Hypoxia couples growth and developmental timing by decoupling steroid synthesis and secretion

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. ..., Drosophila stocks and maintenance
We used the following fly strains: the isogenic RNAi control (VDRC 60,000), UAS Atet RNAiv42751 (VDRC 42751), phm-GAL4 (a gift from Michael O’Connor), P0206-Gal4 (a gift from Christen Mirth), UAS-Syt-GFP (BDSC 6925), UAS-Atet RNAi50563 (BDSC 50563), and UAS-Bnl RNAi (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% O2 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 (25oC, 21% O2) and staged into 2h cohorts at ecdysis to the third instar before being maintained at 25°C, 21% O2 (normoxic flies) or moved to 25°C, 10% O2 (hypoxic flies) for the remainder of their de..., # 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

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