Neuroblast formation and patterning during early brain development inDrosophila

Neuroblast formation andpatterning during early braindevelopment in DrosophilaRolf Urbach and Gerhard M. Technau*

SummaryThe Drosophila embryo provides a useful model systemto study the mechanisms that lead to pattern and celldiversity in the central nervous system (CNS). TheDrosophila CNS, which encompasses the brain and theventral nerve cord, develops from a bilaterally sym-metrical neuroectoderm, which gives rise to neural stemcells, called neuroblasts. The structure of the embryonicventral nerve cord is relatively simple, consisting of asequence of repeated segmental units (neuromeres), andthe mechanisms controlling the formation and specifica-tion of the neuroblasts that form these neuromeres arequite well understood. Owing to the much higher com-plexity and hidden segmental organization of the brain,our understandingof its development is still rudimentary.Recent investigations on the expression and functionof proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes haveprovided new insight into the principles of neuroblastformation and patterning during embryonic developmentof the fly brain. Comparisons with the same processes inthe trunk help us to understand what makes the brain

different from the ventral nerve cord. Several parallels inearly brain patterning between the fly and the vertebratesystems have become evident. BioEssays 26:739–751,2004. � 2004 Wiley Periodicals, Inc.

Introduction

The central nervous system (CNS) has the highest diversity of

cell types and structural complexity of all organs. Thus,

uncovering the mechanisms leading to pattern and cell

diversity in the CNS is one of the major challenges in

developmental biology. The developing CNS of Drosophila is

an ideal model system to study these processes. The insect

CNS is composed of the ventral nerve cord (VNC) and the

brain.Until recently, investigations onCNSdevelopment in the

Drosophila embryo have mainly focused on the VNC due its

simpler organization compared to the brain. The VNC arises

from a bilaterally symmetrical, two-dimensional sheet of cells,

the ventral neurogenic region (vNR) of the ectoderm. Through

the expression of proneural genes of the Achaete–Scute–

Complex at precise locations, groups of neuroectodermal

cells, called proneural clusters, acquire the potential to

become neural precursor cells, termed neuroblasts (NBs,

see, for example Refs. 1, 2). Cell–cell interactions, mediated

by the neurogenic genes, ensure that, in each proneural

cluster, only a single cell with the highest level of proneural

gene expression adopts a NB fate, while the others remain in

the periphery to develop as epidermoblasts (reviewed by

Ref. 3). The singling out of NBs follows a stereotypical spatial

and temporal pattern.(4,5) Upon delamination, NBs typically

undergo repeated asymmetric divisions, budding off smaller

ganglion mother cells, which divide once to produce neurons

and/or glial cells (reviewedbyRef. 6). By this process, eachNB

produces a nearly invariant and unique cell lineage.(7,8) The

fate of the individual NBs is specified by positional information

within the neuroectoderm (reviewed by Refs. 9, 10), temporal

cues,(11) and the combination of developmental control genes

that they express.(4,12) In each thoracic and abdominal hemi-

segment, about 30NBsdelaminate from the vNR.NBs that arise

at corresponding positions and times but in different segments

(called serially homologous NBs) acquire the same or very

similar fate. In total, these 30 NBs produce about 350 progeny

cells (30 glial cells, 30 motoneurons and about 290 inter-

neurons)(7,8,13,14) which build a so-called hemineuromere. In

BioEssays 26:739–751, � 2004 Wiley Periodicals, Inc. BioEssays 26.7 739

Institute of Genetics, University of Mainz, Germany

Funding agencies: Deutsche Forschungsgemeinschaft, grant

numbers: Te130/7-4, Te130/8-1; EC; grant number: QLG3-CT 2000-

01224.

*Correspondence to: Gerhard M. Technau, Institute of Genetics,

University of Mainz, Saarstrasse 21, 55122 Mainz, Germany.

E-mail: [email protected]

DOI 10.1002/bies.20062

Published online in Wiley InterScience (www.interscience.wiley.com).

Abbreviations: AP, anterioposterior; AS– C, Acheate–Scute Complex;

ase, asense; btd, buttonhead; CNS, central nervous system; dpn,

deadpan; DV, dorsoventral; EGFR, epidermal growth factor receptor;

ems, empty spiracles; en, engrailed; gsb–d, gooseberry–distal; hb,

hunchback; hh, hedgehog; hkb, huckebein; ind, intermediate neuro-

blast defective; lab, labial; lbe, ladybird early; l’sc, lethal of scute; MHB,

midbrain–hindbrain boundary; msh, muscle segment homeobox; NB,

neuroblast; otd, orthodenticle; pNR, procephalic neurogenic region;

repo, reversed polarity; S1–5, neuroblast segregation wave 1–5; slp1,

sloppy paired 1; sog, short gastrulation; svp, seven up; tll, tailless; vnd,

ventral nervous system defective; VNC, ventral nerve cord; vNR,

ventral neurogenic region; wg, wingless.

Review articles

each segment, two mirror-symmetrical hemineuromeres

together with the derivatives of the ventral midline form a unit

termed neuromere. Thus, the embryonic VNC consists of a

sequence of rather uniform neuromeres (8 abdominal,

3 thoracic, 3 gnathal) comprising very similar sets of lineages.

