Embryonic Development of theDrosophila Brain. I. Pattern

of Pioneer Tracts

CLAUDE NASSIF, ALEXANDER NOVEEN, AND VOLKER HARTENSTEIN*Department of Molecular Cell and Developmental Biology,

University of California Los Angeles, Los Angeles, California 90095

ABSTRACTThe neuropile of the late embryonic Drosophila brain can be subdivided into a vertical

component (cervical connective), a transverse component (supraesophageal commissure), anda horizontal component for which we propose the term protocerebral connective. The core ofeach neuropile component is formed by numerous axon fascicles, the trajectory of whichfollows an invariant pattern. In the present study we have used an antibody against theadhesion molecule Fasciclin II (FasII) that is expressed in a large number of earlydifferentiating neurons of the Drosophila embryo to follow the development of the axon tractsof the brain. The FasII antigen appears on the surface of clusters of neuronal somata prior toaxon outgrowth. These clusters, for which we propose the term fibre tract founder clusters, arelaid out in a linear pattern that forms an almost uninterrupted longitudinal track reachingfrom the ventral nerve cord to the ‘‘tip’’ of the brain. After expressing FasII on their soma,neurons of the fibre tract founder clusters extend axons that grow along the surface of thefounder clusters and form a simple system of pioneer tracts for each of the components of thebrain neuropile. We have reconstructed the FasII-positive fibre tract founder clusters andtheir axons from optical sections and generated digital 3-D models that illustrate the spatialrelationships of the pioneer tracts. Three fibre tract founder clusters, D/T, P1, and P3m,pioneer the cervical connective. P2l and P2m form a transverse track that pioneers thesupraesophageal commissure. P4m and P4l/P5l/P5m form two tracts that pioneer a medialand a lateral component of the protocerebral connective, respectively. Because FasII expres-sion continues uninterruptedly into the larval period when the ‘‘rudiments’’ of many parts ofthe adult neuropile are readily identifiable, it was possible to assign several of the embryonicpioneer tracts to definitive neuropile components, including the median bundle, antennocere-bral tract, mushroom body, and posterior optic tract. J. Comp. Neurol. 402:10–31, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: neuropile; axon pioneers; digital model

Neurons of the insect form a multitude of functionallyspecialized tracts and neuropile components. A consider-able diversity in the size and pattern of neuropile compo-nents has been documented among different insect groups(Hanstrom, 1928; Bullock and Horridge, 1965). However,it is possible to identify a ground-plan of several prominentbrain structures common to most insects (Fig. 1). Along theanterior-posterior axis (neuraxis), the insect brain is di-vided into the supraesophageal ganglion, comprised of theprotocerebrum, deuterocerebrum, tritocerebrum, and thesubesophageal ganglion, which arises from the fusion ofthree segmental ganglia (labium, maxilla, and mandible).Sensory and motor nerves of the subesophageal ganglionand the tritocerebrum supply the innervation of the mouthparts; the tritocerebrum also sends axons into a group of

peripheral ganglia, the stomatogastric ganglia, which con-trol feeding behavior. The deuterocerebrum receives theantennal nerve and represents the olfactory center of thebrain; the so called posterior slope of the deuterocerebrum(Strausfeld, 1976), a major ‘‘output region’’ of the insectbrain, contains numerous interneurons which project ontothe motoneurons of the ventral nerve cord. The protocere-brum dominates the brain in size and complexity. Among

Grant sponsor: National Institutes of Health; Grant number: NS-29367.*Correspondence to: Dr. Volker Hartenstein, Department of Molecular

Cell and Developmental Biology, University of California Los Angeles, LosAngeles, CA 90095.

Received 16 February 1998; Revised 15 June 1998; Accepted 29 July 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 402:10–31 (1998)

r 1998 WILEY-LISS, INC.

the conserved elements of the protocerebrum are themushroom bodies (corpora pedunculata) and their majorafferent tract (antennocerebral tract), the central complex,pars intercerebralis, optic tubercles, and optic lobes. Thespatial relationship and interconnectivity between theseprotocerebral components is schematically shown in Fig-ures 1, 2, and 11.

The mushroom body (MB), considered to be a center ofcomplex behavioral functions, including learning andmemory, is formed by conglomerations of cell bodies(Kenyon cells) located in the dorsal protocerebrum. Kenyoncell axons form a characteristic branched structure (calyx,peduncle, a, b, and g lobes). Afferent axons from theolfactory lobe and various other sources, among them theoptic lobe, form contacts with Kenyon cell axons in thecalyx neuropile. Medially and anteriorly to the mushroombodies is the pars intercerebralis, which in all insectscontains neurosecretory cells projecting their axons to-ward the corpora cardiaca, neuro-endocrine organs in-volved in molting, cardiovascular, and metabolic functions.Axons to and from the pars intercerebralis, interconnect-ing this brain region with the ventral nerve cord and basalbrain regions, form the crossed median bundle. The cen-tral complex, an unpaired structure implicated in flightcontrol (Ilius et al., 1994) is built of several distinctneuropile components (Power, 1943; Strausfeld, 1976) andlies between the calices of the mushroom bodies. The onlyanatomically defined source of input to the central complexis the ventral body, a spherical neuropile component at the

base of the protocerebrum, between antennal neuropileand lobes of the mushroom body (Power, 1943; Hanesch etal., 1989). All structures mentioned so far form the socalled ‘‘forebrain,’’ which is flanked on either side by theoptic lobe. The two distal neuropiles of the optic lobe,lamina and medulla, receive the highly ordered axons ofthe compound eyes. Lamina and medulla project to theinner optic neuropile (lobula complex), which in turn sendsaxons to its contralateral counterpart, as well as thelateral neuropiles of the midbrain (anterior and posterioroptic tubercles; Strausfeld, 1976; Meinertzhagen and Han-son, 1993).

How do these complex elements of the adult insect brainrelate to the seemingly simple brain structure of theembryo or early larva? The development of the insect brainhas not been studied in much detail. It is clear that some ofthe elements of the ‘‘ground-plan’’ as outlined above,develop quite early. For example, the mushroom body withits characteristic peduncle and lobes has been seen inlarvae of Drosophila (deBelle and Heisenberg, 1994; Ito etal., 1997) and other species (Bierbrodt, 1943; Scholl, 1964).Other structures that have been identified at an earlylarval stage are the optic lobe and antennal lobe (forreviews, see respectively Meinertzhagen and Hanson,1993; Stocker, 1994); however, little is known about theembryonic origin of the brain structures. In the presentstudy we have made use of a marker, the Fasciclin II(FasII) antigen, that is expressed in a large number ofearly differentiating neurons of the Drosophila embryo

Fig. 1. Schematic depiction of adult (A) and larval (B) Dipteranbrain (lateral view). At the brain base is the subesophageal ganglion(sb) that innervates the gnathal segments: mandible (md), maxilla(mx), and labium (lb). The supraesophageal ganglion comprises thetritocerebrum (tr), deuterocerebrum (de), and protocerebrum (pr). Theprotocerebrum has a lateral optic lobe (ol; light shading) and a medial‘‘midbrain’’ (dark shading). Within the midbrain, several neuropilecomponents and fibre tracts that form a ‘‘ground-plan’’ conserved

among all insects are indicated. AGT, antennocerebral tract; an,antennal nerve; aof, anterior optic focus; Bn, Bolwig’s nerve (larvaloptic nerve); cb, central body; lr/hy, labral/hypopharyngeal nerve; mb,mushroom body; MB, median bundle; ncc, nerve to corpora cardiaca;oc, ocellar nerve; pi, pars intercerebralis; pof, posterior optic focus; ps,posterior slope; sns, stomatogastric ganglia; ta, thoraco-abdominalganglion (ventral nerve cord); vb, ventral body.

DROSOPHILA BRAIN DEVELOPMENT 11

(Grenningloh et al., 1991; Goodman and Doe, 1993). Theseneurons form a system of pioneer tracts, both in theventral nerve cord and the brain. For the ventral nervecord, the FasII-positive pioneer tracts were described ingreat detail in previous studies (Goodman and Doe, 1993).By using another marker (MAb22C10) (Zipursky et al.,1984), Therianos et al. (1995) have identified severalelements of the system of pioneer tracts of the brain. Wehave systematically reconstructed the FasII-expressingpioneer tracts from optical sections of successive develop-mental stages and generated digital 3-D models that makeit easier to grasp the spatial relationships of the tracts.FasII expression continues into the larval period, wherethe ‘‘rudiments’’ of many parts of the adult neuropile arereadily identifiable. It was therefore possible to assignsome of the embryonic pioneer tracts to definitive neuro-pile components, such as the median bundle, antennocere-bral tract, mushroom body, and posterior optic tract. Ourfindings provide a basis for subsequent studies of braindevelopment in Drosophila.

