growth cone remains to be elucidated (reviewed in Suter and Forscher, 1998) and has not been well characterized in any motile cell (reviewed in Waterman-Storer and Salmon, 1999). We have used the direct approach of visualizing movements of MTs and actin filaments in living cortical neurons during branching from the growth cone and the axon shaft (Dent et al., 1999a). By microinjecting fluorescent tubulin and phalloidin (which selectively binds filamentous actin) into the same neuron. we were able to image simultaneous changes in both cytoskeletal elements. Preliminary results show that actin filaments in peripheral regions of the growth cone and on the axon shaft are concentrated where new growth is occurring. Further, actin filaments appear to accumulate prior to the invasion of MTs toward sites of axon branching. In fact, the first overt sign of branching along the axon shaft or at the growth cone is a focal accumulation of actin filaments Figure 7 Schematic illustration of a model for reorganization of the MT array during directed axon outgrowth. According to the model, longer bundled MTs in terminal growth cones and developing axon branches locally fragment into shorter MTs. Individual short MTs then explore these developing processes with rapid movements. New growth occurs in regions where MTs become stabilized and elongate. In (A) MTs in the pausing growth cone have formed a looped array. Short MTs move away from the MT loop to explore the growth cone lamellipodium. In (B) a branch is emerging from the axon shaft accompanied by local fragmentation of the MT array and invasion of the nascent branch by short MTs. In (C) the axon branch is extending as individual MTs elongate and move within it. Arrows indicate directions of movement. P and D refer to proximal and distal segments of the axon, respectively. Mechanisms of Axon Branch Formation accompanied by splaying of MTs (Fig. 4). Subsequently, MTs invade regions of high filamentous actin concentration where the two cytoskeletal elements become closely apposed. These results suggest that MTs and actin filaments may be directly coupled. EFFECTS OF TARGET-DERIVED FACTORS ON AXON BRANCHING Although much of our work has been carried out on isolated neurons in the absence of their normal targets, it is known that target-derived factors influence growth cone behaviors and elicit development of axon collaterals. During the past decade different cues have been identified that regulate the guidance of the growth cone (reviewed in Tessier-Lavigne and Goodman, 1996; Cook et al., 1998; Mueller, 1999; Song and Poo, 1999). These cues include neurotrophins and other highly conserved families of guidance molecules. NGF (Letourneau, 1978; Gundersen and Barrett, 1979) and BDNF (Song et al., 1997), for example, attract growth cones of sensory neurons. Basic fibroblast growth factor (FGF-2) has been shown to play a role in targeting retinal axons to the frog optic tectum in vivo (McFarlane et al., 1995, 1996). Families of molecules such as the netrins and semaphorins and most recently the slit proteins are known to exert attractive or repulsive effects on specific growth cones at specific locations in the vertebrate and invertebrate nervous systems (Mueller, 1999). There is increasing evidence that many of these cues affect not only the behavior of the growth cone and the guidance of the primary axon but also the initiation and extension of collateral branches from the axon shaft. For example, in vivo applications of neurotrophin 3 (NT-3) in the spinal cord (Schnell et al., 1994) and brain-derived neutrophic factor (BDNF) in the optic tectum (CohenCorey and Fraser, 1995) promote sprouting of corticospinal axons and arborization of optic axons, respectively. Guidance cues such as the netrins and semaphorins have been characterized primarily for their role in attracting or repelling various growth cones. However, one such molecule, the mammalian Slit 2N protein, which is homologous to slit proteins that repel axons at the midline in Drosophila, was recently found to stimulate formation of collateral axon branches on rat dorsal root ganglion neurons in collagen gels (Wang et al., 1999; reviewed in Van Vactor and Flanagan, 1999). Taken together, previous findings suggest that axon guidance and interstitial branching may be regulated by common factors affecting the growth cone. To investigate this possibility, we have used bath and 155 local application of growth factors on cultured cortical neurons to determine their effects on the development of collateral branches in relationship to behaviors of the growth cone (unpublished results). A survey of a number of growth factors revealed that fibroblast growth factor (FGF)-2 is particularly effective in promoting branching of cortical