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Keywords:

  • Bombyx mori;
  • 20-hydroxyecdysone;
  • programmed cell death;
  • anterior silk gland;
  • early and early late genes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

Programmed cell death (PCD) in Bombyx mori anterior silk glands (ASGs) is triggered by 20-hydroxyecdysone (20E). We examined the expression profiles and effects of 20E on 11 transcription factor genes in the fifth instar to determine whether they demonstrate the hierarchical control seen in Drosophila PCD. Results indicate that EcR-A and usp-2, but not EcR-B1 or usp-1, may be components of the ecdysone receptor complex. Up-regulation of E75A, BHR3, and three BR-C isoforms, but not E75B, appeared to be associated with the induction of PCD. βFTZ-F1 was not expressed during PCD execution. Thus, gene control in B. mori ASGs differs from that in Drosophila salivary glands, despite both tissues undergoing PCD in response to 20E at pupal metamorphosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

Many animal tissues undergo programmed cell death (PCD) after completing their roles, particularly during developmental stages. In insect growth and metamorphosis, PCD is executed by the steroid hormone 20-hydroxyecdysone (20E), which triggers various developmental and physiological processes. The insect prothoracic gland undergoes PCD at the pupal stage after accomplishing the secretion of ecdysone required for adult differentiation (Dai & Gilbert, 1997). A large increase in haemolymph ecdysteroid levels during the prepupal period initiates PCD in tissues that are not needed for adult differentiation, such as the salivary glands of Drosophila melanogaster (von Gaudecker & Schmale, 1974), intersegmental muscles of the giant silkmoth (Lockshin & Williams, 1965), silk glands of Bombyx mori (Chinzei, 1975) and motoneurones of Manduca sexta (Truman & Schwartz, 1982). In these tissues, PCD is accomplished shortly after pupation and can be triggered by 20E in vitro (Chinzei, 1975; Streichert et al., 1997; Terashima et al., 2000).

20E acts via binding the heterodimeric nuclear hormone receptor, ecdysone receptor (EcR), and its partner molecule, ultraspiracle (USP) (Yao et al., 1992, 1993; Thomas et al., 1993). Its hierarchical effects are achieved through the activation and/or inhibition of early and early late genes, which in turn control various effector genes (Ashburner et al., 1974; Ashburner, 1990). In D. melanogaster, early genes include the zinc finger transcription factor Broad-complex (BR-C), the ETS family transcription factor E74 and members of the nuclear receptor superfamily, EcR and E75. Early late genes include Drosophila hormone receptor 3 (DHR3), DHR39 and E78, which are members of the nuclear receptor superfamily. The orphan nuclear receptor gene βFTZ-F1, expressed during the mid-prepupal phase of development, is also regulated by 20E. Although these genes are sequentially activated in 20E-induced PCD, such sequential activation has so far only been elucidated in Drosophila salivary glands.

The B. mori silk gland is a larval-specific tissue that degenerates once the insect has completed the spinning of its cocoon. The silk gland consists of three parts; the anterior, middle and posterior (Akai, 1983). Anterior silk glands (ASGs) begin to exhibit cellular morphology characteristic of PCD 2 days after gut purge (G2) and complete their PCD after pupation (Terashima et al., 2000). In an in vitro study, incubation of ASGs at the day of gut purge (G0) with 1 µm 20E was shown to induce premature PCD (Terashima et al., 2000). Transcriptional events leading to full PCD were completed within 8 h of the 20E challenge, suggesting that early and early late genes might be involved in PCD execution by 20E.

Little is known about the sequential gene activation triggered by 20E in Bombyx ASGs, except that BR-C plays a key role in the execution of PCD (Uhlirova et al., 2003). As the gene expression of E75A, E75B, BR-C and βFTZ-F1 is under the control of 20E in Drosophila salivary glands destined for cell death (Jiang et al., 1997), we examined the developmental profiles and in vitro responses of these genes to 20E in Bombyx ASGs. We also included BHR3 and the alternative isoforms EcR-A, EcR-B1, usp-1, usp-2, BR-C Z1, Z2 and Z4 in our investigation. The alternative isoforms of EcR were of particular interest, as their functional roles depend on the fate of the tissue in which they are expressed. Thus, Drosophila salivary glands about to undergo cell death predominantly express the EcR-B1 isoform, which is involved in the execution of PCD in response to 20E (Talbot et al., 1993; Bender et al., 1997).

