Growth cone form, behavior, and interactions in vivo: Retinal axon pathfinding as a model

Growth Cone Form, Behavior, and Interactionsin Vivo: Retinal Axon Pathfinding as a Model

Carol Mason and Lynda Erskine

Departments of Pathology, Anatomy and Cell Biology, and the Center for Neurobiology andBehavior, Columbia University, College of Physicians and Surgeons, 630 W. 168th Street, New York,New York 10032

Received 20 April 2000; accepted 25 April 2000

ABSTRACT: Studies in vitro have revealed a greatdeal about growth cone behaviors, especially responses toguidance molecules, both positive and negative, and thesignaling systems mediating these responses. Little, how-ever, is known about these events as they take placein vivo.With new imaging methods, growth cone behaviors can bechronicled in the complex settings of intact or semi-intactsystems. With the retinal projection through the optic chi-asm as a model, we examined the hypothesis previouslydrawn from static material that growth cone form is posi-tion-specific: growth cone form in fact reflects specific be-haviors, including rate and tempo of extension, that are

more or less prominent in different locales in which growthcones are situated. Other studies show that growth conesinteract with cells along the pathway, both specialized non-neuronal cells and other neurons, some expressing knownguidance molecules. The present challenge is to bridgedynamic imaging with electron microscopy and molecularlocalization, in order to link growth cone behaviors withcell and molecular interactions in the natural setting inwhich growth cones extend. © 2000 John Wiley & Sons, Inc. J

Neurobiol 44: 260–270, 2000

Keywords:retinal ganglion cell guidance; midline; filop-odia; optic chiasm; growth cone stalling

In the last few years, studies on the biology of growthcones have added to the enormous body of knowledgeon growth cone behaviors on different substrates (e.g.,Burden-Gulley et al., 1995; Hynds and Snow, 1999),cytoskeletal dynamics (e.g., Challacombe et al., 1997;Evans et al., 1997; Suter and Forscher, 1998; Dent etal., 1999), the role of growth cone appendages (e.g.,Isbister and O’Connor, 1999; Steketee and Tosney,1999), and extracellular influences mediating guid-ance behaviors (e.g., McCaig and Erskine, 1996;Grabham and Goldberg, 1997; Condic et al., 1999;Hopker et al., 1999). Advances have been made inthree areas: First, negative or repulsive responses, or

“collapse,” upon meeting negative cues have beendescribed (Luo and Raper, 1994). Second, large fam-ilies of molecular factors that act positively or nega-tively on growth cones, either by contact or by diffu-sion, have been identified and localized to pathwaysin vertebrates and invertebrates (for review, Good-man, 1996; Tessier-Lavigne and Goodman, 1996;Holder and Klein, 1999). Many of these factors arethought to be perceived simultaneously (Rose andChiba, 1999) and elicit both hierarchical and opposingeffects on growth cones (Stoeckli et al., 1997; Varela-Echaverria et al., 1997; Winberg et al., 1998). Third,recent work has defined intracellular signaling sys-tems (Luo et al., 1997; Song et al., 1997; Gitler andSpira, 1998; Kuhn et al., 1998; Gomez and Spitzer,1999) that serve as switches during aversive or posi-tive responses to these families of guidance molecules(e.g., Song et al., 1998).

Most of these advances in our understanding ofgrowth cone function derive from studies of growingneuronsin vitro. Little of this body of knowledge has

Correspondence to:C. Mason ([email protected]).Contract grant sponsor: National Institutes of Health; contract

grant number: EY12736 (formerly NS27615).Contract grant sponsor: National Institutes of Health; contract

grant number: PO NS30532 (CM).Contract grant sponsor: Long-Term Fellowship from the Hu-

man Frontiers Science Program (LE).© 2000 John Wiley & Sons, Inc.

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been verifiedin vivo, mainly because of the difficultyof distinguishing and monitoring individual growthcones, both in simple invertebrate and in more com-plex vertebrate systems. How growth cones behaveand respond to cues in the rather varied and complexenvironments of the intact nervous system is thuspoorly understood. The recent availability of imagingtechniques, along with new ways of molecularly la-beling (and perturbing) individual axons and growthcones (Dynes and Ngai, 1998; Mombaerts, 1999), hasopened the window to glimpsing how growth conesbehave in the context of their actual paths and desti-nations.

This review will address what we have learned inthe last few years about growth cone behaviorsinvivo, focusing on the mammalian visual system, spe-cifically growth through the optic chiasm, while mak-ing parallels with studies on other sites and species.We will discuss how growth cones interact with cellsin their growth pathways, such as other axons orneurons and glia and intermediate targets, reviewingultrastructural studies on unlabeled as well as labeled,identified growth cones. Finally, we discuss some ofthe guidance molecules known to be expressed at theoptic chiasm, their involvement in mediating growthcone guidance decisions at this intermediate target,and the information that can potentially be obtainedon this issue by linking dynamic imaging of growthcone behaviors with ultrastructural analysis.

DYNAMICS OF GROWTH CONEBEHAVIOR IN VIVO

Among the popular systems for studying axon growthand pathfinding, those focusing on the crossing oravoidance of the midline of the CNS have provenuseful models, including the commissural axons fromdorsal spinal cord that cross the floorplate at theventral midline; neurons which cross the midline ofthe insect central nerve cord; and, the subject of ourwork, retinal axons from each eye which divergewithin the optic chiasm to targets on either side of thebrain (for review, Stoeckli and Landmesser, 1998).

