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Keywords:

  • ecdysteroid;
  • metamorphosis;
  • nuclear receptor;
  • pupal development;
  • βFTZ-F1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The nuclear receptor βFTZ-F1 is expressed in most cells in a temporally specific manner, and its expression is induced immediately after decline in ecdysteroid levels. This factor plays important roles during embryogenesis, larval ecdysis, and early metamorphic stages. However, little is known about the expression pattern, regulation and function of this receptor during the pupal stage. We analyzed the expression pattern and regulation of ftz-f1 during the pupal period, as well as the phenotypes of RNAi knockdown or mutant animals, to elucidate its function during this stage. Western blotting revealed that βFTZ-F1 is expressed at a high level during the late pupal stage, and this expression is dependent on decreasing ecdysteroid levels. By immunohistological analysis of the late pupal stage, FTZ-F1 was detected in the nuclei of most cells, but cytoplasmic localization was observed only in the oogonia and follicle cells of the ovary. Both the ftz-f1 genetic mutant and temporally specific ftz-f1 knockdown using RNAi during the pupal stage showed defects in eclosion and in the eye, the antennal segment, the wing and the leg, including bristle color and sclerosis. These results suggest that βFTZ-F1 is expressed in most cells at the late pupal stage, under the control of ecdysteroids and plays important roles during pupal development.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Nuclear receptor type transcription factors play important roles during development and homeostasis in animals (Mangelsdorf et al. 1995). The nuclear receptor αFTZ-F1 in Drosophila has been identified as a transcriptional regulator for the segmentation gene fushi tarazu based on binding to its promoter region (Ueda et al. 1990; Lavorgna et al. 1991). It has been shown that αFTZ-F1 also binds to FTZ and is essential for FTZ function as a segmentation gene product (Guichet et al. 1997; Yu et al. 1997; Suzuki et al. 2001). Although αFTZ-F1 is expressed during early embryogenesis, βFTZ-F1, another isoform of FTZ-F1 carrying a different N-terminal region, is expressed ubiquitously in the late embryo, just before larval ecdysis during the larval period, and slightly before pupation during the prepupal period (Lavorgna et al. 1993; Broadus et al. 1999; Yamada et al. 2000). These periods are always immediately after the ecdysteroid pulses that induce hatching, larval ecdysis and puparium formation (Riddiford 1993). Indeed, it has been shown that βFTZ-F1 is induced after the decline of 20-hydroxyecdysone level (Sun et al. 1994; Woodard et al. 1994; Hiruma & Riddiford 2001). Mutant analyses have revealed that expression at each stage is required for embryogenesis, larval ecdysis and pupation (Broadus et al. 1999; Yamada et al. 2000). Organ-specific knockdown analysis by RNAi has shown that precisely timed βFTZ-F1 expression in Inka cells is necessary for ETH secretion, which induces ecdysis behavior at the end of the larval and prepupal periods (Cho et al. 2014). Furthermore, the legs in a weak hypomorphic mutant are shorter and wider relative to those of control animals and are kinked. This suggests that βFTZ-F1 expression is necessary for leg development (Broadus et al. 1999). It has also been reported that FTZ-F1 function is important for the histolysis of salivary glands (Broadus et al. 1999; Yamada et al. 2000), muscle-driven morphological events (Fortier et al. 2003), fat body remodeling (Bond et al. 2011) and the determination of cell fate in specific neurons during the early metamorphic period (Boulanger et al. 2011; Redt-Clouet et al. 2012). On the other hand, it has been shown that either isoform of FTZ-F1 can mediate juvenile hormone signal by interacting with juvenile hormone receptor candidates MET and GCE in cultured Drosophila S2 cells (Bernardo & Dubrovsky 2012). In addition, βFTZ-F1 is expressed in the prothoracic gland of the late third instar larva and regulates the gene encoding some ecdysone synthetic enzymes (Parvy et al. 2005; Talamillo et al. 2013), and is required for lipid uptake in the prothoracic gland and follicle cells of adult ovary (Talamillo et al. 2013). In contrast to the detailed analysis of βFTZ-F1 function before the early pupal stage, the expression pattern and function of FTZ-F1 during the pupal stage have not been well studied, although a large ecdysteroid pulse occurs during the early to mid-pupal stage (Handler 1982).