The situation is much more complex in the developing

brain. The brain originates from a bilaterally symmetrical

region of head ectoderm, called the procephalic neurogenic

region (pNR). In the adult fly brain, highly organized neuropil

structures have been described, including the mushroom

bodies, central complex, optic lobes and antennal lobes, as

well as other specialized neuropils and major fibre tracts

required for complex behavioural functions; these have no

counterparts in the VNC (Fig. 1D).(15,16) Although approxi-

mately 95% of the neurons in the adult brain are generated

postembryonically, the main structural characteristics of the

bauplan of the adult brain are already laid down during

embryogenesis,(17–19) but it is largely unclear how these

structures evolve from the neuroectoderm and corresponding

NBs.Whichdevelopmentalmechanisms lead to the significant

differences in the specification and differentiation of structures

between the brain and VNC, as well as among regions within

the brain itself?What is the evolutionary origin of brain-specific

structural and functional complexity? An important basis for

approaching these and other questions related to brain

development is the clarification of the composition, develop-

mental origin and segmental organization of the various brain

structures on the cellular level, and the identification of genes

expressed in the respective structures and their progenitor

cells. Here,we review recent advances in our understandingof

early brain patterning in Drosophila on the cellular level.

Formation of neuroblasts

In the thorax and abdomen, the NBs form an invariant, almost

orthogonal subectodermal pattern which comprises seven

anterioposterior rows and three dorsoventral columns.(4,5) A

more derived, but still quite similar NB pattern develops in the

three segments lying anterior to the thorax (termed gnathal

segments: labial, maxillary and mandibular segment). How-

ever, in the pre-gnathal head,which forms the embryonic brain

proper, an orthogonal patterning ofNBs in columnsand rows is

not apparent. This is mainly due to massive morphogenetic

movements that take place during gastrulation in the head

anlagen including the pNR.(20,21) Early studies on the pro-

cephalic NB pattern applied morphological criteria in whole-

mount embryos, and uncovered a population of 70–80 brain

NBs per hemisphere.(5) Based on the expression of the

molecular markers lethal of scute (l’sc), asense (ase) and

seven-up (svp), thesewere subdivided into 23groups of one to

five NBs each.(22) Recently, using a different preparation

technique, the development of the brain NB pattern has been

described at higher resolution at the level of individually

identified NBs. Applying general NB markers (like deadpan

[dpn], ase) andmarkers expressed in subsets of NBs (such as

engrailed [en], svp) as well as morphological criteria, about

100 brain NBs were identified, and, based on their positional

relationships and segmental assignment (see below),

they were subjected to a new systematic nomenclature

(Fig. 1A–C).(23) These cells presumably represent the

complete population of embryonic brain NBs (not including

the progenitor cells of the optic lobes; see below). The spatial

arrangement of the brain NBs is largely invariant, i.e. their

positions relative to each other and to the outer ectoderm do

not change a great deal from one preparation to another. In

addition, the temporal sequence of formation from the pNR

followsa reproducible patternwith eachNBbeinggenerated at

a characteristic time between embryonic stages 8 and late 11.

However, in contrast to theVNC,NBs in the brain appear not to

segregate in waves, but are continuously added.(23)

Modes of brain neuroblast formation are related toneuroectodermal mitotic domainsReproducible spatiotemporal mitotic patterns have been

shown to arise in the Drosophila embryo upon onset of gas-

trulation (from stage 7), defining groups of cells, termedmitotic

domains, which enter mitosis (cycle 14) in close synchrony

with each other, but out of synchrony with cells in other mitotic

domains. The borders of the domains have been found to be

precisely specified and their arrangement to be conspicuously

different between head and trunk. Based on this reproducible

pattern and the comparison with fate maps, it was suggested

that these mitotic domains represent units of morphogenetic

function and similar cell fate.(24–26) Using four-dimensional

microscopy, the origin of brain NBs has recently been traced

back to the ectoderm and linked to particular procephalic

mitotic domains (Fig. 2A,C).(23)

Furthermore, it was reported that the formation of brainNBs

is achieved through several different modes that are related to

the mitotic domain of origin (Fig. 2B). For example, cells in

mitotic domain B do not divide in the peripheral ectoderm and

most of them delaminate as early NBs, which is analogous to

the behaviour of early NBs (S1/S2) in the trunk.(7,8) In contrast,

neuroectodermal cells in mitotic domains 1, 2 and 5 divide

parallel to the ectodermal surface and usually one of the

daughter cells in mitotic domains 1 and 5 (and most likely

mitotic domain 2) subsequently delaminates as a NB. This is

similar to precursors of late delaminating NBs (S3–S5) in the

trunk, which divide once in the neuroectoderm to generate one

NBandoneepidermoblast.(8) Cells inmitotic domain9normally

divide perpendicular to the ectodermal surface to produce a

NB and an epidermoblast. However, some cells in domain 9

delaminate as NBs without a previous division. This indicates

that not all cells within this mitotic domain strictly follow the

same mitotic pattern.(23) Although most parts of the brain

derive from NBs, recent data have shown that particular brain

regions are not formed by typical NBs: small ‘placode’-like

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740 BioEssays 26.7

Figure 1. Spatial organization of neuroblasts and brain structures in Drosophila. A: Brain neuroblasts are individually identifiable by

marker gene expression, size and position relative to each other. A subpopulation of brainNBs in a flat preparation of an embryo at stage 10

