RESEARCH ARTICLE 1117
Development 137, 1117-1126 (2010) doi:10.1242/dev.037218
© 2010. Published by The Company of Biologists Ltd
A role for juvenile hormone in the prepupal development of
Drosophila melanogaster
Lynn M. Riddiford*, James W. Truman, Christen K. Mirth and Yu-chi Shen†
SUMMARY
To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which
produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at
head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe,
downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of
these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or
by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null
mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the
organization of their optic lobe neuropils. Feeding Met27 larvae on the JHM diet did not rescue the premature EcR-B1 expression
or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways
besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused
their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the
precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the
development of the visual system that is likely to result from some cells ‘misinterpreting’ the ecdysteroid peaks that drive
metamorphosis.
INTRODUCTION
Insect molting and metamorphosis are governed primarily by
ecdysone (used in the generic sense) and juvenile hormone (JH),
with ecdysone causing molting and JH preventing
metamorphosis. Juvenile hormone has a classic ‘status quo’ action
in preventing the program-switching action of ecdysone during
larval molts (Riddiford, 1994; Riddiford et al., 2001) and in
maintaining the developmental arrest of imaginal primordia
during the intermolt periods (Truman et al., 2006). Its effects at
the outset of metamorphosis, though, are more complex. Studies
mainly on Lepidoptera show that for selected tissues JH needs to
be present to allow them to undergo pupal differentiation, rather
than undertaking a precocious adult differentiation (Williams,
1961; Kiguchi and Riddiford, 1978; Champlin and Truman,
1998a; Champlin et al., 1999).
The mechanism through which JH maintains the status quo and
directs early development at metamorphosis is still poorly
understood. Whether JH has one or multiple receptors (Wheeler
and Nijhout, 2003; Riddiford, 2008), and the nature of these
receptors, is still controversial. The best candidate for a receptor
is the product of the Methoprene-tolerant (Met) gene, a PAS
domain protein that was originally isolated in Drosophila
melanogaster (Ashok et al., 1998). In vitro transcribed and
translated Met protein has been shown to bind JH with high
Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix
Drive, Ashburn, VA 20147, USA.
†
Present address: Department of Cell and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109-2200, USA
*Author for correspondence ([email protected])
Accepted 26 January 2010
affinity (Miura et al., 2005), and RNAi knock-down experiments
in Tribolium castaneum show that Met is essential for mediating
the status quo action of JH in this beetle (Konopka and Jindra,
2007; Parthasarathy et al., 2008).
In D. melanogaster, JH is thought to have no role in the onset
of metamorphosis, as exogenous JH only delays but does not
prevent pupariation (Riddiford and Ashburner, 1991; Riddiford et
al., 2003). Although it has no apparent effect on the development
of the imaginal discs, JH prevents normal adult development of
the abdominal integument when given at pupariation (for a
review, see Riddiford, 1993) (see also Zhou and Riddiford, 2002).
Internally, JH at this time affects normal reorganization of the
central nervous system and development of the thoracic
musculature (Restifo and Wilson, 1998). These effects of JH on
metamorphosis do not occur in Met mutants, unless at least 100
times the dose is given (Wilson and Fabian, 1986; Restifo and
Wilson, 1998). The Met27-null mutants proceed through larval
development and metamorphosis apparently normally (Wilson
and Ashok, 1998). However, if in addition, RNAi is used to
suppress expression of Germ-Cell Expressed (Gce), a related
bHLH protein with a high similarity to Met that heterodimerizes
with it (Godlewski et al., 2006), Met-null mutants die as pharate
adults (Liu et al., 2009). In the Met-deficient mutant, the adult eye
shows a few (<12) defective ommatidia in the posterior region
(Wilson et al., 2006). Also, the females mature fewer eggs at a
slower rate than do wild-type females (Wilson and Ashok, 1998),
indicating that Met is also important for JH effects in egg
maturation (Soller et al., 1999). While this paper was under
review, Liu et al. (Liu et al., 2009) showed that the fat body
underwent precocious cell death beginning in the third larval
instar in larvae that were allatectomized using the same protocol
as we describe below.
DEVELOPMENT
KEY WORDS: Juvenile hormone, Optic lobe, Methoprene-tolerant, Ecdysone receptor-B1
1118 RESEARCH ARTICLE
Development 137 (7)
Here, we have genetically allatectomized Drosophila larvae by
targeting expression of a cell death gene to the corpora allata (CA),
the gland that produces JH. These larvae formed smaller puparia and
showed precocious maturation of the visual system, but died around
head eversion.
MATERIALS AND METHODS
Animals
Juvenile hormone treatments
Larvae or white puparia were given the JH mimic (JHM) pyriproxifen
(Sumitomo Chemical Company, Osaka, Japan) or 7R-JH III (Toong et al.,
1988) (gift of Dr Y. Toong) topically or in the diet. For topical application,
0.2 ml cyclohexane containing a specified amount of JH III or JHM was
applied to wandering larvae under CO2 anesthesia or to white puparia. For
the feeding experiments, 5 mg JHM in 50-100 ml ethanol was mixed with
5 ml of standard diet to give a diet containing 1 part per million (ppm)
JHM.
Immunocytochemistry and imaging
Brains and other tissues were dissected and fixed in 3.9% formaldehyde
(Fisher Scientific, Fairlawn, NJ, USA) in phosphate-buffered saline
(PBS; Mediatech, Manassas, VA, USA) for 30 minutes, then rinsed and
incubated in PBS-1% Triton X-100 (TX) with 2% normal donkey serum
for 30 minutes. They were then incubated in PBS-1% TX with primary
antibody for 1-2 days at 4°C, rinsed, then incubated in secondary antibody
overnight at 4°C. Primary antibodies used were: ecdysone receptor, B1
isoform (EcR-B1; mouse ascites fluid AD 4.4; 1:10,000; from C. S.