MATERIALS AND METHODS

Markers

The neuropile of embryonic and larval brain was labeledwith a monoclonal antibody against the FasII protein(Grenningloh et al., 1991; kindly provided by Dr. C.Goodman). In addition, several antibody markers andreporter genes carrying P-element constructs (identified inthe following as ‘‘gene PlacZ Code#’’) were used. In em-bryos expressing these constructs, the reporter gene lacZis expressed in the same pattern as the corresponding genein which it is inserted and can be visualized immuno-histochemically with an antibody against the gene prod-uct, b-Galactosidase. Antibodies used in this study in-cluded anti-Elav (Robinow and White, 1991) which labelsnuclei of neurons, and anti-HRP (horseradish peroxidase;Sigma, St. Louis, MO), which labels neuropile (Campos-Ortega and Hartenstein, 1997). Reporter gene constructsincluded PlacZ rhx25 (Hama et al., 1990) which labels theexpression domain of the engrailed gene and PlacZ H162(Doe, 1992) which shows expression of the seven-up (svp)gene in subsets of neuroblasts.

Fly stocks and egg collections

As wile-type stock we used Oregon R. Flies were grownunder standard conditions at room temperature or at25°C. Egg collections were done on yeasted apple juiceagar plates. Embryonic stages are given according toCampos-Ortega and Hartenstein (1997).

Immunohistochemistry and histology

Expression of b-Galactosidase (b-Gal) in PlacZ lines andpromoter constructs was detected with a polyclonal anti-b-Galactosidase antibody (Cappel; dilution 1:2,000). Theanti-FasII and anti-Elav antibody were diluted 50 fold;dilution of anti-HRP was 1:2,000. For staining, embryoswere collected, dechorionated, and fixed for 30 minutes ina mixture of 4% formaldehyde in PEMS (0.1 M Pipes, 2mM MgSO4, 1 mM EGTA, pH 7.0) with heptane. They weredevitellinized in methanol and further prepared for anti-body labeling following standard procedures (e.g., Ash-burner, 1989). For double stainings, preparations weresequentially processed first for one and then for the secondantibody. Straight diamino-benzidine (DAB) was used for

the first color reaction, giving a brown reaction product.Nickel chloride (0.5%) was added to DAB in the secondcolor reaction, giving a purple reaction product.

Generation of 3-D digital models

Staged Drosophila embryos labeled with anti-FasII andother suitable markers were viewed as wholemounts un-der Nomarski optics (Zeiss Axiophot, Thornwood, NY, 403immersion oil lens, NA 1.3). While focusing through theembryos at increments of 2 µm, digitized images werecaptured with a Sony 3CCD camera (Sony Instruments,Mikron Instruct, San Deigo CA.). Digitized images (‘‘rawsection files’’) were imported into Adobe Photoshop (AdobeSystem, San Jose, CA). Bezier curves (‘‘paths’’) were drawnmanually around the labeled structures which were to beincluded in the model. The paths obtained from eachsection file (‘‘section path files’’) were imported into theRayDream Studio program (Ray Dream Inc., MountainView, CA). Because the section path files were taken fromfocal planes of one and the same embryo, there was noneed for alignment of different sections. In RayDreamStudio, section path files are stacked at the proper inter-vals. A ‘‘skin’’ was synthesized by triangulation to create aspace-filling object in three dimensions. Lighting, cameraangle, transparency, reflection, and other parameters wereadjusted in a straightforward manner in the RayDreamStudio software to display each object clearly with respectto all others.

Variability

The pattern of FasII-positive pioneer neurons appears tobe essentially invariant. Thus, in the neuromeres of theventral nerve cord and the brain, at a stage when onlyrelatively few cells express FasII, the number of these cellswas the same in more than 10 embryos analyzed. Theexact pattern of these cells is very similar, yet may vary indetails: Two labeled cells may lie at precisely the sameantero-posterior level in one neuromere, whereas theirserial homologues in another neuromere may be staggereda few micrometers. The reconstructions shown in thispaper are all based on individual embryos representing agiven stage. Only in the model shown in Figure 4A (earlystage 13), was each individual FasII-positive neuron de-picted separately. In all other models, the whole fibre tractfounder clusters, typically comprising two to six neurons,were outlined. In view of the above mentioned variabilityin exact placement of individual cells, the outlines, whentaken from different specimens, will vary slightly. A clustermeasuring 6 µm in length and 12 µm in width in oneembryo may be 8 µm in width and 10 µm in width inanother. However, the variability rarely exceeds the diam-eter of a single cell (4–6 µm).

RESULTS

Overview of the composition of the neuropileof the late embryonic Drosophila brain

Morphological studies of the of the larval brain ofDiptera have been conducted in the past (e.g., Hertweck,1931; Bullock and Horridge, 1965), and we will use theclassical terminology as much as possible for the struc-tures that were previously identified. The central nervoussystem of Dipteran larvae consists of the paired supra-esophageal ganglion (frequently called ‘‘brain’’ in the re-cent literature) and the ventral nerve cord (Fig. 1). The

12 C. NASSIF ET AL.

subesophageal ganglion is included in the brain only in theadult fly; during larval stages, it forms the anterior part ofthe ventral nerve cord. Given the continuity of neuralelements from embryonic to adult stages one shouldinclude both supra- and subesophageal ganglion under theterm brain, even in the larva and embryo. Both supra-esophageal ganglion and ventral nerve cord have an outerlayer of neuronal and glial cell bodies (cortex) and a centralneuropile. In the ventral nerve cord of a mature embryo(Fig. 2A,F), the neuropile is formed by a longitudinalcomponent, the connective, and segmentally reiteratedpairs of commissures. Strictly speaking, the term connec-tive denominates bundles of axons that interconnect theneuropiles of neighboring ganglia. In Dipterans, the CNShas condensed to such a degree that one cannot distin-guish between areas of neuropile (characterized by termi-nal axonal branches and synapses) and connectives inter-connecting neuropile areas; instead, long range(longitudinal) axonal tracts and terminal branches withsynapses are intermingled and are jointly referred to asconnective or longitudinal neuropile. At the level of thecommissures, nerve roots carrying axons to and from theperiphery enter the connectives. In thoracic and abdomi-nal neuromeres, two roots join to form the so-calledintersegmental nerve (ISN), a single root forms the segmen-tal nerve (SN; Fig. 2A; schematically depicted in Fig. 2E,F).

The neuropile of the subesophageal ganglion closelyresembles the one of the thoracic or abdominal neuromeres(Fig. 2B,E). The labial neuromere possesses a pair ofcommissures and two nerves that show serial homologywith the ISN and SN of thoracic and abdominal neuro-meres. The anterior nerve of the labial neuromere corre-sponds to the ISN because it has a single root entering theneuropile at the level of the posterior maxillary commis-sure and is called the lateropharyngeal nerve (Schmidt-Ott et al., 1994) containing afferent axons from andefferent axons to the pharynx. The posterior, SN nerve ofthe labial neuromere, or labial nerve, enters the neuropilevia a root at the level of the anterior labial commissure;this nerve mainly carries sensory axons from the numer-ous labial sensilla and some efferent axons to the ventralpharyngeal musculature. The maxillary neuromere has apair of commissures and contains the maxillary nerve thatenters the connective at the level of the anterior maxillarycommissure; this nerve carries axons from the maxillarysensory complex and is serially homologous with thesegmental nerve of abdominal and thoracic segments (seebelow). There are no further peripheral nerves enteringthe subesophageal ganglion anterior to the maxillarynerve. The subesophageal commissure, a massive fibrebundle crossing the midline at the boundary betweensubesophageal and supraesophageal ganglia, carries axonsof mainly tritocerebral origin (see below), although someaxons of neurons located in the rudimentary mandibularneuromere also contribute to this commissure.

