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Development 121, 3111-3120 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
3111
Genetic determinants of sense organ identity in Drosophila: regulatory
interactions between cut and poxn
Michel Vervoort1, Daniele Zink3, Nathalie Pujol2, Kathleen Victoir1, Nathalie Dumont1, Alain Ghysen2,4 and
Christine Dambly-Chaudière1,4
1Laboratoire
de Génétique du Développement and 2Laboratoire de Neurobiologie, Université Libre de Bruxelles, 67 rue des
Chevaux, 1640 Rhode-St-Genèse, Belgium
3Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, 6900 Heidelberg, Germany
4Laboratoire de Neurogénétique, Unité 432 INSERM, Université de Montpellier II, place E. Bataillon, 34095 Montpellier Cedex,
France
SUMMARY
Two genes involved in defining the type of sense organ have
been identified in Drosophila. The gene cut differentiates
the external sense organs (where it is expressed) from the
chordotonal organs (where it is not); among the external
sense organs poxn differentiates the poly-innervated organs
(where it is expressed) from the mono-innervated organs
(where it is not). Here we show that the expression of poxn
in normal embryos does not depend on cut, and that poxn
is capable of inducing the expression of cut. We have identified a small domain of the very large cut regulatory region
as a likely target for activation by poxn.
INTRODUCTION
Another proneural gene, atonal (ato), has been shown by a
similar LOF/GOF analysis to be responsible for the formation
of the ch organs (Jarman et al., 1993).
The AS-C and ato genes encode transcriptional regulators
(Villares and Cabrera, 1987; Cabrera and Alonso, 1991;
Jarman et al., 1993) and are assumed to act by regulating the
expression of a battery of target genes. Among the potential
targets are genes expressed both in ch and es precursors, which
are thought to confer properties shared by all mother cells (Bier
et al., 1989). Other potential target genes are expressed only in
subsets of mother cells. One such gene is cut (Jack, 1985),
which is expressed in the AS-C-dependent precursors and in
their progeny, but not in ato-dependent mother cells
(Blochlinger et al., 1990). LOF and GOF analysis of cut have
demonstrated that the absence of the gene leads to the transformation of es into ch organs (Bodmer et al., 1987) while its
ubiquitous expression results in the opposite transformation
(Blochlinger et al., 1991). This implies that cut directs the
differentiation of es organs.
If both the AS-C genes and cut are involved in the determination of es organs, what is the relationship between these
genes? The expression of achaete and scute is transient and is
turned off before the mother cell divides, well before its
progeny will differentiate (Ruiz-Gomez and Ghysen, 1993). It
is believed that cut is activated by the AS-C genes, and is maintained by self-activation through the lineage until the daughter
cells differentiate (Blochlinger et al., 1991). In other words, the
AS-C genes would determine the es specificity by activating
cut which itself directs the acquisition of es properties.
The gene poxn (Bopp et al., 1989; Dambly-Chaudière et al.,
1992) is also expressed in a subset of mother cells, those that
A major goal in the analysis of development is to understand
how specific cell fates are established. Progress along that line
has been made recently in the case of the peripheral nervous
system of Drosophila, which comprises different types of
sense organs and is particularly well suited for a genetic
analysis. In this paper, we will concentrate on three types of
sense organs: the mono-innervated external sense organs (mes), the poly-innervated external sense organs (p-es) and the
chordotonal internal sense organs (ch). The three types of
organs differ in their lineage, morphology and neuronal connectivity, thereby providing a useful system to examine the
genetic determinants of these differences.
Each sense organ comes from a single ectodermal precursor,
the sensory mother cell (Bate, 1978; Hartenstein and Posakony,
1989; Bodmer et al., 1989). The competence of ectodermal
cells to become mother cells depends on the expression of
‘proneural’ genes (Ghysen and Dambly-Chaudière, 1989). A
first set of proneural genes are those of the achaete-scute
complex (AS-C; Garcia-Bellido, 1979). Mutations that result
in a loss of function (LOF) of these genes lead to the disappearance of all m-es and p-es organs (Garcia-Bellido and Santamaria, 1978; Dambly-Chaudière and Ghysen, 1987), while a
gain of function (GOF) of the AS-C genes promotes the
formation of supernumerary external sensory organs (GarciaAlonso and Garcia-Bellido, 1986; Rodriguez et al., 1990).
These opposite LOF and GOF phenotypes are typical of
‘selector genes’ which direct developmental choices (GarciaBellido, 1975; Lewis, 1978), and suggest that the AS-C genes
are responsible for the formation of external sense organs.
