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First published online 2 December 2004
doi: 10.1242/dev.01568

Development 132, 155-164 (2005)
Published by The Company of Biologists 2005
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Drosophila retained/dead ringer is necessary for neuronal pathfinding, female receptivity and repression of fruitless independent male courtship behaviors

Lynn M. Ditch1,2,3, Troy Shirangi1, Jeffrey L. Pitman1,2, Kristin L. Latham4, Kim D. Finley2,*, Philip T. Edeen2,{dagger}, Barbara J. Taylor4 and Michael McKeown1,2,{ddagger}

1 Molecular Biology, Cell Biology, and Biochemistry Department, Brown University, Providence, RI 02912, USA
2 Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
3 Department of Biology, University of California, San Diego, La Jolla, CA 92093, USA
4 Department of Zoology, Oregon State University, Corvallis, OR 97331, USA



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  		Fig. 1. Mutations and structure of the retn gene. The structure of the retn gene is shown (introns not to scale), including the previously undetected exon 1-4 and exon 1-6 splice variants. Positions and structures derived from Gregory et al. (Gregory et al., 1996Go) and from our analysis of the genomic sequence of the region. The regions shown in white encode the extended ARID box DNA-binding domain. The viable ('retn class') alleles retnRO44, retnz2-428 and retnRU50 encode missense mutations within the ARID box. Embryonic lethal ('dri class') alleles retndri1 and retndriB142 encode nonsense mutations. P-element insertions retndri7 and retndri8 map in or near retn exon 1. retn-Gal4 insertions were created by targeted transposition into the retndri7 and retndri8 positions (Shandala et al., 1999Go).

 


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  		Fig. 2. retn mutations affect viability. (A) Eclosion rates for retn heteroallelic combinations. retnz2-428/retndri2 (1) and retnz2-428/retndri1 (2) offspring eclose with 8% and 25% of expected rates; retnRU50/retndri2 (3) and retnRU50/retndri1 (4) eclose with 65% and 68% of expected numbers; retnz2-428/retndri8 (5) and retnRU50/retndri8 (6) eclose with 71% and 100% of expected numbers. retnz2-428/retnRU50 (7) is completely viable. (B) retn cDNA rescues partial lethality of retn-Gal4/retnz2-428. retn-Gal489/retnz2-428; +/+ trans-heterozygotes (1) eclose with 33% of expected numbers, while retn-Gal489/retnz2-428; UAS-retn/+ individuals (2) eclose at 100% of expected rates.

 


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  		Fig. 3. retn female behaviors. (A) retn female resistance to male courtship increases with allelic strength. (1) Wild type (Canton-S) (CS), (2) retndri2/+, (3) retnRU50/+, (4) retnz2-428/+, (5) retnz2-428/retnRU50, (6) retnRU50/retndri2, (7) retnz2-428/retndri2. Average time of courtship prior to copulation for 20 females per genotype is shown. Error bars indicate s.e.m. Resistance behavior to a maximum of 1 hour was measured. Wild-type females (1), and retndri2/+ (2), retnRU50/+ (3) and retnz2-428/+ females (4) copulate in 2-4 minutes (P>0.05 relative to wild type). retnRU50/retnz2-428 females (5) average 8.8 minutes (P=0.013). retnRU50/retndri2 females (6) average 34 minutes (P=5 x10-5), and retnz2-428/retndri2 (7) females average 58 minutes (P=5 x10-17). (B) retn cDNA rescues female resistance behavior. (1) Wild type (CS), (2) retn-Gal489/retnRU50; +/+, (3) retn-Gal489/retnRU50; UAS-retn/+. retn-Gal489/retnRU50; +/+ females resist courtship for an average of 25 minutes (P=4 x10-5 relative to wild type). retn-Gal489/retnRU50; UAS-retn/+ females copulate in 4.8 minutes (P=3 x10-4 compared with mutant without construct), comparable with wild type (P=0.077). (C) Courtship chain of retn (retnz2-428/retndri8) females (arrow). (D) Female wing extension performed at another female (arrow). Additional chaining can be seen towards the right (arrowhead). (E) Female (arrow) extends wing at courting male. (F) Bisexual behavior is increased in retn females. (1) retnz2-428/+, (2) retnz2-428/retndri8, (3) retndri8/+. retndri8/+ and retnz2-428/+ females average fewer than three courtship-like behaviors per observation period. retnz2-428/retndri8 females average 42±7 courtship events (P=0.0001 relative to controls). (G,H) retnz2-428/retndri8; fru4-40/fruAJ96u3 females generate male-like courtship, including both following behavior and wing extension.

 


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  		Fig. 4. Courtship behaviors in retn males. (A,B), (1) Wild type (CS), (2) retnz2-428/retnRU50, (3) retnz2-428/retndri8. (A) retn male courtship of females is comparable with wild type. CS males copulate on average 2.7±0.4 minute after initiation of courtship; retnz2-428/retnRU50 males copulate on average in 1.0±0.3 minutes (P=0.1 relative to CS); retnz2-428/retndri8 males average 0.9±1.0 minutes (P=0.05 relative to CS). (B) retn males show low levels of bisexual courtship, comparable with wild-type bisexual courtship. CS males average 5.5±2.8 male-by-male courtship events per 5-minute observation period; retnz2-428/retnRU50 males average 5.5±2.6 courtship events (P=1 relative to CS); retnz2-428/retndri8 males average 1.5±0.8 events (P=0.2).

