Download Temporal and Spatial Expression of Homeotic Genes Is Important for

Mol. Cells, Vol. 21, No. 3, pp. 436-442
Molecules
and
Cells
©KSMCB 2006
Temporal and Spatial Expression of Homeotic Genes Is
Important for Segment-specific Neuroblast 6-4 Lineage
Formation in Drosophila
Sun-Young Kang, Su-Na Kim, Sang Hee Kim1, and Sang-Hak Jeon*
Department of Biology Education, Seoul National University, Seoul 151-748, Korea;
1
Department of Chemistry, Konkuk University, Seoul 143-701, Korea.
(Received May 12, 2006; Accepted June 19, 2006)
Different proliferation of neuroblast 6-4 (NB6-4) in the
thorax and abdomen produces segmental specific
expression pattern of several neuroblast marker genes.
NB6-4 is divided to form four medialmost cell body
glia (MM-CBG) per segment in thorax and two MMCBG per segment in abdomen. As homeotic genes determine the identities of embryonic segments along the
A/P axis, we investigated if temporal and specific expression of homeotic genes affects MM-CBG patterns
in thorax and abdomen. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation,
whereas abd-A and Abd-B caused the transformation
of abdominal MM-CBG to their thoracic counterparts.
On the other hand, gain-of-function mutants of Ubx,
abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG
resembled abdominal MM-CBG. However, mutations
in Polycomb group (PcG) genes, which are negative
transregulators of homeotic genes, did not cause the
thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few percent of embryos were partially reduced or abnormally
patterned. Our results indicate that temporal and spatial expression of the homeotic genes is important to
determine segmental-specificity of NB6-4 daughter
cells along the anterior-posterior (A/P) axis.
Keywords: abd-A; Abd-B; MM-CBG; Neuroblast; PcG
Genes; Ubx.
* To whom correspondence should be addressed.
Tel: 82-2-880-1409; Fax: 82-2-886-2117
E-mail: [email protected]
Introduction
The homeotic genes of the Antennapedia complex (ANTC) and bithorax complex (BX-C) specify segmental identity along the anterior-posterior body axis in Drosophila
(Duncan, 1987; Kaufman et al., 1990). The ANT-C genes
specify fates within head and anterior thoracic segments
and the BX-C genes control the identity of posterior thoracic and abdominal segments. The ANT-C genes include
labial (lab), proboscipedia (pb), Deformed (Dfd), Sex
combs reduced (Scr), and Antennapedia (Antp), and the
three BX-C genes are Ultrabithorax (Ubx), abdominal-A
(abd-A) and Abdominal-B (Abd-B) (Carroll et al., 1986).
Molecular genetic studies have revealed that homeotic
genes are strongly expressed in the nervous system, in
ectoderm, and in visceral mesoderm. Ubx is expressed in
parasegments (PS) 5-13, abd-A in PS7-13, and Abd-B in
PS10-14 (Beachy et al., 1985; Celniker et al., 1989;
Karch et al., 1990). Initially, spatial domains of homeotic
gene expression are regulated by gap gene and pair-rule
gene products (Irish et al., 1989). This expression pattern
is maintained by the cross-regulation of homeotic genes,
the positive regulation of trithorax group (trxG) genes,
and the negative regulation of Polycomb group (PcG)
genes.
Mutations on PcG genes lead to homeotic transformations of segments into a more posterior identity (Jurgens,
1985) and ectopic expression of the homeotic genes
(McKeon and Brock, 1991; Simon et al., 1992). In addition
to developmental regulation, PcG proteins also have some
other roles. Recent studies have focused on their roles as
Abbreviations: MM-CBG, medialmost cell body glia; NB6-4,
neuroblast 6-4; PcG, Polycomb group.
Sun-Young Kang et al.
cell cycle regulators. Several genetic evidence shows that
some PcG proteins control cell proliferation and are involved in hematopoiesis and human cancer (Jacobs et al.,
2002; Mahmoudi et al., 2001).
Although the homeotic genes are strongly expressed in
the nervous system, few reports are available on the roles
of homeotic genes in development of the nervous system.
Functional nervous system development in all higher eukaryotes requires the production of two cell types: neurons and glia. In particular, glia play a number of roles in
the development and function of the nervous system
(Jones, 2001). Glia provide structural support, wrap, and
insulation of neurons, and also regulate neurons by generating cytokines and growth factors. Moreover, developing
glia undergo extensive migrations and cell shape changes,
and act as cues and substrata for neuronal migrations and
axon pathfinding (Lemke, 2001).
