BIOLOGICAL OVERVIEW

Hr78 mutant larvae display four phenotypes: asynchronous development, reduced size during the third instar, tracheal defects, and a failure to pupariate. Hr78 mutants develop synchronously with control siblings through the middle of the second instar but show the first signs of asynchrony at the second-to-third instar larval molt. Wild-type animals that have been synchronized within a 2 hr interval at hatching undergo the second-to-third instar molt during a 6 hr interval, between 44 and 50 hr after hatching. In contrast, Hr78 mutants can remain as second instar larvae for up to an additional day, molting at some time between 56 and 80 hr after hatching. Gut clearing and wandering, two ecdysteroid-triggered changes that occur in mid-third instar larvae, also occur asynchronously in Hr78 mutants. Most mutant third instar larvae survive for as long as 7 days without initiating puparium formation. The few mutants that do pupariate are significantly delayed, initiating puparium formation as long as 5 days after the second-to-third instar molt (Fisk, 1998).

The expression of Hr78 in larval salivary glands allowed for the identification of potential regulatory targets by antibody staining of polytene chromosomes. Hr78 protein can bind to a subset of Ecdysone receptor binding sites in vitro, suggesting that Hr78 might function at the top of the ecdysteroid regulatory hierarchies. The majority of polytene chromosome sites that bind Ultraspiracle (Usp), one indication of Ecdysone receptor binding sites, are bound by Usp and Hr78, consistent with an overlap in their binding specificities. In newly formed white prepupae, over 100 Hr78 binding sites have been identified, many of which correspond to ecdysteroid regulated puff loci (Fisk, 1998).

The binding of Hr78 protein to ecdysteroid-regulated puff loci, combined with the failure of Hr78 mutants to pupariate in response to the late larval pulse of 20E, suggests that Hr78 may play a critical role in the ecdysteroid regulatory hierarchies that direct the onset of metamorphosis. As a step toward determining the role of Hr78 in ecdysteroid signaling, the temporal expression of representative ecdysteroid-regulated genes has been examined during third instar larval development in control larvae and two different transheterozygous combinations of Hr78 mutant alleles. All genes examined were expressed normally in +/Df control animals, in patterns that closely parallel those seen in wild-type third instar larvae. The expression of usp is not affected in Hr78 mutants, consistent with its relatively modest transcriptional regulation by ecdysteroids. In contrast, all other ecdysteroid-regulated transcriptional responses examined are disrupted in Hr78 mutants. The coordinate induction of EcR, E74B, and the BR-C in mid-third instar larvae is significantly reduced, and E74A is not expressed. Fbp-1, which is induced directly by 20E in fat bodies, is not expressed either in these mutants. Finally, mid-third instar larval development is characterized by an ecdysteroid-triggered switch in salivary gland gene expression, from the ng (new glue) genes to the Sgs glue genes. This switch fails to occur in Hr78 mutants. ng-1 is not repressed and Sgs-4 is not induced. Interestingly, one Hr78 mutant animal isolated 44 hr after the molt displayed a pattern of ecdysteroid-regulated gene expression that resembled the pattern seen about 24 hr after the molt in control animals. This animal may represent one of the escapers that would have survived through puparium formation (Fisk, 1998).

Two ecdysteroid-triggered regulatory hierarchies have been defined during the third larval instar in Drosophila. The first of these hierarchies is of concern here. Taking place in mid-third instar larvae, a coordinate induction of EcR, E74B, and the BR-C takes place in apparent synchrony with a low-titer pulse of ecdysteroids. Mutations in these regulatory genes lead to defects in puparium formation, indicating that they play a critical role in preparing the animal for the onset of metamorphosis about a day later. One level at which this function is provided is through EcR and the Br-C facilitating early gene induction in response to the high-titer late larval pulse of the hormone 20-hydroxyecdysone (20E). The Br-C and E74B mediate a switch in salivary gland secondary-response gene expression, repressing the new glue (ng) genes and inducing the glue genes. Glue (salivary gland secretion) genes (Sgs1 and Sgs3-8) are a group of seven genes encoding proteins that are components of the secretion produced by the larval salivary glands during the third instar. This switch in gene expression prepares the salivary gland for its role at puparium formation, when the polypeptide glue is expelled and used to affix the animal to a solid surface. The mid-third instar hierarchy thus appears to represent the first steps that prepare the animal for the onset of metamorphosis (Fisk, 1998 and references).

Hr78 mutants are blocked in their ability to trigger the mid-third instar regulatory hierarchy. The induction of EcR, E74B, and Br-C transcription is significantly reduced in DHR78 mutant larvae. This defect cannot be attributed to a reduction in hormone titer, since Hr78 mutant organs cultured with a high concentration of 20E show low levels of early gene transcription, similar to the levels seen in vivo. The Fbp-1 fat body-specific primary-response gene also fails to be induced in Hr78 mutant third instar larvae, and the switch in salivary gland secondary-response gene expression, from ng genes to glue genes, fails to occur. In addition, genes induced by the high-titer late larval pulse of 20E, such as E74A, are not expressed. Given these effects on gene expression, Hr78 is accordingly positioned at the top of the mid-third instar regulatory hierarchy. The effects on salivary gland gene expression are consistent with the expression of Hr78 protein in this tissue. Polytene chromosomes sites bound by Hr78 include genes that are affected by the Hr78 mutations: 2B5 (the Br-C), 3C (ng-1 and Sgs-4), 42A (EcR), and 74EF (E74) (Fisk, 1998).

Interestingly, the available evidence indicates that EcR and Usp do not function in mid-third instar larvae, when Hr78 plays its critical role in gene regulation. Both EcR and Usp proteins are expressed at very low or undetectable levels in mid-third instar larvae. Recent genetic studies have led to the surprising conclusion that EcR-B1 and usp do not function at this stage of development. Studies of the puffing patterns of the polytene chromosomes in EcR-B1 mutant third instar larvae have revealed that the Br-C and glue gene puffs are present, but the 74EF and 75B early puffs fail to form (Bender, 1997). This observation suggests that the Br-C and glue genes are induced normally in EcR-B1 mutants, but that the response to the high-titer late larval ecdysteroid pulse is selectively blocked. Mutations in usp show a similar stage-specific effect on ecdysteroid-regulated gene expression, where the mid-third instar regulatory hierarchy occurs normally but the late third instar hierarchy is blocked. It is possible that EcR-A could perform a function in mid-third instar larvae, although its level in the salivary gland is very low (Talbot, 1993), and it would have to exert this function independent of Usp. Rather, the current studies suggest that Hr78 is the critical regulator that triggers the mid-third instar regulatory hierarchy, preparing the animal for puparium formation in response to the high-titer late larval pulse of 20E (Fisk, 1998).

PROTEIN STRUCTURE

Amino Acids - 601

Structural Domains

The first third of the Hr78 protein contains a two zinc-finger DNA-binding domain, while the C-terminal half contains a ligand-binding/dimerization domain. In the DNA binding domain (DBD), Hr78 and TR2 are 74% identical. Testicular receptor 2 (TR2) receptors were isolated from human testis and prostate libraries and belong to the Coup subfamily on nuclear receptors. The Tr2 subfamily of nuclear receptors is distinct from the subfamilies represented by Ecdysone receptor. Hr78 also shares homology with the murine RXRbeta DBD (68%) as well as with the DBDs of Coup-TF and Seven-up (63%). The ligand-binding domain is more divergent and cannot be clearly placed within one receptor subfamily (Zelhof, 1995 and Fisk, 1995).

date revised: 1 March 2024

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