To understand the function of FTZ-F1 during the pupal stage, we analyzed the expression pattern of FTZ-F1 during the pupal period using Western blotting and immunohistological staining, as well as by observing the phenotypes of genetic and RNAi-induced mutants during the pupal stage. The results suggest that FTZ-F1 expression during the pupal stage is necessary for the development of many parts of the body, including the eye, antenna, leg and wing.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Fly stocks

Fly stocks were raised at 25°C or as otherwise indicated on standard food containing 0.7% agar, 10% glucose, 8% cornmeal, 4% dry yeast and the anti-fungal agents propionic acid and butyl-p-hydroxybenzoate. Oregon R was used as the wild-type line, and yw was used as a control line for RNAi induction experiments using heat shock. The GMR-GAL4, Dll-GAL4 (Calleja et al. 1996), Sd-GAL4 (sdSG29.1) (Klein & Arias 1998), tub-GAL4 and tub-GAL80ts (McGuire et al. 2003) lines were obtained from Dr Nakagoshi. hsFFi-24 (Lam & Thummel 2000) was obtained from Dr Tsujimura.

Plasmid construction and establishment of fly lines for ftz-f1 RNA interference

A DNA fragment spanning the end of the first exon to the beginning of the second exon of the βFTZ-F1 gene was obtained by polymerase chain reaction (PCR) from βFTZ-F1 cDNA using the primers 5′-GCTCTAGATTCCCGACAGGCTACCAG, which contains a KpnI site and 5′-GGGGTACCGGACTCACCCTCTAGCTTC, which contains an XbaI site, and was digested with KpnI and XbaI. The obtained fragment was ligated into the KpnI and XbaI sites of the pUAST vector. The resulting plasmid was digested with BglII and KpnI and ligated with a BglII and KpnI-digested DNA fragment spanning the end region of the first intron and the beginning of the second exon, which was obtained by PCR from genomic DNA using the primers 5′-GAAGATCTTGAAACGAATAGGATGCTAAC, which contains a BglII site, and 5′-CGGGTACCGCATCACTTGCAACTTC, which contains a KpnI site. The obtained plasmid was digested with EcoRI and BglII and ligated to an EcoRI and BglII-digested DNA fragment spanning the end region of the first exon and the beginning of the first intron, which was obtained by PCR on genomic DNA using the primers 5′-GAAGATCTTGAAACGAATAGGATGCTAAC, which contains a BglII site, and 5′-GGAATTCCCGACAGGCTACCAG, which contains an EcoRI site. The acquired plasmid was injected into fly embryos with the delta2-3 plasmid, and fly lines carrying the gene expressing ftz-f1 dsRNA were established using a P-element mediated gene transfer method as described previously (Yamada et al. 2000). The inducible dsRNA region is different from hsFFi-24 (Lam & Thummel 2000).

Western blotting analysis

A staged pupa was frozen in liquid nitrogen and homogenized in a 1.5 mL microtube containing 50 μL of 1× Laemmli sample buffer. The samples were heated at 95°C for 5 min and then electrophoretically separated on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were transferred to a PROTRAN nitrocellulose membrane (Whatman, Germany). The membranes were blocked with TBST (10 mmol/L Tris–HCl pH 8.0, 150 mmol/L NaCl and 0.05% Tween-20) containing 5% skim milk for 2 h at room temperature or overnight at 4°C, followed by incubation with a polyclonal antibody against FTZ-F1 (1:5000 dilution) (Yamada et al. 2000) for 1 h at room temperature. After being washed with TBST three times, the membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (1:5000 dilution) for 1 h at room temperature. After the secondary antibody incubation, the membranes were washed and processed for the chemiluminescent reaction using Immobilon (Millipore, USA). The signals were detected with a LAS-4000 mini luminescent image analyzer (Fuji film, Japan).

