Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, 28049 Madrid, Spain
Author for correspondence (e-mail:
gmorata{at}cbm.uam.es)
Accepted 27 August 2003
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SUMMARY |
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Key words: Drosophila, Imaginal discs, btd, Sp1, Zinc-finger transcription factors
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Introduction |
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Within the domains defined by the activity of the Hox genes there are other
genes that discriminate the identity of more restricted parts. For example,
pannier (pnr) appears to be responsible for distinguishing
between medial and lateral regions of all thoracic and abdominal segments
(Calleja et al., 2000;
Herranz and Morata, 2001
). The
gene apterous (ap) distinguishes between the dorsal and
ventral regions of the wing appendages
(Díaz-Benjumea and Cohen,
1993
), and Distal-less (Dll) specifies the
growth and identity of ventral appendages
(Cohen and Jurgens, 1989
;
Gorfinkiel et al., 1997
).
A major morphological distinction in the embryonic, larval and adult body
is that between dorsal and ventral regions. There is evidence that the dorsal
and ventral adult structures of the thorax share a common lineage in early
development (Steiner, 1976;
Wieschaus and Gehring, 1976
;
Lawrence and Morata, 1977
),
but by late embryogenesis the dorsal and ventral primordia have different
lineages. It is not clear whether this restriction corresponds to a genuine
compartment segregation or whether it is the result of the physical separation
of the two primordia, which can be observed during embryogenesis
(Goto and Hayashi, 1997
;
Kubota et al., 2000
).
Nonetheless there is a clear difference between dorsal and ventral patterns,
which is also reflected in the activity of different developmental genes.
There are identity conferring genes which are restricted to either the dorsal
or the ventral regions, such as pnr, Dll or vestigial
(vg) (reviewed by Mann and
Morata, 2000
).
We have developed a method (Calleja et
al., 1996) which allows the visualisation of gene expression
patterns in living flies. It is a modification of the Gal4/UAS procedure of
Brand and Perrimon (Brand and Perrimon,
1993
): mobilisation of the pGalw element yields a collection of
insertion lines, which are tested using an UAS-y construct.
Individual adult flies showing dark (y+) patches of
interest were used to establish new Gal4 lines. We have used this method for a
systematic screen of genes showing restricted expression in adult flies. Among
those, the line MD808 was found to confer y+ expression in
ventral adult derivatives, proboscis, antennae, legs and genitalia, suggesting
the gene driving its expression may have a general function in the formation
of these structures.
We report a functional characterisation of the gene driving MD808
expression. It turned out to be buttonhead (btd), which
encodes a zinc-finger transcription factor
(Wimmer et al., 1993) and is
known to be required for the formation of the intercalary, antennal and
mandibular head segments (Cohen and
Jürgens, 1990
). A related adjacent gene, termed Sp1
(previously known as D-Sp1), has been previously characterised
(Wimmer et al., 1996
), the
function of which is partially redundant with that of btd
(Schock et al., 1999
). We find
that in addition to their role in head development btd and
Sp1 are involved in the development of the ventral imaginal discs.
Their expression is under the control of the Wg and Dpp signals and is also
regulated by other factors such as the bithorax complex genes. Once activated,
they induce the function of genes such as Dll and headcase
(hdc), which are involved in the specification of adult ventral
structures. Our results also show that the Btd product is able to trigger the
entire process necessary for leg and antennal development, including the
activation of the wg and dpp signalling genes.
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Materials and methods |
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Fly stocks
The btdXG81 is a strong allele of btd with the
same phenotype as the deletion of the gene
(Cohen and Jurgens, 1990). The
Df(1)C52 is a deletion of the 8E4-9C region,
deficient for btd, Sp1 and about 60 other genes
(Cohen and Jurgens, 1990
)
(FlyBase). The Dp(1;Y)lz+ covers the
8B-9A region (Schock et al.,
1999
) and also rescues the btd and Sp1 larval
phenotypes, indicating the genes responsible for the latter are within the
8E4-9A interval. This interval contains about 44 genes (FlyBase). The
Ubx1 allele has been described by Kerridge and Morata
(Kerridge and Morata, 1982
)
and DllSA1 by Cohen and Jürgens
(Cohen and Jürgens,
1989
). The reporter genes used were dpp-lacZ (Blackman,
1991), wg-lacZ (Ingham,
1991
), Dll-lacZ (Dll01092)
(Spradling et al., 1999
),
esg-lacZ (Whiteley et al.,
1992
) and hdc-lacZ
(Weaver and White, 1995
)
Production of mutant btd clones
Heat shocks were given to larvae of different stages of genotype
btdXG81 f36a FRT18A/y w FRT18A; hsFLP/+.
Mitotic recombination in the first chromosome would produce clones of mutant
btdXG81 cells marked with f36a and
twin btd+ control clones marked yellow.
RNA interference
The GAL4-inducible constructs for RNA interference were made as follows:
for btd, a 400 bp fragment was amplified by PCR using a
5'-gaaggatccgccgccaccgccgccgct-3' upper primer and a
5'-cggggtaccgtaactggctgttcccgcacc-3' lower primer. For
Sp1, a 813 bp fragment was amplified using a
5'-gccggatcctggctggatatggg-3' upper primer and a
5'-gccggtaccggccccgcccgtctgtggg-3' lower primer. Each PCR product
was independently cloned as a BamHI-KpnI fragment in the
pHIBS vector (Nagel et al.,
2002), to make the pHIBS-btd or pHIBS-Sp1
constructs. The BamHI-SacI fragments from pHIBS-btd
or pHIBS-Sp1 were subcloned in the Bluescript vector, generating the
BS-btd and BS-Sp1, respectively. SalI and
KpnI fragments (containing the intron and the gene fragment) from
pHIBS-btd and pHIBS-Sp1 constructs were cloned in the
opposite direction in the BS-btd and BS-Sp1 vector, thus
forming the final RNAi constructs BS-btd RNAi and BS-Sp1
RNAi. The RNAi constructs were cloned in the pUAS KpnI site and
injected in y w1118 embryos by standard procedures. They
are referred to as UAS-btdi and UAS-Sp1i respectively.
