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doi: 10.1242/10.1242/dev.00460

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The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila

Géza Ádám1,*, Norbert Perrimon2 and Stéphane Noselli1,*,{dagger}

1 Institute of Signaling, Developmental Biology and Cancer, Centre de Biochimie-UMR 6543-CNRS, Parc Valrose, 06108 Nice cedex 2, France
2 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA



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  		Fig. 1. Rotation of genitalia and spermiduct looping in wild-type and spin males. (A) Scanning electron micrograph (SEM) of a wild-type male abdomen (ventral view; posterior is upwards), showing the position of the anus and penis. The direction and extent of genitalia rotation is schematized by a looping arrow (bottom right). (B) Schematic view of a wild-type abdomen showing the internal looping of the spermiduct around the gut, after clockwise rotation of the genital plate. (C) SEM of a spinGAL4/Y; UAS-Fas2 male showing complete rescue of rotation. (D-F) SEM of representative spin males showing varying degrees of rotation. (G,G') Dissected wild-type abdomen and its schematic representation showing the rightward looping of the spermiduct. (H,H') Dissected spin male (similar to E), and its schematic representation showing an under-rotation phenotype. sp, spermiduct; g, gut; p, penis.

 


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  		Fig. 2. spin is a novel, rotation-specific Fas2 allele. Schematic view of the Fas2 gene and the Fas2spin p{GawB} P-element insertion (A). Expression of Fas2 in stage 9 (B) and 14 (C) wild-type embryos. In a Fas2spin lethal revertant allele (Fas2spinRM1), Fas2 expression is lost (D), while Fas2 expression in the viable Fas2spin allele is strongly reduced (E), when compared with wild type (compare D with B and E with C). Expression of Fas2 (green) in a wild-type eye imaginal discs starts anterior to the morphogenetic furrow (F, arrowhead), while it is strongly reduced in a Fas2spin eye imaginal disc (G). (H) Histogram showing the percentage of males with normal genitalia, after expression of Fas2 using a UAS-Fas2 transgene under the control of a heat-inducible GAL4 line. For each time-point, the phenotype of the external genitalia has been determined and the extent of rotation rescue plotted. Heat-shocked males have the following genotype: Fas2spinR5/Y; UAS-Fas2/HS-GAL4. Fas2spinR5 is a Fas2spin excision allele that retains the original Fas2spin rotation phenotype but lacks GAL4 activity.

 


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  		Fig. 4. Expression of Fas2 in neurons innervating the corpora allata rescues Fas2spin rotation defects. (A) Summary of rescue experiments using lines expressing GAL4 in different subsets of neurons in the brain. Rescued males had the following genotype: Fas2spinR5/Y; UAS-Fas2; GAL4. +++, full rescue; +, partial rescue; –, no rescue. (B) Ring gland expressing a synaptobrevin-GFP fusion protein (green; to mark terminal boutons) in neurons innervating the corpora allata (nCA), using the Kurs21-GAL4 line. The ring gland (outlined) is double stained with anti-Fas2 (red). (C) Ring gland expressing EGFP (green) in neurons innervating the prothoracic gland (nPT), using the Feb211-GAL4 line. The ring gland (outlined) is double stained with anti-Fas2 (red).

 


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  		Fig. 3. Ring gland defects in Fas2spin. (A) Third instar larval brain and ring gland (outlined) stained using the Kurs21-GAL4; UAS-synaptobrevinGFP (green) line and anti-Fas2 (red). nCA, neurons innervating the corpora allata and expressing GAL4. (B) The ring gland with its three different regions (corpora cardiaca, corpora allata and prothoracic gland). The axon processes of the neurosecretory cells controlling juvenile hormone synthesis are shown in yellow. These neurons express Fas2 in the axon processes and project into the corpora allata. (C,D) Anti-Fas2 staining of a wild-type (C) or spin (D) ring gland (green). Phalloidin-TRITC is in red. (E,F) Close-up of the corpora allata synapse from a wild-type (E) or Fas2spin (F) ring gland.

 


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  		Fig. 7. Model for Fas2 and JH action in Drosophila organ looping. (A) Fas2 is specifically expressed and required in neurosecretory cells innervating the corpora allata, to control JH titers. JH is released into the circulating hemolymph and reaches the genital disc to control its rotation non-autonomously. (B) Parallels between the vertebrate and the Drosophila pathways controlling asymmetric organ looping. In vertebrates, two conserved pathways have been implicated in LR asymmetry, the retinoic acid (RA) and the nodal pathways. RA plays a dual role as it is also involved in organ looping. In Drosophila, mutants exist that can lead to a reversion of genitalia rotation (Milani, 1955Go; Milani, 1956Go) (G.Á. and S.N., unpublished), suggesting that they are involved in LR asymmetry (collectively represented by a question mark). The analysis of Fas2spin shows a role for JH, a RA analog, in the control of organ looping and suggests an evolutionary conserved function of terpenoids in the control of asymmetric organ looping.

 


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  		Fig. 5. Fas2 controls genitalia rotation cell non-autonomously. Morphology of the corpora allata nerve terminals in a wild-type (A), Fas2spin (B) and mosaic Fas2spinR5/Y; D2-3/+ (C) males. The number of terminal boutons is reduced in Fas2spinR5, and is restored in mosaic males. (D,E) Phenotype of adult ring glands from non-rescued (D) or rescued (E) males, after generation of random, `flip-out' clones expressing Fas2 (not shown) and EGFP (green). Only rescued males express GFP and Fas2 in the ring gland.

 


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  		Fig. 6. Effects of JH levels on Fas2spin and genitalia rotation. (A) Phenotype of adult or pharate adult male genitalia after topical application of pyriproxyfen (a JH analog) at the white pupae stage. The use of a half-lethal dose of pyriproxyfen (LD50) induces mis-rotation phenotypes in both the hatched and non-hatched populations (16% and 68%, respectively). In the non-hatched flies, the partial and no rotation phenotypes were four and six times higher than in the hatched population, respectively. At the lethal dose of pyriproxyfen (97% lethality; LD97) the pharate adult males showed a higher proportion of partial (44%) and non rotated (44%) genitalia with only 12% showing a wild-type phenotype. (B) Genetic interaction between Fas2spin and the Methoprene-tolerant (Met) [Rst(1)JH] gene. The loss of Met function (MetK17) completely suppresses the Fas2spin rotation defects, while two extra copies of the Met gene aggravated the extent of genitalia rotation in Fas2spinR5 flies. In Fas2spinR5 males, the rotation of the genitalia stopped most frequently (51%) between 180° and 270°, and the non rotated phenotype was not observed. The Fas2spinR5/Y; Met+ (3x) males died as pharate adults and the extent of rotation was dramatically reduced. In most cases (64%), rotation stopped between 0° and 90°, whereas 26% of the males showed non rotated genitalia.

 

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