Rfx expression was analyzed during embryogenesis. Rfx mRNAs are detected only in the peripheral nervous system and in the brain of the embryo (Durand, 2000).
Drosophila Rfx immunoreactivity is distributed in type I sensory neuron lineage of the peripheral nervous system throughout Drosophila development and thus represents the first described type I lineage characteristic marker in Drosophila. In addition, Rfx is also detected in the brain throughout development and in spermatids in adult flies (Vandaele, 2001).
In embryos, Rfx is first detected at the onset of segmentation in the sensory organ precursors (SOPs) located in the gnathal segment and in the two first SOPs of each thoracic and abdominal hemisegments described as the A (anterior) and the P (posterior) SOPs. At stages 12-14 of embryogenesis, when A and P SOPs have generated new precursors by division or recruitment, Rfx is detected in these secondary precursors. At stage 15, Rfx is found predominantly in nuclei of all chordotonal (ch) and external sensory (es) organ neurons, and, at a lower level, in the accessory sister cells resulting from the last asymmetric division of the precursors. At stage 16 of embryogenesis, Rfx progressively disappears in the accessory sister cells and is only maintained in neuron nuclei at the end of embryogenesis. These results are in agreement with Rfx mRNA expression described previously (Durand, 2000) and provide new important information on Rfx stability. Whereas Rfx messenger RNAs disappear in all cells of the peripheral nervous system except ch neurons at the end of embryogenesis, the protein still persists in es neurons. Two types of sensory neurons can be distinguished in Drosophila: (1) type I neurons that innervate es and ch organs bear a ciliated sensory process at the tip of a unique dendrite and are surrounded by specialized supporting cells, and (2) type II neurons are nonciliated multidendritic neurons. In the embryo, both types of neurons are also characterized by their relative position and shape. Using an antibody that labels all sensory neurons (mAb 22C10), Rfx is found to be restricted to type I neurons of the thoracic and abdominal segments. In the head, Rfx is detected in all sensory neurons, except Bolwig's organ, which represents the future larval visual organ (Vandaele, 2001).
An accumulation of Rfx in the brain is also observed starting from stage 12 of embryogenesis in two small bilateral cell clusters in the procephalic neurogenic region. From stage 14 to 17 of embryonic development, Rfx persists in a small number of cells abutting the optic lobe invagination. Rfx is detected in the brain throughout larval and pupal development in a restricted number of cells in each brain lobe (Vandaele, 2001).
During larval and pupal life, Rfx is absent in early SOPs of the imaginal discs, but appears later in development, i.e. after puparium formation (APF). Rfx is found in all adult type I sensory organ lineage analyzed (antennae, leg and wing discs). No expression is detected in the eye disc. Expression is first detected in the leg discs, at the beginning of eversion, in the innermost SOPs of the femoral ch organ, that are ready to differentiate. Similarly, Rfx is detected in the Johnston organs of the second antennal segment of the antennal discs only once they are fully everted (around 24 h APF) (Vandaele, 2001).
Finally, Rfx is very transiently present in adult males during spermatogenesis in syncitial bundles of 64 spermatids. According to their shape and position within the testis lumen, these spermatids are in the elongation phase of their flagellum. The protein is not detectable in spherical spermatid nuclei nor in late condensing spermatid nuclei. Rfx was not detected in the female germline and in other tissues of whole stained dissected larvae (Vandaele, 2001).
Rfx is located on the third chromosome at 86A1-2 and is flanked by two genes transcribed on the opposite strand: jumeaux (also described as Domina) and CG17100. CG17100 has been suggested to be affected in stich1 mutants. Available deficiencies were investigated in the 86A-B domain that would uncover Rfx. Df(3R)hth uncovers Rfx, as visualized by polytene chromosome in situ hybridization with a Rfx probe. Moreover, no trace of RFX protein is detected in Df(3R)hth homozygous embryo tissues by immunostaining. All together with complementation results these data indicate that Df(3R)hth is a deficiency uncovering the entire 85F-86C region (Dubruille, 2002).