RT-PCR
We used larvae of genotype btd-Gal4>UAS-btdi and
btd-Gal4>UAS-Sp1i to measure the transcripts levels in comparison
to btd-Gal4 larvae used as control.
To isolate RNA for RT-PCR, anterior halves of larvae were lysed in Trizol
(Invitrogen), and extracted RNA was dissolved in water. Synthesis of first
strand cDNA was primed by oligo (dT) and random hexamers. RT-PCR was performed
using published primers to amplify the ribosomal protein 49 (RP49) mRNA
(Foley et al., 1993), which
serves as internal control, and two pair of primers outside the region cloned
in the UAS RNAi to amplify btd and Sp1 mRNA.
Gal4/UAS experiments
ey-Gal4 was provided by Walter Gehring and directs expression
identical to the eyeless gene. The nub-Gal4,
omb-Gal4 and Ubx-Gal4 lines were isolated in our laboratory
and drive expression essentially in the domains defined by the genes in which
the pGalw element is inserted. ptc-Gal4 is described elsewhere
(Wilder and Perrimon, 1995).
The MD743 line directs expression in the hinge and the pleural region of the
wing (M.C., unpublished). The 444-Gal4 line gives an overall uniform embryonic
expression (Azpiazu and Morata,
1998
). The mata-Gal4VP16 Vp15 lines (a gift from Daniel
St Johnston) yield maternal Gal4 expression. The UAS-btd stock
(Schock et al., 1999
) was
provided by Herbert Jackle.
To induce marked clones of cells ectopically expressing btd we
used three different chromosomes as convenient: y w hs FLP122
f36a; abx<f+<Gal4-lac-Z
(de Celis and Bray, 1997),
hsFLP act<CD2<Gal4 (Pignoni
and Zipursky, 1997
) and y w hsFLP122;
act<y+<Gal4 UAS-GFP
(Ito et al., 1997
). They were
crossed to UAS-btd flies and the F1 larvae were given heat shocks at
different stages.
Histochemical methods
Embryos were stained using standard procedures for confocal microscopy
(Gonzalez-Crespo et al.,
1998); secondary antibodies were coupled to Red-X and FITC
fluorochroms (Jackson Inmunoresearch) and were analysed under a laser-scan
BioRad microscope. For double fluorescent in situ/antibody label we followed
the method as described by (Knirr et al.,
1999
). For the in situ label we used a btd RNA probe reported
previously (Wimmer et al.,
1996
) and provided by Herbert Jackle. As probe for Sp1 we
used an RNA transcribed from a cloned 2 kb DNA fragment obtained by PCR using
primers from the 5' and 3' UTR.
Imaginal discs were dissected in PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. They were blocked in PBS, 1%BSA, 0.3% Triton for 1 hour, incubated with the primary antibody over night at 4°C, washed four times in blocking buffer, and incubated with the appropriate fluorescent secondary antibody for 1 hour at room temperature in the dark. They were then washed and mounted in Vectashield (Vector Laboratories).
The primary antibodies used were: rabbit and mouse anti-ß-gal (Capel
and Promega), mouse anti-CD2 (Serotec), rabbit anti-vestigial
(Williams et al., 1991), mouse
anti-dachshund (Mardon et al.,
1994
), guinea pig anti-homothorax (a gift of Natalia Azpiazu),
rabbit anti-Distal-less (a gift of Grace Panganiban), mouse anti-Distal-less
(Duncan et al., 1998
), mouse
anti-engrailed (a gift of Peter Lawrence) and mouse anti-wingless (Hybridoma
Center).
Preparation of larval and adult cuticles
Adult flies were by the standard methods for microscopic inspection. Soft
parts were digested with 10% KOH, washed with ethanol and mounted in Euparal.
Embryos were collected overnight and aged an additional 12 hours. First instar
larvae were dechorionated in commercial bleach for 3 minutes and the vitelline
membrane removed using heptano-methanol 1:1. Then, after washing with methanol
and 0.1% Triton X-100, larvae were mounted in Hoyer's lactic acid 1:1 and
allowed to clear at 65°C for at least 24 hours.
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Results |
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Further to the genetic analysis and the expression data we cloned DNA
fragments at the insertion site to map the position of the P-element on the
genome (see Material and methods). It is located 753 bp 5' of the
btd gene (FlyBase). We note that the related gene Sp1, the
Drosophila homologue of the human SP1
(Wimmer et al., 1993;
Wimmer et al., 1996
) is
immediately adjacent (FlyBase). It is likely that btd and
Sp1 have originated by a tandem duplication of a primordial
btd-like gene.
btd and Sp1 expression domains in the thoracic
segments
The expression patterns of btd and of Sp1 during
embryogenesis have already been described
(Wimmer et al., 1993;
Wimmer et al., 1996
). In early
embryos btd is expressed in the head region, but by the extended germ
band stage the expression domain has expanded to the ventral region of
cephalic, thoracic and abdominal segments. During germ band retraction most of
the abdominal and thoracic expression is lost, except in derivatives of the
peripheral nervous system and the primordia of the imaginal discs.