The neuropile of the larval supraesophageal ganglionhas three major components: a vertical component, thecervical connective; a transverse component, the supra-esophageal commissure; and a horizontal component whichwill be called ‘‘protocerebral connective’’ in the following(Fig. 2C–F). Forming the anterior continuation of theconnectives of the ventral cord, the cervical connectivesare thick axon bundles that curve around the foregut. Atthe basal part of the supraesophageal ganglion, correspond-ing to the tritocerebrum, axons of the subesophageal

(tritocerebral) commissure and the frontal connectivebranch off the cervical connective (Hartenstein et al.,1994). The frontal connective carries peripheral axons toand from the stomatogastric nervous system. Furtherdorsally, the point of entry of the antennal nerve into thecervical connective defines the position of the deuterocer-ebrum (Tissot et al., 1997). After passing the foregut, eachcervical connective branches into the massive supraesopha-geal commissure and the protocerebral connective; thesestructures constitute the neuropile of the embryonic proto-cerebrum. Two peripheral nerves enter the protocerebrum:The optic nerve (or Bolwig’s nerve), carrying sensory axonsfrom the larval photoreceptors (Steller et al., 1987), andthe nerve to the corpora cardiaca (Fig. 2F; Hartenstein etal., 1994).

Development of the neuropile of the thoracicand abdominal neuromeres

FasII is a membrane-bound adhesion molecule ex-pressed by strategically positioned neurons which buildpioneer tracts for the neuropile. An antibody against thisprotein therefore serves as an ideal marker to reconstructthe formation of the neuropile. The FasII expressionpattern in the thoracic and abdominal neuromeres hasbeen described in considerable detail in previous works(reviewed in Goodman and Doe, 1993); however, we willbriefly survey this pattern and establish a segmental‘‘plan’’ of pioneer tracts which will make it easier tounravel the pattern of pioneer tracts of the brain.

FasII expression starts during stage 11 in segmentallyreiterated clusters of cells that foreshadow the position ofthe commissures and connectives; we will call them ‘‘fibretract founder clusters’’ in the following (Figs. 3A,B, 4A).The central group of cells of the fibre tract founder clustersare the two sibling neurons, anterior corner cell (aCC) andposterior corner cell (pCC) and a third neuron, ‘‘friend of ’’posterior corner cell (fpCC). These cells are located adja-cent to the median neuroblast (MNB), which puts theminto the posterior part of the neuromere (Doe, 1992).Posterior and slightly more lateral than aCC/pCC are twoto three additional cells whose axons pioneer the segmen-tal nerve (SN); we will call these cells, which have not beendescribed hitherto, ‘‘SN-pioneers.’’ During stage 12 a thirdgroup of FasII-positive neurons located anterior and me-dial to the aCC/pCC/fpCC triplet complete the fibre tractfounder clusters. These cells are the two midline precursor(MP) 2 neurons (MP2d, MP2v), MP1, and a lightly stainedpair of neuron pioneers that includes the previously de-scribed SP1 (Goodman and Doe, 1993). As can be appreci-ated in Figure 3 and the digital models shown in Figure 4,the cell bodies of the fibre tract founder clusters form analmost uninterrupted ‘‘track’’ of FasII-positive cells. Fromlate stage 12 onward, a group of glial cells called longitudi-nal glia (LG) align at the dorsal surface of the fibre tractfounder clusters (Jacobs and Goodman, 1989).

Starting at late stage 12, neurons of the fibre tractfounder clusters form axons which grow in contact withthe FasII-positive somata and with the LG glia cells (Figs.3C,D, 4A). These axons form two pioneer tracts, called thevMP2 tract (located medially) and MP1 tract (locatedlaterally; for details of these pioneer tracts, see Goodmanand Doe, 1993). The aCC axon grows posteriorly and thenlaterally and pioneers the ISN; SN-pioneer axons follow a

DROSOPHILA BRAIN DEVELOPMENT 13

Figure 2

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similar trajectory, i.e., posteriorly and then laterally. TheSP neurons pioneer the anterior commissure; anothergroup of neurons, as yet unidentified, pioneer the posteriorcommissure (Klambt et al., 1991).

During stage 13, several additional groups of cells whichare smaller in diameter and which are located close to theventral surface of the CNS (a fact that indicates that theyrepresent neurons born later than those close to the core ofthe CNS, such as aCC and pCC) become FasII-positive(Figs. 4B,C, 5A,B). Most of these neurons represent moto-neurons, because they contribute axons to the ISN or SN.There is one medial cluster, closely associated with aCC/pCC, which presumably represents the ‘‘U-neurons’’ (Good-man and Doe, 1993). Another cluster which also sendsaxons into the ISN finds itself more laterally; it probablycorresponds to the ventral intersegmental motoneurons(VIN) cluster described by Sink and Whitington (1991). Acluster which lies close to the SN-pioneers and sends axonsalongside the SN-pioneer axons probably constitutes thelateral segmental motoneurons (LSN) cluster of Sink andWhitington (1991).

In the ventral nerve cord of more mature embryos, fivelongitudinal axon tracts gradually accrue, starting fromthe original vMP2 and MP1 tracts still visible at stage 15(Fig. 5E) over the next two stages (Fig. 5F,G). The dorso-medial and ventro-medial tract develop from the vMP2and MP1 pioneer tract, respectively. The other tracts are oflater origin and carry axons from as yet unidentifiedneurons. A major component of the lateral tract areafferent axons entering the nerve cord through the roots ofthe SN and ISN.

Development of the neuropile of thesubesophageal ganglion

FasII expression in the three gnathal neuromeres whichmake up the subesophageal ganglion resembles the meta-meric pattern described above for thoracic and abdominalneuromeres, although reduction of pattern elements isevident from early stages onward. Three fibre tract founderclusters appear at the boundaries between labial/protho-racic neuromere, maxillary/labial neuromere, and man-dibular/maxillary neuromere, respectively (Figs. 3B,D,4A). Neurons of the labial/prothoracic cluster pioneer thelabial commissures and connectives in the same way asdescribed above for thoracic and abdominal segments. Themaxillary/labial cluster is slightly reduced, lacking thegroup of MP neurons, although it cannot be excluded thatthese neurons appear at a later stage when they cannot bedistinguished from other FasII-positive elements. TheaCC and SN cluster of the maxillary/labial fibre tractfounder cluster pioneer the latero-pharyngeal nerve andlabial nerve, respectively; on the basis of this ontogeneticrelationship it is possible to homologize the labial nervewith the SN of other segments, the latero-pharyngealnerve with the ISN.

The mandibular/maxillary fibre tract founder cluster isextremely reduced, consisting only of SN-pioneers whichpioneer the maxillary nerve (Fig. 3B,D: anterior mostsmall arrow; Fig. 4A). Further anteriorly a large cluster ofFasII-positive neurons appears at the boundary betweendeuterocerebrum and tritocerebrum (D/T fibre tract foundercluster, see below); between D/T and the maxillary fibretract founder cluster exists a relatively wide gap in theotherwise continuous track of FasII-positive pioneer neu-rons (Figs. 3B, 4A). This gap is bridged by the anteriorlyprojecting axon of the maxillary pCC neuron which meetsand fasciculates with posteriorly projecting axons of theD/T cluster. The subesophageal commissure is pioneeredby neurons of the D/T cluster (see below).

The late embryonic pattern of FasII-expressing axontracts of the subesophageal ganglion resembles the pat-tern described above for thoracic and abdominal neuro-meres. The vMP2 and MP1 fascicles pioneer the dorso-medial and ventro-medial tracts, respectively. The lateraltract terminates anteriorly in the subesophageal ganglion.All remaining tracts approach each other at the base of thecervical connective (‘‘convergence zone’’; Fig.10B, arrow-head;10E) where considerable mixing of axons betweenthe individual tracts may take place. As a result, onecannot establish a continuity between the FasII-express-ing tracts of the ventral nerve cord and those ones of thebrain.

