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doi: 10.1242/10.1242/dev.00532
Institut für Genetik, Universität Mainz, D-55099 Mainz, 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, Neuroblasts, Segment polarity genes, Dorsoventral patterning genes, Segmentation, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hartenstein and Campos-Ortega,
1984). Segmental patterning defines functional units, which can
then be refined during further development. For example, within a segment
(neuromere) each NB acquires a unique identity and produces a specific cell
lineage (for a review, see Doe and
Technau, 1993). Genetic mechanisms of neuromere and NB formation
in the ventral nerve cord are quite well understood (for a review, see
Campos-Ortega, 1993), and some
advances in understanding the processes that lead to the specification of
individual NB identities have recently been made. Specific identities appear
to be conferred to presumptive NBs in the neuroectoderm by positional cues.
For example, the segment polarity genes subdivide the neuroectoderm into
transverse rows along the anteroposterior axis and the dorsoventral patterning
genes in longitudinal columns along the DV axis. The superimposition of the
expression patterns of both gene groups establishes a Cartesian coordinate
system of positional cues in which the fate of a particular NB depends on the
respective `quadrant' in which it is formed (for reviews, see
Bhat, 1999;
Skeath, 1999).
The situation is much more complex in the procephalic neuroectoderm and the
brain. The insect brain develops highly organized neuropil structures, such as
the mushroom bodies and the central complex (e.g.
Bullock and Horridge, 1965;
Hanesch et al., 1989;
Hanström, 1928;
Strausfeld, 1976), that are
required for behavioural functions such as olfactory learning and memory or
the control of locomotor activity (e.g.
Heisenberg, 1998;
Strauss and Heisenberg, 1993);
these structures have no equivalents in other ganglia. The key towards
elucidating the origin of these structures lies in an understanding of the
segmental organization of the brain. However, the segmental pattern in the
head is highly derived and its metameric organization has been intensely
debated (e.g. Boyan and Williams,
2000; Haas et al.,
2001; Hirth et al.,
1995; Jürgens et al.,
1986; Rempel,
1975; Rogers and Kaufman,
1996; Schmidt-Ott et al.,
1994). In Drosophila the expression of engrailed
and wingless argues for the existence of four pregnathal segments:
the intercalary, antennal, ocular and labral segments
(Schmidt-Ott et al., 1994;
Schmidt-Ott et al., 1995;
Schmidt-Ott and Technau,
1992). Although it has been suggested that each head segment
contributes to the brain (Schmidt-Ott and
Technau, 1992), the arrangement and boundaries of the
corresponding neuromeres, and the origin and identities of their progenitor
cells are largely unknown.
Based on a detailed description of the entire population of brain NBs and
their spatiotemporal pattern of segregation from the neuroectoderm
(Urbach et al., 2003), we have
investigated the expression of segment polarity genes and dorsoventral
patterning genes in the procephalic neuroectoderm, as well as in the
individually identified brain NBs through to stage 11, when the full
complement of NBs has formed. The work provides new insight into the
positional cues expressed in the procephalic neuroectoderm and the segmental
organization of the evolving brain. The data strongly support the view that
the pregnathal Drosophila head is composed of four segments, and we
now attribute to each of the four pregnathal segments a corresponding
neuromere. Furthermore, we provide evidence that the protocerebrum consists of
two neuromeres, which derive from the ocular and labral segment. The segmental
character of these neuromeres is less conserved compared with the trito- and
deutocerebrum, deriving from the intercalary and antennal segment. Finally, we
discuss similarities in the expression of dorsoventral patterning genes
between the Drosophila and vertebrate brain.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), hedgehog-lacZ (16E)
(Mohler et al., 1995) (kindly
provided by J. Mohler), mirror-lacZ
(Broadus et al., 1995;
McNeill et al., 1997) (kindly
provided by H. McNeill and M. Simon), muscle segment homeobox-lacZ
(rH96) (Isshiki et al., 1997)
(kindly provided by A. Nose), ventral nervous system defective-lacZ
(kindly provided by F. Jimenez) and wingless-lacZ
(Broadus et al., 1995).
Staging, flat preparation and mounting of embryos
Staging of the embryos was carried out according to Campos-Ortega and
Hartenstein (Campos-Ortega and
Hartenstein, 1997); additionally, we used the trunk NB pattern
(Doe, 1992) as a further
morphological marker for staging. Flat preparations of the head ectoderm of
stained embryos and mounting were carried out as described previously
(Urbach et al., 2003).
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), rabbit-anti-Deadpan (1:300)
(Bier et al., 1992) (kindly
provided by H. Vaessin), mouse-anti-ß-Galactosidase (1:500, Promega),
rabbit-anti-ß-Galactosidase (1:2500, Cappel), rat-anti-Gooseberry-distal
(16F12 and 10E10, 1:2) (Zhang et al.,
1994) (kindly provided by B. Holmgren), mouse-anti-Invected
(4D9,1:4) (Patel et al., 1989)
(Developmental Studies Hybridoma Bank), mouse-anti-Ladybird early (1:2)
(Jagla et al., 1997) (kindly
provided by K. Jagla), rabbit-anti-Muscle segment homeobox (1:500; kindly
provided by M. P. Scott), rabbit-anti-Ventral nervous system defective
(1:2000) (McDonald et al.,
1998) (kindly provided by F. Jimenez) and mouse-anti-Wingless
(1:10, Developmental Studies Hybridoma Bank), anti-DIG-AP (1:1000, Roche). 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 diluted 1:500.
