20-Hydroxyecdysone

Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori

Daiki Fujinaga 1, Junjie Gu, Hajime Kawahara, Mari H. Ogihara 2, Ikumi Kojima, Mika Takeshima, Hiroshi Kataoka *

Abstract

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

Keywords:
Embryonic development Ecdysteroidogenesis
Ecdysteroid-phosphate
Bombyx mori RNAi
LC-MS/MS

1. Introduction

Insect development is regulated by a wide variety of hormones. Especially, ecdysteroids are known to induce developmental transitions such as molting and metamorphosis (Thummel, 1996; Yamanaka et al., 2013). During larval development, a prohormone ecdysone is synthesized from cholesterol through a series of tandem steroid conversion reactions known as the ecdysteroidogenic pathway in the prothoracic glands (Iga and Kataoka, 2012; Lafont et al., 2012). In this pathway, cholesterol is converted into 7-dehydrocholesterol (7dC) by a Rieske oxygenase Neverland (Nvd) (Yoshiyama et al., 2006; Yoshiyama-Yanagawa et al., 2011). The conversion steps from 7dC to 5β-ketodiol (KD) have not been clearly characterized, but cytochrome P450 (CYP) 307A1/A2 (Spook: Spo/Spookier) and a short chain dehydrogenase/reductase Non-molting glossy (Nm-g, also known as Shroud in Drosophila melanogaster) have been reported to be involved in the conversion (Namiki et al., 2005; Ono et al., 2006; Niwa et al., 2010). The later conversion steps from KD to ecdysone are well characterized. CYP306A1 (Phantom: Phm), CYP302A1 (Disembodied: Dib), and CYP315A1 (Shadow: Sad) convert KD into 5β-ketotriol (KT), KT into 2-deoxyecdysone (2dE), and 2dE into ecdysone, respectively (Chavez et al., 2000; Warren et al., 2002, 2004; Niwa et al., 2004, 2005). Synthesized ecdysone is secreted into hemolymph and converted into an active ecdysteroid, 20-hydroxyecdysone (20E), in peripheral tissues by CYP314A1 (Shade: Shd) (Petryk et al., 2003). Synthesized 20E is known to regulate expression of a wide variety of genes by activating ecdysone receptor to trigger molting and metamorphosis (Riddiford et al., 2000).
Ecdysteroids are also involved in embryonic development. In Bombyx mori, it has been reported that diapause eggs which are characterized by a cessation of embryonic development have low amounts of ecdysteroids (Sonobe, 1997). Since 20E injection into diapause eggs induces embryonic development (Makka et al., 2002), 20E is considered to trigger embryonic development in B. mori. Moreover, in D. melanogaster, mutant flies of ecdysteroidogenic enzymes exhibit embryonic lethal phenotypes during late embryogenesis due to defective cuticle differentiation, indicating that ecdysteroid biosynthesis is required during embryogenesis in D. melanogaster (Chavez et al., 2000; Petryk et al., 2003). Furthermore, tritium-labeled KD injected into non-diapause eggs of B. mori was converted into labeled-ecdysone and 20E (Horike and Sonobe, 1999). These results suggest that the ecdysteroidogenic pathway is activated to produce 20E during embryogenesis, which induces embryonic development.
In lepidopteran and orthopteran insects, another pathway to produce 20E has been reported. In these insects, phosphorylated conjugates of ecdysteroids were detected in the ovaries and eggs (Ohnishi et al., 1977, 1989; Isaac et al., 1983; Warren et al., 1986). Among these conjugates, C-22 phosphorylated 2dE (2dE-22P), ecdysone (Ecdysone-22P), and 20E (20E-22P) are considered as storage forms of ecdysteroids, whereas C-2 and C-3 phosphorylated ecdysteroids are thought as inactivated metabolites in B. mori (Yamada and Sonobe, 2003; Sonobe and Yamada, 2004). Ecdysteroid 22-kinase (Eck) and Ecdysteroid-phosphate phosphatase (EPPase) have been identified in B. mori as enzymes that phosphorylate and dephosphorylate ecdysteroids, respectively (Yamada and Sonobe, 2003; Sonobe et al., 2006). During adult development of B. mori, Eck is highly expressed in the ovaries, and Eck coverts ecdysteroids into phosphorylated conjugates, which are stored in the yolk in the germ cells (Ito et al., 2008; Sonobe and Ito, 2009). During embryogenesis, EPPase is expressed and converts inactive ecdysteroid conjugates into the free form of 2dE, ecdysone, and 20E (Yamada and Sonobe, 2003). Since homologous genes of Eck and EPPase have been found in various insect species such as D. melanogaster (Ito et al., 2008; Sonobe and Ito, 2009), this dephosphorylation pathway is thought to be common in insects.
Although 20E is produced through both ecdysteroidogenic and dephosphorylation pathways during embryogenesis of the silkmoth, the importance of each pathway remains unclear. To elucidate the details of the ecdysteroid production during embryogenesis, it is necessary to accurately measure the change in the amount of ecdysteroids and the expression levels of enzymes involved in 20E production during embryogenesis. Although some previous studies focused on the change in 20E amount during embryogenesis in B. mori, those measurements were conducted by radioimmunoassay (RIA) using antibody against ecdysteroids (Yamada and Sonobe, 2003; Sonobe, 1997). Because the antibody cannot distinguish between 20E and other ecdysteroids, their results likely represent the amounts of total ecdysteroids. More specific measurement of 20E has also been conducted by the combination of purification by high performance liquid chromatography (HPLC) and RIA; however, a large number of eggs are necessary to detect ecdysteroids with this method (Horike and Sonobe, 1999). Recently, we established a method for measuring ecdysteroids using the liquid chromatography tandem mass spectrometry (LC-MS/MS) (Hikiba et al., 2013), which enabled us to determine the accurate amounts of 20E from fewer numbers of eggs.
In this study, we investigated the activation timing of the ecdysteroidogenic and dephosphorylation pathways during embryogenesis in non-diapause eggs of B. mori. To determine the exact time of activation, we collected eggs every 6 h post-oviposition (hpo). Using these eggs, we observed morphological development of the embryos, quantified the amounts of 20E and 20E-phosphate, and measured mRNA amounts of EPPase and ecdysteroidogenic enzymes. We also analyzed the activities of Phm and Shd by in vitro enzymatic assays using egg extractions. Furthermore, the contribution of each pathway on embryonic development was investigated by knocking down of Shd and EPPase. Our results suggest that the dephosphorylation pathway is primarily activated first, and then the ecdysteroidogenic pathway is activated later to produce 20E during early embryogenesis in B. mori.