Development of the neuropile of thesupraesophageal ganglion

Expression of FasII in the head begins around the samestage as in the trunk, i.e., stage 11 (Fig. 6A). Ventrallythere is one large cluster which contains cells in thesurface ectoderm as well as neural cells segregated from it.With respect to spatial markers such as engrailed (Hamaet al., 1990; Schmidt-Ott and Technau, 1992; Younossi-Hartenstein et al., 1996) this cluster overlaps with part ofthe deuterocerebral as well as of the tritocerebral neurecto-derm (Fig. 6B,C); we have labeled it the deutero/tritocere-bral (D/T) fibre tract founder cluster. The remaining brainfibre tract founder clusters are located further dorsally

Fig. 2. Neuropile of late embryonic brain. A–D show whole mountsof stage 16 embryos labeled with anti-HRP and anti-FasII. E and Fshow schematic ventral and lateral view, respectively, of the nervecord and supraesophageal ganglion, summarizing the elements of theneuropile and peripheral nerve of a late embryo. A: Ventral view ofventral nerve cord with connective (vcn), anterior and posteriorcommissures (ac, pc), segmental and intersegmental nerve (SN, ISN).Anterior root of intersegmental nerve (arrowheads), posterior root(circles). B: Lateral view of anterior part of ventral nerve cord(subesophageal ganglion), showing segmental nerves (dotted lines) ofgnathal segments and first thoracic segment (T1). Intersegmentalnerves enter the connective (vcn) at its dorsal face (black arrows),segmental nerves at its ventral face (white arrows). The intersegmen-tal nerve of the labial segment is the latero-pharyngeal nerve (lpn), thesegmental nerve is the labial nerve (ln). The maxillary segment hasbut a segmental nerve (mn, maxillary nerve). C,D: Lateral view (C)and dorsal view (D) of supraesophageal ganglion. The cervical connec-tive (ccn) forms a continuation of the connective of the ventral nervecord (vcn). Two nerves, the antennal nerve (an) and frontal connective(fc) branch off the cervical connective. Dorsally, the cervical connectivebranches into the supraesophageal commissure (sec) and the protocere-bral connective (pcn). Bolwig’s nerve (Bn) carries axons of the larvalphotoreceptor to the optic lobe (ol). E,F: ac, anterior commissure; an,antennal nerve; Bn, Bolwig’s nerve; ccn, cervical connective; De,deuterocerebrum; es, esophagus; fc, frontal connective; ISN, interseg-mental nerve; Lb, labial segment; ln, labial nerve (segmental nerve oflabial segment); lpn, latero-pharyngeal nerve (intersegmental nerve oflabial segment); lrn, labral nerve (branch of frontal connective,carrying sensory fibres to tritocerebrum; Md, mandibular segment;mn, maxillary nerve (segmental nerve of maxillary segment); Mx,maxillary segment; ncc, nerve to corpora cardiaca; pc, posteriorcommissure; pcn, protocerebral connective; Pr, protocerebrum; sco,subesophageal commissure; sec, supraesophageal commissure; SN,segmental nerve; T1, thoracic segment 1; Tri, tritocerebrum; Scalebar 5 20 µm.

DROSOPHILA BRAIN DEVELOPMENT 15

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than D/T and belong to the protocerebrum. The first ofthese clusters, P3m, appears in the ectoderm that givesrise to the dorsomedial protocerebrum of the larva; thiscluster, like D/T, also reaches from the surface ectoderminto the underlying brain primordium. All other fibre tractfounder clusters are small (two to four cells per cluster)groups of neurons located at the internal surface of theprotocerebral primordium (Fig. 6A). P1 and P2l lie inbetween D/T and P3m; P3l appears laterally adjacent toP3m; P4l is further posterior.

By using double labeling experiments with antibodiesagainst FasII and the seven-up gene product, which isexpressed in subsets of neuroblasts and their progeny, wecould assign the fibre tract founder clusters of the brain todiscrete positions in the brain neuroblast map (Younossi-Hartenstein et al., 1996; see inset in Fig. 6A). P4l appearsto contact cells from the lineages of the central protocere-bral neuroblasts Pc3 (Fig. 6D,E). P3l develops furtheranterior, in contact with neurons of the anterior protocere-bral neuroblast group Pa3 and/or Pa4. P2l is located at thejunction between proto- and deuterocerebrum (neuroblastgroup Dc1).

At late stage 12, several additional clusters join thepattern. The gap between D/T and P3m is bridged by acontinuous track of FasII-positive neurons (small arrowsin Fig. 7B); it is not clear whether these cells migrate outfrom D/T, or express FasII de novo. A small cluster, P2m,appears medially, adjacent to P2l (Figs. 7C, 8B,C). Thecells of P2m, which originate from within the fold betweenthe procephalic lobe and the clypeolabrum, serve as thepioneers of the supraesophageal commissure (see below).P4m is a relatively large group of neurons situated poste-rior toP3m in the medial cortex of the supraesophagealganglion (Figs. 8A–C, 9B). During late stage 15, the fibretract founder clusters P5m and P5l appear in the posterior-ventral region of the protocerebrum, adjacent to the opticlobe (Figs. 8G,H, 10A). Double-labelings with MAb22C10revealed that the P5m neurons correspond to the so-calledoptic lobe pioneers (Tix et al., 1989; Campos et al., 1995).

During stages 12– 15, neurons of the brain fibre tractfounder clusters extend axons that pioneer the cervicalconnective, protocerebral connective, and supraesopha-geal commissure. The following pioneer tracts, docu-mented in microphotographs in Figures 7, 9, 10 and thedigital models shown in Figure 8, can be distinguished.

Medial cervical tract. The D/T cluster emits axonsventrally and dorsally. The ventral axons, which fascicu-

late with the incoming maxillary pCC axon, pioneer theventral segment of the cervical connective (arrow in Fig.7B); a subset of these axons that express the 22C10 epitopewas previously identified by Therianos et al. (1995). Otherventral D/T axons grow along the floor of the stomodeumand pioneer the subesophageal commissure (Fig. 8E,F).Dorsally directed D/T axons converge upon the P2l clusterand pioneer the dorsal segment of the cervical connective(Figs. 8E,F, 9B).

Lateral cervical tract. This tract is formed by axonsof the P3m cluster which extend anteriorly and ventrallyto reach P1 (Figs. 8D–F, 9A). Fasciculating with P1 axons,they continue ventrally toward the subesophageal commis-sure and subesophageal ganglion.

Posterior cervical tract. This fascicle of the cervicalconnective develops later than the other two vertical tracts(stage 16; Figs. 8G, 10B); it carries mainly afferents fromthe ventral nerve cord to the ipsilateral brain hemisphere.

Medial protocerebral tract. This is one of the twoaxon fascicles pioneering the protocerebral connective. Itis formed by anteriorly directed axons of the P4m clusterwhich project toward P3m (Figs. 8D,E, 9B).

Lateral protocerebral tract. Axons of P5l and P4lconverge and project anteriorly toward P3l (Figs. 7A,8D,E, 9B). At a later stage, axons of the P5m (optic lobepioneer) also join this tract. Toward the end of embryogen-esis, a small bundle of fibres, the origin of which could notbe determined, extends alongside the lateral protocerebraltract.

Supraesophageal commissural pioneer tracts. Con-tinuing axons from the medial cervical, lateral cervical,and lateral protocerebral pioneer tracts form three fas-cicles that pioneer the supraesophageal commissure. First,axons of P2l and D/T (medial cervical tract) grow mediallytoward P2m (Figs. 7C, 8C,E,F, 9C). Following the outlineof the P2m cells, these axons reach the dorsal midlinewhere they meet and fasciculate with their contralateralcounterparts and form a bundle that we call the anteriorventral commissural tract (VCT). It could not be resolvedfrom light microscopic analysis whether the P2m cellsthemselves also contribute axons to the commissuralpioneer tract. In the study by Therianos et al. (1995), basedupon MAb22C10 stained material, a subset of the anteriorVCT axons was identified as ‘‘pioneer of the supraesopha-geal commissure.’’

A second discrete FasII-positive commissural tract isformed just posterior to the anterior VCT by collaterals ofthe lateral cervical tract; we call it the posterior ventralcommissural tract (Fig. 10B,C). Finally, axons of P4l andP3l grow anteriorly, make a sharp turn medially, and forma commissural bundle dorsally of the one formed by P2l(dorsal commissural tract, DCT; Fig. 8H,I).

In addition to the central axon tracts, FasII labels motorcomponent of various peripheral nerves (Fig. 10A), thefrontal connective that leads axons from the tritocerebrumto the stomatogastric nervous system, and the neurosecre-tory nerve to the corpora cardiaca, a neurohemal structurelocated between the brain hemispheres (Fig. 10D).