Our results indicate that the temporal expression profiles of these genes in ASGs are not the same as in Drosophila salivary glands. This suggests that despite both tissues undergoing PCD at pupal metamorphosis, the hierarchical gene control for PCD is likely to be very different between the two species.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

Developmental profiles of transcription factor genes in ASGs during the fifth instar

We determined the changes in expression levels of 11 transcription factor genes: EcR-A, EcR-B1, usp-1, usp-2, E75A, E75B, BHR3, BR-C Z1, BR-C Z2, BR-C Z4 and βFTZ-F1 from day 3 (V3) of the fifth instar to day 2 of gut purge (G2) (Fig. 1). During this period, the ASGs of B. mori lose sensitivity to juvenile hormone (JH), become responsive to 20E (Kakei et al., 2005), and initiate PCD in response to the large increase in the haemolymph ecdysteroid titre (Terashima et al., 2000).

image

Figure 1. Developmental profiles of transcription factor gene expression in anterior silk glands. Total RNA was isolated from 10 ASGs of staged B. mori larvae and individual gene mRNA levels were analysed by real-time Q-PCR using the primers listed in Table 1. The results are expressed as copy number of mRNA in 20 ng total RNA. Each datum point is a mean ± SD (n = 3). V3 and G0 indicate day 3 of feeding fifth instar larvae and day of gut purge, respectively. G2 corresponds to 60 h after gut purge (see Fig. 2). The red line in each panel represents the haemolymph ecdysteroid titre (Sakurai et al., 1998; Terashima et al., 2000). The expression levels of EcR-A and usp-2 are represented twice within their panels, with two different scales in order to show the detailed expression levels (open circles) by an extension of the scale on the left. The ordinate for open circles is shown on the right-hand x-axis. Measurements were performed on three different samples of total RNA, and Q-PCR was performed in duplicate for individual total RNAs.

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Both EcR isoforms were expressed during the fifth instar at fluctuating levels. EcR-A expression gradually increased from V4 and peaked slightly on V6 as haemolymph ecdysteroid titres exhibited a small peak that induced gut purge. EcR-A expression greatly increased on G2 as the ecdysteroid titre attained its highest level. By contrast, EcR-B1 expression level peaked on V6 and gradually decreased, reaching its lowest level on G2. Interestingly, the EcR-A expression profile mirrored the changes in ecdysteroid titres while EcR-B1 was reciprocal to the changes in titre. From V6 to G2, EcR-A was expressed at a 10-fold higher level than EcR-B1. The developmental profiles of usp-1 and usp-2 were similar to those of EcR-B1 and EcR-A, respectively, except for an observed up-regulation of usp-1 on G2.

The expression profiles of the remaining transcription factors can be divided into three groups. The first includes E75A, BR-C Z1, BR-C Z2 and BR-C Z4, which exhibited increased mRNA levels from V6. Their expression levels greatly increased after gut purge in a step-wise manner. The second group of transcription factors (E75B and BHR3) demonstrated low expression levels until G1 and then abruptly increased on G2. Low expression levels of βFTZ-F1 were maintained throughout the fifth instar, followed by a transient peak on V5 and a decline in expression thereafter.

Developmental profiles of gene expression after gut purge

Larvae were observed every hour in V6 scotophase for the behaviour of gut purge and ASGs were dissected out at 6 h intervals following gut purge until a final time point of 78 h. This corresponds approximately to the beginning of G3 photophase, a day before pupation.

EcR-A expression increased at 48 h, peaked at 66 h and abruptly decreased to a very low level at 78 h (Fig. 2). By contrast, EcR-B1 expression rapidly increased at 72 h and high levels were maintained until 78 h. EcR-A mRNA levels were 80-fold higher than EcR-B1 at 6 h and 1700-fold higher at 66 h. usp-2 expression increased at 42 h, remained at a plateau until 60 h, increased again at 66–72 h, then decreased to a very low level at 78 h. usp-1 mRNA levels remained low until 60 h, then increased to reach a sharp peak at 72 h. During the period of analysis, usp-1 mRNA levels were always lower than usp-2.

image

Figure 2. Precise profiles of transcription factor gene expression after gut purge. Larvae were observed for gut purge every hour during the scotophase of V6, and gut purged larvae were segregated at the time point designated 0 h after gut purge (AGP). A total of 12–18 ASGs were isolated every 6 h until 78 h AGP for the extraction of total RNA. Q-PCR was performed as in Fig. 1. The red line in each panel represents the haemolymph ecdysteroid titre (Sakurai et al., 1998; Terashima et al., 2000). The expression levels of BR-C isoforms and usp-1 are represented twice, as before, in order to show the detailed expression levels (open circles) before 60 h. The ordinate for open circles is shown on the right-hand x-axis. Each datum point is a mean ± SD (n = 3).

