In the paof insecgations nisms oanalyzed1985; Doe et al., 1985). Insight into the molecular mechanismsthat control these developmental processes has also beengained, both through the use of hybridoma technology (Bastianiet al., 1987; Harrelson and Goodman, 1988; Snow et al., 1988)and through molecular genetic analyses of neuronal develop-ment in
Drosophila (for reviews see Grenningloh et al., 1990;Campos-Ortega and Knust, 1990; Goodman and Doe, 1994).
Most of the investigations of embryonic development in theinsect CNS have been carried out on the accessible and easilyidentifiable cells in the segmental ganglia. The brain, bycontrast, is an enormously complex structure which in largeinsects can consist of over a million neurons (Farrel and Kuh-lenbeck, 1964). While an initial analysis of neurogenesis in thegrasshopper brain has described neuroblasts from which thebrain develops (Zacharias et al., 1993), virtually nothing isknown about the mechanisms by which the progeny of theseneuroblasts produce the complex axonal projections of theadult brain (Boyan et al., 1993).
ortl sehopcti
ndihe ns
neurogenesis, the axons from these neurons initially navigatealong glial-bound aggregates of proliferating neuroblasts,termed proliferative clusters, that are established before axo-genesis begins. Through axonal outgrowth and fasciculation,the identified pioneering neurons rapidly establish a primaryaxon scaffold that interconnects all of the proliferative clustersin the embryonic brain. This scaffold is used for fasciculationby many of the subsequently differentiating neurons and, bythe 40% stage of embryogenesis tracts, commissures and con-nectives of the mature brain become apparent.
MATERIALS AND METHODS
Animals Schistocerca gregaria eggs were kept in moist aerated containers at30°C. Embryo staging was according to Bentley et al. (1979). Thismethod stages embryos at time intervals equal to percentage ofembryogenesis completed.
DevelopmePrinted in G
Axogenesis in the embryonic brain was studied at the singlecell leve
set of ina primabeginnialong aproliferaxonal neuronsbrain mmissurapioneersecond pairs oftracts alasts in
cerebrum; thy ainur
lishfasd, the ssenxpula
Dro
xogcen
SUMMA
Axog ho
an id m
George inr1Labora 512Max-Pl 9 S
, we analyse the cellular processes that give riset of axonal projections in the embryonic brainper. We use immunocytochemical, intracellu-
on and electron microscopical techniques toviduals the neurons that pioneer the initial pro-embryonic brain and to determine the lineage offrom identified brain neuroblasts. During early
75
e postoral tritocerebral commissure is pair of tritocerebral neurons. All of the pio-
neurons express the cell adhesion moleculeing initial axon outgrowth and fasciculation.ed, the primary axon scaffold of the brain is
ciculation by subsequently differentiatingby the 40% stage of embryogenesis, axonalat characterize the mature brain become
ingle cell analysis of grasshopper brain devel-ted here sets the stage for manipulative cell
eriments and provides the basis for compar-r genetic studies of embryonic brain devel-sophila.
Boyan1, Stavros Therianos1, J. Leslie D. Williams2 and He
tory of Neurobiology, Department of Zoology, University of Basel, CH-40anck-Institut für Verhaltensphysiologie, Arbeitsgruppe Kaissling, D-8231
DUCTION
st decade, the embryonic central nervous system (CNS)ts has become an important model system for investi-of neuronal development. Many of the cellular mecha-f neurogenesis and axonal pathfinding have been in detail (i.e. Goodman et al., 1984; Bastiani et al.,
In this repto the initiaof the grasslar dye injeidentify as ijections in tthese neuro
l in the grasshopper Schistocerca gregaria. A smalldividually identifiable pioneer neurons establishesry axon scaffold during early embryogenesis. At theng of scaffold formation, pioneering axons navigatend between glial borders that surround clusters ofating neuroblasts. In each brain hemisphere, anoutgrowth cascade involving a series of pioneer establishes a pathway from the optic ganglia to theidline. At the midline the primary preoral com-l interconnection in the embryonic brain ised by a pair of midline-derived pioneer neurons. Apreoral commissural connection is pioneered by two pars intercerebralis pioneer neurons. Descendingre pioneered by the progeny of identified neurob-the pars intercerebralis, deutocerebrum and trito-
pioneered bneering brafasciclin I dOnce estabused for neurons anprojectionsevident. Thopment prebiological eative molecopment in
Key words: agrasshopper,
76
Whole-mWhole-mothe stomosections odissected
ImmunoImmunoc(1993). Prfollows: a1982; SnoZinn et al1991); an(see Basti
MicroscFor lightwere vierescence tion, micrecordedcessing uImaging)as descriwere as a
Cell lineBrain ne(1993). Tpioneer pioneer neurons iorigin. Seinjected dye coupit was poblast. Thlasts of oporation iZachariasall of the on one orpreparatio
RESULT
FormatineuroblIn the emat approstage, cooccurredally symas midlinnon-neurcells in
G
ount histology and histological sectionsunt preparations (dorsal side up) were dissected to remove
dium and reveal the interior of brain structures. For serialf osmium-ethyl gallate stained brains, embryos were staged,and treated according to Zacharias et al. (1993).
