Insulin signaling under hypoxic conditions.

<p>[A–C] Dilp2 antibody staining (white) of the larval brain was hardly detected in the IPCs of wildtype larvae reared in normoxia (A) but accumulated in those of wildtype larvae reared in hypoxia (B). The increased Dilp2 retention under hypoxic conditions phenocopied that observed during starvation (C). IPCs in B were reared in 2.5% O₂ from 48–72 hAH. [D] Under hypoxic conditions, ubiquitous overexpression of the wildtype form of the <i>Drosophila</i> insulin receptor (InR-WT), using the driver da-GAL4, led to a statistically significant rescue in larval size (n = 52), as compared to the hypoxic wildtype control (da>) (n = 49). Ubiquitous overexpression of the InR-WT led to a minor reduction in larval size under normoxic conditions (n = 37), as compared to the normoxic wildtype control (n = 33), most likely due to an increased growth rate and shortened duration of growth due to insulin-stimulated ecdysone production in the prothoracic gland <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115297#pone.0115297-Colombani2" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115297#pone.0115297-Mirth1" target="_blank">[24]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115297#pone.0115297-Boulan1" target="_blank">[26]</a>. [E-F] InR-WT overexpression in the larval trachea, using the trachea-specific driver btl-GAL4, led to a statistically significant rescue of larval size during hypoxia (n = 20), as compared to the hypoxic wildtype control (btl>) (n = 30) (F; quantified in E). Under normoxic conditions, tracheal-specific overexpression (n = 20) of the insulin receptor leads to a statistically significant mild decrease in larval growth, as compared to the normoxic wildtype control (<i>btl></i>) (n = 20). Tracheal-specific downregulation of the insulin receptor (n = 46) with its respective control (n = 54) was carried out at 29°C to enhance InR knockdown and leads to a statistically significant mild reduction in larval length. [G] Overexpression of Dilp2 in the larval trachea (btl>dilp2) under hypoxic conditions (n = 35) did not rescue growth restriction, as compared to the hypoxic wildtype control (btl>) (n = 20). Volumetric analysis also demonstrated no statistical significance (data not shown). Scale bar in A applies to A–C: 10 µm. Scale bar in F represents 0.50 mm. Statistical significance was determined using a Student's t-test.