Thummel, University of Utah, Salt Lake City, UT, USA); Fasciclin II
[mouse monoclonal antibody (MAb) 1D4; 1:50; from C. Goodman,
University of California, Berkeley, Berkeley, CA, USA]; Deadpan (rat
MAb; 1:1; from C. Q. Doe; Boone and Doe, 2008); phosphohistone H3
(PH3; 1:1000; Upstate, Millipore, Billerica, MD, USA); and chaoptin
(mouse MAb 24B10; 1:50), N-cadherin (rat MAb MNCD2; 1:50) and
neuroglian (mouse MAb BP104; 1:40; all from the Developmental
Studies Hybridoma Bank, Iowa City, IA, USA). Secondary antibodies
were fluorescein, Texas Red or Cy5 conjugated and were used at 1:500
(Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Oregon
Green 488 phalloidin (Molecular Probes, Invitrogen, Carlsbad, CA, USA)
was sometimes used as a counterstain.
Immunostained preparations were typically dehydrated through a graded
ethanol series, cleared in xylene and mounted in DPX (Fluka BioChemika,
Sigma-Aldrich, St Louis, MO, USA). Eye discs that were counterstained
with phalloidin were mounted directly into Vectashield (Vector Laboratories,
Burlingame, CA, USA) to preserve the phallodin immunofluorescence.
The preparations were imaged on a Zeiss 510 confocal microsocope
and processed with Image J. Counts of the number of dividing
cells, as indicated by PH3 labeling, were performed using V3D
software developed by H. Peng (Janelia Farm Research Campus, HHMI,
Ashlam, VA, USA; http://penglab.janelia.org/proj/v3d/v3d2.html).
Fig. 1. Genetic allatectomy and its effect on puparial size. (A) The
ring gland of control CyO, UAS-grim (above) and allatectomized (CAX)
Aug21>grim (below) wandering larvae showing the loss of the corpora
allata (CA) in the latter. a, aorta; PG, prothoracic gland. Scale bar: 25
mm. (B,C) Sample puparia (B) and the pupal weights (C) of CyO, UASgrim (control) and Aug21>grim (allatectomized) larvae raised on normal
diet containing 1% ethanol or 1 ppm pyriproxyfen (JHM) in 1%
ethanol. Bars represent the average ± s.d. for 50-60 puparia (one day
after wandering). Different letters denote average weights that are
significantly different (P≤0.0001, using one-way ANOVA and TukeyKramer HSD comparison of means).
RESULTS
Effects of genetic allatectomy on larval
development and metamorphosis
We confirmed that Aug21-GAL4 drove UAS-GFP expression in the
CA (T. Siegmund and G. Korge, personal communication) and
found that it was also expressed strongly in the salivary glands.
When Aug21-GAL4 drove the cell death gene grim (Wing et al.,
1998) in these tissues, both sets of glands were either missing or in
a state of advanced degeneration by the onset of the third instar (data
not shown), and all remnants of the CA had disappeared by the onset
of wandering (Fig. 1A). Therefore, the larvae had been
allatectomized (CAX).
The Aug21>grim (CAX) larvae developed slightly slower than
did their CyO, UAS-grim counterparts, and pupariated about 8-12
hours later. They were also about 75% of the weight of their CyO,
UAS-grim siblings (Fig. 1B,C). Survival to pupariation was similar
for both the CAX and the control (CyO, UAS-grim) larvae; of a total
of 1300 puparia scored, 51% were from CAX larvae and 49% from
CyO, UAS-grim larvae. When fed a diet containing 1 ppm JHM
during the third instar, the CAX larvae grew to the normal size (Fig.
1B,C), showing that the effects on growth were due to the lack of the
CA and not to the degeneration of the salivary glands. Both the
control CyO, UAS-grim and the CAX larvae on the JHM diet
delayed pupariation by 1-2 days.
Although the CAX larvae showed normal viability up to
pupariation, they typically died around the time of head eversion.
Video imaging showed that they initiated head eversion at the
normal time but were unable to eject the mouth hooks completely
and evert the pupal head (six out of seven animals). One of these did
complete head eversion. Such ‘escapers’ were not found at 29°C, but
occurred 20% of the time when the white puparia were placed at
DEVELOPMENT
Aug21-GAL4 [a stock obtained by T. Siegmund and G. Korge but not
reported in the screen for neurosecretory neurons (Siegmund and Korge,
2001)], UAS-grim (Wing et al., 1998), and the Met27-null mutant (Wilson
and Ashok, 1998) stocks were used. The GAL4 lines R15E10, R17A12,
R23C06, R25B08 and R27G05 were constructed as described by Pfeiffer
et al. (Pfeiffer et al., 2008). The UAS-EcR-B1 (Lee et al., 2000) and UASMetRNAi (45852 and 45854 from the Vienna Drosophila RNAi Center)
lines were used. The Aug21-GAL4/CyO, actin-GFP, the R27G05; UASmCD8::GFP and the Met27; Aug21-GAL4, UAS-GFP/CyO stock were
constructed at the Janelia Farm Fly Core facility.
The Met27 fly stocks and the GAL4>UAS-EcR-B1 and the GAL4>UASMetRNAi crosses were maintained at 25°C. The Aug21-GAL4/CyO, actinGFP⫻UAS-grim crosses were maintained at 29°C until the late third instar,
then transferred to 25°C. The Aug21>grim larvae were separated from the
CyO, UAS-grim larvae by the expression of a GFP transgene on the CyO
chromosome.