Whole-mount in situ hybridization
DIG-labelled intermediate neuroblast defective (ind) RNA
probe (kindly provided by M. P. Scott) was synthesized with T7 polymerase and
HindIII linearized pNB40-ind as a template according to the
manufacturers protocol (Roche). The hybridization on embryos was performed as
described previously (Plickert et al.,
1997; Tautz and Pfeifle,
1989).
Documentation
Embryos were viewed under a Zeiss Axioplan equipped with Nomarski optics
using 40x, 63x and 100x oil immersion objectives. Pictures
were digitized with a CCD camera (Contron progress 3012) and different focal
planes were combined using Adobe Photoshop 6.0. Semi-schematic presentations
are based on camera lucida drawings.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Broadus et al., 1995;
Chu-LaGraff and Doe, 1993;
Skeath et al., 1995;
Zhang et al., 1994). In the
pregnathal head region the expression domains of segment polarity genes are
less obvious, but previous analysis of engrailed and
wingless expression in the head peripheral ectoderm, and of PNS
mutant phenotypes, support the existence of four pregnathal segments in
Drosophila: the intercalary, antennal, ocular and labral segments
(Schmidt-Ott et al., 1994;
Schmidt-Ott et al., 1995;
Schmidt-Ott and Technau,
1992). However, the identity and organization of brain structures
deriving from these segments is still obscure. In order to obtain evidence
concerning the number and extent of the brain neuromeres, and to map the
position of their boundaries, we analysed the expression of segment polarity
genes, including wingless, hedgehog, gooseberry-distal, engrailed,
invected and mirror. The spatiotemporal pattern of their
expression was traced in the neuroectoderm and in the NB-layer until stage 11,
when all brain NBs are formed. The data (detailed in Figs
1,
2 and
4) show that segmental
expression is retained for most of the investigated segment polarity genes in
both the developing head ectoderm (procephalon) and brain NBs, providing
landmarks for the definition of segmental domains within the developing brain
NB pattern.
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; Poole et al.,
1985), from which NBs of row 6 and 7 and NB1-2 derive
(Broadus et al., 1995). In the
pregnathal head (neuroectoderm: Fig.
1B,C,E,F; Fig.
2A,C,E,J,K,L) (NBs: Fig.
1A,D; Fig.
2B,D,F,I,M) we find en expression as follows: from late
stage 8 in the posterior ectoderm of the antennal segment (en
antennal stripe; en as) from which four deutocerebral NBs (Dv8, Dd5, Dd9,
Dd13) delaminate; from stage 9 in a small ectodermal domain in the posterior
part of the ocular segment, the en head spot (en hs), from which two
protocerebral NBs (Ppd5, Ppd8) evolve; and from stage 10 in an ectodermal
stripe in the posterior intercalary segment (en intercalary stripe;
en is), which gives rise to three to four tritocerebral NBs (Tv4, Tv5, Td3,
Td5). Furthermore, from stage 11 onwards, En is weakly detected in the
anteriormost ectoderm of the procephalon corresponding to the region of the
`anterior dorsal hemispheres' (en dh)
(Rogers and Kaufman, 1996;
Schmidt-Ott and Technau, 1992)
(Fig. 2I-N) (see also
Urbach et al., 2003). We
identified about 10 weakly En-positive NBs, which delaminate from the
en dh (Fig. 2I,M).
Thus, consistent with earlier results
(Schmidt-Ott and Technau,
1992), we find that all four pregnathal head segments contribute
to the early embryonic brain. The spatial distribution of the En-positive NBs
closely corresponds to the en domains of their origin in the
ectoderm. This suggests they demarcate the posterior borders of the respective
brain neuromeres (Fig. 4).
In the trunk, hedgehog (hh) matches en
expression (Mohler and Vani,
1992; Tabata et al.,
1992). This is also the case for the intercalary segment in the
pregnathal head ectoderm (Fig.
1C,F; Fig. 2K). By
contrast, the En-positive antennal stripe and head spot are only subfractions
of the large hh-lacZ domain, which, between stage 9 and 10,
encompasses the antennal segment and the posterior part of the ocular segment.
We find that all NBs delaminating from this domain express hh-lacZ
(Fig. 1A,D). From stage 10
onwards, en expressing NBs maintain a strong hh-lacZ signal,
whereas hh-lacZ subsequently diminishes in the neuroectoderm and in
NBs between the en antennal stripe and head spot (compare
Fig. 1D,F with
Fig. 2I,J). Additionally,
hh-lacZ-expressing NBs positioned dorsally to the
en/hh-lacZ-co-expressing Ppd5 and Ppd8 (both NBs demarcating part of
the posterior border of the ocular neuromere), appear to prolong the boundary
between the deuto- and protocerebrum in the dorsal direction
(Fig. 1D,
Fig. 2I,
Fig. 4).
From late stage 8 onwards, Wingless (Wg) protein is expressed in a
neuroectodermal domain spanning a broad area of the ocular and the anterior
antennal segment (and in the invaginating foregut) (see also
Baker, 1988;
van den Heuvel et al., 1989).