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E75A, E75B and BHR3 expression profiles exhibited two distinct peaks around 48–54 h and at 72 h, while the expression profiles of BR-C Z1, Z2 and Z4 exhibited a single, sharp peak at 72 h. βFTZ-F1 expression remained at a very low level until the peak at 72 h.

In vitro responses of transcription factor genes to 20E

G0 ASGs were incubated with or without 1 µm 20E for various periods (Fig. 3). In the presence of 20E, EcR-A mRNA levels increased gradually until 8 h and then decreased to the starting level by 24 h, while EcR-B1 expression rapidly increased from 1 to 4 h, then gradually decreased to a low level. In the absence of 20E, EcR-A mRNA levels decreased throughout the 24 h period to less than half the starting level. EcR-B1 expression in the absence of 20E remained at a very low level.

image

Figure 3. In vitro effects of 20E on transcription factor gene expression in ASGs. G0 ASGs were incubated in the presence (closed circles) or absence (open circles) of 1 µm 20E for 24 h. CHX (50 µg/ml) was added at the start of incubation with 20E and ASGs were harvested at 8, 16 and 24 h (open triangles). Eight pieces of ASGs were incubated individually in Grace's insect culture medium to obtain one total RNA sample. Q-PCR measurements were performed as in Fig. 1. Data are expressed as copy number of mRNA in 20 ng total RNA. Each datum point is a mean ± SD (n = 3).

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The translation inhibitor, cycloheximide (CHX) did not significantly up-regulate EcR-A expression, but increased EcR-B1 expression levels by fivefold following an 8 h incubation. By 16 h, EcR-B1 expression was still significantly higher than the control level.

usp-1 and usp-2 exhibited reciprocal responses to the presence or absence of 20E. Control usp-2 levels remained low throughout the culture time, but were up-regulated threefold within 1 h of incubation with 20E. By contrast, control usp-1 levels increased after 6 h of culture, but levels remained low in the presence of 20E. The addition of CHX to the culture medium up-regulated usp-1 levels after 8 h of incubation, but had no effect on the 20E-stimulated level of usp-2.

In the absence of 20E, E75A mRNA was almost undetectable by Q-PCR. Its expression was greatly induced after 1 h of incubation with 20E, peaking at 2 h. The addition of CHX maintained E75A expression at levels corresponding to or higher than the peak induced by 20E. E75B mRNA was not detected in the absence of 20E, but increased gradually throughout the culture period in its presence. CHX increased E75B mRNA levels sixfold.

BHR3 mRNA levels were undetectable in the absence of 20E, but were up-regulated after 6 h of incubation with 20E, peaking at 12 h. CHX did not exhibit significant effects on 20E-induced BHR3 expression.

The expression profiles of the BR-C Z1, Z2 and Z4 isoforms and BHR3 were very similar to each other. Expression levels were barely detectable in the absence of 20E, were increased in its presence, and were unaffected by CHX.

βFTZ-F1 expression profiles were identical during the first 6 h of incubation irrespective of the presence or absence of 20E. At 6 h, βFTZ-F1 levels increased in 20E-free medium, but declined in the presence of 20E. CHX increased βFTZ-F1 mRNA levels after 8 h of incubation, but expression decreased at 16 h.

Effect of 20E concentration on gene expression

G0 ASGs were incubated with various concentrations of 20E and the expression levels of the transcription factor genes determined after 2 h (E75A) or 8 h of incubation (all other transcription factors) (Fig. 4).

image

Figure 4. Concentration responses of transcription factor genes to 20E. G0 ASGs were incubated with different concentrations of 20E for 8 h to measure mRNA levels of EcR-A, EcR-B1, usp-1, usp-2, E75B, BHR3, BR-C Z1, Z2 and Z4 or for 2 h to measure E75A. Incubation periods were selected according to the data in Fig. 3. Eight ASGs were used to obtain one total RNA sample. Q-PCR was performed as in Fig. 1. Each datum point is a mean ± SD (n = 3).

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EcR-A expression was stimulated by 0.01 and 1 µm 20E, while EcR-B1 responded maximally to 20E concentrations between 0.04 and 4 µm. Although the responses of the EcR isoforms are similar to each other, EcR-A was fourfold more sensitive to 20E than EcR-B1.

usp-1 expression was high in the absence of 20E (control level) (Figs 3 and 4) and lowered in its presence, except for a small peak at 0.04 µm 20E, at which concentration expression approached control levels. usp-2 expression was up-regulated by low concentrations of 20E (0.01 µm) and peaked at 1 µm 20E. Higher concentrations were less effective in stimulating usp-2.