In the mammalian visual system, retinal ganglioncell axons from the two optic nerves grow toward oneanother either cross the midline or turn away from itand thereby establish an X-shaped fiber intersectioncalled the optic chiasm. In animals with binocularvision such as mammals, but not in lower vertebrates,ganglion cell axons originating from the nasal retinacross the midline to project into the contralateral optictract, whereas ganglion cells from temporal retina donot cross, but grow away from the midline into theipsilateral optic tract. In mouse, ipsilaterally project-

ing ganglion cells are found in a ventral-temporalcrescent of the retina, whereas contralaterally project-ing ganglion cells are located throughout the retina(Guillery et al., 1995).

Growth Cone Form is Position-specific:Information from Static Studies

Over a decade ago, the ability to label growth coneswith intracellular markers, either horseradish peroxi-dase (HRP) or DiI, provided considerable evidencethat growth cone morphology reflects the nature of theimmediate local environment (for review, Mason andWang, 1997). These earlier studies were based onviews of selectively labeled growth cones in staticpreparations, either sections or whole mounts. Inpaths such as nerves or tracts within the CNS, growthcones display simple streamlined forms, with thebody of the growth cone longer than wide. If presentat all, short filopodia protrude from the tip. In regionswhere direction of growth changes or at the entry to anew environment, such as at the optic nerve head orentry from the tract into the tectal target, collectivelytermed “decision regions,” growth cones are complex,with an expanded body and filopodia extending inmultiple directions.

In our first studies on retinal axon growth cones inthe chiasm, injections of HRP were made in the wholeretina to ensure that all fibers were effectively labeled(Bovolenta and Mason, 1987). In contrast, PierreGodement analyzed growing axons by applying smallcrystals of the carbocyanine dye, DiI, to selectedregions of the retina. Many of the growth cones la-beled with DiI resembled the HRP-labeled growthcones seen in our previous study, that is, growth conesin the optic nerve near the chiasm were long andstreamlined, and the majority of growth cones in thechiasm were more broad and complex, with multiplefilopodia (Godement et al., 1990). However, somegrowth cones were large and Y-shaped and spreadwithin a zone 150–200mm proximal to the midlineand were observed only after injections into the ven-trotemporal retina, the exclusive site of origin of un-crossed fibers. Further, many of these highly complexendings had a curved profile, with the tip pointingback toward the ipsilateral optic tract.

Growth Cone Forms in Action: DynamicImaging Studies

The profiles arrested in action in the dye-labeled prep-arations suggested a sequence of steps during diver-gence of uncrossed fibers from ventrotemporal retinaaway from the midline and crossing of contralaterallyprojecting fibers. These steps were verified in a living

Growth Cone Forms and Behaviors in vivo 261

preparation of retina, optic nerves, chiasm, and tractsin which retinal axons were labeled with DiI andimaged with time-lapse video microscopy (Godementet al., 1994). The dynamic chronicles confirmed thecorrelations predicted by the static studies: the simpleforms reflect elongation and complex forms developwhen growth cones rest or pause (Fig. 1, and seesupplementary material for video clip at http://journals.wiley.com/neu), agreeing with studies on other brainregions in the vertebrate nervous system (e.q., Hallo-ran and Kalil, 1994; Brittis and Silver, 1995). More-over, in addition to showing the behaviors duringvarious maneuvers, the time-lapse video accountsclearly demonstrated thetempoof growth of individ-ual axons in the chiasm, a feature that is impossible tograsp in static preparations. Growth cone advance issaltatory, with frequent advances and pauses. Stallingis especially lengthy in the midline zone, occurring inboth crossed and uncrossed axons, but growth conesremain motile during pauses and make minute ad-vances and retractions. During pausing or stallingnear the chiasm midline, extensive remodeling of thegrowing tip occurs, especially on the growth cones

that will turn. Ventrotemporal growth cones developmultiple growth cones on branches of the originalgrowth cone; these branches condense and retract,only to spread and become complex again. Many suchcycles can occur within a single pause period. More-over, the spread, Y-shaped morphologies as well ascurved profiles and filopodia pointing backwards de-velop exclusively on uncrossed axons prior to turning.Such morphologies, then, are predictive of an un-crossed trajectory, both in static and living prepara-tions. These and other imaging studies on the chiasmprovided novel views of growth cone maneuvers in adecision region and suggested that crossed and un-crossed axons respond differentially to cues associ-ated with the chiasmatic midline.

Once axons reach their destination, growth coneshave forms associated with growth along paths as theyenter and arborize in their target region. As growthcones choose target cells, they cease advancing but gothrough dynamic “displays” of complex growth coneforms (Kaethner and Stuermer, 1994), rather like theforms seen during the long pauses seen in decisionregions. Arborization follows the loss or resorption of

Figure 1 (A) A single growth cone undergoes successive shape changes according to its behavior:images from dynamic chronicles. DiI crystals were placed in the dorsal temporal retina of a livingpreparation taken from an embryonic day 14 embryo. This growth cone (arrow, A) adopts astreamlined form when advancing (B,D) and spreads when pausing (C,E). This growth cone wasmonitored in the optic chiasm, but the same form–behavior relationships would be seen in othersegments of the pathway. (A) time 0; (B) 20 min; (C) 2 h, 30 min; (D) 3 h, 30 min; (E) 12 h. (Seesupplementary material for video clip at http://journals.wiley.com/neu)

262 Mason and Erskine

the primary growth cone, with smaller growing tipsand branchlets advancing and retracting (Witte et al.,1996). The activities of the branches and tips ofdeveloping axon arbors resemble the dynamic motil-ity of dendritic spines (e.g., Dunaevsky et al., 1999),in that the moving tips are actin-filled and muchsmaller than the lead growth cones, which can be20–30mm in their longest axis.

