Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université, IBDM-CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France
* Author for correspondence (e-mail: perrin{at}ibdm.univ-mrs.fr)
Accepted 16 September 2005
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SUMMARY |
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Key words: Reprogramming, Hox, Steroid, Heart
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Introduction |
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Drosophila melanogaster provides an invaluable model to analyse complex biological mechanisms in the context of an intact developing organism. In particular, metamorphosis offers the opportunity to study the genetic control of an in vivo occurring remodelling process in which the adult organism is, in part, reconstructed from already differentiated, functional organs and tissues.
Drosophila is a holometabolous insect whose metamorphosis is
triggered by three peaks of the steroid hormone 20-hydroxyecdysone (hereafter
referred to as ecdysone) (Riddiford,
1993). Ecdysone exerts its effects through a heterodimer of two
members of the nuclear receptor superfamily, the Ecdysone Receptor (EcR) and
the fly RXR orthologue Ulstraspiracle (Usp). Ecdysone binding directly
impinges on the transcription-regulatory activity of the heterodimer (reviewed
by Riddiford et al., 2000
;
Thummel, 1996
). During
metamorphosis, most Drosophila larval tissues are eliminated by
programmed cell death, and adult structures develop from imaginal cells that
remain quiescent and have no functional requirement during larval life.
However, some larval cells are not eliminated and are subjected to
ecdysone-dependent changes in their shape and physiology. Such remodelling
occurs mainly in the nervous system
(Truman, 1990
). For example,
in mushroom bodies, the olfactory learning and memory centre in insects,
remodelling involves the pruning of larval projection and the subsequent
acquisition of adult specific projections
(Lee et al., 1999
).
The cardiac tube of D. melanogaster is a simple linear tube that
pumps and delivers haemolymph through the organism in an open circulatory
system, as invertebrates have no vessels
(Rizki, 1978;
Rugendorff et al., 1994
).
Despite its simplicity, it is formed by highly conserved molecular mechanisms
(Zaffran and Frasch, 2002
). In
the embryo and the larva, the cardiac tube extends from thoracic segment T1 to
abdominal segment A7. Intra-segmental myocyte diversification is revealed in
the abdominal segments by two transcription factors, Seven Up (Svp, the
CoupTFII orphan nuclear receptor orthologue) and Tinman (Tin, the Nkx2.5
orthologue), which are respectively expressed in the two anterior and four
posterior myocytes (Fig. 1C).
Cooperating with this segmental information, the Hox genes Ubx and
abdA trigger cardiac tube differentiation along the anteroposterior
axis: Ubx expression is restricted to segments A1 to A4, which form
the posterior aorta, while abdA is expressed in segments A5 to A7 and
is required for heart differentiation (Lo
et al., 2002
; Lovato et al.,
2002
; Perrin et al.,
2004
; Ponzielli et al.,
2002
).
It has been reported that the D. melanogaster cardiac tube
undergoes extensive changes during metamorphosis
(Curtis et al., 1999;
Molina and Cripps, 2001
;
Rizki, 1978
), but both the
precise origin of adult cardiac tube myocytes and the underlying genetic
control had not been established until now. Using both time-lapse analysis of
in vivo developing adult heart and cell tracing experiments, we demonstrate
that myocytes in the larval cardiac tube are remodelled without proliferation
to form the adult organ. In addition, we show that the organ remodelling
involves a transcriptional reprogramming of each cell type. Finally, we
investigate the genetic control of this process and show that it is cell
autonomously controlled by the Ecdysone Receptors (EcRs), in a large part
through the regulation of Hox gene expression and of the function of their
protein products. In particular, EcR activation switches AbdA towards a new
activity that leads abdA-expressing myocytes to be reprogrammed, and
to acquire functions, morphology and transcriptional activity specific to the
adult. This analysis provides several unexpected molecular insights into a
naturally occurring cellular reprogramming process.