Immunohistological analysis

Pupae were fixed in 4% paraformaldehyde in PEM (100 mmol/L Pipes pH 7.0, 2 mmol/L EGTA pH 8, and 1 mmol/L MgSO4) for 12 h at 4°C after the pupal case had been removed in PBS. Paraformaldehyde was replaced with PEM, and the pupae were incubated for 10 min. The fixed pupae were dehydrated through a series of graded ethanol washes (70%, 80%, 90%, and 100%) 1 h for each. After dehydration, the pupae were cleared in xylene and embedded in Paraplust (Leica Biosystems, USA). The embedded pupae were oriented and sectioned at a thickness of 7 μm. The sections were deparaffinized by washing twice in xylene for 5 min and twice in 100% ethanol for 5 min; then, they were subsequently rehydrated in 90%, 80%, and 70% ethanol and distilled water for 5 min each. The slides were placed in pre-heated Tris–ethylenediaminetetraacetic acid (EDTA) pH 9.0 buffer (10 mmol/L Tris–HCl, 1 mmol/L EDTA and 0.05% Tween-20) at 100°C for 20 min, were allowed to cool for 15 min at room temperature, and were washed with PEM for 10 min. The slides were washed with PEM containing 1% NP-40 for 30 min and washed with TBS-TB (TBST containing 0.2% bovine serum albumin [BSA]) for 10 min. Then, the slides were blocked with 5% BSA in TBS-TB for 2 h at room temperature and incubated with primary antibody (anti-FTZ-F1 1:500 dilution) (Yamada et al. 2000) in TBS-TB for 2 h at room temperature. The slides were washed with TBS-TB three times for 10 min each and incubated with secondary antibody (anti-rabbit-Cy3, 1:250 dilution) in TBS-TB for 2 h at room temperature. The slides were washed with TBS-TB three times for 10 min, then rinsed several times with PBS before mounting with VECTASHIELD mounting medium with DAPI (Vector Laboratories, USA).

Injection of 20-hydroxyecdysone into pupae

To inject 20-hydroxyecdysone, a small hole was made in the center portion of the pupal case using forceps, and through the hole, approximately 40 nL of 0.1, 0.5 or 2 μg/mL 20-hydroxyecdysone in Ringer's solution were injected using a glass needle connected to an electric microinjector IM-31 (Narishige, Japan).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

βFTZ-F1 is expressed in a dynamic pattern during pupal development

To determine how FTZ-F1 contributes to pupal development, we first examined the temporal expression profile of FTZ-F1 during late prepupal and pupal development by Western blotting analysis using a FTZ-F1 antibody. As shown in Figure 1A, a band corresponding to the expected size of βFTZ-F1 was detected at the prepupal stage, indicating that βFTZ-F1 is also expressed in pupa. The expression level of βFTZ-F1 declined to a very low level soon after pupation and then increased 60 and 66 h after puparium formation (APF) in female and male animals, respectively, reaching a high level. The high level expression continued until 96 and 102 h APF in female and male animals, respectively, and declined again before eclosion, which occurs approximately 4.5 h earlier in females than in males (Fig. 1B), as previously reported (Handler 1982). Moreover, the FTZ-F1 expression levels in females were higher than in males, especially at 66 and 72 h APF (Fig. 1A).

image

Figure 1. βFTZ-F1 is expressed throughout pupal development, with especially high levels during the late pupal stage. (A) The expression profile of FTZ-F1 in two individual female or male animals at the indicated times from the prepupa to adult were detected by Western blotting. Samples were collected every 6 h except after 96 h after puparium formation (APF) to detect rapid changes near the time of eclosion. The positions of βFTZ-F1 are indicated by the arrows. (B) Eclosion timing of females and males was compared. Av, average of eclosion timing; n, number of animals examined. Female (Av = 95.7 h APF, n = 63); Male (Av = 100.2 h APF, n = 37).

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βFTZ-F1 is induced after the ecdysteroid titer declines to a low level

