a PATTERNS & PHENOTYPES

Single-Cell Analysis of Somatotopic MapFormation in the Zebrafish Lateral Line SystemAkira Sato, Sumito Koshida, and Hiroyuki Takeda*

The zebrafish lateral line is a simple sensory system comprising a small number of neurons in addition totheir sensory organs, the neuromasts. We have adopted this system as a model for single-cell level analy-ses of topographic map formation and examined when and how the lateral line topographic map is estab-lished. Single-neuron labeling demonstrated that somatotopic organization of the ganglion emerges by54 hr postfertilization, but also that this initial map is not as accurate as that observed at 6 days postferti-lization. During this initial stage, individual neurons exhibit extensively diverse behavior and morpholo-gies. We identified leader neurons, the axons of which are the first to reach the tail, and later-appearingaxons that contribute to the initial map. Our data suggest that lateral line neurons are heterogeneousfrom the beginning of lateral line development, and that some of them are intrinsically fate determined tocontribute to the somatotopic map. Developmental Dynamics 239:2058–2065, 2010. VC 2010 Wiley-Liss, Inc.

Key words: lateral line; zebrafish; somatotopy; topographic map; single-cell; live imaging; neural circuit formation

Accepted 14 April 2010

INTRODUCTION

The establishment of appropriateneuronal circuits is essential for theeventual proper functioning of thenervous system in higher organisms.During embryogenesis, a populationof newly born neurons is seeminglyhomogeneous and tightly packed in asmall region of the neural tissue, forexample, in a ganglion. However, ifall neurons were to differentiate iden-tically and innervate similar targets,the nervous system would fail to cre-ate the necessary diversity of circuitscrucial to the developing functionalnervous system. This heterogeneitymay arise from a subtle but signifi-cant difference at the single-cell levelbetween adjacent neurons throughcell–cell communication and/or bysensing positional cues. Single-cell

level analyses of neural circuit forma-tion focusing on this early heterogene-ity are, therefore, expected to provideinsights into the mechanisms thatcreate diverse functional neuralnetworks.

Axon guidance has been long stud-ied in many model organisms and isknown to be regulated by a combina-tion of repellants, attractants, andtheir corresponding receptors (Dick-son, 2002; Guan and Rao, 2003). It is,however, still not fully understoodhow individual neurons adopt certainfates and extend their axons towarddistinct targets. To further under-stand these processes, the behavior ofindividual neurons and their axonsneeds to be observed in vivo duringneural circuit formation. Typically,however, this is not an easy taskmainly because of the complexity of

neural systems which normally con-sist of millions of neurons with com-plex networks of entangled axons anddendrites. In view of this complexity,the zebrafish lateral line system, asensory system that responds towater movement, is potentially one ofthe best models with which to studyhow individual neurons select theirappropriate targets at a single-cellresolution.The lateral line system comprises a

set of discrete sensory organs, knownas the neuromasts, and a small num-ber of sensory neurons that innervatethem (Metcalfe et al., 1985). The neu-romasts each have 20 to 30 superficialmechanosensory hair cells (Nagielet al., 2008; Faucherre et al., 2009),and are aligned on the body surface inboth the head and the trunk regions.An array of six to eight neuromasts

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Additional Supporting Information may be found in the online version of this article.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, JapanGrant sponsor: Japan Society for the Promotion of Science; Grant-in-Aid for JSPS Research Fellows; Grant number: 21-6524.*Correspondence to: Hiroyuki Takeda, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail:htakeda@biol.s.u-tokyo.ac.jp

DOI 10.1002/dvdy.22324Published online 19 May 2010 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 239:2058–2065, 2010

VC 2010 Wiley-Liss, Inc.

extends along the lateral midline inthe trunk and tail and is known asthe posterior lateral line (PLL) sys-tem (Metcalfe et al., 1985; Nunezet al., 2009). The PLL system isunique among the nervous systemsanalyzed thus far as the cell bodiesand axons of PLL sensory neuronsare located just beneath the skin(Metcalfe et al., 1985), the number ofneurons involved is less than 30 atthe end of embryogenesis, and theneuromasts they innervate are dis-persed along the body. Because ofthese features, we have been able tocomprehensively observe the full rep-ertoire of events during neural circuitformation in the zebrafish in vivousing a combination of live imagingand transgenic lines.

The primary PLL develops as aPLL placode located just posterior tothe otic vesicle (Kimmel et al., 1995).This placode becomes subdivided intotwo parts at 20 hours postfertilization(hpf). One is a small compartment of�20 cells which will give rise to thePLL ganglion, and the other is alarger compartment of �100 cellswhich forms the PLL primordium, aprecursor cell cluster of the neuro-masts. The size of the ganglionincreases as development proceeds,indicating that some undifferentiatedcells in the ganglion actively prolifer-ate to produce nascent neurons. Theprimordium begins to migrate cau-dally at 22 hpf, and completes migra-tion by 48 hpf, at the tip of the tail(Metcalfe, 1985). As soon as the pri-mordium begins to migrate, bipolarsensory neurons in the ganglionextend their axons both centrally andperipherally toward their targets, thehindbrain and neuromasts, respec-tively (Gompel et al., 2001b). As theprimordium migrates, it is accompa-nied by the peripheral nerve extend-ing from the ganglion (Metcalfe, 1985;Gilmour et al., 2004). On its route, theprimordium sequentially deposits sixto eight groups of cells (Gompel et al.,2001a), each of which will eventuallydifferentiate into a functional neuro-mast. PLL neurons innervate the nas-cent neuromasts upon deposition.