2. Materials and methods

2.1. Animals

A bivoltine strain of the silkmoth, B. mori (Kosetsu), was used in all experiments. Insects were fed with the artificial diet of the silkmoth “Silkmate” (Nihon Nosan Kogyo, Yokohama, Japan) and reared at 25 ◦C under a 4 h-light and 20 h-dark photoperiod to induce non-diapause eggs as described previously (Tsuchida and Yoshitake, 1979). Mated females were put on egg boards to lay eggs at 25 ◦C, and laid eggs were kept at 25 ◦C under a 12 h-light and 12 h-dark photoperiod. Eggs were collected at each time point ±10 min.

2.2. Observation of embryogenesis

The thionine staining was performed to observe morphogenesis of embryos from 0 to 96 hpo. The eggs at each time point were fixed in the Carnoy’s fluid (ethanol: chloroform: glacial acetic acid = 6: 3: 1) for 1 day at 4 ◦C. The fixed eggs were treated with a gradient concentration of ethanol (100%, 95%, 90%, 80%) for 10 min each. Following 20 min boiling, eggs were dechorionized by dissection in the 80% ethanol and transferred into the thionine solution (0.07% of thionine and 0.3% of phenol dissolved in 80% ethanol) for 2 h. The stained eggs were rinsed by 80% ethanol 4 times and dehydrated with a gradient concentration of ethanol (90%, 95%, 100%) for 10 min each. Finally, the eggs were soaked in xylene to make the yolk transparent. Embryos were photographed by Nikon D-5000 (Nikon, Tokyo, Japan). Besides, the eggs after 96 hpo were dissected in physiological saline and photographed.

2.3. Measurement of ecdysteroids

Twenty eggs at each time point were homogenized in 350 μl of sonic buffer (20 mM ethylene glycol tetra-acetic acid, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and sonicated at 4 ◦C for 10 min. After mixed with 700 μl 1-butanol vigorously, the extract was centrifuged at 20,400×g for 10 min, then the upper layer was collected and dried up. Dried extract was dissolved in 200 μl of methanol and centrifuged at 3000×g for 10 min. The supernatant was used to measure ecdysteroid concentration by the LC-MS/MS method with Prominence gradient HPLC (Shimadzu Corporation, Kyoto, Japan) and QTRAP 5500 (AB SCIEX, Framingham, MA, USA) as described previously (Hikiba et al., 2013).