In Drosophila, glial cells missing (gcm) is the primary
regulator of glial cell determination. gcm is transiently
expressed in all embryonic glia except for midline glia
(Hosoya et al., 1995), and differentially regulates the expression of over 400 genes (Egger et al., 2002). gcm also
regulates the expression of genes, such as, reverse polarity (repo) (Halter et al., 1995; Xiong et al., 1994), which
encodes a homeodomain protein and is expressed exclusively in almost every developing glia with few exceptions. MM-CBG, which are a type of glia, have segmentspecificity determined by different neuroblast identity.
NB6-4, the precursor of MM-CBG, is first formed in S3
(stage 10) and its position is identical in all segments
(Doe, 1992). However, during further development, neuroblasts proliferate to form different patterns according to
their positions. NB6-4 in the thoracic segment (NB6-4T)
is divided into a neural lineage and a glial lineage, forming two MM-CBG and one M-CBG with several neurons
per hemisegment, whereas NB6-4 in abdominal segment
(NB6-4A) adopts only the glial lineage, forming one MMCBG and one M-CBG per hemisegment (Akiyama-Oda et
al., 1999; Ito et al., 1995; Klambt, 1993). MM-CBG are
first detected at stage11 and are located laterally along
VUM neurons. Moreover, these segment-specific MMCBG patterns suggest that MM-CBG may be determined
by the homeotic genes.
Recently, abd-A and Abd-B genes were reported to
specify the lineage of NB6-4A by down-regulating CycE
(Berger et al., 2005). Loss of the functions of these two
genes causes transformation of NB6-4A to NB6-4T. In
this work, we describe the effect of homeotic transformation caused by mutations of the homeotic genes and its
transregulators, PcG genes, on the segment specificity of
MM-CBG (Fig. 1). We demonstrate that segment-specific
MM-CBG are regulated by BX-C genes. Both loss-offunction and gain-of-function experiments showed that
MM-CBG pattern formation is closely related to the temporal and spatial function of the homeotic gene.
437
Materials and Methods
Fly culture and strains Flies were reared in 20 mm-diameter
vials containing a standard cornmeal/yeast medium seeded with
live yeast. Stocks were maintained at 20°C, but experimental
flies were reared at 25°C and eggs were also collected at 25°C.
Oregon-R, Ultrabithorax9.22 (Ubx9.22), abdominal-A MX1 (abdMX1
A ), and Abdominal-BD5 (Abd-BD5) (kindly provided by G.M.
Technau) single mutants, and Ubx abd-A double mutants
(Df(3R)Ubx109/Dp(3;3)P5) were used to examine homeotic
gene loss of function. Sca-GAL4 was crossed with UAS-Ubx,
UAS-abd-A or UAS-Abd-B for gain-of-function mutation experiments. Polycomb group gene mutants including Polycomb
(Pc3), extra sex combs (esc2, esc5), and pleiohomeotic (phocv)
were used. w1118; gcm+1.7/+4.5-hs43–lacZ (kindly provided by
B.W. Jones) showed gcm expression only in the abd-A expression domain. This was crossed with PcG mutants to determine
whether glial cell misformations occurred along the anteriorposterior axis.
Antibodies and immunocytochemical staining Embryos were
collected, dechorionated, fixed, and devitellined using standard
procedures. After embryos were preincubated, they were treated
with primary antibody. Primary antibody dilutions were 1:10 for
mouse anti-Repo (8D12; DSHB), 1:10 for mouse anti-Ubx (provided by R. White), 1:30 for mouse anti-Abd-B (1A2E9; DSHB),
1:50 for mouse anti-eg (provided by C. Doe), 1:10 for mouse
anti-en (4D9; DSHB) and 1:1000 for mouse anti-β-gal primary
antibodies (Promega). Immunocytochemical staining was carried
out as described in Jeon (Choi et al., 2000). Biotinylated secondary antibodies (1:500) and ABC kits (Vectorlabs) were used. If
necessary, nickel chloride was added to the final coloring reactant to enhance the signal. Whole-mount embryos were viewed
and photographed using an Olympus microscope BX51.
Whole-mount in situ hybridization RNA in situ hybridization
on whole-mount embryos was performed using digoxigeninlabeled antisense RNA probes, according to Tautz and Pfeifle
(1989). The 0−18 h embryos from Pc3 and Oregon-R stocks
were collected, fixed in formaldehyde, and prepared for hybridization.
