1 Institute of Signaling, Developmental Biology and Cancer, Centre de
Biochimie-UMR 6543-CNRS, Parc Valrose, 06108 Nice cedex 2, France
2 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical
School, 200 Longwood Avenue, Boston, MA 02115, USA
Author for correspondence (e-mail:
noselli{at}unice.fr)
Accepted 17 February 2003
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SUMMARY |
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Key words: Juvenile hormone, Retinoic acid, Left-right asymmetry; Fasciclin 2, Genitalia, Organ looping
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INTRODUCTION |
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Given the harmful consequences of heterotaxia
(Kosaki and Casey, 1998), it
is crucial for animals to genetically control LR asymmetry in order to avoid
organ randomization. A breakthrough in the molecular analysis of LR asymmetry
was the discovery of side-specific expression of nodal, a BMP family
molecule, in chick embryos (Levin et al.,
1995
). The study of nodal homologs in other vertebrate
embryos (Collignon et al.,
1996
; Lowe et al.,
1996
) indicates that nodal is a central and evolutionarily
conserved regulator of LR asymmetry
(Burdine and Schier, 2000
;
Capdevila et al., 2000
;
Mercola and Levin, 2001
;
Wright, 2001
). In addition to
nodal, retinoic acid (RA), a terpenoid compound, has been shown to
participate in LR asymmetry in all vertebrates examined so far
(Niederreither et al., 2001
;
Tsukui et al., 1999
;
Wasiak and Lohnes, 1999
).
Indeed, either an excess or a reduction of RA activity can lead to LR
asymmetry defects (Tsukui et al.,
1999
). Interestingly, RAinduced LR defects are associated with
abnormal expression of the asymmetric markers nodal or
pitx2, indicating that RA interacts with the nodal pathway
and is an important conserved component of the LR program in vertebrates.
Although there is considerable information about how LR asymmetry is
established, very little is known about the mechanisms and molecules
controlling looping morphogenesis of LR asymmetric organs. It is important to
note that in the absence of LR determination, organs still loop but in a
random direction. This indicates that looping morphogenesis lies downstream of
the LR pathway and produces a stereotyped directional looping based on
interpretation of positional information. One clear candidate in the looping
process in vertebrates is RA. Indeed, depending on the species, mode of
administration and stage, exogenous RA can induce partial heart looping in
mouse and Xenopus (Chazaud et al.,
1999; Drysdale et al.,
1997
; Iulianella and Lohnes,
2002
; Niederreither et al.,
2001
; Tsukui et al.,
1999
; Wasiak and Lohnes,
1999
; Zile et al.,
2000
). Furthermore, loss of the Raldh2 gene, which is
essential to convert vitamin A into active RA, leads to an incomplete looping
of the heart in mouse (Niederreither et
al., 2001
). These data thus indicate that RA has a dual role,
being required both in the establishment of LR asymmetry upstream of
nodal and in the downstream morphogenetic control of looping per se.
Whether the mechanisms controlling and coupling LR asymmetry and organ looping
are conserved is unknown.
In contrast to the prominent sidedness found in vertebrates, LR asymmetry
in invertebrates is much less pronounced
(Hobert et al., 2002). For
example, the Drosophila heart is a symmetrical structure lying at the
dorsal midline and running along the AP axis. There are a few stereotyped LR
markers in the fly gut, but no mutations so far have been isolated that
specifically affect any of these (Hayashi
and Murakami, 2001
; Lengyel
and Iwaki, 2002
; Ligoxygakis
et al., 2001
). In order to establish a genetic model of LR
asymmetry and organ looping in Drosophila, we have screened for
mutations affecting the asymmetric looping of the spermiduct and genitalia in
adult flies (Fig. 1A,B). We use
this process as a model to dissect the genetic pathways involved in the
looping of LR asymmetric organs.