cytochemistry ytochemistry was carried out according to Meier et al.imary antibodies were diluted in preincubation solution asnti-horseradish peroxidase (HRP), 1:1 (see Jan and Jan,w et al., 1987); anti-fasciclin I, 1:1 (see Bastiani et al., 1987;., 1988); anti-REGA-1, 1:200 (see Carpenter and Bastiani,ti-engrailed, 1:1 (see Patel et al., 1989); anti-annulin, 1:1ani et al., 1992).
ular dye filling cells were impaled with glass microelectrodes, filled elec-cally with lucifer yellow dye and processed for lighty according to Raper et al. (1983). Alternatively, injected
. Boyan and others
Fig. 1. Formation of glial-bound proliferative clusters in thepreaxogenesis brain. REGA-1 immunoreactivity. (A) Schematic ofproliferative clusters in one hemisphere of the embryonic brain.Neuroblast 12 of lateral protocerebrum where the first REGA-1immunoreactive glial processes appear is highlighted. (B) REGA-1immunoreactive glial processes (white arrow) surround neuroblast 12of lateral protocerebrum. No other neuroblasts or neurons in thebrain hemispheres have REGA-1 immunoreactive glial processes atthis stage (26%). (C) At 30% stage REGA-1 immunoreactive glialprocesses (black arrows) completely surround individual PIneuroblast. (D) Glial processes delimit future axonal pathway insidedeveloping PI proliferative cluster. Dorsal layer of PI neuroblasts hasbeen removed revealing numerous neuronal cell bodies among whichthe labelled glial process (black arrows) is found. 29% stage.(E) REGA-1-immunoreactive glial processes (white arrows) delimitborders of PI (upper right) and PI(ALDL) (lower left) proliferativeclusters. Overlying dorsal sheath removed. 30% stage.(F) Preparation in E at higher magnification. REGA-1immunoreactive glial processes delimit proliferative cluster border
portant to characterize thein.ore 25%), the bilaterally sym-rogenic region of the futuren a sheet-like array, which isnce. Between 26% and 28%,appears and the neuroblastscome arranged into distinctdaries of these clusters arend the differentiation of theseout embryonic development.regates of proliferating neu-and neurons in each of the
(white arrows). Glia also extend processes into proliferative clustersroblasts and progeny (black apply to all figures: GMC,
t; PI, pars intercerebralis proper;e of pars intercerebralis; PC,rotocerebrum; DC,a, lamina; Me, medulla; Re,uent figures, a rectangular box
e brain region being analysed.otherwise stated. Anterior, A, is 6 µm; D, 14 µm; E, 20 µm.
photoconverted and processed for electron microscopy to Sandell and Masland (1988).
opy microscopy, histological material or whole-mount embryoswed in a Zeiss Axioskop microscope equipped for epifluo-and DIC (differential interference contrast). For documenta-roscopic images were either photographed or, alternatively, by a CCD camera, digitized and subjected to image pro-sing the GenIIsys (MTI) and Image 1 systems (Universal. For immunoelectron microscopy, embryos were processedbed by Meier et al. (1993). The primary antibody dilutionsbove.
age analysisuroblast identification was according to Zacharias et al.hree methods were used to determine the cell lineage of
neurons. First, an initial identification of the lineage ofneurons was possible by determining the position of then the glia-ensheathed columns relative to their neuroblast ofcond, to confirm this initial analysis, lucifer yellow dye was
into the neuroblast of origin of the pioneer neuron. Due toling among the neuronal precursors and their early progeny,ssible to assign a given pioneer neuron to an identified neuro-ird, to analyse the generation of neurons from their neurob-rigin in vivo and in situ, 5-bromodeoxyuridine (BrdU) incor-
outgrowth is initiated, it is imfeatures of the preaxogenesis bra
At early embryonic stages (befmetrical neuroblasts in the neubrain hemispheres are arranged ilargely homogeneous in appearathis structural homogeneity distogether with their progeny beclustered aggregates. The boundelineated by glial support cells aglial borders continues throughWe refer to the glial-bound aggroblasts, ganglion mother cells
and surround columnar arrays of neuarrows). The following abbreviationsganglion mother cell; NB, neuroblasPI(ALDL), anterior lateral dorsal lobprotocerebrum proper; LPC, lateral pdeutocerebrum; TC, tritocerebrum; Lretina; M, midline. In this and subseqin the summary diagram indicates thEmbryos are viewed dorsally unless to the top. Scale bar: B, F, 10 µm; C,
nto proliferating neuroblasts was performed according to et al. (1993). For each pioneer neuron investigated, this andother experimental analyses reported here were carried out both contralateral homologs of a minimum of 5 differentns.
S
on of glial borders around proliferatingasts precedes axogenesis bryonic brain of the grasshopper, axogenesis begins
ximately the 29% stage of embryogenesis. At thisnsiderable development of the brain has already
, giving rise to a highly structured set of large bilater-metrical neuroblasts (Zacharias et al., 1993), as welle progenitors (see below), developing neurons and
al cells. Since initial axogenesis relies heavily on thethe brain that are in place before the first axon
embryonic brain hemispheres as proliferative clusters (Fig.1A). The proliferative clusters prove to be important for axo-genesis within the brain hemispheres because the earliest out-growing axons navigate in the space between these clusters aswell as along their glial borders.
