The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila

Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. Here, we show that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. We propose that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period

Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster.

Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation

Chaperonin TRiC/CCT supports mitotic exit and entry into endocycle in Drosophila.

Endocycle is a commonly observed cell cycle variant through which cells undergo repeated rounds of genome DNA replication without mitosis. Endocycling cells arise from mitotic cells through a switch of the cell cycle mode, called the mitotic-to-endocycle switch (MES), to initiate cell growth and terminal differentiation. However, the underlying regulatory mechanisms of MES remain unclear. Here we used the Drosophila steroidogenic organ, called the prothoracic gland (PG), to study regulatory mechanisms of MES, which is critical for the PG to upregulate biosynthesis of the steroid hormone ecdysone. We demonstrate that PG cells undergo MES through downregulation of mitotic cyclins, which is mediated by Fizzy-related (Fzr). Moreover, we performed a RNAi screen to further elucidate the regulatory mechanisms of MES, and identified the evolutionarily conserved chaperonin TCP-1 ring complex (TRiC) as a novel regulator of MES. Knockdown of TRiC subunits in the PG caused a prolonged mitotic period, probably due to impaired nuclear translocation of Fzr, which also caused loss of ecdysteroidogenic activity. These results indicate that TRiC supports proper MES and endocycle progression by regulating Fzr folding. We propose that TRiC-mediated protein quality control is a conserved mechanism supporting MES and endocycling, as well as subsequent terminal differentiation

Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in <i>Drosophila melanogaster</i>

<div><p>Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in <i>Drosophila melanogaster</i>. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation.</p></div

RagA and InR facilitate endocycling in the PG.

