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doi: 10.1242/10.1242/dev.00528
1 Institut für Genetik, Universität Mainz, D-55099 Mainz,
Germany
2 Institut für Genetik, TU Braunschweig, D-38106 Braunschweig,
Germany
* Author for correspondence (e-mail: technau{at}mail.uni-mainz.de)
Accepted 4 April 2003
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: CNS, Brain development, Neuroectoderm, Neuroblasts, Proneural genes, Mitotic domains, Lateral inhibition, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The NBs, which form the truncal CNS (ventral nerve cord and suboesophageal
ganglion), delaminate from the ventral neurogenic region, whereas the NBs that
form the brain derive from the procephalic neurogenic region
(Poulson, 1950). Owing to its
much simpler organization, studies on mechanisms that control early
neurogenesis in Drosophila have mainly focused on the ventral nerve
cord (VNC). 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 NBs
(Cabrera, 1987;
Jimenez and Campos-Ortega,
1990; Skeath et al.,
1992). Cell-cell interactions, which are 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
Campos-Ortega, 1995). The
singling out of NBs follows a stereotypical spatial and temporal pattern
(Doe, 1992;
Hartenstein and Campos-Ortega,
1984). Upon delamination, NBs typically undergo repeated
asymmetric divisions, budding off smaller ganglion mother cells, which divide
once to produce neurones and/or glial cells (reviewed by
Goodman and Doe, 1993). In
this way, each NB produces a specific cell lineage
(Bossing et al., 1996;
Schmidt et al., 1997). The
fate of the individual NBs depends on their position within the neuroectoderm
(reviewed by Bhat, 1999;
Skeath, 1999), time of
delamination (Berger et al.,
2001) and the combination of genes they express
(Broadus et al., 1995;
Doe, 1992).
In contrast to the VNC, our understanding of brain development is still
very rudimentary. Which developmental mechanisms lead to the significant
differences between the specification and differentiation of structures in 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 questions is the clarification of the
composition and developmental origin of the various brain structures at the
cellular level, and the identification of genes expressed in the respective
structures and individual cells. The insect brain is traditionally subdivided
into the tritocerebrum, deutocerebrum and protocerebrum
(Bullock and Horridge, 1965;
Hanström, 1928), which
derive from the intercalary, antennal and ocular/labral head segments,
respectively (e.g. Hirth et al.,
1995; Rempel,
1975; Schmidt-Ott and Technau,
1992; Younossi-Hartenstein et
al., 1996). In the adult fly brain, highly organized neuropil
structures have been described, such as the mushroom bodies, central complex,
optic lobes, antennal lobes and other specialized neuropils and major fibre
tracts, which have no counterparts in the VNC (e.g.
Hanesch et al., 1989;
Power, 1943;
Strausfeld, 1976). Main
structural characteristics of the bauplan of the adult brain are already laid
down during embryogenesis (Hassan et al.,
2000; Kurusu et al.,
2000; Nassif et al.,
1998; Noveen et al.,
2000), but it is largely unclear how these structures evolve from
the neuroectoderm and corresponding NBs.
In this and the accompanying papers
(Urbach and Technau, 2003a;
Urbach and Technau, 2003b) we
have undertaken a comprehensive survey of Drosophila early brain
development (stages 8-11), including the pattern of NB formation, the
segmental organization of the brain, and the genes expressed in the
procephalic neuroectoderm as well as in the individual NBs. We provide a
detailed description of the spatiotemporal development of the entire
population of about 100 NBs forming the trito-, deuto- and protocerebrum
(including glial and sensory precursors), and assign a systematic nomenclature
to the individual NBs. We describe in detail the expression patterns of
proneural genes of the Achaete-Scute-Complex and atonal in
the procephalic neurogenic ectoderm and in the brain NBs. We show that at
least four of the procephalic mitotic domains described by Foe
(Foe, 1989) contribute to the
embryonic brain. Using 4D microscopy we demonstrate that brain NB formation is
achieved in distinct ways related to the respective mitotic domain.
Furthermore, we show that in a central part of the procephalic neuroectoderm
several NBs originate from adjacent cells in contrast to the trunk where only
one cell of each proneural cluster adopts a NB fate. This and the patterns of
proneural gene expression indicate that modes of NB formation differ between
head and trunk.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), seven up-lacZ (H162)
(Mlodzik et al., 1990),
A37-lacZ (Ghysen and O'Kane,
1989) and scute-lacZ
(Martinez and Modolell, 1991)
(kindly provided by J. Modolell).
Staging of embryos
Staging of the embryos was carried out according to Campos-Ortega and
Hartenstein (Campos-Ortega and
Hartenstein, 1997); additionally, we used the well-characterized
trunk NB pattern (Doe, 1992)
as a further reference system for staging.