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Materials and methods |
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dsRNA-abdA and dsRNA-Ubx constructs
Inverted-repeat constructs were generated in order to produce genomic cDNA
fusions predicted to form hairpin dsRNA molecules following splicing
(Kalidas and Smith, 2002).
Genomic and cDNA fragments were PCR amplified with primers containing unique
restriction sites. The cDNA fragment was selected to avoid splice donor
sequences (/GTNNGT) within the inverted repeat sequence. The dsRNA constructs
were cloned into the pUAST transformation vector
(Brand and Perrimon, 1993
).
Primer sequences are available upon request. Transgenic flies for UAS-IR
constructs were generated as previously described using a w1118
strain as a recipient stock (Rubin and
Spradling, 1982
).
Control of Gal4 induction
UAS and P(tub-GAL80[ts]) (McGuire et
al., 2003) transgenes were combined in the same lines and crossed
with appropriate Gal4 lines. Development was allowed to proceed at 18°C
until the late third instar larval stage and individuals were then shifted to
the restrictive temperature (29°C).
Timing of pupal development
The onset of pupal development corresponds to white pupae that were
selected on the basis of spiracle eversion, absence of reaction following
forceps contact and absence of tanning. Individuals were kept for further
development in an air incubator at 25°C.
Antibody staining
Dissections were done as described by Molina and Cripps
(Molina and Cripps, 2001). For
antibody staining, individuals were fixed for 1 hour in 1xPBS, incubated
for 1 hour with 1xPBS, 1% Triton X-100, washed three times for 10
minutes each with BBT (1xPBS, 0.1% Tween, 0.1% BSA), incubated for 30
minutes with saturation medium (1xPBS, 0.1% Tween, 10% BSA) and
overnight at 4°C with primary antibodies in BBT. After four 15-minute
washes with BBT, samples were incubated for 30 minutes with saturation medium,
incubated for 1 hour at room temperature with secondary antibodies in BBT and
washed four times for 15 minutes each in BBT. Primary antibodies used: rabbit
anti-ß-galactosidase (Cappel), 1:500; mouse anti-GFP (Molecular Probes),
1:500; rabbit anti-Tin (Azpiazu and Frasch,
1993
), 1:800; rabbit anti-D-Mef2
(Nguyen et al., 1994
), 1:1000;
mouse anti-Ubx (FP3.38) (White and Wilcox,
1985
), 1:100; rat anti-Abd-A
(Macias et al., 1990
), 1:1000;
rabbit anti-Synaptotagmin (Littleton et
al., 1993
), 1:1000; mouse anti-Wg, 4D4 (1:50) and mouse 22C10
(1:100), obtained from the Developmental Studies Hybridoma Bank.
Affinity-purified secondary antibodies were coupled to alkaline phosphatase or
biotin (Jackson ImmunoResearch Laboratories), or were Alexa-488 or Alexa-546
conjugated (Molecular Probes). All secondary antibodies were used at a
dilution of 1:500. In the adult, better signals were obtained for Ubx with the
aid of a Tyramide Signal Amplification kit (NEN Life Sciences).
Observations and photographs were carried out using a Leica MZ12 fluorescent binocular microscope, an Axiophot Zeiss microscope or a LSM 510 Zeiss confocal microscope.
In situ hybridisation
In situ hybridisation was performed as described previously
(Ponzielli et al., 2002). For
Ih and Ndae1 detection, it was necessary to amplify the
signal with the aid of the Tyramide Signal Amplification kit (NEN Life
Sciences). In situ hybridisation on adults was performed as follows:
individuals were dissected in PBS, fixed for 15 minutes in 4% paraformaldehyde
in 1xPBS, rinsed three times for 10 minutes each in 1xPBS and
rinsed three times in ethanol. Subsequent steps were identical to
hybridisations on whole-mount embryos. Digoxigenin (Dig)-labelled antisense
RNA probes specific for Ih were produced by transcription of an
EcoRI-digested full-length cDNA (clone number GH 23838, BDGP release
1).