(left half) is shown double stained for seven up-lacZ (brown) and engrailed expression (blue; immunopositive NBs are indicated by white,

others by black inscription; anterior is towards the top and dorsal is left). B: Segmental topology of the embryo at the phylotypic stage of

development (stage 11). The scheme represents a flat preparation, in which the head capsule has been opened dorsally. The pregnathal

head segments (dark blue; from anterior to posterior: labral [LR], ocular [OC], antennal [AN] and intercalary [IC]) and the gnathal head

segments (blue; mandibular [MD], maxillary [MX], labial [LA]), as well as the thoracic (prothoracic [PT] and abdominal segments (first

abdominal segment [1.AB]; light blue) are indicated on the right side. On the left side, the primordium of the CNS is highlighted in grey (P,

protocerebrum;D, deutocerebrum; T, tritocerebrum).C:TheDrosophila embryonic brain develops from the procephalic neurogenic region

of the ectoderm, which gives rise to a bilaterally symmetrical array of about 105 neural stem cells (neuroblasts). The left half of a head flat

preparation including the complete brain NB pattern (NBs of the mandibular segment [MD] are indicated additionally) is shown. Brain NBs

are named according to their assignment to the tritocerebrum (T, green), deutocerebrum (D, blue) and protocerebrum (P, red colours),

based on the reconstruction of segmental borders (see also Fig. 4A). Roughly reflecting their origin from distinct mitotic domains,

protocerebral NBs are subdivided into an anterior (Pa), central (Pc) and posterior (Pp) group (see also Fig. 2). Each of these protocerebral

groups as well as the deutocerebral and tritocerebral NBs are further subdivided into a dorsal (d) and a ventral (v) subgroup (indicated by

blue stippled line) based on vnd expression in ventral NBs. Within each subgroup NBs are numbered from anterior to posterior and from

ventral to dorsal.D:Surveyof the adultDrosophila brain in a frontal viewshowing the structural organization of the large protocerebrum (red

colours), the deutocerebrum (blue colours) and the smallest part, the tritocerebrum (which in this perspective is hidden by the overlying

protocerebrum and deutocerebrum, and therefore indicated by the green-shaded region). Note that the brain fuses secondarily with the

subesophageal ganglion (SOG; light grey) deriving from gnathal segments. The adult protocerebrum represents the anterior part of the

brain and comprizes highly organized neuropile structures like the mushroom bodies, the central complex (both in light red) and the optic

lobes (OL, dark grey; these derive from separate anlagen [OA]). The deutocerebrum comprises the antennal lobes (light blue).

A–C: Adapted from Ref. 23; a, anterior; d, dorsal; p, posterior; v, ventral; as, antennal en stripe; is, intercalary en stripe; CL, clypeolabrum;

FG, foregut; hs, en head spot; ML, ventral midline; OA, optic lobe anlagen.

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BioEssays 26.7 741

groupsof ectodermal cells close to theheadmidline invaginate

during stage13 (longafter brainNB formation has ceased) and

contribute subpopulations of cells to the brain.(19,22,27)

Distinctmodesof neuronal precursor formationalso appear

to exist in the developing vertebrate brain. Although neuro-

genesis in vertebrates generally does not involve delamination

of precursors from the neuroectoderm (reviewed by Ref. 28),

in the zebrafish neuronal progenitors have been observed to

delaminate from the neuroepithelium of the inner ear.(29)

Furthermore, it was shown for part of the chick neural plate that

neighbouring cells can adopt neural or epidermal fate. A func-

tional homologue of the fly proneural genes (chick acheate

scute homologue 4) is expressed heterogeneously within

these cells raising the possibility that, like inDrosophila, neural

precursors are specified on a cell-by-cell basis through high

levels of proneural gene expression.(30)

Figure 2. Modes of brain NB formation differ betweenmitotic domains.A:Nomarski picture representing most parts of the blastodermal

head region froma lateral view (anterior towards the left). Thedifferent coloursmark thespatial arrangement ofmitotic domains1, 2, 5, 9 and

B. B: In mitotic domain B, NBs form by basally oriented delamination from the neuroectoderm. In domains 1 and 5, neuroectodermal

progenitors divide parallel to the ectodermal surface; usually one of the two daughter cells stays in the peripheral ectoderm as an

epidermoblast, whereas the second delaminates as a NB. Neuroectodermal cells in domain 9 move apically (red arrow in Ba) and

subsequentially reintegrate into the ectodermal layer to delaminate as NBs (Ba1) or remain in the ectoderm to develop as epidermoblasts

(EB in Ba2). Other cells in domain 9 divide perpendicular to the ectodermal surface (as indicated by the mitotic spindle; Bb); one daughter

cell moves apically but later reintegrates into the neuroectoderm as an epidermoblast, the other is deposited basally to become a NB. C:Origin of brainNBs fromdifferent procephalicmitotic domains. The scheme (orientation as in Fig. 1C) illustrates thatmitotic domains 1, 2, 5,

9 and B (and perhaps 20) contribute NBs to the embryonic brain. Coloured hatching marks subpopulations of NBs that derive from the

respectivemitotic domains (comparewithA), andstippledblue lines indicate thebordersbetween tritocerebrum (T), deutocerebrum (D)and

protocerebrum (P). A–C: Adapted from Ref. 23; AN, antennal appendage; NE, neuroectoderm.

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742 BioEssays 26.7

Efficiency of lateral inhibition differs amongneuroectodermal cells of the head and trunkIn the neuroectoderm, the singling out of NBs is controlled by

the proneural and neurogenic genes. Expression of proneural

genes (Achaete–Scute Complex, AS–C) defines so-called

proneural clusters, which represent equivalence groups in

which all cells have the primary fate to become NBs.(31,32)

Based on cell–cell interactions, a lateral inhibition process

mediatedby theneurogenic genes (Notch signalling pathway),

progressively restricts proneural gene expression to a single

cell, the future NB (reviewed by Ref. 3). In the trunk, genes of

the AS–C are expressed in small segmentally reiterated,

proneural clusters. Their position and size is governed by the

combined activity of dorsoventral patterning genes and pair-

rule genes.(32,33) In the procephalic neuroectoderm, the size of

‘‘proneural clusters’’ is variable. AS–C-expressing domains in

proneural clusters are generally much larger compared to

those in the trunk.(22,23) There is no indication of a segmental

patterning of proneural domains in the procephalon, which is

presumably due to the lack of pair-rule gene expression. It has

been suggested that, instead of pair-rule genes, head-gap

genes activate proneural gene expression (e.g. of l’sc).(34) The

extended expression of cephalic gap genes (e.g. of orthoden-

ticle [otd] and tailless [tll])(34,35) would explain the large size of

most of the procephalic proneural domains.