It has also been reported that the ecdysteroid titer in pupa starts to increase around 20 h APF and declines to a low level until 60 h APF in females and 66 h APF in males (Handler 1982). The ecdysteroid titer decreases to a low level at the same time that the βFTZ-F1 expression level starts to increase in both females and males, suggesting that βFTZ-F1 is induced after the decline of ecdysteroids, as observed for other developmental stages (Lavorgna et al. 1993; Woodard et al. 1994; Yamada et al. 2000). To examine this, we injected 20-hydroxyecdysone at different doses into female pupae 60 h APF and measured the βFTZ-F1 expression level 66 h APF by Western blotting. As shown in Figure 2A, the expression level of βFTZ-F1 66 h APF increased approximately fourfold compared to that at 60 h APF. Similar increases in the βFTZ-F1 levels were observed by injection of Ringer's solution or 20-hydroxyecdysone in low doses. However, the increase was inhibited when 20-hydroxyecdysone was injected in high dose. To examine whether the inhibition of the βFTZ-F1 induction is dependent on the developmental stage, 20-hydroxyecdysone was injected at different time points of the pupal stage and the expression level of βFTZ-F1 was observed by Western blotting. As shown in Figure 2B,C, 20-hydroxyecdysone dependent inhibition of the βFTZ-F1 induction was observed, when 20-hydroxyecdysone was injected at 54 or 72 h APF in addition to the injection at 60 h APF. These results indicate that 20-hydroxyecdysone inhibits the induction of βFTZ-F1, as in other developmental stages (Sun et al. 1994; Woodard et al. 1994; Hiruma & Riddiford 2001), and thus βFTZ-F1 may also be induced after the decline of ecdysteroid levels during the pupal stage.

image

Figure 2. FTZ-F1 is expressed after a decrease in ecdysteroid levels. (A) A dose dependent effect of 20-hydroxyecdysone injections on the FTZ-F1 expression. Different concentrations of 20-hydroxyecdysone (20E) in Ringer's solution (RS) were injected into pupae 60 h after puparium formation (APF), and the expression level of FTZ-F1 was examined at 66 h APF by Western blotting. The average level obtained from non-injected pupae at 60 h APF was set as 1. (B, C) A stage-dependent effect of 20-hydroxyecdysone injections on the FTZ-F1 expression. A high dose of 20-hydroxyecdysone (20E) (2 μg/mL) or Ringer's solution (RS) was injected into pupae 54 (B) or 72 h (C) APF, and the expression level of FTZ-F1 was examined 6 h later by Western blotting. The average level obtained from 60 h APF in (A) was set as 1. The error bars represent the standard error of the mean (SEM) of three independent animals. *< 0.05 by Student's t-test.

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FTZ-F1 is expressed in a dynamic pattern during pupal development

Next, the expression pattern of FTZ-F1 in the different organs of the pupa was analyzed by immunohistological analysis at the late pupal stage. As shown in Figure 3, an obvious FTZ-F1 signal was detected in the nuclei of many cells including salivary glands; thoracic ganglions; fat bodies; epithelial cells in the leg, wing, antenna and trachea; and the epidermis of body trunk. An especially strong signal was observed in the nuclei of the photoreceptor cells in the ommatidium and optic lobe in the eyes. In the ovary, however, the signal was detected mainly in the cytoplasm in both oogonia and follicle cells. We could not detect a signal in the testes (data not shown).

image

Figure 3. Organ-specific expression pattern of FTZ-F1 at the pupal stage 84 h after puparium formation (APF). FTZ-F1 was detected by immunohistological staining in sections of whole animals. The right and left pictures in each panel show FTZ-F1 (red) and DAPI (blue) staining, respectively. The merged picture is also shown under the two pictures in (I′). (A) salivary glands (sg) and thoracic ganglion (tg); (B) fat body (fb); (C) leg (l); (D) wing (w); (E) antenna (a) and trachea (t); (F) epidermis (e); (G) ommatidium (o) and optic lobe (ol); (H) photoreceptor cells (pc); (I) ovary; (I') high magnification view of the boxed region in (I), oogonia cells (oc) and follicle cells (fc). Scale bars: 100 μm (A, B, C, E, F, G), 50 μm (D, H, I).

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Functional analysis of FTZ-F1 during the pupal stage

