RESEARCH REPORT 1493
Development 138, 1493-1499 (2011) doi:10.1242/dev.058958
© 2011. Published by The Company of Biologists Ltd
Apoptosis controls the speed of looping morphogenesis in
Drosophila male terminalia
Erina Kuranaga1,2,3,*, Takayuki Matsunuma1, Hirotaka Kanuka4, Kiwamu Takemoto5,6, Akiko Koto1,
Ken-ichi Kimura7 and Masayuki Miura1,2
SUMMARY
In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental
timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct)
loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia,
indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping
morphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of male
terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed a
full 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced.
Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded the
male genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a ‘moving walkway’ to
accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with
single-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, and
upregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ring
movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia
within a limited developmental time frame.
KEY WORDS: Caspase, Apoptosis, In vivo imaging, Drosophila
1
Department of Genetics, Graduate School of Pharmaceutical Sciences, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 2CREST, JST,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 3Laboratory for Histogenetic
Dynamics, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
4
National Research Center for Protozoan Diseases, Obihiro University of Agriculture
and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
5
Department of Physiology, Yokohama City University Graduate School of Medicine,
Fuku-ura 3-9, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. 6JST, PRESTO,
Saitama 332-0012, Japan. 7Laboratory of Biology, Sapporo Campus, Hokkaido
University of Education, 3-1 Ainosato 5jo, Kita-ku, Sapporo, Hokkaido 002-8502,
Japan.
*Author for correspondence ([email protected])
Accepted 2 February 2011
defective; W – FlyBase) (Abbott and Lengyel, 1991), a proapoptotic gene; drICE (Muro et al., 2006) (Ice – FlyBase), the
Drosophila ortholog of caspase 3; and dronc (Krieser et al., 2007)
(Nc – FlyBase), the Drosophila ortholog of caspase 9. Caspase
family proteases are the central executioners for the genetically
encoded apoptosis in animals (Degterev et al., 2003). However, the
physiological roles of apoptosis in completing the morphogenesis
of male terminalia remain to be elucidated.
We herein used live in vivo imaging to determine the dynamics
of the looping morphogenesis and spatiotemporal apoptosis during
male genitalia development. Our observations suggest that
apoptosis drives the acceleration of rotation, enabling the complete
genitalia morphogenesis to occur within the developmental
timetable.
MATERIALS AND METHODS
Fly stocks and temporal gene expression
Flies were raised on standard Drosophila medium at 25°C. The following fly
strains were used in this study: en-GAL4, UAS-mCD8-EGFP, UAS-lacZ,
Histone2Av (His2Av)-mRFP, tub-GAL80ts (Bloomington Drosophila Stock
Center); AbdB-GAL4LDN (de Navas et al., 2006); UAS-p35 (Zhou et al.,
1997); UAS-PVR DN (Duchek et al., 2001); UAS-JNK DN (Adachi-Yamada
et al., 1999): UAS-SCAT3 (Takemoto et al., 2007; Kanuka et al., 2005;
Kuranaga et al., 2006); and UAS-Histone2B (H2B)-ECFP, UAS-nls-SCAT3
(Koto et al., 2009).
Using the TARGET system, we bred flies at the permissive
temperature (18°C) of GAL80ts until the time when the head of the pupae
had just everted, to suppress the activity of GAL4. After head eversion,
the flies were moved to the restrictive temperature (29°C) of GAL80ts
(McGuire et al., 2003) for 12 hours. Time-lapse imaging using a
stereomicroscope (M205FA, Leica) was performed at 22°C after the heat
shock.
DEVELOPMENT
INTRODUCTION
During animal development, dynamic cell behaviors are precisely
orchestrated to accurately complete morphogenesis. However, the
mechanisms that determine precisely how cell behaviors regulate
morphogenesis according to the developmental timetable are still
uncharacterized. Programmed cell death or apoptosis not only
functions in sculpting and deleting structures in developing
animals, but also it plays dynamic roles in coordinating organ
morphogenesis (Stenn and Paus, 2001; Toyama et al., 2008). The
Drosophila male terminalia is an asymmetric looping organ; the
internal genitalia (spermiduct) loops dextrally around the hindgut.
