Download Ectopic expression of either the Drosophila

1151
Development 120, 1151-1161 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Ectopic expression of either the Drosophila gooseberry-distal or proximal
gene causes alterations of cell fate in the epidermis and central nervous
system
Yu Zhang, Anne Ungar*, Catalina Fresquez† and Robert Holmgren
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA
*Present address: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
†Present address: Department of Biology, Barry University, Miami Shores, FL 33161, USA
SUMMARY
Previous studies have shown that the segment polarity
locus gooseberry, which contains two closely related transcripts gooseberry-proximal and gooseberry-distal, is
required for proper development in both the epidermis and
the central nervous system of Drosophila. In this study, the
roles of the gooseberry proteins in the process of cell fate
specification have been examined by generating two fly
lines in which either gooseberry-distal or gooseberryproximal expression is under the control of an hsp70
promoter. We have found that ectopic expression of either
gooseberry protein causes cell fate transformations that are
reciprocal to those of a gooseberry deletion mutant. Our
results suggest that the gooseberry-distal protein is
required for the specification of naked cuticle in the
epidermis and specific neuroblasts in the central nervous
system. These roles may reflect independent functions in
neuroblasts and epidermal cells or a single function in the
common ectodermal precursor cells. The gooseberryproximal protein is also found in the same neuroblasts as
gooseberry-distal and in the descendants of these cells.
INTRODUCTION
active both before and during the time that neuroblasts (NBs)
are delaminating to generate the CNS. In a systematic study of
CNS alterations in different segment polarity mutant embryos,
Patel et al. (1989) have shown that five of the segment polarity
genes (gooseberry, patched, Cell, wingless and hedgehog)
cause specific defects in neurogenesis, while two other
segment polarity mutants (fused and armadillo) have relatively
normal CNS patterns. Only mutations in the patched and
gooseberry genes cause CNS defects that appear to mirror the
defects observed in the epidermis. These results suggest that
segmentation of the CNS is not controlled by epidermal segmentation per se. Some of the segment polarity genes (patched
and gooseberry) may play homologous roles in epidermis and
CNS segmentation, perhaps by functioning in common ectodermal precursor cells, while others (wingless and Cell)
function during neurogenesis in a second more restricted
fashion. This suggestion is further supported by a more recent
report showing that the repeated expression pattern of achaete
(ac) in NBs is affected only in naked and gooseberry mutants
and not in the other segment polarity mutants (Skeath et al.,
1992).
Here we report our studies on the segment polarity gene,
gooseberry (gsb), which plays a critical role in specifying cell
fates in the epidermis as well as of NBs and their descendants.
The gsb locus has been found to contain two highly homologous transcripts, gooseberry-proximal (gsbp) and gooseberry-
In Drosophila, the anteroposterior body axis is divided into a
series of repeating units called segments. The process of segmentation is initiated at the beginning of embryogenesis by
maternally deposited morphogens in the fertilized egg. Subsequently, a hierarchy of three groups of zygotic segmentation
genes are sequentially activated to refine the maternal field of
positional information (for review see Ingham and Martinez
Arias, 1992).
In addition to patterning the epidermis, the segmentation
genes are also required for the development of the Drosophila
central nervous system (CNS). In gap and pair-rule gene
mutants, lost epidermal segments are associated with elimination of the corresponding segmental ganglia. While these segmentation defects were expected, a second role for gap and
pair-rule genes in the specification of particular neurons was
not. A number of gap and pair-rule genes are reexpressed in
segmentally repeating patterns in the developing CNS. From
the analysis of conditional mutants, the expression of the pairrule genes, fushi tarazu (ftz) and even-skipped (eve) (Doe et al.,
1988a,b), has been shown to be critical to the proper specification of certain neuronal identities.
The segment polarity genes occupy an intriguing position
within the segmentation hierarchy. They are required in the
epidermis to specify cell fate within each segment and are
Key words: gooseberry, cell fate, segmentation, neurogenesis,
Drosophila, ectopic expression, central nervous system
1152 Y. Zhang and others
distal (gsbd) (Baumgartner et al., 1987; Cöté et al., 1987). Both
gsbd and gsbp encode putative transcription factors, each containing a homeodomain and a paired domain. In mutants deficient for both gsb genes, the naked cuticle of each epidermal
segment is eliminated and replaced with a mirror-image duplication of the denticle belt (Fig. 8B; Nüsslein-Volhard and
Wieschaus, 1980). The alterations in the CNS include the deletion of the posterior commissure and the CQ neurons as well as
the duplication of aCC, pCC and RP2 neurons (Fig. 8H; Patel et
al., 1989). The regions expressing the gsb genes closely correspond to the positions where structures are eliminated in gsb
mutants. In the epidermis, gsbd has been localized to the region
bracketing the parasegmental boundary of each segment and, in
the CNS, gsbd and gsbp are expressed in a subset of NBs and
neurons in the same region (Baumgartner et al., 1987; Ouellette
et al., 1992; Gutjahr et al., 1993b).