Results
Effects of the loss-of-function BX-C gene mutations on
the segment-specific MM-CBG formation The expression pattern of repo has been well described (Halter et al.,
1995). repo begins to appear from early stage 10 and is
expressed in MM-CBG at stage 11 (Fig. 2A). Two MMCBG per segment in thorax appear in the CNS of stage 12
embryos. Four MM-CBG per segment in the thorax and
two per segment in the abdomen are present at the medial
region of stage 14 embryos (Fig. 2B). The different pattern of MM-CBG along the anterior-posterior (A/P) axis
438
Effects of Homeotic Transformation on MM-CBG Formation
A. Wild type B. Homeotic gene loss C. Homeotic gene gain
of function mutant
A
B
C
D
E
F
of function mutant
Fig. 1. Hypothetical models of segment-specific MM-CBG pattern formation. Segment-specificity of MM-CBG may be determined by homeotic genes and maintained by their transregulators, PcG. A. MM-CBG pattern of a wild type embryo. Four
MM-CBG are present in each segment of the thorax and two
MM-CBG in each segment of the abdomen. B. Hypothetical
MM-CBG patterns of abd-A mutant embryo. Loss-of-function
mutation in abd-A may cause transformation of the abdominal
MM-CBG pattern to a thoracic pattern. C. Hypothetical MMCBG pattern of Sca;;abd-A or PcG mutant embryo. Ectopic
expression of abd-A in thorax may cause transformation of the
thoracic MM-CBG pattern to an abdominal MM-CBG pattern.
suggests that the MM-CBG pattern formation may depend
on the homeotic genes.
In order to test whether homeotic genes affect segmentspecific MM-CBG patterns, we examined the effect of
mutation in BX-C genes, whose expressions are restricted
to thorax and abdomen. repo expression in Ubx, abd-A,
Abd-B single mutants, and a Ubx abd-A double mutant
was investigated. In the nervous system, Ubx primarily
controls the identity of the third thoracic segment although it is expressed in PS5 through PS13 (Carroll et al.,
1986). Its mutation causes the transformation of PS5 and
PS6 to PS4. abd-A is expressed in PS7 through PS13 and
its mutation causes the transformation of these segments
to PS6. In Ubx abd-A double mutant (Df109) embryos,
PS5-PS9 are transformed to PS4 with a mixed pattern in
PS10-PS13 (Casanova et al., 1987). Finally, Abd-B is expressed in PS10 to PS15 and its mutation causes transformation of PS13 and PS14 to PS12 pattern.
In Ubx mutant embryos, the thoracic MM-CBG pattern
was not present in the first abdominal segment. However,
we found a reduced number of MM-CBG in the second
(2.5 MM-CBG per segment on average) and/or third thoracic segments (2 MM-CBG per segment) (Fig. 2C and
Table 1), which was not reported by previous studies
(Berger et al., 2005). In abd-A mutant embryos, more
than two MM-CBG per segment were present from segments A1 to A4 segments (Table 1), indicating the transformation of A1 through to A4 to thoracic segments in
terms of MM-CBG pattern (Fig. 2D). There were even
embryos whose sixth abdominal segment had an MMCBG thoracic pattern and the increased number of MMCBG in these segments varied (Table 1). Embryos with
Fig. 2. REPO expression in wild type and BX-C gene mutant
embryos. A. Wild type at stage 11. repo is expressed in just one
pair of MM-CBG per segment, which is not migrated to the
medial region yet. B. Wild type embryo at stage 14. repo is
exclusively expressed in most glia. Four MM-CBG per segment
are present in the medial region of the thorax (arrow), whereas
two MM-CBG per segment are present in the abdomen (arrow
head). C. Ubx mutant embryo. MM-CBG are absent in the thoracic segment in a small number of embryos (arrowhead). D.
abd-A mutant embryo. Three or four MM-CBG per segment are
frequently present in segments A1 to A4 (arrows). E. Abd-B
mutant embryo. Three MM-CBG per segment are frequently
present in the A7 segment (arrow). F. Ubx abd-A double mutant
embryo. MM-CBG are frequently missing in the thorax (arrowhead) and three or four MM-CBG per segment are present in A1
to A5 (arrow). T3, third thoracic segment; A1, first abdominal
segment.
these mutant phenotypes were more dramatically increased in Ubx abd-A double mutant embryos (Fig. 2F).