Phalloidin staining of F-actin
Dissected individuals were fixed in 1xPBS, 3.7% formaldehyde for 15
minutes at room temperature, washed three times for 10 minutes each in BBT,
incubated for 20 minutes in saturation medium, incubated either with
TRITC-phalloidin (Sigma; 1:60 in BBT, 10 minutes) or Alexa350-phalloidin
(Molecular Probes; 1:20 in BBT, overnight) and rinsed three times for 10
minutes each in 1xPBS.
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Results |
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In order to unambiguously determine the origin of the adult cardiac myocytes, we analysed cardiac tube-specific expression of GFP throughout metamorphosis. This approach indicated that the adult organ arises by the remodelling of the larval cardiac tube by a continuous and progressive process (see Movie 1 and Fig. S1 in the supplementary material for a detailed timetable description of cardiac tube remodelling). Time-lapse analysis of cardiac tube remodelling, performed from 27 to 64 hours after puparium formation (APF), demonstrates that adult myocytes directly derive from larval myocytes without cell proliferation (see Movie 2 in the supplementary material). In addition, cell tracing experiments (see Fig. S2A-C in the supplementary material) show that the adult heart is formed by the myocytes that constituted the larval posterior aorta (segments A1 to A4), and by segment A5 myocytes, which are a part of the larval heart and which constitute the posterior tip of the adult heart (the terminal chamber). Both approaches thus indicate that the adult heart is formed by persisting larval myocytes that are remodelled without proliferation or the addition of new cells. One identity for each adult cardiac myocyte can therefore be inferred from its larval origin. Fig. 1C summarises the fate of the larval myocytes during the remodelling process.
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Cardiac tube metamorphosis is accompanied by an anterior shift of wg, Ih and Ndae1 expression. wg is transiently expressed at 30-36 hours APF in svp-expressing cells in segments A1-A5 (Fig. 2B,C). Likewise, Ih is activated in Tin-expressing myocytes in segments A1-A4, which constitute the adult heart contractile myocytes (Fig. 2E,F), but, here again, is not activated in svp-expressing cells (Fig. 2E,F). Strikingly, Ih expression is turned off in segment A5 during remodelling (Fig. 2E,F). The same switch of expression pattern is observed for Ndae1, which becomes expressed in Tin-expressing myocytes in segments A1-A4 at adulthood, but which is repressed in segment A5 (Fig. 2H).
In conclusion, morphological and functional transformation of the cardiac tube myocytes is accompanied by transcriptional reprogramming during metamorphosis.
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In addition, the ventral heart imaginal muscles that develop beneath A1-A4 cardiac myocytes (see Fig. S2J-L in the supplementary material) are absent when Ubx overexpression is driven by NP5169-Gal4 in cardiac myocytes (not shown), indicating that their development depends on correct cardiac myocyte reprogramming.
Ubx overexpression does not, however, affect the differentiation of adult ostiae that develop from A1-A5 svp-expressing cells (arrowhead in Fig. 3L), suggesting that Ubx activity is either positive or neutral with regard to ostiae differentiation. To distinguish between these two possibilities, Ubx expression was inhibited by targeted, specific dsRNA in the cardiac tube during metamorphosis, and Wg expression was monitored as a landmark of ostiae differentiation. Targeted Ubx-dsRNA driven in the cardiac tube during metamorphosis consistently reduces Ubx expression (not shown). Under these conditions, Wg expression in svp-expressing cells is abolished (Fig. 3N), suggesting that Ubx function is required for adult ostiae development. In conclusion, the regulation of Ubx expression appears to be required to drive the remodelling of the posterior aorta into adult heart.
