Download Homeotic regulation of segment-specific

Mechanisms of Development 74 (1998) 99–110
Homeotic regulation of segment-specific differences in neuroblast numbers
and proliferation in the Drosophila central nervous system
Andreas Prokop a ,*, Sarah Bray b, Emma Harrison b, Gerhard M. Technau a
a
Institut für Genetik-Zellbiologie, Becherweg 32, D-55128 Mainz, Germany
b
Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK
Received 9 February 1998; revised version received 20 April 1998; accepted 20 April 1998
Abstract
The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo is identical in
thoracic and abdominal segments. However, during late embryogenesis differences in the numbers of neuroblasts and in the extent of
neuroblast proliferation arise between these regions. We show that the homeotic genes Ultrabithorax and abdominal-A regulate these late
differences, and that misexpression of either gene in thoracic neuroblasts after segregation is sufficient to induce abdominal behaviour.
However, in wild type embryos we only detect abdominal-A and Ultrabithorax proteins in early neuroblasts. Furthermore, transplantation
experiments reveal that segment-specific behaviour is determined prior to neuroblast segregation. Thus, the segment-specific differences in
neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts,
and executed in later programmes controlling neuroblast numbers and proliferation.  1998 Elsevier Science Ireland Ltd. All rights
reserved
Keywords: BrdU; Central nervous system; Drosophila; Homeotic genes; Proliferation; Transplantation
1. Introduction
The central nervous system (CNS) is composed of a huge
variety of cells, most of which are unique in their properties.
In insects these individual neurons are arranged in stereotyped patterns with reproducible differences between the
segments corresponding to the diverse regional requirements of the CNS, such as the control of the locomotion
apparatus in the thorax or the reproduction organs in the
abdomen. It is thus important to understand how regional
differences are programmed during nervous system development. The ventral nerve cord in insects derives from
neural stem cells, the neuroblasts (NBs), which segregate
in reproducible segmental patterns from the ventral neurogenic region of the ectoderm (Bate, 1976; Hartenstein and
Campos-Ortega, 1984; Doe, 1992). In Drosophila, individual NBs give rise to lineages of specific size and cell composition (Udolph et al., 1993; Bossing and Technau, 1994;
* Corresponding author. Tel.: +49 6131 394328; fax: +49 6131 395845;
e-mail: [email protected]
0925-4773/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved
PII S0925-4773 (98 )0 0068-9
Bossing et al., 1996; Schmidt et al., 1997). However,
although the pattern of segregating NBs is identical in thoracic and abdominal segments (Doe, 1992), reproducible
differences occur between the thoracic and abdominal versions of some NB lineages (Prokop and Technau, 1994a;
Bossing et al., 1996; Schmidt et al., 1997). This is also
reflected in the proliferation patterns in the CNSs which
show marked differences between thorax and abdomen at
stages 15 and 16 (Prokop and Technau, 1991).
Segment-specific differences are also evident in the number of NBs that persist beyond the end of embryogenesis and
proliferate during larval stages. At stage 17, all NBs have
stopped dividing but can still be monitored by NB-specific
expression of grainyhead (grh) (Bray et al., 1989). Analyses
of GRH expression patterns in the CNSs of wild type
embryos and of mutant embryos where cell death is suppressed strongly suggest that a number of NBs normally die
towards the end of embryogenesis (White et al., 1994). The
degree of cell death shows segment-specific differences, in
that many more NBs die in the central abdomen than in the
thorax and anterior abdomen. As a consequence, when NBs
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resume proliferation as postembryonic NBs in the larval
stages, 47 NBs are detected in each thoracic, about 12 in
the two anterior abdominal neuromeres, but only six in central abdominal segments. Furthermore, postembryonic NBs
in the thorax and anterior abdomen produce hundreds of
daughter cells each, whereas those in abdominal neuromeres A3–A7 give rise to only five to 15 cells (Truman
and Bate, 1988; Prokop and Technau, 1994b). In summary,
there are three major factors regulating the segment-specific
proliferation of NBs: (1) the period and frequency of
embryonic NB proliferation, (2) the number of NBs eliminated at the end of embryogenesis, and (3) the frequency
and period of postembryonic proliferation.
The development of segment-specific characteristics is
programmed during development by the homeotic genes
which encode homeodomain transcription factors and are
conserved from nematodes to vertebrates, both with respect
to their function and to their organisation within gene complexes (for reviews see Lewis, 1978; Duncan, 1987; Beachy, 1990; Kaufman et al., 1990; McGinnis and Krumlauf,
1992; Morata, 1993). Homeotic selector genes are in principle expressed in those body regions in which they are
required to select the developmental pathways specific for
each particular segment. In Drosophila, mutations in any of
the homeotic genes result in the transformation of the morphological characteristics of the segments, where they are
normally expressed, into those of other (in general more
anterior) segments. Following these principles, segment
identities in the head and anterior thorax of Drosophila
are controlled by genes of the Antennapedia-complex,
whereas the posterior thorax and the abdomen are controlled
by genes of the bithorax-complex, including Ultrabithorax
(Ubx) and abdominal-A (abd-A).