Antibodies and immunohistochemistry
Embryos were dechorionated, fixed and immunostained according to previously
published protocols (Patel,
1994). The following primary antibodies were used:
rabbit-anti-Asense (1:5000) (Brand et al.,
1993) (kindly provided by Y. N. Yan), mouse-anti-Achaete (mAb
984A11C1) (1:3) (Skeath and Carroll,
1992) (kindly provided by J. Skeath), rabbit-anti-Atonal (1:5000)
(Jarman et al., 1993) (kindly
provided by A. Jarman), anti-DIG-AP (1:1000, Roche), rabbit-anti-Deadpan
(1:300) (Bier et al., 1992)
(kindly provided by H. Vässin), mouse-anti-Invected (4D9) (1:4)
(Patel et al., 1989)
(Developmental Studies Hybridoma Bank), mouse-anti-ß-galactosidase
(1:500, Promega), rabbit-anti-ß-galactosidase (1:2500, Cappel),
mouse-anti-Ladybird early (1:2) (Jagla et
al., 1997) (kindly provided by K. Jagla), rat-anti-Lethal of scute
(1:500) (Martin-Bermudo et al.,
1991) (kindly provided by J. Skeath), rabbit-anti-Repo (1:100)
(Halter et al., 1995) and
mouse-anti-alpha-Tubulin (1:100, Sigma). The secondary antibodies (Dianova)
were either biotinylated (goat anti-mouse, goat anti-rabbit) or alkaline
phosphatase conjugated (goat anti-mouse, goat anti-rabbit, goat anti-rat), and
were diluted 1:500.
Whole-mount in situ hybridization
DIG labelled glial cells missing (gcm) RNA probe (kindly
provided by Y. Hotta) was synthesized with T7 RNA polymerase and XbaI
linearised pBlue-gcm as a template according to the manufacturer's protocol
(Roche). The hybridization of embryos was performed as described previously
(Plickert et al., 1997;
Tautz and Pfeifle, 1989).
Flat preparation
The abdomen and yolk of stained embryos were removed in 70% glycerol in 0.1
M PBS, and the head capsule was opened along the dorsal midline. Each
dissected embryo was placed in a small drop of 80% glycerol in between two
coverslips (upper coverslip 18x18 mm, lower 60x22 mm), carefully
flattened and sealed with nail-polish. Flat preparations embedded in this way
can be viewed from both sides, and allow for significantly better microscopic
resolution compared with wholemounts (compare
Fig. 3C,E,G with 3D,F,G).
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4D microscopy
Wild-type eggs were collected and mechanically dechorionated at the
blastoderm stage. Single embryos were fixed to the surface of a coverslip
(22x60 mm, coated with glue) in an anterolateral orientation, so that
the main part of the procephalic ectoderm becomes attached to the coverslip in
one focal plane. Each embryo was covered with about 5 µl fluorocarbon oil
(10S). The coverslip with the mounted embryos was transferred onto a second
coverslip (22x60 mm; carrying thin distance brackets at both ends) so
that the embryos are oriented upside down between both coverslips, allowing
subsequent examination under an upright microscope.
For in vivo tracing and documentation of early embryonic development of the
procephalic region (at about 25°C) 4D microscopy was applied. The basics
of this technique to record a three dimensional time-lapse movie are described
by (Schnabel et al., 1997).
The instrumentation was now improved (R.S., unpublished), and allows images of
very high quality to be stored on the computer. The temperature-controlled
stage of a Zeiss Axioplan microscope was moved by a piezo focusing device
(Physik Instrumente D-76337 Waldbronn) to record the z-series (<50
focal levels, typically 1 µm per focal level; depending on the number of
focal levels, recording is repeated every 30 to 60 seconds). The analogue
pictures are collected with a Hamamatsu Newvicon camera, digitised with an
Inspecta-3 frame grabber (Mikroton, D-85386 Eching) and finally compressed to
40 kb per picture with a wavelet function (Lurawave, D-10587 Berlin). The
microscope and the accessories are controlled with a PC using a specially
designed software (4DDM, AK Schulz and RS) programmed in C++. The 4D-records
are replayed and cell positions and cleavages are documented with the database
SIMIBiocell (SIMI D-85705 Unterschleißheim).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or
asense (ase) (Brand et
al., 1993). NB identities are indicated by their position relative
to the cephalic furrow, invaginating foregut, dorsal and ventral midline,
their relative position within the NB pattern, their time of segregation, and
the expression of cell-specific markers
(Fig. 3)
(Urbach and Technau, 2003a;
Urbach and Technau, 2003b).
Following the nomenclature introduced by Younossi-Hartenstein et al.
(Younossi-Hartenstein et al.,
1996), brain NBs are named according to their assignment to the
trito- (T), deuto- (D) and protocerebrum (P), and within the protocerebrum to
an anterior (Pa), central (Pc) and posterior (Pp) group. Assignment of
individual NBs to particular neuromeres is based on the reconstruction of
segmental borders as detailed by Urbach and Technau
(Urbach and Technau, 2003a),
and the three protocerebral groups roughly reflect their origin from distinct
mitotic domains (see below). Differing from the nomenclature by
Younossi-Hartenstein et al.
(Younossi-Hartenstein et al.,
1996), we further subdivide each of the protocerebral groups, as
well as the deuto- and tritocerebral neuroblasts, into a dorsal (d) and a
ventral (v) subgroup based on the expression of the D/V patterning gene
vnd (see Urbach and Technau,
2003a). Finally, within each of these subgroups, individual
neuroblasts are numbered (1,2, etc.) from anterior-to-posterior and from
ventral-to-dorsal sites (so that numbers reflect relative positions along the
DV axis; see Fig. 1). Our
nomenclature is based on the complete late stage 11 NB array, and is also used
for corresponding NBs in embryos younger than late stage 11
(Fig. 2). Although for better
resolution the pictures and semi-schematic NB maps shown in the following
generally correspond to flat preparations, they can be also applied to the
identification of NBs in whole mounts, as demonstrated in
Fig. 3.