A P-element inserted 5' of Rfx (A143.1F3) was mobilized to generate more aberrations affecting the Rfx locus. Several chromosomes were selected by molecular screening, including Rfx49, a small deficiency uncovering the 5' end of Rfx. Molecular characterization of Rfx49 breakpoints revealed that the deletion does not uncover the adjacent jumu gene. Indeed, Rfx49 complements the lethal phenotype of all jumu lethal alleles tested. Moreover, no RFX immunostaining was found in the peripheral nervous system of Rfx49 mutant embryos compared with wild-type embryos (Vandaele, 2001; Dubruille, 2002).
Complementation analysis with Rfx49 and Df(3R)hth chromosomes identifies two complementation groups in the 86A1-2 region that have been identified as jumu and Rfx. All stich1 alleles fail to complement jumu loss of function alleles. S143702 is actually a deficiency uncovering both jumu and Rfx loci. All together these results imply that stich1 mutations affect the jumu gene but not CG17100 (Dubruille, 2002).
The more distal breakpoint of Rfx49 maps 100 bp upstream of the 5' end of CG17100. It is therefore theoretically possible that Rfx49 also affects this gene. An EMS non-complementation screen was undertaken to isolate other Rfx alleles. The Rfx253 chromosome was isolated that defines the same complementation group as Rfx49. Sequencing analysis revealed a mutation causing an amino acid substitution, S435F, in the DNA-binding domain. Serine at position 65 of the DBD is replaced by a phenylalanine. Remarkably, the mutated serine is absolutely conserved in all RFX proteins described from yeast to mammals and, in a crystallographic structure of the mammalian RFX1 DBD, is located in the 'wing' loop that mediates most DNA contacts (Gajiwala, 2000). Electromobility shift assay confirmed that Rfx253 is no longer able to bind its target sequence. Thus, based on its lack of DNA binding, Rfx253 represents a hypomorphic or null allele of Rfx (Dubruille, 2002).
Rfx is necessary for olfactory and gustatory perception in Drosophila larvae. Under standard rearing conditions, most Rfx49 and Rfx253 homozygotes die as larvae. Mutant pupae could not be observed on the walls of the culture vials, regardless of allelic combination. However, in uncrowded conditions, a small proportion of homozygous pupae were detected on vials, indicating that a few larvae can pupate. Approximately 15% of first instar larvae give rise to pupae. Mutant larvae lag their heterologous siblings by more than 3 days in development to pupae. Close observation of larvae show a peculiar foraging behavior with weak food digging efficiency. This could result from an altered peripheral sensitivity, a hypothesis that was suggested by the fact that Rfx is expressed in sensory neurons during embryogenesis (Vandaele, 2000). The gustatory and olfactory-driven behavior of mutant larvae was therefore investigated (Dubruille, 2002).
An olfactory test was performed using two pure odors (butanol and n-octyl-acetate) that induce characteristic attractive and repulsive responses. Whereas control larvae responded efficiently, mutant larvae showed no sensitivity to either odor. Also, repulsion was assayed by high NaCl concentrations: while control larvae are repulsed by concentrated salt, mutant larvae do not avoid this medium, showing that Rfx mutant larvae are defective in gustatory chemotaxis as well. To demonstrate that these responses are not due to a defect in the larval motility, phototaxis behavior was tested. This is based on the finding that Rfx is not expressed in the Bolwig organs, the larval visual system (Vandaele, 2001). Rfx mutant larvae should therefore not be affected in visual perception. This was clearly demonstrated by phototaxis behavioral assay. The phototaxis behaviors of Rfx mutant and control larvae are identical, demonstrating that Rfx mutants are not affected in their capacity to move. These data led to the conclusion that Rfx is necessary for olfactory and gustatory perception in Drosophila larvae (Dubruille, 2002).