During the maturation of the internal genitalia, the male terminalia
rotates 360° clockwise (Gleichauf, 1936). The orientation defect of
adult male terminalia is thought to occur when this rotation is
incomplete (Adam et al., 2003). Apoptosis is thought to contribute
to the completion of genitalia rotation, because an orientation
defect of the adult male terminalia is observed in mutants of
apoptotic pathway components, including: hid (head involution
Development 138 (8)
Fig. 1. In vivo imaging and quantitative analysis of genitalia rotation. (A)An image (left) and schematic drawing (right) of the male
genitalia of His2Av-mRFP flies at 24 hours APF. Each segment is highlighted in a different color: A8 (green), A9 (orange) and A10 (yellow).
(B,D,E) Time-lapse series of genitalia rotation in (B) His2Av-mRFP/+, (D) en-GAL4 UAS-H2B-ECFP/+ and (E) en-GAL4 UAS-H2B-ECFP/UAS-p35
flies are shown. Ventral is towards the top in all figures. (C)Image (left) and schematic drawing (right) of genitalia in en-GAL4 UAS-H2BECFP/+ at 24 hours APF. The posterior region of the A8 segment is highlighted in green. (F,G)Scanning electron micrograph of the adult male
genitalia of en-GAL4/UAS-lacZ (F) and en-GAL4/UAS-p35 (G). (H)The genitalia angle () in control (black) and p35-expressing flies (red) was
measured every 30 minutes, and the mean angle is shown. Error bars indicate s.d. (control, n10; p35, n8). (I)Velocity (Vd/dt) and
(J) acceleration rate (adV/dt) were quantified by measuring  and V as a function of time t in control (black) and p35-expressing flies (red).
The initiation of genitalia rotation (>1 hour) in p35-expressing flies was similar to control flies (indicated by the gray area). Genotypes of the
control flies were as follows: en-GAL4 UAS-mCD8-GFP/+, en-GAL4 UAS-H2B-ECFP/+ and en-GAL4 UAS-nls-ECFP-venus/+. (K)The mean
genitalia angle () in flies expressing dominant-negative JNK (JNK-DN; blue) and dominant-negative PVR (PVR-DN; green) were plotted every
30 minutes. Error bars indicate the s.d. (JNK-DN, n12; PVR-DN, n11). control and p35 in Fig. 1H are represented for reference as black and
red lines, respectively. (L)The average velocity (Vd/dt) was quantified by measuring  as a function of time t in control (black), p35 (red),
JNK-DN (blue) and PVR-DN (green) flies (mean±s.d.) (**P<0.01, *P<0.05).
DEVELOPMENT
1494 RESEARCH REPORT
Apoptosis controls speed of morphogenesis
RESEARCH REPORT 1495
Sample preparation for time-lapse imaging and scanning electron
microscopy
Staged pupae (24 hours APF) were washed in water and mounted on a
glass slide using double-sided tape. The pupal case covering the caudal part
of the abdomen was removed. A very wet filter paper was placed around
the pupae to keep them hydrated. The pupae were covered with a
coverglass in a small drop of water to avoid desiccation. Silicon (Shinetsu)
was used to seal the chamber. In most cases, the animal survived the data
acquisition and developed into an adult. Time-lapse images were captured
using an SP5 confocal microscope (Leica) or an inverted microscope
(Olympus) with a spinning disc-type confocal unit (CSU10, Yokogawa,
Tokyo) equipped with the Aquacosmos/Ashura system (Hamamatsu
Photonics) (Kuranaga et al., 2006). The FRET analysis was performed
using the Aquacosmos (Hamamatsu Photonics) and MetaMorph software
(Molecular Devices) programs. For the scanning electron microscopy, we
used the VE-8800 microscope system (Keyence).