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Procephalic neuroblasts develop in a stereotypical spatial and
temporal pattern
We traced the pattern of brain NBs through the entire period of NB
formation (stage 8-11) in fixed flat preparations of staged embryos. We
subdivide NB formation into seven stages
(Fig. 2). Some of them
correspond to stages where NB patterns have been previously described in the
trunk (Broadus et al., 1995;
Doe, 1992;
Hartenstein et al., 1987),
allowing a comparison of the development of NB patterns in the trunk and
procephalon. Camera lucida drawings were prepared showing the typical
arrangement of NBs at the respective stages
(Fig. 2). The spatial
arrangement of NBs is largely invariant. In addition, the temporal sequence of
formation from the neuroectoderm follows a reproducible pattern, although the
time point at which particular NBs are formed can vary to a certain degree, as
was described for NBs in the trunk (Bossing
et al., 1996; Schmidt et al.,
1997). Intermediate brain NB patterns between the illustrated
stages can therefore be observed.
The procephalon consists of four fused segments: the labral, ocular,
antennal and intercalary segment (from anterior to posterior)
(Schmidt-Ott et al., 1994;
Schmidt-Ott and Technau,
1992). Neurogenesis in the procephalic ectoderm, as in the trunk,
initiates at early stage 8. At this stage antibody staining reveals Dpn
expression in neuroectodermal domains in the antennal and ocular segment
(Fig. 3A). By mid-stage 8 these
domains give rise to first brain NBs, which can be uniquely addressed in flat
preparations by their absolute position in the overlaying procephalic
neuroectoderm and relative position within the NB pattern
(Fig. 2A,
Fig. 3B). As the NB pattern
becomes more complex in the later stages, we examined molecular markers that
are expressed in subsets of brain NBs, such as engrailed
(en, revealed by an en-lacZ line or an antibody against 4D9
recognizing the products of the closely related en and
invected genes) (Coleman et al.,
1987) and seven up (svp, revealed by
svp-lacZ enhancer trap line H162)
(Mlodzik et al., 1990), as
well as an array of other markers (see
Urbach and Technau, 2003a;
Urbach and Technau, 2003b).
en expression allows for a clear distinction of gnathal and
pregnathal segments. In the pregnathal head, it is expressed in several
ectodermal domains and descending NBs, thus demarcating boundaries between
head segments (Schmidt-Ott and Technau,
1992) and corresponding trito-, deuto- and protocerebral
neuromeres. During stages 9-11, svp and en are continuously
expressed in an increasing amount of single NBs or clusters of brain NBs.
Thus, Svp- and En-positive NBs present stable reference points for the
identification of surrounding NBs. The onset of svp expression is
characteristic for each NB. It is generally initiated in NBs during or shortly
after formation, but in a few exceptions svp expression begins quite
some time after formation (e.g. Pcv1 develops at early stage 9, but Svp cannot
be detected before stage 10; Figs
2,
3). Additionally, the level of
svp-lacZ expression appears to differ significantly and specifically
among NBs of the same stage (e.g. at late stage 9 it is higher in Dd1 or Dv6
compared with Pcv3 or Pcv6; Fig.
3D). We find that some new NBs are added at the borders of the NB
array, but that others become interspersed between existing NBs (also at later
stages). This is in contrast to earlier reports suggesting that brain NBs
become sequentially added only in a centrifugal way
(Younossi-Hartenstein et al.,
1996). Until late stage 9 in the procephalon (as in the trunk)
(Doe, 1992), approximately
half of the total number of brain NBs is formed, encompassing 12 deuto- and 34
protocerebral NBs (Fig. 2C,
Fig. 3C,D). An orthogonal
patterning of brain NBs in columns and rows, as described for the trunk
(Doe, 1992;
Hartenstein and Campos-Ortega,
1984), is not apparent. This is corroborated by the expression of
dorsoventral patterning genes and segment polarity genes (see
Urbach and Technau,
2003a).
Until late stage 11 about 106 brain NBs have formed on either side
(Fig. 2G). As we do not find
additional NBs to be formed during stage 12 (for NB identification see above),
we conclude that by late stage 11 the pattern of embryonic brain NBs is
complete (consistent with the situation in the trunk)
(Doe, 1992). It comprises
about 72 protocerebral, 21 deutocerebral and 13 tritocerebral NBs. Svp is
reproducibly expressed in about 39 of all NBs, En is strongly expressed by
about 10 NBs [Tv4, Tv5, Td3, Td5 emerging from the engrailed intercalary
stripe, `en is'; Dv8, Dd5, Dd9, Dd13 from the engrailed antennal stripe, `en
as'; Ppd5, Ppd8 from the engrailed head spot, `en hs'; for nomenclature of
en expression domains in the procephalic ectoderm see Schmidt-Ott and
Technau (Schmidt-Ott and Technau,
1992)] and weakly by a cluster of about 10 NBs in the anteriomost
part of the protocerebral primordium (Fig.