To elucidate the function of ftz-f1 during the pupal stage, we examined the effect of ftz-f1 knockdown in the major ftz-f1 expressing organs using the GAL4/UAS system. Because it has been shown that ftz-f1 is essential for development before the pupal stage (Broadus et al. 1999; Yamada et al. 2000; Gangishetti et al. 2012), the tub-GAL80ts gene was introduced (McGuire et al. 2003), and the culture temperature was shifted from 18 to 29°C immediately after pupation. Although whole body knockdown using the tub-GAL4 driver induced eclosion failure, animals with knockdown driven by tissue-specific GAL4 eclosed, and the phenotypes of the adults were observed. We found that ftz-f1 knockdown in the eye using the GMR-GAL4 driver during the pupal stage showed a rough phenotype, and the effect in females was more striking than in males (Fig. 4A–D). In animals with ftz-f1 knockdown in the wing using the Sd–GAL4 driver (Fig. 4E–H), the adult wings were not completely expanded and curved inward. The effect in females was more drastic than in males. The ftz-f1 knockdown animals using the Dll–GAL4 driver (Fig. 4I–L) exhibited swelled arista with a reduction in the length and branch number in both females and males. The color of the spines in the third antennal segment was lighter in both sexes than in the controls. In the legs of adults with knockdown driven by Dll–GAL4, the distal portion of all of the legs was missing. Thus, the phenotype of the pharate adults was observed (Fig. 4M–P). In females, disruption of the distal portion of all of the legs, including the end of the tibia through the tarsus, was observed, although the proximal region including the coxa, trochanter, femur and the most parts of the tibia were formed normally. In the disrupted regions, no segments were observed, the color change to brown had not occurred, bristles were not formed, and the structure itself was fragile. In males, the development of bristles was inhibited in the distal portion of the legs as in females, but the overall structure was still established as in control animals. The color change was also disrupted, but less than in females.

image

Figure 4. Phenotypes of ftz-f1 knockdown using the GAL4/UAS system in the eye, wing, antenna and leg during the pupal stage. Animals were reared at 18°C until pupation and then shifted to 29°C. The phenotypes in the eye, wing and antenna were observed after eclosion, and the phenotypes in the leg were observed at the pharate adult stage due to loss of the distal parts during eclosion. Photographs of pharate adults were taken after removal of the pupal cases. White arrowheads indicate the end part of the tibia in the first leg and arrows indicate the tarsus in the third legs. Scale bars: 100 μm.

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To support the idea that these observed phenotypes are caused by specific knockdown of ftz-f1, we also observed the phenotype of the ftz-f1 mutant transheterozygote between the null mutant ftz-f1ex7 and the hypomorphic mutant ftz-f103649. Because the mutant animals are prepupal lethal, the animals were rescued by induction of βFTZ-F1 during the mid- to late prepupal period by introducing the hsβFTZ-F1 gene as described previously (Yamada et al. 2000). The rescued animals pupated at a high efficiency, and many became arrested in development at different stages during the pupal stage and failed to eclose, except for a few male escapers. When we observed well-developed pupa, some of them showed the rough eye phenotype (Fig. 5A,B) similar to what was observed for RNAi induction using the GAL4/UAS system in the eye. In the animals that eclosed successfully, the wings were not expanded completely in the proximal region and a severe expansion defect was observed in the distal portion (Fig. 5C,D). Moreover, the length and branch number of arista in most mutant animals were reduced, and the color of the spines in the third antennal segment (Fig. 5E,F) and the bristles on the leg (Fig. 5G,H) were not completely dark brown. The mutant phenotypes of the females were stronger than that of the males as observed using the GAL4/UAS system (data not shown). As another way to confirm the observed phenotype, ftz-f1 was knocked down during the late pupal stage by induced dsRNA under control of the heat shock promoter from the hsFFi-24 transgene (Lam & Thummel 2000) by administering a 1-h heat shock three times every 12 h beginning at 59 APF. Animals failed to eclose, and thus, we observed their phenotype at the time the control animal eclosed. The arista was short, and its color was again reduced (Fig. 5I,J). Darkening of the body color was inhibited, and the entire body including the legs was fragile (Fig. 5K, L). The observed phenotypes by heat-shock induced RNAi were stronger than that by the other methods, and difference between males and females was not obvious. The similar phenotypes observed in the ftz-f1 mutant and the animals treated with RNAi induced by heat shock support the idea that ftz-f1 plays important roles in pupal development.