RESULTS AND DISCUSSION
To visualize the genitalia rotation in living animals, we first used
the His2Av-mRFP Drosophila line whose nuclei are ubiquitously
marked by a fluorescent protein (Pandey et al., 2005). The genital
disc is a compound disc comprised of cells from three different
embryonic segments: A8 (male eighth tergite), A9 (male
primordium) and A10 (anal). During metamorphosis, the genital
disc is partially everted, exposing its apical surface, and adopts a
circular shape (Fig. 1A) (Keisman et al., 2001). Our results
captured the male genitalia undergoing a 360° clockwise rotation
(Fig. 1B; see Movie 1 in the supplementary material). Inhibiting
apoptosis by expressing the baculovirus pan-caspase inhibitor p35
driven by engrailed-GAL4 (en-GAL4), which is expressed in the
posterior compartment of each segment, results in genital misorientation at the adult stage (Macias et al., 2004).
In flies expressing nuclear fluorescent protein driven by enGAL4, we observed that the posterior part of the A8 segment (A8p)
formed a ring of cells surrounding the A9-A10 part of the disc (Fig.
1C). First, we recorded the images at a low resolution (10⫻
objective lens) to measure the rotation speed accurately in control
and p35-expressing flies, because long-term time-lapse imaging at
a high resolution can cause photodamage, and thus alter pupal
development. Most of the cells in the A8p that seem to disappear
at the end of Movies 2 and 3 in the supplementary material actually
moved out of the plane of focus. In our imaging results, the rotation
started around 24 hours APF (after puparium formation) and
stopped about 12 hours later (12 hours 5 minutes±58 minutes;
n10) (Fig. 1D; see Movie 2 in the supplementary material). To
confirm whether the mis-oriented genital phenotype in the caspaseinhibited flies was caused by incomplete rotation, we observed the
rotation in flies expressing p35 under the en-GAL4 driver. In the
p35-expressing flies, the rotation began, but it stopped before it was
complete, after about 12 hours (12 hours 8 minutes±1 hour 27
minutes; n8), i.e. with the same timing as in control flies (Fig. 1E;
see Movie 3 in the supplementary material). This suggested that the
reduced caspase activation in A8p prevented the genitalia from
completing the rotation, resulting in mis-oriented adult genitalia
(Fig. 1F,G).
To compare complete rotation with incomplete rotation, we
calculated the rotation speed by measuring the angle (control and
p35) of the A9 genitalia every 30 minutes on time-lapse images.
The normal rotation was composed of at least four steps: initiation,
acceleration, deceleration and stopping (Fig. 1H). We calculated
the velocity of rotation Vd/dt by measuring  as a function of
time t. At first, the genitalia rotated at an average velocity (Vcontrol)
of 7.67±3.72°/hour by 1 hour after initiation, then the rotation
DEVELOPMENT
Fig. 2. Cellular behaviors and apoptosis in the A8p region. (A)A time-lapse series of genitalia rotation in en-GAL4 UAS-nls-ECFP-venus/+. The
posterior compartment is visible in this fly. Cells represented by magenta rotated with A9-A10, and cells colored green rotated later. (B)Image (left)
and schematic drawing (right) of genitalia in en-GAL4 UAS-nls-ECFP-venus/+ at 24 hours APF. A8p was divided into two parts, A8pa and A8pp.
Each part is highlighted in a different color: A8pa (green) and A8pp (magenta). (C)Caspase activity was examined by the imaging of a FRET-based
probe, nls-ECFP-venus (nls-SCAT3), and is shown in pseudo-color. White circles indicate the cell that underwent apoptosis, pseudo-color gradually
changed from red to blue. (D)Result of cell tracing in the A8p region. Cells that underwent apoptosis are marked by yellow dots. Magenta dots
show cells located in A8pp, which moved with A9, and green dots represent cells in A8pa, which rotated later. Genotype was en-GAL4 UAS-nlsSCAT3/+; His2Av-mRFP/+. Three flies were examined and a typical example is shown.