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MATERIALS AND METHODS |
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Fas2e86, Fas2EB112 and
UAS-Fas2 were kind gifts of C. Goodman
(Grenningloh et al., 1991).
Fas2rd1 was a gift of R. Davis
(Cheng et al., 2001
).
UAS-synaptobrevin-GFP flies were provided by M. Ramaswami
(Estes et al., 2000
).
Met flies were kindly provided by T. Wilson. Transgenic lines
expressing GAL4 in different subsets of neurons (Aug21, Feb78, Feb170, Feb211,
Kurs21) were kindly provided by T. Siegmund
(Siegmund and Korge, 2001
).
All other stocks can be found at FlyBase
(http://flybase.bio.indiana.edu/).
Rescue experiments
Short egg collections (2 hours) were raised on rich medium at 25°C and
subjected to a 1 hour heat-shock at 37°C. Only one heat-shock per life
cycle was applied. After HS treatment, flies were grown at 25°C until
eclosion, and the extent of rotation rescue was scored in adult males. The
genotype of the heat-shocked males is Fas2spinR5/Y;
UAS-Fas2/HS-GAL4.
Fas2-expressing clones, marked with GFP, were generated using the
`flip-out' technique (Struhl and Basler,
1993). Larvae with the genotype
Fas2spinR5/Y; act>y+>GAL4,
UAS-EGFP/UAS-Fas2; hsFLP/+ were heat-shocked at 37°C for 1 hour.
Histology
Third instar larvae were dissected, inverted inside-out with forceps and
antibody stained using standard protocols. After staining, the ring glands
were dissected and mounted. The anti-Fas2 (Developmental Studies Hybridoma
Bank) primary antibody was used at 1:10; Secondary antibodies used in this
study are anti-mouse-FITC (1:400; Molecular Probes), anti-mouse CY3 (1:400;
Molecular Probes), anti-rabbit-CY3 or FITC (1:400; Molecular Probes).
Phalloidin-TRITC was used at 1:2000 (Molecular Probes). Confocal images were
taken using Leica TCS-NT or TCS-SP1 confocal microscopes. Images were
processed using Photoshop 6.0 (Adobe).
For scanning electron microscopy (SEM) imaging, adult flies were critical-point dried and coated with 25 nm gold using standard methods.
Topical application of pyriproxyfen
A JH analog, pyriproxyfen
{2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxy]-pyridine} stock solution (10 mg/l in
acetonitrile from the laboratory of Dr Ehrenstorfer-Schafers, Germany) was
diluted with acetone. White prepupae were collected and 0.25 µl of acetone
containing the desired amount of pyriproxyfen was applied to each pupa on the
dorsal side, as described by Riddiford and Ashburner
(Riddiford and Ashburner,
1991).
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RESULTS |
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The reversible nature of looping genitalia indicates that this structure represents a suitable LR marker that can be used as a novel genetic model to study both LR asymmetry and looping morphogenesis in Drosophila.
spin affects organ looping
To initiate a genetic characterization of LR asymmetry and organ looping in
flies, we screened for viable mutations showing defective genitalia rotation
(G.Á. and S.N., unpublished). We focus on a novel viable P-element
mutation, spin, for which all the adult males show a characteristic
mis-rotation of genitalia and are sterile. In spin males, the extent
of rotation varies from 30° to 320°, with a large proportion of
males (84%; n=195) having their genital plate in a position
corresponding to rotation of 135-225°.
Fig. 1 shows some
characteristic examples of spin genital plates. Because external
mis-rotation does not allow discrimination between under-, hyper- or
counter-rotation of the genitalia, males of different phenotypes were
dissected and the looping of their spermiduct analyzed. All dissected males
showed a clear under-rotation phenotype
(Fig. 1G,H), indicating that
spin is required for the genital plate and spermiduct to undergo
complete looping, but has no role in directionality. Dissection of several
hundred wild-type males did not reveal any rotation defect (data not shown),
indicating the robustness of this process in normal males.
spin is a novel, looping specific, fasciclin 2 allele
After remobilization of the P-element insert in spin (see
Materials and Methods), three different w
populations were found. In the major class (51/73; 70%), flies reverted to a
fully wild-type phenotype. The two other classes included lethal alleles
(18/73; 25%) and viable alleles (4/73; 5%), which retained their original
spin-like phenotype. Altogether, these results indicate that the
P{GAL4} transposon present in spin is responsible for the looping
phenotypes and is inserted in or close to an essential gene.