In this study, we generated hs-gsbd and hs-gsbp fly lines to
misexpress the gsb genes throughout the embryo. We have
found that ectopic expression of either gsb gene causes cuticle
and CNS alterations that are nearly reciprocal to that of a gsb
mutant. Our results suggest that, in the epidermis, gsbd
organizes pattern elements around the parasegmental boundary
by regulating the expression of wingless (wg) and engrailed
(en). In the CNS, gsbd may function as a selector gene to
specify the cell fate of a subset of NBs. gsbp is expressed in
the descendants of the gsbd-positive NBs and may provide
continued gsb function in these cells.
MATERIALS AND METHODS
Fly strains
The OrR, rosy506, Df(2R)SB1(gsb,Kr), Df(2R)IIX62(zip,gsb) are
described in Lindsley and Zimm (1992). The A31 enhancer trap line
was obtained from A. Ghysen and the wg enhancer trap line
CyO/wg17en40 was obtained from N. Perrimon. gsbp/lacZ is a P
element transformant in which lacZ coding sequences have been fused
in frame to gsbp genomic sequences at an EcoRI site in the third exon.
The expression of β-galactosidase in gsbp/lacZ embryos precisely
coincides with gsbp expression in the CNS (Fresquez, unpublished
data).
Production of gsb proteins and generation of antibodies
To make gsbd protein, a 1 kb HincII fragment, which contains most
of the gsbd coding region, was cloned into the T7 expression vector
pET8c at the BamHI site. The protein was induced in pBL21(DE3)
bacteria cells with IPTG and partially purified from inclusion bodies
(Studier et al., 1990).
A full-length gsbp cDNA clone was isolated by screening a 0- to
16-hour cDNA library (a gift from B. Hovemann) with an appropriate probe and confirmed by partial sequence analysis. An NcoI-NsiI
fragment, which contains the entire gsbp coding region, was cloned
into the T7 expression vector pET8c between the NcoI and XbaI sites.
The gsbp protein was subsequently induced and partially purified
from inclusion bodies.
Standard techniques for generating antibodies (Harlow and Lane,
1988) were used to make both rat polyclonal and monoclonal antibodies specific to gsbd and gsbp, respectively. In both cases, the obtained
antisera also cross react with the other gsb protein. To remove the cross
reactivity, the antisera were passed through a sepharose column coupled with the other gsb protein (for example, the anti-gsbd antiserum
was passed through a gsbp coupled column and vice versa). The elimination of the cross reactivity in preabsorbed antisera was confirmed by
western blots and embryo stainings. For monoclonal antibodies,
immunized rat spleen cells were fused to mouse NSO-1 cells. Two
monoclonal lines, 10E10 and 16F12, were isolated which specifically
recognize gsbd. One monoclonal line, 9A1, was found to be specific to
gsbp. Preabsorbed antisera and monoclonal antibodies give the same
results in all the embryo stainings described in this paper; we refer to
them generally as gsbd and gsbp stainings.
Antibody stainings
The antibody staining patterns were visualized using indirect
immunofluorescence or horseradish peroxidase (HRP) techniques as
previously described (Patel et al., 1989). All the fluorescent stainings
were examined by confocal microscopy using a BioRad MRC-600
confocal microscope. For double labeling experiments with HRP
coupled secondary antibodies, NiSO4 and CoCl2 were added to one
of the reactions to give a blue-gray color (Lawrence et al., 1987). The
rabbit anti-β-gal antiserum was generated in our laboratory
(Holmgren, unpublished), the rabbit anti-eve antiserum was a gift
from M. Frasch, the rabbit anti-wg antiserum was a gift from R. Nusse
and the anti-ac antibody was a gift from S. Carroll.
Cuticle preparations
Cuticles were mounted in a 1:1 mixture of Hoyer’s solution and lactic
acid (van der Meer, 1977) and photographed under phase contrast.
hs-gsbd and hs-gsbp fly lines
A genomic clone containing the entire gsbd coding sequence and a
cDNA clone containing the entire gsbp coding sequence were independently inserted into modified Carnegie 20 P element vectors
(Rubin and Spradling, 1983) containing a 0.4 kb hsp70 promoter
(from S. Lindquist). Each construct was then coinjected with the
wings clipped plasmid into rosy506 embryos as described in Roberts
(1986). Multiple insertion sites for each line were examined and gave
similar phenotypes after heat shock.
Heat-shock treatment
Embryos were collected for 30 minutes and allowed to develop at
room temperature for a specified time period as described in the text.
The heat-shock treatment was performed by placing embryos onto
prewarmed molasses-agar egg collection plates floating in a water
bath at 36.5°C for 30 minutes. Multiple heat shocks were performed
with a 1 hour recovery period following every 30 minute heat shock.
To generate mutant phenotypes in the CNS and cuticles, the optimal
time for heat shocking is 2.5-5 hours. Both heat shocked hs-gsbp and
hs-gsbd embryos give similar phenotypes, though the hs-gsbp lines
respond better than the hs-gsbd lines to late heat shocks. Whether this
reflects differences in the intrinsic activity of the gsbp and gsbd
proteins or more efficient expression from the intronless gsbp
construct is not known. Heat shocks before 2.5 hours disrupt the segmentation of embryos, presumably by interfering with the normal
function of the pair-rule gene, paired, since the gsbp protein and the
paired protein can bind to the same DNA target sites (Treisman et al.,
1991). Later heat shocks (up to 7 hours) can cause small regions
within denticle belts to be transformed into naked cuticle.