The projection into the hindbrain ofzebrafish has been reported to displaya topographic ordering, i.e., the rela-tive positions of the axon terminals inthe hindbrain represent that of the

neuromasts they innervate (Alexan-dre and Ghysen, 1999). This topo-graphic aspect of projection isreferred to as somatotopy. A similarrelationship between the position ofcell bodies of sensory neurons withinthe ganglion, and the position of neu-romasts along the body has also beensuggested (Alexandre and Ghysen,1999). Topographic maps are consid-ered to be important for processingthe sensory information detected bythe peripheral nervous system, andare frequently observed in vertebratenervous systems (Kaas, 1997; Wein-berg, 1997). However, although it hasbeen intensively studied in a varietyof systems such as retino-tectal pro-jection (Luo and Flanagan, 2007;Huberman et al., 2008; Clandinin andFeldheim, 2009), the processes thattake place during map formationhave not yet been comprehensivelydescribed at the single-cell level.

In the present study, we have inves-tigated when and how the topographicrelationship between neuromasts andsensory neurons in the ganglion isestablished in the zebrafish PLL sys-tem. Here, we refer to this relationshipas ‘‘ (somatotopic) ganglion organiza-tion’’ because ‘‘somatotopy’’ is usuallyused in relation to the central projec-tion. The ganglion organization isbetter suited for a quantitative analy-sis because relative positions of cellbodies in the ganglion can be pre-cisely determined. Our analyses showthat the somatotopic ganglion organi-zation is initially unclear, and thatnot all neurons in the ganglion con-tribute to the map. Moreover, duringthis early stage of network formation,the PLL neurons exhibit a wide rangeof diversity in their behavior and alsoin the morphology of their growthcones.

RESULTS

Somatotopic Ganglion

Organization of the PLL Is

Initiated by 54 hpf

The somatotopic ganglion organiza-tion, which is the topographic rela-tionship between cell bodies in theganglion and neuromasts, has beenreported to be established by 6 dayspostfertilization (dpf) in zebrafish,

i.e., the relative positions of the cellbodies in the ganglion correspond tothose of the neuromasts they inner-vate (Alexandre and Ghysen, 1999).To determine when this relationshiporiginates, we used a retrogradelabeling method in earlier-stageembryos. Since most neuromasts areclose to the axons that innervate themore posterior neuromasts, we wereunable to perform retrograde labelingof axons innervating a single neuro-mast by simply injecting dye into aneuromast, because when the dye isinjected into a neuromast, it alsolabels the axons running nearby.Thus, in our experiments, a bundle ofaxons located between the neuro-masts was labeled with rhodamine-dextran by means of a small lesioncreated by a fine needle. Using thismethod, all neurons innervating theneuromasts posterior to the site ofdye application could be retrogradelylabeled. To visualize the PLL nervefor the dye application, we used thetransgenic zebrafish strain Tol047 inwhich the PLL neurons express greenfluorescent protein (GFP). We con-firmed that GFP-positive cells in thisstrain were indeed neurons becausethey were found to express the neuro-nal marker HuC/D during our obser-vation (from 24 hpf to 6 dpf; Supp.Fig. S1, which is available online).Next, to confirm the findings of a

previous report showing that thesomatotopic ganglion organization isestablished by 6 dpf (Alexandre andGhysen, 1999), we first labeled axonsat four different levels along the ante-rior–posterior (AP) axis at this devel-opmental stage. As shown in Figure 1A–D, dye application at the posteriorlevel resulted in labeling of cell bodiesin the dorsal part of the ganglion,while dye application at the anteriorlevel resulted in ventral expansion ofthe labeled cells. This confirmed theestablishment of the ganglion organi-zation by 6 dpf. When we labeled themajority of PLL neurons by dye appli-cation at the anteriormost site, cellbodies in the ventral region of theganglion tended to remain unlabeled(Fig. 1D). These unlabeled neuronsmay include neurons of which axonshave not reached the dye applicationsite at this stage. This is supported byour finding that newly differentiatedPLL neurons tend to be present in the

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ventral region of the ganglion (Supp.Fig. S2). Unlabeled neurons may alsoinclude neurons that innervate dorsal

neuromasts, because it has beenreported that neurons in the ganglioninnervate both lateral and dorsalbranches of the posterior lateral linesystem (Raible and Kruse, 2000).However, labeled and unlabeled neu-rons are not clearly segregated.

We next performed a retrogradelabeling experiment in 54 hpf embryos,just after the completion of primaryPLL development. The migrating PLLprimordium was observed to reach thetip of the tail and complete the deposi-tion of neuromasts by 48 hpf. At54 hpf, the results showed a pattern oflabeling similar to that observed in6 dpf embryos (Fig. 1E–H), indicatingthat the ganglion organization is de-tectable as early as 54 hpf.