Sp1 is not expressed in early embryos, but from stage 11 onwards it
shows the same pattern as btd
(Wimmer et al., 1996
).
We paid special attention to the btd/Sp1 expression domain in the thoracic imaginal discs primordia, as it may suggest a novel function related to imaginal development. Double labelling with Dll and btd probes (Fig. 1A-C) indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is smaller and is included within the btd/Sp1 domain: there are cells expressing btd that do not show Dll activity, although all the cells expressing Dll express btd (Fig. 1B,C).
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The overlap of the btd and of esg domains indicates that
btd is also expressed in the hth domain, which is coincident with
that of esg (Goto and Hayashi,
1999). As the hth/esg domain marks the precursor cells of the
proximal region of the adult leg (reviewed by
Morata, 2001
) the embryonic
expression data indicate that btd and Sp1 are active in the
entire primordia of the ventral adult structures, including the distal and the
proximal parts.
The different expression patterns in third instar leg and antennal imaginal
discs are illustrated in Fig.
2. In the mature antennal disc, btd expression is
restricted mostly to the region corresponding to the second antennal segment,
where it co-localises with both Dll and hth. In the leg disc
btd also overlaps in part with Dll and with hth
(Fig. 2A-C). The latter result
is significant, for the expression of Dll and hth define two
major genetic domains, which are kept apart by antagonistic interactions
(Gonzalez-Crespo et al., 1998;
Abu-Shaar and Mann, 1998
). The
fact that btd is expressed in the two domains suggests that its
regulation and function is independent from the interactions between the two
domains. This observation is consistent with the results obtained in embryos
(Fig. 1) and suggests that the
btd domain includes the precursors of the whole ventral thoracic structures
regions from the beginning of development.
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Larvae mutant for the strong btdXG81 allele are
defective in the formation of KOs (Fig.
3); in 45% of the larvae the KO are absent and in the rest the KO
are defective. The incomplete effect of the btdXG81
mutation in suppressing KOs suggested that its function might be redundant and
shared by another gene, the obvious candidate being Sp1. As no
individual Sp1 mutation is available, we tested the contribution of
Sp1 to KO formation by examining first instar larvae hemizygous for
Df(1)C52
(Schock et al., 1999), which
lacks both btd and Sp1 (see Materials and methods).
Df(1)C52 larvae show btd mutant phenotype
in the head as reported previously (Cohen
and Jürgens, 1990
), but in addition we find that no KO are
present (Fig. 3). Both the
btd phenotype and the lack of KOs of
Df(1)C52 larvae are covered by the duplication
Dp(1;Y) lz+, indicating that the
responsible genes are located in the interval 8E-9A, which contains
btd and Sp1 and about 40 other genes (FlyBase). These
results suggest that both btd and Sp1 play a role in the
formation of the disc primordia and that their functions are partially
redundant, but we cannot rule out the possibility that some other gene(s)
located in the 8E-9C interval may also be involved.
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btd acts upstream of Dll and hdc in the
formation of the disc primordia and appears to mediate the control by Wingless
and the BX-C genes
The results above demonstrate that btd and Sp1 are
necessary for normal Dll, hdc and esg expression. Moreover,
btd expression precedes that of Dll, esg and hdc,
which suggests it might act as an early activator of these genes. We have
tested this possibility by forcing btd activity using the Gal4/UAS
method (Brand and Perrimon,
1993) and examining the effect on Dll and hdc
expression. In Ubx-Gal4>UAS-btd embryos, btd activity
extends from the second thoracic segment to the seventh abdominal one. In this
region, the presence of the Btd product induces ectopic Dll activity
in many places, although not in all of the zones where btd is
expressed (Fig. 4D). Those
embryos also show gain of hdc expression. In
ptc-Gal4/UAS-btd embryos there is a general gain of hdc
activity in the ventral region (Fig.
4G). In the case of Dll, we checked whether there is a
mutual requirement for Dll and btd/Sp1 genes by looking at
btd and Sp1 expression in Dll mutant embryos. The
result was that their expression is normal, supporting the idea that
btd and Sp1 act upstream of Dll.
The activator role of btd (and presumably of Sp1) on
Dll suggests that btd and Sp1 may play a role in
mediating some of the factors known to be involved in Dll regulation
during embryogenesis. Previous work has identified some of these regulators,
e.g. wingless (wg), decapentaplegic (dpp)
and the bithorax complex genes (Cohen,
1990; Vachon et al.,
1992
; Goto and Hayashi,
1997
) and we therefore tested whether their role is mediated by an
effect on btd and Sp1.
The secreted molecule Wg is necessary for the early activation of
Dll in the cephalic and thoracic primordia
(Cohen, 1990;
Goto and Hayashi, 1997
). We
find that in the absence of wg function btd/Sp1
expression in the disc primordia is abolished, although it remains in the
central nervous system (Fig.
5B). In addition, early Dll expression is repressed by
dpp; in embryos deficient for dpp function Dll
expression is expanded (Goto and Hayashi,
1997
). We observe a similar situation with early btd
expression, which extends to the dorsal side in dpp mutant embryos
(not shown). The BX-C genes act as repressors of Dll in the abdominal
region (Cohen, 1990
;
Vachon et al., 1992
). In
Ultrabithorax (Ubx) mutations the first abdominal segment is
transformed into a thoracic one and correspondingly there is an additional
domain of Dll. We find that Ubx mutant embryos also exhibit
an additional btd/Sp1 domain (Fig.
5C) in the first abdominal segment. These results suggest that
btd mediates the regulation of Dll exerted by wg
and Ubx.