In experiments correlating patterns of gene expression with cuticle
phenotypes, each collection of heat shocked embryos was split: one
half was used for antibody staining and the second half was used for
cuticle preparation. In these experiments, at least 50% of the embryos
showed cuticle transformations.
RESULTS
Ectopic expression of either gsbd or gsbp causes
the respecification of the denticle belt to naked
cuticle
In order to express gsbd or gsbp ectopically in fly embryos, we
generated hs-gsbd and hs-gsbp fly lines each of which has a
Ectopic gooseberry changes cell fate 1153
gsb coding sequence under the control of an hsp70 promoter.
Following the heat-shock treatment, there is a ubiquitous
expression of gsbd in hs-gsbd embryos and gsbp in hs-gsbp
embryos, which fades away within the first 60 minutes after
heat shock (data not shown). In our study, the effect of this
ubiquitous expression of gsb on cell fate specifications was
Fig. 1. Cuticle phenotypes of mutant and heat shocked embryos. Anterior is up and the view is ventral. In contrast to the wild-type pattern (A),
gsb mutants (B) have the naked cuticle in each segment replaced by denticle belts. In heat shocked hs-gsbd (C,D) and hs-gsbp (E,F) embryos,
denticles are replaced by naked cuticles. Single heat shocks (given at 2.75-3.25 hours for hs-gsbd and at 4-4.5 hours for hs-gsbp) (C,E)
eliminate entire denticle belts or sections of denticles, while triple heat shocks (D,F) are able to transform the entire ventral epidermis to nearly
naked cuticle. When hs-gsbp (G) and hs-gsbd (H) were crossed into a gsb mutant background (Df(2R)IIX62), triple heat shocks restore a near
wild-type cuticle pattern. Note the characteristic holes of the zipper mutation in Df(2R)IIX62 in the anterior end of the embryos (G,H).
1154 Y. Zhang and others
assessed in the development of both the ventral epidermis and
the CNS.
In gsb mutants, the naked cuticle of each segment is eliminated and replaced by a mirror-image duplication of the
denticle belts (Figs 1B, 8B; Nüsslein-Volhard and Wieschaus,
1980). In the cuticles of heat shocked hs-gsbd and hs-gsbp
embryos, groups of denticles or entire denticle belts are
replaced with naked cuticle (Fig. 1C-F). The severity of the
phenotype correlates with the timing of the heat shock. Heat
shocks from 2.75 to 3.25 hours transform complete segments
while those after 4 hours can transform sections of segments.
Multiple heat shocks generate embryos with completely naked
cuticles. To verify that cell fates are being transformed, we
examined the Keilin’s organs, which are located at the parasegmental border in each thoracic segment. In gsb mutants
Keilin’s organs are deleted, while in heat shocked hs-gsbd and
hs-gsbp embryos, ectopic Keilin’s organs occasionally develop
(data not shown). These results indicate that the cells in the
anterior portion of the segment have been respecified and that
the cuticle phenotype of heat shocked hs-gsbd and hs-gsbp
embryos is nearly reciprocal to that of gsb mutants (Fig. 8C).
It was somewhat surprising that heat shocked hs-gsbp was
able to give pattern transformations similar to heat shocked hsgsbd since gsbp is not normally expressed in the epidermis at
the times that the heat shocks were administered (Ouellette et
al., 1992; Gutjahr et al., 1993b). Therefore, we examined the
ability of heat shocked hs-gsbd and hs-gsbp to rescue the
phenotype of Df(2R)IIX62 embryos which are deleted for the
gsb locus. With a series of three heat shocks both hs-gsbd and
hs-gsbp are able to rescue the formation of naked cuticle (Fig.
1G-H).
Heat shocked hs-gsbd or hs-gsbp induces ectopic
expression of the endogenous gsbd gene and
activates expression of wg and en
To determine whether changes at the molecular level correlate
with the cuticle phenotype of heat shocked hs-gsb (hs-gsbd or
hs-gsbp) embryos, we examined the expression of gsbd and
two other segment polarity genes wg and en. We have found
that hs-gsb embryos given heat shocks between 2.5 and 4 hours
after egg-laying, activate ectopic expression of the endogenous
gsbd gene (Fig. 2A,C). The ectopic stripe of gsbd expression
is maintained 2 hours after heat shock and is dependent on the
presence of a wild-type copy of the gsb locus (data not shown).
Thus the ectopic gsbd expression must be due to the activation
of the endogenous gsbd gene. The activation of ectopic gsbd
is accompanied by the formation of an ectopic parasegmental
groove (data not shown) flanked by inverted ectopic stripes of
wg and en (Fig. 2A,C). Thus, it appears that the ubiquitous
expression of gsb induces a mirror-image duplication of the
region bracketing the parasegmental boundary. Heat shocks
after 4 hours also transform the denticle belts into naked
Fig. 2. Expression of gsbd, wg and en in heat shocked hs-gsbp embryos. Late stage 10 embryos; anterior is to the left and view is ventral.