This becomes clearer in En/Wg double labelling at stage 9, revealing that the
en hs is localized within this Wg domain
(Fig. 1B). In contrast to
earlier observations (Richter et al.,
1998; Younossi-Hartenstein et
al., 1996), we find that, at that stage, Wg is already detectable
in about 4-5 protocerebral NBs (Pcd6, Pcd15, Pcd7, Ppd3;
Fig. 1A), derived from the
region with strongest Wg expression [which later corresponds to the
wg head blob; for nomenclature of wg expression domains in
the procephalic ectoderm, see Schmidt-Ott and Technau
(Schmidt-Ott and Technau,
1992)]. Furthermore, Wg is faintly expressed in the deutocerebral
Dd7 (Fig. 1A) emerging from the
antennal part of the Wg domain (Fig.
1B), which corresponds to the later wg antennal stripe
(Fig. 1E,
Fig. 2G,J). By stage 10, when
the wg head blob is clearly distinguishable from the wg
antennal stripe (Fig. 1E),
about 10-12 Wg-positive NBs have emerged from this domain
(Fig. 1D). In addition, we
found a small, spot-like wg domain in the intercalary segment
(Fig. 1E; wg
intercalary spot) from which a single NB (Td4) delaminates
(Fig. 1D). Thus, all three
wg domains, the intercalary, antennal and ocular (head blob),
contribute to the anlage of the brain. From late stage 9 an additional
wg domain is visible in the ectodermal anlage of the clypeolabrum
(Fig. 1B,E,
Fig. 2G,J), which is the
wg counterpart to the En/Inv-positive region in the `dorsal
hemispheres' [wg labral spot in Schmidt-Ott and Technau
(Schmidt-Ott and Technau,
1992)]. Upon double labelling for either asense or
deadpan (both are general markers for neural precursor cells) and
wg, in embryos between stage 9 and 11 we could not identify any NB
emerging from the wg labral spot. By stage 11 the number of
wg expressing NBs originating from the ocular head blob has increased
to about 16-20 (Fig. 2H,I),
which is more than 25% of the total number of identified protocerebral NBs.
Three Wg-positive NBs are identified in the deutocerebrum and one in the
tritocerebrum (Fig. 2I).
The gooseberry (gsb) locus encodes two closely related
proteins, Gsb-distal (Gsb-d) and Gsb-proximal
(Baumgartner et al., 1987;
Bopp et al., 1986), which are
both expressed in the developing ventral nerve cord
(Gutjahr et al., 1993;
Ouellette et al., 1992). Gsb-d
is segmentally expressed at high levels in all row 5 and 6 NBs, as well as in
a median row 7 NB (NB 7-1) (Broadus et
al., 1995; Zhang et al.,
1994). We analysed the expression of gsb-d during early
neurogenesis in the head region, and found segmental expression of Gsb-d to be
conserved in parts of the pregnathal head ectoderm and deriving NBs (for
details see Fig. 1A,B,D,E;
Fig. 2C,D,I,J). Gsb-d/En double
labelling show that the gsb-d intercalary and antennal stripes are
expressed anteriorly to the corresponding en stripes, and are partly
overlapping with the en stripes
(Fig. 1B,E, Fig. 2C,J). Consequently, NBs
from the posterior part of the gsb-d stripe in the tritocerebrum and
deutocerebrum co-express en (Td3, Dd5;
Fig. 1D,
Fig. 2D,I), and those from the
anterior part co-express wg (Td4, Dd1 and Dd7; as seen in Gsb-d/Wg
double labelling; Fig. 1A,D,
Fig. 2I, and data not shown),
resembling the situation in the ventral nerve cord. However, Dd8 and all
Wg-positive protocerebral NBs do not co-express Gsb-d (except for Ppd3 which,
like Ppd10, transiently expresses gsb-d during stage 10;
Fig. 1D). Gsb-d can also be
detected at a low level in ganglion mother cells of the respective NBs, but
fades away in NBs and their progeny during germ band retraction. Expression of
the protein in the brain is completely downregulated at stage 13 (data not
shown).
In the trunk, mirror (mirr)-lacZ is expressed in
segmental ectodermal stripes giving rise to mirr-lacZ-positive NBs of
row 2 and several NBs that flank row 2 at stage 11
(Broadus et al., 1995;
McNeill et al., 1997). The
pattern of mirr-lacZ expression in the procephalic neuroectoderm and
brain NBs differs significantly from the trunk. We find no evidence of a
segmental arrangement of mirr-lacZ expression in the procephalon (for
details, see Fig. 1A,C,D,F,
Fig. 2E,F,I,K). Interestingly,
regarding the DV axis, mirr-lacZ is mainly limited to the ventral
part of the pNR and corresponding NBs (as confirmed by mirr-lacZ/Vnd
double staining, although there is a faint dorsal mirr-lacZ
expression, in the region of the later invaginating optic lobe anlage;
Fig. 1A,C,D,F,
Fig. 2I,K), and is, at stage
9/10, roughly complementary to en, wg and gsb-d expression,
the domains of which are mainly confined to intermediate and dorsal regions of
the pNR (Fig. 1B,C,E,F). At
stage 11, expression extends towards the dorsal part of the antennal
neuroectoderm (Fig. 2E,K) and
is observed in all NBs of the ventral deutocerebrum, as well as in two
tritocerebral (Tv5, Td8) and four ventral, protocerebral NBs (Pad1, Pcv1,
Pcv2, Pcv3; Fig. 2F,I).