Aspects of glial development can be characterized by theexpression pattern of REGA-1, an antigen present in non-midline glia in the embryo (Carpenter and Bastiani, 1991;Seaver et al., 1993). Earliest REGA-1 expression in the brainhemispheres is seen at the 26% stage and is highly restricted;immunoreactive glial processes begin to surround a singlebrain neuroblast (Fig. 1B). This type of focal initial expressionof an antigen by developing glial support cells is also observedfor annulin (see Bastiani et al. 1992; and below). Subsequentlythe spatial expression of the REGA-1 glial marker rapidlyexpands to surround the surface of other brain neuroblasts (Fig.1C) as well as the columnar arrays of their progeny (Fig. 1F).In addition, the REGA-1 antigen also appears on elongatedglial processes, which extend inside a given proliferative
77Brain development in the grasshopper
clusterwill grtowardAlthouare obthese pof the 30% sprogen
A
B
C
D
E
s
nt
F
and demarcate future pathways along which the neuritesow as they project from their neurons of origin outwards the surface of the proliferative cluster (Fig. 1D).gh REGA-1 immunoreactive glial processes of this typeerved repeatedly, it has not yet been possible to relaterocesses to identified glial cells or determine the identityeurons whose axons project along the processes. By the
age, all aggregates of proliferating neuroblasts and theiry have become bordered by REGA-1 immunoreactive
glial processes (Fig. 1E,F). These proliferative clusterssubdivide the embryonic brain into the different neurogenicregions that will give rise to the pars intercerebralis proper, theanterior lateral dorsal lobe of the pars intercerebralis, the pro-tocerebrum proper, the lateral protocerebrum, the deutocere-brum and the tritocerebrum.
In summary, before axogenesis begins, the bilaterally sym-metrical brain neuroblasts are subdivided into the proliferativeclusters, which give rise to the major brain regions. This sub-
78
division by the gquently baxon out
Brain pscaffoldInitial axsmall subsites in thperipherythe abutoutgrowtlishes a pliferativethe first the neuroof the praccordansystem aal., 1985
We stuby direcDIC optiby immlabels ththe develet al., 19neurons tion is shcan be idof their patterns. possible their linebelong tproduces
How pathwaysthe singlpioneerinbrain to analyse tconnectioFinally wcerebral initial pio
A waveoptic loAt the otiation ocin lateralthe axonproject tgrowth cbetween transientThe firstare foundlamina an
G
is accompanied by the formation of glial borders andlial demarcation of future axon pathways. Subse-oth of these glial structures are used as substrata for
growth.
ioneer neurons construct an embryonic axon
ogenesis in the embryonic brain is carried out by aset of neurons, which are located at discretely spacede developing brain. These neurons are found near the of the proliferative clusters and, in many cases, nearment of two different clusters. Through axonalh and fasciculation, this small subset of neurons estab-rimary axon scaffold that interconnects all of the pro- clusters in the embryonic brain. Given that they areto initiate axogenesis in the insect brain, we refer tons that establish the initial interconnections among all
are still unclear, at least the distal part of the medulla appearsto form from cells differentiating from the most proximalregions of the lamina.) There, at the 29% stage, one or a pairof neurons begin to send their axons towards the protocere-brum. These neurons pioneer a pathway, whose locationsuggests that it may be used later by visual neurons of themedulla (Fig. 2C). The axonal growth cones of these pioneer-ing neurons first grow anteriorly along the border betweenlamina and medulla. Then near the anterior edge of the borderthey turn medially. At the turn, their growth cones passfilopodia from a pioneer neuron located at the anterior medulla-lamina border; subsequently their growth cones encounterfilopodia from a pioneer neuron located in the pars intercere-bralis (Fig. 2B). Following reciprocal filopodial contact at theinterface between the developing optic lobes and the lateraledge of the pars intercerebralis proliferative cluster, the growthcones of all three sets of pioneer neurons fasciculate with one
. Boyan and others
oliferative clusters in the brain as pioneer neurons incn;
tcueo8b
tao.d
e
mh
c
bn
soo
c
another. The axons from the optic lobes then project along the
e with the terminology used in the peripheral nervousd in the segmental ganglia (Bate, 1976; Bastiani et
Caudy and Bentley, 1986). died axonal pathfinding of the brain pioneer neurons observation of developing axonal processes usings, by intracellular injection of lucifer yellow dye andnocytochemistry with an anti-HRP antibody that axons and cell bodies of all outgrowing neurons inping insect nervous system (Jan and Jan, 1982; Snow7). A semi-schematic diagram of the brain pioneerased on a representative anti-HRP stained prepara-own in Fig. 2A. Many of the brain pioneer neuronsentified on the basis of their position, the trajectoryaxonal projections and their molecular expressionFor several of the brain pioneers, it has also beeno identify their neuroblast of origin and determinege; in all of these cases, the brain pioneer neurons the initial set of progeny that a given neuroblast
o the pioneer neurons establish the initial axonal in the embryonic brain? To address this question at cell level, we first investigate the formation of theg axonal pathways that link the peripheral part of theore medial structures in each hemisphere. We then
pars intercerebralis axon into a specific axonal junction zonein the brain hemisphere.