The homeotic selector genes are expressed in a complex
pattern in the Drosophila CNS (Doe et al., 1988) and the
segment-specific development of at least one embryonic NB
lineage is regulated by homeotic genes (Prokop and Technau, 1994a). The bithorax-complex genes are thus likely
candidates for regulating the thoracic and abdominal patterns of NB behaviour. To investigate this possibility we
have analysed whether loss-of function mutations or misexpression of homeotic genes have any effects on (a) patterns of bromo deoxyuridine (BrdU) incorporation which
indicates DNA synthesis and is used to give an indication
of NB proliferation (Truman and Bate, 1988; Prokop and
Technau, 1991), and (b) late embryonic patterns of NBspecific GRH expression, which labels the persisting NBs
at that stage (White et al., 1994; see above). Our analysis
indicates that abd-A and Ubx regulate the differences in cell
number between thoracic and abdominal neuromeres that
are seen in the larval and adult CNS by altering both the
amount of proliferation in embryo and larva, and the number of NBs that are present in post-embryonic stages.
Although segment-specific characteristics of BrdU and
GRH patterns are first observed towards the end of embryogenesis, we only detect the expression of ABD-A and UBX
in NBs during earlier embryonic stages. Furthermore, transplantation experiments indicate that determination of thoracic versus abdominal characteristics occurs prior to NB
segregation, before the homeotic proteins themselves are
expressed. We propose therefore that segment-specific differences in neuroblast behaviour are determined during the
early patterning of the embryo which results in specific
expression of homeotic genes in early NBs. This confers
later programmes of proliferation control, which no longer
require the activity of homeotic genes.
2. Results
2.1. Segment-specific differences in late embryonic NB
proliferation are controlled by abd-A and Antennapedia
Individual NBs in the Drosophila CNS undergo different
patterns of division depending on their position (Prokop and
Technau, 1994a; Bossing et al., 1996; Schmidt et al., 1997).
Patterns of replicating cells can be detected through their
incorporation of BrdU which in late stages of embryogenesis reveals scattered cells in the abdominal neuromeres
compared with regular incorporation patterns in the more
anterior regions of the ventral nerve cord (Fig. 1B,C). In the
subesophageal ganglion approximately 20 groups of two to
four cells in ventral to ventrolateral locations are labelled,
and on either side of each thoracic neuromere one to three
groups of two to four cells are detected in ventrolateral/
lateral (but not in ventral) positions. Given that the initial
number and organisation of NBs is the same in thorax and
abdomen (Doe, 1992), the observed replication patterns
indicate that equivalent NBs behave differently in the thorax
and the abdomen; in the thorax they continue replicating
whereas in the abdomen they do not.
In order to determine whether homeotic genes regulate
these differences between thoracic and abdominal DNA
replication, BrdU was injected into homeotic mutant
embryos at stage 16/17. In mutant embryos lacking Ubx
and abd-A function (Df109), BrdU incorporation appears
normal in the subesophageal ganglion and in the thorax.
However, in the abdominal neuromeres (except for the
terminal region) lateral cells incorporate BrdU in patterns
reminiscent of the thoracic region (not shown, but see Fig.
1E). Mutant embryos lacking Ubx function alone, show no
obvious changes in the pattern of BrdU incorporation (not
shown) in spite of a clear presence of the homeotic cuticle
phenotype (Lewis, 1978). In contrast, embryos lacking abdA function had defects reminiscent of embryos carrying the
deficiency Df109 (Fig. 1E) with extra lateral NBs incorporating BrdU in abdominal segments. The regulation by abd-A
but not Ubx suggests that the NBs lie in the posterior compartment of the segment, because the effects are also seen in
A1 where abd-A is only expressed in the posterior compartment (see Fig. 6A). The posterior localisation of the lateral
thoracic NB in wild type embryos was confirmed by double
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
labelling with antibodies against BrdU and ENGRAILED, a
marker for the posterior compartment (Fig. 1D). In embryos
lacking the Antennapedia gene, thoracic BrdU incorporation associated with ventrolateral/lateral NBs is normal,
however, additional staining is detected in ventral positions
resembling the ventral BrdU patterns of the subesophageal
ganglion (Fig. 1F). Taken together, these results demonstrate that in late embryos abd-A function is needed to
repress DNA replication in some lateral NBs of abdominal
neuromeres, and Antennapedia function is required to
repress DNA replication in ventral NBs of the thorax. As
pulse chase experiments have shown that the pattern of
BrdU incorporation in the thoracic neuromeres of late
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embryos reflects NB proliferation (Prokop and Technau,
1991) we believe that the DNA replication patterns we
observe in these experiments are indicative of effects on
NB proliferation.
2.2. Homeotic genes also control the numbers and
proliferation of postembryonic NBs
During the second larval instar, some of the embryonic
NBs increase in size and resume proliferation as postembryonic NBs (pNBs). The thoracic neuromeres contain 47
pNBs and the abdominal neuromeres A1 and A2 about 12
pNBs, all of which proliferate extensively. In contrast, the
Fig. 1. Homeotic regulation of segment-specific BrdU-incorporation in the embryonic CNS. Late stage 17 embryos in lateral (A,B,J) or ventral view (C–H;
anterior always to the left), labelled with antibodies against ENGRAILED (EN) and/or BrdU as indicated (bottom right). (A) Each engrailed stripe indicates
one neuromere (T, thorax, delimited by black lines; Ab, abdomen; H, hemispheres; S, subesophageal ganglion). (B–D) White arrowheads point at lateral
BrdU incorporation in the thorax, black arrows at ventral proliferation in the subesophageal ganglion, black arrowheads at engrailed positive cells. Note that
BrdU labelled cells lie in one line with engrailed positive cells, thus in the posterior neuromere compartment. (E–G) show mutant embryos as indicated (top
middle), (H,J) are embryos with misexpression of UBX; white arrows indicate lateral BrdU incorporation comparable with wild type (see (B,C)), open
arrowheads indicate ectopic lateral, open arrows ectopic ventral and a bent arrow loss of lateral BrdU incorporation. (K) Ectopic UBX expression appears in
the NB layer about stage 10/11 (white arrow), but not in the peripheral ectoderm (except intrinsic expression in T2 and T3; asterisks). Subesophageal (S) and
thoracic (T1–3) neuromeres are indicated. Scale bar, 50 mm (A–C, E–J); 20 mm (D,K).