Considering the patterns of proneural gene expression and

the various modes of NB formation, one would expect the

mechanisms by which the number and distribution of pre-

sumptive NBs are determined to be modified in the pNR

compared to the vNR. Indeed, whereas in the vNR only a

single cell delaminates as a NB from each proneural cluster,

there is evidence that, at least in some parts of the procephalic

neuroectoderm (e.g. in part ofmitotic domainB), NBsoriginate

from neighbouring neuroectodermal progenitor cells, which

belong to the same ‘‘proneural cluster’’.(23) Although the

procephalic neuroectoderm also gives rise to epidermal

progenitor cells(20) based on the activity of neurogenic genes

(as indicated by the hyperplasic brain in neurogenic mu-

tants),(36) the fact that neighbouringneuroectodermal cells can

become NBs indicates that, in parts of the procephalic neuro-

ectoderm, the lateral inhibition process, which leads to

epidermal fate is less efficient or even absent. This is further

corroborated by experimental data showing that the ratio

between neuronal and epidermal precursors differs signifi-

cantly between the neuroectoderm of the trunk and head, as a

much higher proportion of neuroectodermal cells assumes

a NB fate in the procephalon.(20,37,38) As a consequence of

reduced lateral inhibition in the procephalic neuroectoderm, a

high level of proneural gene expression would be maintained

allowing adjacent cells to develop as NBs.

Interestingly, precursor formation in the midline region of

the procephalic neuroectoderm, which gives rise to the

stomatogastric nervous system, the visual system andmedial

parts of the brain, exhibits parallels. Like their ventral midline

counterparts in the trunk, the headmidline cells do not give rise

to typical NBs by delamination but remain integrated in

the surface ectoderm and express proneural genes for an

extended period of time.(39) Since genes involved in epidermal

growth factor receptor (EGFR) signaling are expressed in the

headmidline, it hasbeenproposed that thenegative feed-back

loop between the concomitantly expressed proneural and

neurogenic genes could be modified by EGFR signaling.(27)

Activated MAP kinase (indicative of EGFR signaling)(40) is

dynamically expressed in the parts of the procephalic

neuroectoderm from which brain NBs derive. For example,

by stage7,MAPkinaseexpression is found inmitotic domainB

and slightly later in the neuroectoderm corresponding to

domains 1, 2, 5 and 9 (R.U. and G.M.T., unpublished

observations). This is compatible with the hypothesis that

EGFR signaling inhibits Notch signaling in ‘‘domain B’’ (and

possibly in other parts of the procephalic neuroectoderm) to

enable neighbouring cells to delaminate as NBs, and thus

produce a higher proportion of CNS progenitors as compared

to the neuroectoderm of the trunk.

Individual identities of neuroblasts

For the VNC, it has been shown that each NB acquires a

unique identity that is reflected by the time and position of its

delamination from the neuroectoderm, by the combination of

the genes that it expresses, and by the production of a specific

cell lineage.(4,7,8,12,41) As outlined above, the formation of

brain NBs also follows a stereotypical spatial and temporal

pattern.(23) However, a particular position and time of for-

mation do not necessarily imply a unique cell fate of a given

NB. Considering the high number of brain NBs and their origin

fromseveral segments (as discussedbelow), similarities in the

specification of particular subsets of brainNBscould exist. The

ultimate clarification of individual NB fates depends on a

systematic analysis of their gene expression patterns and

lineages. For the patterns of gene expression in the pNR and

the brain NBs, such an analysis has been recently con-

ducted(35) (see below).

Individual brain neuroblasts express specificcombinations of developmental control genesAdetailedanalysisof theexpressionofmore than40molecular

markers representing the expression of 34 different genes

(including proneural genes, gap genes, segment polarity

genes, dorsoventral (DV)-patterning genes, early eye genes

and many others) has been performed in the pNR and in the

brain NBs from stage 9 to 11, when the full complement of NBs

has been generated.(35) Most of these genes are expressed in

characteristic domains of the pNR, and specifically in different

but overlapping subsets of brain NBs. Neuroblast maps of

different stages of the early embryo (stages 9, 10 and 11) in

which the expression pattern of each marker has been linked

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BioEssays 26.7 743

to individual NBs show that each NB expresses a specific

combination of molecular markers. This allows each of the

about 100 brain NBs to be identified uniquely throughout early

neurogenesis (Fig. 3A). Furthermore, these specific combina-

tions of marker gene expression suggest that each brain NB

assumes an individual fate. The marker genes and their co-

expression presumably reflect part of themachinery leading to

the specification of brain NBs and components of their

corresponding cell lineages. For example, segment polarity

genes (e.g. wingless [wg], gooseberry-distal [gsb-d], en,

hedgehog [hh]), DV-patterning genes (msh, ind, vnd) and

other genes (e.g. hkb, empty spiracles [ems], sloppy paired

1 [slp1]) are expressed in particular domains of the pNRbefore

brain NBs delaminate, implying that these genes might be

required for providing positional information in the pNR and for

subsequent specification of individual brain NBs in analogy to

the situation in the developing VNC.

