Institut für Genetik, Universität Mainz, D-55099 Mainz, Germany
Author for correspondence (e-mail: technau{at}mail.uni-mainz.de)
Accepted 4 April 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CNS, Brain development, Neuroblasts, Gap genes, Molecular markers, Drosophila
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To shed light on how neural diversity is generated in the
Drosophila brain, it is of importance to know which genes (positional
cues) are expressed within the procephalic neuroectoderm at different stages
of development, and which genes are expressed in each individual brain NB. In
the preceding papers, we have traced the pattern and modes of brain NB
formation (Urbach et al.,
2003), and assigned each NB to a particular brain neuromere by
analysing the expression of segment polarity and dorsoventral patterning genes
(Urbach and Technau, 2003
).
Here, we have analysed in detail the expression of the head gap genes
empty spiracles, hunchback, huckebein, sloppy paired 1 and
tailless; the homeotic gene labial; and many other marker
genes in the procephalic neuroectoderm, as well as in the brain NBs, until
stage 11, when the full complement of NBs has evolved. In total, we provide an
array of more than 40 molecular markers (antibodies, mRNA probes and enhancer
trap lines) that represent the expression of 34 different genes. Most of these
molecular markers are expressed in characteristic domains of the procephalic
neuroectoderm, and all of them are specifically expressed in particular
subsets of brain NBs. Each brain NB forms at a stereotypical time and position
(see also Urbach et al., 2003
)
and each exhibits a reproducible pattern of gene expression. Accordingly, it
is now feasible to identify each brain NB uniquely based on its characteristic
expression of certain molecular markers at different stages of early
neurogenesis (stage 9-11). It is reasonable to assume that these marker genes
are involved in the specification of brain NBs and components of their
corresponding cell lineages. Knowing the individual precursor cells and their
patterns of gene expression under normal conditions, is a prerequisite for the
interpretation of mutant phenotypes, as well as the effects of experimental
manipulations. Furthermore, the combination of molecular markers expressed in
the identified NBs allows the identification of serially homologous NBs in the
brain and ventral nerve cord. Thus, these comprehensive data are useful for
investigating mechanisms leading to cell diversity in the developing embryonic
brain, as well as those mechanisms which make the brain different from the
truncal parts of the CNS.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Staging and mounting of embryos
Staging of the embryos was done according to Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1997); additionally, we used the trunk NB pattern
(Doe, 1992
) as further
morphological markers 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 N. Y. Yan), rabbit-anti-Castor (1:1000;
kindly provided by W. Odenwald), mouse-anti-Dachshund (1:250)
(Mardon et al., 1994
)
(Developmental Studies Hybridoma Bank), rabbit-anti-Deadpan (1:300)
(Bier et al., 1992
) (kindly
provided by H. Vässin), anti-DIG-AP (1:1000, Roche), rat-anti-Empty
spiracles (1:1000) (Walldorf and Gehring,
1992
) (kindly provided by U. Walldorf), rabbit-anti-Eyeless
(1:1000; kindly provided by U. Walldorf), mouse-anti-Fasciclin 2 (1D4, 1:15,
Developmental Studies Hybridoma Bank), mouse-anti-ß-Galactosidase (1:500,
Promega), rabbit-anti-ß-Galactosidase (1:2500, Cappel),
rabbit-anti-Hunchback (1:1000, kindly provided by M. Gonzales-Gaitan),
mouse-anti-Invected (4D9,1:4) (Patel et
al., 1989
) (Developmental Studies Hybridoma Bank),
rabbit-anti-Klumpfuss (1:1000) (Yang et
al., 1997
) (kindly provided by X. Yang and B. Chia), rabbit
anti-Labial (1:100) (Diederich et al.,
1991
) (kindly provided by T. Kaufman), mouse-anti-Ladybird (1:2)
(Jagla et al., 1997
) (kindly
provided by K. Jagla), rat-anti-Orthodenticle (1:20)
(Wieschaus et al., 1992
)
(kindly provided by S. Leuzinger and H. Reichert), rabbit-anti-POU-domain1
(1:500) (Yeo et al., 1995
)
(kindly provided by X. Yang and B. Chia), rabbit anti-Proboscipedia (1:200)
(Cribbs et al., 1992
) (kindly
provided by D. Cribbs), rabbit-anti-Runt (1:500)
(Dormand and Brand, 1998
)
(kindly provided by E. Dormand and A. Brand), rat-anti-Sloppy paired (1:300)
(Cadigan et al., 1994b
)
(kindly provided by W. Gehring) and rabbit-anti-Twin of eyeless (1:400; kindly
provided by U. Walldorf). 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
twin of eyeless (toy) and huckebein
(hkb) RNA probes were synthesized using full-length cDNA of
toy (Czerny et al.,
1999) (kindly provided by T. Czerny and M. Busslinger) and
hkb (kindly provided by G. Brönner); eyeless RNA probes
were generated by PCR amplification using as a 5'primer
5'-CTTGGCTAAAGCTTTCATGAGCAG-3' and as a 3'primer
5'-TGAGTATTTAACAGCCGAAGCTTC-3'; the resulting PCR fragment was
used as a template. DIG-labelled RNA probes were prepared using a
DIG-RNA-labelling mix (Roche) according to the manufacturers protocol. 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.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
tailless (tll) has been shown to be expressed in an
anterior horseshoe-shaped stripe in the cellular blastoderm, which after
gastrulation shows a region of high (`HL domain') and a region of low level of
tll expression (`LL domain'), and at stage 9 covers most of the
protocerebral neuroectoderm (Rudolph et
al., 1997) (see also Fig.
1D). Using a tll-lacZ line
(Rudolph et al., 1997
) at
stage 9 we find tll expression in the developing brain in most
protocerebral NBs (except the dorsoposterior ones;
Fig. 1A). During stages 9-11
tll-lacZ expression expands in the protocerebral neuroectoderm beyond
the En-positive head spot (hs; Fig.
1O,H, Fig. 2I,P).
By stage 11 it is detectable in all protocerebral NBs
(Fig. 2J,M), confirming earlier
data (Rudolph et al., 1997
;
Younossi-Hartenstein et al.,
1997
). In addition, we find tll-lacZ in some ventral and
dorsal deutocerebral NBs (Fig.