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abdominal neuromeres A3–7 contain at most six pNBs (two
sets of vm-, vl- and dl-pNBs), each of which divides only a
few times (Fig. 2A,B; Truman and Bate, 1988; Prokop and
Technau, 1994b, 1995). In order to investigate a possible
involvement of homeotic genes in the regulation of these
postembryonic segment-specific differences we analysed
patterns of BrdU incorporation or of pNB-specific toluidine
blue staining (see Truman and Bate, 1988) in allelic combinations of homeotic genes that allow mutant animals to
survive into larval stages (Lewis, 1978; Ghysen et al.,
1985). Larvae carrying just one copy of the Ubx and abdA genes (Df109/+), show additional large clusters of BrdU
labelled cells in A3, A4 and sometimes also A5 (not shown,
but see Fig. 2G). Most of these clusters are located in a
ventromedial position derived either from the vm-pNBs or
from additional adjacent pNBs. Sometimes further BrdU
labelled large cell clusters are located in more dorsal positions, adjacent to or originating from the vl- or dl-pNB.
Larvae carrying just one copy of the Ubx gene have slight
defects in the BrdU pattern in one-fourth of cases, with one
larger cell group in ventrolateral positions of A3 and/or an
enlargement of the vm-cluster (normally about five cells) to
up to 15 cells in A3 or A4 (not shown). However, these
defects are less severe than in larvae heterozygous for
Df109. In contrast, larvae lacking one copy of the abd-A
gene, all show a BrdU pattern resembling the severe defects
observed in larvae carrying the Df109 deletion (Fig. 2G).
This demonstrates that abd-A is the principle factor required
to regulate segment-specific features of pNB proliferation in
most of the abdominal neuromeres, comparable with the
situation in the embryonic CNS.
This interpretation is supported by analyses of larvae
carrying one copy of the abd-A allele Uab4 (Lewis, 1978)
in combination with an abd-A null allele (Uab4/Df109 or
Uab4/abd-AMX1). Such larvae have about 12 pNBs in each
of the abdominal neuromeres A3–A7 (compared with six
pNBs in the wild type; Fig. 2A,D). Each of these surplus
pNBs produces a hundred or more progeny, whereas in wild
type larvae the pNBs in A3–A7 produce at most 15 progeny
(Fig. 2B,E; Truman and Bate, 1988; Prokop and Technau,
1994b). Both the number and the proliferation behaviour of
these pNBs in the central abdomen of the mutant larvae is
reminiscent of the wild type pattern described for neuromere A2 (Truman and Bate, 1988). The NB pattern defects
can already be detected in the late embryo (stage 16/17)
using the NB-specific marker grainyhead (grh): many
more GRH-expressing pNBs persist in the abdominal neuromeres of Uab4/Df109 mutant embryos than in wild type
(Fig. 2C,F). Thus, abd-A regulates the proliferation of pNBs
both by controlling the number of NBs that are eliminated in
the late embryo and by regulating the number of divisions
the persisting NBs undergo during larval stages.
Fig. 2. Effects of homeotic mutations on the pattern and proliferation of postembryonic NBs. (A,B,D,E,G) BrdU incorporation in nerve cords of late third
instar larvae exposed to BrdU during larval life. (C,F,H,I) Anti-GRH labelled nerve cords at late stage 17. Anterior is to the left. (A) In the wild type larva,
abdominal neuromeres A3–A7 (delimited by lines) have six clusters of BrdU labelled cells derived from two pairs of vm-, vl- and dl-pNBs, respectively (see
higher magnification in (B)), comparable with the abdominal NB patterns in late embryos (C); arrows in (A–C) indicate vm-pNBs. In Uab4/abd-A or Uab4/
DF109 mutant larvae many more NBs persist in A3–A7 (D,F) and produce larger lineages (E). (G) In abd-A/+ mutant larvae A3, A4 and sometimes A5 have
few large NB lineages (black arrowheads). Misexpression of UBX (H) or ABD-A (I) in all NBs removes most thoracic NBs (white arrowheads indicate
remaining NBs; arrows indicate hemispheres). Scale bar, 20 mm (F); 30 mm (B,E); 35 mm (C,H,I); 100 mm (A,D,G).