This transformation was detected even in A7 of UBX abdA double mutant embryos (Table 1). The second and/or
third thoracic MM-CBG were also missing in Ubx abd-A
double mutants compared to the Ubx single mutant (Table
1). Abd-B mutant embryos showed an MM-CBG transformation pattern in the A7 segment (Fig. 2E), as well as
in A5 and A6. In contrast to Ubx, it appears that abd-A
and Abd-B are strongly involved in determining the identities of MM-CBG.
Effects of gain-of-function BX-C gene mutations on
segment-specific MM-CBG formation As the loss-offunction mutants of the homeotic genes caused homeotic
transformations of the MM-CBG pattern, we investigated
whether the ectopic expression of BX-C genes transforms
the thoracic MM-CBG pattern to the abdominal pattern.
When BX-C genes were ectopically expressed under heat
shock control, embryonic transformation was found in
segments anterior to their normal realm of expression
(Lamka et al., 1992).
Ubx, abd-A or Abd-B gene was ectopically expressed
using the Sca-GAL4/UAS-Ubx, Sca-GAL4/UAS-abdA or
Sca-GAL4/UAS-AbdB systems, respectively. sca is expressed in cellular blastoderm at stage 5, in neuroecto-
Sun-Young Kang et al.
439
Table 1. Average number of MM-CBG in each segment of homeotic gene mutants.
Strain
Wild type
Ubx
Segment
T1
T2
4
4
T3
4
A1
2
A2
A3
2
2
A4
2
A5
A6
2
2
A7
2
A8
2
3.25
2.5
2
2
2
2
2
2
2
2
2
abd-A
4
4
3.75
3.25
3.25
3.5
3.5
3.5
3
2
2
Abd-B
4
4
4
2
1.5
2
2
2.5
2.5
2.5
2
Ubx abd-A
4
3.5
2.83
2.5
2.33
2.67
3
3.33
2.67
2.33
2
MM-CBG in 10 embryos of each mutants were counted. “T” means thoracic segments and “A” abdominal segments.
A
C
B
A
B
C
D
E
F
G
H
D
Fig. 3. repo expression in embryos in which Ubx, abd-A or AbdB is ectopically expressed. repo is expressed from early stage11
and exclusively present in glia of stage 14 embryos with few
exceptions. (A) Wild type. (B) Ubx, (C) abd-A, and (D) Abd-B
genes were ectopically expressed by Sca-GAL4/UAS-Ubx, ScaGAL4/UAS-abd-A, and Sca-GAL4/UAS-Abd-B system, respectively. In these mutant embryos, glia expressing repo are frequently missing or misaligned. The embryo clearly shows transformation of a thoracic MM-CBG pattern to an abdominal pattern (arrows).
derm at stage 8 and in neuroblasts at stage 9−11 (Graba et
al., 1992). Under the regulation of the sca regulatory region, Ubx, abd-A or Abd-B was expressed in the nervous
system of thoracic and abdominal segments from the early
embryonic stage.
Ectopically expressed Ubx, abd-A or Abd-B reduced the
numbers of MM-CBG in thoracic segments (Figs. 3B, 3C,
and 3D), which concurs with previously reported data,
whereby NB6-4T was transformed to NB6-4A (Berger et
al., 2005). However, in the present study, Abd-B overexpression caused severe defects in glial development,
which has not been reported previously (date not shown).
Unlike its weak function in loss-of-function mutants,
more than sixty percent of Ubx gain-of-function mutatant
embryos reduced the number of MM-CBG in thoracic
segments. Thus, our data suggest that BX-C gene prod-
Fig. 4. Denticle patterns and expression of BX-C genes in wild
type and Pc mutant embryos. (A, C, E, G) Wild type and (B, D, F,
H) Pc mutant embryos. (A, B) Wild type embryo shows segmentspecific denticle belts, while Pc mutant shows homeotic transformation of all denticle belts to the eighth one. (B−H) Expression of
BX-C genes at stage 11 embryos. Anterior is to the left. Arrows
indicate the anterior boundary shown in wild type. (C, E, G) wild
type embryos. Ubx is expressed from PS5 to 13, abd-A from PS7
to PS13, and Abd-B from PS10 to the rest of segments. (D, F, H)
Pc mutant embryos. Ubx, abd-A and Abd-B are expressed in thoracic segments of Pc mutant embryos.
ucts are important for establishing segment-specific MMCBG.