Key words: poxn, cut, neuronal identity, peripheral nervous system,
Drosophila, neurogenesis, determination genes
3112 M. Vervoort and others
give rise to the p-es organs. The LOF/GOF analysis of poxn
revealed that embryos deleted for this gene have no p-es
organs, while the ubiquitous expression of poxn early during
embryogenesis leads to the formation of supernumerary p-es
organs (Dambly-Chaudière et al., 1992). The ubiquitous
expression of poxn during metamorphosis induces the transformation of adult m-es into p-es organs. This transformation
affects not only the external morphology of the organs but also
the number of neurones, their projection in the central nervous
system and their functional properties (Nottebohm et al., 1992,
1994). Thus poxn is responsible for the establishment of the
morphological and functional properties typical of p-es organs.
In summary, if a SMC expresses neither cut nor poxn, it will
form a ch organ; if it expresses cut but not poxn, it will produce
a m-es organ, and if both cut and poxn are expressed, a p-es
organ will form.
An intriguing result of the GOF analysis of poxn in embryos
was that some of the poxn-induced supernumerary organs
appear at positions where no m-es organs are found in wildtype embryos, suggesting that these supernumeraries do not
originate from m-es precursors. Here we investigate the origin
of the ectopic es organs, and demonstrate that the expression
of poxn can transform ch into es precursors by inducing the
expression of cut.
MATERIALS AND METHODS
Drosophila strains and crosses
KL11.1 and KL11.4 are insertions of the hsp-poxn construct on the
third and second chromosome, respectively (Dambly-Chaudière et al.,
1992). Df(1)260.1 is a complete deletion of the AS-C (Garcia-Bellido,
1979). ct145 is a null mutation of the cut locus (Jack, 1985; Bodmer
et al., 1987). E7 2nd 36 is an enhancer trap insertion in which lacZ is
expressed in most of the md neurones (Bier et al., 1989). A3-lacZ is
an insertion on the third chromosome of a P element containing 2.7
kb of cut regulatory sequence fused to lacZ (Jack and Delotto, 1995).
In order to see the effect of ubiquitous poxn expression in AS-C
mutants, KL11.1/TM6 males were crossed with y Df(1)260.1 f / FM6
virgins. In the case of cut mutants, KL11.1 males were crossed with
y ct145/+; KL11.1/+ females. The effect of poxn on the pattern of lacZ
expression in E7 2nd 36 was assessed in the progeny of KL11.1/+;
E7 2nd 36/+ flies; the effect of poxn expression on A3-lacZ expression
was observed in the progeny of KL11.4/+; A3-lacZ/+ flies.
Heat-shock treatments
Heat-shock experiments in the embryo were done as follows: 3 pulses
of 10 minutes at 37°C separated by 2 intervals of 5 minutes at 25°C
are applied to 4 to 6 hours old embryos that are kept at 25°C before
and after the heat shock. This protocol gives a high frequency of
supernumerary p-es organs. Heat shocks of pupae were done as
described in Nottebohm et al. (1992) and applied between 6 and 12
hours APF. For giant chromosomes immunostaining, third instar
larvae were collected, put at 37°C for 30 minutes and left at 25°C for
24 hours before dissection.
Staining and mounting
22C10 staining was done as described in Ghysen et al. (1986); antiCut as described in Blochlinger et al. (1990); and anti-Poxn as in
Dambly-Chaudière et al. (1992) using the anti-Poxn at a 1:250
dilution. Polyclonal anti-galactosidase was from Cappell and was
used at a 1:200 dilution. X-gal staining of whole pupae was done using
the method described for embryos by Ghysen and O’Kane (1989),
including the heptane-fixative step, except that 2.5% glutaraldehyde
was used for fixation instead of formaldehyde. The reaction was
allowed to continue for 5 days at 37°C. Cuticule preparations of late
embryos were mounted and observed as described in DamblyChaudière and Ghysen (1986).
Giant chromosomes
Immunolabelling was done using the method of Zink and Paro (1989)
using the following conditions: the salivary glands were fixed for 25
seconds, anti-Poxn was used at a 1:10 dilution. In situ hybridisation
on giant chromosomes was performed as described in Langer-Safer
et al. (1982).
RESULTS
The ectopic expression of poxn transforms ch
organs into es organs
The ubiquitous expression of poxn during embryogenesis leads
to the formation of an excess of p-es organs (DamblyChaudière et al., 1992). Most of the supernumerary organs
result from the transformation of m-es into p-es precursors.
Some of these organs, however, appear at positions where no
m-es organs are found in the wild type. One possible explanation is that the ectopic organs result from the transformation of
internal sense organs. We have therefore examined whether
poxn has any effect on the internal sense organs.