(A,B) Double labelings of gsbd (red) and wg (green); (C,D) double labelings of gsbd (red) and en (green). Heat shocks were given at 2.75 to
3.25 hours for A and C, and at 4-4.5 hours for B and D. Early heat shocks induce ectopic stripes of gsbd and wg (A), and of gsbd and en (C)
(marked by `). Note that the ectopic en stripes are located anterior to the ectopic gsbd stripes (C). Late heat shocks do not induce ectopic
stripes of gsbd, wg or en (B,D), though there is a low level of wg expression throughout the embryo (B). Similar results are observed using the
hs-gsbd line. Bar, 20 µm.
Ectopic gooseberry changes cell fate 1155
cuticle. These late heat shocks do not lead to ectopic en
expression but correlate with a low level of wg expression
throughout the segment (Fig. 2B,D).
The expression of gsbd and gsbp in the CNS
In addition to epidermal expression, gsbd is expressed in a
subset of NBs and their progeny in the posterior portion of each
segment (Gutjahr et al., 1993b). gsbp is first expressed in NBs
and is found at high levels in the progeny of these NBs
(Ouellette et al., 1992). Later as the germ band starts to retract,
low level of gsbp becomes detectable in the epidermis. Our
analysis of the expression patterns of gsbd and gsbp generally
agrees with the results by Gutjahr et al. (1993b), who found
that in the CNS gsbd is expressed in row 5, row 6 NBs and
transiently in NB7-1. One exception is that we find gsbd to be
continually expressed in NB7-1 (as in Fig. 3A). gsbp is also
expressed in the NBs of row 5, row 6 and 7-1 (Fig. 3B), and
thus gsbp appears to follow gsbd expression in the CNS.
To confirm that gsbp is indeed expressed in all gsbd-positive
NBs, we used a gsbp/lacZ line in which a lacZ gene is under
the control of a gsbp promoter. Using double label experiments
and confocal microscopy, we have found that the gsbp/lacZ
construct is expressed in the exact same set of NBs and neurons
as is the gsbp protein (data not shown). By double labeling with
anti-β-galactosidase (β-gal) and anti-gsbd antibodies, it can be
seen that β-gal (representing gsbp) is expressed in gsbdpositive NBs and their progeny (Fig. 3C-E).
It should be noted that the level of gsbd expression in GMCs
and neurons is quite low and fades away during germ band
retraction, whereas gsbp retains high levels of expression in
GMCs and neurons as neurogenesis progresses. This suggests
that, in the CNS, gsbd functions primarily in NBs and gsbp in
GMCs and progeny neurons.
NB cell fates are respecified in heat shocked
hs-gsbd embryos
To study the effects of heat-shock-induced ectopic gsbd
expression on NB cell fate, we used the anti-ac antibody. In
stage 9 wild-type embryos, four rows of NBs are present within
each CNS hemisegment and the anti-ac antibody recognizes
NBs in row 3 and 7 (Figs 4A, 8D; Skeath and Carroll, 1992).
In gsb mutants, Skeath et al. (1992) have shown that the ac
protein is additionally expressed in the two NBs at the position
of row 5 (Fig. 8E). In heat shocked hs-gsbd embryos, acpositive NBs are restricted to the NBs of row 7 in each CNS
hemisegment and the staining of the row 3 NBs is eliminated
(Figs 4B, 8F). These reciprocal transformations suggest that,
in gsb mutants, row 5 NBs are transformed into row 3 and that,
in heat shocked hs-gsbd embryos, row 3 NBs are transformed
into row 5.
This conclusion was strengthened by analyzing wg gene
expression in heat shocked hs-gsbd embryos. To monitor wg
expression, we used a wg enhancer trap line CyO/wg17en40 in
which β-gal expression is regulated by the wg promoter
(Perrimon et al., 1991). In wild-type embryos, β-gal from the
wg enhancer trap is expressed only in row 5 NBs (Fig. 4C;
Doe, 1992). In heat shocked hs-gsbd embryos, additional NBs
anterior to row 5 NBs express β-gal (Fig. 4D), suggesting the
transformation of row 3 NBs into row 5 NBs.
Ectopic expression of the gsb genes causes CNS
pattern defects reciprocal to that of a gsb mutant
The effect of ectopic gsbd or gsbp on axonal pattern in the
mature embryonic CNS was first examined by anti-HRP
antibody staining. We have found that heat shocks at 2.75 to
3.25 hours of development for hs-gsbd embryos and 4.5-5.0
hours for hs-gsbp embryos give the most consistent alterations
Fig. 3. Expression of gsbd and gsbp in the developing CNS. (A,B) Anterior is up and the view is ventral. (A) gsbd is expressed in four NBs of
each hemisegment at early stage 9, including NB7-1. (B) By early stage 11, gsbp is expressed in the NBs of row 5 and 6 and NB7-1, where
gsbd is also expressed (Gutjahr et al., 1993b). (C-E) Lateral views of a section of a gsbp/lacZ embryo (stage 11) double labeled with anti-gsbd
and anti-β-gal (representing gsbp) antibodies. gsbd (green) is expressed in the epidermis, the NBs and their progeny and the mesoderm while
β-gal is expressed at high levels only in the gsbd-positive NBs and their progeny. At this particular focal plane, the normally strong epidermal
expression of gsbd is out of focus. (N, NB; n, neural cells including GMCs and neurons; m, mesoderm; e, epidermis) Bar, 20 µm.