Effects of mutations in the PcG genes on MM-CBG
formation There is another way to misexpress Ubx, abdA and Abd-B in embryos. Mutations in Polycomb group
(PcG) genes cause BX-C genes to be ectopically expressed (McKeon and Brock, 1991; Simon et al., 1992).
The PcG genes comprise at least fifteen members with related phenotypes and in common show homeotic transfor-
440
Effects of Homeotic Transformation on MM-CBG Formation
A
B
A
B
C
D
C
D
Fig. 5. Effects of Pc, esc and pho mutation on gcm expression.
One gcm-lacZ line, gcm+1.7/+4.5-hs43–lacZ, shows gcm expression in MM-CBG in the abd-A domain. This line was
crossed with Pc (B), esc (C) and pho (D) mutants, and embryos
from these reconstructed flies were stained with anti-β-gal antibody. All three mutant embryos did not show gcm expression in
thoracic MM-CBG. Rather, esc and pho mutant embryos at
stage15 showed loss of MM-CBG or M-CBG in certain abdominal segments (arrows), but did not show MM-CBG or MCBG in thoracic segments. Pc mutant embryos showed no MMCBG defects.
mation as seen in gain-of-function mutation of BX-C genes
(Fig. 4). As BX-C genes begin to be ectopically expressed
in PcG mutant embryos at stage 11, which precedes NB
proliferation (Fig. 4), we predicted that ectopic Ubx, abd-A,
or Abd-B expression in PcG mutant embryos would affect
MM-CBG pattern formation (Fig. 1C). As PcG proteins
work in complexes, we chose three PcG mutants to investigate their effects on the formation of MM-CBGs: Polycomb (Pc) (Lewis, 1978) a member of Polycomb repressive
complex 1 (PRC1), extra sex combs (esc) (Struhl, 1981) a
member of Polycomb Repressive Complex 2 (PRC2), and
pleiohomeotic (pho) (Girton and Jeon, 1994) the product of
which has a unique DNA binding motif among PcG proteins. Pc and esc embryos show the most severe phenotypes among PcG mutants, while pho mutant has very weak
maternal effects (Girton and Jeon, 1994).
Pc was balanced with lacZ reporter genes to distinguish
homozygous Pc/Pc mutant embryos from others. esc embryos showing maternal effects were obtained from transheterozygote females produced by crossing esc2 with
esc5. pho maternal effect mutant embryos were obtained
from phocv mutant allele. Most of PcG mutant embryos
had normal MM-CBG pattern, while about two to three
percent of PcG mutant embryos showed a reduced number of thoracic MM-CBG, although Ubx, abd-A or Abd-B
gene was strongly misexpressed in the head, thoracic and
abdominal segments of these PcG mutant embryos (Fig.
4). These results suggest that the BX-C genes overexpressed lately in PcG mutant did not cause transformation
of MM-CGB.
That lately expressed BX-C genes does not transform
MM-CBG was also shown using the gcm-lacZ line. gcm+
1.7/+4.5-hs43–lacZ line shows gcm expression in M-CBG
Fig. 6. Expression of eg (blue) and en (brown) at stage 16 embryos. (A) Wild type, (B) Pc, (C) esc, (D) pho. Pc and esc mutant embryos showed a normal eg expression pattern. pho mutant embryos surprisingly showed a NB6-4T thoracic pattern in
the abdomen. Arrowheads indicate NB6-4T (arrows). En expression was used as a positional marker of NB6-4.
and MM-CBG of the abd-A functional domain (Fig. 5A).
gcm-lacZ was not expressed in abd-A mutant embryos
(Jones et al., 2004), indicating that abd-A is involved in
determining MM-CBG identity. However, when abd-A was
ectopically expressed in the thorax by Pc mutation, gcm
transcripts were not found in MM-CBG of the thorax, indicating that the thoracic MM-CBG pattern had not been
transformed to the abdominal MM-CBG pattern (Fig. 6B).
esc and pho mutant embryos neither showed expression of
gcm-lacZ in the thorax (Figs. 5C and 5D, respectively).
Instead, MM-CBG and M-CBG were frequently missing in
esc and pho mutant embryos (Figs. 6C and 6D, arrows).
This result is contrast with the fact that PcG proteins are
involved in maintaining the anterior boundary of homeotic
gene expression in epidermis and the central nervous system. Other systems, as yet undiscovered, appear to maintain the anterior boundary of gcm+1.7/+4.5-hs43–lacZ
line.