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
2.3. Misexpression of bithorax-complex genes is sufficient
to confer segment-specific behaviour on NBs
Impaired function or reduced levels of bithorax-complex
genes affect NB behaviour in the domain where these genes
are normally expressed, consistent with their direct requirement for the regulation of abdominal NB characteristics. In
order to test whether the presence of ABD-A or UBX is
sufficient to confer abdominal NB behaviour we analysed
the consequences of expressing these proteins in all NBs
including those in thoracic neuromeres using the GAL4expression system (Brand and Perrimon, 1993). When the
transcription of UAS-Ubx or UAS-abd-A was ectopically
induced in most or all segregated embryonic NBs and
their progeny (but not in the neuroectoderm; Fig. 1K) with
the GAL4 driver line MZ1407 the overall shape of the CNS
was not perturbed indicating that most phases of proliferation are not grossly abnormal. However, the pattern of BrdU
incorporation at stage16/17 is altered with misexpression of
either UBX or ABD-A abolishing the lateral replicating
clusters in the thoracic neuromeres (Fig. 1J; bent arrow).
This is what normally occurs in the abdominal NBs suggesting that the thoracic expression of ABD-A or UBX after NB
segregation is sufficient to transform the characteristics of
some thoracic lateral NBs into those of abdominal NBs.
However, we also find ectopic sites of BrdU-incorporation
in the ventral region of thoracic neuromeres (Fig. 1H,J). The
pattern appears like an extension of the subesophageal pattern and resembles the phenotype of Antennapedia mutant
embryos (Fig. 1F vs. H,J; open arrows), as if ectopic
bithorax gene activity in the thoracic neuromeres represses
Antennapedia function. Indeed ectopic UBX has been
shown to repress Antennapedia gene expression (González-Reyes and Morata, 1990). Thus, our results indicate
that specific NBs respond differently to ectopic expression
of bithorax-complex genes, acquiring abdominal characteristics in some cases and subesophageal characteristics in
others. Similar results are obtained in Polycomb mutant
embryos, which have ectopic expression of ABD-A and
UBX in the NB layer from stage 10/11 onwards (Simon et
al., 1992; Prokop and Technau, 1994a). In Polycomb3
mutant embryos at stage 16, both the lack of lateral and
gain of ventral BrdU incorporating cells are observed, similar to the GAL4 experiments (Fig. 1G). Therefore, our data
are consistent with the hypothesis that expression of
bithorax-complex genes after NB segregation is sufficient
to regulate the segment-specific replication of embryonic
NBs. The homeotic genes do not directly interfere with
the cell cycle (e.g. block it completely upon misexpression),
but they appear to determine which segmental proliferation
schedule is carried out.
As only a few NB lineages have segment-specific BrdUincorporation patterns in the embryo we wanted to investigate whether misexpression of ABD-A or UBX is also sufficient to alter the numbers of NBs at the end of
embryogenesis, which is a characteristic relevant to many
103
more NB lineages. Misexpression of either Ubx or abd-A
leads to a reduction in the number of grh-expressing cells in
the CNSs of stage 17 embryos which we take to indicate that
they affect the number of NBs that are eliminated (see Section 1; White et al., 1994). The effects are dramatic in the
thoracic neuromeres where only four to eight grh-expressing cells remain per neuromere at specific locations (Fig.
2H,I). Thus, the effects of misexpressing ABD-A and UBX
on the patterns of BrdU-incorporation and GRH expression
in the late embryo suggest that most, if not all, thoracic NBs
are responsive to bithorax-complex genes, and that the presence of ABD-A or UBX after NB segregation is sufficient
to induce abdominal behaviour.
2.4. Expression of homeotic genes in NBs precedes the
appearance of segment-specific features
To determine when bithorax gene function is required
during embryogenesis to regulate NB behaviour, we investigated when these proteins are present in the abdominal
NBs and therefore likely to impose their regulation. Using
anti-GRH or a grh-lacZ reporter-gene (which recapitulates
the NB expression of grainyhead) to mark the NBs we
detected no co-expression with ABD-A in the persisting
NBs of stage 17 embryos (not shown). In stage 15 embryos
prior to NB elimination, many more cells express grh. However, even at this stage there are no cells which co-express
UBX and GRH and only rarely (one to two ventral cells per
neuromere) are ABD-A and GRH expression detected in the
same cells (Fig. 3D,E). This suggests that neither ABD-A
nor UBX are present in the NBs shortly before segmentspecific differences in BrdU and GRH patterns occur.
During early stages of embryogenesis NB can be detected
either by their position and shape or by expression of
asense. ASENSE is expressed in NBs shortly after their
segregation from the neuroectoderm and so can be used as
an early marker for NBs. UBX was detected in many NBs at
stages 8–12 although there is wide variation between the
levels of UBX present in different NBs and a subset of NBs
contain no detectable UBX (Fig. 3A,C). Similarly, ABD-A
is present in many NBs at early stages (Fig. 3B). Thus, both
UBX and ABD-A are expressed in embryonic NBs, but their
expression fades before segment-specific differences
become detectable. To see whether we could define a precise stage when homeotic gene function is sufficient to confer segment-specific NB elimination and proliferation
patterns we tested the effects of inducing ABD-A or UBX
at different stages via heat inducible promoters. None of the
heat shock regimes were able to confer homeotic changes on
proliferation or GRH patterns (although these experiments
clearly caused homeotic effects in the epidermis), so we
cannot identify the precise stage when the activity of these
genes is required. Nevertheless the timing of ABD-A and
UBX expression in NBs makes it unlikely that the homeotic
genes directly regulate those genes which mediate proliferation cessation or NB elimination. Instead they seem to con-
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Fig. 3. ABD-A and UBX are found in NBs only at earlier embryonic stages. ABD-A or UBX expression (as indicated top right) in embryos at stage 10/11
(A–C) or stage 15 (D,E), or (F) in early third instar larval CNSs (L3; stages indicated bottom right). NBs were identified by position and shape (A,B,F) or
expression of ASENSE (ASE; (C)) or GRH (D,E). (A–C) NBs contain homeotic proteins at early stages (white arrowheads; orange colour in (C) indicates
double labelling). (D–F) NBs lack homeotic gene expression later on (white arrowheads; for L3 see also Truman et al., 1993). Scale bar, 15 mm (A,B,F); 35
mm (C–E).
trol regulatory pathways, which function subsequent to the
expression of the bithorax-complex genes.