1156 Y. Zhang and others
Fig. 4. NB patterns in heat shocked hs-gsbd embryos. Anterior is up and the view is ventral. Anti-ac antibody stainings in a wild-type embryo
(A) and a heat shocked hs-gsbd embryo (B), both at stage 9. In the wild-type embryo, anti-ac antibody recognizes two rows of NBs in every
segment, row 3 and row 7 (as indicated). In heat shocked hs-gsbd embryos, only the row 7 NBs are recognized by anti-ac antibody and row 3
NBs cease to express the ac protein. wg expression is monitored by the expression of β-gal from the wg enhancer trap wg17en40. At late stage 9
in a wild-type embryo (C), the β-gal of the wg enhancer trap is expressed only in the three NBs of row 5 in each hemisegment (arrowheads). In
a heat shocked hs-gsbd embryo (D), besides the three row 5 NBs (arrowheads), wg enhancer trap expression is also detected in NBs anterior to
row 5 (arrows). In both B and D, heat shocks were performed at 2.75-3.25 hours after egg laying. Bar, 20 µm.
to the pattern of axons (Fig. 5A, wild type; Fig. 5B, hs-gsbd
and 5C, hs-gsbp). The defects in heat shocked hs-gsbd and hsgsbp embryos are very similar, typified by the fusion of the
two commissures within each segment and the elimination of
the longitudinal connectives.
In order to follow cell fate changes in heat shocked hs-gsb
embryos, we chose to examine anti-eve antibody staining,
because anti-eve antibodies recognize subsets of neurons with
different behaviors in gsb mutants: CQ neurons are deleted,
aCC, pCC and RP2 neurons are duplicated, and EL neurons
appear to be unaffected (Fig. 8H; Patel et al., 1989). Anti-eve
antibody staining of heat shocked hs-gsbp embryos reveals
pattern alterations nearly reciprocal to that of gsb mutants. Fig.
6B and 6D show that the eve expression characteristic of the
RP2 and EL neurons is eliminated in most segments. In Fig.
6D, the eve expression expected in aCC and pCC neurons also
appears to be eliminated in some segments, and there are
putative duplications of the CQ neurons.
Using anti-eve antibody stainings, it is difficult to distinguish unambiguously the aCC and pCC neurons from the
adjacent CQ neurons in heat shocked hs-gsbp or gsb mutant
embryos. To resolve this problem, we used the A31 enhancer
trap line. The A31 enhancer trap line has a P[lacZ] element
inserted into the fasciclin II gene (Ghysen and O’Kane, 1989;
Grenningloh et al., 1991). In the CNS of A31 embryos, β-gal
is only expressed in the aCC and pCC neurons at early stage
12. The A31 enhancer trap line was crossed into the hs-gsbp,
hs-gsbd and gsb mutant backgrounds and the behavior of aCC
and pCC neurons was examined by anti-β-gal antibody
stainings. Two pairs of aCC and pCC neurons are in each CNS
segment of wild-type embryos (Fig. 6E), and apparent duplication of aCC and pCC neurons occurs in gsb mutant embryos
Fig. 5. The organization of axons in
the CNS (stage 15) visualized by antiHRP antibody labeling. Wild-type
CNS (A) shows the ladder-like
arrangement of axons; longitudinal
connectives run the length of the CNS
and are linked in each segment by pairs
of commissures. In heat shocked hsgsbd (B) and heat shocked hs-gsbp (C)
embryos, the two commissures of each
segment are fused in most of the
segments and the longitudinal
connectives between adjacent
segments are eliminated. Bar, 50 µm.
Ectopic gooseberry changes cell fate 1157
(Fig. 6F). Elimination of β-gal-expressing aCC and pCC
neurons occurs occasionally in heat shocked hs-gsbp embryos
(heat shocking at 4.5±0.25 hours) (Fig. 6G) and occurs much
more frequently in heat shocked hs-gsbd embryos (heat
shocking at 3.0±0.25 hours) (Fig. 6H). Since the aCC and pCC
neurons are never duplicated in heat shocked hs-gsbp embryos,
the duplicated neurons (Fig. 6D) are most likely CQ neurons.
Neurons deleted in gsb mutants normally express
the gsbp protein, whereas neurons duplicated in
gsb mutants do not express the gsbp protein
The neuronal phenotypes observed in gsb mutant and heat
shocked hs-gsbp embryos suggest that gsb function is both
necessary and sufficient for the cell fate specification of a
subset of posterior neurons, like the CQ neurons. It is important
to examine whether gsbp is indeed expressed in these neurons
but not in neurons which are duplicated in gsb mutants and
deleted in heat shocked hs-gsbp embryos. Again, we used eve
expression as our neuronal marker.