In order to further investigate that PcG mutations do not
cause transformation of MM-CBG pattern, we examined
the segment-specific expression of eg in neuroblasts (NBs).
eagle (eg) is expressed in specific NBs, such as, NB2-4,
NB3-3, NB6-4 and NB7-3 (Higashijima et al., 1996). If
PcG mutations affect the identity of NBs, the thoracicspecific expression of eagle would disappear in PcG mutant embryos. To locate NB6-4T more precisely, eg and en
double staining was performed. In esc and Pc mutant embryos, eg expression was normal likely in wild type. However, pho mutant embryos exhibited reverse transformation,
which was not expected (Fig. 7D). About 20% (n = 165) of
total pho mutant embryos showed such enhanced numbers
of eg-expressing NBs in abdominal segments.
Discussion
In the Drosophila embryonic central nervous system (CNS),
about 30 glia are produced in a stereotyped pattern in each
Sun-Young Kang et al.
hemisegment (Ito et al., 1995), and certain of these glia are
arranged in different patterns between segments along the
A/P axis (Halter et al., 1995; Jones et al., 2004). Thus, it is
important to understand how the regional specificity of
certain glia is determined and maintained during nervous
system development. repo is essentially required for the
differentiation and maintenance of glia (Halter et al., 1995;
Xiong et al., 1994). Moreover, some of these repo expressing cells, MM-CBG, show different patterns along the A/P
axis. In the present study, MM-CBG pattern abnormalities
were examined in BX-C and its negative transregulator,
PcG mutant embryos.
Our data showed that Ubx loss-of-function mutation did
not cause the homeotic transformation of the abdominal
MM-CBG pattern to the thoracic one. However, a loss-offunction mutation in the abd-A gene caused the transformation of abdominal MM-CBG into a thoracic pattern. Abd-B
mutant embryos also showed transformation of MM-CBG
in its functional domain. These results indicate that unlike
Ubx, abd-A and Abd-B genes are involved in the segmentspecific MM-CBG pattern formation. The role of BX-C on
MM-CBG formation was confirmed using gain-of-function
BX-C mutation. Ectopic expression of BX-C with scaGAL4/UAS system caused thoracic MM-CBG to follow
the abdominal pattern of MM-CBG. Unlike the result
shown in Ubx loss-of-function mutant embryos, four thoracic MM-CBG was frequenctly reduced to two or three
MM-CBG in Ubx gain-of-function mutant embryos, suggesting that Ubx might be involved in MM-CBG pattern
formation. The abd-A and Abd-B proteins driven by scaGAL4 driver changed the thoracic MM-CBG pattern to the
abdominal one. It was suggested that abd-A and Abd-B
proteins repress the proliferation of MM-CBG through inhibition of CycE in the abdomen, which makes two MMCBG per abdominal segment and four MM-CBG per thoracic segment (Berger et al., 2005).
PcG mutation causes the ectopic expressions of abd-A
and Abd-B genes in the anterior of their functional domains
(McKeon and Brock, 1991; Simon et al., 1992). We presumed that the ectopic thoracic expressions of abd-A and
Abd-B genes would transform thoracic MM-CBG to an
abdominal one as shown in the gain-of-function BX-C mutation, because the thoracic pattern of the epidermis and
central nervous system are transformed to the abdominal
segments in these two mutants. However, PcG mutant embryos showed little evidence of an abnormal MM-CBG
pattern in the thorax because most PcG mutant embryos
showed wild type thoracic MM-CBG pattern. This was
confirmed using a gcm enhancer trap line. In gcm+1.7/+
4.5-hs43-lacZ embryos, gcm-lacZ is expressed only in the
MM-CBG of the abd-A domain (Jones et al., 2004). Although Pc zygotic, esc and pho maternal effect mutations
caused the ectopic expressions of abd-A and Abd-B in the
CNS from head to tail, the anterior boundary of gcm-lacZ
expression did not move to more anterior segments. In ad-
441
dition, thorax-specific eg expression pattern was unchanged in PcG mutant embryos.