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The targeted dsRNA method, when driven in the cardiac tube during metamorphosis by either the pan myogenic driver 24B-Gal4 or the heart-specific driver NP5169-Gal4, proved to be very efficient at inhibiting abd-A expression (not shown). abdA loss of function completely prevents terminal chamber differentiation. In particular, the organisation of the myofibrils in segment A5 is unmodified at metamorphosis. The fibrils remain oriented transversally, as in the larval heart, instead of being remodelled and longitudinally oriented, as observed in the wild-type adult organ [compare phalloidin staining of wild-type (Fig. 4E) and mutant (Fig. 4F) posterior cardiac tubes]. In addition, abdA loss of function prevents the formation of nerve terminations, without affecting axon growth (Fig. 4D). This indicates that abdA function is cell autonomously required in the myocytes of segment A5 for their innervations. By contrast, dsRNA>abd-A expression in the cardiac tube does not affect the histolysis of segments A6 and A7, which are eliminated upon abdA loss of function (Fig. 4D,F), suggesting that the histolysis segment A6-A7 myocytes is independent of abd-A function.
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AbdA cellular function is modified at metamorphosis
Our results demonstrate that, during metamorphosis, abdA drives
the remodelling of segment A5 and thus confers a terminal chamber identity
onto A5 myocytes. This activity is clearly distinct from that previously
described in the same cells during embryogenesis, as abdA is required
there to confer a heart identity to the cardiac myocytes, and is therefore
responsible for the differentiation of ostiae and contractile myocytes
(Lo et al., 2002;
Lovato et al., 2002
;
Perrin et al., 2004
;
Ponzielli et al., 2002
).
Accordingly, abdA dosage manipulation has distinct consequences for Ih expression when analysed during embryogenesis and during metamorphosis. When abdA function is inhibited in the cardiac tube during remodelling, Ih expression is maintained in segment A5 (Fig. 4K). Reciprocally, targeted abdA overexpression within the whole cardiac tube during metamorphosis represses Ih expression in segments A1 to A4 (Fig. 4L). By contrast, during embryogenesis, abd-A is required for Ih expression (Fig. 4I) and abdA misexpression in the whole cardiac tube ectopically activates Ih transcription (Fig. 4J). These differential outputs of abdA activity during embryogenesis and metamorphosis, observed at the level of both cellular differentiation and Ih transcriptional control, highlight that the cellular function of AbdA is modified at metamorphosis.
Ecdysone signalling is required to cell autonomously trigger cardiac tube remodelling
What could be the temporal input that leads to the regulation of
Ubx expression and the switch in AbdA activity? Because the
reprogramming occurs at metamorphosis, we investigated whether ecdysone
signalling has a role in the process. Indeed, the remodelling starts at 30
hours APF, which corresponds to the latest and major peak of ecdysone titre
observed during Drosophila metamorphosis
(Riddiford, 1993).
Ecdysone signalling is mediated by a heterodimer of nuclear receptors,
consisting of one Ecdysone Receptor (EcR) isoform (either EcR-A, -B1 or -B2)
and Ultraspiracle (Usp), which is activated upon ecdysone binding
(Riddiford et al., 2000;
Thummel, 1996
). In order to
evaluate the involvement of ecdysone-signalling in remodelling, we interfered
with the reception of ecdysone signalling in two ways, either with the
targeted dsRNA technique [with an UAS>dsRNA-EcR construct directed against
the core common to all EcR isoforms
(Roignant et al., 2003
)], or
with a dominant-negative form of EcR [UAS>EcRDN
(Cherbas et al., 2003
)]. When
driven in the cardiac tube during metamorphosis with either the 24B-Gal4
(Fig. 5A,B,D,E) or the
NP5169-Gal4 (Fig. 5C) driver,
both constructs dramatically inhibit heart remodelling. The cardiac tube
retains its larval morphology, with the characteristic division into aorta in
segments A1 to A4 and heart in segments A5 to A7
(Fig. 5A). In addition,
inhibition of ecdysone signalling prevents Wg activation in
svp-expressing cells (Fig.