2). In the observed developmental period, the positions of brain
NBs relative to each other and to the outer ectoderm (e.g. taking ectodermal
en domains as reference points) in principal do not change, except
for slight variabilities that might be due to new NBs becoming accommodated
into the pattern.
Cell size varies between NBs. Apparently, most of the early NBs are larger
than later developing NBs (e.g. Dd8 being formed at stage 8 is significantly
larger than the adjacent Ppd5 and Ppd8, which form at late stage 9/early stage
10; Fig. 2D,E,
Fig. 3E). Also in the trunk
early (S1/2), NBs are generally larger than late (S4/5) NBs, and this has been
shown to be correlated with a previous division of late NBs in the
neuroectoderm (Bossing et al.,
1996; Schmidt et al.,
1997).
The procephalic neuroectoderm also forms the anlagen of the adult optic
lobes. These precursors are clearly distinguishable from NBs, as their mode of
formation is different. They invaginate as separate epithelial primordia from
the dorsoposterior ectoderm that subsequently attach to the brain
(Green et al., 1993). By stage
12, when the optic lobe primordia start to invaginate, all identified brain
NBs have already formed. Some of them are located adjacent to the anterior lip
of the optic lobe anlagen, but none is observed to be part of it (data not
shown). The optic lobe anlagen will not be considered further in this
study.
Glial and sensory precursors
To map the positions of putative glial precursor cells, we investigated the
expression pattern of the two glia specific genes, reversed polarity
(repo) (Campbell et al.,
1994; Halter et al.,
1995; Xiong et al.,
1994) (using an anti-Repo antibody) and glial cells
missing (gcm) (Hosoya et
al., 1995; Jones et al.,
1995; Vincent et al.,
1996) (using gcm RNA probes;
Fig. 4A-E). Like in the ventral
nerve cord these two genes are co-expressed in cells of the early brain, with
Repo expression starting slightly later than gcm at late stage 10
(Fig. 4C). Until late stage 11
more than 20 cells express Repo, most of them being part of the proto- and
tritocerebrum (Fig. 4B). Owing
to their small size, many of them may represent progeny cells of closely
associated NBs (Fig. 4A-C,E).
We were able link Repo expression to identified precursor cells in only two
cases. In the tritocerebrum we detect Repo in Td7
(Fig. 4A,B,D,E). Because of its
position (immediate posterior to the `en as'; data not shown) and onset of
Repo expression, Td7 possibly represents the serial homologue of the truncal
longitudinal glioblast (Halter et al.,
1995). A further tritocerebral Repo-positive cell derives from the
Repo-negative Td4, as it co-expresses the marker gene ladybird early
(Fig. 4D). In the tritocerebrum
ladybird early is expressed in Td4 and its progeny
(Urbach and Technau, 2003b).
As co-expression of Repo only occurs in part of the Td4 progeny, Td4 appears
to act as a neuroglioblast, generating glia and neurones. The identification
of all other glia-producing precursors in the brain will require the
application of cell lineage tracers.
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), and the
lacZ pattern in the enhancer trap strain A37
(Ghysen and O'Kane, 1989),
which are indicative of sensory precursor cells
(Fig. 4E-G;
Fig. 7). Ato expression is less
dynamic and appears to be complementary to the genes of the AS-C (see below).
From stage 8 to 10, it is found in four small patches in the antennal and
preantennal ectoderm (Fig.
7A,C,E). Until stage 11 one protocerebral (ocular Ppd19) and six
antennal NBs (Dd9, Dd11, Dd12, Dd13, Dv1, Dv3) derive from these patches.
Except for Dd13, no Ato-positive NB co-expresses any gene of the AS-C
(Fig. 7D,F,H). In Ato/A37
double staining, co-expression was detected in five stem cells by stage 11
(Dd9, Dd11, Dd12, Dd13 and Dv1; co-expression in Dv3 only until stage 10,
later only A37; Fig. 4E-G). Considering their characteristic position within the ectoderm of the antennal
primordium (Fig. 4F,G; which is
also confirmed by Ato/En double staining; data not shown), it is likely that
Dd9, Dd11, Dd12 and Dd13 represent precursors of the larval antennal dorsal
organ (Campos-Ortega and Hartenstein,
1997; Schmidt-Ott et al.,
1994). Being located in the ventralmost position at the level of
the en antennal stripe (Fig.
2G, Fig. 4F,G), Dv1
(and perhaps Dv3) possibly represents the precursor of the
hypopharyngeal/latero-hypopharyngeal organ
(Campos-Ortega and Hartenstein,
1997; Schmidt-Ott et al.,
1994). Ato protein, but not A37, is found in the anteriormost
protocerebral NB Ppd19 (Fig.
4E, Fig. 7D,F,H),
which appears to generate three Ato-positive progeny cells until stage 13,
mapping in the dorsolateral protocerebrum (data not shown). Determining how
far the other Ato/A37-expressing precursors also contribute cells to the CNS
awaits cell lineage tracings.