2.5. Thoracic versus abdominal behaviour of NB is
determined prior to NB segregation
The misexpression data indicate that homeotic genes
determine segment-specific features in the CNS after NBs
have segregated. However, the analysis of the NB1-1 lineage revealed that segment-specificity can be determined in
the peripheral ectoderm, upstream of homeotic gene function (Prokop and Technau, 1994a). We therefore tested
whether the segment-specific postembryonic proliferation
patterns are also determined in the peripheral ectoderm, to
see whether this early determination could be a general
mechanism underlying CNS development. Single cells,
labelled genetically with the gene encoding b-galactosidase
and cytoplasmically with HRP (horseradish peroxidase),
were transplanted between the thoracic and the abdominal
neuroectoderm at the early gastrula stage about 30 min prior
to the onset of NB segregation. As b-galactosidase is genetically expressed by every cell of the clone, both the embryonically and postembryonically derived cells will contain bgalactosidase activity. However, only the embryonically
derived cells will contain HRP, as the enzyme gets diluted
below detectable level when the NB takes up proliferation
in the larva (Fig. 4B,D; for details see Prokop and Technau,
1991). The composition of the clones was analysed in the
late larval ventral nerve cord when the differences between
abdominal and thoracic NB lineages are very distinctive.
We obtained 66 CNS clones from isotopic transplantations in the thorax, 45 of which contained a pNB with a nest
of daughter cells exclusively labelled by b-galactosidase
activity indicating that they were postembryonic progeny
(Figs. 4A,B and 5, T to T). This proportion of 68% is in
accordance with the number of thoracic NBs which are
reactivated during larval life in relation to the total number
of embryonic precursors (47 pNBs versus 60 NBs and seven
to eight midline precursors per neuromere in the embryo;
Truman and Bate, 1988; Doe, 1992; Bossing and Technau,
1994). Isotopic transplantations in the abdomen yielded 32
abdominal CNS clones of which only two contained a larger
peripheral cell (potential pNB) and a small cluster of postembryonic progeny cells. This low rate of NB reactivation
(6%) and their low proliferation rate reflect the typical behaviour of abdominal pNBs (Fig. 5, A to A).
When cells were heterotopically transplanted from thoracic to abdominal sites of the early gastrula neuroectoderm,
67% gave rise to a large nest of postembryonic cells with a
pNB (Figs. 4C,D and 5, T to A) consistent with the characteristics of thoracic NBs. Conversely, when cells were
transplanted from abdominal to thoracic sites, all clones
failed to express thoracic features and contained only
(embryonic) cells strongly labelled by HRP activity (Fig.
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
105
Fig. 4. Examples of late larval NB lineages originating from transplantations at the early gastrula stage. (A–D) Thoracically derived pNBs (arrowheads) with
embryonic (brown cells) and postembryonic (blue cells) progeny can develop in the thorax (A,B) or abdomen (C,D). (E) Lineages without pNBs always lack
large nests of purely blue cells. (F) Schematic representation of an embryo at the early gastrula stage indicating the modes of transplantation that led to the
lineages in (A–E). Scale bar, 70 mm (A,C); 20 mm (B,D).
4E and 5, A to T). Thus, NBs autonomously execute postembryonic proliferation patterns according to their domain
of origin in the neuroectoderm. This indicates that most or
even all neuroectodermal CNS precursors are determined at
the early gastrula stage with respect to their thoracic or
abdominal identity. Since subsequent misexpression of
homeotic genes can alter the behaviour of NBs, our findings
suggest that genetic mechanisms upstream of homeotic gene
function must initially determine the regional identity of
these cells.
3. Discussion
3.1. Homeotic genes regulate proliferation and numbers of
NBs
The development of the nervous system involves the gen-
eration of the correct numbers of neural precursors, their
subsequent division to generate a defined set of progeny
and the acquisition by their progeny of individual neural
cell fates. These processes must be differentially modulated
along the body axis so that the neural structures appropriate
for each region of the body develop. Previous studies in
Drosophila have emphasised the role of homeotic genes in
controlling the types of neural fate that are adopted by the
progeny of the NBs (Doe and Scott, 1988). Our results
demonstrate that the homeotic genes also regulate segment-specific differences in NB numbers and NB divisions.