1496 RESEARCH REPORT
Development 138 (8)
accelerated, with Vcontrol gradually increasing to 53.83±7.11°/hour
by 7 hours after initiation (Fig. 1I). Interestingly, in the p35expressing flies, the rotation normally started at 24 hours APF, and
the average velocity (Vp35) from the initial rotation to 1 hour later
was 7.45± 2.98°/hour, which was not significantly different from
the normal rotation. However, the acceleration of the rotation in the
p35-expressing flies was lower than normal, with Vp35 gradually
increasing to 21.35±7.45°/hour at 5.5 hours after initiation (Fig. 1I).
As shown in Fig. 1J, the first peak of the acceleration rate, which
was defined as the initiation of rotation, was observed in the p35expressing flies (ap35) and was the same as in the control flies
(acontrol). However, the duration of the acceleration period was
shorter in the p35-expressing flies (Fig. 1J). These data suggest a
relationship between apoptosis and the acceleration of genitalia
rotation.
Next, we examined the signaling mechanism(s) involved in the
acceleration of genitalia rotation. The inhibition of JNK (c-Jun Nterminal kinase) and PVF (platelet vascular factor) signaling in
male flies has been shown to result in mis-oriented adult male
terminalia, and it has been hypothesized that the PVF/PVR (PVF
receptor) may affect the genitalia rotation via JNK-mediated
apoptosis (Macias et al., 2004; Benitez et al., 2010). Consistent
with previous reports, the acceleration of genitalia rotation was
significantly impaired in flies expressing dominant-negative forms
of JNK (JNK-DN) and PVR (PVR-DN) (Fig. 1K,L). These data
implied that caspase activation and JNK signaling contribute to
driving the acceleration of genitalia rotation.
To analyze how the genitalia accelerate their rotation, we traced
the movement of A8p at the single-cell level. For this experiment,
we performed live imaging at a high resolution (20⫻ objective
lens), which enabled the cells in A8p to be tracked at single-cell
resolution. As shown in Fig. 2A, cells (magenta) that were
neighbors of A9 rotated with A9, whereas cells (green) located in
the anterior half of A8p rotated later than A9. Based on our
imaging, we divided A8p into two sheets, named A8pa (anterior of
A8p) and A8pp (posterior of A8p), as shown in Fig. 2B. We found
that a part of the cells in A8p underwent apoptosis.
To observe caspase activation in living animals, we generated a
FRET (fluorescence resonance energy transfer)-based indicator,
SCAT3 (sensor for activated caspases based on FRET) (Takemoto
et al., 2003; Takemoto et al., 2007). To precisely evaluate
apoptosis, we used a nuclear localization signal-tagged SCAT3
(nls-SCAT3; UAS-nls-ECFP-venus) (Koto et al., 2009). The nlsSCAT3 signal was clearly observed in A8p (Fig. 2C). Cells
exhibiting high caspase activity were extruded into the body cavity
and disappeared, consistent with their apoptotic death and
engulfment by circulating hemocytes. We tracked each cell in the
A8p region during the first half of the rotation and found that at
DEVELOPMENT
Fig. 3. Two distinct rotations occur in the genitalia rotation, and the outer ring rotation is impaired by caspase inhibition. (A)Time-lapse
series of genitalia rotation in UAS-nls-ECFP-venus/+; AbdB-GAL4LDN/+. Representative paths of cells are shown. Ventral side is towards the top in all
panels. Different colored dots and lines indicate the tracks of three different cells. (B)Schematic drawing of the AbdB-expression region in the male
genitalia based on the image in A. The AbdB-expressing region is highlighted in green (outer ring; A8a and A8pa). AbdB was not expressed in
A8pp (inner ring). (C)Mean of the turning angle of cells in the AbdB-expressing region (AbdB) from the initial point of rotation. Error bars indicate
s.d. (n4 flies). control in Fig. 1H is represented for reference as a gray line. (D)Time-lapse series of genitalia rotation in en-GAL4 UAS-nls-ECFPvenus/UAS-p35; His2Av-mRFP/+. Cells in the inner ring (magenta) rotated only 180° and the rotation of cells in the outer ring (green) was impaired.