Serial homology of neuroblasts amongdifferent neuromeresWithin the trunk, theCartesian grid-like expression of segment

polarity and DV-patterning genes is almost identical in each

hemisegment. Accordingly, NBs developing from correspond-

ing ‘‘quadrants’’ acquire the same fate (reviewed by Refs. 9,

10) and are called serial homologs. Indications for serial

homology of NBs between different segments come from

similarities in the time of formation, the relative position within

the developing NB pattern and absolute position within a

Figure 3. Specific combinations of marker gene expression reflect individual identities of brain NBs. A: Schematic presentation of the

entire populationof embryonic brainNBs (adapted fromRef. 35).NBsaredepictedasequal-sized circlesat positions roughly corresponding

to their positions in situ.More than40 differentmolecularmarkers (representing 34 different genes as listed in the box on the right; for further

information see Ref. 35) have been found to be specifically expressed in subsets of brain NBs. Each brain NB reveals an individual

combinatorial code of marker gene expression, which uniquely identifies each NB. Blue lines indicate the segmental boundaries between

the tritocerebrum (T), deutocerebrum (D) and protocerebrum (P). B: Example for putative serial homology of NBs in the brain and ventral

nerve cord. Semi-schematic presentation of the left half of a flattened stage 11 embryo in which the NB pattern in the brain, the gnathal and

thoracic neuromeres is highlighted.NB5–6 in theprothoracic (PT) andgnathal neuromeres is assumed tobeserially homologous toNBTd4

in the tritocerebrum (T), and to NBDd7 in the deutocerebrum (D) (serially homologous NBs are indicated in blue) for the following reasons.

(1) Position: each of these NBs exhibits a similar AP position within the respective neuromere, anterior to the En-positive NBs (indicated in

red), and similar DV position. (2) Gene expression: these are the only NBs that specifically coexpress the following molecular markers: lbe

(which is generally expressed in only one NB per hemisegment), wg, gsb-d, slp1 (except in Td4),msh, castor, svp (except Td4), the POU-

domain gene pdm-1 (except in Dd7), klumpfuss and ase. (3) Progeny cells: some of the first daughter cells of Td4 andNB5-6 coexpress lbe

and the glia specific marker repo. An, Cl, Md, Mx, La, antennal, clypeolabral, mandibular, maxillary and labial appendage, respectively; Ml,

ventral midline.

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744 BioEssays 26.7

hemisegment, the coexpression of specificmolecularmarkers

and similarities between their generated lineages.

A closer comparison of the combinations of markers

expressed in individual NBs of the brain, as well as their

relative position within the NB layer, suggest that several NBs

exist in the brain that are serially homologous to NBs in the

VNC.(35) This mainly applies to the posterior brain (deuto-

cerebrum and tritocerebrum), which is less derived than the

anterior brain (protocerebrum). For example, according to

these criteria, ‘‘NB5-6’’ in all abdominal, thoracic and gnathal

neuromeres would be serially homologous to ‘‘NB Td4’’ in the

tritocerebrumand to ‘‘NBDd7’’ in the deutocerebrum (Fig. 3B).

These NBs exhibit a similar AP position within the respective

neuromere immediately anterior to the En-positive NBs, and

they specifically coexpress the same subset of molecular

markers. Furthermore, some of the daughter cells of Td4 and

NB5-6 coexpress ladybird early (lbe) and the glia-specific

marker reversed polarity (repo).(23) The existence of serially

homologous NBs is intriguing since the number of NBs in the

tritocerebrum and deutocerebrum is considerably reduced

compared to the VNC neuromeres, the time course of neuro-

genesis within the brain and VNC is different (especially in the

tritocerebrum where the development of NBs is significantly

delayed),(23) and the development of head segments (and

consequently of brain neuromeres) has been assumed to be

differently regulated (reviewed by Ref. 42).

In the VNC, serially homologous NBs expressing the same

combination of molecular markers(4,12) give rise to almost

identical cell lineages,(7,8) suggesting that similar regulatory

interactions take place during the development of these NBs

and their cell lineages. However, some of the serially homo-

logous VNC lineages have been shown to include a subset of

progeny cells that specifically differ between thoracic and

abdominal neuromeres.(7,8,43) One would expect such seg-

ment-specific differences to be evenmore pronounced among

serially homologous lineages within the brain and between the

brain and VNC. Differences in the combination of marker

genes expressed by putative serially homologous NBs may

point to candidate genes that confer segment-specific

characteristics to their lineages. Thus, unravelling the lineages

of serially homologous NBs and the genetic network control-

ling their development should help to elucidate how region-

specific structural and functional diversity in the CNS evolves

from a basic developmental ground state.

Segmental model of the Drosophila brain

The insect brain is traditionally subdivided (from posterior to

anterior) into the tritocerebrum, deutocerebrum and proto-

cerebrum.(44) Yet, the segmental pattern in the insect head is

highly derived and its metameric organization has been

intensely debated.(21,45–48) In Drosophila, based on the

expression of the segment polarity genes en and wg, and on

the analysis of sensory structures in gap gene mutants, it was

suggested that the pregnathal head consists of four segments

(antennal, intercalary, ocular and labral), each contributing to

the brain.(21,49) However, the arrangement and boundaries of

the corresponding neuromeres, and the origin and identities

of their progenitor cells have been obscure. Recent work has

provided new insight into the positional cues expressed in the

procephalic neuroectoderm and the segmental organization

of the developing brain.(50) The view that the pregnathal

Drosophila head is composed of four segments is strongly

supported by these data, and each of the four pregnathal

segments has been attributed to a corresponding neuromere.