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Requirement for btd and Sp1 during imaginal
development
As shown above, btd and Sp1 are necessary for the initial
formation of the disc primordia, but are also expressed in mature ventral
imaginal discs (Fig. 2),
suggesting that they may be required during imaginal disc development. To test
the imaginal function we first induced a large number of marked
btdXG81 clones (see Materials and methods) during larval
period in all body structures, including legs and antennae. All these clones
developed and differentiated normally according to their position, and behaved
like the twin btd+ control clones. The specific function
of Sp1 could not be tested because there is no individual mutation at
this gene. We tried to recover clones homozygous for the
Df(1)C52, in which both btd and
Sp1 are deleted, but as expected (they are deficient for over 60
genes included in the interval 8E4-9C), these clones did not survive.
The negative result obtained with btdXG81 clones was
not conclusive because there was the possibility that the loss of btd
function could be rescued by that of Sp1, which is expressed in the
same cells. Besides, there is evidence that btd and Sp1 have
partially redundant roles (Schock et al.,
1999). We then tested the imaginal requirements for btd
and Sp1 by RNA interference using UAS-btdi and
UAS-Sp1i constructs (see Materials and methods). As shown in
Fig. 6, these are able to
reduce strongly btd and Sp1 transcripts levels. As a rule
the btd-Gal4 driver was used to check on the morphological effects on
legs and antennae. This driver was chosen because it directs expression
specifically in the btd/Sp1 domain. Moreover, it is mutant for btd,
thus diminishing the amount of wild-type function in the combinations. The
general result is that the inactivation of either btd or Sp1
alone (btd-Gal4>btdi or btd-Gal4>UAS-Sp1i) fails to
affect legs and antennae; at most some reduction of the tarsus was observed in
some btd-Gal4>UAS-Sp1i legs. However, when the two genes are
inactivated, as in btd-Gal4>UAS-btdi UAS-Sp1i flies grown at
29°C, these are unable to hatch and the antennae and legs are affected in
all cases, though to a different extent. The morphological effects are
illustrated in Fig. 6A-C. There
is no change of identity, but the growth is severely impaired. In the antennae
the stronger effect is in the second segment, which is very reduced or absent.
The first ant third segments are also affected in size, but the arista is
normal. In the legs there is a strong reduction in size, which appears to
affect all segments, which often fuse. We also observed similar but weaker
effects with the Dll-Gal4 line (not shown).
The pattern abnormalities observed in the double RNAi experiment reflects a requirement during imaginal disc development. Taking advantage that the phenotype of btd-Gal4>UAS-btdi UAS-Sp1i flies is temperature dependent (at 29°C there is strong effect, but at 17°C there is practically no alteration) we performed temperature shifts to ascertain whether btd and Sp1 are required during the larval period. Shifting the temperature from 17 to 29°C during the second or during the third larval period produced morphological alterations in the adult flies that were very similar to those of flies grown entirely at 29°C, indicating a requirement during the late phases of imaginal disc development.
The effects of RNA interference fit very well with the expression pattern in the antennal and leg discs (Fig. 2), and indicate that btd and Sp1 have a imaginal function necessary for the growth of antennae and legs. This function appears to be redundant.
Ectopic btd activity transforms dorsal disc derivatives into
ventral ones.
The developmental potential of the Btd protein was tested by inducing
ectopic btd activity in different adult regions. In some experiments,
we used Gal4 lines to direct activity in specific body regions whereas in
others induced flip out clones of btd-expressing cells all over the
body (see Materials and methods). The principal result is that ectopic
btd activity is able to transform dorsal cephalic and thoracic adult
structures into the corresponding ventral ones according to the segment.
The effect in the eye was studied with the eyeless-Gal4 line, which confers expression in the eye cells, and also inducing marked clones of btd-expressing cells. In ey-Gal4/UAS-btd flies the eye disappears and is replaced by an additional antenna which often fuses with the normal one (Fig. 7A). The same transformation was observed in clones of btd-expressing cells: clones located in the eye differentiate antennal structures (Fig. 7B).
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We note that btd is able to induce a complete set of leg structures even if its activity is induced in different regions of the wing disc. For example, the MD743 and the omb-Gal4 lines drive expression in different and non-overlapping regions, the hinge/pleura and the center of the wing respectively. Yet MD743/UAS-btd and omb-Gal4/UAS-btd flies develop similar sets of leg structures in the wing disc derivatives, suggesting that btd has a capacity to induce leg development independently of the local context.
The transformation of wings and halteres to leg was also observed in the flip out clones expressing btd. Both in the haltere and the wing btd-expressing clones differentiate leg tissue. These clones normally appear as either vesicles of tissue segregated from the wing or the haltere, or as protruding tissue (Fig 7E,F). They tend to form leg patterns resembling the normal ones, which include the formation of various leg segments, suggesting that the positional mechanism to form the proximodistal leg axis is being activated (see below). Moreover, these clones develop nearly complete leg patterns independently of the wing region where they localise. In the example shown in Fig. 7E,F, the clone is located in the posterior compartment but it forms a leg pattern that includes the `edge' bristle, which corresponds to the inventory structures of the anterior leg compartment.