Double stainings of anti-gsbp and anti-eve antibodies in
wild-type embryos show that anti-eve-stained CQ neurons also
stain with anti-gsbp antibody, while the EL neuron cluster is
outside the gsbp staining region (Fig. 7A). At a more dorsal
focal plane of the same embryo (Fig. 7B), the aCC and pCC
Fig. 6. Changes in patterns of protein expression in heat shocked hs-gsbp and heat shocked hs-gsbd embryos. Anterior is up and the view is
ventral. Heat shocks were performed at 4.25 hours after embryo collections for B, D and G, at 2.75 h for H. A wild-type stage 11 embryo (A)
labeled with anti-eve antibodies shows two clusters of neurons composed of aCC, pCC and CQ neurons (arrow), and a pair of RP2 neuron
(arrowhead) in every segment. In a stage 11 heat shocked hs-gsbp embryo (B), the RP2 neurons are eliminated in most of the segments
(arrowhead) while the aCC/pCC/CQ neuron cluster persists. A wild-type stage 13 embryo (C) stained with anti-eve antibody shows four groups
of neurons: the EL neuron cluster (arrowhead), the aCC and pCC neurons (double arrow), the CQ neurons (,) and the RP2 neurons (visible in
some segments). In a stage 13 heat shocked hs-gsbp embryo (D), the EL neuron cluster is usually deleted (arrowhead), the aCC and pCC
neurons are occasionally eliminated (double arrow), and extra CQ-like neurons are detected (,). Anti-β-gal antibody staining of an early stage
12 A31 embryo (E) shows labeling of the aCC and pCC neurons (double arrow). In an early stage 12 gsb mutant embryo carrying the A31
enhancer trap (F), the aCC and pCC neurons are duplicated in some of the segments (double arrow). In an early stage 12 heat shocked hs-gsbp
embryo carrying the A31 enhancer trap (G), the aCC and pCC neurons are occasionally deleted (double arrow). In a similarly staged heat
shocked hs-gsbd embryo carrying the A31 enhancer trap (H), the deletion of the aCC and pCC neurons occurs at a much higher frequency
(double arrow). Bar, 50 µm.
1158 Y. Zhang and others
Fig. 7. Expression pattern of gsbp relative to eve and the enhancer trap A31. (A,B) Different focal planes of a wild-type embryo (stage 13)
labeled with anti-gsbp antibody (in brown) and anti-eve antibody (in blue). (A) At the more ventral focal plane, the eve-positive CQ neurons
(,) are within the gsbp expression region while the EL neurons (arrow head) are outside. (B) At the dorsal focal plane, RP2 neurons
(arrowhead) lie just outside of the gsbp expression region, while aCC and pCC neurons (double arrow) are localized just above the gsbp
expression region. (C-E) Confocal pictures of an A31 embryo (early stage 12) double labeled with anti-gsbp (in green) and anti-β-gal (in red)
antibodies. The view is lateral with dorsal up and anterior to the left. The aCC and pCC neurons, recognized by the anti-β-gal antibody, are
shown in C and the gsbp expression regions in D. In a merged image (E), it is clear that the aCC and pCC neurons are outside the gsbp
expression region. Bar, 50 µm.
neurons are stained by the anti-eve antibody and appear to be
above the gsbp-expressing neurons, while the RP2 neurons lie
just anterior to the gsbp-expressing region.
It is known that the aCC and pCC neurons are progeny from
NB1-1 (Goodman et al., 1984; Udolph et al., 1993). During
neurogenesis, they migrate anteriorly to the region just dorsal
to the gsbp-expressing neurons. To confirm that the aCC and
pCC neurons are not labeled by anti-gsbp antibodies, a double
labeling experiment was done with the A31 enhancer trap line.
In the lateral view of an A31 embryo double labeled by antigsbp and anti-β-gal antibodies (Fig. 7C-E), it is clear that the
aCC and pCC neurons are just outside the gsbp expression
region. Thus, it is confirmed that gsbp is normally expressed
in CQ neurons, but not in aCC, pCC, RP2 or EL neurons.
DISCUSSION
It is known that the lack of the gsb (gsbd and gsbp) activity
results in the cell fate respecification of both epidermal and
neural cells in gsb mutants. In this study, we set out to ask the
converse question: does the ectopic expression of gsb genes
commit cells to a particular cell fate during embryonic development? To this end, we have generated the hs-gsbd and hsgsbp fly lines in which ubiquitous gsb (gsbd or gsbp)
expression can be induced by heat-shock treatment. Our results
show that ectopic expression of either gsbd or gsbp causes
pattern alterations which are approximately reciprocal to that
of gsb mutants. In the epidermis, this results in the duplication
of structures surrounding the parasegmental boundary and, in
the CNS, neuroblasts which do not normally express gsb
appear to be respecified to fates associated with the gsbexpressing NBs.
gsbd regulates cell fates in a region bracketing the
parasegmental boundary
Previous results have shown that within each segment, gsbd is
expressed in a region of the ventral ectoderm bracketing the
parasegmental boundary (Gutjahr et al., 1993b). Eventually all
wg-expressing cells express gsbd and a subset of the anterior
en-expressing cells express gsbd. Epidermal cells in this region
normally give rise to naked cuticle, but develop into denticle
belts in gsb mutants. This transformation is correlated with the
loss of wg expression during stage 11 (Hidalgo, 1991).