2J,M; Table 1),
indicating that tll is not exclusively confined to protocerebral
progenitors.
|
The sloppy paired (slp) locus contains the two related
genes slp1 and slp2
(Grossniklaus et al., 1992).
slp1, which acts as a head gap gene, plays a predominant role in head
formation, while slp2 is largely dispensable
(Cadigan et al., 1994a
;
Grossniklaus et al., 1994
). In
the trunk neuroectoderm, where slp1 has a function as a pair-rule and
segment polarity gene, it is segmentally expressed in neuroectodermal stripes
(Cadigan et al., 1994a
;
Cadigan et al., 1994b
) as well
as in NBs of row 4 and 5 (Bhat et al.,
2000
). We find this segmental appearance of slp1
expression to be conserved in parts of the procephalon. In the blastoderm,
Slp1 protein is detected in a large domain of the procephalon anlage, which
subsequently diminishes in its anterior/ventral part (data not shown). As a
result, only the posterior half of the original slp1 domain remains
as a circumferential ring [`head stripe' according to Grossniklaus et al.
(Grossniklaus et al., 1994
)]
and gets separated from the anterodorsal part [`head cap' according to
Grossniklaus et al. (Grossniklaus et al.,
1992
)]. To follow the dynamics in the Slp1 expression pattern, we
examined Slp1/En double labelling during stages 8-11. We find that the `head
stripe' corresponds to the slp1 stripe of the prospective mandibular
segment (data not shown), and the posterior part of the `head cap' to the
Slp1-positive stripe in the prospective antennal segment (slp1 as;
Fig. 1D). At the beginning of
gastrulation, a new Slp1 ectodermal spot in the anterodorsal procephalon is
observed (Grossniklaus et al.,
1992
); this spot later becomes part of the labral ectoderm (slp1
lr; Fig. 1D). In addition, at
stage 9, three new ectodermal domains become detectable: one stripe anterior
to the en intercalary stripe belonging to the intercalary segment
[slp 1 is, Fig. 1D; `hypopharyngeal stripe' according to
(Grossniklaus et al., 1992
)],
and two small spots in the region of the ocular segment (anterior to the
en head spot; slp1 oc, Fig.
1D). Except for the labral domain, the slp1 domains
contribute NBs to the brain (Fig.
1A,E, Fig. 2M,L;
Table 1). Thus, slp1
is segmentally expressed in the procephalic neuroectoderm and subsets of brain
NBs, resembling the situation in the trunk
(Bhat et al., 2000
;
Cadigan et al., 1994a
;
Cadigan et al., 1994b
). At
stage 11 patchy expression of Slp1 becomes detectable within the ocular and
labral ectoderm (Fig. 2K,P) and
in some underlying ocular and labral NBs
(Fig. 2M). Some of these NBs
initiate slp1 expression after delamination; e.g. Pcv6 and Pcd2
delaminate at stage 9 and do not express slp1 before stage 11
(compare Fig. 1A with
Fig. 2M). Slp1 expression is
observed in the brain until the end of embryogenesis (data not shown).
huckebein (hkb), a terminal gap gene, is first expressed
at the anterior and posterior blastodermal poles, where it is required for the
specification of the endodermal anlagen, and later for the invagination of the
stomodeum (Brönner and Jäckle,
1991; Weigel et al.,
1990
). After gastrulation, hkb becomes transiently
expressed in a repetitive pattern in the trunk neuroectoderm and in eight,
mainly intermediate, NBs per hemineuromere
(Broadus et al., 1995
;
McDonald and Doe, 1997
). In
the procephalic region at the cellular blastoderm stage, we find hkb
expression in a centrally located stripe and a dorsal ectodermal spot (data
not shown). hkb in situ hybridization combined with anti-Inv antibody
staining reveals that during stage 9/10 the hkb stripe covers most of the
antennal ectoderm and reaches into the anterior region of the intercalary
segment, and the hkb spot covers part of the ocular ectoderm
(Fig. 1C,G). During stage 9,
hkb transcript in the ocular spot becomes progressively restricted to
the delaminating protocerebral NBs, Pcv7 and Pcd2
(Fig. 1A,E), and remains
strongly expressed in both NBs until stage 11
(Fig. 2M). In the antennal
domain during stage 10/11 the transcript becomes confined to three to five
deutocerebral NBs. However, using a hkb-lacZ line (5953)
(Broadus et al., 1995
) the
marker is expressed in all deutocerebral NBs at stage 10
(Fig. 1E). At stage 11,
hkb-lacZ was not detectable in Dd8 and Dd11, indicating that
hkb is not a general deutocerebral NB marker
(Fig. 2M). In the
tritocerebrum, hkb is expressed only in Td6 (stage 10;
Fig. 1E) and in Tv1, Td8 (stage
11; Fig. 2M). Thus, although
expressed in a few trito- and protocerebral NBs, hkb expression
appears to be mainly confined to the antennal neuroectoderm and NBs. Compared
with the transcript, which becomes restricted to the NBs during stage 9-11,
hkb-lacZ expression has a longer perdurance in the peripheral
ectoderm and corresponding NBs. By stage 14, most of the hkb
transcript has disappeared and is confined to some deutocerebral cells;
hkb-lacZ is strongly expressed until the end of embryogenesis in
deutocerebral, and at a lower level, in protocerebral cells, the putative
progeny of the identified Hkb-positive brain NBs (data not shown).
hunchback (hb) expression in the anterior half of the
embryo falls below the limit of detection at the beginning of germ band
extension, but accumulates during the extended germ band stage in the CNS
(Tautz et al., 1987), where it
is transiently expressed in early, fully delaminated, trunk NBs (S1 and S2)
and their progeny (Jimenez and
Campos-Ortega, 1990
; Kambadur
et al., 1998
). Antibody staining reveals that, from stage 8
onwards, Hb protein is not detected in the head neuroectoderm, but is very
dynamically expressed in brain NBs. At stage 9, only about half of the
identified deuto- and protocerebral NBs show Hb protein at a detectable level
(Fig. 1A), suggesting that Hb
is not a general marker for early NBs. Correspondingly, we find that Hb
protein is also lacking in particular S1 and S2 NBs of the trunk (data not
shown). In some of the early brain NBs, Hb first becomes detectable at stage
10, after their delamination. For example, the early NBs Pcv9 and Pcd6
delaminate at late stage 8 but do not start Hb expression before stage 10
(Fig. 1A,E). By stage 10, Hb is
expressed in about 26 brain NBs, most of which delaminate between stage 9 and
10 (Fig. 1E). In most of these
NBs, Hb expression is progressively lost, but is observed in an increasing
amount of progeny cells. At stage 11, it is confined to a small subpopulation
of about five tritocerebral and four to six protocerebral NBs
(Fig. 2C,D,M). Thus, as opposed
to the trunk, hb expression in the brain is not limited to early NBs.
