Glue protein production can be triggered by steroid hormone signaling independent of the developmental program in Drosophila melanogaster

Abstract

Steroid hormones regulate life stage transitions, allowing animals to appropriately follow a developmental timeline. During insect development, the steroid hormone ecdysone is synthesized and released in a regulated manner by the prothoracic gland (PG) and then hydroxylated to the active molting hormone, 20-hydroxyecdysone (20E), in peripheral tissues. We manipulated ecdysteroid titers, through temporally controlled over-expression of the ecdysteroid-inactivating enzyme, CYP18A1, in the PG using the GeneSwitch-GAL4 system in the fruit fly Drosophila melanogaster. We monitored expression of a 20E-inducible glue protein gene, Salivary gland secretion 3 (Sgs3), using a Sgs3:GFP fusion transgene. In wild type larvae, Sgs3-GFP expression is activated at the midpoint of the third larval instar stage in response to the rising endogenous level of 20E. By first knocking down endogenous 20E levels during larval development and then feeding 20E to these larvae at various stages, we found that Sgs3-GFP expression could be triggered at an inappropriate developmental stage after a certain time lag. This stage-precocious activation of Sgs3 required expression of the Broad-complex, similar to normal Sgs3 developmental regulation, and a small level of nutritional input. We suggest that these studies provide evidence for a tissue-autonomic regulatory system for a metamorphic event independent from the primary 20E driven developmental progression.

Keywords: Broad Complex; Developmental checkpoint; Drosophila melanogaster; Salivary gland; Salivary gland secretion 3; Steroid hormone.

Fig. 1

Expression of UAS-transgene in the PG by application of RU486. GFP expression of flies carrying single copies of phm-GS-GAL4 and UAS-GFP. (A) GFP fluorescence observed in the PG of L3 larvae by feeding RU486 at 10 μm final concentration. Typical GFP expression at the indicated time after administration of RU486 are shown in the lower panels. (B) Quantitative RT-PCR analysis of the transcriptional levels of GFP. The transcription level of GFP in larvae just after ecdysis is represented as 1. Each value is plotted as a dot (n = 5–6). Box plot shows 25–75% (box), median (band inside) and minima to maxima (whiskers) (light blue: non-RU486-fed larvae; light red: RU486-fed larvae). Boxes with different letters are significantly different at p < 0.05 by Tukey’s HSD test.

Fig. 2

Developmental profiles of animals forced to inactivate ecdysteroids in the PG using phm-GS-GAL4 driver. (A) Development of animals affected by ectopic expression of GAL4 protein in the PG by application of RU486. Overexpression of GAL4 protein in the PG using a high dosage of RU486 caused developmental arrest at early larval stages, which could be partially rescued by administration of E. (B, C) Developmental profiles of animals, steroidogenesis of which was inactivated by overexpression of CYP18A1 (B) or RNAi mediated knockdown of spok (C) by application of RU486. The arrest of development was partially rescued by administration of E. (D) Developmental profile of ecdysteroid-defective animals by administration of RU486 at different concentrations. (E) Comparison of pupariation timing of L2 and L3 larvae. (F) A L2 pharate adult. Pupal case was removed (right). (A–E) Numbers in parentheses in the figures represent the number of animals. L2PP, IP, PP and PA indicate L2 prepupa, incomplete prepupa, prepupa and pharate adult, respectively.