FasII-positive tracts pioneer prominentcomponents of the adult brain

Toward the end of embryogenesis (15–22 hours), thereare some additions to the pattern of FasII-positive neuro-pile structures that foreshadow the profound growth andreorganization of the neuropile during the larval period.

Fig. 3. Fibre tract founder clusters of the ventral nerve cord.Anterior is to the top. A,B: Ventral view of stage 12 embryo labeledwith anti-FasII (black) and anti-Elav (gray nuclear staining). A showsfocal plane close to ventral surface of ventral nerve cord, B shows focalplane near its dorsal surface. Cell bodies of FasII-positive neurons(MP2, individual midline precursor cells; aCC/pCC, anterior cornercell/posterior corner cell [arrowheads], SNp, segmental nerve pioneercluster [arrows]) form elongated fibre tract founder clusters on whichpioneer tracts will later grow. Boundaries between neuromeres (T1–T3, thoracic neuromeres; Lb, labial neuromere; Mx, maxillary neuro-mere; Tri/Md, fused mandibular/tritocerebral neuromere) are indi-cated by lateral indentations of cell body layer (gray arrows); D/T, fibretract founder cluster at deutero/tritocerebral boundary. C,D: Superfi-cial (C) and deep (D) focal plane of central nerve cord of stage 13embryo labeled with anti-FasII and anti-Elav. Fibre tract founderclusters form uninterrupted track on which first pioneer tracts extend.Additional FasII-positive clusters of mostly motoneurons (LSN, U, inC) and some interneurons (SP, in D) have made their appearance. ISN,intersegmental nerve; SN, segmental nerve. Scale bar 5 20 µm.

DROSOPHILA BRAIN DEVELOPMENT 17

Fig. 4. Digital models of fibre tract founder clusters and pioneertracts in the anterior part of the ventral nerve cord of early stage 13(A) and stage 14 (B,C) embryos. Different populations of FasII-positivepioneer neurons are indicated by different colors (see color key atfigure bottom). Surface of ventral nerve cord is rendered semitranspar-ent. Left half of the models in A and C presents a ventral view, andright half shows a dorsal view; B is a frontal view. For details see text.ac, anterior commissure; aCC, anterior corner cell; D/T, deutero/tritocerebral fibre tract founder cluster; fpCC, ‘‘friend of ’’ posteriorcorner cell; ISN, intersegmental nerve; Lb, labial segment; ln, labial

nerve (segmental nerve of labial segment); lpn, latero-pharyngealnerve (intersegmental nerve of labial segment); LSN, lateral segmen-tal motoneurons; Md, mandibular segment; mn, maxillary nerve(segmental nerve of maxillary segment); MP, midline precursor clus-ter; MP1, 2, individual midline precursor cells; Mx, maxillary seg-ment; pc, posterior commissure; pCC, posterior corner cell; SCO,subesophageal commissure; SN, segmental nerve; SNp, segmentalnerve pioneer cluster; SP, pioneer neurons of anterior commissure;T1–T3, thoracic segments 1–3; Tri, tritocerebrum; VIN, ventral inter-segmental motoneurons. Scale bar 5 20 µm.

18 C. NASSIF ET AL.

Fig. 5. Development of the neuropile of the ventral nerve cord.Anterior is to the top. A,B: Ventral view of stage 14 embryonic nervecord labeled with anti-FasII antibody (A, superficial focal plane; B,deep focal plane). The number of FasII-positive axons has increased inthe intersegmental nerve (ISN), segmental nerve (SN), and the twopioneer tracts of the connective (MP1t, vMP2t). Note close apposition ofthese two tracts at segment boundaries (white arrowheads in B, E,and F). C,D: Gnathal neuromeres of same embryo shown in Aand B (lower magnification). Anterior to the SN and ISN of the firstthoracic neuromere (T1 in panel D), three peripheral nerves canbe distinguished in the labial (Lb) and maxillary (Mx) neuromere(ln, labial nerve; lpn, latero-pharyngeal nerve; mn, maxillary nerve).

E–G: Ventro-lateral view of anti-FasII labeled nerve cords of stage 15(E), early 16 (F), and late 16 (G) embryo. The pattern of twoFasII-positive pioneer tracts (MP1t, vMP2t) is still visible at stage 15.During early stage 16, a third tract (IT, intermediate tract) appearslateral to MP1t. The vMP2 tract shifts ventrally to become theventro-medial tract (VMT); MP1t forms the dorso-medial tract (DMT).Later during stage 16 (G), the lateral tract (LT) makes its appearance.ac, anterior commissure; aCC, anterior corner cell; LSN, lateralsegmental motoneurons; MP, midline precursor cluster; pc, posteriorcommissure; pCC, posterior corner cell; SNp, segmental nerve pio-neers; U, U-motoneurons; VIN, ventral intersegmental motoneurons.Scale bars 5 20 µm.

DROSOPHILA BRAIN DEVELOPMENT 19

Fig. 6. Early development of the fibre tract founder clusters of thesupraesophageal ganglion. A: Lateral view of early stage 12 embryolabeled with anti-FasII (gray) and anti-Elav (brown). B,C: Lateralview of mid-stage 12 embryo labeled with anti-b-Gal, visualizingengrailed stripes (light and diffuse cytoplasmic signal in superficialfocal plane shown in B), and anti-FasII (dark membrane bound signalin deep focal plane shown in C). Dotted lines indicate posteriorboundary of engrailed head spot (hs, demarcates boundary betweenprotocerebrum and deuterocerebrum) and of antennal stripe (An,demarcates boundary between deuterocerebrum and tritocerebrum).The fibre tract founder cluster D/T crosses the deutero-/tritocerebralboundary; P2l, from where the supraesophageal commissure will bepioneered, lies at the proto-/deuterocerebral boundary. P3m, P3l, andP4l are protocerebral. D,E: Lateral view of mid-stage 12 embryolabeled with anti-FasII (brown) and anti-b-Gal visualizing seven-up

expression pattern in subsets of neuroblasts and their progeny (gray,nomenclature of neuroblasts in D and inset of A after Younossi-Hartenstein et al., 1996). Clusters of neuroblasts are located at brainsurface (D); their progeny is pushed interiorly (E), with the first-bornneurons occupying the most internal position. Some of these earlyborn neurons differentiate as fibre tract founder clusters. P4l is part ofthe Pc3 lineage (Pc, Central Protocerebral Domain); P3l develops closeto the Pa3 lineage (Pa, Anterior Protocerebral Domain), P3m appearsmedially to this lineage. P2l derives from the Dc1 or Dc2 lineage (Dc,Central Part of Deuterocerebrum). Inset in A shows schematicneuroblast map (gray circles) in which location of fibre tract founderclusters (orange circles) is tentatively indicated. aCC/pCC, fibre tractfounder cluster at labial/thoracic boundary; Lb, labial neuromere; Mx,maxillary neuromere; T1, first thoracic neuromere. Scale bars 520 µm.

Fig. 7. Fibre tract founder clusters of the supraesophageal gan-glion in a stage 13 embryo. A,B: Lateral view of early stage 13 embryolabeled with anti-FasII. Focal plane shown in A depicts seriallyarranged clusters D/T, P1, P2l, P3l, and P4l. P3m is visible at a moremedial focal plane (B). Note continuous track of FasII-positive cellbodies that has formed between D/T and P3m (arrows in B). Thistrack, similar to the fibre tract founder clusters of the ventral nervecord, serves as the substrate for outgrowing pioneer axons. P4l (A) hasgrown an axon bundle toward P3l. C,D: Two focal planes (C, superfi-cial; D, deep) showing a dorsal view of right supraesophageal ganglionof early stage 13 embryo labeled with anti-FasII. In C, the spatial

relationship between P2l and P2m can be clearly seen. At a slightlylater stage, P2l will form axons that cross the midline in contact withP2m; these axons pioneer the supraesophageal commissure (see alsoFig. 9). E: Schematic lateral view of FasII-positive fibre tract founderclusters at stage 13. Bo, Bolwig’s organ; D/T, fibre tract foundercluster; fc, pioneer cluster of frontal connective; fg, frontal ganglion ofstomatogastric nervous system; ISN, intersegmental nerve; ln, labialnerve; lpn, latero-pharyngeal nerve; mn, maxillary nerve; ol, opticlobe; P1, P2l, P2m, P3l, P3m, P4l, Pa3, Pa4, Pc1, Pc3, Pp3, fibre tractfounder clusters of the protocerebrum; SN, segmental nerve; T1, firstthoracic segment. Scale bar 5 20 µm.