The imaging studies allow predictions on growthcone behavior to be made on the basis of static viewsof growth cone shape. A recent study examined staticpreparations and verified the behaviors byin vitroassays. DiI-labeled growth cones from nasal retinashow streamlined forms, thought to reflect rapid elon-gation, as they travel through the anterior tectum toposterior tectal targets and complex forms in the pos-terior tectum where they will contact target cells (Llir-

bat and Godement, 1999).In vitro, membranes fromthe anterior tectum caused growth cones to developelongated forms and accelerate their rate of advance,whereas target posterior tectal membranes had littleeffect on growth rates and led to a spreading ofgrowth cones, mimicking induction of behaviorsinvivo.

Rates of Growth

In addition to revealing that different growth coneforms correlate with extension or pausing behaviors,dynamic imaging has indicated therates of growthalong individual segments of a pathway (Mason andWang, 1997; Fig. 2). DiI-labeled growth cones weremonitored in a semi-intact preparation of retina, optic

Figure 2 Schema of rates and tempo of growth in different parts the retinal axon pathway. Growthcones advance (straight lines; 55mm/h; duration, 15–5 min), and pause (h; duration, 20–30 min)along the path from retina to the optic tract. When growth cones advance, they are streamlined,whereas during pauses, they adopt complex forms. Turning growth cones (*; long pause) are mostcomplex. Note that pausing is more prevalent in the chiasm than in the retina, optic nerve or tract,where the duration of advance periods is quite lengthy. Numbers refer to average overall rates ofadvance for each segment of the path. Ret, retina; on, optic nerve; ch, chiasm; ot, optic tract. Areawith dashed lines represents the midline region containing specialized radial glia.


nerves, chiasm, and diencephalon, including the optictracts. In the segments of the pathway from retina totargets, the actual rate of growth when growth conesare advancing is rather similar, about 55mm/h. More-over, the rest or pause period lengths are similar inthese segments of the path, on the average, from 20 to30 min. The duration of advance periods, however,varies widely with locale, with the longest stints ofadvance in the retina (;100 min), and the shortest,punctuated by pauses, in the optic chiasm (;15 min).Given that the pause periods within the chiasm are thelongest of any of the segments (30 min on the aver-age), the overall growth rate is slowest in the decisionregion of the chiasm and most rapid in the most directpaths of the retina, followed by the optic nerve andtract. Interestingly, both pre- and postmidline, growthcones exhibit extensive pausing behavior, but as theycross the midline, the speed of advance is rapid (Ma-son and Wang, 1997; Chan et al., 1998; Wang andMason, unpublished observations). These analysesshowed that growth cones pause more frequently andhave an overall slower extension rate in decisionregions where multiple positive and negative guid-ance cues are present.

Growth Cone Form is Behavior-, and, inturn, Position-Specific

The dynamic imaging studies showed that growthcones can display the various forms described abovein each loci along a given growth pathway. Thus,whenever growth cones pause in their advance, evenalong a straight path and without a subsequent changein direction, growth cones become complex. When-ever growth cones advance, even in a decision region,they are elongated, as seen in Figure 1. These obser-vations were surprising, given the conclusions fromstatic studies that growth cone form is “position-specific.” We therefore reexamined this hypothesis.

After establishing the relationship between growthcone form and behavior, we related both of thesefeatures with position along the retinal axon pathway(Mason and Wang, 1997). Behaviors are more or lessfrequent at a given site: growth cones move rapidly instraight tracts, spending more time in the advancemode, and thus streamlined form, than in the pausingform. When in the chiasm, growth cones tend to pausealmost as much as they advance (Fig. 2). Thus, thepredominant form seen both in static and dynamicsituations in straight paths is streamlined and tapered,whereas in decision regions, the most prevalent formis the complex growth cone. Therefore, growth coneform is not strictly position-specific, but rather, first,behavior-specific, and, in turn, position-specific. Aswith neuronal phenotypes in general, the behavioral

and morphological repertoire of growth cone in aparticular pathway is specific for each neuronal class(e.g., interneurons vs. motor neurons), as seen, forexample, in theXenopusspinal cord (Gomez andSpitzer, 1999).

Even with these remarkable glimpses into growthcone behaviors, several questions arise, with referenceto relating growth cone behaviors to those observed inconventional in vitro tests. Can the behaviors de-scribed above only be capturedin vivo, that is, arethey difficult to recapitulatein vitro where the settingis more reductionist? Conversely, are there any be-haviors in vitro that are not seenin vivo, such asfull-scale collapse, rarely recordedin vivo? Is collapsea more drastic growth cone response that occurs insituations where the signals are less conflicting andmore unimodal thanin vivo (e.g., Fan and Raper,1995). Future work on growth cone dynamics in thecontext of the nervous system will have to reconcilespecifics of the behavioral repertoire of growth conesto determine which behaviors are equivalent andwhich are unique to thein vivo and in vitro settings.

GROWTH CONE INTERACTIONSWITH CELLS AND MOLECULESIN THEIR PATH

Ultrastructural Studies ofUnlabeled Axons

What do growth cones grow on and contact as theyextendin vivo? Ultrastructural analyses of unlabeledaxons have given information on the cells that growthcones contact as they extend in their path. In a classicultrastructural study based on thin section reconstruc-tions, the follower growth cones exiting the eye of thecrustaceanDaphniawere seen to grow along the leadgrowth cone (Lopresti et al., 1973). In zebrafish, theearliest axons to grow in the CNS from the epiphysisfollow a prescribed path, consisting of neuroepithelialand other more differentiated cells (Wilson and Eas-ter, 1991), and in theDrosophila CNS, extendingaxons of longitudinal pioneer neurons follow a routeconsisting of other neurons, glia, and their processes(Hidalgo and Booth, 2000).