JH and prepupal optic lobe development
RESEARCH ARTICLE 1119
Table 1. Fate of allatectomized (CAX) larvae fed on a 1 ppm
pyriproxifen (JHM) diet
Genotype, stage
Number puparia
Percentage head eversion
64
13
89
100
69
91
Aug21>grim (CAX)
Second, early third
24-hour third
CyO, UAS-grim (Control)
Second, early third
18°C (n=20). When fed a 1 ppm JHM diet during the third instar,
89% (n=64) continued beyond head eversion and arrested as pharate
adults just before eclosion (Table 1). These showed truncated and
missing abdominal bristles, the typical effects of JH application at
pupariation (Postlethwait, 1974; Riddiford and Ashburner, 1991).
When 10 ng JHM was applied to early wandering larvae, 37%
(n=54) formed pharate adults, but only one pharate adult was formed
after the same treatment of white puparia (Table 2). A similar rescue
was obtained after treatment with 500 ng JH III during early
wandering (Table 2).
Effect of allatectomy on development of the
visual system
In the tobacco hornworm, Manduca sexta, lack of JH during the
prepupal molt leads to premature adult differentiation of the eye
(Kiguchi and Riddiford, 1978; Champlin and Truman, 1998a). To
determine whether JH has a similar effect in Drosophila, we looked
at the early development of the visual system in the CAX animals.
We used the appearance of the B1 isoform of the Ecdysone Receptor
(EcR-B1) as a molecular marker for developmental timing in the
retina and the optic lobes. In controls EcR-B1 is absent from the eye
disc at pupariation (Fig. 2A). It becomes evident in the crystalline
cone cells and underlying photoreceptors by 12 hours after puparium
formation (APF). Its expression then transiently fades in these cells
but returns at 18 to 22 hours APF. Although expression is prominent
in the cones, it is absent from most of the surrounding pigment cells.
In the optic lobes, the only neurons to express EcR-B1 at pupariation
are the three larval visual interneurons (Truman et al., 1994). The
first traces of EcR-B1 expression appear in neurons in the inner
proliferation zone at about 6 hours APF (data not shown). By 8
hours, EcR-B1 expression is evident in the lamina, medulla and
lobula; it is missing from the inner and outer proliferation zone stem
cells, which are Deadpan positive (Egger et al., 2007), and the
strongest EcR-B1 expression is in the neurons that are the farthest
from the proliferation zones, suggesting that neurons need to be a
certain time after their birth before they are competent to express
EcR-B1 (Fig. 3A,D). EcR-B1 expression is maintained in optic lobe
neurons through about 50 hours APF (Truman et al., 1994) (see also
Fig. 6C).
The expression of EcR-B1 was significantly advanced in the
CAX prepupae, such that it was evident in both the eye disc and the
optic lobes at the time of pupariation. In the eye disc, it was
expressed in the cone cells and photoreceptors in the posterior region
of the disc (Fig. 2B). It was absent in cells anterior to the furrow
(data not shown) and in the youngest ommatidia that were
organizing just posterior to the furrow (Fig. 2B). In the optic lobes,
EcR-B1 expression was seen in neurons of the medulla and the
‘central plug’ (Fig. 3B), but not in the neuroblasts of the inner or
outer proliferation zones (Fig. 3B). When third instar CAX larvae
were fed on a diet containing 1 ppm JHM, no EcR-B1 was
detectable throughout the prepupal period (Fig. 3C; data not shown).
Therefore, the early appearance of EcR-B1 in the photoreceptors and
optic lobe neurons in CAX larvae is due to the lack of JH during the
late third larval instar.
We assessed the cellular effects of the lack of JH by examining
the patterns of proliferation in the visual system and also the
morphogenesis of the growth cones of the photoreceptors that
project into the medulla. Proliferation was monitored by
immunostaining for PH3 to detect mitotic cells. We saw no obvious
effect of removal of JH on the proliferation pattern in the imaginal
discs (data not shown). We did, however, see a strong effect on
proliferation in the optic lobes. At pupariation both intact and CAX
animals had comparable amounts of proliferation in both the inner
and outer proliferation zones (Fig. 4A,B), although the brains of the
CAX larvae were somewhat smaller because of the overall reduction
in body size. In control animals, proliferation continued at a high
level in the outer proliferation zone through the next 8 hours (Fig.
4C), but had markedly declined by 12 hours APF (data not shown).
In the CAX prepupae, by contrast, proliferation in the outer zone had
dropped down to about 15% of control values by 8 hours APF (Fig.
4D,F). When the CAX larvae were fed on a JHM-containing diet,
the number of mitotic cells in the outer proliferation zone was
elevated to levels seen in intact prepupae (Fig. 4E,F). Proliferation
Table 2. Rescue of head eversion defect of allatectomized (CAX) larvae by topical application of JH III or its mimic pyriproxifen
CyO,UAS-grim (Control)
Hormone, dose, stage
Aug21>grim (CAX)
Number puparia
Percentage head eversion*
Number puparia
Percentage head eversion*
25
ND
ND
88
ND
ND
54
4
31
37
100
3
8
7
9
88
100
100
8
4
12
12
0
0
21
10
95
100
15
8
53
12†
10 ng pyriproxifen
Early wander
Mid-wander
Pupariation
Early wander
Mid-wander
Pupariation
500 ng JH III
Early wander
Pupariation
*These pupae went on to form pharate adults showing typical JH effects when treated with pyriproxifen. The controls when treated with either 100 or 500 ng JH III emerged
as apparently normal adults, whereas only three out of the eight pharate adults formed after 500 ng JH III treatment of CAX larvae eclosed and were apparently normal.