<p>(A) Expression of RagA^DN and InR^DN in the PG causes delay in DNA content increase. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control (<i>phm22 > +</i>), RagA^DN (<i>phm22 > RagA</i>.<i>T16N</i>) and InR^DN (<i>phm22 > InR</i>.<i>K1409A</i>) animals were labeled for Dib (green) and DNA (white) at indicated stages. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (B) The C value of PG cells of control, RagA^DN and InR^DN larvae at indicated stages. Average C value of control at 120 hAEL is normalized to 64C. Error bars represent standard errors. 11–16 PGs were analyzed for each group. (C) Expression of RagA^DN and InR^DN in the PG causes delay in pupariation. Percentages of pupariated control, RagA^DN and InR^DN animals are shown at indicated stages. Numbers of animals tested are in parentheses. (D) Expression of TOR^DN in the PG of InR^CA abolishes acceleration of DNA content increase. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control, InR^CA (<i>phm22 > InR</i>.<i>A1325D</i>) and InR^CA +TOR^DN (<i>phm22 > InR</i>.<i>A1325D</i>, <i>TOR</i>.<i>TED</i>) larvae were labeled for Dib (green) and DNA (white) at 96 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (E) The C value of PG cells of control, InR^CA and InR^CA +TOR^DN larvae at 96 hAEL. Average C value in control at 96 hAEL is normalized to 32C, according to data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#pgen.1006583.g004" target="_blank">Fig 4B</a>. Error bars represent standard errors. Numbers of animals tested are in parentheses. (F) Expression of TOR^DN in the PG of InR^CA abolishes acceleration of pupariation. Percentages of pupariated control, InR^CA and InR^CA +TOR^DN animals are shown at indicated stages. Numbers of animals tested are in parentheses. (G) Knockdown of <i>Rheb</i> and <i>raptor</i>, but not <i>rictor</i>, in the PG causes reduction in DNA content. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control (<i>phm22 > +</i>), <i>Rheb</i> RNAi (<i>phm22 > Rheb RNAi</i>), <i>raptor</i> RNAi (<i>phm22 > raptor RNAi</i>), <i>rictor</i> RNAi-1 (<i>phm22 > rictor RNAi-1</i>), and <i>rictor</i> RNAi-2 (<i>phm22 > rictor RNAi-2</i>) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (H) The C value of PG cells of control, <i>Rheb</i> RNAi, <i>raptor</i> RNAi, <i>rictor</i> RNAi-1, and <i>rictor</i> RNAi-2 larvae at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. (I) Knockdown of <i>Rheb</i> and <i>raptor</i>, but not <i>rictor</i>, in the PG causes arrest at the 3rd instar larval stage. Developmental profiles of control, <i>Rheb</i> RNAi, <i>raptor</i> RNAi, <i>rictor</i> RNAi-1, and <i>rictor</i> RNAi-2 animals are shown. Numbers of animals tested are in parentheses. (J) Percentages of pupariated control, <i>Rheb</i> RNAi, <i>raptor</i> RNAi, <i>rictor</i> RNAi-1, and <i>rictor</i> RNAi-2 animals are shown at indicated stages. Numbers of animals tested are shown in E. (K) Knockdown of <i>Rheb</i> and <i>raptor</i> in the PG of InR^CA abolishes acceleration of DNA content increase. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control, InR^CA (<i>phm22 > InR</i>.<i>A1325D</i>), InR^CA +<i>Rheb</i> RNAi (<i>phm22 > InR</i>.<i>A1325D</i>, <i>Rheb RNAi</i>), and InR^CA +<i>raptor</i> RNAi (<i>phm22 > InR</i>.<i>A1325D</i>, <i>raptor RNAi</i>) larvae were labeled for Dib (green) and DNA (white) at 96 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (L) The C value of PG cells of control, InR^CA, InR^CA +<i>Rheb</i> RNAi, and InR^CA +<i>raptor</i> RNAi larvae at 96 hAEL. Average C value in control at 96 hAEL is normalized to 32C, according to data in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#pgen.1006583.g004" target="_blank">Fig 4B</a>. Error bars represent standard errors. Numbers of animals tested are in parentheses. (M) Knockdown of <i>Rheb</i> and <i>raptor</i> in the PG of InR^CA abolishes acceleration of pupariation. Percentages of pupariated control, InR^CA, InR^CA +<i>Rheb</i> RNAi, and InR^CA +<i>raptor</i> RNAi animals are shown at indicated stages. Numbers of animals tested are in parentheses. (N) Schematic diagram of the temperature-shift experiment. (O) Expression of InR^CA triggers DNA content increase during starvation before the CW checkpoint. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control +Gal80^ts and InR^CA +Gal80^ts animals were labeled for Dib (green) and DNA (white) at indicated stages and temperature. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (P and Q) The C value of PG cells of control +Gal80^ts and InR^CA +Gal80^ts animals starved from 108 hAEL at 18°C (I) and 29°C (J). The CW checkpoint in control +Gal80^ts is indicated by a dashed line. The C value is normalized using that in control +Gal80^ts at 192 hAEL fed on standard <i>Drosophila</i> medium continuously at 18°C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#pgen.1006583.s006" target="_blank">S6E and S6F Fig</a>). Error bars represent standard errors. 13–18 PGs were analyzed for each group. (R and S) Expression of InR^CA in the PG of animals starved before the CW checkpoint triggers pupariation. Percentages of pupariated control +Gal80^ts and InR^CA +Gal80^ts animals starved from 108 hAEL at 18°C (K) and 29°C (L) are shown. The CW checkpoint in control +Gal80^ts is indicated by a dashed line. 30 animals were tested in each group. (T) Control +Gal80^ts animal arrested at the larval stage (left) and pupariated InR^CA +Gal80^ts animal starved from 108 hAEL at 29°C (right). (U) Model for the CW checkpoint mechanism in <i>Drosophila</i>. TOR-mediated endocycle progression in PG cells functions as an intrinsic timer that irreversibly activates ecdysone biosynthesis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#sec009" target="_blank">Discussion</a>). A. A., amino acid.</p

CW attainment is correlated with endocycle activity in the PG.

<p><b>(A)</b> Starvation before CW attainment causes arrest of DNA content increase in the PG. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) were labeled for Dib (green) and DNA (white) at indicated stages of the continuous feeding scheme. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). <b>(B and C)</b> The C value (B) and number (C) of PG cells of control, late starved and early starved larvae at indicated stages. The CW checkpoint in control is indicated by a dashed line in B. Average C value in control at 108 hAEL is normalized to 64C. Error bars represent standard errors. 10–17 PGs were analyzed for each group. <b>(D)</b> Starvation before CW attainment causes decrease in CycE expression and EdU incorporation in the PG. The PGs were labeled for DNA (blue) and CycE or EdU (magenta) at 96 hAEL. The PGs are outlined by dashed lines. Scale bars, 50 μm. <b>(E and F)</b> Percentages of CycE-positive (E) and EdU-positive (F) s-phase PG cells in control, late starved and early starved larvae at indicated stages. All data are shown as box plot, with a box representing lower and upper quartiles, a horizontal line representing median, and bars representing minimum and maximum data points. The CW checkpoint in control is indicated by dashed lines. 19–30 PGs were analyzed for each group.</p

TOR is required for endocycle progression to activate ecdysone biosynthesis.

