Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in <em>Drosophila</em>

<div><p>In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use <em>Drosophila melanogaster</em> as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.</p> </div

Cell Ablation Strategy.

<p>(A) Strategy used to produce Ablating and Non-Ablating larvae. <i>w*; P{Sgs3-GFP}3</i> females were crossed to <i>w*;+;rn-GAL4,UAS-egr,tubGAL80^ts/TM6Tb, tubGAL80</i> males to give rise to the Ablating genotype (<i>w*;+;rn-GAL4,UAS-egr,tubGAL80^ts/Sgs3GFP</i>) and the control Non-Ablating genotype (<i>w*;+;TM6Tb, tubGAL80/Sgs3GFP)</i>. (B) Strategy to induce cell ablation. Embryos of the Ablating and Non-Ablating genotypes were collected at room temperature in four hour intervals and transferred to 18°C. First-instar larvae (48 hours AEL) were transferred to a vial containing standard cornmeal-yeast-agar medium and were allowed to develop at 18°C until the designated time for ablation induction. At the designated time during L3 (130–230 hours AEL) vials were transferred to 30°C for 40 hours, returned to 18°C and monitored daily to document the time to <i>Sgs3GFP</i> expression, pupariation or eclosion. RNA for qPCR and samples for EIA experiments were collected at time points T₀–T₃.</p

Primers Used for qPCR.

<p>Primers Used for qPCR.</p

Damage to Wing Imaginal Discs Delays Pupariation and Adult Eclosion.

<p>(A–F) Timing of pupariation and adult eclosion following induction of cell ablation in the wing disc at the indicated time. Fraction of larvae that had (A, C, E) pupariated or (B, D, F) eclosed as adults are plotted relative to time in hours AEL for Ablating (Red - <i>w*; rnGAL4, UAS-egr, tubGAL80^ts/Sgs3GFP</i>) and Non-Ablating (Blue - <i>w*; TM6, Tb¹, tubGAL80/Sgs3GFP</i>) larvae. n = 3 independent populations (30 larvae each) were assayed for each ablation time. (A–B) Timing of (A) pupariation and (B) adult eclosion for larvae heat-treated at 173 hours AEL. Mean pupariation times are 286 and 227 hours AEL for Ablating and Non-ablating larvae, respectively. Mean eclosion times are 504 and 440 hours AEL for Ablating and Non-ablating larvae, respectively. Similar results were obtained with larvae heat-treated at 150, 162, or 184 hours AEL (data not shown). (C–D) Timing of (C) pupariation and (D) adult eclosion for larvae heat-treated at 198 hours AEL. Mean pupariation times are 279 and 230 hours AEL for ablating and non-ablating larvae, respectively. Mean eclosion times are 488 and 409 hours AEL for ablating and non-ablating larvae, respectively. Similar results were obtained with larvae heat-treated at 190 hours AEL (data not shown). (E-F) Timing of (E) pupariation and (F) adult eclosion for larvae heat-treated at 223 hours AEL. Mean pupariation times are 263 and 265 hours AEL for ablating and non-ablating larvae, respectively. Mean eclosion times are 419 and 411 hours AEL for ablating and non-ablating larvae, respectively. Similar results were obtained with larvae heat-treated at 213 and 220 hours AEL (data not shown).</p

Effects of Tissue Damage on Ecdysone Biosynthesis and Signaling.

<p>qRT-PCR analysis of transcript levels of (A) <i>ptth</i>, (B) genes required for ecdysone synthesis, (C) <i>shd</i>, (D) ecdysone inducible genes, (E) ecdysone receptor components and (F) <i>ecdysone oxidase</i>. Graphs show changes in transcript levels 24 hours after heat treatment (T₃; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g002" target="_blank">Figure 2B</a>) compared to transcript levels immediately before heat treatment (T₀; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g002" target="_blank">Figure 2B</a>). Ablating (Red - <i>w*/w¹¹¹⁸; rnGAL4, UAS-egr, tubGAL80ts/+</i>). Non-Ablating (Blue - <i>w*/w¹¹¹⁸; TM6, Tb¹, tubGAL80/+</i>).</p

Ecdysteroid Titers Following Wing Disc Cell Ablation.

<p>Ecdysteroid titers measured for larvae of Ablating (Red) and Non-Ablating (Blue) genotypes at time points T₀–T₃ (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g001" target="_blank">Figure 1B</a>), as determined by EIA. Values are expressed as the means of 20E equivalents per mg of tissue. Error bars indicate the SEM (n = 2 samples of 15 larvae each). Asterisks indicate differences statistically significant at p≤0.05 (t-test).</p

Early Response to Tissue Damage.