2.4. Semi-quantification of phosphorylated ecdysteroids

One hundred eggs were homogenized in 250 μl of 50 mM Tris buffered saline (pH 7.8). Following by sonication at 4 ◦C for 20 min, homogenates were centrifuged at 2300×g for 10 min. Collected 100 μl of supernatant was mixed with 200 μl of 1-butanol. After centrifugation at 20,400×g for 10 min, the upper layer was transferred to a new tube and dried up by evaporation. Then, the extracts dissolved in deionized water were incubated with 5U alkaline phosphatase (Promega) at 37 ◦C for 6 h. The control sample was incubated without alkaline phosphatase. After dried up again, the extracts were dissolved in 100 μl of methanol, and ecdysteroid concentration was measured by the LC-MS/MS method described above. The difference in the amount of 20E between the dephosphorylated and non-dephosphorylated extract was calculated as the amount of 20E-phosphate.

2.5. Quantitative RT-PCR

RNA was extracted from ten eggs using TRI Reagent® (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the recommended protocol. After incubating with 100 μl RQI RNase-Free DNase (Promega, Madison, WI, USA) at 37 ◦C for 30 min, the RNA extract was purified with PCI solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and dissolved in diethyl pyrocarbonate-treated water. After measuring RNA concentration by a spectrophotometer (Bio-spec Nano, Shimadzu), 100 ng of extracted RNA was incubated with Prime Script RT reagent Kit (Takara Bio, Inc., Shiga, Japan) following the protocol. Synthesized cDNA was diluted 5 times with TE buffer (10 mM Tris-HCl, 1 mM ethylenediamine-tetraacetic acid, pH 8.0).
Amounts of target genes mRNA were quantified by quantitative real- time PCR (qRT-PCR) using specific primers listed in Supplementary Table S2 with a real-time PCR detection system (TP850 Thermal Cycler Dice Real Time, Takara Bio, Inc.) and a THUNDERBIRD SYBR qPCR Mix (TOYOBO CO. Ltd., Osaka, Japan). The PCR was carried out as follows: 30 s at 95 ◦C, followed by 40 cycles of 5 s at 95 ◦C and 30 s at 60 ◦C, and ended with a dissociation of 15 s at 95 ◦C and 30 s at 60 ◦C. Amount of B. mori ribosomal protein L3 (rpL3) mRNA was measured as an internal standard.

2.6. Enzymatic assay using extracted eggs

Two hundred and fifty eggs were homogenized in 1 ml of 20 mM potassium phosphate buffer (pH 7.5). After sonication at 4 ◦C for 10 min, the mixture was centrifuged at 3000×g for 10 min, and 100 μl of the supernatant was used as the extract of the eggs. The extract was mixed with 100 μl reaction solution (10 mM potassium phosphate buffer, pH 7.5, 2 mM Nicotinamide adenine dinucleotide phosphate) containing 50 ng of steroids (KD or ecdysone) in 45% 2-hydroxypropyl-β-cyclodextrin. The mixtures were incubated at 25 ◦C for 3 h. The reaction mixture without steroids was used as the control sample. After incubation, the solution was vigorously mixed with 400 μl of 1-butanol, then centrifuged at 20,400×g for 10 min. The evaporated supernatant was dissolved in 200 μl of methanol and centrifuged at 3000×g for 10 min, and then the supernatant was used for the following LC-MS/MS analysis.

2.7. Preparation and injection of double-stranded RNAs

DNA templates for double-stranded RNA (dsRNA) synthesis were amplified by RT-PCR with T7 promoter site containing primers shown in Supplementary Table S3. dsRNA was synthesized with MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) using 1 μg of PCR products as template DNA. After incubation at 37 ◦C for 4 h, the synthesized RNA was additionally incubated with RQI RNase-Free DNase at 37 ◦C for 30 min, purified by phenol/chloroform extraction and ethanol precipitation, and dissolved in 20 μl of deionized water. The RNA solution was boiled for 5 min and then cooled down to room temperature for annealing. Concentrations of dsRNA were quantified with the spectrophotometer and adjusted to 1 μg/μl.
Injection of dsRNA was completed within 4 hpo. Ten to twenty nl of dsRNA of DsRed, EPPase, or Shd was injected into the eggs using a microinjector (Electrical injector IM-300, NARISHIGE, Tokyo, Japan) and a micromanipulator (Electrical manipulator system, Micro Support, Shizuoka, Japan). The dsRNA-injected eggs were incubated at 25 ◦C in a wet chamber.