Although expression is also found in the clypeolabrum
(Fig. 2E,K), we did not
identify mirr-lacZ-positive labral NBs.
Expression of dorsoventral patterning genes during early brain
development
In addition to the segment polarity genes, the dorsoventral patterning
genes ventral nervous system defective (vnd),
intermediate neuroblast defective (ind) and muscle
segment homeobox (msh) have been shown to confer positional
information to the truncal neuroectoderm, which also contributes to the
specification of NBs (reviewed by Skeath,
1999). For the head and brain, a detailed analysis of the
expression of these genes has not yet been undertaken. In order to elucidate
their putative role in patterning the head and brain, we analysed the
expression of vnd, ind and msh in the procephalic ectoderm
and NBs in the early embryo (until stage 11). Although our data are consistent
with their role in dorsoventral patterning being principally conserved in the
procephalon, we also find significant differences in their patterns of
expression compared with the trunk (as outlined in the following and in Figs
3,
4).
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; McDonald et al.,
1998; Mellerick and Nirenberg,
1995), and is by stage 11 detected in all ventral and two
intermediate NBs of the ventral nerve cord. Interestingly, the latter
co-express en and are located in the posterior compartment of each
truncal neuromere (Chu et al.,
1998; McDonald et al.,
1998; Shao et al.,
2002; Uhler et al.,
2002). At gastrulation the ventral longitudinal vnd
domain reaches anteriorly across the cephalic furrow into the procephalic
neuroectoderm (data not shown). By stage 9, vnd maps in the ventral
neuroectoderm of the prospective intercalary, antennal and ocular segment
(which is confirmed by En/Vnd double labelling;
Fig. 3C) and is observed in
ventral NBs of the antennal (Dv2, Dv3, Dv6) and ocular neuromere (Pcv1, Pcv3,
Pcv6, Ppv2) (Fig. 3A). It
appears as if the dorsal part of the Vnd-positive antennal neuroectoderm
partly co-expresses ind at that stage
(Fig. 3C), but the NB Dd1,
which emerges from this ectodermal region expresses only ind and not
vnd (Fig. 3A). This is
possibly due to the transient expression of vnd in most parts of both
the ventral antennal ectoderm (compare Fig.
3C with 3D) and corresponding NBs: by stage 10 Vnd is detected in
the ventral Dv2, Dv4 and Dd5, but is already downregulated in Dv3 and Dv6, and
by stage 11 it is confined to Dd5 and the new Dv8
(Fig. 3B,E,L). As a consequence
of the downregulation of vnd (compare
Fig. 3C,D,F,K), some ventral
deutocerebral NBs, which delaminate between stage 9 and 11 from this domain
were not observed to express vnd (e.g. Dv1, Dv5, Dv7;
Fig. 3B,D,E,L). By stage 11 Vnd
is seen in four tritocerebral (Tv2, Tv3, Tv4, Tv5), in two deutocerebral (Dd5,
Dv8), and in a cluster of about 13 protocerebral NBs
(Fig. 3E,L). Interestingly,
vnd expression expands along the posterior border of the en
intercalary stripe (en is), and is also significantly extended dorsally into
the en antennal stripe (Fig.
3D,F,K) and the NBs delaminating from there. The fact that
vnd and en are co-expressed in Tv5 and in Dd5, Dv8
(Fig. 3L,
Fig. 4) is in agreement with
findings in the ventral nerve cord, where these genes are co-expressed in two
intermediate NBs (Chu et al.,
1998; Shao et al.,
2002). This indicates that vnd demarcates the ventral
part of the posterior border in trunk as well as in brain neuromeres.
Furthermore, the posterior border of the ocular vnd domain (including
the NBs Pcv1, Pcv2, Pcv3, Ppv1, Ppv2, Ppv3) abutts dorsally the En-positive
NBs Ppd5 and Ppd8 (deriving from the en head spot;
Fig. 1D, Fig. 3E,K,L), supporting the
view that these NBs demarcate the posterior border of the ocular neuromere
(Fig. 4).
intermediate neuroblast defective (ind) is expressed in
the blastoderm in a bilateral longitudinal column (intermediate column
neuroectoderm) just dorsal to the vnd domains. In the trunk, at stage
9 (when ind mRNA is no longer present in the neuroectoderm), it is
expressed in all intermediate NBs and finally, at stage 11, it is confined to
the NB 6-2 (Weiss et al.,
1998). In the head, at stage 9, ind is detected in an
intermediate longitudinal ectodermal domain in the intercalary segment
(ind is; Fig. 3C), and
weakly in an intermediate ectodermal patch in the antennal segment
(ind as; Fig. 3C) as
well as in the deutocerebral NB Dd1 which develops from this patch
(Fig. 3A). At the same stage,
we observed a further signal in a dorsal ectodermal patch of the ocular region
(ind oc, Fig. 3C). The
ectodermal ind patches in the intercalary, antennal and ocular
segments are both separate from each other and from the ind domain in
the trunk (Fig. 3C,D,I,F).
