Hormone-receptor-like in 78

DEVELOPMENTAL BIOLOGY

Larval

Antibodies raised against the N-terminal region of Hr78 were used to stain organs dissected from staged third instar larvae and prepupae. At all stages examined, Hr78 protein was detected in the nucleus of expressing cells. Hr78 can be detected in tracheae dissected from the earliest time points examined, in early third instar larvae, and was expressed at higher levels in late third instar larvae and prepupae. In contrast, no Hr78 protein could be detected in salivary glands dissected from early third instar larvae. Hr78 expression increases in salivary glands dissected from late third instar larvae and prepupae, in parallel with the high titer pulse of 20E that triggers puparium formation. No expression can be detected in tracheae and salivary glands dissected from animals deficient for Hr78, indicating that the antibody is specific for Hr78 protein. Hr78 protein was also detected in garland cells, the peripodial membrane of imaginal discs, sections of the gut, and Malpighian tubules in wild-type prepupae. Although no nuclear staining is detected in the central nervous system or the ring gland, it should be noted that the levels of Hr78 expression are low, and thus, expression in these and other tissues may be below the detection threshold (Fisk, 1998).

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular and tissue level morphogenesis. A gene expression screen was carried out to identify candidate genes that functioned in wing and wing hair morphogenesis. Pupal wing RNA was isolated from tissue prior to, during and after hair growth and used to probe Affymetrix Drosophila gene chips. 435 genes were identified whose expression changed at least 5 fold during this period and 1335 whose expression changed at least 2 fold. As a functional validation, 10 genes were chosen where genetic reagents existed but where there was little or no evidence for a wing phenotype. New phenotypes were found for 9 of these genes providing functional validation for the collection of identified genes. Among the phenotypes seen were a delay in hair initiation, defects in hair maturation, defects in cuticle formation and pigmentation and abnormal wing hair polarity. The collection of identified genes should be a valuable data set for future studies on hair and bristle morphogenesis, cuticle synthesis and planar polarity (Ren, 2005).

The HR46 gene (also known as DHR3) encodes a nuclear receptor and is an essential gene known to be important for the ecdysone cascade. Large clones of loss of function alleles result in wing (folded and curved) and notum defects (rough short bristles and pale pigmentation). The expression of this gene increased 250 fold from 24 to 32 hr and then decreased 4.3 fold from 32 to 40 hr. Moderate sized wing clones of cells lacking HR46 were examined, but no clear cut phenotype was seen. In pupal wing clones examined a couple of hours after hair formation mutant hairs appeared somewhat thicker but this alteration was transient (Ren, 2005).

The Eip78CD gene encodes a related nuclear receptor. The expression of this non-essential gene increased 3 fold from 24 to 32 hr followed by a three fold drop from 32 to 40 hr (but the differences were not significant) suggesting it might be functionally redundant with HR46. To test this hypothesis Eip78CD mutants, which also contained HR46 mutant clones, were examined. No mutant phenotypes were seen in the clones, suggesting either that there is an alternative redundant gene or that HR46 is not essential for hair morphogenesis. Since the level of HR46 expression fell dramatically between 32 and 40 hrs it seemed possible that declining HR46 expression could be important for hair development. To test this the overexpression of HR46 from a transgene containing a hs promoter was induced. This resulted in a dramatic loss of hair formation leading to wings with extensive bald regions. The strongest phenotype was seen when the transgene was induced by heat shocking 6-8 hrs prior to the time of hair initiation. The phenotype was dose sensitive and directly related to the number of transgenes and length and temperature of transgene induction (Ren, 2005).

Effects of Mutation or Deletion

An orphan nuclear receptor, HR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in Hr78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. All combinations of Hr78 alleles resulted in animals that progressed into the third larval instar, but no more than 7% (ranging from 0 to 7%) of any allelic combination were able to undergo puparium formation. The animals that pupariate appear normal; however, they fail to progress beyond early prepupal development. The severity of the lethal phenotypes caused by the EMS-induced mutations is identical to that found in deficiencies covering Hr78, indicating that the EMS-induced mutations represent null alleles (Fisk, 1998)

Hr78 mutant larvae display four phenotypes: asynchronous development, reduced size during the third instar, tracheal defects, and a failure to pupariate. The size and appearance of Hr78 mutants is normal through the early third instar. However, while wild-type animals increase dramatically in size about 24 hr into the third instar, Hr78 mutants fail to show this increase in size. Hr78 mutants contain defective tracheae, with complete penetrance and variable expressivity. The formation and branching of the tracheal system appears normal in Hr78 mutants. However, the cuticular structure of the tracheae is defective, and some tracheal branches fail to molt properly. In the least severe cases, the taenidial folds of the tracheal cuticle are distorted, or small segments remain filled with fluid. In the more severe cases, segments of cuticle from earlier larval instars fail to molt properly. This obstruction of the tracheal lumen is often accompanied by necrosis and breaks in the dorsal trunks (Fisk, 1998).

Consistent with these phenotypes, HR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. HR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that HR78 directly regulates puff gene expression. In addition, ectopic expression of HR78 has no effects on development, indicating that its activity is regulated post-translationally. It is proposed that HR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis (Fisk, 1998).

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date revised: 15 July 2008

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