(E)Means of the turning angle of cells in the outer ring (p35_ outer) and the inner ring (p35_ inner) from the initial point of rotation are shown. Error
bars indicate s.d.
Apoptosis controls speed of morphogenesis
RESEARCH REPORT 1497
least three types of cellular behavior were observed, as shown in
Fig. 2D: cells located in A8pp (magenta) moved with A9, cells
underwent apoptosis (yellow) and cells located in A8pa (green)
rotated later (Fig. 2D).
Thus, to observe the behavior of the cells in A8pa, we used
Abdominal B (AbdB) as an A8 marker. AbdB is a homeotic gene
that is required for the correct development of the genital disc
(Estrada et al., 2003; Gorfinkiel et al., 2003), and AbdB-GAL4LDN
is expressed in the segment A8 (in A8a and A8p) of the genital
disc during the 3rd instar larval stage (de Navas et al., 2006;
Benitez et al., 2010; Rousset et al., 2010). At 24 hours APF, AbdB
was expressed in A8 and formed a ring (Fig. 3A,B). We took
time-lapse images, and unexpectedly found that most of the cells
in the AbdB-expressing region underwent a 180° clockwise
movement, suggesting that AbdB was not expressed in the A8pp
region that moved 360° with A9 (Fig. 3A; see Movie 4 in the
supplementary material). To determine the speed of the AbdBexpressing cells, we traced three individual cells in each fly
(Nfly4), and calculated the value of the turning angle of the cells
(AbdB) (Fig. 3C). Our findings confirmed that the AbdBexpressing region moved halfway around. Although cells in the
AbdB-expressing region moved only 180°, the A8pp (inner ring),
which was encircled by the AbdB-expressing region (outer ring),
still moved 360°. Furthermore, our imaging data indicated that
the movement of the outer ring started 1-2 hours later than that
of the A9 region (Fig. 3C), when the acceleration of the genitalia
DEVELOPMENT
Fig. 4. Initiation of outer ring rotation correlates with apoptosis. (A)Acceleration of the rotation of the AbdB-expressing region was
quantified by measuring AbdB and VAbdB as a function of time t (green line). Histogram showing the frequency of apoptosis every 10 minutes from
0 hours, when rotation started, to 8 hours. Rapoptosis was normalized to the total number of apoptotic cells in each individual. Error bars indicate s.d.
(n3 flies). (B)Data points represent the relationship between aAbdB and Rapoptosis for 0-3 hours using linear regression (R20.951). (C)The genitalia
angle () in lacZ-expressing flies (black) and Rpr-expressing flies (gray) was measured every 30 minutes and the mean angle is shown. Error bars
indicate s.d. (lacZ, n8; Rpr, n8). Genotypes of flies were as follows: en-GAL4 UAS-nls-ECFP-venus/UAS-lacZ; tub-GAL80ts/+ and en-GAL4 UAS-nlsECFP-venus/UAS-Rpr; tub-GAL80ts/+. (D)The average velocity (Vd/dt) was quantified by measuring  as a function of time t in lacZ- (black) and
Rpr-expressing (gray) flies (mean±s.d.) (**P<0.01, *P<0.05). (E)Model of acceleration of genitalia rotation. The inner ring (magenta) rotates in
concert with A9 genitalia (light gray), then the outer ring (green) that encircles inner ring begins to move, which functions like a ‘moving walkway’
to accelerate the speed of the inner ring. The initiation of outer ring movement strongly correlated with apoptosis (yellow); moreover, this
movement was impaired by the inhibition of apoptosis. Therefore, the apoptosis increases the rotation of genitalia faster in the direction it is already
moving, enabling the full 360° rotation to occur with the correct timing.