2.8. Statistical analyses

All data were presented as means + standard deviations (SD). Student’s t-test was used to compare two groups. Dunnett’s test was used for parametric multiple comparison. Steel’s test was used in Table 1 as a non-parametric multiple comparison test.

3. Results

3.1. Morphological changes of the embryos during early development

To investigate the relationship between embryogenesis and 20E production, embryos in non-diapause eggs were observed every 6 h from immediately after oviposition to 96 hpo by thionine staining (Fig. 1). Embryos after 96 hpo were dissected every 12 h and photographed (Fig. S1). Developmental stages of the stained embryos were identified based on the stage divide principles of the silkmoth (Miya, 2003; Nagy et al., 1994).
The embryos from 0 to 12 hpo were hardly visible. Fertilization, cleavage, and blastoderm and germ-anlage formation occur during Stage 0–3 (Miya, 2003). The embryonic cells became visible at 18 hpo when the germband formation begins (Stage 4; germband formation I stage). The embryos at 24 hpo were at the germband formation II stage, in which the embryos became narrower and exhibited “pyriform-shape” (Stage 5). The embryos at 30 hpo were between the germband formation III stage (Stage 6) and the germband formation IV stage (Stage 7), in which the germband has elongated but primitive segmentation has not happened yet. Since the stages from the diapause stage I (Stage 8) to the critical stage II (Stage 15) were characterized in the diapause eggs, embryos corresponding to these stages were not observed in non-diapause eggs. Segmentation in the embryos was detectable at 36 hpo, which corresponds to the appearance of neural groove stage (Stage 16). The embryos at 42 hpo were between the appearance of neural groove stage (Stage 16) and the appearance of abdominal appendage stage (Stage 17). The semicircular appendages on the abdominal and thoracic segments started to appear at these stages. After 48 hpo, the embryos shortened. The tip of the posterior part of the embryos shrank at 48 hpo, which marks the appearance of processes of labrum stage (Stage 18). The embryos at 54 hpo were between the appearance of processes of labrum stage (Stage 18) and the shortening stage (Stage 19), when the thoracic appendages were more clearly visible. The embryos at 60 hpo reached the shortening stage (Stage 19), when the embryos kept shortening but became thicker. The embryos after 66 hpo were transitioning to the cephalothoracic segmentation stage (Stage 20). During this timeframe, embryos became much thicker and their segments of the future head, thorax, and abdomen were clearly visible. The embryos at 72 and 78 hpo reached the cephalothoracic segmentation stage (Stage 20), the last developmental stage before blastokinesis. The embryos at 84 hpo were the early stage of blastokinesis (Stage 21A), in which the ventral part of the embryos detach from ventral region of eggs. At 96 hpo, the embryos were in the middle of blastokinesis (Stage 21B) and exhibited the twisted shape. After the blastokinesis, the appearance of setae and taenidium and the pigmentation of the head and body were observed. The embryos went through further stages and finally hatched around 240 hpo.

3.2. Changes in ecdysteroid amounts during embryogenesis

In the silkmoth, the continuous supply of 20E is considered necessary for embryogenesis (Makka et al., 2002). However, in previous studies, detailed quantification of ecdysteroids has not been performed because of technical difficulties. The LC-MS/MS method that we recently established (Hikiba et al., 2013) enabled us to determine the exact amount of 20E from only 20 eggs. Therefore, we determined the amount of 20E in early embryogenesis every 6 h from 0 to 96 hpo with this method (Fig. 2A).
The amount of 20E was about 0.07 ng/egg in newly laid eggs, and it continuously decreased until 24 hpo. Thereafter, the amount of 20E increased and reached the plateau at approximately 0.1 ng/egg at 42 hpo. After a transient drop at 60 hpo, it rose again to ~0.17 ng/egg at 96 hpo.
We also semi-quantified phosphorylated 20E with the alkaline phosphorylation assay every 24 h from 0 to 96 hpo (Fig. 2B). Phosphorylated 20E titer was particularly low at 48 and 72 hpo. This result suggests that 20E-phosphate is dephosphorylated during early embryogenesis, mainly from 24 to 48 hpo. It should be noted that semi- quantified 20E-phosphate contains 20E-2P and 20E-3P which are considered as inactivated metabolites, as well as 20E-22P which is converted into free form of 20E by EPPase (Sonobe and Yamada, 2004), because the semi-quantification method using alkaline phosphatase could not distinguish among these conjugates. The increased 20E-phosphate from 72 hpo to 96 hpo was considered the inactivated materials.

