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doi: 10.1242/10.1242/dev.00148

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Drosophila Regulatory factor X is necessary for ciliated sensory neuron differentiation

Raphaelle Dubruille1,*, Anne Laurençon1,*, Camille Vandaele1,*, Emiko Shishido2, Madeleine Coulon-Bublex1, Peter Swoboda3, Pierre Couble1, Maurice Kernan2 and Bénédicte Durand1,{dagger}

1 Centre de Génétique Moléculaire et Cellulaire, CNRS UMR-5534, Université Claude Bernard Lyon-1, 69622 Villeurbanne, France
2 Department of Neurobiology and Behavior, The State University of New York at Stony Brook, Stony Brook, New York 11794, USA
3 Karolinska Institute, Department of Biosciences, Södertörn University College, Section of Natural Sciences, S-14189 Huddinge, Sweden



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  		Fig. 1. Rfx locus and genetic elements. (A) The organization of the Rfx genomic region on chromosome III, centromere to the left. The four P-element insertions and the two deficiencies described in this study are reported. Two elements (jumu6142, jumu6439) are located in the jumu gene. The element of the A143.1F3 line is inserted 5' of Rfx and 5' of the CG17100 gene transcription start site. The S143702 P element is inserted in the jumu gene. The insertion event has generated a deficiency uncovering Rfx and jumu-coding regions. The remaining PlacW element is situated at the deficiency breakpoints. Rfx49 is a small deficiency uncovering the first three exons of Rfx created by A143.1F3 mobilization. 4 kb of the A143.1F3 P(lArB) element remain in Rfx49 at the deficiency breakpoints. (B) Rfx in situ hybridization of Df(3R)hth/TM2 salivary gland polytene chromosomes. Rfx hybridization (arrow) allows the visualization of the loop on the third chromosome because of a complete deficiency of the 85F to 86C regions on chromosome Df(3R)hth. (C) Alignment of RFX DNA-binding domain from different species. Rfx253 corresponds to a point mutation changing an absolutely conserved serine to a phenylalanine within the DNA-binding domain. Species abbreviations: H.s., Homo sapiens; M.m., Mus musculus; D.m., Drosophila melanogaster; C.e., Caenorhabditis elegans; S.c., Saccharomyces cerevisiae; S.p., Schizosaccharomyces pombe. (D) Electromobility shift assay with in vitro translated wild-type RFX or RFX253, and an X box-labeled oligonucleotide. Lane 1, no protein; lanes 2-4, RFX; lanes 5-7, RFX253; lanes 2,5, no competitor; lanes 3,6, X box oligonucleotide as competitor; lanes 4,7, mutated X box oligonucleotide as competitor; RFX253 is not able to bind an X box oligonucleotide. The arrowhead indicates the free probe. The bracket indicates RFX-DNA complex.

 


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  		Fig. 2. Sensory behaviors of Rfx mutants. Three different assays were performed and the corresponding data are plotted. Each point is the response index obtained with ~30 larvae per individual test. Dots represent Rfx253/TM6B, Tb larvae; stars indicate Rfx49/Rfx253 larvae; diamonds indicate Rfx49/Rfx49 larvae. (A) Data for olfactory assays to attractive (butanol), repulsive (n-octyl-acetate) or non odorant (NaCl) control substances. (B) Data for chemosensory assays to high NaCl concentrations or control NaCl concentrations. (C) The result of a typical phototaxy experiment with Rfx49/TM6B control larvae on top and Rfx49/Rfx253 mutant larvae on the bottom. The plot shows the quantification of phototaxis assay data.

 


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  		Fig. 3. Electrophysiological recordings from adult Drosophila external mechanosensory organs. (A) Diagram of a Drosophila mechanoreceptor bristle, adapted from Walker et al. (Walker et al., 2000Go). The bristle sensory organ is composed of a hollow hair shaft and three cells: the socket cell, the sheath cell and a ciliated mechanosensory neuron. The recording configuration consisted of two electrodes: a reference electrode placed in the abdomen of the fly and a recording/stimulation electrode slipped over the cut end of a bristle. (B-E) Mechanically elicited changes in transepithelial potential (TEP) recorded from single macrochaete bristles in Rfx49/TM6B (B,C) and Rfx253/Rfx49 (D,E) flies. (B,D) Representative single traces; the 10 µm stimulating displacement is shown in B. In C,E, the maximum amplitude of the mechanoreceptor potential (MRP) in each bristle is plotted against its resting TEP. Circled points correspond to the traces shown in B,D. (F,G) Transepithelial currents (TEC) recorded from the same bristles, with apical holding potentials of +76 mV and +55 mV, respectively. The stimulus displacement is as in B. No mechanically elicited currents were observed in Rfx mutant flies. (H) Transepithelial resistance in Rfx49/TM6B (n=27, mean=243±16 M{Omega}) and Rfx mutant (n=24; mean=80±12 M{Omega}) bristles. Points are values for individual bristles; lines indicate the mean resistance. Transepithelial resistance is significantly reduced (P<10-9) in Rfx mutants. (I) Diagrams of a Drosophila head showing the position of the electrodes used to record sound-elicited potentials, and of an antenna indicating the different segments. The Johnston's organ is situated in the second antennal segment (2). Chordotonal organs attached to the joint between the second and third segments are stimulated by vibration of the arista and third segment (3). (J) Sound-elicited potentials recorded from Rfx49/TM6B control or Rfx253/Rfx49 mutant antennae in response to pulse sound stimulation. Each trace is an average of the responses to ten consecutive stimulus trains. No sound-elicited potentials were detected in mutant antennae.