It is important to note that the gan-glion organization is not as accurateat 54 hpf as it is at 6 dpf, because‘‘exceptional’’ cells isolated away fromthe cluster of labeled cells (arrows inFig. 1) were frequently detected at54 hpf. These cells do not seem tocontribute to the somatotopic gan-glion organization. Map precision wasquantified by counting exceptionalcells and calculating the proportion ofthese cells among all labeled cells(Fig. 1I and Supp. Fig. S3). We alsoincluded data obtained at 7 dpf (label-ing at L3–L4 and L4–L5) because ter-minal neuromasts are 1 day youngerthan neuromasts anterior to them.Retrograde labeling at anterior sitesrevealed that the proportion of excep-tional cells tended to decrease at thelater stages (Fig. 1I, L1–L2 and

L2–L3). This finding suggests that arefinement of the ganglion organiza-tion occurs within five days afterprimary PLL formation, at least inneurons innervating anterior neuro-masts. On the other hand, after ret-rograde labeling at posterior sites(Fig. 1I, L3–L4 and L4–L5), therewas no significant difference inthe proportion of exceptional cellsbetween 54 hpf, 6 dpf, and 7 dpf, sug-gesting that newly born exceptionalcells were added between thesestages. Indeed, the number of totallabeled cells also increased (data notshown). The lower percentage ofexceptional cells in L4–L5 labelingappears contradictory to data ob-tained in the following single-celllabeling experiment, a more reliablemethod (Fig. 4), probably owing totechnical difficulties of our retro-grade labeling method at the poste-rior tip of the embryo.

PLL Neurons Exhibit Diverse

Behavior When Extending

Their Axons

The above results suggested that atopographic relationship betweenneuromast position along the body,and neuron position within the gan-glion, begins to be established beforethe completion of primary PLL forma-tion. We thus focused our analyses onan earlier developmental stage duringwhich the axons are extending towardtheir targets, the neuromasts, some of

Fig. 1. Somatotopic ganglion organization in the posterior lateral line (PLL) ganglion of a zebrafish embryo. A–H: Confocal images of the PLLganglion of 6 days postfertilization (dpf; A–D) and 54 hours postfertilization (hpf; E–H) Tol 047 embryos that had been retrogradely labeled from dif-ferent inter-neuromast regions; between the fourth (described as ‘‘L4’’ on the top row) and fifth (L5) PLL neuromast (A, E), third (L3) and fourth (L4)neuromast (B, F), second (L2) and third (L3) neuromast (C, G), and first (L1) and second (L2) neuromast (D, H). In some cases, exceptional cellswere observed (white arrows). I: Quantification of somatotopic map precision at 54 hpf, 6 dpf, and 7 dpf. The number of total labeled cells andexceptional cells were counted and the proportion of exceptional cells was thus calculated. Sample numbers are shown in each column. Errorbars are the SEM. **P < 0.05, *P < 0.2 (student’s t-test corrected for multiple comparisons). Scale bars ¼ 10 mm.

Fig. 2. Diversity of posterior lateral line (PLL)neurons in terms of the position of theirgrowth cone. A–C: Three different mosaicembryos expressing DsRed (magenta) in asingle PLL neuron in Tol047 embryos. A–C:The scheme on the top indicates the regionswhere the images were taken, i.e., near thePLL ganglion (A), on the PLL nerve (B), and atthe leading edge of the PLL nerve, that is, inthe primordium (C). All images were taken at32 hpf. D: Distribution of individual growthcones. Black bars indicate the relative posi-tion of each growth cone. The relative positionwas quantified according to the followingequation: relative position ¼ somite number oflabeled growth cone / somite number of tip ofPLL nerve (n ¼ 12). Gray area shows the pri-mordium. Scale bars ¼ 10 mm.

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which have yet to be formed. We ana-lyzed the behavior and morphology ofindividual neurons that were visual-ized using a previously describedtransient-expression method (Nagielet al., 2008; Faucherre et al., 2009).For this, we injected Tol047 embryoswith the HuC:DsRed construct andscreened for larvae expressing DsRedin a single PLL neuron under a fluo-rescence microscope. This was fol-lowed by detailed live observationsduring axonal extension.

Although it has been reported thatthe lateral line nerve is guided by thelateral line primordium (Gilmouret al., 2004), the behavior of individ-ual neurons during axonal projectionis still largely unknown. It has beenreported that some PLL neurons havea growth cone that lags behind theprimordium (Gompel et al., 2001b),suggesting that PLL neurons behavediversely during their elongation.We first assessed the position of indi-vidual growth cones and their distri-bution. The position of individualgrowth cones at a single time point, 32hpf, was examined in independentlyinjected and screened embryos. Theresults showed that the positions of thegrowth cones relative to the primor-dium are extensively diverse, althoughwe observed neurons at the same stage(Fig. 2). The growth cones of some neu-rons (15/42 samples) were found to co-migrate with the primordium (Fig. 2C),whereas other growth cones laggedbehind the primordium at variouspositions along the PLL nerve (Fig.2A,B). Of interest, some neurons hadextremely short axons and theirgrowth cones were still proximal tothe ganglion (Fig. 2A). This clearlyindicates that the axons of PLL neu-rons behave differently during theirextension. We could not detect anybias in the distribution of growthcones along the PLL nerve (Fig. 2D;n ¼ 12). We identified the neuromaststhat were eventually innervated bythe labeled neurons (see below andFig. 4), and found that the axonsextending at 32 hpf innervate theneuromasts deposited by the primaryprimordium, but not the neuromastsderived from a later-developing, sec-ondary primordium (Sapede et al.,2002). The primary neuromasts canbe recognized from secondary neuro-masts based on size and position (Vil-

lablanca et al., 2006; Ghysen andDambly-Chaudiere, 2007).