Hb is expressed in the brain until stage 15, when it is detected in a few
cells of the protocerebrum (data not shown).
Taken together, among the cephalic gap genes, slp1 appears to
respect segmental boundaries during early neurogenesis of the brain. By
contrast, we find that in the considered period of development (stage 9-11),
the expression of ems, otd and tll does not seem to respect
these borders, which has been claimed in previous reports (for otd
and ems) (Hirth et al.,
1995) (for tll)
(Younossi-Hartenstein et al.,
1997
). All three genes are expressed in NBs deriving from
ectodermal domains that are part of two or three neighbouring segments. For
example, ems is expressed in a small number of NBs comprising about
six posterior ocular and four anterior deutocerebral NBs (all of which derive
from the same ems domain, except Dv3 and Pcv5), and one tritocerebral
NB. Accordingly, ems mutants show defects in the intercalary,
antennal (Hirth et al., 1995
;
Younossi-Hartenstein et al.,
1997
), and the ocular segment (e.g. the en hs is missing)
(Schmidt-Ott et al., 1994
).
Considering that ems is expressed in only a few trito- and
deutocerebral NBs it is remarkable that ems mutants show a deletion
of the tritoand deutocerebrum (Hirth et
al., 1995
). An explanation for this could be that ems
expression, which during earlier development covers the neuroectoderm of the
respective segments, possibly confers specific identities to the arising
trito- and deutocerebral NBs. The lack of these NBs might be responsible for
the loss of NB-specific gene expression
(Hartmann et al., 2000
), and
(secondarily) for the gross morphological defects seen in the ems
mutant brain (Hirth et al.,
1995
). A similar proposal has been made to explain the brain
defects that occur in buttonhead (btd) mutants, although
btd is not expressed in NBs of the corresponding brain regions
(Younossi-Hartenstein et al.,
1997
).
Expression of the homeotic gene labial completely covers the
anlage of the tritocerebrum
We analysed the expression of the homeotic genes proboscipedia
(pb) and labial (lab), both members of the ANTC and
known to be expressed in the head ectoderm and in the brain after
mid-embryogenesis (Hirth et al.,
1998; Pultz et al.,
1988
). Antibody staining against Pb reveals that at stage 11, the
protein is restricted to internal cells of the mandibular segment (presumably
mesodermal cells) and to dorsal ectoderm of the maxillary and labial
appendages (Pultz et al.,
1988
) (data not shown). We did not detect Pb protein in brain
NBs.
lab was described to be expressed in the posterior tritocerebrum
at stage 14 (Hirth et al.,
1998). Using an antibody, we investigated the expression of Lab
protein during early neurogenesis. From stage 9 onwards, Lab is detected in
the ectoderm of the intercalary segment, and presumably in a small part of the
posteroventral antennal segment (Fig.
3B,D,F). At that stage, the only NB expressing Lab protein is Dv2
(Fig. 3A). Double labelling
against En reveal that at stage 11 Lab is expressed throughout the ectoderm of
the intercalary segment (Fig.
3F,G), supporting previous reports
(Diederich et al., 1989
;
Diederich et al., 1991
;
Mahaffey et al., 1989
). The
Lab domain overlaps posteriorly with the en intercalary stripe (en
is), indicating that posterior borders of lab expression and of the
intercalary segment coincide (Fig.
3F,G). The character of the anterior border of the lab
domain is less clear. Dorsally, it runs along the posterior border of the
en antennal stripe (en as); ventrally, however, it reaches the
anterior border of the en as. This suggests, that the anterior border of the
lab domain is segmental in the dorsal region and parasegmental in the
ventral region (Fig. 3F,G).
Interestingly, also for scr and dfd, which are other members
of the ANT-C, it was reported that they initiate expression in a jagged stripe
resolving into a pattern that is dorsally segmental and ventrally
parasegmental (Gorman and Kaufman,
1995
; Rogers and Kaufman,
1996
). All NBs arising from the Lab-positive neuroectoderm express
lab, among them all tritocerebral NBs and two ventral NBs, which we
attributed to the deutocerebrum (Dv2 and Dv4) because they are located on the
same anteroposterior level as the en-expressing Dv8 and Dd5
(Fig. 3A,C,E,H;
Table 1) (see also
Urbach and Technau, 2003
).
|
|
|
Further marker genes expressed in the head neuroectoderm and subsets
of brain NBs
In order to establish further molecular markers that are specifically
expressed in subsets of brain NBs, we investigated the expression of
castor, Fasciclin 2, klumpfuss, ladybird early, POU-domain 1 gene,
runt and unplugged. For details of the spatiotemporal expression
pattern of these genes in the neuroectoderm and brain NBs (stages 9, 10 and
11), we refer to Figs 5,
6,
7 and
Table 1.
|
|
In the ventral nerve cord castor (cas, previously known
as ming), encoding a zinc-finger protein, has been shown to be
expressed in 18 NBs per hemineuromere
(Doe, 1992), including early
(S1-S2) and late delaminating (S3-S5) NBs, and to be involved in cell fate
control within NB lineages (Cui and Doe,
1992
; Mellerick et al.,
1992
). In the procephalon, cas expression is not
detectable before stage10. It is dynamically expressed in the central and
dorsal neuroectoderm of the ocular segment, in the median antennal segment,
and, by stage 11, in the labral segment
(Fig. 5F,I;
Fig. 6A), which is surprising
as cas is not expressed in the neuroectoderm of the trunk
(Cui and Doe, 1992
;
Kambadur et al., 1998
;
Mellerick et al., 1992
). A
proportion of Cas-positive protocerebral and deutocerebral NBs derive from
these domains. Most NBs appear to delaminate from Cas-negative neuroectoderm
(Fig. 5D,G), and start to
express cas at the time of formation, or show a reproducible delay in
the onset of cas expression. The latter may already have produced a
part of their lineage, which likewise has been proposed for early trunk NBs
(e.g. NB7-4) (Cui and Doe,
1992
). At late stage 11, Cas is expressed in about 60% of the
total number of identified brain NBs (Fig.