†
This pupa died soon after head eversion.
ND, not determined.
DEVELOPMENT
100 ng JH III
1120 RESEARCH ARTICLE
Development 137 (7)
Fig. 2. Developmental appearance of the
ecdysone receptor EcR-B1 in the
photoreceptors in CyO, UAS-grim
(control), Aug21>grim (allatectomized,
CAX), and Met27 prepupae and pupae.
(A) Confocal sections of developing
ommatidia at the levels of the crystalline
cone cells (top) and the photoreceptors
(bottom) showing the time course of
appearance of EcR-B1 (red) in wild-type
prepupae and pupae. Cellular outlines are
delineated by phalloidin staining (green).
Times shown are hours after puparium
formation (APF). Note that EcR-B1 is first
evident at 12 hours APF. Scale bar: 5 mm.
(B) Confocal sections showing the
precocious presence of EcR-B1 in the
photoreceptors of allatectomized (CAX) and
Met27 eye discs at the time of pupariation.
N-cadherin (green) stains developing
photoreceptors. Scale bar: 20 mm.
(C) Ommatidia from the eye disc of a Met27
prepupa at 2 hours APF showing the
precocious appearance of EcR-B1. In A and
C, the asterisks indicate an example of a
cone cell (top) or a photoreceptor (bottom).
outgrowth of photoreceptors R7 and R8, which extend axons into
the medulla. For a given ommatidium, the R8 growth cone is the first
to arrive, followed many hours later by the R7 growth cone, which
extends slightly beyond it (e.g. Fig. 5, Control). The growth cones
start to separate from each other at 18 hours APF and are clearly
separated by 24 hours (Ting et al., 2005). Ingrowing lamina
interneurons fill much of the space in between. In the CAX
prepupae, we already see the distinct separation between R7 and R8
soon after pupariation (Fig. 5). When CAX larvae were fed on a 1
ppm JHM-containing diet in the third instar, the premature
separation did not occur (Fig. 5).
Fig. 3. Precocious appearance of the ecdysone receptor EcR-B1 in the optic lobes (OL) of Aug21>grim allatectomized (CAX) prepupae
and its suppression by feeding larvae on a diet containing 1 ppm pyriproxyfen (JHM). (A-E) OL confocal sections at different times (X
hours; hr) APF of (A,D) CyO, UAS-grim (control) prepupae, (B) CAX prepupae after feeding on normal diet, (C) CAX prepupa after feeding on the
JHM diet, and (E) Met27 prepupae. Arrows indicate EcR-B1-positive neurons. Insets in A-C show endogenous EcR-B1 in the mushroom body
neurons. (D,E) Deadpan-positive neuroblasts (green); EcR-B1 neurons (red). cp, ‘central plug’ neurons produced by the neuroblasts of the inner
proliferation zone (ipz); la, lamina; m, medulla; opz, outer proliferation zone. Scale bar: 25 mm.
DEVELOPMENT
in the inner proliferation zone in the CAX prepupae showed only a
slight, but significant (P=0.003), reduction, which was also restored
by feeding on a JHM-containing diet (Fig. 4C-F).
The adult optic lobe depends on incoming photoreceptors from
the eye to direct its development (Meinertzhagen and Hanson, 1993;
Ting and Lee, 2007). At mid-third instar, the morphogenetic furrow
begins to move across the eye disc, behind which the photoreceptors
in each ommatidium begin to differentiate (Wolff and Ready, 1993).
This process continues through the prepupal period until head
eversion, when all the ommatidia have formed. We examined the
effects of JH on neuronal morphogenesis by focusing on the
JH and prepupal optic lobe development
RESEARCH ARTICLE 1121
hours APF. Also, in the JHM-treated animals, proliferation was
extended by about 12 hours (Fig. 6B) and EcR-B1 expression in the
optic lobe was severely suppressed (Fig. 6C).
Processes that occur prematurely in CAX animals are delayed or
suppressed in wild-type larvae that are treated with JHM. As seen in
Fig. 6A, puparia treated with JHM show only a partial separation of
R7 from R8 at 24 hours, although separation looks normal by 30
Fig. 5. Dorsal views of the medulla
showing the effects of JH manipulations
on the timing of separation of the R7
and R8 growth cones. (Left) Control
prepupae showing that growth cones
become completely separated by 18 to 24
hours (h) APF. (Middle) Allatectomized (CAX)
prepupae show complete separation by 6
hours APF. (Right) Feeding CAX larvae on a
JHM diet blocks the precocious separation.
Scale bar: 10 mm.
DEVELOPMENT
Fig. 4. Precocious decline in proliferation in the OL in
Aug21>grim allatectomized (CAX) prepupae and its prevention
by feeding larvae on diet containing 1 ppm pyriproxyfen (JHM).
(A-E) The OL at different times (hours) APF of (A,C) CyO, UAS-grim
(control) prepupae, (B,D) CAX prepupae after feeding on normal diet,
and (E) a CAX prepupa after feeding on the JHM diet. (C-E) Frontal zprojection of the optic lobe (left) and a 90° projection along the x-axis
(right), showing an end-on view to discriminate the inner (ipz) and
outer (opz) proliferation zones; (A,B) x-axis projections. Aqua represents
PH3 staining of mitotic cells; red is chaoptin staining of the incoming
photoreceptors, except in E where it was omitted. Scale bar: 50 mm.