Detailed ultrastructural studies of the mouse opticnerve and chiasm in unlabeled material has aimed tofollow the course of fiber growth, both with regard tocellular relations of growth cones, and chronotopicorder (Colello and Coleman, 1997; Colello andGuillery, 1998). In ferrets and primates, retinal gan-glion cell growth cones gather at and enter the opticstalk, preserving little in the way of retinal topography(Fitzgibbon and Reese, 1996), interpose themselves


among neurite fascicles, and do not always associatewith glia or nonneuronal surfaces (Williams et al.,1991). As in the primate, mouse retinal axon growthcones are intermingled with more mature axons, butsegregate and accumulate beneath the pia as the opticnerve meets the brain. Then, however, growth conesmove away from subpial surfaces toward the ventric-ular zone, redistributing through the depth of thepathway and then coursing subpially again as theyenter the optic tract. As retinal fibers course throughthe optic chiasm, they are organized retinotopicallywithin the nerve in a quadrant-specific manner; theposition of each quadrant, however, shifts near themidline, where growth cones distribute more loosely(Colello and Coleman, 1997; Colello and Guillery,1998; Chan and Chung, 1999).

Growth Cone–Intermediate TargetInteractions in the Optic Chiasm

Analyses of dye-labeled growth cones have suggestedthat growth cones change behavior, primarily the di-rection of extension, by interacting with cellular andmolecular cues in their path; these cues are often inintermediate targets (e.g., Bovolenta and Dodd, 1990;O’Connor et al., 1990). In the case of commisssuralneurons in the spinal cord, the intermediate target isthe floor plate, whereas in the insect limb, after en-countering a zone of high fasciclin IV expression,sensory growth cones turn, aided by guidepost cells(Kolodkin et al., 1992; Isbister and O’Connor, 1999).

Our dynamic imaging of retinal growth cones pausingjust proximal to the midline and subsequently eithercrossing the midline or turning away from it sug-gested that cellular specializations might also be at thediencephalic midline. At the site of divergence, glialand neuronal cellular specializations were in factfound (Mason and Sretavan, 1997). In mouse, a pal-isade of radial glia and an early-born population ofneurons, occupy the midline region where the ventro-temporal retinal axons diverge (Fig. 2). The radial gliastraddle the midline in a configuration similar to thefloor plate and have molecular expression patternsdistinct from other surrounding radial glia (Marcus etal., 1995). In other species, growth cones meet radialglia soon after they begin to form the chiasm, lateralto the midline (Reese et al., 1994). The early-bornneurons form a V- or chevron-shaped array caudal tothe optic chiasm, and express carbohydrate epitopes[stage-specific embryonic antigen-1 (SSEA-1; Marcusand Mason, 1995; Marcus et al., 1999] and immunesystem antigens such as CD44 (Sretavan et al., 1994).

Labeling growth cones with HRP or DiI and iden-tifying them in fixed preparations allowed particularforms of growth cones to be related to different cel-lular interactions. Elongated growth cones have con-cave lamellar surfaces. After additional sectioning ofthe preparation and examination it in the electronmicroscope, it can be seen that the curves result fromenfolding fascicles of other neurites (Bovolenta andMason, 1987; Marcus et al., 1995). The dynamicimaging demonstrated that growth cones with this

Figure 3 Growth cone interactions in the optic chiasmin vivo. Inset: Streamlined diI-labeledgrowth cone (arrow). Electron micrograph of the same growth cone indicating that it is apposed toother neurites (*) and enfolds radial glial processes (g). Micrograph5 1 mm; inset5 50 mm;.


form extend rapidly, and the ultrastructural analysis,in turn, indicated that rapid extension can occur alongother fascicles of neurites (Fig. 3). The complex growthcones are more contorted and contact a number of pro-files, belonging both to other axons and to radial glia.

Several observations from the dynamic chroniclesand combined axon tracing and guidance factor local-ization suggest that an intimate relationship with theglial palisade and early neuronal array might reflectthe decision-making process during divergence: First,axons pause at the midline for extended periods(30–45 min), compared to rapid growth in the opticnerve and tract (Godement et al., 1994; Mason andWang, 1997). Second, growth cones expand into com-plex forms and then condense and become more slen-der (Godement et al., 1994). This occurs repeatedlyand may reflect contact with the radial glial palisadeor other neurites and growth cones from the contralat-eral projection (see below for a discussion of the latterpossibility). Third, ultrastructural studies of DiI-la-beled growth cones with complex shapes at the chi-asm midline indicate that radial glial profiles can bevirtually surrounded by such complex growth cones(Marcus et al., 1995). Dynamic imaging indicates thatduring the cycles of spreading and condensing,“holes” appear in the spread growth cones (see Fig. 8in Godement et al., 1994). These holes are predictedto form from the fusion or contact of lamellar exten-sions of the growth cone as they enfold radial glialprocesses. Finally, omega profiles, considered to rep-resent endocytosis, are frequently seen within the glialcytoplasm, with a finger-like process emanating fromthe growth cone or axon (Colello and Coleman,1997). These structures suggest that there is commu-nication between growth cones and midline glia.

With reference to the early born CD44/SSEA neu-rons, combined DiI labeling and immunostaining withantibodies against SSEA-1 show that crossing axonstraverse the midline raphe of early neurons, whereasuncrossed axons approach but never cross the raphe.Analysesin vitro and in vivo indicate that the earlyneurons comprise a zone in which axons do not enter,yet whose borders retinal axons appear to follow,especially in the early stages of growth (Marcus et al.,1995; Marcus and Mason, 1995; Sretavan et al., 1995;Wang et al., 1995, 1996; Mason and Sretavan, 1997;Marcus et al., 2000). Ultrastructural analyses are inprogress to determine whether uncrossed or crossedretinal growth cones ever come into contact with thesecells.