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Brain neuroblasts originate from ectodermal domains with distinct
mitotic behaviours
Foe (Foe, 1989) subdivided
the procephalic ectoderm into several mitotic domains which are characterized
as discrete groups of cells synchronously entering the 14th mitotic cycle. As
these domains were suggested to represent units of morphogenetic function
(Foe, 1989), we attempted to
link populations of identified brain NBs to specific mitotic domains. Because
time of entry into mitosis varies considerably between mitotic domains, each
domain is only recognizable during its period of mitosis but not before or
thereafter. Furthermore, almost all procephalic mitotic domains have already
completed the 14th mitotic cycle (by early/mid-stage 8) before they give rise
to NBs. Therefore, assigning NBs to particular mitotic domains is a demanding
task. To trace the arrangement of procephalic mitotic domains during early
neurogenesis and the populations of NBs they give rise to, we used a 4D
microscope system (Schnabel et al.,
1997), which permits continuous following of cell positions, cell
divisions and cell fates in the living embryo (see Materials and Methods).
During stages 6-11, the relative positions of ectodermal regions corresponding
to particular mitotic domains do not change in principal
(Fig. 5C). Brain NBs derive
from essentially four or five mitotic domains: domain 1, 5, 9 and B [and
possibly domain 2; nomenclature of mitotic domains according to Foe
(Foe, 1989)]. We provide a
correlation between these domains and subpopulations of brain NBs as
summarized in Fig. 5 and
Table 1.
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) (see below). By
stage 7/early stage 8, the ectodermal cell layer in this region appears
thicker and most of the cells are characterized by an apically narrowed and
basally enlarged shape. By middle stage 8, first NBs emerge from this domain
(Fig. 5A,
Fig. 8A-C). We find that all
NBs (about 25) arising from domain B contribute to central parts of the
protocerebrum (Fig. 5B). The
posteroventrally adjoining domain 9 encompasses about 50 neuroectodermal
cells, giving rise to about 10 posterior protocerebral NBs, most (if not all)
deutocerebral and presumably some anterior tritocerebral NBs
(Fig. 5B,C). Domain 1 covers
the anterior part of the procephalon, adjoining domain B anteriorly
(Foe, 1989)
(Fig. 5A,C). Consistent with
its position in the procephalic neuroectoderm and relative to domain B, we
find that the anterior population of (about 15) protocerebral NBs originates
from domain 1 (Fig. 5B). Domain
5 covers the dorsal part of the procephalon anterior to the cephalic furrow,
abutting domains 2 and 9 ventrally, and domains B and 20 anteriorly
(Foe, 1989)
(Fig. 5A,C). A dorsoposterior
subset of about 15 protocerebral NBs develops from domain 5
(Fig. 5B). As neurogenesis in
the intercalary segment is strongly delayed (starting at about stage 10; Figs
2,
3), it is hard to ascertain
from which mitotic domain tritocerebral NBs arise. However, because during
stages 8-11 the relative positions of the mitotic domains do not change, and
of NBs to each other and to the outer ectoderm appear to be maintained, it is
likely that the posterior portion of tritocerebral NBs originates from the
ectoderm posteroventral to domain 9. Thus, we conclude that part (or all) of
the tritocerebral NBs develop from domain 2
(Fig. 5A-C).
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), and from which most of the brain NBs derive.
Four-dimensional microscopy data show that cells in mitotic domain B, as
opposed to all other procephalic mitotic domains, do not divide prior to NB
delamination (as confirmed by anti 
-Tubulin antibody staining;
Fig. 8A-C) supporting earlier
observations (Foe, 1989). By
stage 7/8 neuroectodermal cells in domain B gradually enlarge on the basal end
and delaminate successively as NBs, thereby losing their slender contact to
the apical ectodermal surface (Fig.
6A, Fig. 8A-C).
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). Thus, one
daughter cell becomes located apically, reintegrates into the ectoderm and
presumably develops as an epidermoblast. The sibling cell is deposited in the
basal direction to become a NB.
In mitotic domains 1 and 5 all cells undergo a division in parallel to the
ectodermal surface (Foe, 1989)
before first NBs delaminate from these domains. Most of these divisions appear
to result in one daughter cell which subsequently delaminates from the
ectoderm as a protocerebral NB, and a second precursor which remains within
the outer ectoderm and presumably acts as an epidermoblast
(Fig. 5C).
Taken together, we find different modes according to which brain NBs arise
from the neuroectoderm, and which are correlated with distinct mitotic
domains. Whereas the modes of NB formation we find in mitotic domain B
(Fig. 6A) and 1/5
(Fig. 6C) correspond to the
behaviour of cells in the truncal neuroectoderm
(Bossing et al., 1996;
Hartenstein and Campos-Ortega,
1984), those in domain 9 appear to be brain specific.
The pattern of proneural gene expression in the procephalic ectoderm
and brain neuroblasts
Considering the differences in the patterns and modes of NB formation
between the developing ventral nerve cord and the brain, and the fact that NB
formation is promoted by proneural genes, we investigated in detail the
expression of members of the Achaete-Scute-Complex (AS-C) (for a review, see
Campos-Ortega, 1995)
achaete (ac), scute (sc), lethal of
scute (l'sc) during early brain development (stages 8-11). In
double labelling with engrailed expression as a segmental marker, we
precisely determined the relative position of proneural gene expression
domains within the procephalic neuroectoderm, as well as the expression in the
descending NBs (summarized in Fig.