During the formation of the pioneering connections betweenoptic lobes and pars intercerebralis, an axonal junction zone isestablished in the brain at the interface between the pars inter-cerebralis, anterior lateral dorsal lobe of the pars intercerebralisand protocerebrum (Fig. 3A). There, the axon of a pioneerneuron from the lateral protocerebrum grows out of its prolif-erative cluster of origin and extends into the intercluster space.At the interface between the three proliferative clusters, itsgrowth cone contacts and fasciculates with the growth cone ofthe pars intercerebralis neuron which pioneers the pathwayextending peripherally towards the optic lobes (Fig. 3B,C).(Note that this is the pathway used by the axons from the opticlobes to grow into the brain hemisphere, see above.) Thispioneer neuron is one of the first two neuronal progeny of parsintercerebralis neuroblast 14; its putative sibling neuronpioneers a pathway projecting centrally between proliferativeclusters towards the brain midline (Fig. 3D). In this case, thelineage analysis of neurons is based on the position of theneurons in the glial-ensheathed columns relative to their neuro-blast of origin; in other cases, intracellular dye-injection exper-iments and BrdU incorporation were also used (see methods).After fasciculation, the growth cone of the protocerebral
e cellular processes through which the commissuralns between the two hemispheres are established.e characterize the formation of descending and trito-onnections. In each case, we limit our analysis toneering neurons and pathfinding processes.
of axonal outgrowth interconnects brain andesset of axogenesis in the brain, a wave of differen-
curs in which the axons from pioneer neurons locatedand more peripheral parts of the brain extend towards of more centrally located pioneers, which thenwards the midline. Throughout this process, thenes of all of the pioneer neurons extend along andproliferative clusters and their filopodia makeontact with border cells of the proliferative clusters.
pioneer neurons that initiate axogenesis in the brain in the developing optic lobes at the interface betweend medulla (Fig. 2A,B). (While the exact mechanisms
pioneer neuron first grows centrally along the axon of theperipherally projecting neuron of the pars intercerebralis. Sub-sequently (not shown) the axon of the protocerebral pioneerswitches its growth cone to the axon of the other sibling parsintercerebralis neuron, fasciculates with this cell’s axon andthen continues to grow along the axon of this centrally pro-jecting neuron until it reaches the medial edge of the pars inter-cerebralis. In this way, a pathway is established from the parsintercerebralis-protocerebral junction zone to the medial edgeof the brain hemisphere.
Thus, in each hemisphere, the growth cones of brain pioneerneurons extend along the borders of the proliferative clustersof the brain and carry out a series of axonal fasciculationprocesses at the interfaces between proliferative clusters.Through these fasciculation processes, initial axon pathwaysare established from the optic lobes into the pars intercerebralisas well as from the lateral protocerebrum via the pars inter-cerebralis towards the brain midline. With the linkage of theseaxon pathways at the pars intercerebralis-protocerebral
Fig. 2. primaryimmunis growgrows pmedullneuron with bocells of
A
B
C
rom the optic lobes into the brain. (A) Semi-schematic diagram of neurons that establish aP immunocytochemistry of 29-34% stages. (B) Digitized montage of anti-fasciclin Iack arrow) of a visual pioneer neuron (white asterisk) from the lamina-medulla interfacento the brain. En route the growth cone (open arrowhead) of the visual pioneer neuron the brain from a neuron (partly visible, white star) located at the edge of the anteriorlopodia (black arrowheads) directed peripherally into the optic region from another
Pioneer neurons establish an axonal pathway f axon scaffolding; summary based on anti-HR
ocytochemistry at the 29% stage. The axon (bling along the border of a proliferative cluster iast filopodia (white short arrow) growing into
a-lamina border and subsequently encounters fi
n zone, a set of interconnecting neuronal pathways areshed, which extend from the developing optic lobess the midline of the brain. As soon as these initialys in the brain are pioneered, they are used by numerouseurons which project their axons along them.ng the construction of the axonal pathways from theobes to the midline, the growth cones and axons of allse reciprocally fasciculating brain pioneer neuronss the homophilic cell adhesion molecule fasciclin Ini et al., 1987; Snow et al., 1988; Zinn et al., 1988).in I expression is dynamic; expression of fasciclin I bywth cones and axons of the pioneer neurons is strong
the formation of the primary axon scaffold early ingenesis; expression of fasciclin I is no longer evident
in these neurons at 40% of embryonic development. Interest-ingly, the filopodia of the pioneering growth cones oftencontact cells located at the borders of the proliferative clustersthat they grow along and some of these contacted cells alsoexpress fasciclin I (see Figs 2B,3B).