Fiber–Fiber Interactions

In addition to contacting heterologous cells in theirpath, growth cones may require interactions with

other like neurons (fibers), especially in decision re-gions or when crossing the midline. In the embryonicgrasshopper CNS, the Q1 growth cone crosses themidline and fasciculates with its contralateral Q1 ho-mologue. Dynamic imaging reveals that when it firstreaches the midline, the growth cone retracts, thenadvances again until it touches the contralateral Q1and continues to advance (Myers and Bastiani,1993a,b). Dye coupling accompanies this interaction,perhaps mediating exchange of information. A similarsituation exists in the leech CNS (Wolszon et al.,1995). In the embryonic mouse chiasm, several ex-periments argue for a mechanisms of growth involv-ing fiber–fiber interactions, although this has not beendirectly observed. Given that hundreds of neurons arecrossing at any one time, the likelihood of labelingbilateral partner growth cones is slim, compared withobserving identified cells in the invertebrate systems.In fact, the critical interactions in the mouse opticchiasm appear to be between ipsilateral axons on oneside and crossing fibers from the contralateral side. Ifone eye is ablated, fibers from the remaining eye donot cross, but stall in the optic chiasm (Godement etal., 1990; but see Sretavan and Reichardt, 1993).Although fibers ultimately cross, there is a reductionin the ipsilateral component (Chan et al., 1999). Whengrowth cones stall proximal to the midline, one pos-sibility is that they wait for contralateral partners;because of surface molecules that are complimentary,crossed and uncrossed fibers will then fasciculate andgrow together.

One argument against growth cone–growth coneinteractions at the point of midline crossing in themouse chiasm is that axons are organized in eye-specific bundles that cross each other at right angles(Colello and Guillery, 1998). However, althoughthese bundles are surrounded by glial leaflets, thewrapping is incomplete, and growth cones tend to beon the outer aspect of the bundles, such that therecould be contact and communication with fibers fromeach eye. Surprisingly, fibers apparently do not leavetheir eye-specific bundles.

Role of Filopodia inCell–Cell Interactions

Filopodia are important for proper growth cone guid-ance (e.g., Chien et al., 1993; Kater and Rehder,1995). One similarity across species is that turningcan be mediated by an individual filopodium (Gode-ment et al., 1994) that dilates and becomes a newgrowth cone, as seen in turning in the grasshopperlimb (O’Connor et al., 1990) andDrosophila CNS(Murray et al., 1998). After filopodia contact an in-hibitory or stimulatory cell, distinct activities involv-


ing veil formation, branching, and neurite consolida-tion follow (Steketee and Tosney, 1999). Althoughadhesion of filopodia is similar whether extendingtoward or away from the correct target, filopodialextension rates toward the correct target are morerapid than those away from targets (Murray et al.,1998; Isbister and O’Connor, 1999). These recentinformative studies indicate that filopodia are key toguidance events, but were focused on a simple systemor were performedin vitro. In the intact mammaliansystems, it is more difficult to sort out which of themultiple cell types filopodia contact, even when agrowth cone is imaged and subsequently analyzed inthe electron microscope.

Interactions with MolecularFactors on Cells

What is known about molecular factors on the cellularcues in the optic chiasm? Again, we have drawn onthe insect midline for comparison. From a large-scalescreen for mutations that affect midline guidance inthe Drosophila CNS, two key genes,roundabout(robo) and commissureless(comm) were identified.Robo appears to be a receptor involved in a negativesignal that prevents midline crossing. InDrosophilarobo mutants, axons that normally grow ipsilaterallyproject across the midline, and all axons aberrantlycross and recross multiple times (Seeger et al., 1993),possibly because filopodia become too attached andtether growth cones (Murray and Whitington, 1999).Comm is a ligand expressed by midline glial cells inthe insect, which is thought to be internalized bycrossing axons, mediating the downregulation ofRobo and therefore required for proper decussation(Tear et al., 1996).

Robo defines a novel family of guidance receptors(Kidd et al., 1998). Slit, an inhibitory extracellularmatrix molecule secreted by the midline glia inDro-sophila, is a ligand for Robo. To date, threerobo andthree slit mammalian homologues have been identi-fied and shown to be expressed within the developingnervous system.In vitro, Slits have been found to bothinhibit CNS axon outgrowth and neuronal migrationand promote sensory axon growth and branching (forreview, Brose and Tessier-Lavigne, 2000). No homo-logue of commhas yet been identified. However, ascommissural axons traverse the midline, material istransferred to the axons from the floorplate cells,indicating that communication also occurs betweengrowth cones and the vertebrate midline glia (Cambelland Peterson, 1993).

In the developing mouse visual system, Robos andSlits are expressed in dynamic patterns both in theretina and in the region of the developing optic chiasm

(Erskine et al., 2000).In vitro Slit2 inhibits retinalaxon outgrowth with both ipsilaterally and contralat-erally projecting axons being equally affected. Exper-iments are ongoing to test whether Slit is a generalguidance cue for retinal axons or one that, by a gradedresponse by crossed and uncrossed cells, might me-diate divergence. One challenge in the future will beto determine if the Robo receptors are localized to thegrowth cone (see Gershon et al., 1998 for growth conelocalization of another class of molecule) and, if so,what the relative distribution is on the body of thegrowth cone compared to the filopodia of ipsilaterallyand contralaterally projecting axons. Combining suchstudies with ultrastructural analyses and dynamic im-ages will provide important insights into the functionof these and other guidance moleculesin vivo.