Emerging adults are not able to walk and fly and thus stick to the medium and cannot be retrieved in culture vials. In order to recover adult mutants, third instar larvae and pupae were selected and raised until hatching on wet filter paper. The 15% to 30% of Rfx49/Rfx253 or S143702/Rfx253 adults that emerge from pupae, all present a characteristic uncoordinated phenotype. Mutant flies cannot stand or walk: legs are crossed and wings are held up. However, mutants do react to light stimulation by moving legs and wings in an effort to right themselves and walk, but uncoordination limits their movement to a few steps or a flip from one side to another. This transient activity indicates that general neuromuscular excitability is retained. To ascertain that Rfx is responsible for the observed phenotype, an Rfx cDNA was expressed in all neurons in Rfx49/Rfx253 mutants. The transgene expression indeed completely rescued the Rfx49/Rfx253 uncoordinated and low viability phenotypes (Dubruille, 2002).
The uncoordination of Rfx mutant flies is similar to the phenotype of mutants with defects in mechanosensory transduction. Type I external mechanosensory bristles include three support cells and one neuron. The innermost support cell (sheath cell) forms a tubular extension around the sensory process and produces an extracellular dendritic cap that covers the cilium tip. The two outer support cells (shaft and socket cells) construct the cuticular bristle socket and shaft and in mature organs generate the receptor lymph. To test if Rfx mutants also have defects in mechanosensory bristle electrophysiology, transepithelial potentials (TEPs) and currents were recorded from macrochaete bristles. Unstimulated wild-type bristles have a positive TEP, due to electrogenic K+ transport by sensory support cells into the restricted apical extracellular space. This creates the electrical driving force for a receptor current that is triggered by bristle deflection: current flows from the apical extracellular space into the sensory neuron, reducing the TEP and depolarizing the neuron so that it fires a series of action potentials. The response adapts to a sustained deflection (Dubruille, 2002).
The average resting TEP in Rfx mutants (12±9 mV) is significantly lower than in their heterozygous sibs (61±4 mV), although the mutant and control distributions overlap. More strikingly, no mechanically elicited changes in TEP were detected in any Rfx mutant bristle, even in bristles with TEP in the normal range. Holding the TEP constant while stimulating the bristle enables a mechanoreceptor current to be recorded. No receptor currents were elicited by mechanical stimulation in Rfx mutants, even when the TEP was clamped at a large positive value (+100 mV). Finally, no action potentials were observed in mutant neurons. Thus, the adult uncoordination phenotype probably reflects a failure of bristle mechanotransduction. Maintenance of the TEP requires both active ion transport by sensory support cells, and high cuticular and transepithelial resistance. The resistance at Rfx mutant bristles was found to be, on average, one-third that at control bristles, suggesting some reduction in the integrity of the intercellular junctions connecting the neuron and the surrounding support cells (Dubruille, 2002).
Most mechanosensory mutations affect chordotonal organs as well as bristles. Chordotonal organs are type I stretch receptors that transduce proprioceptive and auditory stimuli. To test chordotonal organ operation, extracellular sound-elicited potentials were recorded from the antennal nerve projecting from the auditory chordotonal organ (Johnston's organ) in wild-type and mutant antennae. Johnston's organ comprises over 100 individual sensory units or scolopidia located in the second antennal segment, that normally respond when distal antennal segments are vibrated by near-field sound. A series of sound pulses elicited a corresponding response from Rfx49/TM6B heterozygotes, but Rfx49/Rfx253 mutants showed no response to stimuli of the same or greater amplitude, indicating a complete loss of auditory chordotonal organ function (Dubruille, 2002).
Taken together, the broad spectrum of behavioral and electrophysiological defects suggest that all type I organs are affected in Rfx mutants. This idea was further pursued using light and electron microscopy (Dubruille, 2002).
To examine the morphology of sensory neurons in Rfx mutants, a series of molecular markers for the peripheral nervous system (PNS) was used. With the 22C10 antibody, which stains a microtubule-associated protein in all sensory neurons, no differences were observed between mutant and wild-type embryos in the number and position of sensory neurons. By contrast, the anti-HRP antibody, which stains all neuron surfaces in wild-type embryos, revealed a very weak staining of the PNS in the mutant embryos regardless of the Rfx allelic combination used. An identical weak anti-HRP staining of sensory neurons was also reported for S143702 embryos (a deficiency mutation uncovering Rfx), confirming that this phenotype is specifically due to loss of Rfx function (Dubruille, 2002).