Our observations indicate that temporal and spatial homeotic gene expression is important in MM-CBG pattern
formation. The homeotic gene products driven by scaGAL4 driver are present in the neuroectoderm from embryonic stage 8, which clearly changes the thoracic MM-CBG
pattern. However, derepressed BX-C gene products caused
by PcG mutations do not affect MM-CBG pattern. Ubx,
abd-A and Abd-B genes begin to be weakly misexpressed
from stage 11 and shows strong ectopic expression at stage
13 in Pc and esc mutant embryos (McKeon and Brock,
1991; Simon et al., 1992). In wild type embryos MM-CBG
appears to proliferate once between stage 11 and 12, and
become four cells per segment in the thorax, while there is
no cell division of MM-CBG in the abdomen because abdA and Abd-B proteins repress CycE expression (Berger et
al., 2005; Halter et al., 1995). So PcG mutants seems to
cause the ectopic expression of the BX-C genes after MMCBG are already determined to be prolifered in the thorax.
Early segment-specific commitment of NB6-4 progeny
cells also supports our conclusion. When BX-C genes are
overexpressed from stage 10 using eg-GAL4, thoracic
MM-CBG pattern was not changed. Taken together, temporal and spatial expression of the homeotic genes is important to determine segmental-specificity of MM-CBG along
the anterior-posterior (A/P) axis.
Acknowledgments We thank G. M. Technau for the Abd-B
mutant, B. W. Jones for gcm-lacZ line, R. White for anti-Ubx
antibody, C. Doe for anti-Eg antibody. We also thank Chung-Min
Oh and Keuk Il Jung for their technical support. This research was
supported by grants from the Korean Science and Engineering
Foundation (KOSEF) (R01-2004-000-10585-0).
References
Akiyama-Oda, Y., Hosoya, T., and Hotta, Y. (1999) Asymmetric
cell division of thoracic neuroblast 6-4 to bifurcate glial and
neuronal lineage in Drosophila. Development 126, 1967−1974.
Beachy, P. A., Helfand, S. L., and Hogness, D. S. (1985) Segmental distribution of bithorax complex proteins during Drosophila
development. Nature 313, 545−551.
Berger, C., Pallavi, S. K., Prasad, M., Shashidhara, L. S., and
Technau, G. M. (2005) A critical role for cyclin E in cell fate
determination in the central nervous system of Drosophila
melanogaster. Nat. Cell Biol. 7, 56−62.
Breen, T. R. (1999) Mutant alleles of the Drosophila trithorax
gene produce common and unusual homeotic and other developmental phenotypes. Genetics 152, 319−344.
Carroll, S. B., Laymon, R. A., McCutcheon, M. A., Riley, P. D.,
and Scott, M. P. (1986) The localization and regulation of
Antennapedia protein expression in Drosophila embryos. Cell
47, 113−122.
442
Effects of Homeotic Transformation on MM-CBG Formation
Casanova, J., Sanchez-Herrero, E., Busturia, A., and Morata, G.
(1987) Double and triple mutant combinations of bithorax
complex of Drosophila. EMBO J. 6, 3103−3109.
Celniker, S. E., Keelan, D. J., and Lewis, E. B. (1989) The molecular genetics of the bithorax complex of Drosophila:
characterization of the products of the abdominal-B domain.
Genes Dev. 3, 1424−1436.
Choi, S. H., Oh, C. T., Kim, S. H., Kim, Y. T., and Jeon, S. H.
(2000) Effects of Polycomb group mutations on the expression
of Ultrabithorax in the Drosophila visceral mesoderm. Mol.
Cells 10, 156−161.
Doe, C. Q. (1992) Molecular markers for identified neuroblasts
and ganglion mother cells in the Drosophila central nervous
system. Development 116, 855−863.
Duncan, I. (1987) The bithorax complex. Annu. Rev. Genet. 21,
285−319.
Egger, B., Leemans, R., Loop, T., Kammermeier, L., Fan Y., et al.
(2002) Gliogenesis in Drosophila: genome-wide analysis of
downstream genes of glial cells missing in the embryonic
nervous system. Development 129, 3295−3309.
Girton, J. R. and Jeon, S. H. (1994) Novel embryonic and adult
homeotic phenotypes are produced by pleiohomeotic mutations in Drosophila. Dev. Biol. 161, 393−407.
Graba, Y., Aragnol, D., Laurenti, P., Garzino, V., Charmot, D., et
al. (1992) Homeotic control in Drosophila; the scabrous gene
is an in vivo target of ultrabithorax proteins. EMBO J. 11,
3375−3384.
Halter, D. A., Urban, J., Rickert, C., Ner, S. S., Ito, K., et al.