Positional cues and segmental boundaries during early brain

development (stages 9–11) have been identified through a

detailed analysis of the expression of segment polarity genes

(including wg, hh, gsb-d and en) and three DV-patterning

genes (vnd, ind and msh) in the pNR and in the entire

population of brain NBs derived from this region. All segment

polarity genes are segmentally expressed in the pNR as well

as in brain NBs. However, the expression of these genes is

eithermainly confined to intermediateanddorsal regionsof the

antennal and ocular segment (in the case of en,wg and gsb-d)

or is at least stronger (in the case of hh) in these parts of the

pNR. Consequently, segment polarity genes provide a clear

segmental demarcation in the intermediate and dorsal parts of

the respective neuromeres, but not in their ventral parts

(except in the tritocerebrum). Surprisingly, the DV-patterning

genes vnd and msh endorse an arrangement of brain

neuromeres along the AP axis. vnd expression demarcates

the ventral part of the posterior border of the tritocerebrum,

deutocerebrum and ocular neuromere, and msh demarcates

the dorsal anterior border of the deutocerebrum. Thus, based

on the expression of segment polarity genes (en, hh) and

DV-patterning genes (vnd, msh), it has been possible to

reconstruct the segmental boundaries in the developing brain

on the level of identified cells (Fig. 4A). Furthermore, evidence

has been provided that the protocerebrum consists of two

neuromeres, a large ocular neuromere (comprising more than

60 NBs) and a smaller labral neuromere (comprising about

10 NBs), which appears to be confined to the posterior

compartment. The segmental character of these neuromeres

is less conserved compared to the tritocerebrum and deuto-

cerebrum, deriving from the intercalary andantennal segment.

The orthogonal expression of segment polarity and DV-

patterning genes is principally conserved in the posterior part

of the pregnathal head neuroectoderm and corresponding

regions of the early brain, but becomes obscure towards

anterior sites. The tritocerebrum behaves like a reduced trunk

neuromere. Although the orthogonal pattern of expression of

these genes appears to be essentially retained in the antennal

neuroectoderm and deutocerebrum, it seems to be less

conserved compared to the tritocerebrum. The pattern is

conserved to a minor extent, if at all, in the posterior half of

the ocular neuromere, and it is not evident in the labral

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BioEssays 26.7 745

segment.(50) Thus, in the ocular and labral neuroectodermand

NBs, segmental features are largely obscure. Accordingly, the

existenceof putative serially homologousNBs in these regions

of the brain is less evident.(35)

For a part of the segmented head (mandibular, intercalary

and antennal), it was proposed that a combinatorial expres-

sion of the cephalic gap genes otd, ems and buttonhead

(btd)(51,52) mediates metamerization by acting directly on

segment polarity genes, thereby omitting the intermediate

function of pair-rule genes (reviewed by Ref. 53). More recent

data indicate that other, intermediate regulators are involved in

the segmental patterning of this head region. One of these

is collier, which is already expressed in the blastoderm and is

required for the formation of the intercalary segment. It is

controlled by the combinedactivity ofemsandbtdand thepair-

rule gene even skipped, thus integrating inputs from both the

head and trunk segmentation systems.(54) Such factors might

help to explain why trunk-specific segmental characteristics

are more conserved in the intercalary and antennal neuroec-

toderm and NBs, as compared to the ocular and labral

neuroectoderm and NBs.

How do adult brain structures evolve

from the embryonic pattern?

Development of holometabolous insects involvesanextensive

morphological transformation of the crawling larva into the

flying adult during metamorphosis. The adult fly exhibits a

behavioural repertoire that is much more complex than that

of the larva. Accordingly, the larval brain, which is formed in

the embryo, also has to be substantially remodelled during

postembryonic development (Fig. 1B–D) to fulfill the require-

ments for the control of adult behaviour. In adult Drosophila,

discrete brain structures have been linked to specific

behavioural functions, like olfaction or control of locomotion

(e.g. reviewed by Refs. 55, 56). Furthermore, considerable

advances have recently been made in bringing the resolution

Figure 4. A: Segmental model of the early embryonic brain. Semi-schematic presentation of the brain NB pattern at stage 11 (the

mandibular NBs are included in addition). Based on the expression of segment polarity genes (en, hh) and DV-patterning genes (msh, vnd;

as indicated by the colour code), the embryonic brain is proposed to consist of four neuromeres (according to Ref. 50). From posterior to

anterior, thebrainanlagenencompass the tritocerebrum (T), thedeutocerebrum(D), theocular part of theprotocerebrum (Oc-P, comprising

the largest fraction of brain NBs), and themuch smaller labral part of the protocerebrum (Lr-P). Blue lines indicate the borders between the

respective neuromeres. Intensities of colours indicate low (�) and high (þ) expression levels of en and hh. B: Comparison of expression

domains of DV-patterning genes in the embryonicDrosophila andmouseCNS. Schematic diagramof DV gene expression (as indicated by

colour code) in theembryonicCNSofDrosophilaat developmental stage11 (top; comparewithA) andmouseat approximately 10daysafter

gestation (bottom). Anteriorly, the extent of expression is specific for each gene. Expression data for mouse are from Ref. 84 (Nkx-2.2),

Ref. 85 (Gsh-1) and Ref. 81 (Msx-3). A,B: adapted from Ref. 50; AN, CL, MD, antennal, clypeolabral and mandibular appendage,

respectively; FG, foregut; ML, ventral midline; Md, Mx, La indicate mandibular, maxillary and labial neuromere; Te, Di, Me, telen-, dien-,

mesencephalon; rh1–8, rhombomeres 1–8.

Review articles

746 BioEssays 26.7

of adult brain structures to the level of individual cells. For

example, the components and connectivity of the post-

embryonic Drosophila olfactory system have been traced

from the antennal receptor neurons via the deutocerebral

neurons of the antennal lobe to prominent protocerebral

structures like the mushroom bodies (involved in olfactory

learning and memory; Fig. 1D) and the lateral horn (for

example, Refs. 57–60).