Formation of the embryonic brain commissureBy the time the wave of outgrowing axons in each brain hemi-sphere reaches the medial border of the hemisphere (31%stage), an initial brain commissural connection has alreadybeen established. How is this embryonic brain commissureformed? A central role in pioneering the brain commissure isplayed by neurons that are generated by a small set of midlineprogenitor cells. These midline progenitors do not stain with
at the PC/PI(ALDL) interface. All growth cones extend between proliferative clusters; their filopodia (long white arrows) make contactrder cells within the proliferative cluster. (C) The axon of the visual pioneer neuron establishes a pathway that may later be utilized by medulla (black arrow). 45% stage. 16 µm horizontal section. Scale bar: B, 15 µm; C, 60 µm.
80
the anti-Hengrailedwhere laprogeny aneuroblas
G hers
Fig. 3. Comontage, a(white astebetween thasterisk) insummary dneuroblastLPC. The Neuroblasprotocereb
A
B
C
. Boyan and ot
D
RP antibody. Moreover, they do not express the protein. This contrasts with the segmental ganglia,rge engrailed-positive midline neuroblasts andre seen (data not shown). However, as is the case forts in the segmental ganglia, the midline progenitors
in the brain do become surrounded by glial sheath cells whichexpress annulin in correlation with the mitotic activity of thecells they surround (Bastiani et al., 1992; Singer et al., 1992).Thus, before the midline progenitors differentiate and becomeactive, no annulin staining is seen in this region of the brain
ntact and fasciculation of pioneer neurons at PI/PI(ALDL)/PC interface. (A) Semi-schematic summary diagram. (B) Digitizednti-fasciclin I immunocytochemistry of 30% stage. Fasciculation of two outgrowing axons, one from the LPC proliferative clusterrisk), the other from the PI proliferative cluster (partly visible, white star). Axonal growth cones (black arrows) meet in the spacee two clusters. (C) Digitized montage showing fasciculating growth cones from PI neuron (white star) and LPC neuron (white B at higher magnification. Filopodia (black arrows) extend from growth cones to proliferative cluster borders. (D) Schematicata describing lineage relationships of the neuron indicated by white star in B. One of the first born neurons (white star) of
14 projects its axon laterally along a proliferative cluster border to fasciculate with the growth cone of a neuron (white asterisk) fromother neuron pioneers a pathway projecting centrally towards brain midline along posterior proliferative cluster border of PI.t 14 and its first born progeny are in solid blue; other progeny outlined in blue, GMC outlined in black. Unlabelled neuroblasts areral. Scale bar: B, 20 µm; C, 10 µm.
The most pexpresconnecneuronout a dpioneergenesisneurongrowthdial strextendsin size growthjust posthen coeral hoeral heacross
Oncepathwamediopeach b(SCN),pathwa
ne progenitors in the brain.in immunoreactivity; (D)ration. (A) At 26% stagession is seen in developing
ve clusters (asterisks) butl brain midline. Annulin also seen in optic lobes,m, tritocerebrum and ventral
) At 30% stage annulin seen in preoral midlinells (arrow) and in the PIclusters (asterisks).agnification showingssion surrounding preoral
enitor cells between the PIclusters (asterisks). Notenulin immunoreactiveheads) delimiting thee progenitor cells from
A
C
Brain development in
Fig. 4. Midli(A-C) AnnulBrdU incorpoannulin exprePI proliferatinot in preoraexpression isdeutocerebrunerve cord. (Bexpression isprogenitor ceproliferative (C) Higher mannulin expremidline progproliferative prominent anborder (arrowcluster of larg
B
D
contacts and fasciculates. 5F) just like the PCP
ch SCN growth cone thenateral homolog and growsre.
missural pathways in the embryonic development),axons onto them, somet directions. The lateral
l body at the medial edge (Fig. 5D). The LP neuronection to the PCP neuronand establishes a lateraline commissure peripher-D,E). Both the secondary
pithelial cells. (D) BrdUhree midline progenitor cellste progeny (star) of thee progenitor. BrdU is also seen in cells of PIclusters (upper left andbar: A, 166 µm; B, 120 µm; 25µm.