Fig. 3

Sgs3-GFP expression is triggered by application of 20E at any larval stage even before attainment of MVW. (A) Schematic representation of 20E titer from the L2 stage to pupariation. (B) Predicted 20E titer of larvae fed with RU486 just after L2-L3 molting. 20E was applied at around the time of the onset of pupariation. Larvae did not initiate pupariation, but showed Sgs3-GFP expression. (C) Predicted 20E titer of larvae fed with RU486 just after L1-L2 molting. 20E was applied at 24 h AL2E, the timing of which corresponded to L2-L3 molting. Larvae did not molt to the L3 stage, but showed Sgs3-GFP expression at the L2 stage. (D) Predicted 20E titer of larvae fed with RU486 just after hatch. 20E was applied at 48 h after hatch, the timing of which corresponded to L2-L3 molting. Larvae did not molt to the L2 stage, but showed Sgs3-GFP expression at the L1 stage. (E) Percentage of animals fed with normal food that underwent pupation after starvation at a given size (n = 12–20 for each interval). The MVW (vertical dashed line) was estimated from a weight which corresponds to the 50% threshold (horizontal dashed line) for pupation after starvation. The vertical solid line indicates the average weight of L2-arrested larvae expressing Sgs3-GFP (0.48 ± 0.013 mg, n = 14, error is SE). (F) Developmental profile of L1 arrested larvae fed with RU486 after hatch. Larvae were administered 20E at 24 h or 48 h AH. ^** p < 0.00001. P-values were calculated in Chi-square test using residual analysis. (G) RT-PCR analysis of induction of endogenous glue genes, Sgs3 and Sgs5, in ecdysteroid depleted L2 larvae. Sgs3 and Sgs5 expressions were induced at 6 and 8 h after application of 20E, respectively. (H) Administration of a JH analog does not inhibit Sgs3-GFP expression. Percentage of L3 larvae showing Sgs3-GFP fluorescence. No significant difference in the frequency of Sgs3-GFP expression was detected between methoprene-fed larvae and unsupplied larvae at each stage (Fisher’s exact test: p > 0.05). (I) Immunostaining of EcR or Br-C in SGs of L2 larvae at 28 h AL2E. L2 larvae forced to inactivate ecdysteroids were administered 20E or none at 18 h AL2E. Scale bar: 100 μm. (J) Transcriptional levels of EcR and Br-C in SGs of L2 larvae at 28 h AL2E. L2 larvae forced to inactivate ecdysteroids were administered 20E or none at 18 h AL2E. The expression level of unsupplied-larvae is represented as 1. Each value is plotted as a dot (n = 6). Box plot shows 25–75% (box), median (band inside) and minima to maxima (whiskers). Student’s t-test: *p < 0.05, **p < 0.001. (F, H) Numbers in parentheses in the figures represent the number of animals.

Fig. 4

Developmental profiles of animals without manipulation of ecdysteroidogenesis under nutritional defective conditions. Developmental profiles of animals carrying a transgene Sgs3:GFP without manipulation of ecdysteroidogenesis were examined at 25 °C. (A, B) Developed stages of animals. Larvae were transferred to an agar medium containing 20E (B) or none (A), after feeding on normal food until the time indicated in the panel. AL2E and AL3E indicate after L1-L2 ecdysis and after L2-L3 ecdysis, respectively. Numbers in parentheses in the figure represent the number of animals. (C) Percentage of animals showing Sgs3-GFP fluorescence. Larvae were transferred to an agar medium containing 20E, after feeding on normal food until the indicated time. No larva transferred to an agar medium without 20E-supplementation showed Sgs3-GFP fluorescence.

Fig. 5

Developmental fates of ecdysteroid-defective L2 larvae administered 20E at the different times. Percentage of developmental stages of phm-GS > CYP18A1: Sgs3-GFP larvae. L2 larvae just after ecdysis were fed food containing RU486 at 0 h AL2E to inactivate ecdysteroids in the PG cells. These larvae were fed 20E at a different time indicated in the panel. Numbers in parentheses in the figures represent the number of animals.

Fig. 6

Time requirement for Sgs3-GFP expression after 20E-treatment due to requirement for preliminary activation of Br-C in SG cells. (A) Percentage of L2 larvae showing Sgs3-GFP fluorescence. Larvae were fed RU486 just after L1-L2 molting, and then supplied 20E at 18 h (red) or 24 h (blue) AL2E. (B) Percentage of L3 larvae showing Sgs3-GFP fluorescence. Larvae fed with normal food (green) or normal food with 20E supplemented at 12 h AL3E (red) or larvae fed RU486 just after L2-L3 molting, and then supplemented with 20E at 12 h AL3E (blue). (C) Immunostaining of EcR and Br-C in SGs of L3 larvae at 0, 6, or 12 h AL3E. In each set of staining, the first three panels are minus RU486, while the last panel shows the results of treating with RU486 starting at L2/L3 ecdysis. Scale bar: 100 μm. (A, B) Numbers in parentheses in the figures represent the number of animals.

Fig. 7

A model of heterochronic expression of Sgs3-GFP in a tissue autonomous manner. While 20E triggers developmental transitions including molting and metamorphosis, depletion of 20E causes failure of larval or metamorphic molt. However, a metamorphic event independent from the primary developmental progression initiates in a tissue autonomous manner, i.e. another 20E independent clock or process continues in a temporally unabated fashion in the absence of 20E so that competence to express Sgs3-GFP responding to exogenous 20E has developed. Therefore, when 20E is added at a subsequent time, Sgs3-GFP expression is induced at a stage-aberrant time.