(1995) The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic
nervous system of Drosophila melanogaster. Development 121,
317−332.
Higashijima, S., Shishido, E., Matsuzaki, M., and Saigo, K.
(1996) eagle, a member of the steroid receptor gene superfamily, is expressed in a subset of neuroblasts and regulates
the fate of their putative progeny in the Drosophila CNS. Development 122, 527−536.
Hosoya, T., Takizawa, K., Nitta, K., and Hotta, Y. (1995) glial
cells missing: a binary switch between neuronal and glial determination in Drosophila. Cell 82, 1025−1036.
Ingham, P. W. (1985) A clonal analysis of the requirement for the
trithorax gene in the diversification of segments in Drosophila.
J. Embryol. Exp. Morphol. 89, 349−365.
Irish, V. F., Martinez-Arias, A., and Akam, M. (1989) Spatial
regulation of the antennapedia and ultrabithorax homeotic
genes during Drosophila early development. EMBO J. 8,
1527−1537.
Ito, K., Urban, J., and Technau, G. M. (1995) Distribution, classification, and development of Drosophila glial cells in the late
embryonic and early larval ventral nerve cord. Roux’s Arch.
Dev. Biol. 204, 284−307.
Jacobs, J. J. and van Lohuizen, M. (2002) Polycomb repression:
from cellular memory to cellular proliferation and cancer. Biochim. Biophys. Acta 1602, 151−161.
Jones, B. W. (2001) Glial cell development in the Drosophila
embryo. Bioessays 23, 877−887.
Jones, B. W., Abeysekera, M., Galinska, J., and Jolicoeur, E. M.
(2004) Transcriptional control of glial and blood cell development in Drosophila: cis-regulatory elements of glial cells missing. Dev. Biol. 266, 374−387.
Jurgens, G. (1985) A group of genes controlling the spatial expression of the bithorax complex in Drosophila. Nature 316,
153−155.
Karch, F., Bender, W., and Weiffenbach, B. (1990) abdA expression in Drosophila embryos. Genes Dev. 4, 1573−1587.
Kaufman, T. C., Seeger, M. A., and Olsen, G. (1990) Molecular
and genetic organization of the antennapedia gene complex of
Drosophila melanogaster. Adv. Genet. 27, 309−362.
Klambt, C. (1993) The Drosophila gene pointed encodes two
ETS-like proteins which are involved in the development of
the midline glial cells. Development 117, 163−176.
Lamka, M. L., Boulet, A. M., and Sakonju, S. (2002) Ectopic
expression of Ubx and ABd-B proteins during Drosophila embryogenesis: competition, not a functinal hierachy, explains
phenotypic suppression. Development 116, 841−854.
Lemke, G. (2001) Glial control of neuronal development. Annu.
Rev. Neurosci. 24, 87−105.
Lewis, E. B. (1978) A gene complex controlling segmentation in
Drosophila. Nature 276, 565−570.
Mahmoudi, T. and Verrijzer, C. P. (2001) Chromatin silencing and
activation by polycomb and trithorax group proteins. Oncogene 20, 3055−3066.
McKeon, J. and Brock, H. W. (1991) Interactions of the Polycomb
group of genes with homeotic loci of Drosophila. Roux’s Arch.
Dev. Biol. 199, 387−396.
Orlando, V. and Paro, R. (1995) Chromatin multiprotein complexes involved in the maintenance of transcription patterns.
Curr. Opin. Genet. Dev. 5, 174−179.
Sedkov, Y., Tillib, S., Mizrokhi, L., and Mazo, A. (1994) The
bithorax complex is regulated by trithorax earlier during Drosophila embryogenesis than is the Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early
trithorax transcripts. Development 120, 1907−1917.
Simon, J., Chiang, A., and Bender, W. (1992) Ten different Polycomb group genes are required for spatial control of the abdA
and AbdB homeotic products. Development 114, 493−505.
Struhl, G. (1981) A gene product required for correct initiation of
segmental determination in Drosophila. Nature 293, 36−41.
Struhl, G. and White, R. A. (1985) Regulation of the Ultrabithorax
gene of Drosophila by other bithorax complex genes. Cell 43,
507−519.
Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals transcriptional control of the segmentation gene hunchback. Chromosoma 98, 81−85.
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A., and Montell,
C. (1994) repo encodes a glial-specific homeo domain protein
required in the Drosophila nervous system. Genes Dev. 8,
981−994.