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doi: 10.1242/10.1242/dev.00157

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The Drosophila Pox neuro gene: control of male courtship behavior and fertility as revealed by a complete dissection of all enhancers

Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

View larger version (38K): [in a new window]   Fig. 1. Map of the Poxn gene and its enhancers with associated functions identified by Poxn rescue constructs and Poxn-Gal4 reporter genes. (A) Map of chromosomal region at 52D1, including the Poxn gene. Transcribed regions of Poxn (red bar), and annotated genes CG8249 and CG8253 (gray bars) are shown with arrows indicating their directions of transcription below a scale that indicates distances in kb from the `upstream' transcriptional start site of Poxn. The insertion P{Lac-W}M22 and the Poxn deficiency Df(2R)PoxnM22-B5 (open bar) are mapped. The deficiency deletes about 17 kb and extends from 131 bp upstream of the Poxn start codon in exon 2 to 730 bp proximal of the duplicated insertion site of the excised P element. In addition, the location of a genomic fragment is indicated that was used as transgene Resdistal (blue bar) to rescue the annotated genes affected by the Poxn deficiency. (B) Map of the Poxn gene, Poxn rescue constructs and Poxn-Gal4 constructs driving the GFP reporter gene. Below a restriction map (only selected restriction sites are indicated; numbers in parentheses refer to distances in base pairs from the `upstream' transcriptional start site marked 0) of the region including the Poxn gene, the composition of Poxn rescue constructs and of Poxn-Gal4 driver constructs, as listed on the right, is illustrated with respect to the Poxn upstream region (green), 5' leader and 3' trailer (orange), coding region (black), introns (yellow) and downstream region (light blue). In Poxn-Gal4 constructs, the position of the Gal4-coding region is shown as open box. (C) Map of Poxn enhancers and their functions. Double-headed arrows indicate location of Poxn enhancers, which regulate Poxn expression in specific spatiotemporal patterns and the associated functions listed on the right. Enhancers regulating expression during embryonic and larval stages not mapped in this study (W. B. and M. N., unpublished), some of which overlap with, or even contribute to, adult functions, are also shown. In few cases, enhancers delimited by arrows have only been tested to be essential, not necessarily also sufficient, for the control of a specific Poxn function (see text).

View larger version (100K): [in a new window]   Fig. 2. Phenotypes of wing and leg bristles and of leg chemosensory neurons of wild type and Poxn null mutants. (A-D) Chemosensory wing and leg bristle phenotypes of wild type and Poxn null mutants. Bristles on the dorsal anterior wing margin (A,B) and the first male prothoracic tarsal segment (C,D) of wild type (A,C) and PoxnM22-B5 mutants (B,D) are compared by bright field microscopy at a resolution of 20x and 25x magnification. (E-G) GFP expression in chemosensory neurons of wild-type and PoxnM22-B5 male prothoracic legs. GFP expressing neurons are visualized in tarsal segments 4 and 5 of prothoracic legs of w; Poxn-Gal4-13-1-101 UAS-GFP/+ (E), w; PoxnM22-B5; Poxn-Gal4-13-1-101 UAS-GFP/BasiK-109 (F) and w; PoxnM22-B5; Poxn-Gal4-13-1-101 UAS-GFP (G) males by confocal fluorescence microscopy at a resolution of 40x magnification and with maximum projection of Z-stack. Note that tarsal segments 4 and 5 of PoxnM22-B5 mutants endowed with (F) or without (G) the BasiK transgene are fused. Arrows indicate a cluster of four chemosensory neurons innervating a taste bristle (E) and a pair of bipolar neurons associated with a degenerated chemosensory organ (F,G), Dorsal side of legs is upwards (C,D) or towards the right (E-G).