5G, Fig. 6B).
Using an antibody against the cell membrane glycoprotein Fasciclin 2 (Fas2)
(Grenningloh et al., 1991), we
find that in the procephalic region Fas2 is first expressed by late stage 10
in an ectodermal patch at the border between the intercalary and antennal
segment (Fig. 5E). Later it
also covers the posterodorsal ocular neuroectoderm (including the optic lobe
anlage) (Schmucker et al.,
1997
) and part of the labral ectoderm
(Fig. 5H,
Fig. 6C). Fas2 is also detected
in brain NBs emerging from the antennal and intercalary neuroectoderm
(Fig. 5D,G,
Fig. 6D), and at a low level in
a few dorsal ocular NBs (Fig.
5G,H). It has been found that Fas2 controls proneural gene
activity in the eye/antennal imaginal disc
(Garcia-Alonso et al., 1995
),
raising the possibility that it functions likewise in the procephalic
neuroectoderm. However, we find that Fas2 expression in almost all identified
brain NBs is initiated after delamination from Fas2-negative neuroectoderm,
suggesting that Fas2 in the procephalic neuroectoderm is not involved in the
regulation of proneural genes. It has been shown that Fas2 appears on the
surface of neural somata prior to axon outgrowth; these neurones belong to
`fibre tract founder clusters' that pioneer the main axonal tracts in the
brain (Nassif et al., 1998
).
Considering position and time point of development, we suggest that the
identified Fas2-positive deuto- and tritocerebral NBs (Tv1, Tv2, Td1, Td2,
Td6, Td8; Dv2, Dd9, Dd11) are the precursors of the `D/T fibre tract founder
cluster' (Nassif et al.,
1998
).
In the trunk, the zinc-finger transcription factor Klumpfuss (Klu) is
expressed from stage 10 onwards in an increasing number of NBs, and at stage
11, almost all NBs (except NB2-3 and NB6-4) show nuclear Klu staining
(Yang et al., 1997). We
analysed the expression of Klu in the procephalon using an antibody against
Klu and the P-lacZ enhancer trap strain klu P212
(Yang et al., 1997
) which
basically show an identical expression pattern. Klu is not expressed in the
neuroectoderm. Similar to the situation in the trunk CNS, we first find Klu
protein at a detectable level at stage 9, in a subset of (about 17) brain NBs
(Fig. 5A) and at late stage 11
in almost all brain NBs (Fig.
5G, Fig. 6E,F). For
most NBs, there is a significant delay between birth and onset of klu
expression. Klu also appears to be expressed in ganglion mother cells, as was
shown for the trunk (Yang et al.,
1997
).
ladybird (lb), a tandem of the homeobox genes
ladybird early (lbe) and ladybird late
(lbl), both of which encode transcription factors, show a similar
expression pattern, with lbe activity slightly preceding that of
lbl (Jagla et al.,
1994). At stage 11, both genes are expressed in segmental
repetitive patches in the laterodorsal trunk ectoderm
(Jagla et al., 1997
) and
specifically in one NB per hemineuromere, the lateral NB 5-6 (J. Urban,
personal communication). Using an antibody against Lbe
(Jagla et al., 1997
) we first
observed the protein by stage 10 in three small procephalic patches in the
labral, ocular and antennal ectoderm (Fig.
5E), and at stage 11 in an additional patch of the intercalary
ectoderm (Fig. 5H,
Fig. 6G). Lbe is selectively
expressed in only four brain NBs on either side: one in the tritocerebrum
(Td4), one in the deutocerebrum (Dd7) and two in the protocerebrum (Ppv3,
Pcv8; Fig. 5D,G,
Fig. 6H). Wg/Lbe double
labelling demonstrates that Lbe and Wg expression are colocalized in the
intercalary, antennal and labral ectoderm, and in Td4 and Dd7; remarkably, the
ocular Lbe-positive domain and corresponding NBs (Ppv3 and Pcv8) are Wg
negative [see Fig. 2G,H by
Urbach and Technau (Urbach and Technau,
2003
)]. Lbe protein is detected in the progeny of the identified
brain NBs until the end of embryogenesis (data not shown).
The two closely related Drosophila POU-domain genes, pdm1
(nub FlyBase) and pdm2, are co-expressed in the
developing CNS (before stage 13) and have been shown (at least with respect to
the specification of the first ganglion mother cell of the truncal NB4-2) to
be functionally redundant (Dick et al.,
1991; Yang et al.,
1993
; Yeo et al.,
1995
). pdm1 is expressed in the trunk neuroectoderm
during the first and second wave of NB segregation (stage 8/9), and
transiently in most NBs at stage 10 and 11
(Dick et al., 1991
;
Kambadur et al., 1998
). In the
procephalon the expression of the Pdm1 protein is highly dynamic
(Fig. 5C,D,F,G,I;
Fig. 6I,J). Until stage 10,
Pdm1 is roughly restricted to the neuroectoderm of the antennal and ocular
segments (as confirmed by Pdm1/En double labelling;
Fig. 5C,F). Later, it is also
found in the intercalary and labral ectoderm
(Fig. 5I). At stage 9, NBs
derived from Pdm1-positive neuroectoderm appear to be Pdm1 negative
(Fig. 5A,C) and initiate
pdm1 expression at stage 10 (Fig.
5D) or stage 11 (Fig.
5G). At late stage 11, approximately one half of the brain NBs
(about 52 NBs) express pdm1, including most deuto- and tritocerebral
NBs, as well as central ocular NBs and part of the labral NBs
(Fig. 5G).