1498 RESEARCH REPORT
possibility that local apoptosis acts as a brake release to regulate
genitalia rotation, coupled with left-right determination (Suzanne
et al., 2010). However, it has been reported that the cell shape
change by apoptosis enables not only the extrusion of dying cells,
but also the reorganization of the actin cytoskeleton in neighboring
cells (Rosenblatt et al., 2001). Therefore, apoptosis could affect the
behavior of neighboring cells, to act as a main driving force of the
cell-sheet movement. Taken together, apoptosis may generally
participate in the morphogenetic process of cell-sheet movement
during morphogenesis.
Acknowledgements
We thank E. Sanchez-Herrero, B. Hay, the Bloomington Drosophila Resource
Center and the Drosophila Genetic Resource Center (Kyoto) for fly strains; A.
Tonoki, Y. Fujioka, K. Tomioka, A. Isomura and A. Tsukioka for technical
support; all members of the M.M. laboratory for helpful discussions; Y.
Takahashi, K. Matsuno, S. Hayashi, A. Bergmann, H. Okano and A. Kakizuka
for kind support and encouragement; and M. Sato for helpful discussion and
technical support. We especially thank S. Kuroda for the generous suggestion
for the quantification analysis. We thank the University of Tokyo and Leica
Microsystems Imaging Center for imaging. This work was supported by grants
from the Japanese Ministry of Education, Science, Sports, Culture, and
Technology (to E.K. and M.M.) and by grants from the Takeda Science
Foundation (to E.K.), the Naito Foundation (to M.M.), the Cell Science
Research Foundation (to M.M.), and a RIKEN Bioarchitect Research Grant (to
M.M.).
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058958/-/DC1
References
Abbott, M. K. and Lengyel, J. A. (1991). Embryonic head involution and rotation
of male terminalia require the Drosophila locus head involution defective.
Genetics 129, 783-789.
Adachi-Yamada, T., Nakamura, M., Irie, K., Tomoyasu, Y., Sano, Y., Mori, E.,
Goto, S., Ueno, N., Nishida, Y. and Matsumoto, K. (1999). p38 mitogenactivated protein kinase can be involved in transforming growth factor beta
superfamily signal transduction in Drosophila wing morphogenesis. Mol. Cell.
Biol. 19, 2322-2329.
Adam, G., Perrimon, N. and Noselli, S. (2003). The retinoic-like juvenile
hormone controls the looping of left-right asymmetric organs in Drosophila.
Development 130, 2397-2406.
Benitez, S., Sosa, C., Tomasini, N. and Macias, A. (2010). Both JNK and
apoptosis pathways regulate growth and terminalia rotation during Drosophila
genital disc development. Int. J. Dev. Biol. 54, 643-653.
de Navas, L., Foronda, D., Suzanne, M. and Sanchez-Herrero, E. (2006). A
simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines
allows obtaining Gal4 insertions in the bithorax complex of Drosophila. Mech.
Dev. 123, 860-867.
Degterev, A., Boyce, M. and Yuan, J. (2003). A decade of caspases. Oncogene
22, 8543-8567.
Duchek, P., Somogyi, K., Jekely, G., Beccari, S. and Rorth, P. (2001). Guidance
of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107, 17-26.
Estrada, B., Casares, F. and Sanchez-Herrero, E. (2003). Development of the
genitalia in Drosophila melanogaster. Differentiation 71, 299-310.
Gleichauf, R. (1936). Anatomie und variabilitat des geschlechtapparates von
Drosophila melanogaster. Z. Wiss. Zool. 148, 1-66.
Gorfinkiel, N., Sanchez, L. and Guerrero, I. (2003). Development of the
Drosophila genital disc requires interactions between its segmental primordia.
Development 130, 295-305.
