BIOLOGICAL OVERVIEW

The transcription factor Rfx (Regulatory factor X) is an essential regulator of ciliated sensory neuron differentiation in Drosophila. Ciliated neurons play an important role in sensory perception. Modified cilia at dendrite endings serve as sites of sensory signal capture and transduction. Mutations are described that affect the transcription factor Rfx and genetic rescue experiments 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 (Dubruille, 2002).

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 (Dubruille, 2002).

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, 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 (Dubruille, 2002).

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 (Dubruille, 2002).

Several mutants affecting sensory perception by type I sensilla have been isolated in Drosophila in screens for loss of mechanosensation, audition or olfaction. Those that have been molecularly characterized include nompC, which encodes a member of the TRP channel superfamily, and nompA, a component of the dendritic cap that ensheaths the sensory cilium. In nompA mutants, defects in mechanosensory behavior and electrophysiology are associated with disconnection of dendritic caps from the sensory cilia. Two other mutants specifically affecting chordotonal organs, btv and tilB, have axonemal defects illustrating the importance of axoneme integrity for chordotonal organ function (Dubruille, 2002 and references therein).

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 but are highly conserved in other phyla. An intraflagellar transport (IFT) mechanism required for ciliary assembly is also widely conserved. 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 (Swoboda, 2000). 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. 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, 2001; Qin, 2001; Swoboda, 2000: Dubruille 2002 and references therein).

RFX transcription factors are defined by a 76 amino acid DNA-binding domain with a characteristic wing-helix structure. The yeasts S. pombe and S. cerevisiae each have a single RFX factor (Huang, 1998; Wu, 1995), while five RFX proteins have been identified in mammals. Mammalian RFX5 is essential for the transcription of MHC class II genes in the immune response (for a review, see Reith, 2001), but little is known about the cellular functions of the other mammalian RFX proteins. Two Rfx genes can be identified in Drosophila (Durand, 2000). 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. Rfx is expressed in the peripheral nervous system (PNS), in the brain and in the testis during Drosophila development (Vandaele, 2001; Dubruille, 2002).

Two Rfx mutations have been described that define a novel pseudolethal complementation group. One of these mutations, Rfx²⁵³ alters an absolutely conserved serine in the DNA-binding domain. This mutation is the first one affecting the RFX DNA-binding domain, thus confirming its importance for the biological function of RFX transcription factors. It supports predictions from crystallographic studies (Gajiwala, 2000) showing that this serine residue contacts DNA (Dubruille, 2002).

Rfx function in ciliated sensory neurons is essential for the proper signaling in type I sensory organs. Rfx mutant larvae have severe chemosensory and olfactory defects, being insensitive to attractive and repulsive odors. These sensory defects could explain the peculiar, potentially insufficient foraging and feeding behavior of the mutants, and could thus be responsible for major developmental delays, leading to lethality at significant frequencies. Moreover, consistent with their adult uncoordinated phenotype, Rfx mutant bristles show clear electrophysiological defects: they are completely unable to generate mechanoreceptor potentials or currents. Reductions in resting transepithelial potentials and transepithelial resistance are not sufficient to explain the complete loss of transduction, because mutants with Transepithelial mechanoreceptor potentials (TEPs) in the normal range still show no receptor current, and receptor currents can not be restored by voltage clamping the TEP at wild-type levels. Although recordings were limited to mechanosensory macrochaete bristles, the combination of mechanosensory and chemosensory behavioral phenotypes suggests a global defect in type I sense organ function, consistent with the pattern of Rfx expression (Dubruille, 2002).

Remarkably, the sensory defects are correlated with abnormal morphogenesis of the dendrite, which affects primarily the organization of its ciliated sensory endings. Indeed Rfx mutants show sensory dendrites that have severe cilium defects in femoral chordotonal organs as well as wing campaniform sensilla. This probably reflects a general defect in cilium assembly, since both the proximal and the distal parts of the organelle are disorganized -- that is, both rootlet apparatus and axonemal structures are absent from scolopidia in Rfx mutant antennae as observed by electron microscopy. All together, these observations show that the dendrite cilium is required for signal transduction in these neurons (Dubruille, 2002).

The observed mutant sensory defects are probably not restricted to only cilium disorganization. Indeed, electron microscopy reveals that the scolopale cell of chordotonal organs is also modified in the mutant antennae: the scolopale rods and membranes are disorganized compared with wild type. A similar effect on the sheath cell (the homolog of the scolopale cell in bristle organs) and other supporting cells could underlie the reduced epithelial resistance and TEP observed in Rfx mutant bristles. Rfx is expressed as early as the first neural precursor and is maintained throughout successive asymmetric division in the type I neuron lineage. Rfx is transiently expressed in the accessory scolopale cell (Vandaele, 2001). It could thus be necessary for proper accessory cell differentiation and in particular for cell junction formation between these cells (Dubruille, 2002).

