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Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis

View Article: PubMed Central - PubMed

ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.


Autophagy is activated at spinning stage.(a) qRT-PCR analysis of BmATG8 mRNA levels in midgut tissues; (b) Western blot analysis of BmATG8–PE; (c,d) immunofluorescence staining of BmAtg8 showing the presence of autophagosomes (arrows) in midgut tissues at SD2 (d) compared to L5D5 (c) stage; (e) TEM images of autophagosomes in midgut cells at SD2 stage (A: autophagosome; arrows: autophagosome membranes); (f) analysis of acid phosphatase activity in midgut cells. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).
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f1: Autophagy is activated at spinning stage.(a) qRT-PCR analysis of BmATG8 mRNA levels in midgut tissues; (b) Western blot analysis of BmATG8–PE; (c,d) immunofluorescence staining of BmAtg8 showing the presence of autophagosomes (arrows) in midgut tissues at SD2 (d) compared to L5D5 (c) stage; (e) TEM images of autophagosomes in midgut cells at SD2 stage (A: autophagosome; arrows: autophagosome membranes); (f) analysis of acid phosphatase activity in midgut cells. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).

Mentions: In previous work from our group, the presence of autophagic features in the midgut of Bombyx mori during metamorphosis was reported7. In that study, however, we did not assess the precise timing of autophagy activation. To clarify this aspect, we first analysed the expression of the gene BmATG8 at different time points by using quantitative reverse transcription PCR (qRT-PCR), starting from the fifth larval instar up to the pupal stage. BmATG8 mRNA levels increased at the end of the last larval instar and peaked at the SD1 stage (Fig. 1a).


Roles and regulation of autophagy and apoptosis in the remodelling of the lepidopteran midgut epithelium during metamorphosis
Autophagy is activated at spinning stage.(a) qRT-PCR analysis of BmATG8 mRNA levels in midgut tissues; (b) Western blot analysis of BmATG8–PE; (c,d) immunofluorescence staining of BmAtg8 showing the presence of autophagosomes (arrows) in midgut tissues at SD2 (d) compared to L5D5 (c) stage; (e) TEM images of autophagosomes in midgut cells at SD2 stage (A: autophagosome; arrows: autophagosome membranes); (f) analysis of acid phosphatase activity in midgut cells. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016986&req=5

f1: Autophagy is activated at spinning stage.(a) qRT-PCR analysis of BmATG8 mRNA levels in midgut tissues; (b) Western blot analysis of BmATG8–PE; (c,d) immunofluorescence staining of BmAtg8 showing the presence of autophagosomes (arrows) in midgut tissues at SD2 (d) compared to L5D5 (c) stage; (e) TEM images of autophagosomes in midgut cells at SD2 stage (A: autophagosome; arrows: autophagosome membranes); (f) analysis of acid phosphatase activity in midgut cells. Values represent mean ± s.e.m. (*p < 0.05; **p < 0.01 compared to L5D2 using ANOVA followed by Tukey’s HSD test).
Mentions: In previous work from our group, the presence of autophagic features in the midgut of Bombyx mori during metamorphosis was reported7. In that study, however, we did not assess the precise timing of autophagy activation. To clarify this aspect, we first analysed the expression of the gene BmATG8 at different time points by using quantitative reverse transcription PCR (qRT-PCR), starting from the fifth larval instar up to the pupal stage. BmATG8 mRNA levels increased at the end of the last larval instar and peaked at the SD1 stage (Fig. 1a).

View Article: PubMed Central - PubMed

ABSTRACT

We previously showed that autophagy and apoptosis occur in the removal of the lepidopteran larval midgut during metamorphosis. However, their roles in this context and the molecular pathways underlying their activation and regulation were only hypothesized. The results of the present study better clarify the timing of the activation of these two processes: autophagic and apoptotic genes are transcribed at the beginning of metamorphosis, but apoptosis intervenes after autophagy. To investigate the mechanisms that promote the activation of autophagy and apoptosis, we designed a set of experiments based on injections of 20-hydroxyecdysone (20E). Our data demonstrate that autophagy is induced at the end of the last larval stage by the 20E commitment peak, while the onset of apoptosis occurs concomitantly with the 20E metamorphic peak. By impairing autophagic flux, the midgut epithelium degenerated faster, and higher caspase activity was observed compared to controls, whereas inhibiting caspase activation caused a severe delay in epithelial degeneration. Our data demonstrate that autophagy plays a pro-survival function in the silkworm midgut during metamorphosis, while apoptosis is the major process that drives the demise of the epithelium. The evidence collected in this study seems to exclude the occurrence of autophagic cell death in this setting.

No MeSH data available.