There are two major types of internal sense organs, ch and
multidendritic (md; Bodmer and Jan, 1987). The ch and md
neurones can be distinghuished from each other and from the
es neurones by using the 22C10 antibody, which recognises an
antigen in the cytoplasmic membrane of all sensory neurones
(Zipursky et al., 1984): es neurones have a long and thin
dendrite that extends to the surface of the embryo; ch neurones
have a stubby dendrite that extends under the epidermis and
md neurones develop their multiple dendrites much later than
the other neurones, after hatching (Bodmer and Jan, 1987).
Besides the difference in morphology, ch also differ from
other neurones in that ch neurones or their precursor cells often
migrate away from their original position while most other
neurones are found near the position where their precursors
form. For example, the precursors to the five lateral ch organs
in the abdominal segments (lch5, Fig. 1A) form dorsolaterally
but later migrate to a lateral position (Ghysen and O’Kane,
1989; Bier et al., 1990; Salzberg et al., 1994). This migration
event is ch-specific: if the precursors to the lch5 are transformed into es precursors due to the ubiquitous expression of
cut, the transformed neurones keep a dorsolateral position
(Blochlinger et al., 1991).
We have labelled embryos with 22C10 after heat-induced
expression of poxn. Since the hsp-poxn chromosomes are
homozygous lethal (Dambly-Chaudière et al., 1992), crosses
were made between heterozygotes and therefore one half of the
progeny is expected to carry one copy of the hsp-poxn
construct. In about one half of the embryos, we observe the
displacement of the lch5 neurones to a more dorsal position
(Fig. 1B, arrow). These neurones have es-like dendrites (Fig.
1C), confirming that they have acquired an es identity. About
one fourth of the embryos show a disorganisation of the peripheral nervous system, with a loss of ch neurones and a large
excess of es ones. We believe that this phenotype corresponds
to the embryos that carry two copies of the heat-shock
Regulatory interactions between cut and poxn 3113
construct. The large excess of es neurones in these embryos
probably reflects the transformation of many ch and m-es into
p-es organs.
Fig. 1. Effect of the ubiquitous expression of poxn on sensory
neurones. Lateral and dorsal clusters of neurones in the fifth
abdominal segment of a wild-type (A) and in a hsp-poxn (B,C)
embryo after heat-shock treatment. The arrowheads in A and B point
to the v’ch1 chordotonal organ which arises from the ventral cluster.
The thick arrow indicates the lateral chordotonal organs (lch5) in A.
In the heat-shocked embryo, no neurones are present at the position
of the lch5 (asterisk in B), but a new dorsal cluster is present (arrow).
These neurones have es-like dendrites that extend into a more
superficial focal plane (arrows in C). Anterior is left, dorsal is up.
construct, as we do not observe it in experiments where
embryos have at most only one copy of the heat-shock
poxn can form es organs in AS-C− embryos
If the expression of poxn can transform ch into es precursors,
one would expect to observe ectopic es organs even in embryos
where only ch precursors are present. Embryos deficient for the
AS-C genes lack all es organs and most md neurones, but their
ch organs develop normally (Dambly-Chaudière and Ghysen,
1987). We have found that the expression of poxn can lead to
the formation of external structures typical of es organs in ASC− embryos (Fig. 2C).
In the thoracic segments, where the external structure formed
by the p-es organ (kölbchen; Hertweck, 1931) is easy to detect
even in an otherwise naked embryo, we detected a mean
number of 0.2 kölbchen per hemisegment in heat-shocked ASC−; hsp-poxn/+ embryos. Some hemisegments bear up to 2
kölbchen. These kölbchen appear at ventral and dorsolateral
positions that correspond to the positions where the ventral and
dorsolateral ch precursors arise. Using 22C10 labelling, we
observed that some of the ch neurones are morphologically
transformed into es-like neurones (not shown). Taken together,
these results show that the ubiquitous expression of poxn can
lead to the transformation of ch into es organs.
In contrast, we have never observed a clear transformation
of the few md neurones that remain in AS-C− embryos. Since
most md neurones are absent in such embryos, we have used
another method to decide whether poxn affects the md
neurones. We induced poxn in an enhancer-trap line E7 2nd 36
strain, where lacZ is expressed in all md neurones but not in
ch or es neurones (Bier et al., 1989). We did not detect any
change in the pattern of lacZ-expressing cells (not shown), thus
confirming the lack of effect of poxn on md neurones.
Fig. 2. Induction of p-es organs by ubiquitous poxn expression in AS-C− embryos. (A) Ventral view of the third thoracic segment of a wildtype embryo after heat-shock treatment. The arrow points to the ventral kölbchen (poxn-dependent organ), the arrowhead to the Keilin organ
(poxn-independent organ). (B) Third thoracic segment of an AS-C− embryo after heat-shock treatment; no sensory organ are present. (C) Third
thoracic segment of an AS-C−; hsp70-poxn embryo after heat-shock treatment. The arrow points to a kölbchen in ventral position. This organ is
slightly displaced relative to the normal kölbchen (A) and probably arises from the transformation of the ventral ch organ (vch1; Ghysen and
Dambly-Chaudière, 1986).