Signaling

A major advance in our understanding of how growthcones respond to cues has come from experimentsshowing that differences in levels of cyclic nucleo-tides within the growth cone can induce oppositeturning of growth cones in response to the sameguidance cue. Depending on the molecular factor andthe neuron in question, alterations in cAMP or cGMPcan effect these changes (Song et al., 1998). Calciumhas also been a focus of recent studies showing thatalterations in calcium levels are associated with aslowing or acceleration of growth cone advance(Kuhn et al., 1998; Gomez and Spitzer, 1999). Thishas been elegantly studied in a simple system of theearly developing spinal cord, where large neurons(Rohon-Beard cells), interneurons, and motor neuronscould be identified by position. The correlations ofgrowth cone form and behavior hold true for thissystem, and in addition, calcium transients serve as anicon of signaling that can be correlated with growthcone behavior. Unfortunately, the complexity of theoptic chiasm with orders of magnitude greater num-bers of growth cones transiting through this locus atany one time makes such an analysis difficult. How-ever, as with the growth cone shape–behavior corre-lations, these data will be useful in indirectly analyz-ing signaling.

CONCLUSIONS

Dynamic imaging has led to a greater understandingof how growth cones behave in their growth context.The changes in morphology and rates of growth dur-ing maneuvers through distinct cellular milieus haveprovided clues to the mechanisms of axon navigationtoward targets. Analyses that have shown that the


forms of growth cones take reflect behavior and be-haviors depend on the site, explaining previous ob-servations that growth cone form is position-specific.Ultrastructural studies have explained how shapes ofgrowth cones relate to interactions and define whatdifferent cellular interactions are. These distinct be-haviors and interactions can now be interpreted in thecontext of multiple guidance cues, currently beingelucidated by molecular localization andin vitro tests.The challenge ahead is to look at growth cones cel-lularly and subcellularly with the electron micro-scope, after dynamic imaging, thereby linking behav-ior with cellular and molecular components, tounderstand how growth cones navigate in complexenvironments.

We thank Dr. L.-C. Wang for use of unpublished data,Drs. R. Marcus and P. Godement for many useful discus-sions during the course of the work, and Dr. Q. Zhang, R.Blazeski, and N. Tulino for help with data analysis andimaging.

REFERENCES

Bovolenta P, Dodd J. 1990. Guidance of commissuralgrowth cones at the floor plate in embryonic rat spinalcord. Development 109:435–447.

Bovolenta P, Mason CA. 1987. Growth cone morphologyvaries with position in the developing mouse visual path-way from retina to first targets. J Neurosci 7:1447–1460.

Brittis PA, Silver J. 1995. Multiple factors govern intrareti-nal axon guidance: a time lapse study. Mol Cell Neurosci6:413–432.

Brose K, Tessier-Lavigne M. 2000. Slit proteins: key reg-ulators of axon guidance, axonal branching, and cellmigration. Cur Opin Neurobiol 10:95–102.

Burden-Gulley SM, Payne HR, Lemmon V. 1995. Growthcones are actively influenced by substrate-bound adhe-sion molecules. J Neurosci 15:4370–4381.

Cambell RM, Peterson AC. 1993. Expression of a lacZtransgene reveals floor plate cell morphology and mac-romolecular transfer to commisssural axons. Develop-ment 119:1217–1228.

Challacombe JF, Snow DM, Letourneau PC. 1997. Dy-namic microtubule ends are required for growth coneturning to avoid an inhibitory guidance cue. J Neurosci17:3085–3095.

Chan SO, Chung KY, Taylor JSH. 1999. The effects ofearly prenatal monocular enucleation on the routing ofuncrossed retinofugal axons and the cellular environmentat the chiasm of mouse embryos. Eur J Neurosci 11:3225–3235.

Chan SO, Chung KY. 1999. Changes in axon arrangementin the retinofugal pathway of mouse embryos: confocalmicroscopy study using single and double-dye label.J Comp Neurol 406:251–262.

Chan SO, Wong KF, Chung KY, Yung WH. 1998. Changes

in morphology and behaviour of retinal growth conesbefore and after crossing the midline of the mouse chi-asm—a confocal microscopy study. Eur J Neurosci 10:2511–2522.

Chien CB, Rosenthal DE, Harris WA, Holt CE. 1993.Navigational errors made by growth cones without filop-odia in the embryonic Xenopus brain. Neuron 11:237–251.

Colello SJ, Coleman LA. 1997. Changing course of growingaxons in the optic chiasm of the mouse. J Comp Neurol379:495–514.

Colello SJ, Guillery RW. 1998. The changing pattern offibre bundles that pass through the optic chiasm of mice.Eur J Neurosci 10:3653–3663.

Condic ML, Snow DM, Letourneau PC. 1999. Embryonicneurons adapt to the inhibitory proteoglycan aggregan byincreasing integrin expression. J Neurosci 15:10036–10043.

Dent EW, Callaway JL, Szebenyi G, Baas PW, Kalil K.1999. Reorganization and movement of microtubules inaxonal growth cones and developing interstitial branches.J Neurosci 19:8894–8908.

Dunaevsky A, Tashiro A, Majewska A, Mason CA, YusteR. 1999. Developmental regulation of spine motility inthe mammalian central nervous system. Proc Nat AcadSci 96:13438–13433.

Dynes JL, Ngai J. 1998. Pathfinding of olfactory neuronaxons to stereotyped glomerular targets revealed by dy-namic imaging in living zebrafish embryos. Neuron 20:1081–1091.

Erskine LE, Williams SE, Brose K, Kidd T, Rachel R,Goodman CG, Tessier-Lavigne M, and Mason CA. 2000.Retinal ganglion cell axon guidance in the mouse opticchiasm: expression and function of Robos and Slits.J Neurosci 20:4975–4982.

Evans LL, Hammer J, Bridgman PC. 1997. Subcellularlocalization of myosin V in nerve growth cones andoutgrowth from dilute-lethal neurons. J Cell Sci 110:439–449.

Fan J, Raper JA. 1995. Localized collapsing cues can steergrowth cones without inducing their full collapse. Neuron14:263–274.