The P-element in spin is inserted in the 5' UTR of the
fasciclin 2 (Fas2) gene
(Fig. 2A; see Materials and
Methods) (Goodman et al.,
1997; Grenningloh et al.,
1991
), suggesting that spin is a novel Fas2
allele (hereafter referred to as Fas2spin). This
conclusion is supported by the following points. First, the expression of the
Fas2 protein in the original Fas2spin allele and in a
lethal revertant (Fas2spinRM1) is strongly reduced or
absent in embryos, respectively (Fig.
2B-E). Significantly, in Fas2spin third instar
larvae, the expression of the Fas2 protein in eye imaginal discs or in whole
brain extracts is also strongly reduced
(Fig. 2F,G; data not shown).
Second, we show that expression of a UAS-Fas2 transgene under the
control of the original spinP{GAL4} line can fully rescue
the rotation and sterility phenotypes (Fig.
1C).
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Taken together, these results indicate that spin is a novel viable, looping-specific Fas2 allele. Fas2spin is the first fully penetrant defective looping mutant to be described, and thus represents an important tool to study organ looping in Drosophila.
Stage requirement for Fas2 function in organ looping
Rotation of genitalia was reported by Gleichauf
(Gleichauf, 1936) to take
place in 2- to 3-day-old pupae over a period of 24 hours. To establish the
temporal requirement for Fas2 function in genitalia rotation, we used
a heat-inducible GAL4 line to express Fas2 under the UAS promoter. In
this experiment, we rescued the Fas2spinR5 allele, a
viable Fas2spin excision allele retaining the rotation
phenotypes but lacking the GAL4 activity associated with the original
Fas2spin allele (data not shown). Short egg collections
were subjected to a single one hour heat-shock (HS) at 37°C. Adult males
were then analyzed and the extent of genitalia rotation rescue determined. As
shown in Fig. 2H, a single 1
hour HS at day 7 of development is sufficient to rescue genitalia rotation.
Indeed, flies that received a HS at day 7 of development showed a very high
degree of rescue (up to 90%), while HS applied either before or after this
period had little or no effect (Fig.
2H). Control males which have not been heat-shocked show no rescue
(data not shown). These results indicate that Fas2 is required during
a limited period of time during pupal development for rotation to take place
normally. The timing of the rescue is consistent with the previously described
period of rotation (Gleichauf,
1936
).
Fas2spin affects the corpora allata synapses
In order to identify the tissue(s) and cells that require Fas2
function for genitalia rotation, we used the GAL4-UAS system
(Brand and Perrimon, 1993) to
drive tissue-specific expression of a UAS-Fas2 transgene in
Fas2spinR5 males. Because Fas2 is required in
many aspects of neuronal development
(Brunner and O'Kane, 1997
;
Goodman et al., 1997
), and is
expressed mostly in neurons, we first asked whether Fas2 function was
required in the nervous system for rotation. Surprisingly, we found that the
elav-GAL4 line, which drives expression specifically in the CNS
during development, is able to rescue Fas2spinR5 rotation
defects fully (Fig. 4A). This
result prompted us to examine in detail the expression pattern of Fas2 protein
in the brain and to look for potential nervous system phenotypes in
Fas2spin. Our analysis uncovers a previously unknown
function of Fas2 in the ring gland (RG). The RG is a composite
neuroendocrine organ made of three different specialized regions: the
prothoracic gland (PT), the corpora cardiaca (CC) and the corpora allata (CA;
Fig. 3B). The CC probably plays
a role in the regulation of blood sugar levels in larvae through adipokinetic
hormones (Noyes et al., 1995
;
Rulifson et al., 2002
). The PT
and CA are specialized cells responsible for the secretion of the two primary
insect hormones ecdysone and juvenile hormone (JH), respectively
(Riddiford, 1993
).