7). The rapidly changing pattern of L'sc expression roughly
foreshadows the spatiotemporal development of brain NBs
[Fig. 7B,D,F,H; for description
of l'sc expression see also Younossi-Hartenstein et al.
(Younossi-Hartenstein et al.,
1996)]. About 60% of all NBs formed until stage 11 express L'sc,
including almost all NBs formed during stages 8 and 9. The pattern of Ac
expression during stage 8 and 9 is largely complementary to L'sc
(Fig. 7A-D). sc is not
expressed before stage 10 (Fig.
7E-H).
Co-expression of proneural genes in brain NBs appears to be rare and
transient; e.g. by stage 8, four out of 16 NBs show co-expression of two
proneural genes (ac and l'sc), by stage 9 co-expression
occurs in only one out of 27 NBs (Fig.
7B,D,F,H). Despite the general correspondence between the pattern
of proneural gene expression in the neuroectoderm and deriving NBs, some NBs
express proneural genes at detectable level only after their formation, i.e.
upon delamination from the neuroectoderm, which at that time does not express
the respective gene (e.g. ac in Dd3, Dv6, Pad4, Pcd15, Pcd16 and
Pcv3, or l'sc in Pcd17, Pcd21;
Fig. 7B-H). Likewise, in the
trunk l'sc expression was found in NB3-5, but not in the
corresponding proneural cluster (Skeath et
al., 1994). However, a subset of brain NBs (about 25%) does not
express any of the investigated proneural genes at a detectable level. This is
mostly observed in late developing NBs (e.g. for five stage 10 NBs and about
22 stage 11 NBs; Fig. 7F,H),
implying that other proneural genes might exist.
Proneural gene expression in the procephalic neuroectoderm is found in
patches of significantly varying size. ac, sc, l'sc and ato
are all expressed in small proneural clusters (of five to seven cells) as well
as in larger ectodermal domains. The dynamics of gene expression in the small
clusters reflects the process of singling out of the presumptive NBs, i.e.
expression initially occurs in all cells of a cluster, but after segregation
it is only maintained in the respective NB. Proneural gene expression in
larger ectodermal domains appears to be regulated differently. For example,
the large l'sc domain which during stages 7-10 spans most of the
procephalic neuroectoderm, gives rise to more than one NB
(Fig. 7)
(Younossi-Hartenstein et al.,
1996). Accordingly, l'sc expression within this
`proneural cluster' (equivalence group of cells with NB-forming potential)
shows a distinct dynamic: although NBs after segregation express L'sc at high
levels, all surrounding cells do not lose it; thus, presumably retaining their
potential to become a NB. For further details of proneural gene expression see
Fig. 7.
Brain neuroblasts can develop from adjacent neuroectodermal
cells
In the ventral neurogenic ectoderm of the trunk, each proneural cluster of
five to seven cells gives rise to a single NB. A lateral inhibition process
mediated by the neurogenic genes prohibits more than one cell from each
cluster adopting a neural fate (for a review, see
Artavanis-Tsakonas et al.,
1991; Campos-Ortega,
1993). Thus, in the truncal neuroectoderm, immediately
neighbouring cells are very unlikely to develop as NBs. The fact that, in the
head, expression of proneural genes is found in larger domains of the
neuroectoderm and in groups of NBs corresponding to these domains (see above),
raises the possibility that in the procephalic neuroectoderm adjacent cells
may develop as NBs. To test this hypothesis, we traced the segregation of
individual NBs from the procephalic ectoderm more closely. First, we performed
double labelling with antibodies against -Tubulin and Dpn
(Fig. 8A-C). In domain B, most
of the developing NBs transiently show a thin, apically directed process,
which is visible until the NB has completely delaminated
(Fig. 6A). In some cases, we
observe that, consistent with the subectodermal position of the delaminating
NBs, their corresponding apical processes are also in immediate vicinity of
each other (e.g. Pcd2, Pcd4 and Pcv9 in
Fig. 8B,C), suggesting that
these NBs derive from neighbouring neuroectodermal cells. To obtain more
direct evidence for this spatial relationship, we applied 4D microscopic
analysis (see Materials and Methods). This allowed us, in vivo, to trace back
the origin of a subset of identified NBs to their corresponding
neuroectodermal progenitors in the blastoderm (stage 6;
Fig. 8D-G). We focused on early
NBs (stage 8; Fig. 2) most of
which derive from domain B (Fig.
5A). As NBs from domain B do not divide before delamination from
the ectoderm (see Fig. 6A) they
are rather large, facilitating their identification in vivo. We traced the
origin of a group of about 10 identified late stage 8 NBs
(Fig. 8C,F,G). We find that the
spatial relationships of these cells in the NB layer
(Fig. 8D,E) closely correspond
to their previous arrangement in the neuroectoderm, where they represent a
group of adjacent cells (Fig.
