DOI: 10.5061/dryad.w9ghx3g4f
title: Hypoxia couples growth and developmental timing by decoupling steroid synthesis and secretion

description: In almost all animals, low physiological levels of oxygen (hypoxia) reduce
 growth rate and adult body size. Despite the near ubiquity of this
 response, the systemic mechanisms that coordinate growth and development
 under hypoxia remain poorly understood. In Drosophila, hypoxia increases
 circulating levels of the steroid hormone ecdysone to inhibit insulin/IGF
 signaling and slow growth. At the same time, ecdysone biosynthesis is
 reduced to delay pupation and extend development. Traditionally, the
 secretion of lipid-soluble steroids is thought to be regulated at the
 level of biosynthesis. This therefore presents a paradox: how can a single
 environmental factor both increase ecdysone levels yet decrease ecdysone
 synthesis? Our data show that this paradox is resolved by the dual
 regulation of ecdysone at the levels of secretion and biosynthesis. We
 show that, in the short term, hypoxia increases basal levels of ecdysone
 through the Atet-dependent exocytosis of ecdysone-containing vesicles. In
 the long term, hypoxia decreases the expression of ecdysone synthesis
 genes to delay the peak in ecdysone that triggers pupation. We also
 present evidence that both ecdysone synthesis and secretion are regulated,
 in part, by NO-signaling. Collectively, our findings reveal regulated
 steroid secretion as a critical and environmentally-responsive component
 of endocrine control, expanding our understanding of how animals integrate
 growth and developmental timing in response to acute and chronic
 environmental change.
description: Drosophila stocks and maintenance We used the
 following fly strains: the isogenic RNAi control (VDRC 60,000),
 <em>UAS Atet RNAi<sup>v42751</sup></em> (VDRC
 42751), <em>phm-GAL4</em> (a gift from Michael O’Connor),
 <em>P0206-Gal4</em> (a gift from Christen Mirth),
 <em>UAS-Syt-GFP</em> (BDSC 6925), <em>UAS-Atet
 RNAi<sup>50563</sup></em> (BDSC 50563), and
 <em>UAS-Bnl RNAi</em> (BDSC 34572). All constructs were
 backcrossed into VDRC 60,000, for five generations prior to analysis.
 Flies were reared at low density (50-100 larvae per vial), 25°C, 21%
 O<sub>2</sub> in constant light on a sucrose-yeast diet
 containing 13 g of carrageenan, 100 g of yeast extract, and 50 g of
 sucrose in 1000 ml of water. To prevent bacterial and fungal growth, we
 added 3% Nipagin and 0.3% (v/v) propionic acid to the cooled
 mixtures. Body size measurement All
 larvae were reared under standard conditions (25<sup>o</sup>C,
 21% O<sub>2</sub>) and staged into 2h cohorts at ecdysis to
 the third instar before being maintained at 25°C, 21%
 O<sub>2</sub> (normoxic flies) or moved to 25°C, 10%
 O<sub>2</sub> (hypoxic flies) for the remainder of their
 development. We used pupal case size as a measure of body size. We
 collected digital images of the pupal cases and measured the area of the
 pupal case when viewed from the dorsal aspect using ImageJ. Measurements
 were log transformed prior to analysis. Growth rate
 measurements <em>P0206&gt;+</em> and
 <em>P0206&gt;HIF-1α.RNAi</em> larvae were reared under
 standard conditions (25<sup>o</sup>C, 21%
 O<sub>2</sub>) and massed at ecdysis to L3 (72h after
 oviposition) and 24h later (96h after oviposition). Specific growth rate
 (mg/d) was calculated as log M<sub>96</sub> – log
 M̅<sub>72</sub>, where
 <em>M<sub>x</sub></em> is mass at age
 <em>x.</em> Quantitative PCR
 (qPCR) VDRC <em>60,000</em> larvae were
 reared under standard conditions (25<sup>o</sup>C, 21%
 O<sub>2</sub>) and staged into 2h cohorts at ecdysis to the
 third instar before being maintained at 25°C, 21%
 O<sub>2</sub> (normoxic flies) or moved to 25°C, 10%
 O<sub>2</sub> (hypoxic flies). Larvae were subsequently
 collected every four or six hours for 24 hours and preserved in groups of
 ten in RNAlater (Thermo Fischer Scientific). We extracted the RNA using
 Trizol (Invitrogen), treated it with DNase I (Thermo Fischer Scientific),
 quantified it with a NanoDrop One (Thermo Fischer Scientific) and reverse
 transcribed it with a High-Capacity cDNA Reverse Transcription Kit
 (Applied Biosystems). Quantitative RT–PCR was conducted using PowerUp SYBR
 Green Master Mix (Applied Biosystems) and measured on a QuantStudio 3
 Real-Time PCR system (Applied Biosystems). We calculated mRNA abundance
 for three biological replicates of ten larvae, using a standard curve and
 normalizing against the expression of ribosomal protein 49
 (<em>RP49</em>). Standard curves were generated using seven
 serial dilutions of total RNA extracted from the isogenic control line
 (60,000): 5x 1st instar larvae, 5x 2nd instar larvae, 3rd instar larvae
 (male), 5x pupae (male), 5x adult flies (male). Primer sequences used in
 the study are listed in Supplementary Table S1.