Interestingly, Fas2 expression is restricted to the CC and to specific axonal
processes innervating the CA (Fig.
3A,C). These neurosecretory cells (nCA; see
Fig. 3A) control JH level and
can be easily identified using a specific GAL4 line expressed in all CA
neurons (Kurs21-GAL4) (Siegmund and Korge,
2001
). As Kurs21-GAL4 driven GFP expression and the anti-Fas2
staining overlap precisely, we conclude that Fas2 is expressed in all nCA
terminals (Fig. 3A). In
Fas2spin, the overall morphology of the CA synapse is
abnormal (Fig. 3C,D), showing
fused terminal boutons and a reduced number of presynaptic nerve terminals
(compare Fig. 3E,F). This
result is consistent with the previous finding that the bouton number is
reduced in the neuromuscular junction in strong hypomorph Fas2 flies
(Stewart et al., 1996
).
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Role of CA neurosecretory cells in organ looping
In order to demonstrate a direct link between Fas2, the CA, and
genitalia rotation, we used the UAS-GAL4 system to express Fas2 in
specific subsets of neurons innervating the RG. In a recent study, Siegmund
and Korge (Siegmund and Korge,
2001) identified and mapped the few neurons innervating the RG in
Drosophila. The PT is innervated by two neurons from each brain
hemisphere, whereas the CA is innervated by three neurons
(Fig. 3A)
(Siegmund and Korge, 2001
).
Importantly, neurons innervating the PT and CA are different and map to
distinct regions of the brain. We used a collection of GAL4 lines expressed in
different populations of neurons innervating the RG
(Fig. 4)
(Siegmund and Korge, 2001
) to
induce neuron-specific expression of Fas2 in
Fas2spinR5 mutants. When GAL4 is expressed strongly in the
neurons innervating the CA (nCA) using the Kurs21-GAL4 line
(Fig. 4A,B), the rotation of
genitalia is completely rescued, just as observed with an elav-GAL4
driver. Using another line in which GAL4 is only weakly expressed in CA
neurons (Feb78), we found that the rotation, although only weakly and
partially rescued, exceeds that seen in control Fas2spinR5
males. Using another nCA-GAL4 line (Feb170) in which GAL4 shows
variegated expression (i.e. some larvae express GAL4 in nCA, while in others
the expression is absent) (Siegmund and
Korge, 2001
), rescue is either complete or absent, respectively
(Fig. 4A). By contrast, when
GAL4 is expressed in the neurons innervating the prothoracic gland (nPT;
Feb211; Fig. 4A,C) or in the CA
itself (Aug21), no rescue was observed. These results show that Fas2
is required in the nCA for normal genitalia rotation. In addition, they
indicate that the rotation defects associated with
Fas2spinR5, and probably also with other viable
Fas2 alleles, are linked to a defective neuroendocrine function
leading to abnormal synthesis of JH during pupal development
(Fig. 2H).
Fas2spin controls looping non-autonomously
Our data support a model in which Fas2-expressing nCA neurons
control JH titers which in turn remotely control the rotation of the genital
plate (Fig. 7A). This model has
two main predictions: first, if JH is a mediator of Fas2 function
during rotation, then Fas2spin should function cell
non-autonomously; second, JH itself should have the potential to perturb
rotation when its level is altered experimentally.