<p>qRT-PCR analysis of transcript levels of (A) <i>ptth</i>, (B) genes required for ecdysone synthesis, (C) <i>shd</i>, (D)ecdysone inducible genes, (E) ecdysone receptor components and (F) <i>ecdysone oxidase</i>. Graphs show changes in transcript levels mid-way through heat treatment (T₁; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g002" target="_blank">Figure 2B</a>) compared to transcript levels immediately before heat treatment (T₀; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g002" target="_blank">Figure 2B</a>). Ablating (Red - <i>w*/w¹¹¹⁸; rnGAL4, UAS-egr, tubGAL80ts/+</i>). Non-Ablating (Blue - <i>w*/w¹¹¹⁸; TM6, Tb¹, tubGAL80/+</i>).</p

Imaginal Disc Damage Delays the Mid-Third Instar Transition.

<p>(A–F) Timing of the mid-third instar transition (as measured by <i>Sgs3GFP</i> expression) in larvae that were heat-treated for 24 hours at 172 hours AEL to induce tissue damage in wing discs. (A–C) <i>Sgs3GFP</i> expression in Non-Ablating (<i>w*; TM6, Tb¹, tubGAL80/Sgs3GFP</i>) larvae. (A) <i>Sgs3GFP</i> expression is absent in most larvae at 164 hours AEL, just before heat treatment (Sgs3GFP⁺ = 3.6±4.7%; n = 28). <i>Sgs3GFP</i> is expressed at high levels after heat treatment in salivary glands at (B) 197 hours AEL (Sgs3GFP⁺ = 77.3±0.6%; n = 22), at (C) 215 hours AEL (Sgs3GFP⁺ = 91.7±7.9%; n = 12) and at 236 hours AEL (Sgs3GFP⁺ = 100±0.0%; n = 19) – data not shown. (D–F) <i>Sgs3GFP</i> expression in Ablating (<i>w*; rnGAL4, UAS-egr, tubGAL80^ts/Sgs3GFP</i>) larvae. (D) <i>Sgs3GFP</i> expression is absent in most larvae at 164 hours AEL, just before heat treatment (Sgs3GFP⁺ = 2.6±3.1%; n = 38). (E) <i>Sgs3GFP</i> expression is detected at low levels at 197 hours AEL in only a small fraction of larvae examined (Sgs3GFP⁺ = 10.0±13.3%; n = 30). (F) High levels of <i>Sgs3GFP</i> expression is visible at 215 hours AEL (Sgs3GFP⁺ = 53.9±37.9%; n = 26) and at 236 hours AEL (Sgs3GFP⁺ = 75.0±1.3%; n = 28) – data not shown.</p

Binding of CRC to EcR-B2 <i>in vitro</i>.

<p>(A) CRC-A bound in vitro to the 17 amino acid amino-terminus of EcR-B2 and full length EcR-B2, but not to EcR-B1 or EcR-B2-E9K. This interaction was not dependent upon USP or ecdysone. Gels from two experiments are shown; the labeled protein was ³⁵S-GAD-CRC-A in the left gel, ³⁵S-CRC-A in the right gel. Lanes labeled “input” contained 100% of the input labeled protein. The remaining samples contained the EcR or EcR fragment shown above, and other components as indicated. GBT-B2-NS is a fusion of the DNA-binding domain of GAL4 with the amino-terminal 17 amino acids of EcR-B2. In lanes labeled B2-E9K, E9K mutant EcR-B2 or E9K mutant GBT-B2-NS was substituted 1∶1 for the corresponding wild-type protein. Full-length EcRs were precipitated with a mixture of two EcR-common region monoclonal antibodies, and GBT-B2-NS was precipitated with an antibody to the GAL4 DNA-binding domain. (B) A similar experiment, showing the binding of wild-type and mutant CRCs to full-length EcR-B2. Each incubation mixture contained the ³⁵S-radiolabeled CRC protein listed above and the other components listed below, and precipitations were performed with the same mix of EcR common region antibodies as in (A). Wild-type CRC, CRC-R347E, and CRC-R353E bound to EcR-B2 but not EcR-B2-E9K. However, the basic-to-acidic mutation in CRC-R361E permitted binding to EcR-B2-E9K.</p

Switch in Injury Response Coincides with the Mid-third Instar Transition.

<p>Cell ablation was induced as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049105#pone-0049105-g001" target="_blank">Figure 1</a>) at various time points (arrows) during the third larval instar. Cell ablation resulted in delayed pupariation (Red Arrows), no effect on developmental timing (Green Arrows), or a mixed effect (Yellow Arrows) in which some animals delayed development and others developed at the same time as controls.</p