E75A was maximally induced at 1 µm 20E but suppressed at higher concentrations, a similar response to that of EcR-A. E75B was up-regulated at 0.4 µm and peaked at 1 µm. Thus, E75A is more sensitive than E75B although the most effective concentration (1 µm) is the same for the two isoforms. The concentration response for BHR3 was similar to that of E75B, although the curve shifted to a higher concentration range with maximum induction for BHR3 at 4 µm. The BR-C Z1, Z2 and Z4 isoforms exhibited two distinct peaks at 0.04 and 4 µm 20E, similar to the expression profile of EcR-A.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

The sequence of PCD in Bombyx ASGs can be roughly divided into four phases with respect to the ASG response to JH and 20E (Fig. 5). During the initial phase from V4 to V5, ASGs lose their sensitivity to JH, then become able to respond to 20E over the next 24 h period from the evening of V5 to the evening of V6 (Kakei et al., 2005). During the period between V6 and G2, ASGs are responsive to 20E but haemolymph ecdysteroid levels are insufficient to induce PCD. By the evening of G2, ASGs are sufficiently stimulated to complete PCD, and a large increase in haemolymph ecdysteroid triggers pupal metamorphosis (Terashima et al., 2000). G2 is day 9 (V9) of the fifth instar in gate I larvae.

image

Figure 5. Schematic representation of 20E-regulated transcription in Bombyx fifth instar ASGs. Top panel: Developmental changes in haemolymph ecdysteroid titre represented with two different scales. The ordinate for thick line is shown on the right-hand x-axis (Sakurai et al., 1998; Terashima et al., 2000). Middle panel: Developmental changes in ASG responsiveness to JH and 20E. Lower panel: Developmental expression of individual transcription factor genes. Feeding period and prepupal period are indicated as V3-6 and G0-2, respectively. White and black boxes for each day represent 12 h photophase and 12 h scotophase, respectively. Gene expression levels are indicated by the thickness of the solid lines and black boxes. Dashed lines represent low expression levels, while no line indicates negligible expression levels.

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The hierarchal control of gene expression in PCD of Drosophila salivary glands has been extensively examined using loss-of-function and gain-of-function mutants. By contrast, application of such mutations to lepidopterans is not yet available, so we have compared in vivo and in vitro gene expression profiles in Drosophila salivary glands with those in Bombyx ASGs in order to elucidate the mechanism of gene control in the silkworm.

The unique expression profiles exhibited by individual transcription factor genes during the period from V3 to G2 are schematically represented in Fig. 5. Of the 11 mRNAs examined, the expression levels of nine were greatly increased at 72 h after gut purge (AGP). If ASGs are transferred to a hormone-free medium at 72 h, they complete PCD without additional exogenous 20E stimulation, but this is not the case if they are transferred at 48 h (Terashima et al., 2000). This suggests that, by 72 h, they have received sufficient stimulation from 20E and that execution of PCD has begun. If ASGs are undergoing PCD by 72 h, it is doubtful that gene expression peaks at this time are meaningful. We have therefore excluded examination of gene expression levels at 72 h AGP from the following discussion.

EcR and usp

In Drosophila, EcR-B1 mRNA predominates in tissues destined to undergo PCD at pupal metamorphosis including salivary glands, some muscles, fat body and midgut (Talbot et al., 1993), suggesting that this EcR isoform is involved in metamorphic changes in larval-specific tissues that undergo cell death. Indeed, an EcR-B1 loss-of-function Drosophila mutant fails to activate ecdysone-inducible genes in its larval salivary glands (Bender et al., 1997). Moreover, an EcR-A-specific deletion mutant shows that low levels of EcR-A mRNA in the salivary glands are indispensable for the PCD of this tissue (Davis et al., 2005). By contrast, the role of EcR isoforms in lepidopteran insects appears to differ from that in Drosophila. EcR-A is the predominant isoform in Bombyx ASGs and in the silk glands of the wax moth, Galleria mellonella, during the prepupal period (Jindra & Riddiford, 1996; Kamimura et al., 1997; present study). In Bombyx pupal wings, EcR-A is predominantly expressed on the outside of the bordering lacuna, which is eliminated by PCD shortly after pupation, while EcR-B1 is the predominant isoform expressed inside the bordering lacuna, which differentiates into the adult wing (Suyama et al., 2003). This selective expression of EcR-A in lepidopteran tissues destined for cell death is in contrast to Drosophila.