image

Figure 5. ftz-f1 knockdown phenotypes induced by the GAL4/UAS system were reproduced in ftz-f1 mutant animals and in pupa after heat shock induction of ftz-f1 knockdown. Prepupae of the genotype ftz-f1ex7 hsβFTZ-F1/ftz-f103649 (Yamada et al. 2000) were heat shocked for 1 h at 7 h after puparium formation (APF) at 34°C, and the phenotypes were observed at the late pupal (A, B and E–H) or adult stage (C and D). hsFTZ-F1i pupae were heat shocked for 1 h at 59, 72 and 85 h APF at 37°C, and the phenotypes were observed at the late pupal stage (I–L). Photographs of pharate adults were taken after removal of the pupal cases. Scale bars: 100 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We showed that βFTZ-F1 is expressed in the late pupal stage in almost all organs immediately after an ecdysteorid pulse (Figs 1, 3). It has been reported that βFTZ-F1 is expressed in almost all organs after ecdysteroid pulse during embryogenesis, larval stages and the prepupal stage (Yamada et al. 2000). These observations represent a common pattern for βFTZ-F1 expression. However, βFTZ-F1 expression during the pupal stage is prolonged for 40 h, whereas the gene is expressed only several hours before pupation (Yamada et al. 2000). This suggests that the mechanism controlling gene expression during the pupal stage is not identical to that during other stages of development. Recently, a new ftz-f1 transcript, ftz-f1-C, which also encodes βFTZ-F1 and has a transcriptional start site 1.3 kb upstream of the previously identified start site of the transcript encoding βFTZ-F1 (Kageyama et al. 1997), was annotated in FlyBase, and the detection of the ftz-f1-C transcript during the pupal stage has been reported (Vorobyeva et al. 2012). We expect that the differences between the promoters might explain the different regulation mechanism.

We observed cytoplasmic localization of FTZ-F1 in oogonia and follicle cells (Fig. 3). We did not observe any morphological defect in the ovary, even after RNAi was induced in the ovary using the GAL4/UAS system. On the other hand, females subjected to RNAi induction were sterile (A. S. Sultan, unpubl. data, 2013). However, we could not determine the importance of FTZ-F1 in the ovary during pupal development because RNAi induced in the ovary during the pupal period might affect on FTZ-F1 expression in the adult stage that is necessary for early embryogenesis in their progenies (Guichet et al. 1997; Yu et al. 1997).

We showed that one of the phenotypes of βFTZ-F1 loss of function is inhibition of the hardening of the cuticle. There are several results to indicate a relationship between FTZ-F1 and cuticle formation in Drosophila and other insects. In Drosophila, Gangishetti et al. (2012) reported that several genes responsible for cuticle formation are regulated by βFTZ-F1 during embryogenesis. We reported that two pupal cuticle genes, EDG78E (Kawasaki et al. 2002) and EDG84A (Murata et al. 1996), are induced by βFTZ-F1 during the prepupal period and that premature expression of βFTZ-F1 at the larval stage induces abnormal cuticle structures. Moreover, Lestradet et al. (2009) reported that ACP65A, an adult cuticle gene, is expressed beginning approximately at 60 h APF. This timing is consistent with when βFTZ-F1 is expressed at a high level (Fig. 1). In other insects, it has been reported that the cuticle gene is one of the targets of the ftz-f1 gene, such as in the last nymphal stage of Blattella germanica (Cruz et al. 2008) and in the last larval stage of the silkworm Bombyx mori (Nita et al. 2009). It has been also reported that some chitin synthase genes are expressed when FTZ-F1 is expressed during the prepupal period in Drosophila (Gagou et al. 2002) and the pupal stage in Spodoptera litura (Gu et al. 2013). These results support the idea that one of the targets of βFTZ-F1 during the pupal stage in Drosophila is related to the formation of the adult cuticle. We also observed defects in cuticle color change (Figs 4, 5). It has been suggested that expression of dopa decarboxylase, one of the important enzymes for cuticular melanization (Davis et al. 2007), is regulated by FTZ-F1 during the larval molt in Manduca sexta (Hiruma & Riddiford 2009). Our results support the idea that βFTZ-F1 controls the gene for cuticular melanization. Identification of the βFTZ-F1 target genes during the pupal stage is a future experiment that will further elucidate the function of βFTZ-F1 during the pupal stage.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank all members of our laboratory for helping us and giving useful comments during our research, especially Drs K. Akagi and M. Sarhan. We are grateful to Drs Nakagoshi and Tsujimura and Drosophila Genetic Resource Center at Kyoto Institute of Technology for providing fly lines. This work was partially supported by Grants in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. Abdel-Rahman Sultan is supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho Scholarship).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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