Another observation suggesting that mutant defects are probably not exclusively restricted to the cilium is the reduced anti-HRP staining in mutant embryos. Anti-HRP has been shown to bind to neuronal membranes. It is known to recognize an epitope of carbohydrate origin, an alpha1,3-fucosylated, N-linked glycan associated with proteins such as Nervana-2, an Na⁺K⁺ ATPase expressed in all neurons, and with cell adhesion molecules, Fasciclin I and II, neurotactin and neuroglian. In Rfx mutants, either the expression of these proteins is downregulated or the relevant post-translational modification does not operate efficiently. Interestingly, a reduction in anti-HRP staining is seen in both type I and non-ciliated type II PNS neurons, even though no morphological defects of type II neurons are observed by anti-HRP labeling. Type I and type II neurons arise from common precursors expressing Rfx, but the latter lose Rfx expression upon differentiation and only type I neuron lineage express Rfx throughout embryonic development (Vandaele, 2001). Anti-HRP staining phenotype may suggest that no PNS neurons reach full terminal differentiation in Rfx mutant. Rfx transient expression in type II precursors could also be necessary for their differentiation. The anti-HRP staining phenotype could thus reflect a more general function for Rfx during neuronal differentiation (Dubruille, 2002).

Cilium defects are associated with dendrite morphogenesis defects such as incorrect positioning in the wing, swollen shape in the femur and the wing. It is not known if the cilium defect is responsible for these abnormal dendrite differentiations. The swollen dendrite could be a consequence of a defect in outward transport of intraflagellar transport, resulting in an accumulation of unassembled cilium components. The cilium defect could be responsible for incorrect dendrite positioning by disrupting close interactions between the cilium and structures of the sheath and the hair cell. Interestingly, the dendritic cap in the Johnston's chordotonal organ is not affected by cilium defects: this suggests that its production by the scolopale cell does not depend on the cilium. Analyzing Rfx regulatory pathways with the available mutants will allow these question to be clarified (Dubruille, 2002).

Cilia are assembled through a process called intraflagellar transport (IFT) first described in the unicellular green alga Chlamydomonas and highly conserved throughout evolution. osm-1, osm5 and osm-6, which encode proteins involved in IFT are under the control of daf-19, the sole RFX gene in C. elegans (Swoboda, 2000; Haycraft, 2001; Qin, 2001). osm-1 and osm-6 encode homologous proteins to Chlamydomonas IFT polypeptides p172 and p52. osm-5 encodes a protein involved in cilium assembly and is homologous to Chlamydomonas IFT88 protein and to a mouse protein called Tg737/Polaris that is required for assembly of the primary cilia in mouse kidney and node. Thus, IFT involves conserved proteins in many different organisms and plays fundamental roles in a variety of physiological functions. The conservation of function of the two invertebrate homologous genes as essential regulators of sensory ciliogenesis will allow a fruitful comparative identification of novel target genes likely to be involved in IFT (Dubruille, 2002).

In C. elegans, the only ciliated cells are the sensory neurons; sperm cells are not flagellated and do not express daf-19. Conversely, in Drosophila, spermatozoids have an unusually long flagella. Interestingly, Rfx is also expressed in spermatids but after flagellar elongation during a stage in which only a few genes have been reported to be transcribed, suggesting a different role in spermatogenesis. Rfx mutant sperm are normally elongated and motile, and preliminary electron microscopy data show no gross defects in spermatid flagellar 9+2 ultrastructure. Because Rfx mutants are too uncoordinated to mate, it is not known if mutant spermatozoids are functional in fertilization. Rfx may have a function in spermatogenesis other than ciliary assembly. This would imply a different mode of flagellar assembly in Drosophila spermatozoids that could reflect structural and functional differences between sensory cilia and sperm flagella. Rfx is also expressed in subdomain-specific regions of the brain. No ciliated cells have been described in Drosophila brain at the present time, whereas ciliated cells are present in mammalian brain. Studying Rfx function in Drosophila will thus bring important information regarding the diversification of the function of RFX transcription factors in evolution. Whether the ciliary function of RFX genes has been conserved in vertebrates remains an unanswered question and is of great interest regarding human pathologies associated with cilium defects (Dubruille, 2002).

GENE STRUCTURE

cDNA clone length - 3939

Bases in 5' UTR - 595

Exons - 11

Bases in 3' UTR - 550

PROTEIN STRUCTURE

Amino Acids - 897

Structural Domains

The RFX family of transcription factors is characterized by a unique DNA binding domain. Five genes have been isolated in mammals, one gene in Caenorhabditis elegans and in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Whereas the roles of the RFX genes are beginning to be understood in yeasts, no clear function has been reported in multicellular organisms, except for RFX5, the most divergent member of the family. To study the physiological role of RFX transcription factors using an alternative multicellular model, the Drosophila RFX gene (Rfx) has been isolated and characterized. The fruit fly protein shares highly conserved domains with the mammalian factors RFX1, RFX2 and RFX3 and is more closely related to this subgroup. It binds DNA with the same target specificity as mammalian factors RFX1 to 3. Drosophila Rfx is located on chromosome III and the entire locus has been characterized (Durand, 2000).

date revised: 5 December 2002

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