In the current study, EcR-B1 mRNA levels increased abruptly at V6 in Bombyx (Fig. 1), corresponding to the time at which ASGs become responsive to 20E (Kakei et al., 2005) and suggesting that EcR-B1 could be involved in the heightening of responsiveness to 20E. In Drosophila salivary glands, EcR-A mRNA levels are transiently increased at the same time as EcR-B1 mRNA disappears several hours before puparium formation (Huet et al., 1995). EcR-A is indispensable for PCD in the salivary glands (Davis et al., 2005) and might therefore be involved in the preparatory phase of cell death. EcR-B1 is extensively expressed 6 h after puparium formation (APF), corresponding to the time at which PCD begins (Huet et al., 1995). These data indicate the possible involvement of a particular EcR isoform, which differs between the two species, in the hours before and after puparium formation in Drosophila salivary glands and on V6 in Bombyx ASGs.

In the current study, the similarity between the expression profiles of usp-1 and EcR-B1 suggest their potential involvement as partner proteins; likewise, USP-2 might be a partner protein of EcR-A. Similar partnerships exist in the epidermis of M. sexta larvae, in which the DHR3 homolog, MHR3, involved in sclerotization is up-regulated by the 20E/EcR-B1/USP-1 complex and down-regulated by 20E/EcR-A/USP-2 (Lan et al., 1999; Riddiford et al., 2003). When G0 ASGs are incubated in the continuous presence of 20E, the gene expression required for PCD is complete after 8 h (Terashima et al., 2000). In this study, EcR-A mRNA levels were high prior to incubation with 20E and could be sufficient for mediating the 20E signal for downstream gene regulation. usp-2 expression tripled in the first hour of incubation and remained high thereafter. Such responses support the above-mentioned hypothesis that EcR-A/USP-2 might play a key role in PCD.

Taken together, the results indicate that EcR-B1 and usp-1 expression levels are increased on V5 when ASGs acquire the capacity to respond to 20E, probably brought about by the 20E signal at the small ecdysteroid peak. After the gain of sensitivity, the 20E stimulus (which might be received by the EcR-A/USP-2 complex) during the large ecdysteroid peak on G2 triggers the execution of PCD.

E75A and E75B

In Drosophila salivary glands, haemolymph ecdysone peaks occur around −4 h and 10 h APF (Riddiford, 1993), which approximately correspond to the expression times of E75A and BR-C. E75B is also expressed around 10 h APF (Woodard et al., 1994; Huet et al., 1995; Jiang et al., 2000). Changes in ecdysteroid titre and expression profiles of E75A and BR-C are similar in Bombyx, with a small ecdysteroid peak observed at V6 and a larger increase at G1 and G2, which causes pupal ecdysis. This suggests that the roles of these genes in Bombyx ASGs could be related to those in Drosophila salivary glands.

In vivo profiles of E75A and E75B after gut purge (Fig. 2) show that E75A expression peaks at 48 h AGP, followed by a E75B peak 6 h later. In Drosophila salivary glands, E75A (as well as E75B) suppresses Inhibitor of Apoptosis Protein 2 (diap2) expression (Jiang et al., 2000), thereby inducing PCD. As a 2 h exposure to 1 µm 20E was sufficient to activate E75A in Bombyx ASGs, E75A might be similarly involved in PCD execution in these tissues. Although E75B was up-regulated following an 8 h incubation with 20E in Bombyx ASGs, this in vitro response differs from the in vivo profile, indicating that E75B regulation does not simply depend on 20E stimulation. In the in vitro challenge, ASGs are exposed to continuous 20E, while in vivo they are subject to daily changes in haemolymph ecdysteroid titres. The continuous presence of 20E might be less effective for E75B up-regulation than a variable ecdysteroid titre. Exposure to 20E in the presence of the translation inhibitor CHX caused delayed activation, indicating the action of a suppression factor that has thus far remained elusive. The responses of E75A to 20E and CHX were the same as those of the early genes in Drosophila, suggesting that E75A is directly induced by 20E and that its repression is controlled by negative feedback. Early genes are controlled in this manner: the coexistence of CHX and 20E inhibits down-regulation causing expression levels to remain high (Ashburner et al., 1974).