<p><b>(A)</b> Expression of TOR^DN in the PG causes arrest in DNA content increase at the 3rd instar larval stage. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) of control (<i>phm22 > +</i>) and TOR^DN (<i>phm22 > TOR</i>.<i>TED</i>) animals were labeled for Dib (green) and DNA (white) at 78 and 114 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). <b>(B and C)</b> The C value (B) and number (C) of PG cells of control and TOR^DN larvae at indicated stages. The CW checkpoint in control (81 hAEL, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#pgen.1006583.s004" target="_blank">S4O Fig</a>) is indicated by a dashed line in B. 11–19 PGs were analyzed for each group. Average C value in control at 114 hAEL is normalized to 64C. Error bars represent standard errors. <b>(D)</b> Expression of TOR^DN in the PG causes reduction in CycE expression and EdU incorporation at the 3rd instar larval stage. The PGs of control +GFP (<i>phm22 > mCD8</i>::<i>GFP</i>) and TOR^DN +GFP (<i>phm22 > mCD8</i>::<i>GFP</i>, <i>TOR</i>.<i>TED</i>) larvae were labeled for GFP (green) and CycE or EdU (magenta) at indicated stages. The PGs are outlined by dashed lines. Scale bars, 50 μm. <b>(E and F)</b> Percentages of CycE-positive (E) and EdU-positive (F) PG cells in control +GFP and TOR^DN +GFP larvae at indicated stages. All data are shown as box plot (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006583#pgen.1006583.g002" target="_blank">Fig 2E and 2F</a>). The CW checkpoint in control is indicated by dashed lines. 17–26 PGs were analyzed for each group. <b>(G)</b> Expression of <i>CycE</i> in the PG of TOR^DN restores CycE protein expression. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in TOR^DN +GFP, TOR^DN +CycE-1 +GFP (<i>phm22 > mCD8</i>::<i>GFP</i>, <i>TOR</i>.<i>TED</i>, <i>CycE-1</i>) and TOR^DN +S6K^TE +GFP (<i>phm22 > mCD8</i>::<i>GFP</i>, <i>TOR</i>.<i>TED</i>, <i>S6K</i>.<i>TE</i>) larvae were labeled for GFP (green) and CycE (magenta) at 84 hAEL. Scale bars, 50 μm. <b>(H)</b> Percentages of CycE-positive PG cells in TOR^DN +GFP, TOR^DN +CycE-1 +GFP and TOR^DN +S6K^TE +GFP larvae at 84 hAEL. All data are shown as box plot. Numbers of animals tested are in parentheses. <b>(I)</b> Expression of <i>CycE</i> in the PG of TOR^DN rescues reduction in DNA content. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in TOR^DN, TOR^DN +CycE-1 (<i>phm22 > TOR</i>.<i>TED</i>, <i>CycE-1</i>) and TOR^DN +S6K^TE (<i>phm22 > TOR</i>.<i>TED</i>, <i>S6K</i>.<i>TE</i>) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). <b>(J)</b> The C value of PG cells of TOR^DN, TOR^DN +CycE-1 and TOR^DN +S6K^TE larvae at 120 hAEL. Average C value in TOR^DN is normalized to 11C, according to data in B. Error bars represent standard errors. Numbers of animals tested are in parentheses. <b>(K)</b> Expression of <i>CycE</i> in the PG of TOR^DN restores expression of ecdysone biosynthetic genes. Expression of ecdysone biosynthetic genes in TOR^DN, TOR^DN +CycE-1 and TOR^DN +S6K^TE larvae at 120 hAEL was measured using qPCR. Average values of three independent data sets are shown with standard errors. <b>(L)</b> Expression of <i>CycE</i> in the PG of TOR^DN rescues decrease in ecdysteroid level. Whole-body ecdysteroid levels in TOR^DN, TOR^DN +CycE-1 and TOR^DN +S6K^TE larvae at 120 hAEL were measured using ELISA. Average values of five independent data sets are shown with standard errors. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (* <i>P</i> < 0.05). <b>(M)</b> Expression of <i>CycE</i> in the PG of TOR^DN rescues developmental arrest. Percentages of pupariated TOR^DN, TOR^DN +CycE-1 and TOR^DN +S6K^TE animals are shown at indicated stages. Numbers of animals tested are in parentheses. <b>(N)</b> TOR^DN animal arrested at 3rd instar larval stage (left) and pupariated TOR^DN +CycE-1 animal (right).</p

CW attainment is coupled with activation of ecdysone biosynthesis.