Anti-HRP staining in mutant embryos is, however, strong enough to visualize a specific dendrite defect, particularly visible in the lateral chordotonal organ clusters (1ch5). Indeed, this array of chordotonal organs is disorganized in Rfx embryos. Most dendrites are shorter, and vary in length within a cluster. The cilia present in wild type or control embryos are always either shorter or missing in Rfx mutants (Dubruille, 2002).
To visualize neuronal dendrites in more detail, mutant flies were engineered expressing a membrane-localized GFP in all neurons. Focus was placed on two types of adult sensory organs: campaniform sensilla of the wing and chordotonal organs of the femur (Dubruille, 2002).
Campaniform sensilla of the wing are composed of a single neuron and three accessory cells. The neuronal cell body and the dendrite are situated in the same focal plane and are thus easily visible when marked with GFP. The cilium at the tip of the dendrite is located exactly under the dome-shaped sensilla in wild-type or in rescued wings. In Rfx mutant wings, the dendrite does not reach the dome and the cilium is either absent, or shorter (Dubruille, 2002).
In the femoral chordotonal organ, each scolopidium is innervated by two ciliated neurons. Instead of external cuticular structures, a rigid scolopale structure encloses the ciliary outer segments. By confocal microscopy analysis of GFP distribution, three ciliary regions can be distinguished. These regions correlate with an ultrastuctural organization that has been described by transmission electron microscopy: the axoneme, the ciliary dilation and the tubular body enclosing the tubular bundle (a dense array of microtubules). These subregions are clearly distinguished in control or rescued Rfx mutant flies. By contrast, the cilium is always shorter and even absent in mutant flies. When present, no clear cilium subregions can be distinguished indicating that the architecture of the cilium is very disorganized. Moreover, the inner dendritic segment appears swollen. These defects are specifically rescued when the Rfx cDNA is expressed in all neurons, thus demonstrating that Rfx expression in the neuron is necessary for differentiation of the ciliated sensory ending in adult sense organs (Dubruille, 2002).
Investigation of the ultrastructure of sensitive neurons was carried out on the Johnston's organ in the second antennal segment. Each scolopidium of the Johnston's organ includes two neurons, a scolopale cell and an elongated conical dendritic cap that encloses the distal tips of the two cilia. The tips of a pair of cilia are attached via an extracellular cap to a distal attachment cell (cap cell), and their bases are anchored within the neurons by a long axial filament that extends through the neuronal dendrites and cell bodies. Cross-sections performed at different proximodistal positions reveal the three different typical ultrastructural subregions of chordotonal cilia. The sensory cilium is composed of 9+0 microtubule doublets. The ciliary dilation contains a paracrystalline inclusion and the tubular body is composed of microtubule arrays that are not assembled in a stereotyped fashion as in the cilium. The two cilia are surrounded by electron dense structures (scolopale rods) produced by the scolopale cell. The cilium at the tip of the dendrite contacts an extracellular matrix (dendritic cap) (Dubruille, 2002).
Examination of sectioned antennae of Rfx mutants by light microscopy showed no gross defects in Johnston's organ, but the ultrastructure of individual scolopidia was profoundly altered. Sections of several scolopidia at different proximodistal positions clearly show the 9+0 axonemal structure in wild-type antennae, but no axoneme profiles were found in serial sections of Rfx253/Rfx49 antennae. In longitudinal sections the anchoring axial filament can be clearly observed in wild type, but not in mutant scolopidia. Its absence indicates that Rfx mutant defects extend beyond the cilium proper and affect associated structures in the neuron. In addition, a non-neuronal defect is noted: the electron dense scolopale rods enclosing the cilium are not as regularly arrayed as in wild-type flies. By contrast, the dendritic cap, also produced by the scolopale cell appears equally well organized in wild type and in Rfx mutants. These results demonstrate that Rfx is necessary for proper cilium assembly in chordotonal organs (Dubruille, 2002).
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date revised: 20 January 2004
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