With heat shocks from 2.5 to 4.0 hours after egg laying, both
hs-gsbd and hs-gsbp cause a transformation of the denticle
belts into naked cuticle. Associated with this transformation is
the generation of a new mirror-image duplicated parasegmental boundary with ectopic expression of wg, en and the endogenous gsbd genes. Our explanation of this result is that the gsb
expression can activate inappropriate wg transcription that
establishes the feedback loop normally found between the wgand en-expressing cells (DiNardo et al., 1988; Bejsovec and
Martinez Arias, 1991). Establishing this ectopic feedback loop
is dependent upon the endogenous gsbd gene since heat shocks
with hs-gsbd or hs-gsbp in a gsb mutant background do not
lead to ectopic expression of either wg or en and the anterior
denticle belts are never transformed into naked cuticle.
Heat shocks after 4 hours of development are ineffective in
establishing the wg-en cell feedback loop but still cause transformation of the denticle belt into naked cuticle. In these
embryos, high level ectopic expression of wg, en and the
Summary Diagram
Ectopic gooseberry changes cell fate 1159
Fig. 8. Summary
A. wild type
B. gsb mutant
C. hs-gsb
diagram. The upper
panels illustrate the
cuticle phenotypes in
wild-type (A), gsb (B)
and hs-gsb embryos (C).
The trapezoidal box
represents the wild-type
abdominal type denticle
belt (the first row of
denticles at the posterior
margin of the segment is
not shown) and the
three-hair structure
depicts the thoracic
D. wild type
F. hs-gsb
Keilin’s organ. In the
E. gsb mutant
gsb embryo, naked
cuticle is replaced by a
A m
A m
l
i
A m
mirror-image
i
l
i
l
duplication of the
2
2
2
denticle belts while, in
hs-gsb embryo, denticle
3
3
5?
belts are replaced by
naked cuticle, including
3?
5
5
an occasional
duplication of the
7
7
7
Keilin’s organ. The
P
P
middle panels (D-F)
P
depict the NB pattern in
hemisegments at early
G. wild type
I. hs-gsb
H. gsb mutant
stage 9. In wild-type
embryos (D), the
anteroposterior axis has
4 rows of NBs: row 2, 3,
5 and 7 (Doe, 1992). In
the dorsoventral axis,
there are three columns:
medial (m),
intermediate (i) and
}en
}en
}en
lateral (l) (Hartenstein
and Campos-Ortega,
1984). Each NB can be
given a row and column
designation in this grid.
The correspondence of
ac positive NB
wg positive NB
Keilin's organ
the grid designations
with NB identities
MNB progeny
RP2
aCC
described in Doe (1992)
EL neuron cluster
pCC
CQ
en positive neuron
is: 2m, 2-2; 2i, 3-2; 2l,
2-5; 3m, MP2; 3l, 3-5;
5m, 5-2; 5i, 5-3; 5l, 5-6;
7m, 7-1 and 7l, 7-4. The
L-shaped outline encloses the NBs that express gsbd. A key at the bottom of the figure shows the code used to depict the expression
patterns of the markers used in this study. Small black symbols show the sites of NB formation at later stages: diamond, NB 4-2 (gives rise
to RP2 neurons); triangle, NB 6-2 (gives rise to CQ neurons) and square, NB 1-1 (gives rise to aCC and pCC neurons). In gsb mutants (E),
ac is also expressed in what would be row 5. This may represent a transformation of the row 5 NBs into row 3 NBs, though the row 5 NBs
continue to express wg. In heat shocked hs-gsbd embryos (F), gsbd and gsbp expression expands throughout the segment and usually has
the appearance of a rough mirror-image duplication. This is shown by the dotted outline. ac expression is eliminated in row 3 NBs and is
replaced by the expression of wg. This may represent a transformation of row 3 into row 5. (The deletions and duplications of NB 4-2, NB
6-2 and NB 1-1 are inferred from the alterations to their progeny.) (G-I) The neuronal patterns in mature CNS segments. The axon bundles
are shown as hatched areas. The gsbp-expressing neurons are positioned in the boxed L region, partially overlapping the en-expressing
neurons. The labels for the EL, CQ, aCC, pCC and en neurons are shown at the bottom of the figure. In gsb mutants (H) the posterior
commissure is eliminated along with the CQ neurons, while there are duplications of the RP2, aCC and pCC neurons. In heat shocked hsgsb embryos (I), the longitudinal connectives are eliminated and the commissures are fused, the EL, RP2, aCC and pCC neurons are
deleted, and the CQ neurons are duplicated.
1160 Y. Zhang and others
endogenous gsbd is not observed but low level wg expression
is seen throughout the segment. Experiments with wgts mutants
and HS-wg flies have shown that wg function is required
through 11 hours of development for the formation of naked
cuticle (Baker, 1988; Noordermeer et al., 1992). With later heat
shocks, gsbd and gsbp are able to induce low levels of ectopic
wg and this presumably leads to the formation of naked cuticle.