4A). When the midline progenitors generate theiry, their surrounding glial sheath cells become annulin-oreactive and immunoreactive processes surround therating precursors as well as their progeny (Fig. 4B,C).ve identified a set of three midline progenitors based onosition at the midline, on the annulin immunoreactivity glial sheath cells and on the incorporation of BrdU intocursors and their progeny (Fig. 4D). Additional, as yettified midline progenitors may exist. first pair of neurons that are generated by the lateral-rogenitors on each side of the midline are fasciclin Ising neurons and these pioneer the first commissuraltion in the embryonic brain (Fig. 5A,B). We call theses the primary commissural pioneers (PCP). We carriedetailed analysis of the axonal outgrowth process of these
growth cone of each SCN neuron with its contralateral homolog (Figneurons did previously (Fig. 5C). Eaextends along the axon of its contralinto the contralateral brain hemisphe
Soon after these two initial combrain are established (within 1-2% ofnumerous other neurons project extending growth cones in differenpioneer (LP), for example, has a celof the developing pars intercerebralisextends its axon in the opposite diralong the initial midline pathway pathway that extends from the midlally into the brain hemisphere (Fig. 5
neurons by intracellular dye filling. At the onset of axo-, a single small process extends from each pioneer towards the midline (Fig. 5B). Shortly thereafter a large cone with numerous filopodia separated by lamellipo-uctures is generated (not shown). This growth cone towards the midline and, in doing so, becomes smaller
and gives rise to a trailing axonal process (Fig. 5B). The cones of these pioneer neurons extend over the midlineterior to the soma of the medial midline precursor. Theyntact and fasciculate with the axons of their contralat-mologs and grow along these axons into the contralat-misphere, thus establishing an initial axonal bridgethe brain midline (Fig. 5A,C). the PCP neurons have established the first axonaly across the midline, another pair of neurons at theosterior edge of the developing pars intercerebralis inrain hemisphere, the secondary commissural neurons extend their growth cones medially onto this axony and fasciculate with it (Fig. 5D,F). At the midline, the
commissural neurons and the lateral pioneer express fasciclinI during axonal outgrowth. The results of an ultrastructuralanalysis of such a nascent commissural fascicle are shown inFig. 6. In this experiment, the lateral pioneer and the paired(contralateral) primary commissural pioneers have been filledwith dye in a 33-34% stage embryo (Fig. 6A). Closer to thebrain midline, the axons of the primary commissure pioneersare tightly apposed to one another and still at some distancefrom the axon of the lateral pioneer (Fig. 6B). Closely associ-ated with these pioneer axons are numerous other unstainedaxonal profiles. These unstained axons have projected onto theinitial pioneering pathway and together now form the devel-oping commissural fascicle. Interestingly, a large putativemidline progenitor cell is in close association with the fascicle,suggesting that this precursor cell might play a role in the com-missural pathfinding processes. Further away from the midline,the primary commissure pioneers have fasciculated with thelateral pioneer as they run laterally along the posterior marginof the pars intercerebralis proliferative cluster (Fig. 6C,D).
82
Once agassociateaxons rebefore it can be idsurroundThis gliain the fa
G
A B
C
D
F
. Boyan and others
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G
ain, numerous unstained axonal profiles are closelyd with the commissural pioneer axons. Together thesepresent the developing commissural fascicle justenters the brain hemisphere. A large glial cell, whichentified by the typical involuted membrane structureing its nucleus, is in close contact with the fascicle. cell extends processes that envelop all of the axonsscicle. Numerous glial cells of this type are found
wherever axonal outgrowth occurs in the brain. They are seenat the midline and in the spaces between the proliferativeaggregates of the brain hemispheres; they can be seenextending processes along pathways that are subsequently usedby pioneer neurons for axonal outgrowth. These glia are likelyto play important roles in early axogenesis, however, a moredetailed understanding of their function awaits further investi-gation.
83Brain development in the grasshopper
Formation of embryonic descending pathways andtritocerebral commissureWith the establishment of the primary preoral commissure, theaxon pathways in the two brain hemispheres become inter-connected. Concurrently, descending pathways are pioneeredthat connect the brain hemispheres with the ganglia of theventral nerve cord (Figs 5G,7A). This is a multistage process.In the protocerebrum, a descending ipsilateral pathway ispioneered at 31-32% of embryonic development by a pair ofcells derived from neuroblast 3 of the pars intercerebralis (Fig.5G). Within 1% of embryonic development, the axons ofneurons derived from other pars intercerebralis neuroblastsproject onto this pioneer pathway and through the protocere-brum proper towards the deutocerebrum. Other pioneerneurons derived from neuroblast 1 of the pars intercerebralisproject axons laterally towards the optic lobes (Fig. 5G). In thedeutocerebrum, neurons derived from neuroblast 5 pioneer adescending ipsilateral pathway along the medial edges of thedeutocerebrum tocircumesophagea7B,C). The axonfasciclin I.
In the tritocepioneered by thea descending ipsicommissure (Figing both pathwaexpress fasciclinside of the brain.identical route throne sibling direcsophageal connetowards the mid
Fig. 5. Formation of the primary brain commissure and of otherprotocerebral pathways. (A) A pair of neurons that originate from alateral progenitor cell on each side of the midline pioneer the primarypreoral commissure. 30-31% stage. Top: camera lucida drawing ofthese primary commissure pioneer (PCP) neurons (arrowheads, cellbodies). Bottom: anti-HRP immunocytochemistry showing PCPneurons (white arrows) and axons extending across midline (openarrows). (B) Details of the primary commissural pioneers followingintracellular lucifer yellow labelling. A PCP neuron initiating processoutgrowth at 30% stage (top). Digitized montage (bottom) showsaxon extension across midline towards contralateral hemisphere at31% stage of a different preparation. (C) Intracellular lucifer yellowlabelling of a PCP neuron from each brain hemisphere at 32-33%stage. Arrowhead indicates brain midline. Digitized montage.(D) Details of the secondary commissural neurons (SCN), digitizedmontage of fasciclin I immunoreactivity. The cell body of one SCNneuron in left PI is shown (asterisk). At midline (white short arrow)the fascicle bends around midline cells. The lateral pioneer (LP)(star) located at the medial border of the PI proliferative clusterdirects a process laterally along the axon fascicle. Growth cones(white arrow) extending into left PI express fasciclin I as theycontact surrounding neurons and proliferative cluster borders. 31%stage. (E) Intracellular lucifer yellow labelling of lateral pioneerneuron (star) described in D; different preparation. Arrow indicatesgrowth cone. (F) Intracellular labelling of SCN neuron (asterisk)from each brain hemisphere. 31-32% stage. Arrowhead indicatespreoral brain midline. Digitized montage. (G) Summary diagramshowing lineage relationships of neurons involved in early pathwayformation in the brain. 32% stage. Progeny of PI neuroblast 3contribute to commissural and descending pathways; progeny ofneuroblast 1 contribute to laterally projecting pathway. Progeny fromother unidentified PI neuroblasts contribute to commissural anddescending pathways. Arrowheads indicate direction of initial axonoutgrowth; lineage-related progeny are coloured the same. Scale bar:A,B, 8 µm; C, 14 µm; D, 10 µm; E, 11 µm; F, 17 µm.