Figure 8

22 C. NASSIF ET AL.

Figure 8 (legend on following page)

DROSOPHILA BRAIN DEVELOPMENT 23

Most important among these is the emergence of themushroom body and a prominent tract that may actuallycorrespond to the pioneer of the antennocerebral tract. Thelatter develops from an interchange of axons between themedial/lateral cervical tracts and the lateral protocerebraltract (Figs. 8G, 10B, 11B). Thus, before approaching themidline, bundles of axons branch off the lateral protocere-bral tract and join the medial and lateral cervical tracts,respectively. In the following, this interchange of axonswill be called central anastomosing tract (CAT). Wedged inbetween the lateral protocerebral tract and the centralanastomosing tract appears a tangle of FasII-positivefibers which, around the time of hatching, has adopted atrilobed configuration, with one lobe projecting posteriorly,the other medially, and the third dorsally (Fig. 11A,B).These lobes, as can be seen from their later larval appear-ance, correspond to the peduncle, b-lobe, and a-lobe of themushroom body. It should be noted that in case of themushroom body, as well as other neuropile structuresdiscussed below, FasII is clearly not expressed on theentire neurons (somata plus axons), but only part of theaxons. Even the more proximal part of the peduncle, closeto the mushroom body somata, does not express FasII.From the medial lobe of the mushroom body primordium(b/g lobe) emerges a commissural bundle which crosses themidline between the ventral commissural tracts and thedorsal commissural tract. This ‘‘mushroom body commis-sure’’ persists throughout larval and adult development(Strausfeld, 1976).

The identification of the mushroom body primordiumhas greatly helped in following the surrounding FasII-positive fascicles throughout larval development and as-signing them to neuropile components of the larval andadult brain. Although the postembryonic development ofFasII-positive axon tracts will be documented in detailelsewhere, it seems appropriate to point out briefly therelationship between embryonic/larval and adult neuro-pile structures. The brain region anterior and medial tothe peduncle of the mushroom body represents the primor-

dium of the superior medial protocerebrum. The medialand lateral cervical tracts of the embryo remain FasII-positive in the larva (Fig. 11). They send crossed anduncrossed axons to the superior medial protocerebrum andare connected to the corpora cardiaca nerve, which alsocontinues to express FasII. On the basis of their connectionwith the superior medial protocerebrum and the corporacardiaca, as well as their anterior-ventral position in thebrain commissure, we postulate that the medial/lateralcervical tracts seen in the embryo and larva are theforerunner of the adult median bundle.

The lateral protocerebral tract shows a complex rear-rangement during larval development. Already during lateembryonic stages its central region stands out by itsincreased diameter and strong FasII expression (Fig. 11).Thus, beside the earlier described long axons extendingfrom the P5m/P4l toward the supraesophageal commis-sure (long distance component of lateral protocerebraltract), there appear other FasII-expressing elements along-side the central segment of the lateral protocerebral tract;similar to the lobes and peduncle of the mushroom body(see above), these later developing elements are masses ofshort axon segments whose cell bodies of origin andfurther trajectory cannot be determined because they don’texpress the marker (short distance component of lateralprotocerebral tract). During the larval period, the shortdistance component of the lateral protocerebral tract fansout as an expanding tangle of fibres that overlay thepeduncle of the mushroom body and therefore may corre-spond to part of the calyx neuropile. Beside the calyxneuropile, the long distance component of the lateralprotocerebral tract of the embryo gives rise to a prominentlarval tract that we postulate corresponds to the forerun-ner of the adult posterior optic tract. The P5m cluster ofneurons from which the lateral protocerebral tract of theembryo originates, become part of the developing medullaof the larval optic lobe (Fig. 11I). Their axons projectdorso-medially, pass right behind the calyx neuropile, andthen cross the midline in the brain commissure. The onlycrossed axon tract of the adult brain that originates in themedulla is the posterior optic tract (Strausfeld, 1976); allother optic commissures are formed by neurons of thelobula complex.

The central anastomosing tract of the embryonic brain,based on its topology, likely represents the forerunner ofthe antennocerebral tract. Throughout larval developmentthe anastomosing tract forms a conspicuous bundle thatextends parallel to the peduncle of the mushroom body(Fig. 11). Ventrally, the anastomosing tract maintains itscontinuity with FasII-expressing fiber bundles originatingin the ventral nerve cord; toward later stages, additionalfiber bundles from basal brain regions (antennal lobe ofdeuterocerebrum) feed into this tract (Fig. 11G). Dorsally,the anastomosing tract feeds into the growing calyx neuro-pile and areas laterally adjacent to it.

DISCUSSION

In this study we have described the pattern of FasII-expressing axons in the developing brain and ventralnerve cord of Drosophila embryos. The FasII antigenappears on the surfaces of clusters of neuronal cell bodiesprior to axon outgrowth; these fibre tract founder clustersare laid out to form an almost uninterrupted longitudinaltrack that reaches from the ventral nerve cord to the ‘‘tip’’

Fig. 8. (overleaf ) Digital models of brain hemispheres of late stage12 (A–C), late stage 14 (D–F), and stage 16 (G–I) embryos, illustratingthe pattern of fibre tract founder clusters and pioneer tracts (color key,top left) in different views (first column A,D,G, lateral view; secondcolumn B,E,H, dorsal view; third column C,F,I, frontal view). In panelsshowing dorsal and frontal view, only one hemisphere was modeledand then duplicated and mirrored. Surface of brain is renderedsemi-transparent. Diagram on left side below color key schematicallyshows pattern of fibre tract founder clusters. For details see text.aCC/pCC, Anterior-Posterior corner cell cluster of ventral nerve cord(in maxillary segment); CC, corpora cardiaca; D/T, fibre tract foundercluster at boundary between deutero- and tritocerebrum; DCT, dorsalcommissural tract(s); DMT, dorso-medial tract (of ventral cord connec-tive); es, esophagus; FC, frontal connective; fg, frontal ganglion; IT,intermediate tract; lc, late developing FasII-positive neuronal clustersfeeding into posterior cervical tract; lp, late developing FasII-positiveneuronal clusters feeding into lateral protocerebral tract; LPT, lateralprotocerebral tract (of protocerebral connective); LT, lateral tract ofcervical connective; mc, late developing FasII-positive neuronal clus-ters feeding into medial cervical tract; MPT, medial protocerebraltract; MT, medial tract of cervical connective; ol, optic lobe; P1, P2l,P2m, P3l, P3m, P4l, P4m, P5l, P5m, fibre tract founder clusters of theprotocerebrum; pc, late developing FasII-positive neuronal clustersfeeding into posterior cervical tract; PT, posterior tract of cervicalconnective; SCO, subesophageal commissure; VCT, ventral commis-sural tract(s); VMT, ventromedial tract (of ventral cord connective).Scale bar 5 20 µm.

24 C. NASSIF ET AL.

Fig. 9. Fibre tract founder clusters of the supraesophageal gan-glion in a stage 15 embryo. A,B: Lateral view of early stage 15 embryolabeled with anti-FasII (A, superficial focal plane; B, deep focal plane).Axons of the fibre tract founder clusters D/T, P1, P2l, and P3m formthe medial (MT) and lateral (LT) pioneer tract of the cervical connec-tive, as well as the subesophageal commissues (out of focal plane; seediagram E). All axons grow in contact with the continuous trackformed by the cell bodies of fibre tract founder clusters (see Fig. 7).Axons of the P4l (A) and P4m (B) cluster form the lateral (LPT) andmedial (MPT) pioneer tracts of the protocerebral connective. The gapbetween cervical connective and ventral nerve cord is bridged by axon

bundle (arrow in B). C,D: Dorsal view of early stage 15 embryo (C,superficial focal plane; D, deep focal plane) showing pioneer tracts ofthe cervical connective (LT, MT), protocerebral connective (LPT, MPT),and supraesophageal commissure (SEC). Axons between the tritocere-brum and frontal ganglion (fg) form the frontal connective (fc);posterior axons connecting the frontal ganglion to more posteriorstomatogastric ganglia (sns in D) form the recurrent nerve (rn).E: Schematic lateral view of FasII-positive fibre tract founder clustersat stage 15. ol, optic lobe; SPO, subesophageal commissure. Scalebar 5 20 µm.