The diverse positioning of thegrowth cones can be caused by diver-sity in the axonal growth rate and/orin the timing of axonal extension.Indeed, our time-lapse analysesrevealed diversity in the timing ofaxonal outgrowth because we coulddetect neurons that had commencedaxonal extension around 26 to 30 hpf(n ¼ 4), well after the outgrowth ofleading axons. Diversity was alsoobserved in axonal growth rates. At32 hpf, the growth rate, measured by30 min time-lapse observations,showed considerable variationsamong neurons (maximum rate: 125mm/hr, minimum rate: �38 mm/hr,n ¼ 39). Surprisingly, some neuronsexhibited a mild retraction but even-tually elongated their axons and in-nervated neuromasts at later stagesin a normal manner. This finding sug-gests that the rate of axonal elonga-tion of individual neurons is notconstant.

In zebrafish commissural axons,the leading axons that guide followerneurons were shown previously to ex-hibit a significantly lower axonalgrowth rate than that of the followers(Bak and Fraser, 2003). To examinewhether a similar difference in axonalgrowth rate exists also among PLLneurons, we divided these neuronsinto two groups based on the positionof the growth cones at 32 hpf, andcompared the axonal growth rate ofthese two groups. One group con-tained neurons whose growth cone co-migrates with the primordium,named type A neurons. The othergroup (type B neurons) comprisedneurons with growth cones along thenerve, behind the primordium. Theaxons of type A neurons wereexpected to behave as leading axonsand those of type B neurons as fol-lowers. The axonal growth rate oftype A neurons was 80 6 7.6 mm/h(n ¼ 15) on average, which is compa-rable to the migration rate of the pri-mordium (Metcalfe, 1985). On theother hand, the axonal growth rate oftype B neurons was 32 6 11 mm/h (n¼ 24) on average, a significantly lowerrate than that of type A neurons (P <0.005). Thus, in contrast to commis-sural axons, the axonal growth oftype A neurons (leaders) is faster

than that of type B neurons (fol-lowers) in the PLL.It has been shown that the mor-

phology of growth cones can berelated to their behavior (Mason andWang, 1997) or to their function, i.e.,role as a pioneer or follower (Loprestiet al., 1973; Bastiani et al., 1984; San-chez-Soriano and Prokop, 2005). Aprevious study has revealed diversemorphology of growth cones in thePLL neurons (Gompel et al., 2001b).We analyzed the morphology of indi-vidual growth cones and the relation-ship between morphology and posi-tion of the growth cones. The PLLneurons were found to have diversemorphologies in their growth cones at32 hpf (Fig. 3A) as previouslydescribed. For quantitative analysis,we measured the area and the perim-eter of these growth cones and calcu-lated the complexity index P^2/A(square of the perimeter divided bythe area) (Bowie and Young, 1977).The complexity index of type A neu-rons (index ¼ 126 6 21, n ¼ 15) wassignificantly higher than that of typeB neurons (index ¼ 72 6 6.3, n ¼ 24)(Fig. 3B; P < 0.005). Hence, type Aneurons tend to exhibit not only a dis-tinct axonal growth rate but also adifferent growth cone morphologyfrom type B neurons.

Somatotopic Ganglion

Organization Is Established

by the Follower Neurons

In our final experiments of this study,we re-examined the establishment ofthe ganglion organization at a single-cell resolution. A previous study hasdescribed the relationship betweengrowth cone morphology and positionof innervation, revealing that axonswith complex growth cones innervateposterior neuromasts and that latermigrating axons innervate more ante-rior neuromasts (Gompel et al.,2001b). In the present study, wefocused on the contribution of type Aand type B neurons to the ganglionorganization because these types ofneurons were evidently different intheir morphology and behavior.Embryos were injected with theHuC:DsRed construct, and were pre-screened at 32 hpf and classified intotwo groups depending on whether

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TOPOGRAPHIC MAP FORMATION IN ZEBRAFISH PLL 2061

they had a type A or type B neuronexpressing DsRed. Classified embryoswere then allowed to develop untiltheir labeled axons reached the targetneuromasts (2 to 3 dpf). In theseembryos, we determined the relation-ship between the relative positions of

each cell body in the ganglion alongthe dorsal–ventral (DV) axis, and thepositions of the neuromast they inner-vated along the AP axis.

The relationship between the posi-tion of the cell body and its targetneuromast exhibited a different tend-

ency between type A and type B neu-rons. Type A neurons tended to inner-vate the posterior neuromasts,irrespective of the position of theircell bodies in the ganglion (Fig. 4B),and thus did not contribute to thesomatotopic ganglion organization. Incontrast, the majority (13/16) of typeB neurons contributed to the estab-lishment of the somatotopic ganglionorganization, i.e., the more ventrallythe neurons were located in the gan-glion, the more anteriorly their targetneuromasts were positioned (Fig. 4B).A few neurons plotted in the lowerright corner of the graph did not con-tribute to the ganglion organization.Such unordered cells were detectedalso in our retrograde experiment asnamed ‘exceptional’ (Fig. 1).Finally, we examined the respective

distribution of the cell bodies of typeA and B neurons in the PLL ganglion.They were found to be intermingledand not segregated (Fig. 4C), suggest-ing that a relationship does not existbetween the position in the ganglionand the type of neuron.