than (B). Axon of the PCP neurons and the LP neuron are now fasciculated (arrowhead). The of the developing commissure are surrounded by processes of a large glial cell (asterisk). (D) in C. Scale bar: B,C, 3 µm; D, 1 µm.
A
B
C D
the tritocerebrum and from there through thel connectives into the ventral nerve cord (Fig.s of all of these descending pioneers express
rebrum, two very different pathways are early progeny of tritocerebral neuroblast 12:lateral pathway and the postoral tritocerebral. 7B,C). The neurons responsible for pioneer-ys are putative sibling cells. Their axons
I and are arranged symmetrically on either Initially the axons of both siblings follow anough the tritocerebrum but they soon part andts its axon posteriorly towards the circume-ctive. The other directs its growth coneline where it meets the growth cone of its
Fig. 6. Ultrastructuralanalysis of commissuraldevelopment.(A) Schematic summary.Two primary commissuralpioneer neurons (PCPs)and the contralateral
lateral pioneer (LP) werestained in the samepreparation (34% stage) byintracellular injection andphotoconversion of luciferyellow. Dashed linesindicate plane of sectionsin B, C. (B) Section showsthe PCP axons (openarrowhead) separated fromthe LP axon (filledarrowhead). All profilesare closely associated withthe developingcommissure (arrow) andwith a large midlineprecursor (asterisk).(C) Section more lateral
labelled axon profiles and other unlabelled axonsEnlargement of labelled axon profiles (star) seen
84
homologthen fasctralateraltocerebra
Aspectlater emOnce thelished, thof the insure, ove37% stagshaped c
G
Fig. 7. Focommissuthe tritoctwo pairsregion anschematicneuroblasdeutocereone of thebrain andT, tritocer
A
B
C
. Boyan and others
tritocerebral commissure. (A) Summary diagram. (B) Formation of the tritocerebralimmunoreactivity. 33-34% stage. Two bilaterally symmetrical tritocerebral neurons (stars),ct axons (white arrows) towards postoral midline (open arrow). Axons (arrowheads) frompathways through the tritocerebrum. Inset (white rectangle) is a magnification of midlines have made contact and fasciculated at the midline (black arrow). (C) Summaryns described in B. TCCP neuron and sibling (white asterisks) derive from tritocerebraling cell forms a descending axonal projection (black asterisk). Descending pathway from
D
rmation of descending pathways and of there. Digitized montage based on fasciclin I
erebral commissure pioneers (TCCP), proje of deutocerebral neurons form descending d shows that axons from both TCCP neuron showing lineage relationships of the neurot 12. TCCP projects across midline; its sibl
from the other brain hemisphere; these two axonsiculate and grow along their homologs into the con- tritocerebrum (Fig. 7B). This initial fascicle of the tri-l commissure is pioneered at the 33-34% stage.
s of the early axon scaffold remain evident inbryonic stages axonal scaffolding in the embryonic brain is estab-e axonal projections rapidly grow in size. Within 3%itial pioneering of the primary preoral brain commis-r 100 axons have crossed the midline. Indeed, by thee, some fascicles of the adult brain, such as the U-
ommissure PC20 (Boyan et al., 1993), can already be
recognized. By the 40% stage, a massive amount of axonaloutgrowth and fasciculation has occurred in the brain. Tracts,commissures and descending pathways have increased enor-mously in size and complexity. Moreover, nerves that extendfrom the peripheral nervous system into the brain have formed.Nevertheless, some aspects of the early orthogonal scaffold ofbrain axons are still visible at this stage (Fig. 7D). Lateral axontracts extend from the pars intercerebralis and protocerebrumregions into the optic lobes; descending axon tracts extendfrom the pars intercerebralis towards the deutocerebrum andtritocerebrum and from there through the connectives into theventral nerve cord; preoral and postoral brain commissuresformed around the gut link the brain hemispheres. Interest-
brum into ventral nerve cord (more lateral axons in B) is pioneered by progeny (white triangle) of DC neuroblast 5 (33% stage). Only sibling progeny contributing axons is shown. Other lineage-related cells are colored alike. (D) Horizontal section (16µm) through part of subesophageal ganglion showing orthogonal axonal scaffold at 45% stage. Osmium-ethyl gallate staining. P, protocerebrum;ebrum; S1, first subesophageal segment. Scale bar: B, 17 µm; inset, 11 µm; D, 108 µm.
the orthogonal scaffold described above is formed in thece of all higher brain structures such as the mushroom and the central body. These structures only take shape0% of embryonic development.