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Pyriproxyfen, a JH agonist, induces looping defects
The second prediction of our model is that JH, which is proposed to control
rotation, should induce looping defects when its level is modified during
pupal development. Interestingly, it has been shown that the JH analogs
methoprene and pyriproxyfen produce rotation defects at low doses, after
topical application to white pre-pupae
(Riddiford and Ashburner,
1991). Higher doses of these JH analogs induce abdominal defects
and lethality. Because the extent and direction of rotation in the previous
study were not determined, we applied varying doses of pyriproxyfen to white
pre-pupae and monitored the effects on genitalia rotation. Application of
half-lethal doses of pyriproxyfen (0.25 pM/pupae) to wild-type pupae induces
rotation defects that are very similar to Fas2spin
phenotypes. Indeed, dissection of the posterior abdomen of unhatched adults
(pharate adults) indicates that pyriproxyfen-treated males have an
under-rotated phenotype, with some males showing a complete absence of
rotation (Fig. 6A). At higher
doses (0.4 pM/pupae), the treatment is lethal; as animals die as pharate
adults, their morphology can be analyzed. Dissection reveals a shift toward a
no rotation phenotype, with a larger proportion of males having their genital
plate in its initial position. Thus, increasing the level of a JH analog
produces looping defects ranging from partial to a complete loss of
circumrotation.
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DISCUSSION |
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Conserved role of terpenoids in organ looping
We show that asymmetric organ looping in Fas2spin males
is impaired and is due to an abnormal endocrine activity of the ring gland
during the pupal stage. The effects on genitalia and spermiduct are mediated
by an excess of JH, which then modifies looping morphogenesis downstream of LR
determination (Fig. 7A). How do
these results relate to vertebrate organ looping and LR asymmetry? The fact
that JH affects looping morphogenesis in Drosophila suggests an
important evolutionary conservation of the role of terpenoids in this process,
downstream of LR determination. Like the retinoid hormones, JH is synthesized
from the common isoprenoid precursor farnesyl diphosphate via the mevalonate
biosynthetic pathway (Harmon et al.,
1995). Furthermore, JH is a sesquiterpenoid that is chemically
related to the vertebrate terpene group, represented by retinoic acid
(Jones and Sharp, 1997
). The
common terpenoid nature of JH and RA has thus led to the proposal that these
molecules may bind a common family of nuclear hormone receptors that might
play similar functions in different organisms
(Moore, 1990
). In this
respect, it is important to note that the JH analog methoprene, the topical
application of which leads to genitalia rotation defects
(Riddiford and Ashburner,
1991
), can specifically bind and activate the RXR receptor in
mammalian cultured cells (Harmon et al.,
1995
). RA signal transduction in vertebrates requires the binding
to and the activation of heterodimers composed of RAR and RXR nuclear
receptors isoforms. Interestingly, the only insect homolog of vertebrate RXR
is encoded by the Drosophila ultraspiracle (usp) gene, which
has been shown to bind to JH in vitro
(Jones and Sharp, 1997
;
Jones et al., 2001
).
Altogether, these data thus suggest that JH and RA have a related
activity.
In addition to sharing common chemical features, both RA and JH, when
present in excess, have strikingly similar effects on organ looping. In
conditions of excess RA, a series of heart defects has been observed,
including reversal of symmetry or incomplete looping. In Xenopus, the
heart tube fails to loop after continuous exposure to low doses of RA
(Drysdale et al., 1997), and
incomplete looping of the heart is also observed in mice treated with RA over
a long period (Chazaud et al.,
1999
). It is important to note that the effects of excess RA on
heart looping are dose sensitive and stage specific, as are the effects of JH
analogs (methoprene and pyriproxyfen) on the looping of genitalia in flies
(Fig. 6A)
(Riddiford and Ashburner,
1991
).
In addition to blocking organ looping, excess RA can also induce a reversal of LR asymmetry in several vertebrate models (see Introduction). Such a reversal of asymmetry has not been observed after topical application of pyriproxyfen in Drosophila (Fig. 6). This apparent discrepancy may be explained by species- and/or stage-specific responsiveness to excess terpenoids, as is found among vertebrates for RA. Another possibility is that JH in flies may have a function restricted to organ looping, not sharing the dual role of RA seen in vertebrates (Fig. 7B).