5B), and A5 myofibrils retain their larval circular orientation
(Fig. 5D,E). Furthermore,
segment A5 innervations are prevented following EcR inhibition
(Fig. 5D), segments A6 and A7
are not eliminated (Fig. 5A)
and ventral imaginal muscles do not form. These results demonstrate that
ecdysone signalling is cell autonomously required for all aspects of cardiac
tube remodelling.
Ecdysone signalling impinges on Ubx regulation and on abdA activity
We next investigated whether ecdysone signalling acts upstream of
Ubx expression and AbdA activity. Inhibition of ecdysone signalling
through dsRNA>EcRcore expression in the Tin-expressing myocytes with the
5169-Gal4 driver results in the maintenance of Ubx expression during
metamorphosis (Fig. 5C),
indicating that Ubx repression, which is required for the correct
remodelling of the larval posterior aorta into adult heart, is regulated by
the ecdysone pathway. Interestingly, despite maintenance of Ubx
expression, A1-A4 Tin-expressing myocytes are not reprogrammed. This contrasts
with the above described effect of Ubx ectopic expression during
metamorphosis, which affects Tin-expressing myocyte remodelling and induces
the formation of longitudinal myofibrils
(Fig. 3J,L). This result might
indicate that the action of Ubx expression in Tin-expressing myocytes during
metamorphosis requires the ecdysone signalling pathway. In agreement with this
hypothesis, we failed to detect any formation of longitudinal myofibrils in
cardiac myocytes in which Ubx and EcRDN were co-expressed during
metamorphosis (data not shown).
AbdA expression is maintained in A5 myocytes after EcR inhibition (Fig. 5E). Its expression is therefore not dependent on ecdysone signalling. However, A5 myocytes that do express abdA are unable to remodel when EcR activity is depressed, suggesting that ecdysone signalling is instrumental in driving the switch of AbdA activity at metamorphosis. Indeed, the consequences of inhibiting EcR function during remodelling are very similar to those observed upon loss of abdA function, including a lack of innervations and of myofibril orientation remodelling (Fig. 5D,E). Consistent with this, ecdysone signalling downregulation inhibits the effects of AbdA overexpression. Indeed, although it efficiently induces terminal chamber-like structures in segments A1 to A4 when expressed alone, targeted AbdA overexpression is unable to induce terminal chamber-like structures in the cardiac tube when EcR function is inhibited, not only in A1-A4 segments (Fig. 5F), but also in A5 (Fig. 5G), where myofibrils remain transversal. Together, these results indicate that the reprogramming of segment A5 myocytes requires the modulation of AbdA activity (but not of its expression) by the ecdysone-mediated metamorphosis program.
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Discussion |
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Like myocytes in segment A5, those in segments A1-A4 also gain a number of
novel properties during remodelling. For example, Tin-expressing myocytes
increase their myofibrillar content, activate Ih transcription and
acquire contractile activity; svp-expressing cells transiently
express wg and differentiate into functional ostiae. However, this
may not represent transdifferentiation per se, as these cells do not appear to
lose any of the differentiated characteristics they would have acquired during
embryonic or larval life. Rather, we consider that larval A1-A4 myocytes are
committed to differentiate into adult heart myocytes, but do not represent, on
their own, a fully differentiated state. Interestingly, their final
differentiation relies on an ecdysone-dependent modulation of Ubx
expression: Ubx expression has to be downregulated in Tin-expressing
myocytes, while its maintenance, and probably its overexpression, is required
in svp-expressing cells. Ubx function, however, appears to
be unchanged in these cells. Indeed, when Ubx is overexpressed in the
embryonic cardiac tube, it is able to activate wg expression in A1-A4
svp-expressing myocytes (Perrin
et al., 2004), and to repress Ih transcription in
Tin-expressing myocytes (B.M., unpublished). This clearly indicates that, at
least at the wg and Ih transcriptional level, Ubx activity
is unmodified during remodelling.