Interestingly, ind mRNA is significantly longer present in the
ectoderm of the intercalary and mandibular segment, when compared with the
antennal segment and the trunk ectoderm (data not shown). This presumably
mirrors the delayed onset of neurogenesis in both segments (see also
Urbach et al., 2003). Until
stage 10, five NBs derive from the three ind patches: Td1, Td2, Td3,
from the intercalary, Dd1 from the antennal and Ppd13 from the ocular
ind patch (Fig. 3B,D).
Subsequently, the ocular ind patch enlarges but never reaches the
ocular vnd domain (Fig.
3F), and by stage 11 about four additional Ind expressing NBs
(Pcd7, Pcd13, Ppd6, Ppd9) are identifiable
(Fig. 3D,E).
muscle segment homeobox (msh) expression is first
detected at the blastoderm stage in discontinuous patches in the dorsolateral
part of the neuroectoderm, which later extend and form a bilateral
longitudinal stripe (D'Alessio and Frasch,
1996); this domain gives rise to the lateral NBs of the ventral
nerve cord (Isshiki et al.,
1997). We detected at stage 7 msh expression anterior to
the cephalic furrow (data not shown), which expands until stage 9 to cover, as
a broad domain, the dorsal ectoderm of the intercalary and the antennal
segment (Fig. 3C). As evidenced
by Msh/Inv double labelling during stage 9 and stage 11, the anterior border
of the msh domain coincides with the posterior border of the en hs
(Fig. 3C,D,F,G). This suggests
that msh expression in the pregnathal region is restricted to the
intercalary and antennal segments, and matches the border between the antennal
and ocular segment. This is further supported by Msh/hh-lacZ double
labelling (data not shown) in stage 11 embryos, using hh as a marker
for the posterior border of the ocular segment (for hh expression,
see above and Figs 1,
2). All identified brain NBs
delaminating from the dorsal intercalary and antennal neuroectoderm express
msh (Fig. 3A,B,E,H).
This suggests that during early neurogenesis, msh controls dorsal
identities of the procephalic neuroectoderm and brain NBs, as was shown for
the ventral nerve cord (Isshiki et al.,
1997). In the ventral nerve cord, most glial precursor cells
(glioblasts and neuroglioblasts) derive from the dorsal neuroectoderm
(Schmidt et al., 1997), and
express msh (Isshiki et al.,
1997). In the intercalary segment of the early brain, we
identified two glial precursors (Td4 and Td7) (see
Urbach et al., 2003).
Interestingly, both precursors are also located dorsally and express
msh. At least until stage 11 we do not find msh expression
in the preantennal segments.
Expression of DV patterning genes differs in the head and trunk
neuroectoderm
Comparing the expression of DV patterning genes in the trunk and
procephalic region we observed the following significant differences.
), it is
not expressed in the preantennal head ectoderm
(Fig. 3C-H). )]. Yet,
this ectodermal region gives rise to Dd1 which expresses ind but not
vnd (Fig. 3A). ). This implies that other, still unknown factors might be involved in the DV patterning of the anterior head neuroectoderm and protocerebrum.
Segmental boundaries in the early embryonic brain
With regard to the expression of the segment polarity genes en, hh,
wg and gsb-d, as well as the DV patterning genes msh
and vnd, we propose that the procephalic (pregnathal) neuroectoderm
gives rise to four brain neuromeres: the tritocerebrum, the deutocerebrum, the
ocular and the labral neuromere. These tightly fused neuromeres form a
supraoesophageal brain hemisphere on either side. The ocular and labral
neuromeres represent the most prominent part of the brain which is
traditionally referred to as the protocerebrum.
The detailed analysis of the dynamic expression of these genes in the
procephalic neuroectoderm and in the identified brain NBs allows us to map the
boundaries of the brain neuromeres (summarized in
Fig. 4). The posterior border
of the tritocerebrum is clearly represented by the en- and
hh co-expressing NBs Tv4, Tv5, Td3, Td5. In the antennal and
preantennal neuroectoderm the expression of en, hh, wg and
gsb-d is largely restricted to intermediate and dorsal regions, and
NBs deriving from there. Thus, regarding segment polarity genes, a clear
demarcation of the antennal and preantennal neuromeres is only possible for
the intermediate and dorsal, but not for the ventral domains. vnd is
observed to be co-expressed with en in some tritocerebral (Tv5) and
deutocerebral NBs (Dv8 and Dd5), located at intermediate DV positions. This is
consistent with observations in the trunk, where vnd expression is
dorsally expanded into each en domain in the neuroectoderm, as well
as at the level of NBs (Chu et al.,
1998). We therefore suggest that the (transiently) vnd
expressing NBs Dv2 and Dv4, which follow Dd5 and Dv8 ventrally, demarcate the
ventral part of the posterior border of the deutocerebrum. The intermediate
part of this border is defined by the en/hh/vnd-co-expressing Dv8,
Dd5, and the dorsal part by the en- and hh-co-expressing Dd9
and Dd13. For the posterior border of the ocular neuromere, we propose the
following. Under the assumption that vnd expression also marks the
posterior compartment in this neuromere, the vnd expressing NBs Pcv1,
Pcv2, Pcv3, Ppv1, Ppv2 and Ppv3 would demarcate the ventral part of this
border. The intermediate part is defined by the en/hh-co-expressing
Ppd5 and Ppd8, and the dorsal part by the Hh-lacZ-positive NBs Ppd10,
Ppd11, Ppd15 and Ppd16. Interestingly, the anterior border of the msh
domain abutts exactly on the posterior ocular segmental border, indicating
that msh expression is confined to the trito- and deutocerebrum.