USSION
igations on insect axogenesis have become increasinglytant for analyzing the cellular and molecular mechanismsed in guided outgrowth and pathway formation in the
us system (for recent reviews see Reichert, 1993;an and Doe, 1994). Virtually all of this work has,
er, been carried out on the simple segmental ganglia orripheral nervous system of insects. In consequence, verys known about the development of more complex struc-uch as the insect brain. In this study, we investigate axo-
is in the early embryonic brain of the grasshopper Schis-
The primary scaffold of axons in the embryonic brain isestablished in the absence of higher brain structures such as thecentral body, the protocerebral bridge or the mushroom bodies.These structures are established as part of a second stage ofdevelopment that leads to the brain acquiring the anatomicalorganization characteristic of the adult. In hemimetabolousinsects like the grasshopper, this second stage of developmentoccurs in the embryo once the first stage has been achieved(Boyan et al., unpublished data). Thus, the early embryonicbrain is not constructed as a miniature model of the adult brain.It initially acquires features that are reminiscent of the devel-oping ventral ganglia. Only later does the embryonic brainassume the features that are specific for the mature suprae-sophageal ganglion. In consequence, for a complete under-standing of axogenesis in the insect brain, the formation of thenerve tracts and connections in these later developing, higherbrain structures must also be investigated.
The single cell analysis of early brain axogenesis described
a gregaria. Our results show that, despite the enormousexity of the adult brain, a set of simple developmentalses involving individually identifiable cells and axons
lies early axogenesis in the brain. genesis in the brain commences in a restricted group ofnd leads to a simple orthogonal scaffolding of axonalays. The events that occur in the formation of this scaf-g resemble those that are seen in the segmental nervous in several ways. First, identifiable pioneering neurons
ish a simple axonal scaffold (Bate, 1976; Bentley andshian, 1982; Ho and Goodman, 1982). In the brain,rs are involved in the construction both of the commis-
that link the two hemispheres and of the tracts and con-es that interconnect the different parts of the brain inemisphere. Second, in the brain and in the segmentala, all of the initially outgrowing axons derive from thel nervous system itself (Goodman et al., 1984). Axonsow into the brain from structures on the head such as theae, head hairs or the ocelli grow into the axonal scaf-
g later and appear to depend on the existence of thel nervous scaffolding for proper navigation. Third, theh cones of neurons pioneering the preoral and postoralcommissures meet and then fasciculate with their con-ral homologs in the midline as described for commissuretion in the segmental ganglia (Myers and Bastiani, 1993).
here lays the foundation for further cell biological studies inwhich the many experimental advantages of the grasshopperembryo, such as large size of embryonic neurons, easy acces-sibility for intracellular labelling and ablation, existence of arobust embryo culture system and established antibody-blockprotocols (Raper et al., 1984; Bastiani et al., 1985; Kolodkinet al., 1993; Xie et al., 1994) can be exploited. Moreover, thisanalysis sets the stage for following molecular genetic inves-tigations of embryonic brain development inDrosophila.(Therianos et al., unpublished data). The merger ofwork on the grasshopper with its highly accessible identifiedembryonic neurons with work on Drosophila with its powerfulgenetics and molecular biology has led to significant advancesin our understanding of neuronal development in general(Thomas et al., 1984; Goodman et al., 1984; Zinn et al., 1988;Grenningloh et al., 1990). Indeed, the expectation that mecha-nisms and molecules, first discovered in insects, have equiva-lents in the developing vertebrate nervous system has alreadybeen fulfilled in several cases (Thomas and Capecchi, 1990;McGinnis and Krumlauf, 1992; Goodman and Shatz, 1993;Kolodkin et al., 1993).
Anti-fasciclin I, anti-annulin and anti-engrailed antibodies weregenerous gifts of C. S. Goodman and N. Patel; anti-REGA-1 anti-bodies were generous gifts of M. J. Bastiani and E. C. Seaver. We
, glia play an important role in axonal pathfindingani and Goodman, 1986; Meyer et al., 1987; Jacobs and
an, 1989; Klämbt and Goodman, 1991; Carpenter andni, 1991). The pioneering axons of the brain follow glialrs during their outgrowth and glia are also important inishing connections across the midline. Interestingly, glial neuronal aggregates are likely to play important roles instembryonic development of the insect brain (Tolbertland, 1989) as well as in embryonic brain developmenttebrates (see Steindler, 1993). Fifth, some cell bodies inveloping brain may have a function equivalent to that ofpost cells’ in the peripheral nervous system (Bentley andshian, 1982; Ho and Goodman, 1982). Such cell bodiesserved near the borders of proliferative clusters. Thesexpress the cell adhesion molecule fasciclin I and areted by outgrowing axons that also express fasciclin I. cells, therefore, might play an important role in directingutgrowth.
thank T. Meier, F. Xie, D. Sanchez, L. Ganfornina and E. Ball fortechnical advice. Supported by the Swiss NSF, the FAG Basel andthe Max-Planck Gesellschaft, Seewiesen.
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