BIOLOGICAL OVERVIEW

Induction of molting in Drosophila coincides with release from the ring gland of 20-hydroxyecdysone, also known as ecydsone. Prior to each of the larval molts, at pupariation, at pupation and during metamorphosis, hormone is released in carefully timed spurts, coinciding with major morphological transitions. (A description of these stages is give in Developmental origin of adult structures ).

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. For more information see the section entitled "Bombyx and Manduca prothoracicotropic hormone" below.

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. A small number of genes react by puffing within minutes of exposure to ecdysone, and a much larger number (>100) react within hours. It is hypothesized that the time sequence of puffing represents a genetic hierarchy of gene activation. Early puffs are independent of protein synthesis while late puffs require prior protein synthesis (Ashburner, 1990).

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. Although ECR can bind ecdysone on its own, binding is greatly stimulated by the addition of USP. 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. Binding sites for EcR exist in the promoter of E75, and EcR is required for the induction of the early response. The E75 response is self delimiting, as transcription is terminated soon after it initiates.

Several other genes act as delayed early genes, including hormone receptor superfamily genes E78B and DHR3, both of which are induced in a delayed fashion after EcR induction. Both require ecdysone-induced protein synthesis for their maximal levels of transcription, and appear to function as monomers to control expression of target genes (Horner, 1995). The delayed timing of E78B and DHR3 induction may allow EcR and E75 to perform regulatory functions before the delayed early genes become active, but the function of these genes is still unknown (Thummel, 1995).

During a second wave of puffing 4 to 6 hours after puparium formation, ßFTZ-F1 is induced as a mid-prepupal gene. FTZ-F1 maps to the 75CD mid-prepupal puff. It has been shown to interact with elements of the alcohol dehydrogenase gene and has also been implicated as an activator of fushi tarazu. ßFTZ-F1 is another complex gene with multiple isoforms; antibodies to ßFTZ-F1 detect binding to 166 loci in late prepupal salivary gland polytene chromosomes, 51 of which represent ecdysone-regulated puffs. Of 33 puffs that show increased activity after the peak of the 75CD puff, 17 show reproducible staining for ßFTZ-F1 (Lavorgna, 1993).

Experiments with cultured larval salivary glands have demonstrated that ßFTZ-F1 transcription is negatively regulated by ecdysone. In the absence of ecdysone, ßFTZ-F1 is induced. Repression is overcome as the levels of both ecdysone and EcR decrease during early-prepupal development. Thus ßFTZ-F1, through its interaction with EcR, provides a molecular mechanism for stage-specific responses to steroid hormones (Woodard, 1994).

In late prepupae, the midprepupal puffs regress and the early puffs are reinduced. In addition, a few stage-specific early puffs, typified by E93, are directly induced by ecdysone in late prepupae, but not in late larvae. The early puffs cannot be induced by ecdysone in early-prepupal salivary glands. Rather, a preceding period of protein synthesis and low ecdysone concentration is required before these puffs become competent to respond to hormone. It is suggested that one or more proteins encoded by the mid-prepupal puff genes provide the compentence for the early puffs to be induced by the prepupal ecdysone pulse; FTZ-F1 is a good candidate for this required gene (Woodard, 1994). Negative regulation is required in molting, as much as positive effects. For example, DHR78, an orphan nuclear receptor expressed throughout the early stages of metamorphosis, cannot heterodimerize with either ECR or USP but can bind to an Ecdysone receptor response element of a downstream gene in the hierarchy inhibiting the ability of ECR and USP to induce transcription (Zelhof, 1995).

In conclusion, each of these gene dyads and triads; EcR and USP, E75A, E78B and DHR3, ßFtz-F1, DRH78 and E93, is required in a sequential genetic hierarchy for the the timing of metamorphosis and the induction and repression of genes required for the differentiation process (Thummel, 1995). The complexity of the insect molting hierarchy serves as a warning for a generation of scientists who would unravel the hierarchies of mammalian development.

GENE STRUCTURE

Genomic length - 36 kb cDNA length - 5534

Bases in 5' UTR -1068

Exons - 6

Bases in 3' UTR - 1819

PROTEIN STRUCTURE

Three protein isoforms are encoded by EcR, designated ECR-A, ECR-B1 and ECR-B2. These proteins differ in their amino-terminal sequences but contain identical DNA binding domains and ligand binding domains. The A and B1 isoforms are encoded by overlapping transcription units that have different promotors and can be separately controlled. The N-terminal amino acids of ECR-A are coded for by three exons specific to that isoform, while both the DNA binding and ligand binding domains of ECR-A are coded for by exons shared with the other two isoforms. The B1 and B2 isoforms are encoded by mRNAs that derive from the EcR-B primary transcript by alternative splicing (Talbot, 1993).

Amino Acids - 878

Structural Domains

There are two conserved domains characteristic of steroid receptor superfamily members. The more N-terminal domain is a DNA-binding domain and the more C-terminal domain is a hormone-binding domain also implicated as a protein interaction domain (Koelle, 1991). EcR is a Class II member of the nuclear receptor superfamily, classified as such on the basis of its ability to heterodimerize with RXR (in Drosophila Ultraspiracle) and a its ability to bind to direct repeats. EcR is most closely related to the vertebrate Farnesoid X receptor (Mangelsdorf, 1995).

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

date revised: 28 MAY 97  

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