3114 M. Vervoort and others
poxn cannot promote es development in the
absence of cut
In the absence of cut, no es organ is formed (Bodmer et al.,
1987). Yet we observed in AS-C− embryos that ch precursors
(which do not express cut) can form es organs if they are
forced to express poxn. One way to explain this result is that
poxn can substitute for cut. We tested this explanation by
inducing the ubiquitous expression of poxn in cut− embryos.
These embryos are easily distinguished from the cut+
embryos produced in the same cross because their normal
complement of external sense organs is deleted. We
examined 96 heat-shocked cut−; hsp-poxn embryos containing on the average one copy of the hsp-poxn construct, and
did not detect a single external sense organ, either at the
positions where the normal es organs should have appeared,
or at the positions where transformed ch organs would be
expected, or in any other position. By contrast, the cut+
embryos produced in the same cross form about 0.6 supernumerary organ per hemisegment, as reported previously
(Dambly-Chaudière et al., 1992).
It might be argued that the heat shock is for some reason
less efficient in cut− than in cut+ embryos. We could however
rule out this remote possibility by relying on another phenotype
resulting from the heat-induced expression of poxn. We have
noticed that the ectopic expression of poxn modifies the morphology of the antennal organ, a complex sensory structure that
does not depend on AS-C and cut. The poxn-induced transformation of this organ is fully observed in heat-shocked cut−;
hsp-poxn embryos, demonstrating that poxn is indeed
expressed ectopically in these embryos. We conclude that, in
the absence of cut, poxn is not capable of imposing an es fate
to the ch precursors.
poxn expression is independent of cut but depends
on AS-C
All the data so far suggest that the presence of an active cut
gene is necessary for poxn to impose an es fate to ch precursors. Does this conclusion hold true for the normal p-es
precursors? It has been observed that p-es organs are absent
in cut− mutants (Bodmer et al., 1987). This result is consistent with the hypothesis that poxn requires cut to promote the
p-es fate. Alternatively, it might be that the expression of
poxn in p-es precursors depends on cut activity. We have
therefore asked whether or not poxn is expressed in cut−
mutants.
Fig. 3A shows the pattern of expression of poxn in cut−
embryos. The p-es precursors and their progeny express poxn
normally but never give rise to an es organ (as monitored by
the absence of any external structures and the presence of morphogically ch-like neurones; Bodmer et al., 1987; our own
observations) while they give rise to a poly-innervated organ
(M. V. and A. G., unpublished data). We conclude that cut is
necessary for poxn to confer their complete fate to the normal
p-es precursors.
The observation that the expression of poxn does not depend
on cut raises the possibility that it is also independent of ASC activity, and relies directly on other positional cues. Fig. 3B
shows an embryo homozygous for a complete deletion of the
AS-C labelled with the anti-Poxn antibody. The expression of
poxn is abolished in these embryos except in the central
nervous system (arrowheads).
Fig. 3. Expression of poxn in cut− and AS-C− embryos. (A) The
distribution of Poxn protein in cut− embryos is indistinguishable
from the wild-type pattern (as described in Dambly-Chaudière et al.,
1992). The arrows and arrowheads show, respectively, the ventral
and dorsal lines of p-es organs. In the second and third thoracic
segments, the dorsal p-es organs are displaced laterally (asterisks).
(B) In AS-C− embryos, no cell expresses poxn in the peripheral
nervous system. Labelling can be observed, however, in the central
nervous system (slightly out of focus, empty arrowheads).
Fig. 4. Effect of the ubiquitous expression of poxn on cut expression.
Lateral (l) and dorsal (d) clusters of three adjacent abdominal
segments of a wild-type (A) and of a hsp-poxn (B) embryo after
heat-shock treatment are shown. Arrows in B point to two new
dorsolateral clusters of cut-expressing cells. The position of these
cells corresponds to that of the es-like neurones shown in Fig. 1B).
poxn induces cut
We have shown so far that the formation of normal and ectopic
p-es organs under the control of poxn requires cut. Since the
expression of poxn can cause ch precursors to form es organs,
one has therefore to postulate that the expression of cut has
Regulatory interactions between cut and poxn 3115
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wing margin
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embryonic es organs
posterior
spiracle
malpighian tubules
been activated in the transformed precursors. We have
examined this hypothesis by observing the pattern of cutexpressing cells after ubiquitous activation of poxn. In conditions that lead to the formation of ectopic es organs, we
observe the presence of ectopic cut-expressing cells in the
dorsolateral position that is typical of transformed ch organs
(Fig. 4).