How is the transformation of the larval into the adult brain

achieved? There is plenty of evidence that formation of the

adult brain involves massive production of new cells during

postembryonic development as well as integration, reorgani-

zation and degeneration of cells or neuropil structures formed

during embryogenesis.(18,19,61–64) However, whereas the

major segmental subdivisions of the adult brain (protocere-

brum, deutocerebrum and tritocerebrum) can be roughly

assigned to the embryonic NB pattern (Fig. 1C,D), it has not

yet been possible to trace the development of the various adult

brain structures all through development on the level of

identified cells or cell lineages. Instead, the analysis so far has

been restricted to particular stages. As outlined above, cell-

specificmolecularmarkers have beenused to follow the fate of

individual brain NBs through early embryonic stages.(19,35) In

the early larva, after a period of mitotic silence, a second

generation of NBs (postembryonic NBs) becomes visible and

starts proliferation to add large numbers of progeny cells

required for the adult CNS.(63,65) In the VNC, postembryonic

NBs originate from embryonic NBs,(66) and it has been

suggested that the 80–85 postembryonic NBs that proliferate

in each larval brain hemisphere,(67) likewise represent a

subpopulation of the about 100 embryonic brain NBs.(23)

Except for the four progenitor cells that give rise to the

mushroom bodies,(17,19,65,67–69) postembryonic brain NBs

havenot yet been identified individually. Not only have they not

been linked to the identified embryonic NBs, but it is also not

known to which adult brain structures they contribute.

Thus, uncovering the links between the individual embryo-

nic NBs, their lineages, the postembryonic NBs, and the

various structures in the adult brain is a major challenge for

future work. Tracing the fate of identified cells continuously

through all stages of the developing CNSwill provide the basis

for an understanding of the interrelation between develop-

mental, structural and functional units of the brain.

Parallels in brain patterning between

Drosophila and vertebrates

DV patterningIn the fly, the border between the neurogenic and non-

neurogenic ectoderm along the dorsoventral body axis

becomes defined by two antagonistically acting proteins

encoded by the genes short gastrulation (sog) and decapen-

taplegic.(70) The vertebrate homologs of these factors,

Chordin and Bone Morphogenetic Protein 4, basically

serve the same function.(71) In both Drosophila and verte-

brates, the region in which sog/Chordin is expressed forms

the neuroectoderm. Since the neuroectoderm is located

ventrally in insects but dorsally in vertebrates, this supports

the hypothesis of a dorsoventral body axis inversion after

the separation of protostomes and deuterostomes during

evolution.(72,73)

In Drosophila, the trunk neuroectoderm is further sub-

divided by DV-patterning genes into longitudinal columns

(reviewed byRefs. 10, 74); vnd is required for the specification

of the ventral neuroectodermal columnandNBs,(75–77) indand

msh have analogous functions in the intermediate and dorsal

neuroectodermal columns and NBs.(78–80) Remarkably,

homologousgenesare found tobeexpressed in the vertebrate

neural plate and subsequently in the neural tube. In the neural

tube, the order of expression along the DV axis is analogous

to that of Drosophila: like vnd the vertebrate homologs of the

Nkx family are expressed in the ventral portion, the ind

homologs, Gsh-1/2, in the intermediate and the msh homo-

logs, Msx-1/2/3, in the dorsal portion of the neural tube

(reviewed by Refs. 28, 74).

Asmentioned above, theseDV-patterning genes have also

been found to be expressed in the procephalic neuroectoderm

and developing brain of Drosophila, and it has been observed

that the anterior extent of expression is specific for each gene:

msh is confined tomore posterior regions, and vnd expression

extends into anterior regions of the brain. Moreover, the

expression borders of msh and vnd coincide with neuromeric

borders.(50) A comparison of the anterioposterior sequence of

DV-patterning gene expression in the early brain ofDrosophila

with that published for the early mouse brain, reveals striking

similarities (Fig. 4B). Msx3, which presumably represents

the ancestral msh/Msx gene, becomes restricted to the

dorsal neural tube during later development (in contrast to

Msx1/2).(81,82) The anterior border of the Msx3 domain is

positioned within the rostral region of the dorsal rhombence-

phalon,(82) thus showing the shortest rostral extension of all

vertebrate DV-patterning genes. This is analogous tomsh, the

expression domain of which coincides with the anterior border

of the dorsal deutocerebrum, thus representing the shortest

anterior extension of DV-patterning genes in Drosophila.

Mouse Nkx2.2 extends ventrally into the most-rostral areas

of the forebrain.(83,84) vnd is expressed ventrally in anterior

parts of the ocular and labral protocerebrum. Thus, the

expression of the respective homologs in both species

displays the most-anterior extension among DV-patterning

genes. Moreover, Nkx2.2 expression in the mouse forebrain

suggests that it may be involved in specifying diencephalic

neuromeric boundaries.(83) Similarly, in Drosophila, dorsal

expansions of the vnd domain appear to correspond to the

tritocerebral and deutocerebral neuromeric boundaries.