3.3. Changes in mRNA amounts of enzymes related to 20E production

In order to determine the correlation among morphological development, ecdysteroid levels, and activity of biosynthetic enzymes during embryogenesis, mRNA amounts of EPPase and ecdysteroidogenic enzymes were measured (Fig. 3). EPPase produces 20E through the dephosphorylation pathway, whereas the other enzymes, Nvd, Spo, Nm- g, Phm, Dib, Sad, and Shd, synthesize 20E through the ecdysteroidogenic pathway in this order.
The amount of EPPase mRNA was low until 18 hpo and increased at 24 hpo onward. The amount reached the highest peak at 48 hpo but dropped at 60 hpo and remained low until 72 hpo. It was increased again at 78 hpo and fluctuated thereafter (Fig. 3A). The first peak of Nvd mRNA appeared around 12 and 18 hpo, although it was still low. The substantial increase started at 48 hpo and peaked at 72 hpo (Fig. 3B). The amount of Spo mRNA was extremely low from 0 to 48 hpo, then it rapidly increased subsequently from 54 hpo (Fig. 3C). The amount of Nm-g mRNA was high at 0 hpo and increased rapidly, reached the highest peak at 6 hpo, then sharply dropped. The amount remained relatively low level after 18 hpo but slightly increased at 66 hpo onward (Figs. 3D and S2). The amount of Phm mRNA was low at 0 hpo and gradually increased afterward, with peaking at 78 hpo (Fig. 3E). Similar to the Nm-g, the amount of Dib mRNA was also high immediately after oviposition and peaked at 6 hpo, then gradually decreased and remained relatively low level but slightly increased after 66 hpo (Figs. 3F and S2). The amount of Sad mRNA had the same pattern as Nm-g and Dib, although Sad showed highest amount at 0 hpo (Figs. 3G and S2). The change in Shd mRNA amount was similar to Phm; it was low after oviposition, gradually increased afterward and reached the peak at 78 hpo (Fig. 3H).
In summary, during 0–96 hpo, the change in the amount of EPPase mRNA had two peaks around 48 and 78 hpo. In contrast, mRNA amounts of ecdysteroidogenic genes showed two different patterns; The first pattern maintained relative low levels immediately after oviposition and then increased. The second pattern gradually decreased from relatively high levels at the beginning of development and remained low afterward. Then, they slightly increased after 66 hpo. Nvd, Spo, Phm, and Shd belonged to the first pattern, whereas Nm-g, Dib, and Sad belonged to the second.

3.4. Activity of Phantom and Shade during embryogenesis

Although the determination of ecdysteroid amounts and mRNA amounts of 20E production-related genes suggested that 20E production started from 24 hpo onward, it remains uncertain when 20E is produced by the ecdysteroidogenic pathway. A previous study showed that tritium-labeled KD injected into the eggs at 72 hpo was converted to labeled 20E (Sonobe et al., 1999), suggesting that the ecdysteroidogenic pathway is activated at that time. To determine detailed timing of activation of the ecdysteroidogenic pathway, extracts of eggs collected every 24 h from 0 to 72 hpo were incubated with ecdysteroid substrates (KD or ecdysone). The product (KT or 20E, respectively) was then determined by the LC-MS/MS method (Fig. 4).
The KT levels in KD-added samples were not significantly different compared to control at 0 and 24 hpo. However, the levels became significantly higher than that in control at 48 hpo, and the difference became even larger at 72 hpo (Fig. 4A). In contrast, the 20E titer in the egg extract incubated with ecdysone was higher than that in control at 0 hpo. There were no significant differences at 24 and 48 hpo, but it became significantly higher again at 72 hpo (Fig. 4B). These results indicate that Phm and Shd are inactive at 24 hpo, and become activated thereafter. Due to technical difficulties of the LC-MS/MS method, products of other enzymes we tested (7dC, KD, 2dE, and ecdysone) could not be detected.