The chemical, and, as shown in this study, the functional and phenotypic similarities associated with JH and RA in flies and vertebrates, respectively, show that terpenoids play an evolutionarily conserved role in handed looping (Fig. 7B).
LR asymmetry and organ looping in Drosophila
In Drosophila, genetic control of the establishment of the two
major body axes has been well described. LR asymmetry has attracted little
interest and thus remained an elusive process for several reasons. First,
there are only few and mostly transient (i.e. present during embryonic stages
only) LR organs (Hayashi and Murakami,
2001; Lengyel and Iwaki,
2002
; Ligoxygakis et al.,
2001
), leading to the view that flies may not represent a good
model to study LR axis like vertebrates. In the case of genitalia rotation,
only one dedicated study has been published
(Gleichauf, 1936
), and, yet,
this or another LR process have not been clearly validated as candidate LR
markers. Second, to our knowledge no mutations have been isolated so far that
showed a fully penetrant and rotation-specific defect. Though some studies
have reported rotation defects in specific allelic combinations, the rotation
phenotypes are poorly penetrant and are associated with other developmental
defects (e.g. Abbott and Lengyel,
1991
; Holland et al.,
1997
; Santamaria and
Randsholt, 1995
; Yip et al.,
1997
).
In this study, we developed an approach to identify genes that are involved
in two main processes underlying directional organ looping: the determination
of directionality (LR asymmetry) and looping morphogenesis. Interestingly, our
work reveals the existence of a zygotic control for genitalia LR asymmetry
that is apparently distinct from the maternal control of LR development during
embryogenesis (Hayashi and Murakami,
2001; Lengyel and Iwaki,
2002
; Ligoxygakis et al.,
2001
). Additional work will be necessary to understand the
mechanisms underlying asymmetric development in different tissues and
developmental stages. Together with previously described mutations
(Milani, 1955
;
Milani, 1956
) (see
Introduction) and our recent identification of a situs inversus mutation
leading to a complete reversion of genitalia rotation (counterclockwise)
(G.Á. and S.N., unpublished), these data suggest that just like in
vertebrates, the earliest steps of symmetry breaking are accessible to genetic
analysis in flies. (Mochizuki et al.,
1998
; Morgan et al.,
1998
; Yokoyama et al.,
1993
). Altogether, our results indicate that Drosophila
has all the basic elements to make it a genetic model to study organ looping
in the context of LR asymmetry. In this respect, it is interesting to note
that asymmetric organ looping, rather than asymmetric organ localization, is a
more general and common LR marker among animals. For example in vertebrates,
the first appearance of LR asymmetry is indicated by embryo turning and heart
tube looping. Furthermore, organ looping can also be observed in plants
through the helical growth of stalks and stems
(Thitamadee et al., 2002
).
We have shown a novel parallel between the programs underlying
Drosophila and vertebrate asymmetric organ looping. Is this parallel
more general? In order to address this question, future work will have to be
focused on the identification of new genes involved in genitalia rotation in
Drosophila, using genetic screens and reverse genetic approaches. One
major goal of future work in Drosophila will be the identification of
asymmetrically expressed genes and/or proteins. The fact that no such gene has
been identified so far may be due to the lack of appropriate data on the
developmental aspects of LR asymmetry in Drosophila. In this respect,
our study now allows identification of the male genital disc
(Sanchez and Guerrero, 2001)
as a clear candidate tissue for looking at asymmetrically expressed molecular
markers. The use of Drosophila and the comparative analysis of the LR
asymmetry programs in vertebrates and invertebrates will help provide insights
into the molecular mechanisms that underlie the question of symmetry breaking
in animals.
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ACKNOWLEDGMENTS |
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Footnotes |
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