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Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program

James D. Baker^* and James W. Truman

Department of Zoology, University of Washington, Box 351800 Seattle, WA 91895, USA

View larger version (36K): [in a new window]   Fig. 1. The DLGR2 coding region is disrupted in noncomplementing deficiencies that end within the rk gene and in two strong rk alleles. (A) Non-complementing deficiencies, Df(2L)b-L and Df(2L)A376, were mapped by testing for the presence of sequence tagged sites, STS1, STS2 and STS3, in a mixed population of homozygous balancer and homozygous deficiency flies. (B) Df(2L)A376 breaks in the 5' end of DLGR2 removing STS1, the initiator ATG and most of the first exon. (C) Df(2L)b-L removes STS2 and STS3 and breaks in 3' end of the gene removing at least the entire transmembrane domain. 20 embryos from each cross were tested. (D) Electropherogram from a rk1 homozygote. The wild-type sequence of codon 698, TAC, is converted to TAA, changing a tyrosine residue to a stop codon. (E) In rk4 the wild-type sequence of codon 954, TGG, is changed to TGA, altering a tryptophan to a termination codon.

View larger version (112K): [in a new window]   Fig. 2. Homozygous rk4 flies show delayed post-ecdysial tanning. Photomicrographs show the dorsal abdominal cuticle dissected from (A) wild-type males or (B) females and (C) rk4homozygous males or (D) females at 3 h after eclosion. Cuticles are mounted to show the extent of melanization.

View larger version (38K): [in a new window]   Fig. 3. Homozygous rk4 flies show normal release of eclosion hormone at ecdysis and are responsive to injections of ecdysis triggering hormone (MasETH). (A,B) The eclosion hormone immunoreactivity in the ventral nervous system of rk4 flies before (A) and after (B) adult eclosion. The insets show the loss of large immunoreactive swellings after eclosion. (C) The latency to adult ecdysis when pharate adult rk4 homozygotes were injected with MasETH. The advance in the time to ecdysis is identical to that seen in normal flies. 5 nervous systems from each stage were assayed.

View larger version (22K): [in a new window]   Fig. 4. Bioassays of the blood from wild-type (wt) and rk4 flies showing that bursicon is released by the mutants. Blood was collected donors at either 0 min or 20 min after ecdysis. It was assayed for bursicon activity by injecting into either wild-type or rk4 hosts that had been neck ligatured immediately after eclosion. Hatched bars, injection of blood from wild-type into wild-type; black bars, injection of rk4 blood into wild-type; gray bars, partial melanization responses.

View larger version (51K): [in a new window]   Fig. 5. The effects of the rk mutation on the ability of flies to respond to bursicon or its putative second messenger, cyclic AMP. Neck-ligatured wild-type (wt) flies were injected with (A) an extract containing bursicon or (B) saline. Neck-ligatured rk4 flies were injected with (C) a bursicon extract, (D) saline or (E) 8-Br-cAMP. The arrows indicate the abdominal tergites where darkening has occurred.

View larger version (13K): [in a new window]   Fig. 6. A summary of the post-ecdysial behavior in wild-type and rk mutants. Data were collected by repeated observations during the early post-ecdysial period. Where given, behavioral durations are derived from continuous observation. Bars represent the activation of behavior. Triangles are used to represent the increasing likelihood of behavior during a period. In contrast to wild-type, rk4 mutants do not become quiescent after eclosion and subsequently fail to initiate the expansional behaviors. A label on a red line marks behaviors missing in mutants.

View larger version (32K): [in a new window]   Fig. 7. Ligature experiments link eclosion hormone with the bursicon pathway and separate wing expansion from tanning. (A) Pharate adults were prepared by dissecting away the puparial case and pupal cuticle surrounding the neck region. These flies were staged using morphological markers (Kimura and Truman, 1990) and neck-ligatured. Following ligature, treated flies were maintained in a humid chamber and observed at intervals for up to 5 h. Ligature of wild-type (wt) flies prior to eclosion induces premature tanning and wing expansion behavior upon eclosion. In EH cell knockouts, tanning and wing expansion are eliminated. Flies lacking rk function (rk4) did not melanize but did expand their wings. Ligature-induced wing expansion is accompanied by tonic contraction of the lateral abdominal muscles in both wild-type and (B) rk4 mutants.