DISCUSSION

In the present study, a combination ofsingle-cell analysis and of our newtransgenic line was successfullyapplied to the study of neural networkformation in the zebrafish PLL sys-tem. Our analyses identified twotypes of neurons within the PLL gan-glion, type A and type B, and showedthat the axons of type A neurons first

Fig. 3. Morphology of the growth cones of individual posterior lateral line (PLL) neurons. A: Individual growth cones in nine different mosaicembryos. The type of each growth cone (type A or B) is denoted in the lower left corners. The numbers in the lower right corners indicate thecomplexity index (P^2/A; square of the perimeter divided by the area) of each growth cone. All images were taken at the same original magnifica-tion. B: Average complexity index 6 SEM for the growth cones of type A (n ¼ 15) and type B (n ¼ 23) neurons (*P < 0.005; Student’s t-test cor-rected for multiple comparisons). Scale bar ¼ 10 mm.

Fig. 4. Positions of individual cell bodies and their innervation. A: Method for measurement ofthe relative cell body positions in the posterior lateral line (PLL) ganglion. The angles of eachimage were corrected by matching the directions of the peripheral nerve extending from theganglion (arrow). B: Relationship between the relative positions of the cell bodies (along the dor-sal–ventral axis) and the positions of the neuromasts innervated by these neurons. The type Aand type B neurons were separately analyzed. L1, L2, L3, L4, and L5 represent the anteriormost, second, third, fourth and fifth neuromast, respectively. Two or three neuromasts on the tipof the tail are considered to be the same target (‘‘term’’ in the plots). The neurons within the bro-ken line contribute to the somatotopic ganglion organization. C: Distribution of the cell bodies oftype A and B neurons in the PLL ganglion. The color of each dot indicates the target neuromastas shown in the right corner.

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2062 SATO ET AL.

establish a connection with the pri-mordium and co-migrate to the poste-rior end, while those of type B growtoward their targets behind type Aneurons. Moreover, only type B neu-rons were found to be involved inestablishing the somatotopic ganglionorganization.

Leader Neurons in the

PLL System

We speculated that type A neurons inthe ganglion could be leader neuronsin the PLL system as they are thefirst to extend their axons, and arefollowed by later appearing axonsmigrating along their axonal path-way. In both vertebrate and inverte-brate nervous systems, the first neu-rons that extend their axons oftenfunction as leaders that guide fol-lower axons to establish the correctprojections (Raper et al., 1984;Kuwada, 1986; Klose and Bentley,1989; Pike et al., 1992; Hidalgo andBrand, 1997; Whitlock and Wester-field, 1998; Williams and Shepherd,2002; Pittman et al., 2008). Such lead-ing axons are known to exhibit char-acteristic behaviors and/or morpholo-gies. For example, a slower growthrate and the complex shape of growthcones may help each individualgrowth cone to sense guidance cuesand navigate in an environment thatis devoid of other axons. Indeed, inthe zebrafish commissural axons, onlyleader axons slow down at the mid-line, and the average growth conewidth to length ratio of leader axonsis significantly higher than that of fol-lower axons (Bak and Fraser, 2003).Consistently, in the PLL, the growthcones of type A neurons exhibit thegreatest complexity in shape withinthe migrating primordium. However,type A neurons are distinct fromother typical pioneers in that theiraxonal growth rate is much fasterthan that of type B neurons (fol-lowers). This could be explained bythe fact that the PLL primordiumnavigates itself by sensing positionalcues using the CXCR4-SDF1 pathway(David et al., 2002; Li et al., 2004;Haas and Gilmour, 2006), whereastype A neurons simply co-migrateonce the connection is established.

In several systems, leader neuronsare known to undergo apoptotic cell

death after the axonal growth of fol-lower neurons (Klose and Bentley,1989; Whitlock and Westerfield,1998). In the PLL system, however,type A (leader) neurons maintaininnervation to the posterior neuro-masts at 6 dpf (four of five neurons),suggesting that these neurons con-tinue to function at later stages.

Origin of Somatotopic

Ganglion Organization

Our retrograde labeling experimentsconfirmed that the ganglion organiza-tion is established by 6 dpf as previ-ously reported (Alexandre and Ghy-sen, 1999). Labeling at this stage didnot distinguish between the earlyneurons that innervate the embryonicline, and later-forming neurons thatfollow a secondary primordium dur-ing early larval development. How-ever, our early labeling clearlyrevealed that a somatotopic mapestablished by initially appearingaxons. The ganglion organization inthe PLL system thus begins to emergeas early as 54 hpf, i.e., much earlierthan 6 dpf. Our single-cell resolutionanalysis confirmed this finding; a por-tion of the PLL neurons (type B neu-rons) in the ganglion already exhibitsa topographic relationship with theirtargets at this stage. Because ourobservations were performed soon af-ter the differentiation of these neu-rons, it is reasonable to assume thatthis relationship is formed withoutrefinement of the neural connections,i.e., it is intrinsically determined.Thus, most type B neurons probablyrecognize their targets’ position by atleast the time at which they outgrowtheir axons.