Expression of the homeodomain gene unplugged (unpg) in
the trunk starts at stage 8 in the ventral midline
(Chiang et al., 1995) and
becomes detectable in NBs of the ventral nerve cord at late stage 11
(Doe, 1992
). Using an
unpg-lacZ line (1912) (Cui and
Doe, 1995
; Doe,
1992
), we observed unpg expression in the head at stage 9
in a large domain encompassing the intercalary, antennal and most of the
ocular ectoderm (Fig. 5B).
Until stage 11, the expression is gradually lost in the intercalary ectoderm,
but upregulated in the dorsal part of the antennal and adjacent ocular
ectoderm (Fig. 5E,H,
Fig. 6K). In contrast to trunk
NBs, which have already divided several times before expressing unpg
at late stage 11 (Cui and Doe,
1995
), we find unpg-lacZ to be weakly expressed already
at stage 9 in all deutocerebral and almost all protocerebral NBs
(Fig. 5A). At late stage 11, it
is strongly expressed in almost all deutocerebral NBs (except for some ventral
ones), and in some ocular NBs close to the deutocerebral/ocular border
(Fig. 5G, Fig. 6L). Until the end of
embryogenesis, unpg expression is observed in the putative progeny
cells of the unpg-lacZ-positive deuto- and protocerebral NBs (data
not shown).
All embryonic brain neuroblasts are uniquely identified
For thoracic and abdominal segments, it has been previously shown that each
NB acquires a unique identity, which corresponds to a particular position in
the neuroectoderm and (upon delamination) in the subectodermal NB layer, to a
certain time point of its delamination, and to the combination of genes
expressed (Broadus et al.,
1995; Doe, 1992
;
Hartenstein and Campos-Ortega,
1984
). These descriptions have provided an important basis for the
elucidation of mechanisms controlling cell fate specification during early
neurogenesis in the trunk region. In contrast to the truncal CNS, in which the
segmental organization is obvious and the composition of the neuromeres is
almost identical, the brain neuromeres are much more diverse and complex.
Accordingly, information on identified brain cells and their gene expression
is hardly available so far, and thus essential tools for investigating the
mechanisms underlying pattern formation and cell diversity in the brain are
lacking.
In this and the preceding studies
(Urbach et al., 2003;
Urbach and Technau, 2003
), we
provide an array of more than 40 different molecular markers (enhancer trap
lines, antibodies, mRNA probes) characterizing the expression of 34 different
genes in the early procephalic neuroectoderm and in the brain NBs emerging
from it. We show that these marker genes are expressed in specific subsets of
more than 100 brain NBs on either side (as summarized in
Fig. 7 and
Table 1). Based on the
expression of specific combinations of molecular markers, morphological
criteria as well as time and position of NB formation (see accompanying
papers), each NB of the developing embryonic brain is now uniquely identified.
Furthermore, as we analysed the expression of these markers in the
neuroectoderm and in the NBs during various developmental stages, the fate of
the individual brain progenitor cells can be followed through early
neurogenesis. The unambiguous identification of NBs is a prerequisite for
future work on mechanisms that control the specification and lineages of
individual brain NBs.
Implications for regulatory interactions among genes expressed in the
procephalic neuroectoderm and identified neuroblasts
Work on early neurogenesis in the trunk has provided evidence that the set
of genes expressed within a particular proneural cluster of ectodermal cells
specifies the individual fate of the NB it gives rise to. This is especially
the case for segment polarity genes (for a review, see
Bhat, 1999) and DV patterning
genes (for a review, see Skeath,
1999
). We show that these and other genes are also expressed in
specific domains of the procephalic neurogenic region before brain NBs
delaminate (e.g. wg, gsb-d, en, hh, msh, ind, vnd, hkb, ems and
slp1). This implies that these genes might likewise be required for
providing positional information in the procephalic neuroectoderm and for
subsequent specification of individual brain NBs. Furthermore, all of the
molecular markers assayed are expressed in different but overlapping subsets
of brain NBs, and some of them we also observed to be expressed in (part of)
their progeny cells (e.g. en, hh-lacZ, msh-lacZ, ey, ems, hkblacZ,
lbe). Co-expression might hint to regulatory interactions among these
genes within the neuroectoderm, particular NBs, and their lineage. Thus, in
addition to being useful as molecular markers, the linking of these markers to
specific regions of the neuroectoderm and to identified NBs uncovers candidate
genes and putative molecular interactions that might be part of the machinery
leading to cell diversity in the brain.
Serially homologous neuroblasts in the brain and ventral nerve
cord
A comparison of NB patterns between neuromeres of the brain, as well as
between the brain and ventral nerve cord, allows us to address the question of
whether serial homologies exist among NBs of the different segmental regions.
Within the trunk, the Cartesian grid-like expression of segment polarity and
DV patterning genes is almost identical in each hemisegment and, accordingly,
NBs developing from corresponding `quadrants' acquire the same fate (reviewed
by Bhat, 1999;
Skeath, 1999
). Indications for
serial homology of NBs between different segments come from similarities in
the time of formation, the relative position within the evolving NB pattern
and absolute position within a hemisegment, the co-expression of specific
molecular markers, and similarities between their lineages.
Some of the molecular markers analysed here for brain NBs have been
previously mapped in trunk NBs (e.g. Doe,
1992; Broadus et al.,
1995
). In addition to these, we analysed the expression of many
other genes in the brain (this work) as well as in the gnathal, thoracic and
abdominal segments (R.U. and G.M.T., unpublished). Owing to the large number
of markers analysed, we find expression patterns that are unique to particular
NBs. Comparison of the combination of markers expressed in individual NBs as
well as their relative position within the NB layer of each segment suggests
that several NBs exist in the brain that are serially homologous to NBs in the
ventral nerve cord (VNC). This mainly applies to the posterior brain (deuto-
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 (R.U. and G.M.T., unpublished)
would be serially homologous to Td4 in the tritocerebrum and to Dd7 in the
deutocerebrum (Fig. 7). These
NBs exhibit a similar posterodorsal position within the respective neuromer
immediately anterior to the En-positive NBs, and are the only NBs which
specifically co-express the following molecular markers: lbe (which
is generally expressed in only one NB per hemisegment), wg, gsb-d,
slp1 (except Td4), msh, cas, seven-up (except Td4), pdm1,
klu and asense. Furthermore, some of the daughter cells of Td4
and NB5-6 co-express ladybird and the glia-specific marker
reversed polarity [see Fig.