inv expression is observed in about 10 NBs deriving from the most
anterior part of the protocerebral anlage, a region that corresponds to the
En-positive `dorsal hemispheres' (en dh)
(Schmidt-Ott and Technau,
1992). We suggest that these NBs represent the neural correlate of
the labral segment. The existence of a labral neuromere deriving from the
en dh has already been discussed by Schmidt-Ott and Technau
(Schmidt-Ott and Technau,
1992). This fourth brain neuromere seems to be of rudimentary
character as it is confined to the posterior segmental compartment
(considering that en/inv is normally expressed in the posterior
compartment), and we did not find NBs anterior to en dh. Thus, the
wg domain in the clypeolabral ectoderm, which is located immediately
anterior to the en dh does not give rise to brain NBs
(Fig. 2I,J). The existence of
four brain neuromeres, in the spatial orientation shown, is furthermore
substantiated by the segmental expression of other genes like gsb-d
(Fig. 1D,
Fig. 2J), sloppy paired
1 and ladybird (see Urbach
and Technau, 2003).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Schmidt-Ott et al., 1995;
Schmidt-Ott and Technau,
1992). However, a detailed description of related brain neuromeres
was still lacking. In order to identify positional cues and segmental
boundaries during early brain development, we analysed the expression of five
different segment polarity genes (en, wg, hh, mirr and
gsb-d) and three DV patterning genes (msh, ind and
vnd) in the procephalic neurogenic region of the ectoderm, as well as
in the entire population of brain NBs derived from this region. We focused our
analysis on the developmental stages 9-11 for the following reasons: (1) the
complex morphogenetic reorganization during the process of head involution has
not yet taken place; (2) late stage 11 represents a phylotypic stage at which
the head pattern is most clearly displayed
(Jürgens and Hartenstein,
1993) and the full complement of brain NBs has formed
(Urbach et al., 2003), except
the primordia of the optic lobes (which develop after stage 11 and fuse
secondarily with the brain) (Green et al.,
1993); (3) it is possible to work on the level of identified brain
NB; and (4) it is also possible to correlate gene expression in the outer
ectoderm and in the evolving NBs. Our data clearly support the view that the pregnathal head consists of four segments (antennal, intercalary, ocular and labral). Furthermore, we were able to attribute to each of the four pregnathal head segments a corresponding neuromere. All segment polarity genes are segmentally expressed in the pNR as well as in brain NBs, except mirr, the segmental expression of which is not overt. wg and gsb-d are partly overlapping, and are expressed anterior to the respective en domains, which are colocalized with hh. The expression of these genes is either mainly confined to intermediate and dorsal regions of the antennal and ocular segment (in case of en, wg and gsb-d) or is at least stronger (hh) in these parts of the pNR. Consequently, with regard to segment polarity genes there is a clear segmental demarcation, which is limited to intermediate and dorsal parts of the respective neuromeres, but it remains unclear in their ventral parts (except in the tritocerebrum). Surprisingly, we find that the DV patterning genes vnd and msh endorse a separation of brain neuromeres in AP axis. As outlined above, vnd expression demarcates the ventral part of the posterior border of the tritocerebrum, deutocerebrum and ocular neuromere, and msh the dorsal anterior border of the deutocerebrum. Thus, based on the expression of segment polarity genes (en/inv, hh) and DV patterning genes (vnd, msh) we provide for the first time a reconstruction of segmental boundaries in the developing brain on the level of identified cells (Fig. 4).
The protocerebrum is formed by the ocular segment and the posterior
compartment of the labral segment
The segmental organization of the anterior head, in particular the origin
of the labrum, the existence of a corresponding segment and its position at
the anterior pole, are central issues of a long-lasting debate concerning head
segmentation (e.g. Boyan et al.,
2002; Haas et al.,
2001; Jürgens and
Hartenstein, 1993; Rogers and
Kaufman, 1996; Schmidt-Ott et
al., 1994; Scholz,
1998) (reviewed by Rempel,
1975). Consequently, the segmental origin of the protocerebrum,
the largest and most anterior portion of the brain, has been a matter of
debate and there is disagreement about whether it can be assigned to the
labral and/or the ocular segment (equivalent to the acron). en
expression in the en dh has been attributed to the labral segment
(Schmidt-Ott and Technau,
1992), the existence of which is further substantiated by PNS
phenotypes in head gap mutants
(Schmidt-Ott et al., 1994). We
identify about 10 NBs that derive from this domain and weakly express
en. Immediately anterior to the en dh, within the
clypeolabral ectoderm, we find the genes wg (see also
Schmidt-Ott and Technau,
1992), gsb-d, lbe and slp1 (see
Urbach and Technau, 2003) to
be expressed, but we observed that these domains do not contribute to the
brain. The spatial pattern of expression of these genes confirms the
following: the anteroposterior orientation of a labral segment, as proposed by
Schmidt-Ott and Technau (Schmidt-Ott and
Technau, 1992); and a parasegmental character of the border
between the en dh and the labral wg domain, supporting the
view that the en dh is the en-expressing part of the labral
segment. We therefore conclude 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). As en expression
delimits the posterior compartment of each segment
(Kornberg et al., 1985), the
labral neuromere appears to be confined to the posterior compartment.