This regulation is achieved both by controlling the proliferation period and frequency and by regulating whether NBs
persist at the end of embryogenesis. Because of the latter,
the number and pattern of postembryonic NBs depend on
homeotic gene function unlike the embryonic NBs where
the pattern of segregating NBs is the same between thoracic
and abdominal neuromeres (Doe, 1992). Thus in the larval
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Fig. 5. Isotopic and heterotopic transplantation experiments. Schematic
representations of cell transplantations in embryos at the early gastrula
stage (top), of late larval CNSs with schematic NB lineages (middle)
and a table summarising the results (bottom). Numbers 1–4 indicate the
transplantation modes and are reflected in the table below (T, thorax; A,
abdomen). Only modes 1 and 4 (explantation from the thorax) give rise to
large nests of postembryonic cells (with pNB; white part in schematic
lineage; about two-thirds of cases) regardless of whether they develop in
the thorax (1) or abdomen (4). Potential pNBs (?) in modes 2 and 3
(explantation from the abdomen) are rare and have very small lineages.
Only a third of thoracically derived lineages (1, 4) but about 90% of
abdominally derived lineages (2, 3) consist of embryonically born cells
only (black in schematic lineage).
nervous system there are dramatic differences between the
numbers of pNBs present in the thoracic versus the abdominal neuromeres, where the homeotic genes Ubx and abd-A
are expressed. The A1 and A2 neuromeres have an intermediate number of pNBs (Truman and Bate, 1988), and in
the absence of Ubx the A1 neuromere contains more pNBs
indicating that Ubx regulates the pattern of pNBs within the
A1 neuromere (Truman et al., 1993). Our findings of supernumerary NBs in larvae heterozygous for Ubx suggest that
UBX may also influence the behaviour of pNBs in more
posterior neuromeres (similar transformations were
observed in larval cuticles; Beachy, 1990). However, the
main differences in the distribution and proliferation of
pNBs in more posterior abdominal neuromeres are regulated by abd-A, as is evident from the aberrant pNB proliferation seen in neuromeres A3–A7 in Uab4 mutant larvae
which is more reminiscent of the A2 segment and thus is
analogous to transformations observed in the cuticle (Lewis,
1978). The regions where ectopic proliferation occurs when
abd-A function is reduced (e.g. in larvae hemizygous for
abd-A) correspond to those which have been reported to
give rise to abnormal neuropil structures under the same
conditions (Teugels and Ghysen, 1983; Ghysen et al.,
1985; Ghysen and Lewis, 1986). Thus, these morphological
abnormalities may result from supernumerary progeny of
misregulated NBs. Homeotic regulation of NB proliferation
has also been observed in the moth Manduca sexta (Booker
and Truman, 1989; Miles and Booker, 1993) suggesting that
this is a fundamental mechanism through which the development of the nervous system is regulated in insects and
most likely in other organisms.
The transition from the embryonic stages of neurogenesis
to the postembryonic stages involves programmed cell
death. This transition can be seen in stage 17 embryos
when the number of grh-expressing cells in the abdomen
decreases dramatically, and it is prevented by mutations that
disrupt the cell-death pathway (Bray et al., 1989; White et
al., 1994). In abd-A mutant embryos (Uab4) more grhexpressing cells persist in the abdomen, already reflecting
the NB pattern which is later on detected by BrdU and
toluidine blue in the larval nerve cord. Conversely, when
ABD-A or UBX are ectopically expressed, fewer grhexpressing cells are found, suggesting that the homeotic
genes are controlling whether or not these cells undergo
programmed cell death. Interestingly, the numbers of
abdominal NBs are also reduced upon misexpression of
UBX or ABD-A (Fig. 2C vs. H,I). A possible explanation
is that some NBs in A3–A7 (e.g. vm, vl and dl; Fig. 2A–C)
never express functional amounts of UBX or ABD-A, so
that targeted expression of UBX or ABD-A in those NBs
could trigger their elimination. If so, then regulation of these
NBs should normally be independent of abd-A and Ubx
gene function. For example these NB could have a basal
level of proliferation in all segments or they could undergo
segment specific patterns of proliferation in response to
other homeotic genes such as Antennapedia (see Fig. 1F).
To distinguish these possibilities specific markers are
needed for the individual NBs so that they can be identified
in all segments and studied throughout embryonic and postembryonic stages.
3.2. Cellular memory is implicated in the regulation of
homeotic gene expression and function
The segment-specific effects of homeotic genes on proliferation and pNB fates are first detected quite late in
embryogenesis, at about stage 15, and continue to be manifest throughout larval development (Truman and Bate,
1988; Bray et al., 1989; Prokop and Technau, 1991). We
therefore expected that the late effect of homeotic mutations
on NB behaviour would correlate with late expression of
Ubx and abd-A in these cells. However, neither UBX nor
ABD-A are present in NBs during stage 15–17 (Fig. 3D,E)
nor during postembryonic stages (Fig. 3F; Truman et al.,
1993), so they are unlikely to be directly controlling NB
numbers or proliferation. However, ABD-A and UBX are
present in many NBs at earlier stages and their presence is
sufficient to induce abdominal regulation, as demonstrated
by misexpression experiments. Likewise it has been shown
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
that the NB1-1 lineage requires Ubx and abd-A function
during stage 10/11 of embryogenesis when the proteins
can be detected in this precursor (Prokop and Technau,
1994a). Therefore, the activity of UBX and ABD-A at an
early stage must be able to determine the later proliferation
and persistence of the NBs (Fig. 6C). This is contrary to the
original hypothesis, based on the requirements for Ubx in
the epidermis (Morata and Garcı́a-Bellido, 1976), which
proposed that the expression of a homeotic gene would
need to be stably maintained in the progeny cells for them
107
to retain their identity. In the NBs there must be mechanisms
downstream of homeotic genes through which cells can
retain a stable memory or imprint of having previously
expressed Ubx or abd-A. This could involve the homeotic
genes acting as transcriptional repressors which initiate a
repressed state of their target genes that can be maintained
even after UBX or ABD-A have decayed. Alternatively,
UBX or ABD-A may activate target genes which have the
capacity for autoregulation, so that the targets can maintain
their own expression in the absence of homeotic proteins.