Both hs-gsbd and hs-gsbp are able to transform denticle belts
into naked cuticle in wild-type animals and rescue the development of naked cuticle in gsb mutants. We believe that these
functions are normally carried out by the gsbd gene since we
cannot detect the gsbp protein in the epidermis of wild-type
animals at the times that heat shocks are effective. Therefore,
gsbp protein can partially substitute for the function of the gsbd
protein. This might be expected given that gsbp can bind to the
same DNA target sites as the related prd protein (Treisman et
al., 1991). Redundancy between the prd and gsbd proteins may
in part explain the continued expression of wg through stage
10 in gsb mutants since the prd protein is present until the end
of germ band extension and is expressed in the same cells that
express gsbd (Gutjahr et al., 1993a). We have examined wg
expression in prd gsb double mutants (data not shown) and wg
expression is reduced in the remaining even-numbered
parasegments but not eliminated. Thus, it seems that another
factor contributes to the maintenance of wg expression through
stage 10 in gsb mutants.
of row 3 into row 5. This conclusion is supported by examining
the expression of β-gal from a wg enhancer trap line in hs-gsbd
background. wg is normally expressed only in row 5 NBs (Doe,
1992). In heat shocked hs-gsbd embryos, the wg enhancer trap
is also detected in some row 3 NBs (Fig. 5B). The role of gsbd
in the specification of NB 7-1 is less clear. This NB also
expresses en and it continues to express ac in gsb mutants.
Later in nervous system development, additional NBs are
formed and a complex pattern of neurons is generated. With
the anti-eve antibody, we can follow the progeny from four
different NBs (Doe, 1992). The aCC and pCC neurons are
derived from NB 1-1, an en-expressing NB located just
posterior to row 7 (Fig. 7A). The RP2 neuron is derived from
NB 4-2, which does not express gsb and is located just anterior
to the gsb expression region (Fig. 7A). The lineage of the EL
neurons is not known, but is outside the region of gsb
expression. The CQ neurons are derived from NB 6-2 which
expresses gsb (Fig. 7A). The effects of heat shock hs-gsbp on
aCC, pCC, RP2 (deletion) and CQ (duplication) are the reciprocal to that in gsb mutants. This result again suggests the possibility that there are NBs and neuronal lineages that differ
from one another only in their expression of the gsb genes. This
correlation is not perfect, as the EL neurons do not fit this
pattern. The EL neurons are present in gsb mutants, but do not
appear to be duplicated. In heat shocked hs-gsbp embryos these
neurons are consistently eliminated.
gsb functions as a selector gene in the CNS
In the CNS analysis, we focused on the cell fate transformations of a subset of NBs and neurons. Our assignment of
neuronal and NB transformations is based on changes in the
expression patterns of various proteins. It is possible that these
changes do not reflect complete transformations of one cell
type into another. This will be known only after neuronal phenotypes have been carefully analyzed. We believe that we are
following cell fate transformations, as two neuronal markers
(eve and A31 β-gal expressions) and two NB markers (ac and
wg expressions) used in this study all behave appropriately.
At both neuronal and NB levels, we have found that gsb
(both gsbd and gsbp) is normally expressed in those cells that
are deleted in gsb mutants but not expressed in cells that are
duplicated in gsb mutants. Ectopic expression of gsb leads to
cell fate transformations that are nearly reciprocal to those of
gsb mutants. These results suggest that the gsb locus may
function as a classical selector gene, similar to the homeotic
genes, and choose between different neuronal fates (GarciaBellido, 1975). The simplest model would be one in which
there was a one-to-one correspondence between gsb-expressing and non-expressing NBs and their neurons. If this were the
case, inappropriate expression of the gsb genes would lead to
pattern transformations reciprocal to those in gsb mutants. This
prediction is approximated in the initial patterning of NBs.
It is known that wild-type stage 9 embryos express the ac
protein in row 3 and row 7 NBs but not in row 2 and 5 (Skeath
and Carroll, 1992; Doe, 1992). In gsb mutants, ac expression
is also found in row 5 (Skeath et al., 1992) and may represent
the transformation of row 5 into row 3. (It cannot be a transformation of row 5 into row 7 since the NBs in row 5 of gsb
mutants do not express en, while row 7 NBs normally do
express en.) In heat shocked hs-gsbd embryos, ac expression
is eliminated in row 3, suggesting a reciprocal transformation
gsb is a link between epidermal and CNS patterning
The roles of several segmentation genes in CNS development
have been studied in detail. Some pair-rule genes, such as ftz
and eve, have two independent roles, one in epidermal segmentation and a second in neurogenesis (Doe et al., 1988a,b).
gsb is distinct from this paradigm since the pattern of gsbd
expression in the CNS is always found in segmentally
repeating stripes which begin in the neuroectoderm and are
maintained as NBs delaminate. This may reflect similar independent functions in the epidermis and NBs or a single
function in the common ectodermal precursor cells.
We thank Cindy Motzny, Darlene Buenzow, Jennifer Kennedy and
Andrea Brand for stimulating discussions, and Linda Orlofsky for
generating the hs-gsbp line. We are grateful to Lin Gu for her
expertise and assistance in producing the monoclonals, Jim Skeath for
providing his anti-ac antibody and sharing his expertise in the staining
procedures, A. Ghysen for providing the A31 enhancer trap line, N.
Perrimon for providing the wg enhancer trap line, M. Frasch for the
anti-eve antiserum, R. Nusse for the anti-wg antiserum and C. Doe
for sharing unpublished results of NB maps. This work was supported
by an NSF Graduate Fellowship (A. U.) and an NIH grant no.
NS28472 (R. H.).
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(Accepted 28 January 1994)