We have confirmed this result by using embryos deleted for
the AS-C genes, where the only precursors left are those that
do not express cut. Labelling AS-C− embryos with anti-Cut
antibodies reveals the presence of Cut product in non-neuronal
cells only (Blochlinger et al., 1991), while after ubiquitous
expression of poxn, ectopic clusters of cut-expressing cells can
be observed at positions consistent with those of the sensory
organs seen on the cuticle (not shown).
A regulatory sequence of cut can be activated by
poxn in ch organs
The regulatory region of cut extends over approximately 200
kb. It comprises several independent enhancer regions, each
of which directs the expression of cut in different tissues (Fig.
5; Jack et al., 1991; Liu et al., 1991). Some of these regions
have been shown to direct the expression of the reporter gene
lacZ in the peripheral nervous system (Jack and DeLotto,
1995). Late during embryogenesis, the 2.7 kb fragment called
A3 drives lacZ expression in the same subset of larval sense
organs that also express cut: in all es organs and in some md
neurones, but in none of the ch organs (Blochlinger et al.,
1990).
We have examined the pattern of lacZ expression at earlier
stages of development, at the time that the first mother cells
are formed. The first precursors arise as two pairs of cells in
each segment, an anterior pair, which gives rise to es organs,
and a posterior pair generating ch organs (Ghysen and
O’Kane, 1989). The anterior precursors express achaete and
cut (Ruiz-Gomez and Ghysen, 1993; Blochlinger et al.,
1990), while the posterior ones express only atonal
(Jarman et al., 1993). Consistent with the pattern of
expression of cut, we found that A3 directs lacZ only in the
anterior cells and in their progeny (Fig. 6A). After heatshocking hsp-poxn; A3-lacZ embryos, however, we observe
lacZ expression in posterior cells (Fig. 6B). We conclude
that the A3 fragment can be activated in ch precursors by
poxn.
embryonic central nervous
system
adult es organs
tracheae
anterior spiracle
Fig. 5. Schematic representation of
the organisation of the cut locus.
The map shows a part of the
region upstream of cut. Scale is in
kilobases and is based on data
from Jack (1985). The large arrow
indicates the beginning of the
transcribed region. The boxes
represent regions which, when
fused to lacZ, drive the expression
of the reporter gene in defined
tissues (Jack et al., 1991; Jack and
DeLotto, 1995). The nomenclature
of the boxed regions is according
to Jack and DeLotto, 1995.
The A3 enhancer drives lacZ in adult poxndependent bristles
We found that the A3 fragment also directs the expression of
the reporter gene in a subset of adult es organs, the p-es organs
(chemosensory bristles) and some m-es organs (the campaniform sensilla). The second type of m-es organs, the
mechanosensory bristles, do not express the reporter gene (Fig.
7A). The expression of lacZ becomes detectable only at about
40 hours after pupariation formation (APF). This is a relatively
late stage in the development of these organs, several hours
after all the cells of the lineage have been generated. At earlier
stages, we detect no lacZ expression in the imaginal discs,
Fig. 6. Expression of lacZ in the second thoracic segment of A3-lacZ
and A3-lacZ; hsp70-poxn embryos. (A) The expression of lacZ is
observed in the ‘A’ cells (arrowhead), posterior to the tracheal pit
(asterisk), as well as in the more ventral ‘V’ cells, which appear
slightly later, but never in the posterior ‘P’ cells which appear first.
The P cells are located anterior to the next tracheal pit (doubleasterisks) and are ch precursors (Ghysen and O’Kane, 1989).
(B) After the ectopic expression of poxn, some P cells express lacZ
(arrow). Nomenclature of the cells is according to Ghysen and
O’Kane (1989).
3116 M. Vervoort and others
Fig. 8. lacZ expression in A3-lacZ; hsp70-poxn pupae after heatshock treatment. (A) Wing disc showing expression of lacZ 30
minutes after a heat shock delivered at 6 hours APF. The arrowheads
point to the p-es precursors of the anterior wing margin. Arrows
indicate the precursors of the campaniform sensilla (m-es organs).
(B) Higher magnification of the wing margin of the same disc.
Fig. 7. Expression of lacZ in the tibia of A3-lacZ and A3-lacZ;
hsp70-poxn pharate adults after heat-shock treatment. (A) X-gal
staining of the tibia of a A3-lacZ pharate adult. Labelling is restricted
to cells belonging to the recurved, open-tipped p-es bristles (arrows).
(B) X-gal staining of the proximal region of the tibia of an A3-lacZ;
hsp70-poxn pharate adult after heat-shock treatment. Many p-es
bristles (arrows) are present and are all associated with blue cells.
except a very weak expression in the posterior part of the eye
disc.