Ectopic btd recapitulates the formation of ventral discs
primordia, including the activation of the Wg and Dpp signalling pathways
The transformation of the dorsal into ventral structures described in the
preceding experiments can be visualised before differentiation takes place by
the activation of specific ventral genes, accompanied by the repression of
dorsal ones.
btd-expressing clones in the eye gain expression of Dll
and hth (Fig. 8), the
two selector-like genes that contribute to antennal identity
(Casares and Mann, 1998;
Dong et al., 2002
). They also
lose eyeless expression (not shown). These clones adopt a round
shape, indicative of having acquired different cell affinities, probably owing
to the change of identity. In the wing disc btd-expressing clones
lose expression of vestigial (vg)
(Fig. 8D,H), a gene that
confers wing identity (Kim et al.,
1996
). In parallel these clones acquire hth, dachshund
(dac) and Dll activity
(Fig. 8E-K). The gain of
hth expression is of interest, as it occurs in clones located in the
center of the wing where there is no local hth activity. This
suggests that btd-expressing clones tend to recapitulate the
development of the entire leg disc, including the proximal region where
hth is expressed (Rieckhof et al., 1997). This possibility was
further explored by generating large clones in the first larval period. Their
size facilitates the study of the expression of leg disc marker genes. These
clones often show a spatially discriminated expression of hth, dac
and Dll just like the normal leg disc; some are shown in
Fig. 9. The activity of
hth is always restricted to the periphery of the clone
(Fig. 9A,B), whereas
dac and Dll extend inside. Although the expression of
dac and of Dll partially overlap, they occupy different
parts of the clones (Fig.
9C-F). In some favourable cases
(Fig. 9E,F), their domains
define three different regions within the clone: a region containing only
dac expression, a medial one containing dac and Dll
and yet another only containing Dll activity. This arrangement
resembles closely the normal distribution of these genes products along the PD
axis of the leg disc (Lecuit and Cohen,
1997
).
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The differential deployment of leg markers within the groups of cells
expressing btd suggested that the Btd product triggers the signalling
mechanism, involving the Hh, Dpp and Wg pathways, that normally patterns the
leg disc (Basler and Struhl,
1994; Lecuit and Cohen,
1997
). We have checked this by examining the expression of
engrailed (en), wg and dpp in the
btd-expressing clones.
The activity of en marks the distinction between anterior (A) and
posterior (P) compartment, which is fixed during embryogenesis
(Lawrence and Morata, 1977;
Vincent and O'Farrell, 1992
).
Thus, all the btd-expressing clones, which are initiated during the
larval period, are originally either of A (no cell expresses en) or P
(all cells express en) provenance. We find that many of these clones
exhibit en activity only in part of the clone
(Fig. 10A-D). Thus, depending
on their original assignment they either gain or lose en activity.
Some are located within the P compartment and therefore lose en,
whereas others are in the A compartment and gain en. The formation of
this sharp border of en expression within the clones is expected to
originate a zone of hedgehog (hh) activity, which in turn
induces local activation of wg and dpp
(Basler and Struhl, 1994
).
Indeed, we find that wg and dpp are activated in the
btd-expressing clones. This is illustrated in
Fig. 10E-H, showing that
either gene is activated in part of btd-expressing clones. It is very
likely that the appearance of the Wg and Dpp signals what induces the
spatially discriminate expression of dac and Dll in the
clones.
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Discussion |
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Our work demonstrates a novel and also redundant function of btd and Sp1: they are responsible for the formation of the ventral imaginal discs by activating the genetic network necessary for their development. Furthermore, we show that the Btd protein retains the capacity of inducing the entire ventral genetic network during the larval period. We propose that the activation of btd/Sp1 is the crucial event in the determination of the ventral structures of the adult fly.
btd and Sp1 are responsible for the formation and
required for the growth of the ventral discs
Our argument is based on the finding that btd and Sp1
appear to mediate all events connected with the formation of the ventral
discs. We center the discussion in the leg disc, but there is evidence that
antennal primordium also requires btd
(Cohen and Jürgens,
1990). Moreover, we have also observed that the genital primordium
is lacking in Df(1)C52 embryos (C.E. and G.M.,
unpublished), suggesting that this disc is also under the same control. Most
of our experiments concern the function of btd but given the
expression and functional similarities between the two genes
(Wimmer et al., 1996
;
Schock et al., 1999
) (this
report), we assume that Sp1 encodes the same or very similar role.
Therefore, we will refer to btd/Sp1 as a single function.
One crucial feature is that btd is an upstream activator of
Dll and hdc, which are considered developmental markers of
disc primordia (Cohen, 1993;
Weaver and White, 1995
): (1)
btd expression precedes that of Dll and of hdc; (2)
the btd expression domain includes those of Dll and hdc; (3)
in btd mutants, Dll and hdc activity is much
reduced, and completely absent in Df(1)C52 embryos;
(4) Ectopic btd function induces ectopic activation of Dll
and hdc.
The role of btd in embryogenesis can be illustrated in the light
of the models of Dll regulation
(Cohen et al., 1993;
Kubota et al., 2000
).
Dll is activated by wg and its expression modulated by the
EGF spitz and by dpp, whereas it is repressed in the
abdominal segments by the BX-C genes
(Vachon et al., 1992
). Our
experiments suggest that Dll regulation is mediated by btd:
in wg mutants there is no btd expression and hence neither
Dll nor hdc activity. In dpp mutant embryos,
btd expands to the dorsal region resembling the effect on
Dll (Goto and Hayashi,
1997
). In Ubx- embryos there is an additional
group of cells in the first abdominal segment expressing btd; the
same cells that also express Dll in those embryos. The interpretation
of the role of btd mediating Dll regulation by Ubx
is complicated by previous observations
(Vachon et al., 1992
) showing
direct repression of Dll by the Ubx protein. It is possible that
Ubx regulates Dll both directly and by controlling
btd activity.
We propose that the localisation of btd/Sp1 activity to a group of ventral cells is a major event in the specification of adult structures. btd and Sp1 are activated in response to spatial cues from Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the genes necessary for ventral imaginal development.