View larger version (115K): [in a new window]   Fig. 3. Labellar taste bristle and associated chemosensory neuron phenotypes of wild-type and Poxn null mutant males. (A-C) Labella with chemosensory taste bristles of w1118 males (A), PoxnM22-B5 males (B) and PoxnM22-B5 males rescued with SuperA-207 (C) are compared by bright field microscopy at a resolution of 20x magnification. Arrows in B indicate bizarre shapes of shafts of transformed taste bristles, which are completely rescued to wild-type in C. (D-F) GFP expressing labellar chemosensory neurons of w; Poxn-Gal4-13-1 UAS-GFP/+ (D,F) and w; PoxnM22-B5; Poxn-Gal4-13-1 UAS-GFP/BasiK109 (E) males are visualized by confocal fluorescence microscopy at resolutions of 20x (D,E) and 100x (F) magnification and with maximum projections of Z-stacks. Arrows in D,E indicate a group of GFP-expressing neurons innervating the labral sense organ (LSO) and in E a few remaining GFP-expressing neurons in the labellum. (F) Larger magnification of the labellum shown in D, illustrating the multiple innervation of the labellar taste bristles by chemosensory neurons: shaft of taste bristle (tb) is invaded by the dendrites of three chemosensory neurons (chs n).

View larger version (48K): [in a new window]   Fig. 4. Courting behavior of partially rescued Poxn males. The histogram at the top indicates the average number of recurved taste bristles per prothoracic leg (gray), the percentage of males initiating courtship under daylight (light blue) or red light (orange), and the percentage of copulating males under daylight (dark blue) and red light (red) within the observation period of 15 minutes (see Materials and Methods) for wild-type and partially or completely rescued Poxn males of the genotypes indicated below. The table at the bottom summarizes, as listed on the left, average latency times with standard deviations in parentheses, number of tested couples in single choice courting tests, and the rescue of phenotypic characters affected by the PoxnM22-B5 mutation. Complete and partial rescue are indicated as + in dark-colored and as (+) in light-colored fields. The degree of male fertility is indicated by +++, ++ and + if more than 65%, 35-65% and 5-35% of single crosses result in offspring. In one case, male fertility is indicated as (-) because two out of 90 males produced a very low number of offspring (two and 20 larvae). The number of taste bristles on the male prothoracic leg is indicated with standard deviation in parenthesis, that of taste bristles on the anterior wing margin is given as percentage of the number observed in Oregon-R males.

View larger version (78K): [in a new window]   Fig. 5. Sexually dimorphic pupal expression pattern of Poxn in the genital region and cuticular phenotype of PoxnM22-B5 male genitalia. (A,B) GFP expression is analyzed in ventral views (anterior towards the left) of male (A) and female (B) w; Poxn-Gal4-14-1/TM6B pupae (48 hours APF) by fluorescence microscopy at a resolution of 10x magnification. (C) Details of GFP expression in the genital region of male pupae (72 hours APF), driven by three different Poxn-Gal4 constructs (Poxn-Gal4-581, Poxn-Gal4-13-1 and Poxn-Gal4-14-7; compare with Fig. 1B). (D) Frontal view (with dorsal side up) of cuticle preparations of male genitalia of wild type (w1118; left) and Poxn mutant (right) under bright field microscopy at a resolution of 8x magnification. Arrows indicate anal plate (ap), clasper (cl), lateral plate (lp), penis (pe) and posterior lobe (pl). The four panels below compare details of the penis (left) and the posterior lobe, clasper and lateral plate (right) regions between wild type (wt) and Poxn mutants (Poxn) at a 2- to 2.5-fold greater magnification. Arrows indicate the wild-type penis (pe) and the penis apodeme (ad) in the Poxn mutant without penis. The four panels at the bottom illustrate, at the same magnification as the panels above, the rescue in Poxn mutants of the penis, but not posterior lobe and clasper, by two copies of EvK, which include the upstream genitalia enhancer absent from L1, and of the posterior lobe and clasper, but not penis, by two copies of L1, which include the intron genitalia enhancer not present in EvK. In the eight panels at the bottom, posterior is towards the right.