The mother cells that will produce chemosensory bristles
and campaniform sensilla appear several hours earlier than
those that will form mechanosensory bristles, both in the wing
(Huang et al., 1991; Hartenstein and Posakony, 1989) and in
the leg (Nottebohm et al., 1994). Thus the differential
expression of lacZ driven by the A3 fragment could be due to
the difference in the time of emergence of the different sensory
organs, rather than to the difference in identities.
In order to determine whether A3 responds to precursor
identity or to stages of pupal development, we have examined
the effect of transforming the identity of precursors cells
without changing their time of emergence. The precursors of
the tibial chemosensory bristles (p-es) are determined at about
the time of puparium formation, while the precursors of the
mechanosensory bristles (m-es) appear 12 hours later
(Nottebohm et al., 1994). Inducing the ubiquitous expression
of poxn at 12 hours after puparium formation (APF) transforms
the m-es precursors into p-es precursors (Nottebohm et al.,
1992), making it possible to form chemosensory bristles from
‘late appearing’ precursors and hence to distinguish whether
the A3 enhancer responds to time or to cell type. In the first
case, time dependence, we expect lacZ to be expressed only in
the normal (early appearing) chemosensory bristles while in
the second case, identity dependence, we should see lacZ
expression in both normal (early) and transformed (late)
chemosensory bristles. The result is shown Fig. 7B: the normal
as well as the supernumerary p-es bristles express lacZ. We
conclude that the A3-controlled expression of lacZ in the p-es
organs is correlated to the identity of these organs and not to
their time of emergence. Moreover, this experiment confirms
that the A3 fragment contains at least one element that
responds directly or indirectly to poxn.
Transient activation of A3-lacZ by poxn
The expression of A3-lacZ in supernumerary p-es organs is
detected 40 hours APF, at the same time when this construct
is also expressed in the normal p-es cells. This is long after the
ubiquitous induction of poxn. We examined whether poxn
might have a more immediate effect on A3 by assessing the
expression of lacZ 30 minutes and 60 minutes after induction.
As mentioned previously, the A3 fragment drives no detectable
expression of lacZ before 40 hours APF, except in the eye. The
same is true for heat-shocked pupae bearing only the A3-lacZ
construct and for non-shocked A3-lacZ; hsp-poxn pupae: in
both cases, lacZ is only weakly expressed in eye discs and not
at all in the precursor cells of the other discs. In contrast, when
we heat-shocked pupae of the A3-lacZ; hsp-poxn line at 6
hours APF, we detected a transient expression of lacZ in
several precursor cells (Fig. 8). These include the cells that will
form the p-es bristles of the anterior wing margin and of the
legs as well as the precursors of the campaniform sensilla and
of chordotonal organs. In addition, lacZ is strongly expressed
in the eye disc (not shown). One hour after the heat shock, the
expression of lacZ is not detectable any more in the precursor
cells, and only occasionally and weakly in the photoreceptors.
Regulatory interactions between cut and poxn 3117
Fig. 10. The A3 fragment contains a Poxn-binding site. (Upper
panel) Localisation of the insertion site of the A3 fragment in A3lacZ transgenic flies by in situ hybridisation using P-element DNA as
a probe. Hybridisation band (arrowhead) is located in 73A. (Lower
panel) Immunostaining of A3-lacZ; hsp70-poxn giant chromosomes
with anti-Poxn antibody. Arrowhead points to a Poxn-binding site in
73A.
Fig. 9. Distribution of Poxn protein after heat-shock treatment.
(A) Wing disc, (B) leg disc and (C) salivary glands of A3-lacZ;
hsp70-poxn pupae labelled with the anti-Poxn antibody 30 minutes
after heat shock. The arrowheads and arrows in A point to the
precursors of the p-es organs of the wing margin and to the precursors
of the campaniform sensilla of the wing blade, respectively.
tified these sites and found that one of them corresponds to the
cytological position of the cut locus. This result suggests that
cut may be a direct target of poxn.
The A3 fragment makes the associated lacZ gene inducible
by poxn. If this induction is direct and involves binding of the
Poxn product, one might expect the A3 fragment to comprise
an immunodetectable Poxn-binding site. We have stained the
giant chromosomes of A3-lacZ; hsp-poxn larvae after heat
shock and observed a new binding site at the position where
the A3-lacZ construct is inserted, 73A (Fig. 10). Poxn binding
is never observed at this position in the absence of the A3-lacZ
construct insertion. This result indicates that the A3 fragment
contains a Poxn-binding site and supports the idea that poxn
directly regulates the expression of cut.