4D by Urbach et al. (Urbach et
al., 2003
)]. The existence of serially homologous NBs is
intriguing as the number of NBs in the tritocerebrum and deutocerebrum is
considerably reduced, the timecourse of neurogenesis within the brain and VNC
is different [especially in the tritocerebrum the development of NBs is
significantly delayed; see Urbach et al.
(Urbach et al., 2003
)], and
the development of head segments (and consequently of brain neuromeres) has
been assumed to be differently regulated (for a review, see
Jürgens and Hartenstein,
1993
).
In the VNC, serially homologous NBs that express the same combination of
molecular markers (Broadus et al.,
1995; Doe, 1992
)
give rise to almost identical cell lineages
(Bossing et al., 1996
;
Schmidt et al., 1997
),
suggesting that similar regulatory interactions take place during the
development of these NBs and their cell lineages. However, some of the
serially homologous VNC lineages have been shown to include a subset of
progeny cells that specifically differ between thoracic and abdominal
neuromeres (Udolph et al.,
1993
; Bossing et al.,
1996
; Schmidt et al.,
1997
). We expect such segment-specific differences to be even more
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 conferring
segment-specific characteristics to their lineages. Thus, unravelling the
lineages of serially homologous NBs and the genetic network that controls
their development will help to elucidate how region-specific structural and
functional diversity in the CNS evolves from a basic developmental ground
state.
Conclusions
We provide a comprehensive description of the spatiotemporal expression
pattern of a large number of marker genes in the procephalic neuroectoderm and
the entire population of identified brain NBs. Each of these genes is
expressed in a specific subset of brain NBs. Thus, on one hand these markers
are useful for tracing the fate of particular NBs in different genetic
backgrounds. On the other hand, each of these genes itself is a candidate
factor involved in the formation, specification or further development of
specific NBs. Furthermore, each individual NB expresses a specific combination
of markers that could reflect potential regulatory interactions among these
genes. Finally, many of these genes are also known to be expressed in the
neuroectoderm and/or NBs of the trunk, and to play a role during formation of
the ventral nerve cord. Thus, the clarification of their function during brain
development and comparison with the situation in the trunk would help us to
understand what makes the brain different from the truncal part of the
CNS.
The detailed descriptions of NB formation, segmentation and marker gene
expression presented in this and the accompanying papers
(Urbach et al., 2003;
Urbach and Technau, 2003
) in
itself provide new insight into principles of early patterning of the brain.
However, they should be particularly useful as a basis for approaching the
molecular mechanisms that control the generation 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 brain
NBs.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bhat, K. M. (1999). Segment polarity genes in neuroblast formation and identity specification during Drosophila neurogenesis. BioEssays 21,472 -485.[CrossRef][Medline]
Bhat, K. M., van Beers, E. H. and Bhat, P.
(2000). Sloppy paired acts as the downstream target of
wingless in the Drosophila CNS and interaction between
sloppy paired and gooseberry inhibits sloppy paired
during neurogenesis. Development
127,655
-665.
Bier, E., Vaessin, H., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1992). deadpan, an essential pan-neural gene in Drosophila, encodes a helix-loop-helix protein similar to the hairy gene product. Genes Dev. 6,2137 -2151.[Abstract]
Bossing, T., Udolph, G., Doe, C. Q. and Technau, G. M. (1996). The embryonic central nervous system lineages of Drosophila melanogaster. I. Neuroblast lineages derived from the ventral half of the neuroectoderm. Dev. Biol. 179, 41-64.[CrossRef][Medline]
Brand, M., Jarman, A. P., Jan, L. Y. and Jan, Y. N.
(1993). asense is a Drosophila neural precursor
gene and is capable of initiating sense organ formation.
Development 119,1
-17.
Broadus, J., Skeath, J. B., Spana, E. P., Bossing, T., Technau, G. M. and Doe, C. Q. (1995). New neuroblast markers and the origin of the aCC/pCC neurons in the Drosophila central nervous system. Mech. Dev. 53,393 -402.[CrossRef][Medline]
Brody, T. and Odenwald, W. F. (2000). Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev. Biol. 226,34 -44.[CrossRef][Medline]
Brönner, G. and Jäckle, H. (1991). Control and function of terminal gap gene activity in the posterior pole region of the Drosophila embryo. Mech. Dev. 35,205 -211.[CrossRef][Medline]
Cadigan, K. M., Grossniklaus, U. and Gehring, W. J. (1994a). Functional redundancy: the respective roles of the two sloppy paired genes in Drosophila segmentation. Proc. Natl. Acad. Sci. USA 91,6324 -6328.[Abstract]
Cadigan, K. M., Grossniklaus, U. and Gehring, W. J. (1994b). Localized expression of sloppy paired protein maintains the polarity of Drosophila parasegments. Genes Dev. 8,899 -913.[Abstract]
Campos-Ortega, J. A. and Hartenstein, V. (1997). The Embryonic Development of Drosophila melanogaster. Berlin, Heidelberg, New York: Springer Verlag.
Chiang, C., Young, K. E. and Beachy, P. A.
(1995). Control of Drosophila tracheal branching by the
novel homeodomain gene unplugged, a regulatory target for genes of
the bithorax complex. Development
121,3901
-3912.
Cribbs, D. L., Pultz, M. A., Johnson, D., Mazzulla, M. and Kaufman, T. C. (1992). Structural complexity and evolutionary conservation of the Drosophila homeotic gene proboscipedia. EMBO J. 11,1437 -1449.[Abstract]
Cui, X. and Doe, C. Q. (1992). ming is
expressed in neuroblast sublineages and regulates gene expression in the
Drosophila central nervous system.
Development 116,943
-952.
Cui, X. and Doe, C. Q. (1995). The role of the
cell cycle and cytokinesis in regulating neuroblast sublineage gene expression
in the Drosophila CNS. Development
121,3233
-3243.