BHR3

In Drosophila salivary glands, DHR3 is not expressed until 0–6 h APF, following E75A and BR-C expression at the end of the third instar (Huet et al., 1995; Lam et al., 1999). Here, we observed the highest BHR3 expression at 54 h AGP, 6 h after the E75A expression peak. Whole prepupae analysis shows that DHR3 regulates the EcR/USP-dependent gene transcription of βFTZ-F1, BR-C, E75A, EcR and E93 (White et al., 1997; Lam et al., 1999). Although there is no information available on the role of DHR3 in PCD of the salivary glands, the expression of E93 is suppressed in the Drosophila DHR3 loss-of-function mutant, and E93 has been proposed as the master gene for PCD in Drosophila (Lee et al., 2000; Lee & Baehrecke, 2001). It is therefore possible that BHR3 plays a role in 20E-induced PCD in Bombyx ASGs, although it remains to be seen whether E93 homolog is present in Bombyx. In this study, we observed an in vivo expression peak of BHR3 6 h after an E75A peak. During this 6 h, haemolymph ecdysteroid titre increased from 0.6 to 1.1 µm 20E equivalents (Takaki and Sakurai, unpublished data). In addition, BHR3 was up-regulated 8 h after 20E incubation, and the optimum 20E concentration was 4 µm, fourfold higher than that for E75A. These data suggest that the potential involvement of BHR3 in PCD execution might be a result of the large increase in prepupal ecdysone.

BR-C

In the Drosophila BR-C loss-of-function mutant, most cellular events associated with PCD in the salivary glands are disrupted except for DNA fragmentation (Lee et al., 2000, 2002). Similarly, larvae of the RNAi-mediated Bombyx BR-C loss-of-function mutant retain morphologically intact ASGs 12 h postpupation (Uhlirova et al., 2003), showing that BR-C is closely involved in PCD in the ASGs.

The two optimum 20E concentrations for induction of the three BR-C isoforms (0.04 µm and 4 µm), correspond to the V5–V6 low ecdysteroid titre and high prepupal ecdysteroid titre, respectively. This suggests that BR-C might play additional roles in the late feeding period and in the gain of responsiveness to 20E that occurs between V5 and V6 (Kakei et al., 2005).

βFTZ-F1

In Bombyx ASGs, βFTZ-F1 expression peaked at V5, after which expression levels remained low. By contrast, in Drosophila salivary glands, βFTZ-F1 is expressed after the first ecdysone pulse for puparium formation from 8 to 10 h APF (Horner et al., 1995; Huet et al., 1995; Jiang et al., 2000; Yamada et al., 2000). βFTZ-F1 expression is under the control of DHR3 (which is up-regulated by 20E; Kageyama et al., 1997), and a decrease in 20E is a prerequisite for βFTZ-F1 up-regulation (Sun et al., 1994).

Mid-prepupal expression of βFTZ-F1 in Drosophila salivary glands is crucial for execution of PCD, as mutations in βFTZ-F1 disrupt the cellular changes associated with PCD and DNA fragmentation (Lee et al., 2002). An increase in βFTZ-F1 in Drosophila mid-prepupa also activates the expression of diap2, BR-C, E74A and E75A (Woodard et al., 1994; Broadus et al., 1999; Jiang et al., 2000). In the prepupal period of Bombyx ASGs, however, βFTZ-F1 was not increased prior to the induction of BR-C and E75A (Fig. 2), probably because of the continuous increase in haemolymph ecdysteroid titre. Accordingly, βFTZ-F1 might not play a role in PCD of Bombyx ASGs. Alternatively, the peak in βFTZ-F1 expression at V5 concomitant with the loss of sensitivity to JH (from V4 to V5) and the increase in responsiveness to 20E (from V5 to V6) (Kakei et al., 2005) suggest that βFTZ-F1 might be involved in the early phases of PCD. Although βFTZ-F1 expression is under the control of 20E in Drosophila, we did not observe an increase in haemolymph ecdysteroid titre or in the expression of transcription factor genes prior to the V5 peak, indicating that βFTZ-F1 expression at this time point might be caused by an unknown factor. The increase in expression of βFTZ-F1 caused by incubation with CHX suggests that the inhibitor blocks the down-regulation caused by 20E.

20E titre and gene expression

In vitro EcR-A expression was up-regulated by two distinct 20E concentrations, 0.01 and 1 µm, while in vivo EcR-A expression followed a similar pattern to ecdysteroid titre levels. By contrast, the EcR-B1 expression profile cannot be fully explained by a response to 20E concentration. EcR-B1 expression was stimulated by 0.04 µm 20E, which corresponds to the haemolymph ecdysteroid titre level between V5 and V6. In vivo EcR-B1 expression after V6, however, decreased continuously to a low level until G2, despite its in vitro up-regulation by 20E concentrations attained between G0 and G2. Factor(s) other than 20E are therefore likely to be involved in the EcR-B1 expression dynamics during the prepupal period.