(F) Mean number (±s.d.) of proliferating cells in the opz (black bars) and
the ipz (white bars) of 3-5 animals at 8 hours APF.
The role of the Methoprene-tolerant (Met) gene in
JH regulation of optic lobe development
The Met gene encodes a likely JH receptor. The Met27 null mutants
did not show the small size or prepupal lethality characteristic of the
CAX larvae, but they did show similar effects on the development
of the optic lobe. As in the CAX prepupae, EcR-B1 appeared
precociously in the photoreceptors (Fig. 2B,C) and in the optic lobe
(Fig. 3E, Fig. 7A) of Met27 prepupae. Also, proliferation in both the
outer and the inner proliferation zones showed an early suppression
(Fig. 7B,G), and R7 showed precocious separation from R8 (Fig.
7C).
When third instar Met27 larvae were fed a diet containing 1 ppm
JHM, the hormone mimic was unable to block most of the
precocious changes. EcR-B1 still appeared precociously in the
prepupal optic lobe (Fig. 7D) and proliferation in the outer
proliferation zone was reduced (Fig. 7E,G), just as in prepupae from
Met27 larvae fed on a normal diet (control; Fig. 7A,B). By contrast,
the proliferation in the inner proliferation zone was significantly
increased (P=0.003), although not back to the levels seen in the wildtype prepupae (Fig. 7G). Furthermore, the R7-R8 separation was not
as pronounced (Fig. 7F) as in the Met27 prepupae (Fig. 7C), but was
more extended than in control prepupae (Fig. 5). Therefore, JH
apparently is acting via the Met pathway in controlling EcR-B1
expression and proliferation in the outer proliferation zone, but only
partially so to prevent premature shutdown of proliferation in the
inner proliferation zone and growth cone separation.
In the Met27 mutant, the separation of the R7 and R8 terminals is
evident by 6 hours APF, about 12 hours earlier than normal (Ting et
al., 2005) (Fig. 7). To determine if Met was causing an advancement
of the development of the entire visual system, relative to
pupariation, we used a GAL4 driver line from the Rubin collection
of enhancer tester lines (Pfeiffer et al., 2008). R27G05-GAL4 drives
expression in lamina neurons that project into the medulla. Using a
mouse CD8 (mCD8)::GFP reporter, we counted the number of rows
of lamina axon terminals that had grown into the medulla at various
times after pupariation. Over the 6 to 9 hours of the prepupal period,
both wild-type and Met27 prepupae added approximately one row of
terminals per hour, but the Met27 individuals in the early stages were
two rows ahead of the controls (Fig. 8A,B). Hence, the loss of Met
results in a two-hour advancement in the ingrowth of lamina
1122 RESEARCH ARTICLE
Development 137 (7)
Fig. 6. The effect of 100 ng
pyriproxifen (JHM) applied at
the time of pupariation on OL
development. (A) Dorsal view of
the medulla showing that JHM
treatment retards the separation
of the R7 and R8 growth cones.
(B) Frontal projections of the
optic lobe showing the
prolongation of proliferation in
the JHM-treated animals: red,
chaoptin; aqua, phosphohistone
H3. (C) JHM treatment
suppresses the appearance of
EcR-B1. h, hours APF; MB,
mushroom body. Scale bars: 10
mm in A; 25 mm in B,C.
Cell-cell interactions and the action of JH
Possible interactions between different neuronal populations in
generating some of the Met27 mutant phenotypes were tested using
R27G05-GAL4 (Fig. 9A), which expresses in the lamina, and
R25B08-GAL4, which expresses strongly in photoreceptors R1
through R7 (Fig. 9C). We used UAS-MetRNAi constructs to reduce
Met levels in the photoreceptors or the lamina neurons. We lacked a
reliable antibody to measure the level of Met reduction in response
to the RNAi, but immunostaining for EcR-B1 at 3 hours APF
showed that driving UAS-MetRNAi by R25B08-GAL4 or by
R27G05-GAL4 resulted in the precocious appearance of EcR-B1 in
the photoreceptors or the lamina neurons, respectively (Fig. 9B,D).
Similar results were seen when UAS-MetRNAi was driven in the
photoreceptors by two other GAL4 lines (data not shown). The
levels of EcR-B1 expression in these cells was similar to that seen
in comparably aged, Met27 null mutants, and both RNAi constructs
gave similar results. Therefore, the RNAi constructs give sufficient
Met reduction to mimic at least one aspect of the Met null
phenotype.
Examination of the terminal projections of R7 and R8 at 6 hours
APF showed that UAS-MetRNAi driven in the photoreceptors caused
many of the R7 photoreceptors to separate from the R8
photoreceptors (Fig. 9I), just as in the Met27 mutant (Fig. 9F). By
contrast, when R27G05-GAL4 was used to knock down Met levels
in the lamina neurons, the photoreceptor terminals stayed in their
pre-18 hour APF positions (Fig. 9H). To see if the precocious
expression of EcR-B1 in the photoreceptors could also cause early
separation of R7 and R8, we used R25B08-GAL4 to drive UAS-EcRB1 in the photoreceptors. As seen in Fig. 9G, the two receptor
terminals remained associated despite the expression of EcR-B1 in
R7. Therefore, the loss of Met function must be producing other
effects that are needed for the precocious separation of the growth
cones.
DISCUSSION
Although a number of studies have reported the effects of applying
exogenous JH or JH mimics to Drosophila (for a review, see
Riddiford, 1993; Riddiford and Ashburner, 1991; Restifo and
Wilson, 1998), there are only two very recent studies of the effects
of manipulating endogenous JH on larval growth and
metamorphosis, both of which appeared while this paper was under
review. JH is normally present in the early larval instars, declines
substantially during the last (third) larval stage and then returns
DEVELOPMENT
neurons, relative to the time of puparium formation. However, this
difference in the timing of ingrowth is much less than the 12-hour
advancement seen for the separation of R7 from R8.