This hypothesis is strongly supported by the results obtained inducing
ectopic btd activity in the dorsal discs, as just the presence of the
Btd product alone is sufficient to bring about ventral disc development. In
the wing and the haltere discs, Btd induces a transformation into leg, whereas
in the eye it induces antennal development. This indicates that it specifies
ventral disc identity jointly with other factors, e.g. the Hox genes, possibly
through the activation of subsidiary genes such as Dll, known to
contribute to ventral appendage identity in combination with Hox genes
(Gorfinkiel et al., 1997).
The requirement for btd and Sp1 activity appears to be
restricted only to the ventral discs, even during the early phases of the
thoracic disc primordia. In this context it is worth considering the
observation that in Df(1)C52 embryos there is
esg expression in the wing and haltere disc primordia, even though it
is absent in the leg discs. Thus, the wing and haltere discs are formed in the
absence of btd and Sp1. Because in these embryos there is a
almost complete lack of Dll expression, this observation raises the
question of the origin of the dorsal thoracic discs, which are currently
considered to derive from the original ventral primordium, formed by cells
expressing Dll (see Cohen,
1993; Kubota et al.,
2000
). Although some of the original group of ventral cells may
contribute to the dorsal disc primordia, our data suggest that there may be
cells recruited to form the dorsal discs that do not originate in the initial
ventral primordium. Accordingly, it is worth considering that in the absence
of Dll activity the leg and wing discs are formed
(Cohen et al., 1993
), although
the leg only differentiates proximal disc derivatives. Thus, the activity of
Dll cannot be considered a reliable marker for imaginal discs.
Our RNA interference experiments also indicate that both btd and Sp1 are required for the growth of the antennal and leg discs. When the two gene functions are reduced simultaneously, leg segments fuse and there is an overall reduction in the size of antennae and legs. The reduction of growth affects the proximal and distal regions of the appendage (Fig. 6), and assigns a role to the expression observed in the imaginal discs. The two genes are able to perform this function on their own, for the inactivation of only one is not sufficient to impair growth. This conclusion is also supported by the observation that mutant btd clones do not have any effect, as they still possess Sp1 activity, which is sufficient for normal development. At this point we do not know the mechanism by which btd/Sp1 may affect growth.
btd activity induces the entire genetic network necessary
for leg development
One particularly significant result about the mode of action of
btd comes from the analysis of the ectopic leg patterns observed in
the wing and halteres. The cells expressing btd tend to recapitulate
the complete development of leg and antennal discs. For example, the whole
genetic network necessary to make a leg appears to be activated. btd
induces the activity of hth, dac and Dll, the domains of
which account for the entire disc (Lecuit
and Cohen, 1997; Abu-Shaar and
Mann, 1998
). Furthermore, hth, dac and Dll are
activated in a spatially discriminated manner: in the clone shown in
Fig. 9A,B hth is
expressed only in the peripheral region, resembling the normal expression in
the leg disc; in the clone in Fig.
9E,F the discriminate expressions of dac and Dll
define three distinct regions. The formation of the dac and Dll domains is
dependent on signalling from Wg and Dpp, although they require different
signal thresholds (Lecuit and Cohen,
1997
), but the hth domain is independent from Wg and Dpp
(Abu-Shaar and Mann, 1998
).
The generation of distinct hth, dac and Dll domains within the clones
suggested that btd-expressing cells in the wing and haltere generate
their own signalling process. Indeed, we find that within these clones there
is local activation of en, the transcription factor that initiates
Hh/Wg/Dpp signalling in imaginal discs (reviewed by
Lawrence and Struhl, 1996).
btd-expressing clones also acquire wg and dpp
activity in subsets of cells (Fig.
10). It is probably in the boundary of en-expressing with
non expressing cells where the Wg and Dpp signals are generated de novo;
subsequently, their diffusion initiates the same patterning mechanism which
operates during normal leg development. The result of this process is that the
hth, dac and Dll genes are expressed in different domains
contributing to form leg patterns containing DV and PD axes. One question for
which do not have a clear answer is how the initial asymmetry is generated, so
that a few cells within the group gain (or lose) en activity. We have
observed that the cells expressing en within the clones are
(Fig. 10A-D) those closer to
the posterior compartment cells. It is conceivable that there might be an
external signal, perhaps mediated by Hh, which triggers the initial
asymmetry.
The ability of cells expressing btd to build their own patterning mechanism is also indicated by the observation that inducing btd activity in different parts of the wing disc results in the production of similar sets of leg pattern elements. For example, in MD743/UAS-btd and omb-Gal4/UAS-btd flies, btd is induced in different, non-overlapping wing regions, and yet all leg pattern elements are produced in both genotypes. Thus, the effect of btd is by and large independent of the position where it is induced, e.g. it does not depend on local positional signals.
A relevant issue is whether the ability of the Btd product to induce the formation of the full array of ventral structures has a functional significance in normal development. We believe that this property may be a faithful reflection of the original btd/Sp1 function: the activation of the developmental program to build the ventral adult patterns. We envisage btd/Sp1 function as follows. During the embryonic period, the conjunction of several regulatory factors (Wg, Dpp, EGF, Hox genes) allows activation of btd/Sp1 in a group of cells in each thoracic segment (we assume a similar process takes place in the head). These cells become the precursors of the ventral imaginal discs and will eventually form the ventral thorax of the adult which includes the trunk (the hth domain) and appendage (the Dll domain) regions. The activity of btd/Sp1 is instrumental in segregating these ventral discs precursors from those of the larval epidermis and determining their imaginal fate. It is involved in specifying their segment identity (in collaboration with the Hox genes) and in establishing their pattern and growth. To achieve the latter role btd/Sp1 induces the production of the growth signals Wg and Dpp, probably in response to localised activation of en and subsequent signalling by hedgehog (hh).