At present, there are no data show-ing the origin of somatotopy in thePLL system, where the informationfor this process resides or how neu-rons are predetermined before neuriteextension. The positional informationcould originate in the sensory gan-glion or alternatively in the hindbrain(the target of the central projection ofthe PLL neurons). Although we havenot applied our single-cell analysis tothe central projection, we favor theidea that the majority of type B neu-rons are intrinsically specified accord-ing to their relative position withinthe ganglion rather than that of the

axon terminals in the hindbrain. Ifthe hindbrain were the organizingelement of the map, we would expectthat the cell bodies of type B neuronswould change their relative positionduring a short period of time duringor soon after innervation. Our prelim-inary observations, however, did notreveal any significant repositioningalong the DV axis before (32 hpf) orafter peripheral innervation was com-pleted (48 hpf or 72 hpf; Supp. TableS1), and we further observed that thecentral projection begins to reach thehindbrain at around 32 hpf. It is alsonoteworthy that similar to type Bneurons in the PLL system, thegrowth cone of the primary retinalganglion cell (RGC) axons in the vis-ual systems of frogs and fish stopswhen it reaches its correct termina-tion zone without overshooting orrefinement (Harris et al., 1987;Kaethner and Stuermer, 1992). In ei-ther case, we do not yet know the mo-lecular mechanism allowing an indi-vidual neuron to intrinsically find itscorrect target.We contend that because the lateral

line system in zebrafish provides anexcellent framework to analyze theprocess of map formation in detail,further studies using this system willlikely uncover the mechanisms whichinstruct neurons to find their targetsduring neuronal circuit formation.

Refinement of the Ganglion

Organization

Not only neurons that establish theinitial somatotopic map but also‘exceptional’ cells were observed inour retrograde labeling (Fig. 1).Because our data showed that theproportion of these neurons wasdecreased at 6 dpf (Fig. 1I, L1–L2 andL2–L3), they may change their inner-vation or be eliminated by 6 dpf whenthe ganglion organization completes.In our live imaging experiments, wesometimes observed that neuronswere eliminated during axonal projec-tion or after innervation (data notshown). These data suggest the pres-ence of a fine-tuning mechanism inthe PLL. Indeed, tuning mechanismshave been well studied, mainly in theaxons of RGCs. In the developing vis-ual circuit of chick and mouse, thegradient of axon guidance molecules,

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such as the ephrins and their recep-tors (O’Leary and Wilkinson, 1999),has been implicated in establishingthe initial topographic map as well asin correcting axons that overshoottheir expected termination zone(McLaughlin and O’Leary, 2005). Thisinitial map is subsequently refinedand maintained by activity-dependentmechanisms, both spontaneous andvisually driven (Huberman et al.,2008). Guidance molecules and activ-ity are partly interdependent mecha-nisms, and the refinement weobserved in our present analysesbetween 54 hpf and 6 dpf may alsodepend on these mechanisms.

On the other hand, among the neu-rons innervating posterior neuro-masts, the number of exceptional cellsincreased between 54 hpf and 7 dpf(Fig. S2, L3–L4 and L4–L5) such thattheir proportion remained at thesame level (Fig. 1I, L3–L4 and L4–L5). These neurons may correspond tounordered neurons which innervateposterior neuromasts irrespective ofthe position of their cell bodies, asrevealed by our single-cell resolutionanalysis (Fig. 4B, neurons plotted inlower right corner). We speculate thata portion of neurons born after pri-mary PLL formation innervate poste-rior neuromasts irrespective of theirposition in the ganglion, and that atleast some of them survive until 6 dpf.Although our present study could notreveal the role of these unorderedneurons, we hypothesize that theiraxons could function as guides forother late-appearing type B neuronsby maintaining and reinforcing theaxonal tract initially established bytype A neurons. The small number ofembryonic type A neurons may not besufficient to guide entire PLL neuronsthat are continuously supplementedas development proceeds.

Heterogeneity in

PLL Neurons

PLL neurons are small in numberand seemingly homogeneous bothbefore and during central and periph-eral projection. However, the behaviorand morphology of PLL neurons werefound to be very diverse in our analy-sis. The diverse morphology of thegrowth cones of the PLL neurons waspreviously reported in the zebrafish

(Gompel et al., 2001b), but the rela-tionship between morphology, behav-ior, and the contribution to the soma-totopic ganglion organization in thesecells has not previously beenaddressed. Our present study thusprovides the first detailed and com-prehensive data on the diversity thatexists between individual neurons inthe zebrafish PLL during initial net-work formation. Our single-cell-labelexperiments further demonstratethat diverse neurons are nonran-domly arranged in the ganglion (Fig.4C). Although the differences betweentype A and type B neurons are quiteremarkable, the cell bodies of thesetwo cell types were found to be highlyintermingled in the ganglion. On theother hand, within the population oftype B neurons, the cell bodies werearranged in an orderly manner in theganglion, reflecting the relative posi-tions of the neuromasts they inner-vate. It is not yet known how theseheterogeneities emerge in a smallpopulation of neurons, and by whatmolecular mechanisms neurons adoptone of the two fates, A or B. One pos-sibility is that the intermingled pat-tern can be organized by cell–cellinteractions such as lateral inhibitionthrough the Notch pathway. Theorderly arrangement of type B neu-rons could of course also be achievedby a gradient of signal moleculeswithin the ganglion. Hence, PLL neu-rons could be controlled by distinctmechanisms that function in parallelwithin the small ganglion consistingof less than 30 neurons.

EXPERIMENTAL

PROCEDURES

Zebrafish

Zebrafish were maintained at 28.5�Con a 14/10 hr light/dark cycle.Embryos and larvae were raised atthe same temperature in 1/3 Ringer’ssolution with 200 mM 1-phenyl-2-thio-urea to inhibit pigment formation.The transgenic strain Tol047 was gen-erated in our laboratory using theTol2 transposon-mediated enhancertrap technique (Kawakami, 2005),and expresses GFP in the lateral lineneurons and a portion of the otherneurons.