 


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  		Fig. 5. Sex-non-specific alternative splicing of retn. (A) A lack of sex-specific splicing of retn. RNAs from CNS tissue from late third instar larvae (lanes 1, 2, 5, 6, 9, 10) and mid-stage pupae (lanes 3, 4, 7, 8, 11, 12) from both sexes were analyzed by RT-PCR probing exons 1 to 4 (lanes 1 to 4), 4 to 8 (lanes 5 to 8), and 8 to 11 (lanes 9 to 12). The splice variant of retn joining exon 1 to 4 is present at very low levels and migrates beyond the level shown. (B) An abundant splice variant of retn joining exon 1 to 6 is present in both sexes. RNAs from CNS tissue from mid-stage pupae of both sexes were analyzed as above (A), probing between exons 1 and 8 (lanes 1 and 2), and 4 and 11 (lanes 3 and 4). The faster migrating band in lanes 1 and 2 represent splice variant 1-6.

 


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  		Fig. 6. retn expression during metamorphosis. retn-Gal4/+; UAS-mGFP expression in late larval (A,D,G,J), early pupal (B,E,H,K), and late pupal/early adult (C,F,I,L) stages. (A-L) Anterior is upwards. (A-C) retn expression labels subsets of CNS neurons through metamorphosis: mushroom bodies (arrowheads), subesophageal ganglion (arrows) and ventral abdominal ganglion (asterisks). (D-F) retn labels {alpha} and ß mushroom body processes in the larval CNS (D). These projections are pruned in 24-hour-old pupae (arrow in E). In 48-hour-old pupae, expression can be seen in all MB lobes, but expression subsequently fades in non-{alpha}/ß projecting neurons (F). (G-I) Subesophageal ganglion cells remain constant in number, but show remodeling of projections from larval (G) to early (H) and late (I) pupal patterns. (J-L) Eighteen larval retn-expressing abdominal ganglion cells (arrow, J) reduce to 12 in early pupae (arrow, K). Six neurons are present (arrow, L) in late pupal stages.

 


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  		Fig. 7. retn mutations cause neuronal pathfinding errors. (A) Mushroom bodies in retn-Gal489/+ show a clear separation of ß-lobes (arrows). (B) In retn-Gal489/retnz2-428 larvae, ß-lobe fusion is evident (arrow). Compare with single larval MB in Fig. 6D, which is unlinked to its paired MB. (C) retn-Gal489/retnz2-428 pupae also show ß-lobe fusion (arrow) and poor fasciculation of neuronal projections (arrowhead). (D) ß lobes of Canton-S adult female brain do not cross the midline. In 90% (n=6) Canton-S adult male brains there was no lobe fusion but one animal did have some crossing ß-lobe fibers; a low frequency of ß-lobe fiber crossing has been noted in wild-type animals (Moreau-Fauvarque et al., 1998Go; Michel et al., 2004Go). The gamma lobe fibers label weakly with Fas2 (Crittenden et al., 1998Go). Arrow and arrowhead indicate the midline between the ß-lobes and the {alpha}-lobe, respectively. (E) ß-Lobes of retndri8/retnz2-428 adult female have Fas2-positive axons that cross the midline giving a fused appearance. In this mutant female, the right {alpha}-lobe is also smaller than in wild-type females, as though there are fewer Fas2-postive axons. ß-Lobe fusion of Fas2-positive axons was also found in 75% (n=4) of retndri8/retnz2-428 and 33% (n=3) of retnRO44/retnRO44 adult male brains. Anterior is upwards. Arrow and arrowhead indicate the midline between the ß-lobes and the {alpha}-lobe, respectively. (F,H,J) retn-Gal489/+; UAS-mGFP. (G,I,K) FRTG13, retn-Gal489/FRTG13, retn-Gal489; UAS-mGFP clones following heat shock of hs-FLP; FRTG13, retn-Gal489/FRTG13, Gal80; UAS-mGFP/+. (F) Mid-pupal SOG neurons in retn-Gal489/+ show dense arborization (arrowhead) and projections extending towards the protocerebrum (arrows). (G) Mid-pupal SOG neurons in retn-Gal489 homozygous clones have little dendritic branching (arrowhead) and poor extension of distal processes (arrows). G is at a higher magnification than F. (H) Confocal section of midline-crossing transect (F) shows tight fasciculation of neurites (arrow). (I) Confocal sections of SOG transect (G) shows poor fasciculation of the same neuronal projections (arrow). (J) Mid-pupal photoreceptors R1-R6 project to the lamina (LA), while faint pattern of R8 projections (arrow) is visible in the medulla (ME). (K) In retn-Gal489 homozygous tissue, subsets of R1-R6 cells extend beyond the lamina into the medulla (arrow). A-I, anterior is upwards; J,K, anterior towards the left.

 


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  		Fig. 8. A model for the relationship between retn and fru functions in male behavior. (A) In the absence of retn or fru functions, there is an intrinsic tendency towards development of neural circuitry leading to male-like courtship. In situations in which retn and the fruM products are absent, such as in retn mutant females, this will be revealed as some degree of male-like courtship behavior. (B) The retn products normally act to counter the tendency towards male-like behaviors such that wild-type females do not show male-like behavior. (C) In wild-type males, retn is still active as a repressor, but the presence of fruM product substantially enhances development of the male behavior pathway, such that the male behavior pathway overcomes the negative effect of retn function.

 

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© The Company of Biologists Ltd 2005