 


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  		Fig. 4. Type I sensory neuron defects in Rfx mutant embryos. Embryonic peripheral nervous system was observed by immunostaining in wild-type and Rfx mutant embryos. Nomarski observation of 22C10 staining on wild-type (A) or Rfx49 mutant embryos (B). No differences in the number and position of PNS neurons are observed. (C-H) Anti-HRP staining of wild-type (C,E), control Rfx253/TM6B (G) or Rfx49 mutant (D,F,H) lateral chordotonal organs (lch5). Staining is revealed with a peroxidase-coupled secondary antibody (C-F) and with a fluorescent secondary antibody (G-H). In mutant embryos, staining is strongly affected and neuron groups appear disorganized (D). When staining is strong enough to visualize dendrites in mutant embryos, cilia (arrowhead) are often absent (F) when compared with wild-type embryos (C,E). Note the labeled dendritic cap (arrow) enclosing the cilium also present in wild-type (C) or mutant embryos (F). (G,H) Fluorescent labeling allows accurate visualization of the terminal dendritic structures caused by a faint staining of the dendritic cap. In Rfx mutant embryos (H) the cilium (arrowhead) is always shorter or absent compared with control embryos (G).

 


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  		Fig. 5. Dendrite defects in late pupal or adult type I sensory neurons. All neurons are labeled with mCD8-GFP. (A) A scheme representing the neuron innervation of a campaniform sensillum of the adult wing vein. Arrowhead indicates the dome-shaped structure of the sensillum. (B-E) Dendrite observation in adult wing campaniform sensilla. Arrowheads indicate the position of the dome shaped sensilla. In control (not shown) or rescued Rfx253/Rfx49 mutant wings (B), the cilium is located just under the dome-shaped sensory structure produced by the accessory cell. In all Rfx253/Rfx49 mutant wings (C-E), the dendrite is never situated under the dome-shaped sensilla and sometimes ends well before the sensillum (C). In most cases, the cilium is either shorter or absent (D,E). (F) A schematic representing the two neurons innervating a chordotonal organ of the femur as observed in G-L. In Rfx253/TM6B (G) or rescued Rfx253/Rfx49 mutant pupae (H,I), the three subregions of the cilium are clearly visible. tb, tubular bundle; cd, ciliary dilation; ci, cilium; tz, transition zone. In mutant legs (J,L), the cilium is either absent or shorter (arrows) and distinct subregions cannot be distinguished. The dendritic transition zone appears swollen (arrowheads). (J) Rfx49/S143702; (K) Rfx49/Rfx253; (L) Rfx253/S143702.

 


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  		Fig. 6. Ciliary defects in Rfx mutants. (A) An antennal scolopidium of the Johnston's organ in longitudinal and cross-sections. tb, tubular bundle; cd, ciliary dilation; af, axial filament; dc, dendritic cap; sr, scolopale rods; ci, cilium. (B-J) Transmission electron micrographs of scolopidia of the Johnston's organ of wild-type and Rfx mutant adults. Cross-sections of wild-type (B,D,F) and Rfx253/Rfx49 scolopidia (C,E,G,H). Longitudinal sections of wild-type (I) or Rfx253/Rfx49 scolopidia (J). In B,C, scolopidia (sc, arrows) are sectioned at different levels from distal (up) to proximal (down). (B) Cilia are observed on proximal sections in wild-type antenna. (C) Scolopidia are less well organized and no typical cilia are observed in the Rfx mutant antenna, the arrowhead indicates a noticeably disorganized scolopidium. (D) Enlargement of a typical proximal section of a wild-type scolopidium presents the nine microtubule doublets (ci, arrowhead). (E) Enlargement of a proximal section of a Rfx mutant scolopidium shows that no microtubule doublet is present (arrowhead). (F-H) Enlargement of distal scolopidia sections. No differences are observed between wild-type (F) and Rfx49/Rfx253 mutant (G,H) dendritic cap structures. (I,J) Longitudinal sections through scolopidia. (I) The axial filament clearly appears in the control dendrite process. (J) No typical axial filament is observed at the expected position (arrowheads) in longitudinal section of three Rfx mutant scolopidia.

 

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