Retrograde Labeling of

PLL Neurons

We modified the retrograde labelingmethod for the PLL described in aprevious report (Alexandre and Ghy-sen, 1999). Briefly, before labeling,the neuromasts were visualized byincubating the larvae in 0.2 mg/ml 4-(4-diethylaminostyryl)-N-methylpyri-dinium iodide (4-Di-2-Asp, Sigma D-3418) in 1/3 Ringer’s solution for 1 hr.After several washes, the larvae wereanesthetized in 0.15 mg/ml tricaine(3-aminobenzoic acid ethyl ester,Sigma A5040) in 1/3 Ringer’s solutionand transferred to larva-size slotswith a 1% agarose bottom. Rhoda-mine-dextran solution was allowed todry on a glass slide, and a smallamount of dye paste was picked upwith a fine needle with which the PLLnerves were labeled by creating asmall lesion. The larvae were incu-bated in a humidified chamber for 5min and transferred to 1/3 Ringer’ssolution. After a 6-hr incubation, thelarvae were fixed overnight at 4�C in4% paraformaldehyde in phosphatebuffered saline (PBS), and washed inPBS.

Single-Cell Labeling by

DNA Injection

HuC:DsRed plasmid DNA (gener-ously provided by Dr. S. Higashijima)was linearized with Not1 and puri-fied. Embryos at the one cell stagewere injected with this construct at150–180 ng/ml and then screened at24 hpf for DsRed expression in a sin-gle PLL neuron with a fluorescencemicroscope. The screened embryoswere then allowed to develop for con-focal observation (32 hpf � 6 dpf).

Confocal Imaging and

Image Analysis

Both living and fixed embryos weremounted in low gelling temperatureagarose (Sigma, A9045) for confocalimaging of the PLL neurons (FV500,Olympus or LSM710, Carl Zeiss). Theaverage rate of axonal elongation wascalculated from time-lapse data whichwere recorded for 30 min. Imageswere analyzed with ImageJ (NIH) formeasuring the area, perimeters, andrelative positions of the PLL neurons.

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ACKNOWLEDGMENTSWe thank Dr. Koichi Kawakami forcollaborating in the establishment ofthe zebrafish enhancer trap strain,and Dr. Kazuki Horikawa for generat-ing the Tol047 strain. We also thankDr. Shin-ichi Higashijima for provid-ing the plasmid for the single-celllabeling. The Tg(HuC:Kaede) line wasobtained from the National BioRe-source Project in Japan. This workwas supported in part by Grants-in-Aid for Scientific Research PriorityArea Genome Science and ScientificResearch, Global COE Program fromthe Ministry of Education, Culture,Sports, Science and Technology of Ja-pan. This work was also supported byGrant-in-Aid for JSPS Research Fel-lows (AS).

REFERENCES

Alexandre D, Ghysen A. 1999. Somato-topy of the lateral line projection inlarval zebrafish. Proc Natl Acad SciU S A 96:7558–7562.

Bae YK, Kani S, Shimizu T, Tanabe K,Nojima H, Kimura Y, Higashijima S,Hibi M. 2009. Anatomy of zebrafish cer-ebellum and screen for mutationsaffecting its development. Dev Biol 330:406–426.

Bak M, Fraser SE. 2003. Axon fascicula-tion and differences in midline kineticsbetween pioneer and follower axonswithin commissural fascicles. Develop-ment 130:4999–5008.

Bastiani MJ, Raper JA, Goodman CS.1984. Pathfinding by neuronal growthcones in grasshopper embryos. III.Selective affinity of the G growth conefor the P cells within the A/P fascicle. JNeurosci 4:2311–2328.

Bowie JE, Young IT. 1977. An analysistechnique for biological shape-II. ActaCytol 21:455–464.

Clandinin TR, Feldheim DA. 2009. Makinga visual map: mechanisms and mole-cules. Curr Opin Neurobiol 19:174–180.

David NB, Sapede D, Saint-Etienne L,Thisse C, Thisse B, Dambly-ChaudiereC, Rosa FM, Ghysen A. 2002. Molecularbasis of cell migration in the fish lateralline: role of the chemokine receptorCXCR4 and of its ligand, SDF1. ProcNatl Acad Sci U S A 99:16297–16302.

Dickson BJ. 2002. Molecular mechanismsof axon guidance. Science 298:1959–1964.

Faucherre A, Pujol-Marti J, Kawakami K,Lopez-Schier H. 2009. Afferent neuronsof the zebrafish lateral line are strictselectors of hair-cell orientation. PLoSOne 4:e4477.

Ghysen A, Dambly-Chaudiere C. 2007.The lateral line microcosmos. GenesDev 21:2118–2130.

Gilmour D, Knaut H, Maischein HM,Nusslein-Volhard C. 2004. Towing of

sensory axons by their migrating targetcells in vivo. Nat Neurosci 7:491–492.

Gompel N, Cubedo N, Thisse C, Thisse B,Dambly-Chaudiere C, Ghysen A. 2001a.Pattern formation in the lateral line ofzebrafish. Mech Dev 105:69–77.

Gompel N, Dambly-Chaudiere C, GhysenA. 2001b. Neuronal differences prefig-ure somatotopy in the zebrafish lateralline. Development 128:387–393.

Guan KL, Rao Y. 2003. Signalling mecha-nisms mediating neuronal responses toguidance cues. Nat Rev Neurosci 4:941–956.

Haas P, Gilmour D. 2006. Chemokine sig-naling mediates self-organizing tissuemigration in the zebrafish lateral line.Dev Cell 10:673–680.