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The developmental switch triggering cardiac tube remodelling is mediated by
ecdysone, which impinges on Ubx expression and on AbdA activity (see
Fig. 6 for a summary model).
Interestingly, like retinoic acid (RA) in the developing vertebrate hindbrain
(Gavalas, 2002;
Gavalas and Krumlauf, 2000
),
the outcome of ecdysone activity during the remodelling of segments A1-A4 is a
`posterior transformation': adult A1-A4 myocytes acquire properties
characteristic of larval A5-A7 posterior myocytes. This is the first reported
example of a steroid function in axial patterning in arthropods. In addition,
we show that, like RA, which modulates Hox expression in vertebrates
(Conlon and Rossant, 1992
;
Conlon and Rossant, 1995
;
Dupe et al., 1997
;
Kessel and Gruss, 1991
), the
ecdysone signalling pathway modulates Ubx expression. These
observations strongly suggest that steroid activity on axial patterning is
conserved from flies to vertebrates.
In contrast to its effects on Ubx, ecdysone does not affect abdA expression but impinges on its activity. This is the first observation of such an activity for a steroid hormone and it certainly warrants further study in other animal models. Is this also a property of steroids (such as RA) in vertebrates? To date, the only known activity of RA on Hox gene function is a modulation of their transcriptional expression. The data presented here recommend a re-examination of RA/Hox functional interactions in vertebrates.
Cellular identities along the rostrocaudal axis mainly depend on the Hox
code, i.e. on the combinatorial expression of Hox genes
(Hunt and Krumlauf, 1992;
Kessel and Gruss, 1991
;
Kmita and Duboule, 2003
). The
fact that, as we show here, a steroid hormone can additionally control Hox
activity greatly increases the number of cellular identities that can
potentially be determined by the same Hox code. In addition, the situation
described here concerning AbdA activity indicates the importance of the
developmental context upon Hox specificity
(Brodu et al., 2002
;
Lohmann and McGinnis, 2002
;
Rozowski and Akam, 2002
). An
important unanticipated result from the present study is that the change of
AbdA activity can occur in the same cells, leading to a reprogramming between
two differentiated states that are both controlled by the same Hox gene.
A future challenge will be to understand the molecular basis of the
steroid-dependent Hox activity switch. Ecdysone signalling might change AbdA
transcriptional activity, by specifying new transcriptional targets or by
switching its activity from an activator to a repressor (and vice versa) on
the same transcriptional targets. What could be the molecular mechanism by
which EcR activation impinges on AbdA activity? Given the pivotal role played
by transcriptional cofactors in Hox function
(Mann and Affolter, 1998;
Mahaffey, 2005
), one
hypothesis is that EcR activates or represses such a Hox cofactor. Exd/Hth are
obvious candidates (Mann and Affolter,
1998
) but do not seem to be involved; they are not expressed in
the cardiac myocytes, neither during embryogenesis
(Perrin et al., 2004
) nor
during the remodelling process (B.M., unpublished). Conversely, there is
evidence that Hox transcriptional activity can be modified by phosphorylation
(Jaffe et al., 1997
). An
alternate possibility would therefore be that EcR activates or represses the
expression of some kinases or phosophatases, which would in turn modify AbdA
activity. In any case, elucidation of the molecular mechanisms will rely on
the identification of direct abdA targets in the cardiac tube.
wg and Ih are potential candidates, and are currently under
investigation.
In conclusion, our study may shed light on Hox gene function and regulation
in other cell reprogramming processes, such as pathological remodelling of the
human heart, in which steroids appear to play a key role
(Sun, 2002). In addition,
cellular therapies for inherited myopathies are based on the cellular
plasticity of the donor cells (either satellite cells or muscle-derived stem
cells). Cell transplantations for the treatment of muscular dystrophies appear
promising (Huard et al.,
2003
), and understanding the molecular basis of one example of
myocyte reprogramming should help to unravel the underlying mechanisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5283/DC1
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