8F,G). Also in domains 1, 5 and 9, we found cases in which two or
more neighbouring cells develop as NBs (data not shown). We conclude, that in
contrast to the situation in the truncal neuroectoderm, adjacent cells in the
procephalic neuroectoderm (belonging to the same `proneural cluster') can
adopt neural fate. Thus, the process of lateral inhibition appears to be less
efficient in the procephalic neuroectoderm, allowing more cells to follow
their primary neural fate. In domain B, where many adjacent cells develop as
NBs, lateral inhibition may even be entirely lacking.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) found about 70 brain NBs, and based on the
expression of l'sc, ase and svp, Younossi-Hartenstein et al.
(Younossi-Hartenstein et al.,
1996) subdivided a total of 75-80 brain NBs per hemisphere into 23
groups of one to five NBs each. In this study, we describe for the first time
the development of the procephalic NB pattern at the level of individually
identified NBs. Using general NB markers (dpn, ase) and morphological
criteria, we have identified about 105 brain NBs in each hemisphere and
documented their temporal sequence of formation as well as their positional
relationships between developmental stages 8 and 11. Identities of the
individual NBs at different stages are confirmed by the analysis of about 30
other marker genes expressed in single, or subsets of, brain NBs (this paper)
(Urbach and Technau, 2003a;
Urbach and Technau, 2003b).
The differences in NB numbers, when compared with earlier studies, might be
due to the fact that, by generating flat preparations (see Materials and
Methods) instead of wholemounts (as in previous reports), we gained a
significantly higher resolution of the NB layer (compare
Fig. 3C,E,G with
Fig. 3D,F,H). We find that
during formation of the brain anlage, new NBs are continuously added. We did
not observe a segregation of brain NBs in waves, which has been suggested to
occur in analogy to the trunk
(Younossi-Hartenstein et al.,
1996). Differences in the spatiotemporal pattern of NB development
between trunk and procephalon are not surprising considering the patterns of
expression of proneural genes (see below), and the fact that brain NBs derive
from distinct mitotic domains (see below), the size and proliferation
properties of which (e.g. onset of mitosis, orientation of the mitotic
spindle) are heterogeneous and different to the trunk
(Foe, 1989). The largest
region of the embryonic brain is the protocerebrum, which consists of about 72
NBs. Its extended size, when compared with the deutocerebrum (about 21 NBs)
and tritocerebrum (about 13 NBs), is in agreement with the hypothesis that the
protocerebrum is composed of two neuromeres
(Urbach and Technau;
2003a).
The brain neuroblast map includes glial and sensory progenitor
cells
In the trunk, about a quarter of all embryonic NBs generate both neurones
and glia (they are appropriately called neuroglioblasts) or only glia
(glioblasts) (Bossing et al.,
1996; Schmidt et al.,
1997). In the brain, a complex pattern of glia is formed
(Hartenstein et al., 1998),
but their progenitors have so far not been identified. Our data provide first
evidence for the existence of a neuroglioblast (Td4) in the embryonic brain.
Furthermore, we also have indications for the existence of a glioblast (Td7).
The identification of the other glial progenitor cells will require a
comprehensive cell lineage analysis. Considering the spatiotemporal pattern of
Repo and gcm expression, we speculate that – corresponding to
the situation in the trunk – most of these progenitors represent
neuroglioblasts born at early stages (stage 8/9). Furthermore, in the trito-
and deutocerebrum, most glial cells appear to originate from dorsal sides of
the neuroectoderm, which express the gene muscle segment homeobox
[msh; for procephalic expression of DV patterning genes see Urbach
and Technau (Urbach and Technau,
2003a)], again resembling the situation in the trunk
(Isshiki et al., 1997;
Schmidt et al., 1997). Whether
msh is required for proper development of these brain NBs and their
glial progeny, as has been shown in the trunk
(Isshiki et al., 1997),
remains to be settled. However, in contrast to the trito- and deutocerebral
brain regions, and the ventral nerve cord, the sites of origin of glial cells
in the protocerebrum do not appear to be mainly confined to dorsal positions.
This may be due to the profound differences in the expression pattern of DV
genes in the preantennal neuroectoderm
(Urbach and Technau,
2003a).
Cell lineage tracing in the trunk has indicated that there is a spatial
overlap between proneural clusters that give rise to CNS and ventral PNS
progenitors (the NB 4-3 and 4-4 lineages each include a sensory subclone)
(Schmidt et al., 1997),
implying that both types of progenitors can develop in close vicinity. To find
out if PNS and CNS precursors intermingle in the procephalon, we applied
molecular markers that have been used to label sensory organ precursors (SOPs)
in the trunk (Dambly-Chaudiere and Leyns,
1992; Ghysen and O'Kane,
1989; Jarman et al.,
1993; Younossi-Hartenstein and
Hartenstein, 1997). We identified about six putative SOPs (four
dorsal and two ventral) in the vicinity of CNS precursors. Regarding their
position, these can be assigned to the dorsal organ and the
hypopharyngeal/latero-hypopharyngeal organ
(Campos-Ortega and Hartenstein,
1997). Whether these sensory precursors share common lineages with
CNS cells will have to be clarified by lineage analysis. We identified further
putative SOPs in the procephalon (precursors of the labral sensory organs and
the Bolwig organ) (Campos-Ortega and
Hartenstein, 1997), which we did not include in the NB map as they
are clearly separated from CNS precursors.