As the Met27 mutants became viable adults, we were able to see
the final outcome of the lack of JH signaling on the development of
the optic lobes. The gross anatomy of the lamina and medulla
appeared normal and we did not see a mistargeting of photoreceptor
axons to inappropriate layers (Fig. 8D). The lobula, however,
showed gross abnormalities with irregular lobes that projected
towards, or sometimes penetrated, the medulla neuropil (Fig. 8C).
These projections severely disrupted the layering of the lobula and
the lobula plate, such that neurons that normally had their dendrites
confined to a single layer, now projected in a disorganized fashion
throughout the lobula (Fig. 8D). The beginnings of these irregular
projections were evident at 12 hours APF, the same time that cellular
changes were occurring in the more superficial neuropils (Fig. 8E).
Fig. 7. The Met27 mutant shows precocious OL development and
reduced sensitivity to a JHM (1 ppm pyriproxifen) diet.
(A,D) Frontal sections through the brain and OL showing precocious
EcR-B1 immunostaining in the OL of both Met27 prepupae (A) and
those fed a JHM diet (D). (B,E) An x-projection of the OL showing that
activity in the outer proliferation zone (OPZ) is also precociously reduced
in both groups. red, chaoptin; aqua, phosphohistone H3; IPZ, inner
proliferation zone. (C,F) Dorsal view of the medulla showing that the
prominent separation of the R7 and R8 growth cones is partially
suppressed by feeding the mutant JHM. In the above series, the Met27
examples were at 6 hours APF and the Met27 fed JHM were at 8 hours
APF. (G) Mean number (±s.d.) of proliferating cells in the OPZ (black
bars) and IPZ (white bars) of 4-5 animals at 8 hours APF. The reduced
proliferation in the OPZ is not rescued in the mutants that were fed on
the JHM diet, whereas the number of dividing cells in the IPZ is partially
restored. Scale bars: in A, 25 mm for A,B,D,E; in C, 10 mm for C,F.
transiently around the time of pupariation (Bownes and Rembold,
1987; Sliter et al., 1987). The CAX larvae undergo the expected two
larval molts, but because we sometimes see remains of degenerating
CA cells at the start of the last larval stage, we cannot conclude
anything about the requirements of JH for these larval molts.
Recently, Jones et al., using 3-hydroxy-3-methylglutaryl CoA
RESEARCH ARTICLE 1123
reductase (HMGCR) RNAi to depress the level of JH and its
farnesoid precursors in early larvae, showed that the larvae mainly
die during the molt to the third instar (Jones et al., 2010), indicating
that JH may be required for that molt.
The destruction of the CA by the third instar allowed us to examine
the role of JH during the last instar and early metamorphosis. Our
finding that these larvae were smaller than their CyO, UAS-grim
siblings at pupariation could be explained by either the loss of JH or
by the loss of the salivary glands, as these glands are also destroyed.
Because dietary JH in the final instar rescued these larvae to normal
size, the lack of the CA, rather than the lack of the salivary glands, is
the cause of their reduced growth. Preliminary studies show that CAX
larvae grow more slowly in the third instar (C.K.M. and L.M.R.,
unpublished), but the underlying basis for this retardation is not yet
understood. Similarly, allatectomized third instar larvae displayed
premature apoptosis of the fat body and downregulation of several
enzymes involved in energy metabolism at the onset of wandering
(Liu et al., 2009). These fat body effects could underlie the reduced
larval growth seen in CAX larvae.
A major effect of the removal of JH was on the timing of events
during the prepupal period. Studies on the wild silkmoth
Hyalophora cecropia first showed that removal of the CA in the last
larval stage resulted in the formation of a pupa with adult
characteristics (Williams, 1961). Other moths, like Manduca sexta,
showed more subtle responses to allatectomy, with premature adult
differentiation most evident in the patterned region of the compound
eye, posterior to the morphogenetic furrow (Kiguchi and Riddiford,
1978; Champlin and Truman, 1998a). Subsequent studies on a
variety of tissues in Manduca showed that the eye, the optic lobe and
the ventral diaphragm each had a prolonged period of proliferation
that extended from the prepupal period through early adult
differentiation (Champlin and Truman, 1998a; Champlin and
Truman, 1998b; Champlin et al., 1999). This proliferation was
maintained by a-ecdysone or low levels of 20-hydroxyecdysone
(20E), but was terminated by high levels of 20E, which induced
differentiation. These tissues are exposed to differentiation-inducing
titers of 20E that occur during the larval-pupal transition early in
their growth, but studies on the ventral diaphragm showed that JH
‘protects’ them from these high 20E levels, allowing them to
continue proliferating (Champlin et al., 1999). Removal of JH
resulted in these tissues undergoing premature termination of tissue
growth and precocious adult differentiation.
The response of Drosophila larvae to the loss of JH is in line
with the effects seen in Manduca, and is also most evident in the
developing visual system. In normal individuals, the appearance
of EcR-B1 in the optic lobe (Truman et al., 1994) and the
termination of proliferation in the outer proliferation zone (T. A.