Kanuka, H., Kuranaga, E., Takemoto, K., Hiratou, T., Okano, H. and Miura,
M. (2005). Drosophila caspase transduces Shaggy/GSK-3beta kinase activity in
neural precursor development. EMBO J. 24, 3793-3806.
Keisman, E. L., Christiansen, A. E. and Baker, B. S. (2001). The sex
determination gene doublesex regulates the A/P organizer to direct sex-specific
patterns of growth in the Drosophila genital imaginal disc. Dev. Cell 1, 215225.
Koto, A., Kuranaga, E. and Miura, M. (2009). Temporal regulation of Drosophila
IAP1 determines caspase functions in sensory organ development. J. Cell Biol.
187, 219-231.
Krieser, R. J., Moore, F. E., Dresnek, D., Pellock, B. J., Patel, R., Huang, A.,
Brachmann, C. and White, K. (2007). The Drosophila homolog of the putative
DEVELOPMENT
rotation occurred (Fig. 1H-J). These observations raise the
possibility that the outer ring movement is related to the
acceleration of the genitalia rotation.
We therefore considered that the outer ring movement was
restricted in the p35-expressing flies, resulting in an incomplete
genitalia rotation of about 180°, which mimics the movement of only
the inner ring. To verify this possibility, we examined the movement
of the outer ring in the p35-expressing flies (en-GAL4+UAS-p35).
Although the inner ring rotated normally, the rotation of the outer
ring was impaired in the p35-expressing flies (Fig. 3D). We
determined the turning angles by tracing cells in the p35-expressing
flies and found that p35 _inner increased, while the increase of
p35 _outer was impaired (Fig. 3E). These data suggest that the A8
segment was composed of two independently regulated rings, and
when apoptosis was inhibited, the inner ring could move only 180°
with no outer ring movement, resulting in incomplete genitalia
rotation.
Thus, to determine whether apoptosis correlates with the outer
ring movement, we quantified the apoptosis in A8pa every 10
minutes from 0-8 hours after the start of genitalia rotation. The
frequency of apoptosis (Rapoptosis) was normalized to the total
number of apoptotic cells in each individual. Pulsatile increases in
Rapoptosis were observed, with peaks at 1, 2.5 and 4 hours after the
start of genitalia rotation (Fig. 4A). To verify the participation of
Rapoptosis in the initiation of outer ring movement, we calculated the
acceleration rate of AbdB (aAbdB) by measuring VAbdB as a function
of time t, and compared Rapoptosis with aAbdB. The starting time of
outer ring movement was characterized by the early peaks of aAbdB
(Fig. 4A). Our analysis suggested that the aAbdB was related to the
Rapoptosis, because aAbdB showed its first two peaks at about 1 and
2.5 hours after genitalia rotation started (Fig. 4A). To quantify these
observations, we calculated the correlation between Rapoptosis and
aAbdB. This analysis confirmed that there was a strong correlation
between these parameters (R20.951), because the correlation
between aAbdB and Rapoptosis is approximately linear during this time
(Fig. 4B). Therefore, these data implied a possible mechanism of
apoptosis that facilitates the outer ring movement.
To verify this possibility, we examined whether the upregulation of
apoptotic signals induces an increase in genitalia rotation speed.
Because the expression of apoptotic genes using an en-GAL4 driver,
which is expressed at the embryonic stage, is lethal, we used the
TARGET system to control gene expression temporally (McGuire et
al., 2003). Flies were allowed to develop at 18°C until the head of the
pupae had just everted, to inhibit gene expression. The pupae were
then heat-shocked at 29°C for 12 hours to induce gene expression.
Live imaging was performed at 22°C, after the heat shock. At this
temperature, the genitalia rotation in the control flies was slower than
in control flies bred at 25°C, because a low breeding temperature
affects the rate of fly development, including genitalia rotation.