The relationship between embryonic and postembryonic neuroblasts
In the embryonic CNS, NB size decreases with each division, and (except for
five brain NBs on either side) NBs cease to proliferate by stage 16 when they
are no longer identifiable. After a period of mitotic silence in the late
embryo (stage 17) and first instar larva, a population of large postembryonic
NBs becomes visible in the peripheral CNS cortex and commences proliferation
to produce large numbers of cells of the adult CNS
(Prokop and Technau, 1994;
Truman and Bate, 1988). For
the ventral nerve cord it has been shown that postembryonic NBs originate from
embryonic NBs (members of the same lineages) and may even represent identical
cells (Prokop and Technau,
1991). About 23 postembryonic NBs have been identified per
thoracic hemineuromere in the larva
(Truman and Bate, 1988)
compared with 31 embryonic NBs (Doe,
1992). Thus, about 75% of the thoracic embryonic NBs resume
proliferation in the larva. Interestingly, this coincides with the ratio of
NBs found in the embryonic and larval brain. About 80-85 NBs have been
described to proliferate in each larval brain hemisphere
(Ito and Hotta, 1992).
Compared with the number of about 105 embryonic brain NBs found in this study,
this suggests that about 78% of the embryonic NBs resume proliferation in the
larval brain.
Distinct modes of neuroblast formation are related to mitotic
domains
Foe (Foe, 1989) showed that
spatiotemporal mitotic patterns arise in the Drosophila embryo upon
onset of gastrulation (from stage 7), and she defined groups of cells, termed
mitotic domains, that enter mitosis (cycle 14) in close synchrony with each
other, but out of synchrony with cells of other mitotic domains. She found the
borders of the domains to be precisely specified and their arrangement to be
conspicuously different in head and trunk. Based on this reproducible pattern
and the comparison with fate maps (e.g.
Hartenstein and Campos-Ortega,
1985), Foe suggested that the mitotic domains of cycle 14
represented units of morphogenetic function. In order to trace the origin of
brain NBs back to the ectoderm and to link them to particular mitotic domains,
we used 4D microscopy. As proposed by Foe
(Foe, 1989) we found that NBs
derived from domains 9 and B. In addition, we observed brain NBs descending
from domains 1, 5 and, most probably, 2. However, we cannot exclude the
possibility that other mitotic domains (located more ventrally or dorsally)
may also participate in the formation of the brain anlage. For example, domain
20, which was recently shown to give rise to the Bolwig organ and optic lobe
(Namba and Minden, 1999), may
contribute to some of the most dorsal brain NBs (see
Fig. 5).
Furthermore, we find that the formation of brain NBs is achieved through
several different modes that are related to the mitotic domain of origin. Most
domain B cells do not divide in the peripheral ectoderm and delaminate as
early NBs, which is analogous to the behaviour of early NBs (S1/S2) in the
trunk (Bossing et al., 1996;
Schmidt et al., 1997).
Neuroectodermal cells in domains 1, 2 and 5 divide in parallel to the
ectodermal surface, and usually one of the daughters subsequently delaminates
as a NB. Similarly, precursors of late delaminating NBs (S3-S5) in the trunk
divide once in the neuroectoderm to generate one neuroblast and one
epidermoblast (Schmidt et al.,
1997). Domain 9 cells normally divide perpendicular to the
ectodermal surface (Foe, 1989)
to produce a neuroblast and an epidermoblast. However, we observed that some
cells in domain 9 delaminated as NBs without a previous division. This
indicates that not all cells within this mitotic domain strictly follow the
same mitotic pattern. Although most parts of the brain descend from NBs,
recent data have shown that some parts are not formed by typical NBs: small
`placode'-like groups of ectodermal cells close to the head midline invaginate
during stage 13 (long after brain NB formation has ceased) and contribute
subpopulations of cells to the brain
(Dumstrei et al., 1998;
Noveen et al., 2000;
Younossi-Hartenstein et al.,
1996).
Distinct modes of neuronal precursor formation also appear to exist in the
developing vertebrate brain. Although neurogenesis in vertebrates generally
does not involve delamination of precursors from the neuroectoderm (for a
review, see Arendt and Nübler-Jung,
1999), in the zebrafish neuronal progenitors have been observed to
delaminate from the neuroepithelium of the inner ear
(Haddon and Lewis, 1996).
Furthermore, it has been shown for part of the chick neural plate that
neighbouring cells can adopt neural or epidermal fate. A functional homologue
of the fly proneural genes (cash4) is expressed heterogeneously
within these cells raising the possibility that, as in Drosophila,
neural precursors are specified on a cell-by-cell basis through high levels of
proneural gene expression (Brown and
Storey, 2000).
Expression of AS-C genes differs between head and trunk and does not
cover the entire neuroectoderm
In the trunk, genes of the AS-C are expressed in segmentally reiterated,
proneural clusters. Their position and size are governed by the combined
activity of DV patterning genes and pair-rule genes
(Skeath and Carroll, 1992
) levels of expression are indicated by colour intensities). In the
peripheral ectoderm (A,C,E,G) single cells are outlined when expression
domains comprise less than eight cells. Additionally, the expression of
engrailed (en) in the peripheral ectoderm is highlighted.
Orientation as in 