DROSOPHILA BRAIN DEVELOPMENT 25

Figure 10

26 C. NASSIF ET AL.

of the brain. Gaps separating neighboring clusters are inno case wider than 15 µm. It is possible that cells that donot express FasII lie within these gaps; however, weconsider this unlikely, because none of the markers de-scribed in the literature and investigated by us (see below)labeled fiber tract founder clusters that were not labeledby anti-FasII as well.

After expressing FasII in the cell bodies of fiber tractfounder clusters, these same cells extend axons thatalways grow in contact with the somata. Thus, the pioneerneurons provide both axons and the substrate on whichthese axons grow. It is tempting to speculate that homo-philic adhesion between pioneer growth cones and cellbodies mediated by FasII is involved in pathfinding. Thecomplete loss of FasII leads to defective contacts of axonsamong each other, although at a later stage, no visibleabnormality in axonal connectivity results (Grenninglohet al., 1991; Lin et al., 1994). The same is true for the brain,where loss of FasII function also leads to no detectableabnormalities at the light microscopic level (Hartenstein,unpublished observations). It is possible that there existother adhesion molecules that, like FasII, are expressed atan early stage on both somata and axons of pioneerneurons. Among the adhesion molecules already identi-fied, Fasciclin III (FasIII) appears on some neurons of thefiber tract founder clusters, notably on pCC and commis-sural neurons, possibly the SP neurons (Patel et al., 1987).Fascilin I (FasI) is expressed at a low level quite widely; ahigher level of expression is detected on aCC, the ventralunpaired motoneurons (VUMs), and RP1 neurons (Zinn etal., 1988; McAllister and Goodman, 1992).

Pattern of fiber tract founder clustersin the embryonic brain

The segmentally reiterated fiber tract founder clustersthat express the FasII antigen demarcate the neuraxis of

the ventral nerve cord, including the subesophageal gan-glion, of the Drosophila CNS at an early stage. Theseclusters are formed by serially homologous neurons (MP1,MP2d/v, aCC/pCC/fpCC, segmental nerve pioneer cluster[SNp], and others) the axons of which link up to form twouninterrupted longitudinal connectives on either side ofthe midline. Neuromeres are lined up along the longitudi-nal connective; each neuromere (except for the two termi-nal neuromeres, mandibular and A9) forms a pair ofcommissures which carry axons crossing from one side tothe other. What can be stated about how the neuraxiscontinues anteriorly into the supraesophageal ganglion?The neuropile of the supraesophageal ganglion is alsopioneered by FasII-positive fibre tract founder clusters. Itis not possible to identify cells of these supraesophagealclusters as serial homologues of individual pioneer neu-rons of the ventral nerve cord (e.g., d/vMP2, MP1, pCC).However, it seems plausible to consider the cervical connec-tive, pioneered by neurons of the FasII-positive D/T, P1,and P2l/m clusters, as the anterior continuation of theconnective of the ventral nerve cord. At the level of the P2lcluster, located in the basal protocerebrum, the cervicalconnective branches into supraesophageal commissure(medial) and protocerebral connective (posterior-lateral).Two views might be taken about where to place theanterior tip of the neuraxis.

The first view is that the point of origin of the protocere-bral connective at the level of the optic lobe might repre-sent the anterior tip of the neuraxis (Fig. 12, right side). Inthis arrangement, structures of the protocerebrum (e.g.,optic lobe, mushroom body) would be arranged seriallyalong the neuraxis.

The second view is that the basal protocerebral P2cluster represents the anterior tip of the neuraxis (Fig. 12,left side). According to this view, the protocerebral connec-tive would not represent a connective in the true sense ofthe word (i.e., a tract connecting parts of the nervoussystem that are serially arranged along the neuraxis), butcould be considered as a lateral tract that branches off theconnective near its anterior tip. Protocerebral structureswould be arranged in the medio-lateral axis, with the opticlobe taking the most lateral position. The supraesophagealcommissure would not in that case represent a commis-sure in the same sense as commissures of the ventral nervecord, but a terminal bundle at the anterior tip of theneuraxis where longitudinal axons of the connectivesconverge.

The origin of the glial sheath of the supraesophagealcommissure actually supports the second view. Thus,‘‘true’’ commissures of the ventral nerve cord are associ-ated with a group of so called midline-glia that are derivedfrom the mesectoderm. The connectives are ensheathed byprocesses of the longitudinal glia cells which are quitedifferent from midline glia both in origin from the lateralneurectoderm and in expression of molecular markers(Jacobs and Goodman, 1989). In the brain, midline gliacells (recognizable by specific molecular markers such aspointed; Klambt, 1993) do not form. Instead, a populationof glial cells that is primarily associated with the base ofthe cervical connective, and that therefore might be consid-ered serially homologous with the longitudinal glia of theventral nerve cord, migrates dorsally and ensheathes thesupraesophageal commissure (Hartenstein et al., 1998).

Fig. 10. Fibre tract founder clusters of the supraesophageal gan-glion in a stage 16 embryo. Lateral views (A,B) and dorsal views (C,D)of late stage 16 embryo labeled with anti-FasII (A,C, superficial focalplane; B,D, deep focal plane; inset in B shows supraesophagealcommissure optically ‘‘cut’’ in the sagittal plane). The pattern ofFasII-positive pioneer tracts is complete. Three main tracts can bediscerned in the cervical connective (MT, medial tract; LT, lateraltract; PT, posterior tract). Axons of P4l (A) are now joined by axons ofP5m (the ‘‘optic lobe pioneers’’ of Tix et al., 1989) to form the lateralprotocerebral tract (LPT). This tract shows a conspicuous thickeningin its central segment (arrow in A); medially, it continues as one of thedorsal commissural axon bundles (DCT in inset in B). The medialprotocerebral tract (MPT in B) is continuous with the lateral tract ofthe cervical connective. Axons that branch off the MT and LT form ananastomosis with the LPT, called the central anastomosing tract (CATin B). Also shown in B are the ventro-medial (VMT), dorsomedial(DMT), and intermediate (DIT) tract of the ventral nerve cord.Arrowhead in B points at the position at the base of the cervicalconnective where axon tracts converge, making it difficult to establishunambiguously how tracts of the supraesophageal ganglion connectedwith those of the ventral nerve cord. E: Schematic lateral view ofFasII-positive fibre tract founder clusters at stage 16. an, antennalnerve; Bn, Bolwig’s nerve; DLT, dorso-lateral tract (of ventral connec-tive); D/T, fibre tract founder cluster at boundary between deutero-and tritocerebrum; fc, frontal connective; fg, frontal ganglion; ln, labialnerve; mn, maxillary nerve; ncc, nerve of corpora cardiaca; ol, opticlobe; P3l, P3m, P4l, P4m, P5l, P5m, fibre tract founder clusters of theprotocerebrum; rn, recurrent nerve; SEC, supraesophageal commis-sure; SPO, subesophageal commissure; sns, stomatogastric ganglia;VIT, ventro-intermediate tract (of ventral connective). Scale bar 520 µm.

DROSOPHILA BRAIN DEVELOPMENT 27

Figure 11

28 C. NASSIF ET AL.

Phylogenetic conservation of insect brainpioneer tracts

The pattern of early pioneer tracts in the Drosophilaembryonic brain resembles the pattern described for grass-hopper embryos (Boyan et al., 1995), suggesting that theremay exist in all insects the same simple scaffold of axontracts that serves as a ‘‘skeleton’’ around which the neuro-pile regions are built. In the grasshopper, Boyan et al.(1995) describe a ‘‘transverse’’ pioneer tract, that reachesfrom the lateral optic lobe toward the midline, and avertical pioneer tract. Both tracts converge at the supra-esophageal commissure. By using the terminology pre-sented here, the transverse pioneer tract of Boyan et al.(1995) would correspond to the protocerebral connective,the vertical tract to the cervical connective. The transversetract/protocerebral connective of Drosophila has a moreposterior-to-anterior orientation than its grasshopper ho-mologue; this may be simply a reflection of the fact that theindividual elements of the protocerebrum (i.e., midbrain,inner optic anlage, outer optic anlage) in grasshopperembryos are laid out from medial to lateral, in Drosophilaembryos from anterior to posterior (Younossi-Hartensteinet al., 1996).