Therefore, it was necessary in this experiment to compare the rotation
speeds at the same temperature. The expression of reaper (rpr), a proapoptotic gene, using the TARGET system, showed that the
upregulation of apoptotic signaling significantly increased the timing
of acceleration and speed of genitalia rotation (Fig. 4C,D). These
observations led us to propose that the outer ring functions like a
‘moving walkway’ to accelerate the speed of the inner part of the
structure, including the A9 genitalia, enabling genitalia to complete
rotation within the appropriate developmental time window (Fig. 4E).
According to our observations, we found that apoptosis drives
the movement of cell sheets during the morphogenesis of male
terminalia. Further questions remain with regard to how apoptosis
contributes to the cell sheet movement. A recent study indicated the
Development 138 (8)
phosphatidylserine receptor functions to inhibit apoptosis. Development 134,
2407-2414.
Kuranaga, E., Kanuka, H., Tonoki, A., Takemoto, K., Tomioka, T., Kobayashi,
M., Hayashi, S. and Miura, M. (2006). Drosophila IKK-related kinase
regulates nonapoptotic function of caspases via degradation of IAPs. Cell 126,
583-596.
Macias, A., Romero, N. M., Martin, F., Suarez, L., Rosa, A. L. and Morata, G.
(2004). PVF1/PVR signaling and apoptosis promotes the rotation and dorsal
closure of the Drosophila male terminalia. Int. J. Dev. Biol. 48, 1087-1094.
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and Davis, R. L. (2003).
Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 17651768.
Muro, I., Berry, D. L., Huh, J. R., Chen, C. H., Huang, H., Yoo, S. J., Guo, M.,
Baehrecke, E. H. and Hay, B. A. (2006). The Drosophila caspase Ice is
important for many apoptotic cell deaths and for spermatid individualization, a
nonapoptotic process. Development 133, 3305-3315.
Pandey, R., Heidmann, S. and Lehner, C. F. (2005). Epithelial re-organization
and dynamics of progression through mitosis in Drosophila separase complex
mutants. J. Cell Sci. 118, 733-742.
Rosenblatt, J., Raff, M. C. and Cramer, L. P. (2001). An epithelial cell destined
for apoptosis signals its neighbors to extrude it by an actin- and myosindependent mechanism. Curr. Biol. 11, 1847-1857.
RESEARCH REPORT 1499
Rousset, R., Bono-Lauriol, S., Gettings, M., Suzanne, M., Speder, P. and
Noselli, S. (2010). The Drosophila serine protease homologue Scarface regulates
JNK signalling in a negative-feedback loop during epithelial morphogenesis.
Development 137, 2177-2186.
Stenn, K. S. and Paus, R. (2001). Controls of hair follicle cycling. Physiol. Rev. 81,
449-494.
Suzanne, M., Petzoldt, A. G., Speder, P., Coutelis, J. B., Steller, H. and
Noselli, S. (2010). Coupling of apoptosis and L/R patterning controls stepwise
organ looping. Curr. Biol. 20, 1773-1778.
Takemoto, K., Nagai, T., Miyawaki, A. and Miura, M. (2003). Spatio-temporal
activation of caspase revealed by indicator that is insensitive to environmental
effects. J. Cell Biol. 160, 235-243.
Takemoto, K., Kuranaga, E., Tonoki, A., Nagai, T., Miyawaki, A. and Miura,
M. (2007). Local initiation of caspase activation in Drosophila salivary gland
programmed cell death in vivo. Proc. Natl. Acad. Sci. USA 104, 13367-13372.
Toyama, Y., Peralta, X. G., Wells, A. R., Kiehart, D. P. and Edwards, G. S.
(2008). Apoptotic force and tissue dynamics during Drosophila embryogenesis.
Science 321, 1683-1686.
Zhou, L., Schnitzler, A., Agapite, J., Schwartz, L. M., Steller, H. and Nambu,
J. R. (1997). Cooperative functions of the reaper and head involution defective
genes in the programmed cell death of Drosophila central nervous system
midline cells. Proc. Natl. Acad. Sci. USA 94, 5131-5136.
DEVELOPMENT
Apoptosis controls speed of morphogenesis