Awad, PhD thesis, University of Washington, 1995; Fig. 6B)
coincide with the ecdysteroid peak at head eversion (Handler,
1982) and become more pronounced at 18 hours APF with the rise
of ecdysteroid for adult differentiation (Handler, 1982). The
separation of the R7 and R8 growth cones also begins about this
latter time (Ting et al., 2005) (Fig. 5). The only one of these
tissues that has been directly tested in vitro for 20E sensitivity is
the optic lobe (T. A. Awad, PhD thesis, University of Washington,
1995) and in this case high levels of 20E do indeed suppress
proliferation. We assume that the other processes also respond to
the changing ecdysteroid levels. The lack of JH results in a
heterochronic advance of these events by 10 to 12 hours,
consistent with the tissues now responding to the earlier
ecdysteroid peak that causes pupariation. Although the removal
of JH advances these processes, we find that the application of JH
DEVELOPMENT
JH and prepupal optic lobe development
1124 RESEARCH ARTICLE
Development 137 (7)
mimics delays them (Fig. 6). Consequently, in selective tissues in
Drosophila, JH acts to direct the nature of tissue responses to
ecdysone.
The removal of JH or of one its receptors, Met, has a mixed
effect on the developing visual system. We saw no effect on
proliferation or inductive events in the eye disc itself, in that in
CAX animals the morphogenetic furrow continues to move and
similar rows of ommatidia have sent R8 axons into the medulla
by 6 hours APF, as compared with controls (Fig. 5). Likewise, in
Met27 individuals there is only a slight advance (about 2 hours) in
the schedule of lamina interneuron ingrowth into the medulla
(Fig. 8). However, for some of the cellular and molecular events,
like the appearance of EcR-B1 and the separation of R7 from R8,
there is a 10- to 18-hour advance in their occurrence. Hence, the
lack of JH or of its receptor Met causes a heterochronic shift
within the developing visual system with some differentiation
responses being advanced relative to the normal schedule of
neuronal birth and axon ingrowth. At least in the case of the
photoreceptors, the effect of Met removal is largely cell
autonomous, with the reduction of Met function in just those cells
being sufficient to cause the precocious appearance of EcR-B1
and the early separation of R7 from R8 (Fig. 9D). By contrast, the
reduction of Met in lamina interneurons allowed these cells to
precociously express EcR-B1 but did not affect the behavior of
the R7 and R8 growth cones. This suggests that the separation of
R7 and R8 is an active response of the photoreceptors, which is
likely to be caused by the rising ecdysteroid titer driving adult
differentiation. Although the lack of JH or Met function at the
outset of metamorphosis results in the cell-autonomous
expression of EcR-B1 in the photoreceptors, misexpression
experiments (Fig. 9F) show that the appearance of this receptor
alone is not sufficient to bring about the early separation of R7
and R8. Therefore, although the upregulation of EcR-B1 is a
prominent response to rising ecdysteroid titers, it is not the key
change responsible for the repositioning of the receptor terminals.
As it is viable, the Met mutant allowed us to see the final results
of the mistiming of development in the optic lobes. We could not
detect any permanent effect of the early separation of the R7 and R8
growth cones on the final anatomy of these projections in the
medulla, or on the structure of the later neuropil. However, the
lobula was grossly distorted and the normal layering of dendritic
arbors disrupted (Fig. 8C-E). This aberrant morphogenesis also
starts early, being already evident by 12 hours APF. The cellular
basis for the lobula distortion, however, is not yet known.
DEVELOPMENT
Fig. 8. Comparison of OL development in wild-type and Met27 mutants. (A,B) R27G05-GAL4 driving mCD8::GFP revealed lamina axon
ingrowth into the medulla in wild-type and Met27 backgrounds. (A) The mean number (±s.d.) of rows of lamina neuron terminals seen in the
medulla of wild-type (black bars) and Met27 (white) individuals at various times after pupariation; 3-5 samples per bar. (B) Confocal projections of
rows of lamina neuron terminals in the medulla at various times APF. Scale bar: 10 mm. (C,D) Frontal sections of the adult OL. (C) The lobula (lo) of
the Met27 mutant has abnormal lobes (arrows) that project towards the medulla (m); (D) expression of GFP in the R23C06 line reveals lobula
interneurons whose dendrites are normally confined to defined layers of the lobula, but in the mutant they extend throughout this neuropil: red,
chaoptin; blue, N-cadherin. Insets show a z-projections of the entire dendritic tree. Below are horizontal sections at the level of the arrow; lp, lobula
plate. (E) Sections through the developing optic lobe of Met27 mutants show that lobula irregularities are evident as early as 12 hours APF. Staining
in C and E: N-cadherin, aqua; neuroglian, red. Scale bars: 20 mm in B; 50 mm in C-E.
Fig. 9. Effects of targeted expression of genes involved in the
response to JH. (A,C) Images of wandering larval brains showing that
(A) R27G05 drives expression in lamina neurons (la), and (C) R25B08
drives expression in the photoreceptors, but not in any intrinsic neurons
in the lamina or medulla. (B,D) Confocal projection showing EcR-B1
expression in the brain hemisphere and attached eye imaginal disc (ED)
at 3 hours APF. Expression of Met RNAi under the control of R27G05
and R25B08 results in precocious appearance of EcR-B1 in lamina
neurons (la) and photoreceptors, respectively. Mushroom bodies (MB)
show strong endogenous expression. (E-I) Transverse slices through the
medulla at 6 hours APF, showing the relative position of the terminals
of photoreceptors R7 and R8. (E) Control showing the overlap of R7
and R8. F) Met27 mutant in which R7 appears to have a distinct stalk
(arrowhead) that separates it from R8. (G) Expression of EcR-B1 in the
photoreceptors does not produce an early separation of the two
terminals. (H) Knockdown of Met in lamina neurons by RNAi does not
produce a separation. (I) Knockdown of Met in the photoreceptors
results in early, stalked (arrowheads) terminals for R7. Scale bars: in D,
100 mm for A-D; in I, 10 mm for E-I.