Fig. 6. Control of segment-specific proliferation and survival in Drosophila neuroblasts. The scheme shows (A) the expression domains of Antennapedia, Ubx
and abd-A in relation to (B) the phenotypes observed (prolif., prolonged proliferation of embryonic NB; NB1-1, segmental differences of the NB1-1 lineage,
see Prokop and Technau, 1994a; elimin., late embryonic NB elimination; no diff., possible NBs without segment specific differences, e.g. vm-, vl-, dl-pNB).
(C) The implementation of genetic programmes for the regulation of NB numbers and proliferation depends on earlier patterning events. Mechanisms
regulating persistence and prolonged proliferation of thoracic NB (+ in square) appear to operate after UBX or ABD-A have faded in NBs. Only those NBs
expressing UBX (blue NBs) or ABD-A (red NBs) can prevent installation of these mechanisms (crossed out squares). Ubx (blue) and abd-A (red) are
expressed only in those NBs which contain lineage or cell specific activators of homeotic transcription (e.g. segmentation genes; green NBs), and which have
not expressed anterior gap genes (e.g. hunchback; NBs with grey gradients). The function of gap genes (gap, black) is maintained by Polycomb-group genes
(Pc-group, grey) and both repress the NB-specific activation of Ubx or abd-A (bent green arrow). (D) Upon transplantation, precursors from the thorax (1)
maintain their repressed state mediated by gap and Polycomb-group genes (grey gradient). Precursors from the abdomen (2) have not expressed inhibitory
gap genes and can therefore express UBX and ABD-A (red/blue).
108
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
Although the activity of homeotic genes is required after
NB segregation we found that at the early gastrula stage the
neuroectodermal cells are already autonomously determined as to whether they will give rise to NBs with thoracic
or abdominal proliferation behaviour, and whether they will
persist as pNBs (Figs. 4 and 5). Gastrula cells transplanted
from the thorax to the abdomen display thoracic patterns of
proliferation, whereas cells transplanted from the abdomen
to the thorax retain abdominal characteristics. This is likely
to be the result of domain-specific repression of homeotic
genes mediated by the early expressed gap gene products
such as hunchback, a known repressor of Ubx (White and
Lehmann, 1986). Once the proteins encoded by the gap
genes have decayed, the repressed state of the homeotic
genes is maintained by Polycomb group genes (Paro,
1990), so that the regulatory sequences of bithorax-complex
genes in cells derived from the thorax remain repressed,
whereas homeotic genes in abdominal cells are accessible
to activation (Fig. 6C,D). Experiments in C. elegans have
also demonstrated the importance of lineage rather than
local positional signalling in the regulation of homeotic
gene expression (Cowing and Kenyon, 1996), suggesting
that there could be conservation of the mechanisms underlying this regulation.
We conclude therefore that control of segment-specific
development in the CNS involves a cascade of subsequent
determination steps (Fig. 6C), in which first homeotic gene
function is regionally permitted or not, and subsequently
homeotic genes themselves select a programme, which
can be maintained even after the bithorax-complex genes
cease to be expressed. Reproducible intrasegmental expression patterns could spare some cells from homeotic gene
expression (e.g. the abdominal vm-, vl- and dl-NB) and
thus add further complexity and regulatory potential to
this system.
4. Experimental procedures
4.1. Fly stocks
We used the following mutant strains: AntennapediaW10
(Wakimoto et al., 1984); Ubx6.28 (Kerridge and Morata,
1982); abd-AMX1 (Sánchez-Herrero et al., 1985); the abd-A
allele iab3Uab4 (Lewis, 1978, referred to as Uab4);
Df(3R)Ubx109 (Lewis, 1978; referred to as Df109) and
Pc3 (Lewis, 1978). For misexpression of homeotic genes
in neural cells after NB segregation we used the GAL4
system (Brand and Perrimon, 1993) with the GAL4 driver-line MZ1407 (Sweeney et al., 1995; Urban et al., unpublished data) in conjunction with UAS-Ubx62.1 and UAS-abdA21.6 (Greig and Akam, 1993; Castelli-Gair et al., 1994).
During misexpression experiments embryos were kept at
25 or 29°C. For cell transplantations we used as hosts the
b-Gal-1 strain cq20 (Knipple and MacIntyre, 1984), which
lacks endogenous b-galactosidase activity and therefore
shows no background staining. As donors we used P-lacZ
enhancer trap lines with strong expression of b-galactosidase throughout the nervous system. Either we used A45
(O’Kane and Gehring, 1987) or the line E;B1;Z1 carrying
three different P-lacZ insertions: E is an insertion into the
elav locus (Bier et al., 1989), B1 an unpublished insertion on
the 2nd chromosome (Bier et al., 1989) and Z1 a 3rd chromosome insertion (unpublished data; kindly provided by
J.A. Campos-Ortega).