<p><b>(A)</b> Schematic diagram of the continuous feeding scheme. Wild-type <i>Oregon R</i> larvae were reared on standard <i>Drosophila</i> medium (solid line) either continuously (control) or starved (dashed lines) from indicated time points. The CW checkpoint (78 hAEL) is indicated by a shaded box. <b>(B)</b> Schematic diagram of ecdysone biosynthetic pathway. <b>(C–H)</b> Starvation before CW attainment impairs increase in expression of ecdysone biosynthetic genes. Expression of ecdysone biosynthetic genes in control, late starved and early starved larvae was measured using qPCR. The CW checkpoint in control is indicated by dashed lines. Average values of five independent data sets are shown with standard errors. <b>(I)</b> Starvation before CW attainment causes decrease in expression of ecdysone biosynthetic genes in the PG. Whole-mount <i>in situ</i> hybridization was performed using antisense probes in control and starved larvae (late and early starvation) at 108 hAEL. The PGs are outlined by dashed lines. Scale bars, 50 μm. <b>(J)</b> Starvation before CW attainment causes decrease in ecdysteroid level. Whole-body ecdysteroid levels in control, late starved and early starved larvae at 108 hAEL were measured using ELISA. Average values of five independent data sets are shown with standard errors. Statistical significance was calculated using ANOVA with Tukey’s post hoc test (*<i>P</i> < 0.05). <b>(K)</b> 20E feeding rescues developmental arrest in early starved larvae. Larvae were starved on wet filter paper with or without 1 μg/ml 20E from 72 hAEL. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses. <b>(L)</b> Control starved larva (left) and pupariated animal by 20E feeding (right).</p

Endocycle is required for ecdysone biosynthesis.

<p><b>(A)</b> Knockdown of <i>CycE</i> in the PG causes reduction in DNA content. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) of control (<i>phm22 > dicer2</i>) and <i>CycE</i> RNAi-1 (<i>phm22 > dicer2</i>, <i>CycE RNAi-1</i>) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). <b>(B and C)</b> Knockdown of endocycle regulators in the PG causes reduction in DNA content. Each gene was knocked down using two independent RNAi lines. The C value (B) and number (C) of PG cells of control (black bars) and RNAi (<i>phm22 > dicer2</i>, <i>RNAi</i>; gray bars) larvae at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. Significance was calculated using Student’s <i>t</i>-test (* <i>P</i> < 0.001; n.s., Not significant). <b>(D)</b> Knockdown of endocycle regulators in the PG causes arrest at the 3rd instar larval stage. Developmental profiles of control and RNAi animals are shown. Numbers of animals tested are in parentheses. <b>(E)</b> Pupariated control animal (left) and <i>CycE</i> RNAi-1 larva arrested at the 3rd instar stage (right). <b>(F)</b> Knockdown of <i>Fzr</i> in the PG causes block of mitotic-to-endocycle transition. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control (<i>phm22 > dicer2</i>), <i>Fzr</i> RNAi (<i>phm22 > dicer2</i>, <i>Fzr RNAi</i>) and <i>Fzr</i> RNAi +InR^CA (<i>phm22 > dicer2</i>, <i>Fzr RNAi</i>, <i>InR</i>.<i>A1325D</i>) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). <b>(G and H)</b> The C value (G) and number (H) of PG cells of control, <i>Fzr</i> RNAi and <i>Fzr</i> RNAi +InR^CA larvae at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. <b>(I)</b> Knockdown of <i>Fzr</i> causes reduction in expression of ecdysone biosynthetic genes. Expression of ecdysone biosynthetic genes in control, <i>Fzr</i> RNAi and <i>Fzr</i> RNAi +InR^CA larvae at 120 hAEL was measured using qPCR. Average values of three independent data sets are shown with standard errors. <b>(J)</b> Knockdown of <i>Fzr</i> causes decrease in ecdysteroid level. Whole-body ecdysteroid levels in control, <i>Fzr</i> RNAi, and <i>Fzr</i> RNAi +InR^CA larvae at 120 hAEL were measured using ELISA. Average values of five independent data sets are shown with standard errors. Statistical significance was calculated using Student’s <i>t</i>-test (* <i>P</i> < 0.05). <b>(K)</b> Knockdown of <i>Fzr</i> in the PG causes developmental arrest at the 3rd instar larval stage. Percentages of pupariated animals in control, <i>Fzr</i> RNAi and <i>Fzr</i> RNAi +InR^CA animals are shown at indicated stages. Numbers of animals tested are in parentheses. <b>(L)</b> Pupariated control animal (left) and <i>Fzr</i> RNAi and <i>Fzr</i> RNAi +InR^CA larvae arrested at the 3rd instar stage (middle and right, respectively). <b>(M)</b> 20E feeding rescues developmental arrest in <i>Fzr</i> RNAi. Percentages of pupariated <i>Fzr</i> RNAi animals reared on 20E-containing or control medium from 72 hAEL are shown at indicated stages. Numbers of animals tested are in parentheses.</p