The protocerebrum develops prominent neuropile structures such as the
central complex and the mushroom bodies
(Hanesch et al., 1989;
Strausfeld, 1976). On
comparative morphological grounds, the protocerebrum in arthropods has been
subdivided into the archicerebrum and prosocerebrum. Accordingly, the
archicerebrum, which bears the optic lobes and mushroom bodies, belongs to the
acron (or ocular segment) (Schmidt-Ott and
Technau, 1992), and the prosocerebrum, which comprises the
remainder of the protocerebrum (including the central complex and the
neurosecretory cells of the pars intercerebralis) belongs to the labral
segment (Larink, 1970;
Malzacher, 1968;
Scholl, 1969) (for a review,
see Rempel, 1975). We
identified the progenitor cells of the mushroom bodies to be part of the
ocular neuromere (R.U. and G.M.T., unpublished), supporting the view that the
mushroom bodies are indeed neuropil structures of the ocular segment or
archicerebrum. Consequently, the identified labral NBs would be progenitors of
neurones of the pars intercerebralis. This appears likely because the
en dh during further embryogenesis becomes displaced in a brain
region corresponding to the pars intercerebralis of postembryonic stages
(Fig. 2M). In
Drosophila, little is known about the embryonic origin of the central
complex. In the grasshopper, it was recently documented that NBs in the pars
intercerebralis contribute neurones to the central complex
(Boyan and Williams, 1997).
Taking into consideration that the identified labral NBs presumably represent
the progenitors of cells of the pars intercerebralis and that the fundamental
`bauplan' of the brain is believed to be conserved among insects
(Boyan et al., 1993;
Nassif et al., 1998), we
suggest that, in Drosophila progeny cells of labral NBs participate
in the formation of the central complex.
The segmental character of the tritocerebrum and deutocerebrum is
more conserved than that of the ocular and labral neuromere
In the trunk, the neuroectoderm and NB pattern of each hemisegment is
subdivided by the activity of segment polarity genes into transverse rows and
by the activity of DV patterning genes into longitudinal columns (for a
review, see Skeath, 1999). We
find that this 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 intercalary neuroectoderm and neuromere
are subdivided by en, hh, wg and gsb-d expression into
transverse-like rows and by msh, ind and vnd into
longitudinal columns. Analysis of other genes that are segmentally expressed
in the trunk CNS, e.g. slp1 (Bhat
et al., 2000; Cadigan et al.,
1994a; Cadigan et al.,
1994b), ems (Hartmann
et al., 2000) and lbe (R.U. and G.M.T., unpublished),
provides further support for the notion that the tritocerebrum behaves like a
reduced trunk neuromere (see Urbach and
Technau, 2003). Similarly, this orthogonal pattern of segment
polarity and DV patterning gene expression appears to be essentially retained
in the antennal neuroectoderm and deutocerebrum. However, it appears less
conserved compared with the tritocerebrum because en, wg and
gsb-d (and slp1) expression is confined to
intermediate/dorsal sites, ind is restricted to one NB and
vnd is only transiently expressed. The orthogonal expression pattern
of both gene groups is to a minor extent, if at all, conserved in the
posterior half of the ocular neuromere. Owing to the lack of msh
expression, a dorsoventral polarity is less obvious and most ocular NBs do not
express any DV patterning gene. Finally, conservation of this pattern is not
evident in the labral segment. Although some segment polarity genes are
expressed in the labral ectoderm, expression of DV patterning genes is missing
(except for the two vnd-positive NBs, Pav1 and Pcv4, at the border to
the ocular neuromere).
In this context, it is interesting to note that the head has been claimed
to be composed of two distinct domains, an anterior terminal domain and a
segmented region (Finkelstein and
Perrimon, 1991). Both domains require high levels of Bicoid
protein as an anterior determinant
(Driever and Nüsslein-Volhard,
1988; Struhl et al.,
1989), but the anterior terminal domain, which encompasses the
labral segment and the acron (which is equivalent to the ocular segment)
(Schmidt-Ott and Technau,
1992), is primarily specified by a signalling pathway mediated by
the receptor tyrosine kinase TORSO
(Klingler et al., 1988;
Sprenger and Nüsslein-Volhard,
1992). Zygotic target genes which become activated by this
signalling pathway (reviewed by Perrimon
and Desplan, 1994) are the gap genes hkb and tll
(Brönner et al., 1994;
Pignoni et al., 1990). For
tll, it has been shown that (part of) its anterior, blastodermal
expression is necessary for the development of the protocerebrum, which is
missing in tll mutants (Pignoni
et al., 1990; Rudolph et al.,
1997; Strecker et al.,
1988). tll represses hb and ftz and may
thus function in the head as an `anti-segmentation' gene
(Reinitz and Levine, 1990). We
find that tll expression, which covers the ocular and labral
neuroectoderm (the latter of which coincides with the region of the
en dh) and emerging NBs (Urbach
and Technau, 2003) (see also
Rudolph et al., 1997), closely
corresponds to that part of the early brain where segmental features are
largely obscure. A coordinated, orthogonal expression of segment polarity and
DV patterning genes within the ocular and labral neuroectoderm is not obvious,
and the existence of putative serially homologous NBs in those regions of the
brain is less evident (Urbach and Technau,
2003). This implies that tll might be a component crucial
for the suppression of segmental characteristics in the ocular and labral
neuromere. Furthermore, crossregulatory interactions among the segment
polarity genes in the pregnathal head differ from those in the trunk and are
unique for each pregnathal segment
(Gallitano-Mendel and Finkelstein,
1997).