Harris WA, Holt CE, Bonhoeffer F. 1987.Retinal axons with and without theirsomata, growing to and arborizing inthe tectum of Xenopus embryos: a time-lapse video study of single fibres invivo. Development 101:123–133.

Hidalgo A, Brand AH. 1997. Targetedneuronal ablation: the role of pioneerneurons in guidance and fasciculationin the CNS of Drosophila. Development124:3253–3262.

Huberman AD, Feller MB, Chapman B.2008. Mechanisms underlying develop-ment of visual maps and receptivefields. Annu Rev Neurosci 31:479–509.

Kaas JH. 1997. Topographic maps arefundamental to sensory processing.Brain Res Bull 44:107–112.

Kaethner RJ, Stuermer CA. 1992. Dy-namics of terminal arbor formation andtarget approach of retinotectal axons inliving zebrafish embryos: a time-lapsestudy of single axons. J Neurosci 12:3257–3271.

Kawakami K. 2005. Transposon tools andmethods in zebrafish. Dev Dyn 234:244–254.

Kimmel CB, Ballard WW, Kimmel SR,Ullmann B, Schilling TF. 1995. Stagesof embryonic development of the zebra-fish. Dev Dyn 203:253–310.

Klose M, Bentley D. 1989. Transient pio-neer neurons are essential for forma-tion of an embryonic peripheral nerve.Science 245:982–984.

Kuwada JY. 1986. Cell recognition byneuronal growth cones in a simple ver-tebrate embryo. Science 233:740–746.

Li Q, Shirabe K, Kuwada JY. 2004. Che-mokine signaling regulates sensory cellmigration in zebrafish. Dev Biol 269:123–136.

Lopresti V, Macagno ER, Levinthal C.1973. Structure and development ofneuronal connections in isogenic organ-isms: cellular interactions in the devel-opment of the optic lamina of Daphnia.Proc Natl Acad Sci U S A 70:433–437.

Luo L, Flanagan JG. 2007. Developmentof continuous and discrete neural maps.Neuron 56:284–300.

Mason CA, Wang LC. 1997. Growth coneform is behavior-specific and, conse-quently, position-specific along the reti-nal axon pathway. J Neurosci 17:1086–1100.

McLaughlin T, O’Leary DD. 2005. Molecu-lar gradients and development of reti-notopic maps. Annu Rev Neurosci 28:327–355.

Metcalfe WK. 1985. Sensory neurongrowth cones comigrate with posteriorlateral line primordial cells in zebrafish.J Comp Neurol 238:218–224.

Metcalfe WK, Kimmel CB, Schabtach E.1985. Anatomy of the posterior lateralline system in young larvae of thezebrafish. J Comp Neurol 233:377–389.

Nagiel A, Andor-Ardo D, Hudspeth AJ.2008. Specificity of afferent synapsesonto plane-polarized hair cells in theposterior lateral line of the zebrafish. JNeurosci 28:8442–8453.

Nunez VA, Sarrazin AF, Cubedo N,Allende ML, Dambly-Chaudiere C, Ghy-sen A. 2009. Postembryonic develop-ment of the posterior lateral line in thezebrafish. Evol Dev 11:391–404.

O’Leary DD, Wilkinson DG. 1999. Ephreceptors and ephrins in neural devel-opment. Curr Opin Neurobiol 9:65–73.

Pike SH, Melancon EF, Eisen JS. 1992.Pathfinding by zebrafish motoneuronsin the absence of normal pioneer axons.Development 114:825–831.

Pittman AJ, Law MY, Chien CB. 2008.Pathfinding in a large vertebrate axontract: isotypic interactions guide retino-tectal axons at multiple choice points.Development 135:2865–2871.

Raible DW, Kruse GJ. 2000. Organizationof the lateral line system in embryoniczebrafish. Journal of Comparative Neu-rology 421:189–198.

Raper JA, Bastiani MJ, Goodman CS.1984. Pathfinding by neuronal growthcones in grasshopper embryos. IV. Theeffects of ablating the A and P axonsupon the behavior of the G growthcone. J Neurosci 4:2329–2345.

Sanchez-Soriano N, Prokop A. 2005. Theinfluence of pioneer neurons on a grow-ing motor nerve in Drosophila requiresthe neural cell adhesion molecule homo-log FasciclinII. J Neurosci 25:78–87.

Sapede D, Gompel N, Dambly-ChaudiereC, Ghysen A. 2002. Cell migration inthe postembryonic development of thefish lateral line. Development 129:605–615.

Villablanca EJ, Renucci A, Sapede D, LecV, Soubiran F, Sandoval PC, Dambly-Chaudiere C, Ghysen A, Allende ML.2006. Control of cell migration in thezebrafish lateral line: implication of thegene ‘‘tumour-associated calcium signaltransducer,’’ tacstd. Dev Dyn 235:1578–1588.

Weinberg RJ. 1997. Are topographic mapsfundamental to sensory processing?Brain Res Bull 44:113–116.

Whitlock KE, Westerfield M. 1998. Atransient population of neurons pio-neers the olfactory pathway in thezebrafish. J Neurosci 18:8919–8927.

Williams DW, Shepherd D. 2002. Persis-tent larval sensory neurones arerequired for the normal development ofthe adult sensory afferent projections inDrosophila. Development 129:617–624.

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TOPOGRAPHIC MAP FORMATION IN ZEBRAFISH PLL 2065