Czerny, T., Halder, G., Kloter, U., Souabni, A., Gehring, W. J. and Busslinger, M. (1999). twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell 3, 297-307.[Medline]
Dalton, D., Chadwick, R. and McGinnis, W. (1989). Expression and embryonic function of empty spiracles: a Drosophila homeo box gene with two patterning functions on the anterior-posterior axis of the embryo. Genes Dev. 3,1940 -1956.[Abstract]
Dick, T., Yang, X., Yeo, S. and Chia, W. (1991). Two closely linked Drosophila POU domain genes are expressed in neuroblasts and sensory elements. Proc. Natl. Acad. Sci. USA 88,7645 -7649.[Abstract]
Diederich, R. J., Merrill, V., Pultz, M. A. and Kaufman, T. C. (1989). Isolation, structure, and expression of labial, a homeotic gene of the Antennapedia-Complex involved in Drosophila head development. Genes Dev. 3, 399-414.[Abstract]
Diederich, R. J., Pattatucci, A. M. and Kaufman, T. C. (1991). Developmental and evolutionary implications of labial, Deformed and engrailed expression in the Drosophila head. Development 113,273 -281.[Abstract]
Doe, C. Q. (1992). Molecular markers for
identified neuroblasts and ganglion mother cells in the Drosophila
central nervous system. Development
116,855
-863.
Doe, C. Q. and Technau, G. M. (1993). Identification and cell lineage of individual neural precursors in the Drosophila CNS. Trends Neurosci. 16,510 -514.[CrossRef][Medline]
Dormand, E. L. and Brand, A. H. (1998).
Runt determines cell fates in the Drosophila embryonic CNS.
Development 125,1659
-1667.
Finkelstein, R. and Perrimon, N. (1990). The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development. Nature 346,485 -488.[CrossRef][Medline]
Gao, Q., Wang, Y. and Finkelstein, R. (1996). Orthodenticle regulation during embryonic head development in Drosophila. Mech. Dev. 56, 3-15.[CrossRef][Medline]
Garcia-Alonso, L., VanBerkum, M. F., Grenningloh, G., Schuster, C. and Goodman, C. S. (1995). Fasciclin II controls proneural gene expression in Drosophila. Proc. Natl. Acad. Sci. USA 92,10501 -10505.[Abstract]
Gergen, J. P. and Butler, B. A. (1988). Isolation of the Drosophila segmentation gene runt and analysis of its expression during embryogenesis. Genes Dev. 2,1179 -1193.[Abstract]
Gorman, M. J. and Kaufman, T. C. (1995).
Genetic analysis of embryonic cis-acting regulatory elements of the
Drosophila homeotic gene sex combs reduced.
Genetics 140,557
-572.
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: Fasciclin II functions as a neuronal recognition molecule. Cell 57,45 -57.
Grossniklaus, U., Pearson, R. K. and Gehring, W. (1992). The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev. 6,1030 -1051.[Abstract]
Grossniklaus, U., Cadigan, K. M. and Gehring, W. J.
(1994). Three maternal coordinate systems cooperate in the
patterning of the Drosophila head.
Development 120,3155
-3171.
Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267,1788 -1792.[Medline]
Hama, C., Ali, Z. and Kornberg, T. B. (1990). Region-specific recombination and expression are directed by portions of the Drosophila engrailed promoter. Genes Dev. 4,1079 -1093.[Abstract]
Hartenstein, V. and Campos-Ortega, J. A. (1984). Early neurogenesis in wild-type Drosophila melanogaster. Roux's Arch. Dev. Biol. 193,308 -325.
Hartmann, B., Hirth, F., Walldorf, U. and Reichert, H. (2000). Expression, regulation and function of the homeobox gene empty spiracles in brain and ventral nerve cord development of Drosophila. Mech. Dev. 90,143 -153.[CrossRef][Medline]
Hirth, F., Therianos, S., Loop, T., Gehring, W. J., Reichert, H. and Furukubo-Tokunaga, K. (1995). Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila. Neuron 15,769 -778.[Medline]
Hirth, F., Hartmann, B. and Reichert, H.
(1998). Homeotic gene action in embryonic brain development of
Drosophila. Development
125,1579
-1589.
Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106,511 -521.[CrossRef][Medline]
Jagla, K., Jagla, T., Heitzler, P., Dretzen, G., Bellard, F. and
Bellard, M. (1997). ladybird, a tandem of homeobox
genes that maintain late wingless expression in terminal and dorsal
epidermis of the Drosophila embryo.
Development 124,91
-100.
Jagla, K., Stanceva, I., Dretzen, G., Bellard, F. and Bellard, M. (1994). A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster. Nucleic Acids Res. 22,1202 -1207.[Abstract]
Jimenez, F. and Campos-Ortega, J. A. (1990). Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of Drosophila melanogaster. Neuron 5,81 -89.[Medline]
Jürgens, G. and Hartenstein, V. (1993). The terminal regions of the body pattern. In The Development of Drosophila melanogaster (ed. C. M. Bate and A. Martinez-Arias), pp. 687-746. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J.
and Odenwald, W. F. (1998). Regulation of POU genes by
castor and hunchback establishes layered compartments in the
Drosophila CNS. Genes Dev.
12,246
-260.
Kammermeier, L., Leemans, R., Hirth, F., Flister, S., Wenger, U., Walldorf, U., Gehring, W. J. and Reichert, H. (2001). Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development. Mech. Dev. 103, 71-78.[CrossRef][Medline]
Kania, M. A., Bonner, A. S., Duffy, J. B. and Gergen, J. P. (1990). The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system. Genes Dev. 4,1701 -1713.[Abstract]
Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J.
and Furukubo-Tokunaga, K. (2000). Genetic control of
development of the mushroom bodies, the associative learning centers in the
Drosophila brain, by the eyeless, twin of eyeless, and
dachshund genes. Proc. Natl. Acad. Sci. USA
97,2140
-2144.
Mahaffey, J. W., Diederich, R. J. and Kaufman, T. C. (1989). Novel patterns of homeotic protein accumulation in the head of the Drosophila embryo. Development 105,167 -174.[Abstract]
Mardon, G., Solomon, N. M. and Rubin, G. M.
(1994). dachshund encodes a nuclear protein required for
normal eye and leg development in Drosophila.
Development 120,3473
-3486.
Martini, S. R., Roman, G., Meuser, S., Mardon, G. and Davis, R.
L. (2000). The retinal determination gene,
dachshund, is required for mushroom body cell differentiation.
Development 127,2663
-2672.
McDonald, J. A. and Doe, C. Q. (1997).