For a part of the segmented head (mandibular, intercalary and antennal) it
was proposed that a combinatorial expression of the cephalic gap genes
otd, ems and buttonhead
(Finkelstein and Perrimon,
1990; Wimmer et al.,
1993) mediates metamerization by acting directly on segment
polarity genes, thereby omitting the intermediate function of pair rule genes
(Cohen and Jürgens, 1990)
(for a review, see Finkelstein and
Perrimon, 1991). More recent data indicate that, in the segmental
patterning of this head region, other (intermediate) regulators are involved.
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 combined activity of ems and buttonhead, and the pair
rule gene even skipped, thus integrating inputs from both the head
and trunk segmentation system (Crozatier
et al., 1996; Crozatier et
al., 1999). Such factors might help to explain that trunk specific
segmental characteristics are more conserved in the intercalary and antennal
neuroectoderm and NBs, when compared to the ocular and labral neuroectoderm
and NBs.
Comparison of DV patterning gene expression in the
Drosophila and vertebrate brain
In Drosophila the DV patterning genes subdivide the trunk
neuroectoderm into longitudinal columns (for a review, see
Cornell and Ohlen, 2000;
Skeath, 1999); vnd is
required for the specification of the ventral neuroectodermal column and NBs
(Chu et al., 1998;
Jimenez et al., 1995;
McDonald et al., 1998;
Mellerick and Nirenberg,
1995), ind and msh have analogous functions in
the intermediate and dorsal neuroectodermal columns and NBs, respectively
(D'Alessio and Frasch, 1996;
Isshiki et al., 1997;
Weiss et al., 1998).
Remarkably, homologous genes are found to be expressed in the vertebrate
neural plate and subsequently in the neural tube
(Fig. 5). 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 region; the ind homologs,
Gsh-1/2, are expressed in the intermediate region; and the
msh homologs, Msx-1/2/3, are expressed in the dorsal region
of the neural tube (for a review, see
Arendt and Nübler-Jung,
1999; Cornell and Ohlen,
2000).
|
; Shimeld et al.,
1996; Wang et al.,
1996). The anterior border of the Msx3 domain is
positioned within the rostral region of the dorsal rhombencephalon
(Wang et al., 1996), thus
showing the shortest rostral extension of all vertebrate DV patterning genes.
This displays analogy to msh, 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 (Price et
al., 1992; Shimamura et al.,
1995). 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 Nkx2.2 may be involved in specifying diencephalic
neuromeric boundaries (Price et al.,
1992). 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 homologue Gsh1
show similarities in their expression in the early brain
(Fig. 5). 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 [see Fig.
4 by Valerius et al. (Valerius
et al., 1995)], dorsally to Nkx2.2 [for expression of
Nkx2.2; see Fig. 3 by
Shimamura et al. (Shimamura et al.,
1995)], and in the hindbrain ventrally to Msx3 [see
Fig. 4 by Shimeld et al.
(Shimeld et al., 1996)].
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)
(Valerius et al., 1995).
Correspondingly, in Drosophila we find ind expression in
restricted domains within the gnathocerebrum (R.U. and G.M.T., unpublished),
the tritocerebrum, deutocerebrum and ocular part of the protocerebrum,
demonstrating that the anteriormost extension of ind (and
Gsh1) expression lies between that of msh and
vnd.
Taken together, considering these similarities, we suggest that in the
Drosophila and vertebrate early brain the expression of DV patterning
genes is to some extent conserved, both along the DV axis (as suggested for
the truncal parts of the Drosophila and mouse CNS) and along the AP
axis. Furthermore, in Drosophila we observed that large parts of the
anterodorsal procephalic neuroectoderm and NBs (more than 50% of all
identified brain NBs) lack DV patterning gene expression. Likewise, in the
vertebrate neural tube, gaps between the expression domains of DV patterning
genes have been described, raising the possibility that other genes might fill
in these gaps (Weiss et al.,
1998). How DV fate is specified in the anterior and dorsal part of
the Drosophila procephalic neuroectoderm, and if other genes are
involved, remains to be clarified.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and strong (+)
expression levels of inv, hh-lacZ and wg. Stars indicate
mandibular NBs. (J,K) Segment polarity gene expression in the peripheral
procephalic ectoderm. (L-N) inv expression in the dorsal hemispheres
(dh). (L) Lateral view of late stage 11 head, showing a faint inv
expression in the dh. Note that en expression in the dh is not
detected using en-lacZ and was only observed with anti-Inv antibodies
from late stage 11 onwards. (M) Left half of a head flat preparation. The dh
comprises about 10 Inv-positive NBs (as depicted in I). (N) inv
expression in the brain and sub-oesophageal ganglion (SOG) of a stage 16
embryo (horizontal view). Note, that Inv staining in the dh corresponds to the
pars intercerebralis of postembryonic stages. The secondary head spot (shs)
marks a small group of cells which secondarily separates from the hs (see
Schmidt-Ott and Technau,
1992