3.5. Knockdown of EPPase and Shade

Determination of ecdysteroid levels and changes in mRNA amounts suggested that both the dephosphorylation and ecdysteroidogenic pathways are activated during early embryogenesis. However, the critical timing of 20E production by each pathway, as well as their significance in embryonic development remains unclear. Therefore, we next performed knockdown of EPPase and Shd by injecting dsRNA targeting these genes.
The expression levels of EPPase and Shd in the eggs injected with dsRNAs targeting EPPase and Shd, respectively, were quantified at 72 hpo to evaluate knockdown efficiency of RNA interference (Fig. 5A and B). In the eggs injected with EPPase dsRNA, the expression level of EPPase was significantly lower than that in the negative control injected with DsRed dsRNA. The knockdown efficiency was about 74%. In the eggs injected dsRNA targeting Shd, the expression level of Shd was also significantly lower than that in the negative control. The knockdown efficiency was about 53%. These results indicate that we can efficiently knock down EPPase and Shd in embryos. When the amount of 20E was determined at 72 hpo (Fig. 5C), it was about half in the EPPase-knocked down eggs as compared to that in the negative control. On the other hand, there was no significant difference in the amount of 20E between Shd-knockdown eggs and the negative control. These results indicate that the dephosphorylation is the predominant 20E production pathway at 72 hpo. Besides, Shd does not seem to contribute to 20E production at least at 72 hpo.

3.6. Effects of EPPase and Shade knockdown on embryonic development

To investigate how the dephosphorylation and ecdysteroidogenic pathways contribute to embryonic development through 20E production, developmental stages of embryos were determined with thionine staining after knockdown of EPPase and Shd (Fig. 6 and Table 1). At 48 hpo, embryos in the negative control eggs were at the appearance of abdominal appendage stage (Stage 17 or 18) that corresponds to 42 to 48 hpo in intact eggs (Fig. 1), indicating that embryogenesis progresses normally in dsRNA-injected eggs. At 48 hpo, some embryos in the EPPase-knocked down eggs were still at the germband formation stage (Stage 6 or 7), which corresponds to ~30 hpo in intact eggs. In contrast, most embryos in Shd-knockdown eggs were at Stage 17 or 18, similar to the negative control. At 72 hpo, many embryos in the negative control had characteristics of Stage 18 or 19 (shrinking posterior tips and thicker abdomen) that corresponds to 54 to 66 hpo in intact eggs, indicating that the dsRNA injection caused a slight developmental delay. Although one embryo at Stage 19 was observed in an EPPase-knockdown egg, most of the embryos were at Stage 17 or 18, which corresponds to 42 to 54 hpo in intact eggs. This suggests that after a significant delay in early embryogenesis, morphological development still continues in the EPPase-knockdown embryos with maintaining 12- to 18-h delay. Most embryos in Shd-knockdown eggs were stage 18 or 19 similar to the negative control, indicating that knockdown of Shd did not cause any developmental delay up to 72 hpo. Interestingly, at 96 hpo, developmental delay was observed in both EPPase- and Shd-knockdown eggs. Many embryos in the negative control were during blastokinesis (Stage 21A and B), whereas most embryos in EPPase-knocked down eggs were at Stage 20. Embryos in Shd-knockdown eggs were at the stage of cephalothoracic segmentation or the early stage of blastokinesis (Stage 20 or 21A). These results indicate that EPPase contributes to embryonic development before 48 hpo, whereas Shd contribution is important between 72 and 96 hpo.