DISCUSSION
The transient expression of lacZ occurs only in some
precursor cells including those that already express poxn. One
explanation for this restricted activation is the need for specific
cofactors present only in these cells. Alternatively, it might
result from a non-homogenous distribution of the heat-induced
Poxn protein. We examined this hypothesis by immunolabelling discs with anti-Poxn. We found that 30 minutes after
the heat shock, Poxn is present in a few cell types only. Besides
the p-es precursor cells where it is normally present, Poxn is
found in campaniform sensilla precursors, in some photoreceptors and muscle cells, and in the salivary glands cells (Fig.
9).
Poxn protein binds to the cut regulatory sequence
If poxn activates cut directly, one would expect the Poxn
protein to bind to the cut locus on salivary glands chromosomes (Zink and Paro, 1989). As poxn is normally not
expressed in the salivary glands, we have used the hsp-poxn
construct to induce the expression of poxn in this tissue prior
to the immunolabelling. We found that the Poxn product binds
reproducibly to about forty sites in the genome. We have iden-
The ectopic expression of poxn changes ch into es
fate by activating cut
The ubiquitous expression of poxn leads to the formation of an
excess of p-es organs, mainly arising from the transformation
of m-es organs (Dambly-Chaudière et al., 1992; Nottebohm et
al., 1992). We have shown here that some of the supernumerary organs come from the transformation of internal sense
organs of the ch type.
One obvious explanation for the ability of poxn to transform
ch into es organ is that poxn can substitute for cut. We
observed, however, that in cut− embryos, poxn is normally
expressed although p-es organs do not form. Moreover, the
overexpression of poxn in cut− embryos does not lead to the
formation of es organs. Thus poxn cannot substitute for cut for
the formation of es organs.
An alternative explanation for the fact that poxn is capable
of imposing an es fate to precursors that do not express cut, is
that poxn can activate the expression of cut. We have shown
that the ectopic expression of poxn results in the presence of
Cut product in sensory cells that do normally not express cut,
3118 M. Vervoort and others
indicating that poxn can indeed activate cut, either directly or
indirectly.
Poxn-binding sites on salivary gland chromosomes
The gene poxn encodes a protein which contains a putative
DNA-binding region (the paired domain) and an activation
region (the acidic C-terminal domain), suggesting that it acts
as a transcriptional regulator (Bopp et al., 1989; DamblyChaudière et al., 1992). We have identified the binding sites of
Poxn on giant chromosomes and found that one of them corresponds to the cytological position of cut.
The detection of bound transcriptional regulators on salivary
gland chromosomes (Zink and Paro, 1989; DeCamillis and al.,
1992; Rastelli et al., 1993) has been shown to identify bona
fide targets in several cases, as for example that of Polycomb
(Pc). Anti-Pc immunolabelling was observed at the positions
of several genes known to be regulated by Pc (Zink and Paro,
1989; Zink et al., 1991). In contrast to Pc, poxn is not expressed
in salivary glands and we had therefore to induce its expression
using a heat-inducible construct. This raises the question of
whether the binding sites that we observe correspond to bona
fide targets of poxn.
Our reasons to think that Poxn binds to its normal targets
when ectopically expressed in salivary gland cells are the
following. First, we detected a defined number of sites (forty)
at completely reproducible positions. Second, several of the
sites correspond to putative targets of poxn (M. V., unpublished observations). These include, besides cut, the gene poxn
itself, which is thought to be autocatalytic (C. D-C., unpublished observations), the gene east (Vijayraghavan et al.,
1992), which is specifically expressed in the leg chemosensory
neurones (M. V., unpublished observations) and a new gene
that is specifically expressed in the p-es, but not in the m-es,
organs of the embryo (P. Gautier, C. D-C. and A. G., unpublished observations).
leads to a transient ectopic expression of A3-lacZ in some es
precursor cells, and also at a later stage in the cells of the
mechanosensory sense organ. We conclude that poxn acts,
directly or indirectly on the A3 fragment to activate the
expression of the adjacent reporter gene. Since larvae carrying
the A3 fragment inserted on the third chromosome were shown
by immunolabelling with anti-Poxn antibody to have a new
Poxn-binding site at the position where the fragment is
inserted, we conclude that the activation of A3-lacZ by poxn
is a direct effect due to the binding of the Poxn protein to one
or more sites in the A3 fragment.
The response of A3-lacZ to poxn overexpression is restricted
to sensory cells, suggesting that activation ofA3 also requires
other factors present in these cells. Since the activation of A3
region and the maintenance of Poxn protein after heat-induced
expression of poxn are mostly restricted to cells that already
express cut, one possibility is that the Cut protein is needed for
Poxn-mediated transcriptional activation. This would also
explain the very low efficiency of the transformation of ch into
es organs by poxn ubiquitous expression, as compared to the
high efficiency of the m-es to p-es transformation. The occasional transformation of ch into es may reflect the fact that high
concentrations of Poxn product can exert an effect even in the
absence of Cut protein, or that cut is transiently expressed in
ch precursors.