A problem with this model is that in normal development all the genes
involved, hth, en, hh, wg and dpp, are expressed in embryos
prior to btd/Sp1. Why should a new round of activation be necessary?
Although we cannot offer a totally satisfactory answer, we note that clones of
btd-expressing cells in wing or haltere lose their memory of
en expression. Those that originated in the A compartment had no
previous en expression, but gained it in some cells. Conversely, all
cells in P compartment clones contained en activity but some lose it.
The best explanation for this unexpected behaviour is that btd
provokes a `naïve' cell state in which the previous commitment for
en activity is lost. Later, en activity is re-established.
We believe this phenomenon may reflect the process that occurs in normal
development. The initial btd/Sp1 domain may not inherit the previous
developmental commitments and has to build a new developmental program. It is
worth considering that the btd/Sp1function appears to determine
ventral imaginal fate as different from larval fate. This is a major
developmental decision, which may require de novo establishment of the genetic
system responsible for pattern and growth. A key aspect of this would be the
localised activation of en in part of the btd/Sp1 domain. We
speculate that there might be a short-range signal, perhaps Hh, emanating from
nearby en-expressing embryonic cells, that acts as an inducer in the
btd/Sp1 primordium. There is evidence that Hh can induce en activity
(Guillen et al., 1995).
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ACKNOWLEDGMENTS |
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REFERENCES |
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Abu-Shaar, M. and Mann, R. S. (1998).
Generation of multiple antagonistic domains along the proximodistal axis
during Drosophila leg development.
Development 125,3821
-3830.
Azpiazu, N. and Morata, G. (1998). Functional
and regulatory interactions between Hox and exd genes
Genes Dev. 12,261
-273.
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368,208 -214.[CrossRef][Medline]
Blackman, R. K., Sanicola, M., Raftery, L. A., Gillevet, T. and Gelbart, W. M. (1991). An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-ß family in Drosophila.Development 111,657 -665.[Abstract]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Calleja, M., Moreno, E., Pelaz, S. and Morata, G.
(1996). Visualization of gene expression in living adult
Drosophila. Science 274,252
-255.
Calleja, M., Herranz, H., Estella, C., Casal, J., Lawrence, P.,
Simpson, P. and Morata, G. (2000). Generation of medial and
lateral dorsal body domains by the pannier gene of Drosophila.Development 127,3971
-3980.
Casares, F. and Mann, R. (1998). Control of antennal versus leg development in Drosophila. Nature 392,723 -726.[CrossRef][Medline]
Cohen, B., Simcox, A. and Cohen, S. M. (1993).
Allocation of thoracic imaginal primordia in the Drosophila embryo.
Development 117,597
-608.
Cohen, S. M. (1990). Specification of limb development in the Drosophila embryo by positional cues from segmentation genes. Nature 343,173 -177.[CrossRef][Medline]
Cohen, S. M. (1993). Imaginal disc development. In The Development of Drosophila melanogaster Vol.2 (ed. M. Bate and A. Martinez-Arias), pp747 -842. New York: Cold Spring Harbor Laboratory Press.
Cohen, S. and Jürgens, G. (1989). Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO J. 8,2045 -2055.
Cohen, S. M. and Jürgens, G. (1990). Mediation of Drosophila head development by gap-like segmentation genes. Nature 346,482 -485.[CrossRef][Medline]
de Celis, J. and Bray, S. (1997). Feed-back
mechanisms affecting Notch activation at the dorsoventral boundary in the
Drosophila wing. Development
124,3241
-3251.
Díaz-Benjumea, F. and Cohen, S. M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila.Cell 75,741 -752.[Medline]
Dong, P. D., Dicks, J. S. and Panganiban, G. (2002). Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna. Development 129,1967 -1974.[Medline]
Duncan, D. M., Burgess, E. A. and Duncan, I.
(1998). Control of distal antennal identity and tarsal
development in Drosophila by spineless-aristapedia, a
homolog of the mammalian dioxin receptor. Genes Dev.
12,1290
-1303.
Foley, K. P., Leonard, M. W. and Engel, J. D. (1993). Quantitation of RNA using the polymerase chain reaction. Trends Genet. 9,380 -385.[CrossRef][Medline]
Gonzalez-Crespo, S., Abu-Shaar, S. M., Torres, M., Martinez-Arias, C., Mann, R. S. and Morata, G. (1998). Antagonism between extradenticle function and Hedgehog signalling in the developing limb. Nature 394,196 -200.[CrossRef][Medline]
Gorfinkiel, N., Morata, G. and Guerrero, I.
(1997). The homeobox gene Distal-less induces ventral
appendage development in Drosophila. Genes Dev.
11,2259
-2271.
Goto, S. and Hayashi, S. (1997). Specification
of the embryonic limb primordium by graded activity of decapentaplegic.
Development 124,125
-132.
Goto, S. and Hayashi, S. (1999). Proximal to
distal cell communication in the Drosophila leg provides a basis for
an intercalary mechanism of limb patterning.
Development 126,3407
-3413.
Guillen, I., Mullor, J. L., Capdevila, J., Sanchez-Herrero, E.,
Morata., G. and Guerrero, I. (1995). The function of
engrailed and the development of the wing imaginal disc.
Development 121,3447
-3456.
Hayashi, S., Hirose, S., Metcalfe, T. and Shirras, A. D.
(1993). Control of imaginal cell development by the escargot gene
of Drosophila. Development
118,105
-115.