Identification of primordia of adult neuropilecomponents in the embryonic brain

In this study we have made a first attempt to assign thepioneer tracts that appear in the embryo to definitiveneuropile components of the adult brain. The structuremost easily recognizable is the mushroom body with itscharacteristic axonal branching pattern (a- and b-lobes,commissure between b-lobes, peduncle). FasII expressionin the mushroom body begins very late during embryogen-esis in the two lobes, the b-lobe commissure, and the distalpart of the peduncle. The location of the embryonic mush-room body neuronal cell bodies can be deduced from theposition of the four MB neuroblasts (Ito and Hotta, 1992),as well as the expression of numerous MB specific molecu-lar markers (Noveen and Hartenstein, unpublished obser-vations). According to these markers, MB neurons form alarge cluster in the posterior quadrant of the brain hemi-sphere. Based on the fact that MB neuroblasts are among

the first neuroblasts to appear in the head (Noveen andHartenstein, unpublished observations), it is reasonable toassume that some MB neurons differentiate and emitaxons quite early in the embryo. Unfortunately, no markerlabeling these axons prior to their expression of FasII hasso far been described.

Whereas in shape and orientation, the embryonic/larvalmushroom body closely resembles its adult counterpart,its size relative to other brain structures is much larger inthe embryo/early larva than in the adult. Thus, the ‘‘spur,’’where the peduncle is joined to the two lobes (Heisenberget al., 1985), is located at the very ventrolateral edge of theprotocerebral neuropile. The length of the a- and b-lobescorrespond to a full diameter of the neuropile, as opposedto approximately 40% of the diameter of the adult neuro-pile.

The antennocerebral tract which provides a major sourceof input to the mushroom body may also be pioneered inthe embryo by a tract that we call the ‘‘central anastomos-ing tract.’’ Thus, from embryonic stage 16 through larvaldevelopment, the FasII-positive central anastomosing tractcan be recognized as a branch of the cervical connective; itpasses beneath the b-lobe of the MB and projects posteri-orly, parallel and medial to the peduncle. The tract termi-nates in close proximity to the proximal part of thepeduncle. Primarily, the central anastomosing tract seemsto carry collateral axons that originate in the subesopha-geal neuromeres, or even further posteriorly in the ventralnerve cord. During larval stages, these axons are joined byFasII-positive axons originating in the antennal lobe,recently identified by R. Stocker and colleagues (Stocker,1994; Tissot et al., 1997). In all of these properties thecentral anastomosing tract matches the antennocerebraltract. Work is in progress to follow the central anastomos-ing tract through metamorphosis and establish its identitywith the antennocerebral tract of the adult brain.

The embryonic medial and lateral tracts of the cervicalconnective, that are both continuous with the FasII-positive bundles of the ventral nerve cord, most likelycorrespond to pioneers of the median bundle of the adultbrain. We base this designation on a list of properties thatthe medial/lateral tracts of the cervical connective of the

Fig. 11. Larval development of FasII-positive pioneer tracts. Allpanels show brain hemispheres labeled with anti-FasII in lateral view(A–D) or frontal view (E–I). A,B: Two focal planes (A, 10 µm belowbrain surface; B, 30 µm below brain surface) of a late stage 17 embryo.Pioneer tracts of cervical connective (LT, MT, PT) and of protocerebralconnective (LPT) are labeled. Note origin of LPT at optic lobe (ol; cellbodies of P5m cluster from which LPT arises are not discernible); notealso prominent central anastomosing tract (CAT) connecting LPT withcervical connective. C,D: First instar larva (C, 10 µm below brainsurface; D, 30 µm below brain surface). Pioneer tracts of cervical andprotocerebral connective (LT/MT, PT, LPT) continue expressing FasII.Note prominent staining of peduncle (PED) and lobes (a, b) ofmushroom body. The central anastomosing tract (pioneer of antenno-cerebral tract) passes beneath the b-lobe (D) and extends medial to thepeduncle. The peduncle terminates close to the junction between CATand LPT. E,F: Frontal view of first instar larval brain (E, 5 µm beneathanterior brain surface; F, 20 µm beneath anterior surface), showingspatial relationship between mushroom body and pioneers of cervicalconnective and protocerebral connective. Note commissural bundle(MBC) between b-lobes that lies between the ventral (VCT) and dorsal(DCT) bundle of supraesophageal commissure. G,H,I: Frontal view oflate third instar larval brain (G, approximately 10 µm below anteriorsurface; H, approximately 25 µm below surface; I, approximately 50

µm below surface). H and I represent optical sections that arecomparable to those shown in E and F, respectively. Although theneuropile, including the lobes and peduncle of the mushroom body, hasincreased in volume several fold, the spatial relationship between thevarious FasII-positive tracts has remained similar. The former LT andMT form the median bundle (MB). From the LT/MT branches theformer central anastomosing tract, now imposing as the prominentantennocerebral tract (AGT) extending medially parallel to the pe-duncle (H, I), fanning out in the area where the peduncle endsposteriorly (not shown), thereby defining the position of the calyxneuropile of the mushroom body. J: Schematic model showing theadult and larval brain in antero-lateral view, with several neuropilecomponents of the adult brain that form a ground-plan (after Wil-liams, 1975). Three prominent adult tracts and their larval counter-parts are shown in bright color: the median bundle (MB, red),antennocerebral tract (AGT, blue), and posterior optic tract (POT,green). al, antennal lobe; AL, FasII-positive clusters in antennal lobe;ALC, commissure formed by antennal lobe axons; aof, anterior opticfocus; ca, calyx of mushroom body; cb, central body; la, lamina of opticlobe; lh, lateral horn; lo, lobula; lop, lobula plate; me, medulla of opticlobe; pi, pars intercerebralis; pof, posterior optic focus; POT, posterioroptic tract (emerges from the lateral protocerebral tract of earlierstages). Scale bars 5 20 µm.

DROSOPHILA BRAIN DEVELOPMENT 29

embryonic and larval brain share with the median bundleof the adult. The adult medial bundle connects the supe-rior medial part of the protocerebrum with the subesopha-geal ganglion and the ventral nerve cord (Power, 1943;Bullock and Horridge, 1965; Williams, 1975; Strausfeld,1976). Crossing collaterals of this bundle occupy the mostanterior and basal position within the large complex ofsupraesophageal commissures (Strausfeld, 1976). Axons oflarge neurosecretory neurons located in the pars intercere-bralis join the median bundle, project ventrally, and thensplit off the bundle as a peripheral nerve (Williams, 1975)destined for the corpora cardiaca. The median bundle ofthe adult brain forms but a minor part of the extensivesystem of commissure and neuropile structures located inthe midline of the protocerebrum.

Another adult brain structure for which we can tenta-tively assign an embryonic pioneer is the posterior optic

tract. In the adult brain, this prominent fiber bundle isformed by axons of medulla neurons that run posteriorlyon the inner face of the medulla, turn around in a medialdirection and then cross the midline to terminate in thecontralateral medulla (Strausfeld, 1976). All other fibrebundles that leave the optic lobe to terminate in the opticfoci or the contralateral brain originate in the lobulacomplex (Strausfeld, 1976). A prominent, FasII-positivecommissural fibre tract growing out of the medulla existsin the larval brain, and can be followed back in time to thelateral protocerebral tract of the embryo. As described inthis paper, the tract is formed by a cascade of neurons,including (from medial to lateral) the P3l, P4l, and P5m/lclusters. P5m is situated right adjacent to the optic lobeand in size and projection corresponds to the optic lobepioneers described by Tix et al. (1989) and later by Camposet al. (1995). These neurons may actually receive theterminals of the larval eye (Bolwig’s organ), or at leastfasciculate with Bolwig’s organ axons that enter the brain.At early larval stages, the P5m/optic lobe pioneer cluster issandwiched between the outer and inner optic anlage,right at the position where the medulla neurons willappear. Also, the axons forming the lateral protocerebraltract persist into the larval period as a thin bundle thatextends medially, passing closely behind the calyx of themushroom body, and crosses the midline. At later stages,axons of medulla neurons (produced by the outer opticanlage) join the bundle. Work is proceeding to follow otheraxon tracts from the embryonic stages throughout thelarval period and metamorphosis into the adult brain.

ACKNOWLEDGMENTS

The authors are grateful to Drs. C. Doe, T. Kaufman, S.Robinow, and C. Goodman for providing antibodies and flystocks which facilitated this study.

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