4.2. Generation of transgenic flies containing grh-lacZ
reporter gene
A 4-kb fragment (XhoI-XbaI) from the second intron of
the grainyhead gene (Uv et al., 1997) was cloned into
pBluescript (Strategene), excised using XbaI and KpnI
and inserted into the XbaI-KpnI sites of the enhancer detector P-element vector, HZ50PL (Hiromi et al., 1985) which
carries rosy as a selectable marker. The resulting plasmid
(NBgrh-lacZ) was injected into cinnabar rosy embryos in
the presence of transposase using standard techniques
(Rubin and Spradling, 1982) and transformants were
selected on the basis of rosy + eyes. At least four independent lines were analysed, all of which had expression in NBs
as shown.
4.3. Bromodeoxyuridine (BrdU) labelling
For embryonic BrdU application 5–10 nl of a 15 mM
solution of BrdU (Sigma) in 0.2 M KCl were injected into
embryos about 45 min after the three-portioned midgut
stage (stage16/17 according to Campos-Ortega and Hartenstein, 1997), following procedures described elsewhere
(Prokop and Technau, 1991; Prokop and Technau, 1993).
At late stage 17 embryos were mechanically removed from
the vitelline membrane, washed in PBT, fixed in Carnoy’s
solution (6:3:1 ethanol/chloroform/acetic acid), rehydrated
and their tips cut off to allow subsequent penetration of
antibodies. In larval stages BrdU was applied by feeding
animals with standard medium containing between 1 and
10% of a BrdU-solution (33 mM BrdU in 40% ethanol;
Truman and Bate, 1988). CNSs were removed, fixed in
Carnoy’s solution, and rehydrated.
To stain for BrdU-incorporation we used monoclonal
antibodies against BrdU (Becton and Dickinson, diluted
1:100), HRP-coupled secondary antibodies (Dianova,
diluted 1:500) or biotinylated secondary antibody (Dianova,
diluted 1:500) followed by treatment with ABC elite kit
(Vector Laboratories); blocking steps included 1% low fat
milk powder. Specimens were embedded in Araldite
(Serva) in borosilicate capillaries (Hilgenberg; see Prokop
and Technau, 1993).
4.4. Immunocytochemistry
Further antibody stainings were carried out on whole
A. Prokop et al. / Mechanisms of Development 74 (1998) 99–110
mounts following standard procedures and using biotinylated secondary antibodies (1:300) and ABC elite kit (Vector Laboratories) or fluorescent secondary antibodies from
Jackson laboratories (1:200). Third larval instar CNSs were
briefly post fixed in methanol subsequent to formaldehyde
fixation. For detection of grainyhead (grh) expression, the
CNSs were dissected from stage 17 embryos (approximately
19–22 h after egg lay) and transferred to poly-l-lysine
coated coverslips (Bray et al., 1989). Embryos and larval
CNSs were embedded in Araldite (see above) or mounted in
citifluor. The final dilutions of primary antibodies were
1:1500 for rat polyclonal anti-ABD-A (Macı́as et al.,
1990); 1:20 for rabbit polyclonal anti-ABD-A (Karch et
al., 1990), 1:20 for monoclonal anti-UBX (White and Wilcox, 1984, 1985), 1:3 for monoclonal anti-ENGRAILED
(Patel et al., 1989); 1:5 for monoclonal anti-GRH (Bray et
al., 1989); 1:10 000 for rabbit anti-ASENSE (Brand et al.,
1993); and 1:2000 for rabbit anti-b-galactosidase (Cappell).
4.5. Cell transplantation, preparation and staining of
donors and hosts
For cell transplantations we used as donors enhancer trap
lines with lacZ gene expression throughout the CNS (see
Section 4.1). The lacZ gene is inherited upon cell division so
that embryonic and postembryonic progeny of transplanted
cells produce b-galactosidase cell autonomously. In addition, donors were injected with 5–10 nl of horseradish peroxidase solution (HRP; 5–10% in 0.2 M KCl) at the
syncytial blastoderm stage. Transplantations of single cells
were carried out about 10 min after the onset of gastrulation
(stage 7) in the ventral neurogenic region at either 30%
(abdominal) or at 60% egg length (thoracic, Fig. 5; for
more details see Prokop and Technau, 1991). Late larval
host CNSs were dissected out in PBS (other tissues were
discarded), fixed in 1% solution of glutaraldehyde in PBS,
stained for b-galactosidase and subsequently for HRP activity. Specimens were dehydrated in alcohol, cleared in
xylene, embedded in araldite (see above).
Acknowledgements
We thank José Campos Ortega, James Castelli-Gair, Stephen Greig, Rolf Reuter, Srikala Raghavan, Suzanna
Romani, Ernesto Sánchez Herrero, Joachim Urban and
Rob White for providing fly stocks and antibodies, Rob
White and Ernesto Sánchez-Herrero for comments on the
manuscript. A.P. is grateful to Michael Bate, in whose
laboratory part of the project was carried out. This work
was supported by a grant from the Deutsche Forschungsgemeinschaft to G.M.T. (Te 130/1-4), by a project grant from
the Biotechnology and Biological Science Research Council
to S.J.B. and by a Human Capital and Mobility Fellowship
(EU) and a research fellowship from the Lloyd’s of London
Tercentenery Foundation to A.P.
109
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