Herranz, H. and Morata, G. (2001). The
functions of pannier during Drosophila embryogenesis.
Development 128,4837
-4846.
Ingham, P. (1991). Segment polarity genes and cell patterning within the Drosophila body segment. Curr. Opin. Genet. Dev. 1,261 -267.[Medline]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto. D.
(1997). The Drosophila mushroom body is a quadruple
structure of clonal units each of which contains a virtually identical set of
neurones and glial cells. Development
124,761
-771.
Kerridge, S. and Morata, G. (1982). Developmental effects of some newly induced Ultrabithorax alleles of Drosophila. J. Embryol. Exp. Morphol. 68,211 -234.[Medline]
Kim, J., Sebring, A., Esch, J. J., Kraus, M. E., Vorwerk, K., Magee, J. and Carroll, S. B. (1996). Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382,133 -138.[CrossRef][Medline]
Knirr, S., Azpiazu, N. and Frasch, M. (1999).
The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.
Development 126,4525
-4535.
Kubota, K., Goto, S., Eto, K. and Hayashi, S.
(2000). EGF receptor attenuates Dpp signalling and helps to
distinguish the wing and leg fates in Drosophila.Development 127,3769
-3776.
Lawrence, P. and Morata, G. (1977). The early development of mesothoracic compartments in Drosophila. An analysis of cell lineage and fate mapping and an assesment of methods. Dev. Biol. 56,40 -51.[Medline]
Lawrence, P. and Morata, G. (1994). Homeobox genes: their function in segmentation and pattern formation of Drosophila.Cell 78,181 -189.[Medline]
Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments and pattern: Lessons from Drosophila.Cell 85,951 -961.[Medline]
Lecuit, T. and Cohen, S. (1997). Proximal-distal axis formation in the Drosophila leg. Nature 388,139 -145.[CrossRef][Medline]
Mann, R. and Morata, G. (2000). The developmental and molecular biology of genes that subdivide the body of Drosophila. Annu. Rev. Cell Dev. Biol. 16,243 -271.[CrossRef][Medline]
Mardon, G., Solomon, N. and Rubin, G. (1994).
dachshund encodes a nuclear protein reqired for normal eye and leg
development in Drosophila. Development
120,3473
-3486.
Morata, G. (2001). How Drosophila appendages develop. Nat. Rev. Mol. Cell. Biology 2, 89-97.[CrossRef][Medline]
Nagel, A. C., Maier, D. and Preiss, A. (2002). Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila. Dev. Genes Evol. 212, 93-98.[CrossRef][Medline]
Ng, M., Diaz-Benjumea, F. J. and. Cohen, S. M.
(1995). nubbin encodes a POU-domain protein required for
proximal-distal patterning in the Drosophila wing.
Development 121,589
-599.
Pignoni, F. and Zipursky, S. L. (1997).
Induction of Drosophila eye by decapentaplegic.Development 124,271
-278.
Rieckohf, G., Casares, F., Ryoo, H. D., Abu-Shaar, M. and Mann, R. (1997). Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein. Cell 91,171 -183.[Medline]
Schock, F., Purnell, B. A., Wimmer, E. A. and Jackle, H. (1999). Common and diverged functions of the Drosophila gene pair D-Sp1 and buttonhead. Mech. Dev. 89,125 -132.[CrossRef][Medline]
Spradling, A. C., Stern, D., Beaton, A., Rhem, E. J., Laverty,
T., Mozden, N., Misra, S. and Rubin, G. M. (1999). The
Berkeley Drosophila genome project gene disruption project. Single P-element
insertions mutating 25% of vital Drosophila genes.
Genetics 153,135
-177
Steiner, E. (1976). Establisment of compartments in the developing leg imaginal discs of Drosophila melanogaster. Wilhelm Roux Arch. 180, 9-30.
Vachon, G., Cohen, B., Pfeifle, C., McGuffin, M. E., Botas, J. and Cohen, S. M. (1992). Homeotic genes of the bithorax complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distal-less.Cell 71,437 -450.[Medline]
Vincent, P. and O'Farrell, P. H. (1992). The state of engrailed expression is not clonally transmitted during early Drosophila development. Cell 68,923 -931.[Medline]
Weaver, T. A. and White, R. A. (1995).
headcase, an imaginal specific gene required for adult morphogenesis
in Drosophila melanogaster. Development
121,4149
-4160.
Whiteley, M., Noguchi, P. D., Sensabaugh, S. M., Odenwald, W. F. and Kassis, J. A. (1992). The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes. Mech. Dev. 36,117 -127.[CrossRef][Medline]
Wieschaus, E. and Gehring, W. (1976). Clonal analysis of primordial discs cells in the early embryo of Drosophila melanogaster. Dev. Biol. 50,249 -263.[Medline]
Wilder, E. L. and Perrimon, N. (1995). Dual
functions of wingless in the Drosophila leg imaginal disc.Development 121,477
-488.
Williams, J. A., Bell, J. B. and Carroll, S. B. (1991). Control of Drosophila wing and haltere development by the nuclear vestigial gene product. Genes Dev. 5,2481 -2495.[Abstract]
Wimmer, E. A., Jackle, H., Pfeifle, C. and Cohen, S. M. (1993). A Drosophila homologue of human Sp1 is a head-specific segmentation gene. Nature 366,690 -694.[CrossRef][Medline]
Wimmer, E. A., Frommer, G., Purnell, B. A. and Jackle, H. (1996). buttonhead and D-Sp1: a novel Drosophila gene pair. Mech. Dev. 59, 53-62.[CrossRef][Medline]
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