Furthermore, Drosophila ind and its mouse homolog Gsh1

Review articles

BioEssays 26.7 747

show similarities in their expression in the early brain. In

the posterior parts of the Drosophila brain, ind is expressed

in intermediate positions between vnd and msh. Likewise, in

the posterior part of the mouse brain, Gsh1 appears to be

expressed in intermediate positions,(85) dorsally toNkx2.2,(84)

and in the hindbrain ventrally to Msx3.(81) Gsh1 has been

shown to be expressed in discrete domains within the mouse

hindbrain, midbrain (mesencephalon) and the most anterior

domain in the posterior forebrain (diencephalon).(85) Corre-

spondingly, Drosophila ind is expressed in restricted domains

within the gnathocerebrum, the tritocerebrum, deutocerebrum

and ocular part of the protocerebrum, demonstrating that the

anteriormost extension of ind (and Gsh1) expression lies

between that ofmsh and vnd.(50)

Taken together, the expression of DV-patterning genes

appears to be partly conserved between the Drosophila and

vertebrate early brain, both along theDVaxis and along theAP

axis. Furthermore, it has been observed in Drosophila that

large parts of the anterodorsal procephalic neuroectoderm

and NBs (more than 50% of all identified brain NBs) lack DV-

patterning gene expression.(50) Likewise, gaps between the

expression domains of DV-patterning genes have been

described in the vertebrate neural tube, raising the possibility

that other genes might fill in these gaps.(80)

AP patterningThecephalic gapgenes, a class of segmentationgenes, divide

the anterior part of the blastodermal embryo into broad

circumferential stripes, each encompassing several segmen-

tal primordia. Some of these genes, including btd, ems, otd,

slp1 and tll (and other gap genes such as hkb and hunchback

[hb]) are expressed in the procephalic neuroectoderm and

subsets of brainNBs, and play a crucial role in the patterning of

the peripheral cephalic ectoderm as well as in the regionaliza-

tion of the brain primordium.(34,35,42,46,86,87) The vertebrate

homologs of some of the Drosophila cephalic gap genes

are also involved in embryonic brain patterning. Similarities in

the topology of expression and in mutant phenotypes have

been observed for theDrosophila otd and ems genes and their

murine homologs Otx1/Otx2 and Emx1/Emx2, respective-

ly.(e.g. 46,71,88,89) Furthermore, cross-phylum rescue experi-

ments have been carried out inDrosophila andmouse inwhich

Drosophila otd and ems mutant phenotypes were at least

partially rescued by the expression of the respective mam-

malian homologs, and vice versa, demonstrating that these

genes share conserved genetic functions required in inverte-

brate and vertebrate brain development(86,90,91) (reviewed by

Ref. 92).

Other genes involved in AP patterning and segmental

specification belong to the highly conserved class of homeotic

genes (also called Hox genes).(93) In Drosophila, two Hox

genes of the Antennapedia–Complex, proboscipedia and

labial (lab), are known to be expressed in the pregnathal

head ectoderm and developing brain.(35,94) Lab protein is

expressed throughout theectodermof the intercalary segment

and in NBs arising from this region, i.e. NBs of the

tritocerebrum,(35) and lab loss-of-function prevents normal

differentiation of tritocerebral neurons.(94) Similarly in verte-

brates, Hox genes are expressed in and required for normal

posterior brain patterning.(95) For example, loss-of-function

mutations in the lab orthologs Hoxa-1 and Hoxb-1 lead to

altered segmental identities and differentiation defects in the

developing mouse hindbrain.(96,97)

In vertebrates, the primordium of the CNS is subdivided

along the AP axis into three basic regions by the expression of

distinct groups of genes. The anterior region (forebrain

and anterior midbrain) is characterized by the expression of

the Otx/Emx gene families, the posterior region (spinal

cord and rhombomeres 2–8) by the expression of the Hox

genes, and the intermediate zone by the expression of the

Pax2/5/8 genes (reviewed by Ref. 98). Within the

intermediate zone, a distinct neuroectodermal region, called

the midbrain–hindbrain boundary (MHB), is specified, which

has organizing activity crucial for midbrain and anterior

hindbrain development (reviewed by Refs. 99, 100). The

above-mentioned tripartite organization of the CNS primor-

dium, including aMHBbut without organizer properties, is also

found in the much-simpler organized ascidian tunicate

(urochordate), one of the closest relatives to vertebrates,

implying that the MHB is not a vertebrate innovation.(101)

However, in another invertebrate chordate, Amphioxus, the

expression of key developmental genes, including Pax2/5/8,

Wnt1 and en, is lacking in the intermediate zone, arguing

against the existence of a MHB region(102) (reviewed by

Ref. 101). Interestingly, a comparable tripartite pattern was

recently reported for the embryonic brain of Drosophila,

and mutant analysis suggests that at least some of the

genetic interactions at the deutocerebral–tritocerebral border

are similar to those observed during MHB formation in

vertebrates. These data suggest that the tripartite ground

plan has appeared much earlier in evolution, perhaps already

in the brain of the last common ancestor of proto- and

deuterostomes.(103)

Conclusions

Recent comprehensive descriptions of NB formation, gene

expression and segmentation have provided new insights into

the principles of early brain development inDrosophila. These

observations will be particularly useful as a basis for further

studies on the molecular mechanisms controlling the genera-

tion of cell diversity in the brain, as they make it feasible to

study mutant phenotypes and the effects of genetic and

experimental manipulations on the level of identified cells.

Most of the genes found to be expressed in the pNR and

specific brain NBs are also known to play a role during the

formation of the VNC. Thus, the clarification of their function

Review articles

748 BioEssays 26.7

during brain development and a comparison with their role in

the trunkwill help to understandwhatmakes the brain different

from the VNC.

The topography of expression of a number of genes as well

as aspects of their function appear to be conserved between

Drosophila and vertebrates. Thus, exploring the genetic

mechanisms underlying cell-fate specification and patterning

in the Drosophila brain will facilitate the identification and

clarification of corresponding processes in the developing

vertebrate brain.

Acknowledgments

We thank Heinrich Reichert, Gert Pflugfelder, Joachim Urban

and Ana Rogulja-Ortmann for critical comments on the

manuscript, and we apologize to all whose work we did not

cite due to limitations of space. We also thank the Deutsche

Forschungsgemeinschaft and the EC for support.

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Neuroblast formation and patterning during early brain development inDrosophila