View larger version (120K): [in a new window]   Fig. 6. Poxn expression in the embryonic and adult CNS. (A-C) Poxn expression in the developing embryonic ventral cord. (A) Segmentally repeated expression of Poxn protein in the CNS of a stage 15 w; PoxnM22-B5 embryo rescued by C1-1-86, shown as ventral view under Nomarski optics at a resolution of 20x magnification. The Poxn protein is detected by a rabbit anti-Poxn antiserum. (B,C) GFP expression in the ventral CNS of a w; Poxn-Gal4-13-1 UAS-GFP embryo at stage 17, visualized in a ventrolateral overview (B) or enlarged view (C) by confocal fluorescence microscopy at a resolution of 20x magnification and with maximum projection of Z-stack. (B) Clusters of segmentally repeated GFP expressing neurons in thorax (T1-T3) and abdomen (A1-A8) of the ventral CNS and projections from the ventral and lateral pes organs of the thoracic (t1-t3) and first four abdominal segments (a1-a4) into the ventral cord are clearly visible. (C) The morphology of the cell bodies and projections demonstrate that most, if not all, of the GFP-expressing cells in the ventral cord are neurons. The ventral midline is indicated by a white line. Note that this pattern of Poxn expression, which normally disappears by stage 17, is still clearly visible because of the high stability of Gal4 and GFP. (D,E) Poxn expression in the adult ventral ganglion. Ventral views of the anterior part of a thoracic ganglion dissected from a w; Poxn-Gal4-14-1 UAS-GFP male (D) or female (E) adult. Projections of the chemosensory neurons of the gustatory bristles on legs and wings are labeled by Poxn-driven GFP expression and visualized by confocal fluorescence microscopy, as in B. Note that in females, no projections from the prothoracic chemosensory neurons are crossing the midline. pro ln, prothoracic leg neuromere, wn, wing neuromere, mes ln, mesothoracic leg neuromere. Anterior is towards the left (A,B) or upwards (C-E).

View larger version (107K): [in a new window]   Fig. 7. Poxn expression pattern in the developing brain. (A,B) Bilateral-symmetric expression of Poxn in the two brain hemispheres (arrows) of a stage 16 wild-type embryo (A) or third instar larva (B), shown in dorsal views (anterior upwards) under Nomarski optics at a resolution of 20x magnification. (B) Note that no Poxn is detectable in the ventral ganglion. (C) Bilateral-symmetric Poxn expression in the brain of a wild-type adult male, visualized as frontal view by bright field microscopy at a resolution of 16x magnification. Arrows indicate the dorsolateral cluster of about 200 Poxn expressing cells and the ventral cluster of about 100 Poxn expressing cells, which forms an arc around the region where the antennal nerve (an) enters the brain. (D,E) Frontal views of Poxn expression in brains of 2-day-old w; Poxn-Gal4-13-1 UAS-GFP (D) and w; PoxnM22-B5; Poxn-Gal4-13-1 UAS-GFP (E) males, visualized by GFP expression and confocal fluorescence microscopy at a resolution of 20x magnification and with maximum projection of Z-stacks. (D) The majority of cells in both GFP expressing clusters have the morphology of neurons, which project into different regions of the brain. The ventral clusters project mainly into the antennal lobes. The dorsal clusters have the ellipsoid body (eb) as major target and arborize in the lateral triangle (ltr) and in at least one additional region of the brain. The arborizations in the subesophageal ganglion (sog) of the chemosensory neurons of the labellar taste bristles and the LSO are also labeled by GFP. The arborization in the antennal lobe (al) and the antennal nerve (an) are indicated by arrows. (E) The projection pattern of the dorsal clusters has changed dramatically, the ellipsoid body is not targeted and hence not visible, and the arborizations do not reveal the striking symmetry of the wild-type pattern. Note that the disturbance of the projection pattern, as revealed by the Gal4-driven GFP expression, is completely rescued by two copies, but not by a single copy of PK6. This effect is presumably caused by the presence of Gal4 because the GFP pattern is not entirely wild-type even in a PoxnM22-B5/+ background, whereas the Poxn pattern of a PoxnM22-B5 mutant is completely rescued to wild-type by XBs if visualized by anti-Poxn and histochemical staining. (F,G) Poxn expression in the brain of a 2-day-old w; Poxn-Gal4-9/+; UAS-GFP/+ male, visualized as in D,E, but with average projection of the Z-stack. The Poxn-Gal4-9 driver shows a very strong GFP expression in the Poxn expression domains of the brain with some ectopic expression, mainly in the medulla. The enhanced GFP expression combined with the average Z-stack projection produces a clearer image of the arborizations of the Poxn expressing neuronal clusters. (F) The major targets of the projections from the dorsal cluster (dc) of neurons are marked by asterisks. (G) Same specimen as F, but with inverted Z-stack, which offers a posterior view of the GFP expressing neuronal clusters and of the extensive arborizations of the ventral cell cluster (vc) expressing Poxn. The major neurite bundles are marked by arrows and the target areas by asterisks.