Fitzgibbon T, Reese BE. 1996. Organization of retinal gan-glion cell axons in the optic fiber layer and nerve of fetalferrets. Vis Neurosci 13:847–861.

Gershon TR, Baker MW, Nitabach M, Macagno ER. 1998.The leech receptor protein tyrosine phosphataseHmLAR2 is concentrated in growth cones and is in-volved in process outgrowth. Development 125:1183–1190.

Gitler D, Spira ME. 1998. Real time imaging of calcium-induced localized proteolytic activity after axotomy andits relation to growth cone formation. Neuron 20:1123–1135.

Godement P, Salaun J, Mason CA. 1990. Retinal axonpathfinding in the optic chiasm: Divergence of crossedand uncrossed fibers. Neuron 5:173–196.

Godement P, Wang L-C, Mason CA. 1994. Retinal axondivergence in the optic chiasm: Dynamics of growth conebehavior at the midline. J Neurosci 14:7024–7039.


Gomez TM, Spitzer NC. 1999. In vivo regulation of axonextension and pathfinding by growth-cone calcium tran-sients. Nature 397:350–355.

Goodman CS. 1996. Mechanisms and molecules that con-trol growth cone guidance. Annu Rev Neurosci 19:341–377.

Grabham PW, Goldberg DJ. 1997. Nerve growth factorstimulates the accumulation of beta1 integrin at the tips offilopodia in the growth cones of sympathetic neurons.J Neurosci 15:5455–5465.

Guillery RW, Mason CA, Taylor JSH. 1995. Developmen-tal determinants at the mammalian optic chiasm. J Neu-rosci 15:4727–4737.

Halloran MC, Kalil K. 1994. Dynamic behaviors of growthcones extending in cortical brain slices observed withvideo microscopy. J Neurosci 14:2161–2177.

Hidalgo A, Booth GE. 2000. Glia dictate pioneer axontrajectories in the Drosophila embryonic CNS. Develop-ment 127:393–402.

Holder N, Klein R. 1999. Eph receptors and ephrins: effec-tors of morphogenesis. Development 126:2033–2044.

Hopker VH, Shewan D, Tessier-Lavigne M, Poo M, Holt C.1999. Growth-cone attraction to netrin-1 is converted torepulsion by laminin-1. Nature 401:69–73.

Hynds DL, Snow DM. 1999. Neurite outgrowth inhibitionby chondroitin sulfate proteoglycan: stalling/stopping ex-ceeds turning in human neuroblastoma growth cones. ExpNeurol 160:244–255.

Isbister CM, O’Connor TP. 1999. Filopodial adhesion doesnot predict growth cone steering events in vivo. J Neu-rosci 19:2589–2600.

Kaethner RJ, Stuermer CAO. 1994. Growth behavior ofretinotectal axons in live zebrafish embryos under TTX-induced neural impulse blockade. J Neurobiol 25:781–796.

Kater SB, Rehder V. 1995. The sensory-motor role ofgrowth cone filopodia. Curr Opin Neurobiol 5:68–74.

Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-LavigneM, Goodman CS, Tear G. 1998. Roundabout controlsaxon crossing of the CNS midline and defines a novelsubfamily of evolutionary conserved guidance receptors.Cell 92:205–215.

Kolodkin AL, Matthes DJ, O’Connor TP, Patel NH,Admon A, Bentley D, Goodman CS. 1992. FasciclinIV: Sequence, expression, and function during growthcone guidance in the grasshopper embryo. Neuron9:831– 845.

Kuhn TB, Williams CV, Dou P, Kater SB. 1998. Laminindirects growth cone navigation via two temporally andfunctionally distinct calcium signals. J Neurosci 18:184–194.

Llirbat B, Godement P. 1999. Positional specificities ofretinal growth cones in the mouse superior colliculus. EurJ Neurosci 11:2103–2113.

Lopresti V, Macagno ER, Levinthal C. 1973. Structure anddevelopment of neuronal connections in isogenic organ-isms: Cellular interactions in the development of the opticlamina of Daphnia. Proc Nat Acad Sci USA 70:433–477.

Luo L, Jan LY, Jan Y-N. 1997. Rho family small GTP-

binding proteins in growth cone signalling. Curr Op Neu-robiol 7:81–86.

Luo Y, Raper JA. 1994. Inhibitory factors controllinggrowth cone motility and guidance. Curr Opin Neurobiol4:648–654.

Marcus RC, Blazeski R, Godement P, and Mason CA. 1995.Retinal axon divergence in the optic chiasm: uncrossedaxons diverge from crossed axons within a midline glialspecialization. J Neurosci 15:3716–3729.

Marcus RC, Mason CA. 1995. The first retinal axon growthin the mouse optic chiasm: axon patterning and the cel-lular environment. J Neurosci 15:6389–6402.

Marcus RC, Shimamura K, Sretavan DW, Lai E, Ruben-stein JLR, Mason CA. 1999. Domains of regulatory geneexpression and the developing optic chiasm: correspon-dence with retinal axon paths and candidate signalingcells. J Comp Neurol 403:346–358.

Marcus RC, Matthews GA, Gale NW, Yancopoulos GD,Mason CA. 2000. Axon guidance in the mouse opticchiasm: Retinal neurite inhibition by ephrin “A”-express-ing hypothalamic cells in vitro. Dev Biol 221:132–147.

Mason CA, Sretavan DW. 1997. Glia, neurons and axonpathfinding during optic chiasm development. Curr OpinNeurobiol 7:647–653.

Mason CA, Wang L-C. 1997. Growth cone form is behav-ior-specific and, consequently, position-specific along theretinal axon pathway. J Neurosci 17:1086–1100.