The developmental profiles of usp-1, usp-2 and E75A in Bombyx ASGs are well accounted for by the changes in ecdysteroid concentrations. The haemolymph ecdysteroid titre reaches a level of 0.04 µm at V6 photophase (Sakurai et al., 1998; Terashima et al., 2000) and, accordingly, we observed a transient increase in usp-1 expression on V6 (Fig. 1). Moreover, in vitro incubation of G0 ASGs with 20E showed that usp-1 expression was only up-regulated by 20E at 0 and 0.04 µm. usp-1 expression was decreased to a low level on the following day (G0), when haemolymph ecdysteroid titre increased to induce gut purge. As the haemolymph ecdysteroid titre increases continuously from 0.01 µm on V5 to 0.1 µm on G0, the 0.04 µm-induced up-regulation of usp-1 might give rise to its transient expression on V6.

usp-2 and E75A were both maximally induced by ecdysteroid titres of 1 µm. E75B and BHR3 mRNA levels were up-regulated at 0.4 µm, and peaked at 1 µm and 4 µm, respectively. These data account for the low expression levels of E75B and BHR3 on and before V6 due to haemolymph ecdysteroid titres of less than 0.1 µm. In addition, the sequential expressions of E75A, E75B and BHR3 after gut purge could be interpreted as the result of the different levels of sensitivity of these genes to 20E. That is, E75A is more sensitive and is therefore expressed earlier than E75B or BHR3.

BR-C isoforms appear to be up-regulated in response to the small ecdysteroid increase on V6, and to the larger peak on G2. As the haemolymph ecdysteroid titres found between V6 and G1 (0.04–4 µm) have been shown to be ineffective in stimulating BR-C in vitro, the isoform levels are not further increased until G2.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

As shown in Fig. 5, the in vivo expression profiles of EcR-A, EcR-B1, E75A and BR-C indicate that these genes respond to a very narrow window of 20E concentration in the fifth instar, being up-regulated before gut purge (V6) and again in the prepupal period (G2). The optimum 20E concentrations for stimulation of individual genes differ, but coincide with the haemolymph ecdysteroid titre at the time of in vivo up-regulation. These data indicate that the temporal expression pattern of the transcriptional genes is a result of hierarchical control and depends on the temporal changes in the haemolymph ecdysteroid titre. This may be brought about by the sensitivity of the genes to 20E and/or the 20E/EcR-A/USP2 complex.

Comparison with available data on Drosophila indicated that the transcription factor genes investigated here might be involved in PCD of Bombyx ASGs. The roles of individual genes remain to be determined through functional analysis, but are likely to differ from those in Drosophila salivary glands.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

Animals

Larvae of the silkworm, Bombyx mori (Kinshu × Showa), were reared and staged as previously described (Terashima et al., 2000). Larval ages were counted in days, consisting of a photophase followed by a scotophase; 0 AZT denotes the beginning of a photophase (Truman & Riddiford, 1974). Newly moulted larvae were fed from the beginning of the photophase following the scotophase in which they moulted into the fifth instar. We used only gate I larvae, in which spinneret pigmentation developed by the morning of day 6 and gut purging occurred in the middle of the following scotophase (Sakurai et al., 1998).

In vitro culture of anterior silk glands

The 20E (Sigma, St Louis, MO, USA) was dissolved in ethanol and its concentration determined spectrophotometrically at 243 nm (ɛEtOH = 12 300). An aliquot of the stock solution was evaporated and dissolved in Grace's insect culture medium (Gibco, Grand Island, NY, USA). Cycloheximide (CHX, Sigma) was dissolved in distilled water at 10 mg/ml and stored at −20 °C until required. CHX solution was added to the culture medium at a final concentration of 50 µg/ml (Terashima et al., 2000).

ASGs were dissected out at approximately the mid-point of photophase (06.00 h). They were rinsed in Grace's insect culture medium (pH 6.4, adjusted with 1 N NaOH) and cultured individually in 24-well plates (Greiner, Frickenhausen, Germany) with 0.3 ml of medium at 25 °C.