Heterochronic shifts in the timing of development that extend
beyond the visual system are likely to be the cause of the lethality
seen in the CAX puparia. Puparia appear normal through the first 6
to 7 hours after pupariation but then abruptly undergo tissue
collapse. In normal flies, the early part of metamorphosis is
accomplished by a complicated replacement of histolyzing larval
tissues by the growing adult tissues. Diverse tissues show
individualized times of histolysis that are tied to the ecdysteroid titer.
For instance, the larval midgut cells degenerate in response to the
pupariation peak of ecdysone, whereas the larval salivary gland
degeneration is triggered by the small rise of ecdysteroid at the end
of the prepupal period (Jiang et al., 1997). We suspect that without
JH, some of the histolysis events are mistimed, leading to the rapid
death of the prepupa. Liu et al. have recently shown in CAX larvae
that the fat body undergoes precocious programmed cell death
beginning in the third larval instar (Liu et al., 2009). Interestingly,
this lethal effect was not seen in animals in which Aug21-GAL4
drove RNAi for JH acid O-methyltransferase, the enzyme that
converts JH acid to JH, in the CA (Niwa et al., 2008). Whether this
RESEARCH ARTICLE 1125
indicates that JH acid plays a role in prepupal development or
merely reflects the incomplete loss of JH in these animals is
unknown.
All these effects of allatectomy can be rescued by JH either fed
during the third instar or applied at the time of early wandering, but
not at pupariation. Both Bownes and Rembold (Bownes and
Rembold, 1987) and Sliter et al. (Sliter et al., 1987) found a decline
of JH III in the third instar; this was followed by a peak of JH during
late wandering (Sliter et al., 1987). When JH begins to rise is
unknown, as their measurements were made every 24 hours.
Presumably it is the lack of this JH during wandering when the
ecdysteroid titer is rising and peaking that leads to the optic lobe
anomalies and the premature histolysis.
The finding that the Met27 null mutant has the same defects in
optic lobe development as are found in CAX prepupae strongly
suggests that JH is acting via the Met pathway in controlling the
timing of some events in the optic lobe. Accordingly, JHM treatment
cannot suppress most of the premature development seen in
prepupae lacking Met (Fig. 7). However, a major difference between
the CAX animals and the Met27 mutants is that the CAX prepupae
died before head eversion, whereas the Met27 animals were viable.
This difference is also seen in the precocious cell death of the fat
body caused by allatectomy, which does not occur in the Met null
mutant even in the presence of gce RNAi (Liu et al., 2009). Instead
precocious cell death of the fat body was seen when Met was
overexpressed in that tissue and the death could be suppressed by
exogenous methoprene (a JH mimic). This latter finding suggests
that JH would act in this case to suppress Met-mediated cell death.
We tested this idea by seeing whether the removal of Met would
protect the prepupa from the death caused by early allatectomy.
When Met27; Aug21-GAL4>UAS-GFP/CyO females were crossed
with UAS-grim males, 44% (n=88; 18 males and 22 females)
eclosed, all showing the CyO phenotype. The remainder died at head
eversion, and should have been half CAX, Met-heterozygous
females and half CAX, Met-null males. We then separated another
group by sex prior to pupariation. Forty-nine percent of the females
(n=57) and 48% of the males (n=52) died at head eversion. All of the
adults that emerged were CyO, showing that all the CAX prepupae
died regardless of whether or not they were lacking Met function.
These results together with our findings that JHM treatment of the
Met27 mutant gave a partial rescue of the premature separation of R7
and R8, and of the decreased proliferation in the inner proliferation
zone (Fig. 7), indicate that there may be more than one receptor for
JH. Thus, we support the idea summarized by Wheeler and Nijhout
(Wheeler and Nijhout, 2003) that JH might act through multiple
pathways. A major pathway involves Met, but Gce or some other
mediator may serve as an alternate pathway in some tissues. A
similar protective role of JH at pupation mediated by Met is found
in Tribolium, as injection of Met RNAi into either fourth instar
larvae (Konopova and Jindra, 2007) or final instar larvae
(Parthasarathy et al., 2008) caused the precocious appearance of
adult eyes, adult antennae and other features in the resulting pupae.
These studies show that JH has an endogenous function in
regulating Drosophila metamorphosis, a specific example being in
orchestrating the timing of differentiation events in the developing
visual system. These effects of JH are primarily mediated through
the Met pathway. JH also is necessary for normal larval growth and
has another, as yet undefined, crucial role in prepupal development
that prevents death at head eversion. The latter effect is not mediated
through Met, indicating that JH might act through multiple
pathways.
DEVELOPMENT
JH and prepupal optic lobe development
Acknowledgements
We thank Drs G. Korge, J. R. Nambu, T. G. Wilson and A. Shingleton for gifts
of the Aug21-GAL4, UAS-grim, Met27 and UAS-MetRNAi stocks, respectively;
Dr G. M. Rubin for the R15E10, R17A12, R23C06, R25B08,and R27G05 lines;
Drs C. S. Thummel, C. Goodman and C. Q. Doe for the EcR-B1, Fasciclin II and
Deadpan antibodies, respectively; and Karen Hibbard and James McMahon of
the Janelia Farm Fly Core facility for stock construction. The study was
supported by the Howard Hughes Medical Institute. Early preliminary
experiments were supported by NSF IBN0344933 to L.M.R. Deposited in PMC
for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
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