axons (Szebenyi et al., 1999). Application of FGF-2 for as little as 2 h is sufficient to elicit maximal branching, which is three times greater than for neurons in untreated control cultures. As we had previously determined (Szebenyi et al., 1998) branches had to be at least 30 m long to persist and become stabilized. Also, branches typically occur in clusters, are often tipped by growth cones, and frequently rebranch. Previous observations of growth cone pausing in relation to branching showed that the larger the primary growth cone becomes during pausing the more axon branches develop in the pausing region. We therefore investigated effects of FGF-2 on growth cone size and rates of extension. We found that because of lengthy pausing periods by the growth cones, axons of FGF-2–treated neurons extend at only half the rates of controls and that by 20 h after treatment, growth cones become twice as large as untreated controls. Thus, one mechanism of action of FGF-2 could be a direct effect on growth cones by slowing or arresting their extension and greatly increasing their size. Both of these effects would promote collateral branching. Another possibility is that growth factors can stimulate local regions of the axon shaft to branch. This was demonstrated in a recent study in which application of NGF-coupled beads to the axons of dorsal ganglion neurons elicited filopodial sprouts in close proximity to the bead (Gallo and Letourneau, 1998). Using a similar approach, we coupled heparin to polystyrene beads, coated them with FGF-2, and then applied them at low densities to cortical cultures. We found that in comparison with BSA-coated control beads, which were associated with branches only a fraction of the time, the FGF-2– coated beads often promoted interstitial branching in close proximity (within 10m) to the bead but not at greater distances. Beads contacting or landing close to growth cones also elicit development of branches. In fact, our results suggest that beads acting on localized regions of the axon are more likely to elicit branches on distal as opposed to proximal regions of the axon. In a previous study (Gallo and Letourneau, 1998), application of NGF-coupled beads elicited filopodia that develop within minutes of bead application and are often transient. Moreover, in order for a branch to develop, the growing process had to be in continual contact with 156 Kalil et al. the NGF-coupled beads. In contrast, we found that branches in our cultures develop over a much longer time course (1–3 days), are stable over the entire observation period, grow to lengths averaging over 90 m, and often rebranch. Branches continue to elongate even when the bead moved away from contact with the axon. This shows that continuous contact with the FGF-2– coated beads is not necessary for growth of cortical axon branches. Thus, FGF-2 can act locally to induce axons to branch, but such branches occur preferentially on more labile regions of the axon in the vicinity of the growth cone. Large increases in growth cone size and slowing of growth cone extension also suggest that effects of FGF-2 on axon branching in many cases directly involves the growth cone. This is consistent with a hypothesis that FGFs in the optic tectum may slow growth cone advance and switch axons form a growing mode into an arborizing or branching mode (McFarlane et al., 1996). Because FGF-2 has been shown to increase L-type calcium channels at branch points during branching of cultured hippocampal neurons (Shitaka et al., 1996) and calcium transients are known to slow growth cone advance (Gomez et al., 1995; Gomez and Spitzer, 1999), it is possible that local changes in intracellular calcium and other second messengers may be a common pathway by which growth factors such as FGF-2 induce growth cone pausing leading to axon branching. FUTURE DIRECTIONS The mechanisms by which environmental cues are transduced into the cytoskeletal reorganization that underlies guidance and branching of the axon are poorly understood. Nevertheless, it is becoming clear that these two developmental events are interrelated and may share many of the same cellular and molecular mechanisms. In the future, to understand the mechanisms by which axon guidance and branching are orchestrated, it will be important to determine the effects of extracellular guidance molecules on the cytoskeleton, the role of intracellular second messengers in transducing these effects, the molecular machinery regulating the local splaying and fragmentation of microtubules, the identity of the motor proteins driving anterograde and retrograde microtubule movements, and the nature of the interactions that link the microtubule and actin cytoskeleton. 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