4. Discussion

It is widely accepted that 20E plays important roles in embryonic development as well as in molting and metamorphosis. During embryogenesis in the silkmoth, and possibly a variety of insects, 20E quantities fluctuate in a stage-dependent manner to regulate development. The appropriate amount of 20E must be produced at each time point in embryogenesis. In silkmoth embryos, 20E is produced via dephosphorylation of maternally loaded ecdysteroid conjugates and through ecdysteroidogenesis from cholesterol (Sonobe and Yamada, 2004). The present analyses demonstrate that both pathways contribute to 20E production in Bombyx embryos; the dephosphorylation pathway is activated first, followed by the activation of the ecdysteroidogenic pathway. EPPase- and Shd-knockdown delay embryonic development at different time points, confirming that both pathways are important for producing 20E during normal embryonic development.
From 0 to 96 hpo, the amount of 20E in eggs shows significant fluctuation (Fig. 2A). Although 20E was detected in embryos from 0 to 24 hpo, production of 20E is expected to be low during this time because of low mRNA amounts of EPPase and ecdysteroidogenic enzymes, except for Nm-g, Dib, and Sad (Fig. 3). 20E at 0 hpo is likely provided by the maternal ovaries because ecdysteroidogenic enzymes are expressed in the pupal ovaries to produce the free form of ecdysteroids as well as phosphorylated conjugates (Ito et al., 2008). In addition to 20E, mRNA of Nm-g, Dib, and Sad, and Shd protein are likely loaded as maternal products. Our study shows a high expression of Nm-g, Dib, and Sad in the ovaries of adult females (Fig. S3). Mining of RNA-seq data from unfertilized eggs in B. mori (Zhang et al., 2018) supports that mRNA of these genes are loaded as maternal products. Enzymatic assays show that embryos at 0 hpo convert ecdysone into 20E; however, mRNA of Shd was not detected at this time point (Figs. 3H and 4B), suggesting that Shd protein is loaded maternally into eggs. Maternal mRNA of Nm-g, Dib, and Sad, and Shd protein might function in embryos; however, their activity may not be sufficient to increase the amount of 20E in early embryos. The amount of 20E instead decreases immediately after oviposition to 24 hpo. Maternal 20E might be degraded or inactivated during this timeframe. Several enzymes for 20E inactivation are identified, i. e., CYP18A1 for 26-hydroxylation and ecdysone oxidase for 3-epimerization (Rewitz et al., 2010; Sun et al., 2012). However, involvement of these enzymes in 20E inactivation during embryogenesis is unclear because neither knockout of Cyp18A1 in D. melanogaster nor knockout of ecdysone oxidase in B. mori affects embryonic development (Guittard et al., 2011; Li et al., 2015). Double knockout of these enzymes should be analyzed to elucidate roles of 20E inactivation during early embryogenesis. In conclusion, low activity levels of 20E production and inactivation of 20E may result in a continuous decrease of 20E levels between 0 and 24 hpo.
Amounts of 20E increased after 30 hpo, and then reached the plateau at around 48 hpo, which coincides with the increase of EPPase transcripts (Fig. 2A). Amounts of 20E-phosphate is correspondingly decreased from 24 to 48 hpo (Fig. 2B). These results suggest that 20E is produced through dephosphorylation between 24 and 48 hpo. Knockdown of EPPase delays embryonic development at the germband formation stage. Shd-knockdown did not cause any developmental delay at 48 hpo (Fig. 6), confirming that 20E is produced through dephosphorylation in this timeframe. In contrast, 20E production through the ecdysteroidogenic pathway has less, if any, contribution.
The amount of 20E increases from 60 hpo onward after reaching the plateau around 48 hpo. Production after 48 hpo, and particular after 72 hpo, is likely due to ecdysteroidogenesis. Upregulated expression of ecdysteroidogenic enzymes, such as Phm and Shd, are likely activated after 48 hpo (Figs. 3 and 4). However, Shd-knockdown embryos did not show developmental delay until 72 hpo. Therefore, the contribution of the ecdysteroidogenic pathway is small until 72 hpo. Conversely, 20E might be produced by the ecdysteroidogenic pathway to support embryonic development until 72 hpo. EPPase-knockdown embryos showed developmental delay instead of developmental arrest, implying that the partial rescue caused by 20E produced through the ecdysteroidogenic pathway even before 72 hpo. Developmental delay of Shd-knockdown embryos at 96 hpo supports the necessity of the ecdysteroidogenic pathway during middle embryonic development. Although EPPase mRNA increased again at 78 hpo, amounts of 20E-phosphate changed little from 48 to 72 hpo (Figs. 2B and 3A). Thus, the contribution of the dephosphorylation pathway to 20E production may decline after 48 hpo.