Other functions of poxn do not seem to depend on cut,
however. In cut null mutant, the expression pattern of poxn is
completely normal. This pattern reveals several features that
are dependent on poxn activity, e.g. the number of cells (more
than four) and the shift in the position of the thoracic organs
relative to the abdominal ones (see Fig. 3). Thus it may be that
only a subset of poxn function, among which would be the activation of cut itself, requires both poxn and cut, while another
set, including the lineage and migration properties of the p-es
organs, would be cut-independent.
The complexity of the A3 fragment and the poxn-cut
relationship
During embryogenesis, A3 activates lacZ expression at all
stages of larval es organ development. In contrast, during metamorphosis, A3 is active only at a very late stage of adult es
organ formation and only in a subset of these organs, the campaniform sensilla and the chemosensory bristles. We do not
know whether the latter pattern has any specific function or
role, nor why the embryonic and pupal patterns are so different.
One possibility is that the late expression of A3-lacZ in adult
sense organ development is related to a difference between
larval and adult es organ formation. We note that the patterns
of cut expression at the late stages of sense organ development
differ in the larva (where the trichogen and tormogen cells
express cut at a higher level than the neurone and the thecogen)
and in the adult (where all cells express cut at the same level).
This suggests that there may be separate controls for cut
expression at the latest stage of sense organ development in the
embryo and in the pupa, and that the dissimilar behaviour of
A3 in these two systems may be related to this difference.
The expression of the A3-lacZ construct responds to the
expression of poxn at both stages. During embryogenesis, the
ubiquitous expression of poxn induces the expression of A3lacZ in ch precursors where this construct is normally never
expressed. During metamorphosis, the expression of poxn
Functional significance of the poxn-cut relationship
cut is expressed in all m-es and p-es organs and in most md
neurones (Blochlinger et al., 1990, 1991) while poxn is only
expressed in a subset of these cells, the p-es set (DamblyChaudière et al., 1992). The activation of cut in the m-es precursors (and in the md neurones) could depend on the AS-C
genes, and be subsequently maintained throughout the lineage
by self-activation (Blochlinger et al., 1991). What then could
be the meaning of the activation of cut by poxn, since poxn
itself is expressed only in AS-C-dependent precursors where
cut would be activated anyway?
One possible explanation is that the control of cut by poxn
is a remnant of an ancestral situation where the attributions and
roles of the two genes were different. The p-es bristles of
present-day adult flies are bifunctional: they are innervated by
one mechanosensory and several chemosensory neurones, and
their shaft comprises side by side a closed lumen resembling
that found in mechanosensory bristles and an open-tipped
lumen where the chemosensory dendrites extend. This raises
the possibility that the ancestral es organs were of the p-es type.
In that case, poxn may originally have been a major determinant of es organ formation, directing the expression of
several other genes (one of which was cut) to control and
realise this program of differentiation. The m-es organs may
then be considered as incomplete p-es organs, formed by using
Regulatory interactions between cut and poxn 3119
a subset of the complete p-es program. Accordingly, cut seems
to have only a partial effect on the identity of the sense organs.
We have noticed that, in cut mutants, the organs that correspond to the poxn-expressing lineage still contain more than
one neurone, a property typical of chemosensory organs (M.
V. and A. G.; unpublished observations). Furthemore, even in
null mutants, some es organs (about 5%) are not transformed
into ch and others are often only partially transformed (68% of
the transformed organs lack external structures but do not
produce the typical scolopale structures of the normal ch
neurones; Bodmer et al., 1987). All this suggests that cut may
be primarily a determinant of the external derivatives of the es
organs.
A second explanation, which is not exclusive with the first
one, emphasises the co-operation between cut and poxn. If both
genes are required to promote p-es formation, then coexpression of poxn and cut is essential. A direct activation of
cut by poxn would ensure that the expression of poxn is always
accompanied by the expression of cut.
We are grateful to J. Jack for providing fly stocks and informations
prior to publication, K. Blochlinger for giving us the anti-Cut antibody
and S. Fujita for the 22C10 antibody. M. V. is grateful to Professor
R. Paro for welcoming him in his laboratory in Heidelberg and to the
Fondation Jean Brachet for supporting this stay. This work was
supported by a contract from the Human Frontier Science Programme
Organization and a contract from the Communauté Française de
Belgique. M. V. holds a fellowship from the Institut pour l’encouragement de la Recherche Scientifique dans l’Industrie et l’Agriculture; A. G. is Directeur de Recherche at the INSERM.
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(Accepted 6 June 1995)