Drosophila Regulatory factor X is necessary for ciliated sensory neuron differentiation

Raphaelle Dubruille 2 Anne Laurenon 2 Camille Vandaele 2 Emiko Shishido 1 Madeleine Coulon-Bublex 2 Peter Swoboda 0 Pierre Couble 2 Maurice Kernan 1 Bndicte Durand ) 2 0 Karolinska Institute, Department of Biosciences, Sodertorn University College, Section of Natural Sciences , S-14189 Huddinge , Sweden 1 Department of Neurobiology and Behavior, The State University of New York at Stony Brook , Stony Brook, New York 11794 , USA 2 Centre de Genetique Moleculaire et Cellulaire , CNRS UMR-5534, Universite Claude Bernard Lyon-1, 69622 Villeurbanne , France - Ciliated neurons play an important role in sensory perception in many animals. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. We describe Drosophila mutations that affect the transcription factor RFX and genetic rescue experiments that demonstrate its central role in sensory cilium differentiation. Rfx mutant flies show defects in chemosensory and mechanosensory behaviors but have normal phototaxis, consistent with Rfx expression in ciliated sensory neurons and neuronal precursors but not in photoreceptors. The mutant behavioral phenotypes are correlated with abnormal function and structure of neuronal cilia, as shown by the loss of sensory transduction and by defects in ciliary morphology and ultrastructure. These results identify Rfx as an essential regulator of ciliated sensory neuron differentiation in Drosophila. INTRODUCTION In multicellular organisms, sensory perception relies on cells with specialized sensory structures. In many sense organs these structures are modified cilia: vertebrate examples include the outer segments of the retinal and pineal photoreceptors, the kinocilia associated with the stereocilia of the hair cells and the multiple cilia on the sensory neurons in the main olfactory epithelium. In invertebrates, chemosensory sensilla and many mechanosensory organs, but not photoreceptors, are innervated by ciliated neurons (Eakin, 1972). Cilia are found in most eukaryotes except for fungi and higher plants. They are distinguished by an axoneme, a radially symmetric cytoskeleton of nine microtubule doublets and associated structures, enclosed in an extension of the plasma membrane. The presence or absence of a central microtubule pair classifies cilia into two types. Those with a central pair (9+2 configuration) usually have a propulsive function, while those without a central microtubule pair (9+0 configuration) are found on many animal cell types, where they are known as primary cilia. Some 9+0 cilia [e.g. those on the mammalian embryonic node (Nonaka et al., 1998)], move with a circular, whirling motion. Sensory cilia are derived from primary cilia and have been modified to varying degrees; most are probably non-motile. The importance of cilia for sensory transduction has been demonstrated in the nematode C. elegans, in which mutations affecting ciliary structure have been isolated in screens for a variety of sensory defects. These include defective osmotic avoidance (Osm), chemotaxis (Che), dauer formation (Daf) as well as defective fluorescent dye uptake (Dyf) and poor male mating behavior (Perkins et al., 1986; Starich et al., 1995). In Drosophila, nonvisual sensory perception relies on two major classes of sense organs. Type I organs or sensilla include one or more neurons and several support cells that construct specialized sensory structures such as bristles. They include the olfactory and mechanosensory bristles, as well as chordotonal organs (internally located stretch receptors that transduce auditory or proprioceptive stimuli). Each neuron in a type I organ bears a single sensory dendrite with a modified cilium. Type II sense organs are multidendritic neurons that lack cilia and specialized support cells. Their sensitivities are not known, but they also have been suggested to function as proprioceptors or mechanoreceptors (Jan and Jan, 1993). Several mutants affecting sensory perception by type I sensilla have been isolated in Drosophila in screens for loss of mechanosensation (Kernan et al., 1994), audition (Eberl et al., 2000) or olfaction (Shiraiwa et al., 2000). Those that have been molecularly characterized include nompC, which encodes a member of the TRP channel superfamily (Walker et al., 2000), and nompA, a component of the dendritic cap that ensheaths the sensory cilium (Chung et al., 2001). In nompA mutants, defects in mechanosensory behavior and electrophysiology are associated with disconnection of dendritic caps from the sensory cilia (Chung et al., 2001). Two other mutants specifically affecting chordotonal organs, btv and tilB, have axonemal defects illustrating the importance of axoneme integrity for chordotonal organ function (Eberl et al., 2000). Structural components of cilia such as tubulins, tektins and axonemal dynein subunits have mostly been isolated from the single-celled alga Chlamydomonas reinhardtii and from sea urchin (Dutcher, 1995; Stephens, 1995) but are well highly conserved in other phyla. An intraflagellar transport (IFT) mechanism required for ciliary assembly is also widely conserved (Kozminski et al., 1993; Rosenbaum et al., 1999). Best characterized in Chlamydomonas, IFT is a rapid movement of particles along the axonemal microtubules of cilia and flagella. Although many individual proteins involved in cilium architecture and IFT are well described, factors that regulate and coordinate their expression are poorly understood. In C. elegans, one such factor is DAF-19, a member of the RFX family of transcription factors. Loss of function daf-19 mutations result in the absence of cilia in sensory neurons, the only type of ciliated structures present in the nematode (Swoboda et al., 2000). DAF-19 regulates several genes required for normal sensory cilium formation, including components of the intraflagellar transport complex: che-2, osm-1, osm-5 and osm-6 (Haycraft et al., 2001; Qin et al., 2001; Swoboda et al., 2000). RFX transcription factors are defined by a 76 amino acid DNA-binding domain with a characteristic wing-helix structure (Reith et al., 1990; Emery et al., 1996; Gajiwala et al., 2000). The yeasts S. pombe and S. cerevisiae each have a single RFX factor (Huang et al., 1998; Wu and McLeod, 1995), while five RFX proteins have been identified in mammals (Emery et al., 1996; Morotomi-Yano et al., 2002). Mammalian RFX5 is essential for the transcription of MHC class II genes in the immune response (for a review, see Reith and Mach, 2001), but little is known about the cellular functions of the other mammalian RFX proteins. Two Rfx genes can be identified in Drosophila (Durand et al., 2000) (FlyBase: http://flybase.harvard.edu:7081/). Rfx is homologous to daf-19 and to mammalian Rfx1, Rfx2 and Rfx3, whereas the second gene shares conserved motifs with Rfx5, the most divergent mammalian Rfx (A. L., unpublished). Rfx is expressed in the peripheral nervous system (PNS), in the brain and in the (...truncated)