The activity of EPPase and Shd was assessed in eggs from B. mori every 24 h during embryogenesis in previous studies. A low level of EPPase activity was detected at 24 hpo, and activity increased thereafter (Yamada and Sonobe, 2003). In contrast, injected tritium-labeled KD was converted into 20E at 48, 72, and 96 hpo, but not at 24 hpo in non-diapause eggs (Sonobe et al., 1999). In addition, incubation using the microsomal fraction from eggs shows that ecdysone was converted into 20E after 60 hpo, but not at 24 and 48 hpo (Horike and Sonobe, 1999). These results are consistent with findings in this study that the dephosphorylation pathway starts producing 20E after 24 hpo, while the ecdysteroidogenic pathway becomes activated after 48 hpo.
Knockdown of EPPase and Shd indicate the importance of their respective pathways to progress through embryonic development at specific stages, from the germband formation stage (Stage 6 or 7) to the appearance of neural groove stage (Stage 16) and from the cephalothoracic segmentation stage (Stage 20) to blastokinesis (Stage 21A), respectively (Fig. 6). However, dsRNA injection did not inhibit activation of pathways completely. Shd-knockdown embryos showed non- significant amounts of 20E for negative control (Fig. 5C), which could be due to low knockdown efficiency. Analysis using mutants of EPPase and Shd in the silkmoth is necessary to determine critical timing of activation of dephosphorylation and ecdysteroidogenic pathways.
We also compared mRNA expression between B. mori and D. melanogaster, well-studied models of 20E production. Interspecies comparison for 20E production shows similarities and differences in mRNA expression patterns. Since neither EPPase expression nor function is reported in embryos in species other than B. mori, we compared mRNA expression of ecdysteroidogenic enzymes during embryogenesis. Embryos of D. melanogaster, according to expression data in Flybase (FB2020_03), show transcription of all ecdysteroidogenic enzymes (Thurmond et al., 2019). Most of these enzymes exhibit similar changes in mRNA amounts between B. mori and D. melanogaster, indicating that 20E production by embryos through upregulating ecdysteroidogenic enzymes is common in two species. However, unlike B. mori, the mRNA contents of Shroud, Dib, and Sad are quite low from 0 to 2 hpo in D. melanogaster, indicating that these mRNA are not provided by the maternal ovaries. Rather, these mRNA are expressed in embryos in D. melanogaster, and maternal provision of these mRNA is a unique phenomenon in B. mori.
This difference may cause different phenotypes of Nm-g/Shroud mutants between B. mori and D. melanogaster. Shroud mutant flies exhibit an embryonic lethal phenotype during late embryogenesis, whereas embryos of an Nm-g mutant in B. mori develop normally, although they cannot develop after hatching because of molting failure (Niwa et al., 2010). Thus, maternally loaded Nm-g mRNA is translated, and its protein synthesizes ecdysteroids during embryogenesis in B. mori. However, we show that the amount of Nm-g mRNA rapidly decreased until 24 hpo, but production of 20E through the ecdysteroidogenic pathway is essential for normal embryonic development during mid-embryogenesis. These results and the phenotype of the Nm-g mutant suggest that 20E is produced thorough the ecdysteroidogenic pathway without Nm-g mRNA expression in embryos. A possible explanation is that Nm-g protein translated from maternally loaded Nm-g mRNA produces an intermediate ecdysteroid during early embryogenesis, and this intermediate is stored until mid-embryogenesis. Other enzymes, such as Dib and Sad, may also act during early embryogenesis. Phenotypes and amounts of ecdysteroids in mutants of each ecdysteroidogenic enzyme in the silkmoth need to be analyzed in the future to demonstrate roles of each ecdysteroidogenic enzyme in embryonic development. Further, stability of Nm-g, Dib, and Sad need to be investigated using antibodies to validate the possibility that these enzymes are translated from a maternally loaded mRNA and catalyze the conversion during mid-embryogenesis.
In conclusion, we propose the following hypothesis for the mechanism of 20E production during embryogenesis in B. mori (Fig. 7). During the first 24 h after egg laying, both dephosphorylation and ecdysteroidogenic pathways are not activated or only show minor activity. The amount of 20E decreases continuously between 0 and 24 hpo because of degradation of maternally loaded 20E. At 24 hpo, in parallel with EPPase expression, the dephosphorylation pathway is activated. Produced 20E induces embryonic development from the germband formation to the appearance of neural groove stage. The function of the dephosphorylation pathway declines along with the rapid decrease of EPPase expression, causing the amount of 20E plateau after 48 hpo. However, along with increased expression of ecdysteroidogenic enzymes, 20E is produced through the ecdysteroidogenic pathway after 48 hpo, in particular at 72 hpo onward. This increase is necessary for normal blastokinesis. Proper activation of the two pathways produces the appropriate amount of 20E, thus promoting embryonic development.

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