 Statistical analyses All statistical analyses
 were conducted in <em>R (66).</em> All the data and the
 R-scripts used to analyze them are provided as supplementary information.
 The effects of genotype on the response of body size and specific growth
 rate to hypoxia were tested using two-way ANOVAs (<em>body
 size/specific growth rate = genotype</em>oxygen),* with post hoc
 Šidák-corrected comparisons. The effect of genotype on developmental time
 was tested using a binomial GLMM <em>(pupation =
 age</em>genotype + vial),* where vial was treated as a random
 factor, with post hoc Tukey-corrected pairwise comparisons between
 genotypes. The effect of oxygen level on gene expression was tested using
 two-way ANOVAs <em>(expression = age</em>oxygen*), where
 <em>age</em> is a categorical variable, with <em>post
 hoc</em> Šidák-corrected comparisons within specific ages.
description: # Hypoxia couples growth and developmental timing by decoupling steroid
 synthesis and secretion Dataset DOI:
 [10.5061/dryad.w9ghx3g4f](https://doi.org/10.5061/dryad.w9ghx3g4f) ##
 Description of the data and file structure The data were collected to test
 the hypothesis that atet-mediated secretion of ecdysone from the
 prothoracic gland slows growth in response to hypoxia in *Drosophila*. ###
 Interpretation keys h AEL3: hours after ecdysis to the third larval instar
 Normoxia: 21% O~2~ Hypoxia: 10% O~2~ ### Files and variables #### File:
 Hypoxia_Analysis_Script.Rmd **Description:** The script in this file
 applies all the statistical tests and builds all the charts used in the
 paper [Hypoxia couples growth and developmental timing by decoupling
 steroid synthesis and
 secretion](https://www.biorxiv.org/content/10.64898/2026.01.12.699026v2): bioRxiv 2026.01.12.699026; doi: [https://doi.org/10.64898/2026.01.12.699026](https://doi.org/10.64898/2026.01.12.699026).  #### File: qPCR1.csv **Description:**  ##### Variables * O2: Oxygen level (Normoxia, Hypoxia), categorical * age: Age (h AEL3), continuous * age_cat: Age (h AEL3), categorical * dNOS: Relative expression of *dNOS* * PTTH: Relative expression of *PTTH* * E75B: Relative expression of *E75B* * phantom: Relative expression of *phantom* * disembodied: Relative expression of *disembodied*  * torso: Relative expression of *torso* * dHR4: Relative expression of *DHR4* * Cyp18a1: Relative expression of *Cyp18a1* * shroud: Relative expression of *shroud* * atet: Relative expression of *atet* * ecdysone importer: Relative expression of *ecdysone importer* #### File: qPCR2.csv **Description:**  ##### Variables * O2: Oxygen level (Normoxia, Hypoxia), categorical * age: Age (h AEL3), continuous * age_cat: Age (h AEL3), categorical * branchless: Relative expression of *branchless* * neverland: Relative expression of *neverland* * shadow: Relative expression of *shadow* #### File: NOS_KD_size.csv **Description:**  ##### Variables * Genotype: Genotype larva * Oxygen: Oxygen level (Normoxia, Hypoxia), categorical * Pupal Area: Pupal area (a proxy for body size) (μm^2^) * Log Pupal Area: Log pupal area (μm^2^) #### File: Atet_KD_size.csv **Description:**  ##### Variables * Genotype: Genotype of larva * Oxygen: Oxygen level (Normoxia, Hypoxia), categorical * Pupal Area: Pupal area (a proxy for body size)  (μm^2^) * Log Pupal Area: Log pupal area (μm^2^) #### File: HIF_KD_developmental_time.csv **Description:**  ##### Variables * genotype: Genotype of larva * vial: Rearing vial * age: Age (hours after egg lay) * pupated: Number of pupated larvae in the vial * not_pupated: Number of non-pupated larvae in the vial #### File: Atet_qPCR.csv **Description:**  ##### Variables * O2: Oxygen level (Normoxia, Hypoxia), categorical * age: Age (h AEl3) * Atet:  Relative expression of *atet* #### File: HIF_KD_growth.csv **Description:**  ##### Variables * genotype: Genotype * age: Age (h AEL3) * condition: Oxygen level (Normoxia, Hypoxia), categorical * mass(mg): Larval mass (mg) * log mass: Log larval mass (mg) * sgr: Specific growth rate #### File: NOS_Atet_KD_size.csv **Description:**  ##### Variables * Genotype: Genotype * Oxygen: Oxygen level (Normoxia, Hypoxia), categorical * Pupal Area: Pupal area (a proxy for body size) (μm^2^) * Log Pupal Area: Log pupal area (μm^2^) #### File: HIF_HPH_KD_size.csv **Description:**  ##### Variables * sex: Sex * type: Genotype * size: Log pupal area (μm^2^) #### File: Branchless_KD.csv **Description:**  ##### Variables * type: UAS-reponse element (*UAS-branchless.RNAi* or control) * sex: sex * driver: GAL4 driver * size: Log pupal area (μm^2^) ## Code/software The analysis code is written in R Markdown and can be run using R Studio Version 2025.05.1+513, with R version 4.5.1.