RNA isolation and real-time reverse transcriptase–PCR

Total RNA was isolated from 8 to 18 ASGs by the acid guanidinium thiocyanate–phenol–chloroform (AGPC) method (Chomczynski & Sacchi, 1987) with minor modifications (Tsuzuki et al., 2001). Total RNA concentrations were determined spectrophotometrically at 260 nm.

Early and early late gene mRNA levels were determined using real-time quantitative PCR (Q-PCR), as previously described (Gibson et al., 1996). Briefly, a 20 µl reverse transcriptase reaction was carried out using 1 µg of total RNA, oligo dT primer [5′-TTTTTTTTTTTT(A/C/G)(A/C/G/T)-3′], and 100 U of ReverTra Ace (TOYOBO, Osaka, Japan). The resultant cDNA was diluted fivefold with distilled water. Q-PCR was performed using a SYBR Green PCR Core Reagents Kit (Perkin-Elmer, Applied Biosystems, Shelton, CT, USA) on an Applied Biosystems Prism 7700 Sequence Detector (Perkin-Elmer). The primers used for PCR are listed in Table 1. Ribosomal protein L3 (RpL3) mRNA (Matsuoka & Fujiwara, 2000) was used as an endogenous control. The primers were derived from the sequences of the Bombyx genes (see References in Table 1). The validity of primers was verified by sequencing of PCR products obtained using individual sets of primers. Data were analysed with Sequence Detector Software (Perkin-Elmer). Recombinant plasmids (pGEM 7zf(+)) containing individual RT–PCR products were purified to prepare the absolute standards. The concentration was measured at A260, and the solution was diluted to 500 000 copies/µl for individual genes according to the molecular weight of the recombinant plasmid DNA, which was used as an exogenous control.

Table 1.  Forward and reverse primers used in Q-PCR
GeneForward primer Reverse primerFinal conc. (nm)Accession no.Reference
RpL35′-AGCACCCCGTCATGGGTCTA-3′ 5′-CACGAAGACGCTCCAAAAATGA-3′125AB024901Matsuoka & Fujiwara (2000)
βFTZ-F15′-TGCGTCCAAGCTCATCCTGC-3′ 5′-AGGTGTGCGGCAAGCTGCTGT-3′ 62.5D10953Sun et al. (1994)
BR-C Z45′-CATAGTGGTAGCCGCTGACTTT-3′ 5′-CGGGAAGGTACTGTGCTCAAAG-3′ 62.5AB166727Ijiro et al. (2004)
BR-C Z25′-ACCTTGTGGCAGAGCGTGCA-3′ 5′-GCACCCCCCAAGAAGATTACAG-3′250AB166726Ijiro et al. (2004)
BR-C Z15′-CACTCTCTCGCATACGGTACA-3′ 5′-TTCGGCTCATCGATATCTGGCA-3′ 62.5AB166725Ijiro et al. (2004)
BHR35′-CCTGGACGGTCTCATGTGTACG-3′ 5′-TCAGCCGTCACTCGTGTCGGTA-3′100AB024902Matsuoka & Fujiwara (2000)
E75B5′-TTTGCATCCCTCGCAGGTGAT-3′ 5′-AAGCGACCTTCATCCCTTGTGAC-3′125AB024905Matsuoka & Fujiwara (2000)
E75A5′-TCAGCGCCACACGACATGGT-3′ 5′-GCAACAAGGTCGTTGAACTAA-3′125AB024904Matsuoka & Fujiwara (2000)
usp-25′-GTAGTGGAACCCACTGGCCTTGT-3′ 5′-GTCGAGCGTGGCGAAGAAA-3′125AB182582Takaki (personal communication)
usp-15′-GATATCGTGATAATAAACCTAAGTA-3′ 5′-ATAACGGTGGCTTCCCGCTGCG-3′125U06073Tzertzinis et al. (1994)
EcR-B15′-CAGCCATTGTATATCGAGTTCAA-3′ 5′-TGGAGCTGAAACACGAGGTGGC-3′100L35266Kamimura et al. (1997)
EcR-A5′-CGGTGTTGTGGGAGGCATTGGTA-3′ 5′-TCCCATTAGGGCTGTACGGACC-3′ 62.5D87118Kamimura et al. (1997)

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References

We are indebted to Dr K. Takaki for information on the haemolymph ecdysteroid titre after gut purge, and to Dr S. Harada of the Kanazawa University Medical School, Japan for suggestions about real-time Q-PCR. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) 14360033 awarded to S.S.

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  3. Introduction
  4. Results
  5. Discussion
  6. Summary
  7. Experimental procedures
  8. Acknowledgements
  9. References
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