Studies with other insects shows that release of Ecdysone from the ring gland is triggered by the prothoracicotropic hormone, produced by four dorsolateral neurosecretory cells of brain. Puffing is the term for changes in polytene chromosomes. The idea that puffing represents gene activity is currently 40 years old. A temporal pattern to puffing in the salivary glands of larval flies is inducible by ecdysone injection. It is hypothesized that the time sequence of puffing represents a genetic hierarchy of gene activation.
Ecdysone receptor is induced at the beginning of the gene activation hierarchy.
EcR is induced directly by ecdysone, and provides an autoregulatory loop that increases the level of receptor protein in response to the hormone ligand. EcR exists in three isoforms, each one having an different biological function. Each requires as a partner in heterodimerization the protein Ultraspiracle, the Drosophila homolog of vertebrate RXR proteins. Ligand binding stabilizes the ECR-USP heterodimer and increases its affinity for binding to ecdysone response elements in the promoters of genes. At least five other genes, and probably more than a dozen, are of critical importance to the regulatory hierarchy directed by EcR. E75 is also induced as an early gene, one that codes for another hormone receptor superfamily transcription factor with multiple protein isoforms.