McCaig CD, Erskine L. 1996. Nerve growth and nerveguidance in a physiological electric field. In: McCaig CD,editor. Nerve growth and nerve guidance. London: Port-land Press Ltd, p 151–170.

Mombaerts P. 1999. Seven-transmembrane proteins asodorant and chemosensory receptors. Science 286:707–11.

Murray MJ, Merritt DJ, Brand AH, Whitington PM. 1998.In vivo dynamics of axon pathfinding in the DrosophilaCNS: a time-lapse study of an identified motorneuron.J Neurobiol 37:607–621.

Murray MJ, Whitington PM. 1999. Effects of roundabout ongrowth cone dynamics, filopodial length and growth conemorphology at the midline and throughout the neuropile.J Neurosci 19:7901–7912.

Myers PZ, Bastiani MJ. 1993a. Cell-cell interactions duringthe migration of an identified commissural growth conein the embryonic grasshopper. J Neurosci 13:115–126.

Myers PZ, Bastiani MJ. 1993b. Growth cone dynamicsduring the migration of an identified commissural growthcone. J Neurosci 13:127–143.

O’Connor TP, Duerr JS, Bentley C. 1990. Pioneer growthcone steering decisions mediated by single filopodialcontacts in situ. J Neurosci 10:3935–3946.

Reese BE, Maynard TM, Hocking DR. 1994. Glial domainsand axonal reordering in the chiasmatic region of thedeveloping ferret. J Comp Neurol 349:303–324.

Rose D, Chiba A. 1999. A single growth cone is capable ofintegrating simultaneously presented and functionallydistinct molecular cues during target recognition. J Neu-rosci 19:4899–4906.

Seeger M, Tear G, Ferres-Marco D, Goodman CS. 1993.Mutations affecting growth cone guidance in Drosophila:


genes necessary for guidance toward or away from themidline. Neuron 10:409–426.

Song H-j, Ming G-l, Poo M-m. 1997. cAMP-inducedswitching in turning direction of nerve growth cones.Nature 388:275–279.

Song H-j, Ming G-l, He A, Lehmann M, McKerracher L,Tessier-Lavigne M, Poo M-m. 1998. Conversion of neu-ronal growth cone responses from repulsion to attractionby cyclic nucleotides. Science 181:1515–1518.

Sretavan DW, Feng L, Pure E, Reichardt LF. 1994. Embry-onic neurons of the developing optic chiasm express L1and CD44, cell surface molecules with opposing effectson retinal axon growth. Neuron 12:957–975.

Sretavan DW, Pure E, Siegel MW, Reichardt LF. 1995.Disruption of retinal axon ingrowth by ablation of em-bryonic mouse optic chiasm neurons. Science 269:98–101.

Sretavan DW, Reichardt LF. 1993. Time-lapse video anal-ysis of retinal ganglion cell axon pathfinding at the mam-malian optic chiasm: growth cone guidance using intrin-sic chiasm cues. Neuron 10:761–777.

Steketee MB, Tosney KW. 1999. Contact with isolatedsclerotome cells steers sensory growth cones by alteringdistinct elements of extension. J Neurosci 19:3495–3506.

Stoeckli ET, Landmesser LT. 1998. Axon guidance atchoice points. Curr Op Neurobiol 8:73–79.

Stoeckli ET, Sondregger P, Pollerberg GE, Landmesser LT.1997. Interference with axonin-1 and NrCAM interac-tions unmasks a floor-plate activity inhibitory for com-missural axons. Neuron 18:209–221.

Suter DM, Forscher P. 1998. An emerging link betweencytoskeletal dynamics and cell adhesion molecules ingrowth cone guidance. Curr Op Neurobiol 8:106–117.

Tear G, Harris R, Sutaria S, Kilomanski K, Goodman CS,

Seeger MA. 1996.Commissurelesscontrols growth coneguidance across the CNS midline inDrosophila andencodes a novel membrane protein. Neuron 16:501–514.

Tessier-Lavigne M, Goodman CS. 1996. The molecularbiology of axon guidance. Science 274:1123–1133.

Varela-Echavarria A, Tucker A, Puschel AW, Guthrie S.1997. Motor axon subpopulations respond differentiallyto the chemorepellents netrin-1 and semaphorin D. Neu-ron 18:193–207.

Wang L-C, Dani J, Godement P, Marcus RC, Mason CA.1995. Crossed and uncrossed retinal axons respond dif-ferently to cells of the optic chiasm midline in vitro.Neuron 15:1349–1364.

Wang L-C, Rachel RA, Marcus RC, Mason CA. 1996.Chemosuppression of retinal axon growth by the mouseoptic chiasm. Neuron 17:849–862.

Williams RW, Borodkin M, Rakic P. 1991. Growth conedistribution patterns in the optic nerve of fetal monkeys:implications for mechanisms of axon guidance. J Neuro-sci 11:1081–1094.

Wilson SW, Easter SS. 1991. Stereotyped pathway selectionby growth cones of early epiphysial neurons in the em-bryonic zebrafish. Development 112:723–746.

Winberg ML, Mitchell KJ, Goodman CS. 1998. Geneticanalysis of the mechanisms controlling target selection:Complementary and combinatorial functions of netrins,semaphorins, and IgCAMs. Cell 93:581–591.

Witte S, Stier H, Cline HT. 1996. In vivo observations oftimecourse and distribution of morphological dynamicsin Xenopus retinotectal axon arbors. J Neurobiol 31:219–234.

Wolszon LR, Passani MB, Macagno ER. 1995. Interactionsduring a critical period inhibit bilateral projections inembryonic neurons. J Neurosci 15:1506–1515.


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Growth cone form, behavior, and interactions in vivo: Retinal axon pathfinding as a model