Establishing neuroblast-specific gene expression in the Drosophila
CNS: huckebein is activated by Wingless and Hedgehog and repressed by
Engrailed and Gooseberry. Development
124,1079
-1087.
Mellerick, D. M., Kassis, J. A., Zhang, S. D. and Odenwald, W. F. (1992). castor encodes a novel zinc finger protein required for the development of a subset of CNS neurons in Drosophila. Neuron 9, 789-803.[Medline]
Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C. S. and Rubin, G. M. (1990). The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates. Cell 60,211 -224.[Medline]
Nassif, C., Noveen, A. and Hartenstein, V. (1998). Embryonic development of the Drosophila brain. I. Pattern of pioneer tracts. J. Comp. Neurol. 402, 10-31.[CrossRef][Medline]
Noveen, A., Daniel, A. and Hartenstein, V.
(2000). Early development of the Drosophila mushroom
body: the roles of eyeless and dachshund.
Development 127,3475
-3488.
Patel, N. H. (1994). Imaging neuronal subsets and other cell types in whole mount Drosophila embryos and larvae using antibody probes. In Methods in Cell Biology. Drosophila melanogaster: Practical Uses in Cell Biology, Vol.44 (ed. L. S. B. Goldstein and E. Fyrberg), pp.445 -487. New York: Academic Press.
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C., Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58,955 -968.[Medline]
Plickert, G., Gajewski, M., Gehrke, G., Gausepohl, H., Schlossherr, J. and Ibrahim, H. (1997). Automated in situ detection (AISD) of biomolecules. Dev. Genes Evol. 207,362 -367.[CrossRef]
Prokop, A. and Technau, G. M. (1994). Early
tagma-specific commitment of Drosophila CNS progenitor NB1-1.
Development 120,2567
-2578.
Pultz, M. A., Diederich, R. J., Cribbs, D. L. and Kaufman, T. C. (1988). The proboscipedia locus of the Antennapedia complex: a molecular and genetic analysis. Genes Dev. 2,901 -920.[Abstract]
Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the small eye gene in mice and aniridia in humans. Science 265,785 -789.[Medline]
Reichert, H. (2002). Conserved genetic mechanisms for embryonic brain patterning. Int. J. Dev. Biol. 46,81 -87.
Rogers, B. T. and Kaufman, T. C. (1996).
Structure of the insect head as revealed by the EN protein pattern in
developing embryos. Development
122,3419
-3432.
Rudolph, K. M., Liaw, G. J., Daniel, A., Green, P., Courey, A.
J., Hartenstein, V. and Lengyel, J. A. (1997). Complex
regulatory region mediating tailless expression in early embryonic
patterning and brain development. Development
124,4297
-4308.
Schmidt, H., Rickert, C., Bossing, T., Vef, O., Urban, J. and Technau, G. M. (1997). The embryonic central nervous system lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neuroectoderm. Dev. Biol. 189,186 -204.[CrossRef][Medline]
Schmidt-Ott, U., Gonzalez-Gaitan, M., Jäckle, H. and Technau, G. M. (1994). Number, identity, and sequence of the Drosophila head segments as revealed by neural elements and their deletion patterns in mutants. Proc. Natl. Acad. Sci. USA 91,8363 -8367.[Abstract]
Schmucker, D., Jäckle, H. and Gaul, U.
(1997). Genetic analysis of the larval optic nerve projection in
Drosophila. Development
124,937
-948.
Shen, W. and Mardon, G. (1997). Ectopic eye
development in Drosophila induced by directed dachshund
expression. Development
124, 45-52.
Skeath, J. B. (1999). At the nexus between pattern formation and cell-type specification: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system. BioEssays 21,922 -931.[CrossRef][Medline]
Tautz, D., Lehmann, R., Schnürch, H., Schuh, R., Seifert, E., Kienlin, A., Jones, K. and Jäckle, H. (1987). Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes. Nature 327,383 -389[CrossRef]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Udolph, G., Prokop, A., Bossing, T. and Technau, G. M.
(1993). A common precursor for glia and neurons in the embryonic
CNS of Drosophila gives rise to segment-specific lineage variants.
Development 118,765
-775.
Udolph, G., Lüer, K., Bossing, T. and Technau, G. M. (1995). Commitment of CNS progenitors along the dorsoventral axis of Drosophila neuroectoderm. Science 269,1278 -1281.[Medline]
Urbach, R., Schnabel, R. and Technau, G. M.
(2003). The pattern of neuroblast formation, mitotic domains and
proneural gene expression during early brain development in
Drosophila. Development
130,3589
-3606.
Urbach, R. and Technau, G. M. (2003). Segment
polarity and D/V patterning gene expression reveals segmental organization of
the Drosophila brain. Development
130,3607
-3620.
Walldorf, U. and Gehring, W. J. (1992). empty spiracles, a gap gene containing a homeobox involved in Drosophila head development. EMBO J. 11,2247 -2259.[Abstract]
Weigel, D., Jürgens, G., Klingler, M. and Jäckle, H. (1990). Two gap genes mediate maternal terminal pattern information in Drosophila. Science 248,495 -498.[Medline]
Wieschaus, E., Perrimon, N. and Finkelstein, R.
(1992). orthodenticle activity is required for the
development of medial structures in the larval and adult epidermis of
Drosophila. Development
115,801
-811.
Yang, X., Yeo, S., Dick, T. and Chia, W. (1993). The role of a Drosophila POU homeo domain gene in the specification of neural precursor cell identity in the developing embryonic central nervous system. Genes Dev. 7, 504-516.[Abstract]
Yang, X., Bahri, S., Klein, T. and Chia, W. (1997). Klumpfuss, a putative Drosophila zinc finger transcription factor, acts to differentiate between the identities of two secondary precursor cells within one neuroblast lineage. Genes Dev. 11,1396 -1408.[Abstract]
Yeo, S. L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X., Skonju, S. and Chia, W. (1995). On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor. Genes Dev. 9,1223 -1236.[Abstract]
Younossi-Hartenstein, A., Green, P., Liaw, G. J., Rudolph, K., Lengyel, J. and Hartenstein, V. (1997). Control of early neurogenesis of the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev. Biol. 182,270 -283.[CrossRef][Medline]
Related articles in Development: