The Interactive Fly
Genes involved in tissue and organ development
Peripheral Nervous SystemWhat is the peripheral nervous system?Auditory apparatus of Drosophila Identification and function of thermosensory neurons in Drosophila larvae Integration of complex larval chemosensory organs into the adult nervous system of Drosophila Genetic programs activated by proneural proteins in the developing Drosophila PNS
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The peripheral nervous system consists of sensory neurons. There are two types. Type I neurons innervate the sensory organs to which they are related by lineage. Each of these sensory organs is thought to be derived from a single ectodermal precursor (sensory organ precursor or SOP) which gives rise to one or several monodendritic neurons and several support cells. Type I sensory organs have been classified into two major groups: first, mechano- or chemosensory organs that have external sensory structures in the cuticle such as bristles, campaniform, and basiconical sensilla (external sensory organs), and second, chordotonal organs that are internally located stretch receptors. In addition the larval PNS also contains numerous type II neurons with multiple dendrites. These neurons, with one exception, do not seem to be associated with support cells. Multiple dendrite neurons are thought to function as stretch or touch receptors. Multiple dendritic neurons are derived from three sources, one group from external sensory organ lineages, a second set from chordotonal neurons and a third set is unrelated to sensory organs (Brewster, 1995).
For information about the development of the antennal olfactory sense organs see lozenge.
The Drosophila auditory system is presented as a powerful new genetic model system for understanding the molecular aspects of development and physiology of hearing organs. The fly's ear resides in the antenna, with Johnston's organ serving as the mechanoreceptor. New approaches using electrophysiology and laser vibrometry have provided useful tools to apply to the study of mutations that disrupt hearing. The fundamental developmental processes that generate the peripheral nervous system are fairly well understood, although specific variations of these processes for chordotonal organs (CHO) and especially for Johnston's organ require more scrutiny. In contrast, even the fundamental physiologic workings of mechanosensitive systems are still poorly understood, but rapid recent progress is beginning to shed light. The identification and analysis of mutations that affect auditory function are summarized here, along with a discussion of how analysis of the role of the Drosophila auditory system will further an understanding of both insect and vertebrate hearing (Caldwell, 2002).
The antenna of Drosophila is an asymmetric, flagellar structure composed of three segments [a1 (antennal segment) or scape, a2 or pedicelus, and a3 or funiculus] and a feathery arista extending from the distal-most segment. The arista resonates in the presence of the species-specific courtship song and twists a3 relative to a2. The feather-like arista (antennal segments 4, 5, and 6) extends anterolaterally from a3 and slightly downward. The arista is innervated with three sensilla; these are not physiologically involved in hearing, but rather likely in thermosensation. For hearing, the arista is a component of the mechanical operation of the antenna (Caldwell, 2002).
The only known acoustic stimulus to which Drosophila responds is the courtship song, produced by the courting male. The D. melanogaster species-specific 'love song' is composed of pulse and sine components. The sine song is thought to 'woo' females prior to courtship and is, on average, a 160-Hz sinusoidal sound wave, although there is considerable variation between males. The pulse song is composed of trains of pulses with characteristic 30- to 45-ms interpulse intervals between 5- to 10-ms pulses. The interpulse interval oscillates rhythmically with a period of 50-65 s. Interestingly, this has been described as the most relevant song feature in increasing female receptivity and stimulating both partners in the courtship to expedite copulation (Caldwell, 2002).
Sound intensity is expressed in a simple relationship of the product of pressure and particle velocity. The male stays less than 5 mm away from the female during courtship, less than the wavelength of the sound being produced. At this distance, the acoustic energy is almost entirely in particle velocity rather than in pressure. When the male is positioned within one wavelength of the female, energy dissipation is low and the near-field amplitude is 80-95 dB. In Drosophila, therefore, it is advantageous to use a displacement receptor like the arista rather than a pressure receptor. Interestingly, the male also detects, and responds to the courtship song. This feature has been exploited in the design of a mutagenesis screen for deaf mutants. The roles of courtship stimuli for male courtship are poorly understood; they may be auto-stimulatory or they may be important in competitive situations (Caldwell, 2002).
The arista and a3 together are the fly's sound receivers that oscillate sympathetically when stimulated acoustically. Laser vibrometry has been performed to analyze the biomechanics of the Drosophila antenna. Measurements of oscillations taken from different locations along the arista and a3 indicate that the arista and a3 vibrate together as a stiff unit and rotate about the longitudinal axis of a3; however, a2 and the head capsule remain stationary. Thus, the major articulation is at the a2/3 joint. This articulation causes stretching of the sound transducer, the Johnston's Organ (JO). The JO is a sense organ that mediates hearing in a2 and is a cluster of about 200 scolopidia, which are the functional units of chordotonal organs (CHOs) (Caldwell, 2002).
A useful feature of the antenna, as an asymmetric sound receiver that represents a moderately damped simple harmonic oscillator when presented with sound, is that the resonance frequency of the arista and a3 increases with the sound intensity, permitting the fly to tune a large dynamic range of sound: acute hearing at low intensities and damped sensitivity at high intensities (Göpfert, 2002). This broad tuning ensures that antennal vibrations are detectable both at frequencies below antennal resonance (when elicited at close range) and when the distance of the courting male from the female's receiver increases (Göpfert, 2002). The arista and a3 rotate relative to a2 and vibrate visibly when presented with acoustic stimulation. This vibration, in turn, stimulates the mechanosensitive scolopidia of JO in a2. Indeed, the JO scolopidia are arrayed in such a way as to easily detect acoustic vibrations in the more distal antennal segments and immobilization or loss of antennal segments drastically reduces sound-evoked potentials (Caldwell, 2002).
The nonlinearity represented by the intensity-dependent frequency response of Drosophila antennal vibrations has minimal effects on tuning sharpness and sensitivity (Göpfert, 2002). Therefore, this nonlinearity is in the stiffness of the resonator. This is in contrast to that found in the vertebrate ear (which underlies the cochlear amplifier) and the mosquito antenna where the nonlinearity is introduced by negative damping. Whether the Drosophila nonlinearity in the stiffness is mediated by active processes, such as power generated by the sensory organs, or by passive processes remains to be tested (Caldwell, 2002).
The mechanoreceptive auditory organ of the Drosophila antenna is a large CHO in a2, comprising 150-200 sensory units. The homologous CHO in the mosquito had been proposed as the auditory organ by Christopher Johnston (1855); hence, this CHO is referred to as Johnston's organ (JO). Chordotonal sensory units, called scolopidia, are classified as type I sense organs: they have monodendritic, ciliated neurons associated with accessory cells. By comparison, type II sense organs are multidendritic, nonciliated neurons with no accessory cells. CHOs are not associated with external structures, in contrast to other type I sense organs, such as bristle organs and campaniform sensilla that together are called external sensory (es) organs. Rather, CHOs act as stretch receptors with attachments at two points of cuticle, often at the joints of appendages. In adult flies, each scolopidium has two or three bipolar neurons and several accessory cells. The precise composition of accessory cells in JO has not yet been well defined, but likely includes cap cells, scolopale cells, and ligament cells similar to those in larval CHOs. Cap cells are responsible for apical attachments of scolopidia, although there may also be accessory epidermal cells that secrete specialized cuticular elements for attachment. Larval CHO ligament cells are responsible for basal attachments; the JO equivalents must perform a similar function. The scolopale cell wraps several times around the neuronal outer dendritic segment (the cilium) in myelin sheath-like layers that are joined by extensive septate junctions. The scolopale cell also seals around the inner dendritic segment of the neurons with desmosomal junctions. All these junctions allow for a sealed extracellular receptor lymph space, the scolopale space, which serves as an endolymph-like ionic compartment that can drive receptor potentials. Finally, the scolopale cell also elaborates a prominent series of scolopale rods composed of thick bundles of filamentous actin. It is not yet clear whether these rods are fixed structural components or contribute to adaptation by adjusting their length (Caldwell, 2002).
In general, CHOs develop in the same way as other PNS elements. They arise from epithelia, cells of which are equipotent for CHO fates. A prepattern defined by genes that establish dorso-ventral, anterior-posterior and proximo-distal axes influences the positions at which clusters of cells acquire competence for CHO development. Competence of these proneural clusters is achieved by proneural gene expression; in the case of CHO development, atonal (ato) is the primary proneural gene. From each cluster, a single chordotonal organ precursor (COP) is selected by upregulation of the transmembrane ligand, Delta (Dl). The Delta ligand binds to the Notch (N) receptor on the other cells in the cluster. N activation blocks proneural gene expression and the non-COPs lose their competence for chordotonal fate, via lateral inhibition. In the case of CHO clusters such as the leg CHO, COPs first recruit additional COPs in a reiterative fashion. The first-specified COPs delaminate from the epithelium and activate the rhomboid (rho) gene, which results in signaling through the epidermal growth factor receptor [Egfr] on the N-activated non-COP cells of the cluster. Egfr activation mitigates N signaling and activates Dl and ato, generating additional COPs. Once COPs are specified, they undergo several asymmetric divisions to produce differentiated neuron(s) and the supporting cells of a mature scolopidium (Caldwell, 2002).
In recent years, large strides have been made towards identifying the molecular machinery that underlies JO mechanotransduction as well as the effects mutations of these components have on hearing and other mechanosenses in the fly. Genetic screens for hearing, proprioceptive, and mechanosensory mutants are well suited for isolating the molecular machinery involved in these processes regardless of the abundance or nature of the component. The sole prerequisite is that loss of function mutants will exhibit an easily recognizable phenotype (Caldwell, 2002).
Kernan (1994) isolated numerous mutants in an EMS mutagenesis screen for genes encoding mechanosensory transduction components because they exhibited reduced larval behavioral response to gentle touch. X-linked mutations in uncoordinated (unc), uncoordinated-like (uncl), and touch insensitive larva B (tilB) were isolated in the primary screen. An additional screen for mutants on the second chromosome that exhibited uncoordination resembling that of unc mutants resulted in several no mechanoreceptor potential (nomp) mutants and one reduced mechanoreceptor potential (remp) mutant. Mutations in these nomp genes produce flies with aberrant touch-sensitivity, coordination, and acoustic reception. Bristle function was measured by attaching a voltage recording pipette to the K+-rich receptor lymph of an exposed mechanosensory bristle and using a piezoelectric stage to deliver a precise movement of the bristle shaft. In wild-type flies, a bristle in the resting position exhibits a positive transepithelial potential (TEP: the voltage difference between the apical and basal sides of the sensory epithelium), reflecting the K+-richness of the receptor lymph. Deflections of the bristle towards the body elicit a stereotypic strongly depolarizing current, carried by the flow of K+ions from the receptor lymph into the neurons, presumably through the mechanotransduction channel. This transduction current results in a mechanoreceptor potential (MRP: the voltage change upon deflection). The unc, uncl, nomp, and remp mutations all eithe reduce or abolish the MRP, while the TEP is relatively unaffected (Caldwell, 2002 and references therein).
These mutants were later shown to also disrupt sound-evoked electrophysiologic responses measured from the antennal nerve (Eberl, 2000). A number of mutants were isolated in an EMS mutagenesis screen for mutations on chromosome 2 that disrupt an auditory response in Drosophila adults. Mutant strains were identified based on defects in the vigorous group courting behavior normally seen when males are presented with the pulse song. The previous assay for auditory function was female receptivity as measured statistically by a difference in latency of copulation, an assay prohibitive to screening efficiency. From this screen, 15 mutant lines exhibiting a loss or reduction of male chaining behavior were further characterized. Of these, the 5P1 mutant [later named beethoven (btv)] was the only one to affect JO sensory electrophysiology severely (Eberl, 2000). It has since been found that the 5D10 mutant also has a moderate effect on JO physiology. Finally, Drosophila homologs to vertebrate deafness genes have begun to show promise. Perhaps the best example of this is the crinkled gene, which encodes a myosin VIIa homolog. In addition, one mutant, smetana, was discovered as an unrelated mutation on a chromosome carrying another mechanosensory mutation (Caldwell, 2002 and references therein).
Mutations analyzed from these various sources could potentially disrupt several different structures or processes involved in mechanosensation in JO. (1) The acoustically induced mechanical vibrations must be propagated to the mechanosensory neurons. This is likely achieved by a tensioned system that relies on counteracting forces, namely, elasticity of the cuticle opposing tension from the JO scolopidia. Propagation of the signal requires intact structural linkages of the vibrating distal elements. (2) Given that mechanical vibrations are delivered to the neurons, the mechano-transduction machinery within the neuron must be intact to allow activation of a physiologic sensory response. This must occur somewhere along the outer dendritic segment, which is bathed in the receptor lymph, although the precise location is not yet known. Transduction likely occurs by direct activation of an ion channel, which may be part of a multiprotein complex. Specialized cytoskeletal architecture is likely required not only for localization of transduction components by motor proteins, but also for anchoring the transduction complex to allow direct activation (Caldwell, 2002).
The crinkled (ck) gene of Drosophila encodes myosin VIIa. Vertebrate myosin VIIa is an unconventional myosin expressed primarily in sensory hair cells. Orthologs of crinkled have been studied in humans (MYO7A), mouse (shaker-1) and zebrafish (mariner). In humans, defects in MYO7A primarily underlie Usher syndrome type 1B, characterized by sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa, and specific mutations are responsible for two forms of nonsyndromic deafness, DFNB2 and DFNA11. In Drosophila, strong ck mutants are completely deaf, as determined electrophysiologically by the absence of sound-evoked potentials in the antennal nerve. In preliminary histologic examination of ck mutant antennae, the JO scolopidia appear detached from the a2/a3 joint. This suggests that the mechanical vibrations of the arista and a3 cannot be propagated to the scolopidia. Although this defect would adequately explain the deafness phenotype, it is quite possible that the myosin motor encoded by ck is required not only for apical scolopidial attachments or their maintenance, but also for physiologic function of the scolopidia. Such a dualism appears to be true for vertebrate myosin VIIa as well because mutants not only show gross structural defects in the stereocilia, suggesting a morphologic maintenance role, but also show defects in gating properties of the transduction channel, suggesting an additional more intimate role in transduction (Caldwell, 2002 and references therein).
Bristle shafts in ck adults are also shorter and appear bent when compared with the wild type. Bristle MRP amplitudes are severely reduced in ck mutants. This is consistent with broad sharing of mechano-transduction components between bristle organs and CHOs (Eberl, 2000). The structural or physiological basis of ck bristle dysfunction is not yet clear (Caldwell, 2002).
Like ck, the no mechanoreceptor potential A (nompA) mutants disrupt the physical propagation of the mechanical signal to the sensory neurons. However, in nompA the disruption appears as a detachment of the sensory dendrites from the dendritic cap elaborated by the support cells. Thus, the disruption is farther along the mechanical stimulus chain. Nevertheless, some apical detachment of scolopidia from the a2/a3 joint is also seen. nompA was recovered as a complementation group of two mutant alleles associated with severe adult uncoordination and a third allele was identified later. Mutations in nompA affect both es and CHOs as evidenced by the lack of bristle MRPs and lack of sound-evoked potentials from JO, both of which are rescued with a nompA+transgene. nompA encodes a type I sensilla-specific, single-pass transmembrane protein with extracellular domains that include a Zona Pellucida (ZP) domain and five plasminogen N-terminal (PAN) modules. In CHO, the NompA protein is a component of the dendritic cap produced by the scolopale cell, but is not expressed in the neuron. The ZP domain is necessary and sufficient for incorporation into the cap matrix, while the divergent PAN domains may permit the cap to bind to a diverse array of attachment sites. In es organs, the shaft (trichogen) and socket (tormogen) cells secrete K+-rich endolymph that generates the TEP while the sheath cell (thecogen) ensheathes the sensory cilium and produces the dendritic cap. nompA is expressed primarily in the sheath cell of es organs; in mutants the dendrite is usually detached from the dendritic cap, consistent with uncoupling of the mechanical stimulus from the mechano-transduction machinery. Clearly, nompA encodes a protein essential for organization of the cap and its proper attachment to mechanosensory cilia and to apical cuticular structures (Caldwell, 2002).
no mechanoreceptor potential C (nompC) mutants were of interest because of severe uncoordination, loss of larval touch-sensitivity, and loss of MRPs in bristle organs. The nompC gene has been cloned; it encodes a six-transmembrane domain protein, distantly related to the TRP (transient receptor potential) family of ion channels, and at the cytoplasmic amino terminus it has a long array of 29 ankyrin repeats, which could mediate associations with a variety of cellular components, including the cytoskeleton. Under voltage clamp conditions, three nonsense alleles of nompC, all causing strong uncoordination, exhibit an almost complete loss of mechanoreceptor current (MRC: receptor current evoked by mechanical stimulation) when the bristle is deflected toward the body and a severe reduction in the number of action potentials fired. A fourth missense allele with less severe uncoordination shows retention of the robust mechanically evoked response, but exhibits adaptation to mechanical perturbations much more quickly than in wild-type controls and a twofold reduction of action potentials. Therefore, these flies produce fewer action potentials due to the rapid adaptation that limits the time of receptor depolarization. These features argue strongly that nompC represents the major mechano-transduction channel in bristle organs and mimics very closely the biophysical properties of vertebrate hair cell transduction channels. Surprisingly, nompC mutants show only a modest reduction of sound-evoked potentials recorded from the JO when presented with the pulse song. In fact, of all bristle MRP mutants tested for JO response, nompC was the only one that retained much of the sound-evoked response. Thus, although nompC is absolutely required for meaningful bristle organ function, it appears to play only a minor role in transducing auditory stimuli in JO. Therefore, an additional transduction channel is inferred to operate in CHOs. This additional inferred channel may be CHO-specific, or it may be responsible for the small MRC remaining in bristle organs in nompC mutants (Caldwell, 2002 and references therein).
nompB flies, like nompA, exhibit no bristle MRP and no sound-evoked potentials in response to the pulse song. As with nomp A, nompB mutants show gaps between the tips of sensory dendrites and the external sensory structures of campaniform sensilla. Unlike nompA, it is the sensory ending of the neuron rather than the dendritic cap that is abnormal. This is clear in the long outer segments of CHOs, which are missing or malformed in nompB mutants. Molecular analysis of the nompB locus suggests that the basis of the dendritic detachment phenotype is different from that of nompA. nompB encodes a protein containing ten tetratricopeptide repeats that compose two potential protein interacting domains. It is homologous to the mouse and human Tg737/orpk gene, to osm-5 in C. elgans and to IFT88 in Chlamydomonas. These proteins are all associated with ciliary elongation defects, which, in some of these systems, have been shown to result from defects in intraflagellar transport (IFT). IFT is a mechanism by which proteins required for assembly and maintenance of the axoneme are trafficked along the axonemal microtubules of cilia and flagella microtubules. Thus, the mutant phenotype of nompB is consistent with the role of the putative IFT component it encodes (Caldwell, 2002 and references therein).
The 5P1 mutant, recovered as an auditory behavior mutant, was later named beethoven (btv) because it disrupts JO function but leaves bristle MRPs intact. These flies are slightly uncoordinated and sedentary, consistent with defects in all CHOs. Preliminary electron microscopy reveals ciliary defects in the outer dendritic segment of JO neurons, as well as other abnormalities in the appearance of the scolopidia. Homozygous and hemizygous btv males have motile sperm and are fertile, whereas males deficient for the btv region in overlapping chromosomal deficiency combinations are sterile. It is not yet known whether the male sterility results from complete deletion of btv or from a separate genetic function, although the latter interpretation is favored. The btv gene maps to the 36DE region of chromosome 2. The sequence in the candidate region encodes two compelling candidates for the btv gene. One is a new cadherin gene (CadN2) which is adjacent to CadN, and which likely arose from a tandem duplication-divergence of a single ancestral gene. The other major candidate is a dynein heavy chain, Dhc36C, specifically the 1b isoform implicated in IFT in other organisms (Caldwell, 2002).
The two touch insensitive larva B (tilB) mutations, recovered in a larval touch screen, disrupt CHOs but have no effect on bristle physiology or MRP, mirroring the defect seen in btv. Unlike the nomp genes, tilB mutants only show slight motor uncoordination. tilB sound-evoked antennal nerve potentials are completely absent. In addition to auditory dysfunction, these mutants are also male sterile because of sperm amotility. Ultrastructurally, tilB spermatid axonemes have defects in the outer microtubule doublet arrangement. The axonemes of wild-type spermatids are 9 X 2 + 2. Each microtubule doublet has inner and outer dynein arms extending from the A microtubule and a nexin linkage between AB microtubules. The dynein arms, required for microtubule sliding to effect sperm motility, are absent in tilB mutants (as perhaps is the nexin bridge). tilB maps genetically to an unsequenced gap in the genome on the X chromosome (Caldwell, 2002). Similar to tilB, smetana (smet) flies are defective for hearing and male fertility. This combination suggests that smet is also required for axonemal integrity of sperm flagella and CHOs. smet was discovered fortuitously as an additional unrelated mutation on a nompC mutant chromosome that resulted in complete loss of sound-evoked potentials. smet has been mapped to an unsequenced gap near the histone gene cluster on chromosome 2 (Caldwell, 2002).
The function of unc in Drosophila is also currently under investigation. In addition to their uncoordination and bristle MRP defects, unc mutants are also deaf and male sterile. Males do not produce mature sperm, and the flagella of primary spermatids show gross defects in their axonemal structure. unc encodes a coiled-coil protein that is expressed solely in neurons of type I ciliated mechanoreceptors and in the male germline. It is localized to the centrioles of primary spermatocytes and the junction of the nucleus and flagellum in differentiating spermatids, but not in mature sperm. The inner dendritic segments of leg and JO scolopidia are normal, but the ciliated outer dendritic segments fail to connect to the dendritic cap. Clearly, unc has an important role in axonemal formation in sensory cilia and flagella, because it appears to be involved in the conversion of mitotic centrioles into ciliogenic basal bodies (Caldwell, 2002).
Recently, the role in hearing has been investigated of technical knockout (tko), a nuclear gene encoding mitochondrial ribosomal protein S12. In humans, mitochondrial DNA (mtDNA) mutations are responsible for a large number of pathologic syndromes with which sensorineural deafness is often associated. In some cases, the only physiologic defect in patients is hearing loss. tko flies are bang-sensitive, exhibiting a temporary paralysis from the mechanical perturbation. The paralysis could be explained in part by altered sensory feedback from the mechanosensitive bristles. This sensitivity to mechanical vibration is similarly found in humans as a result of aberrant sensory signaling from mechanoreceptors in the inner ear. In addition to lengthened developmental times, tko males also show a reduced male courtship response upon presentation of the pulse song, suggesting a hearing deficiency. These phenotypes are associated with a single missense mutation (L85H) of tko, and mutant larvae have reduced activity of mitochondrial redox enzymes and mitochondrial small subunit rRNA. Raising wild-type flies on doxycyclin generates a phenocopy of the tko phenotypes. It is likely that hearing is an energetically costly process and, consistent with this, reduced levels of ATP were found in tko flies. Electrophysiologic recordings will be necessary in the future to determine the anatomical location of tko behavioral deafness. Nevertheless, this mutant provides a compelling model for mitochondrial deafness that could be used to study not only the precise role of mitochondria themselves, but perhaps also their intracellular transport to relevant parts of the cell (Caldwell, 2002).
It is concluded that construction of a functional auditory receptor requires proper integration of developmental and mechanical processes. Specification, asymmetric divisions and differentiation of the cells producing sense organs must proceed unperturbed. Then, the sense organs must express the diverse assortment of cellular components that establish the intercellular and intracellular environment for the sense organ to be poised for mechanosensation. Furthermore, the mechanoreceptive cells must be mechanically linked to the acoustically sensitive vibrating structures. The development of JO, the Drosophila auditory organ in the antenna, is understood in many fundamental ways, at least vicariously. Although few studies have focused specifically on JO, it is believed that the basic steps of PNS development, which are generally well understood, will also hold for JO. Thus, further studies on JO development must focus on determining the extent of the variations on a well-known theme. In some cases it has already been seen that the mechanisms can differ in JO, such as in the role of sal/salr. Specifically flies in which sal/salr expression is absent in the antenna are completely deaf to the courtship song because of defects in the JO. In these mutants, the specialized cuticle at the a2/a3 joint where JO is attached is missing. a2 and a3 in these flies are effectively fused, restricting rotation of the a3 relative to a2. This fusion prevents the propagation of the mechanical signal to JO; thus, sal/salr mutants have conductive hearing loss. Furthermore, most or all scolopidia of JO are absent in sal/salr mutants. Mutant scolopidia appear to be specified, but are not maintained (Caldwell, 2002).
Many future prospects remain for research in Drosophila hearing: (1) it is important to understand the developmental issues that make JO like other sense organs, and those that set it apart as a specialized organ for hearing; (2) cloning more transduction components will illuminate understanding of the nature of the mechanosensory machinery. In particular, the transduction channel that operates in JO and acts in parallel with nompC, must be identified. (3) To understand the function of each component, determining the relationship of components to each other will be crucial. Epistasis experiments, through interaction screens or localization of components in genetic backgrounds mutant for various other auditory components, would be useful experimental paradigms. (4) The fundamental role of ciliary action in transduction of CHOs is still not understood. However, analysis of auditory mutants with laser vibrometry may add greatly to this endeavor. (5) For Drosophila to achieve its greatest usefulness in understanding of the relationship between insect and vertebrate auditory mechanisms, continual comparison, and testing of homology with vertebrate auditory genes and mechanisms must be carried out. The Drosophila auditory system is poised to become an important test system for dissecting the function of human homologs, even human-specific auditory components (Caldwell, 2002).
Although the ability to sense temperature is critical for many organisms, the underlying mechanisms are poorly understood. Using the calcium reporter yellow cameleon 2.1 and electrophysiological recordings, thermosensitive neurons were identified and their physiologic responses were examined in Drosophila larvae. In the head, terminal sensory organ neurons show increased activity in response to cooling by ~1°C, heating reduces their basal activity, and different units show distinct response patterns. Neither cooling nor heating affects dorsal organ neurons. Body wall neurons show a variety of distinct response patterns to both heating and cooling; the diverse thermal responses are strikingly similar to those described in mammals. These data establish a functional map of thermoresponsive neurons in Drosophila larvae and provide a foundation for understanding mechanisms of thermoreception in both insects and mammals (Liu, 2003).
To identify neurons responding to changes in temperature, an optical approach using yellow cameleon 2.1 (YC2.1), an engineered, calmodulin-based, Ca2+-sensitive protein, was used. Its two fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), comprise a fluorescence resonance energy transfer (FRET)-capable pair; a conformational change in the protein causes FRET to increase when the Ca2+ concentration rises. Cameleon fluorescence has been used to measure intracellular (or cytosolic) Ca2+ concentration, [Ca2+]i, in vitro and in vivo (in C. elegans). Transgenic Drosophila larvae were developed that express cameleon in their neurons, and FRET was assayed to monitor activity in the peripheral neurons as the temperature was changed. The FRET measurements, plus electrophysiologic and behavioral assays, indicate that the terminal organ is a thermosensitive structure that responds to cool temperatures. Some body wall neurons also showed FRET changes with temperature shifts, and in contrast to the terminal organ, they responded to warm temperatures (Liu, 2003).
The pan-neuronal promoter elav was used to drive expression of the YC2.1 variant of cameleon with the Gal4-UAS system. Heterozygous transgenic larvae (elav-Gal4/+;UAS-YC2.1/+) showed fluorescence signal in all neurons. For example, cameleon fluorescence was distributed in neurons of the lateral pentascolopidial chordotonal organs in a diffuse pattern in the cell bodies and neurites, but not in the cell nuclei. Similar results were obtained in two other lines. To increase fluorescence intensity, homozygous lines (elav-Gal4;UAS-YC2.1) were generated. They produced a more intense fluorescence signal than did the heterozygous line; therefore, homozygous larvae were used for optical recording (Liu, 2003).
The dorsal and terminal organs are the major sensory structures of the larval head. Each organ contains more than 30 bipolar neurons with large dendrites extending to the tip of a dome-like structure where pores open to the environment. Cameleon was expressed in the larval head in terminal and dorsal organ neurons. Terminal and dorsal organ YFP/CFP ratio images were measured at 18, 10 and 40°C. When the temperature fell, the terminal organ YFP/CFP ratio and the calculated [Ca2+]i increased, and when the temperature rose, terminal organ [Ca2+]i fell. In contrast to the terminal organ, temperature variations had little effect on [Ca2+]i in the dorsal organ, salivary gland or trachea (Liu, 2003).
At 18°C, the fluorescence ratio from terminal organ neurons fluctuates spontaneously; this was reflected in the variance of the fluorescence ratio (sigma2). These fluctuations are not an artifact, since the fluorescence ratio in the adjacent dorsal organ and in the salivary gland and trachea did not fluctuate, and the sigma2 was much lower. Cooling increases and heating decreases terminal organ sigma2, but has little effect on sigma2 in the other cell types. These findings suggest that terminal organ neurons have substantial spontaneous activity, even at room temperature, and the large [Ca2+]i fluctuations raise the possibility that some neurons might show oscillating activity (Liu, 2003).
Although the data do not allow distinguishing whether the changes in [Ca2+]i are upstream or downstream of action potential firing, these results indicate that the larval terminal organ contains thermosensory neurons that respond to cooling. Their response profile resembles the behavior of the most commonly observed mammalian cold receptors. The lack of thermoreceptor activity in dorsal organ neurons provides an important control showing the specificity of temperature sensing by the terminal organ: both terminal and dorsal organs contain sensory neurons, they lie adjacent to each other in the larval head; they are exposed to the same temperature stimuli, and cameleon fluorescence from both organs can be examined in the same image. These data also indicate that the cameleon protein itself does not respond to temperature changes (Liu, 2003).
To further test the hypothesis that terminal organ neurons detect cool temperatures and to obtain an independent assessment of the response of thermosensitive neurons, a glass electrode was inserted into early third-instar larval terminal or dorsal organs and extracellular recordings were obtained. Dorsal organ recordings showed no activity at room temperature or any response to warming or cooling. Terminal organ neurons, however, showed spontaneous activity at room temperature. This difference between terminal and dorsal organ basal activity is consistent with the difference in sigma2 in the optical measurements. Depending on the electrode position, recordings were obtained with one or two units. Thirty-six such recordings were analyzed (Liu, 2003).
When larvae are cooled by placing a cold metal block in their vicinity, terminal organ neurons respond in one of three ways. A type-I response is an increased firing frequency that adapts during the time course of the stimulus. This response occurred in 20 of 36 recordings. A type-II response, which occurred in 3 of 36 recordings, showed cold-induced oscillatory activity. This oscillating electrical activity may explain, in part, the increase in FRET sigma2 observed on cooling the terminal organ. In contrast to the type-I response, the onset of the type-II response is slow, and after the cold stimulus is removed, oscillations persist for some time before returning to baseline. A type-III response involves a transient cold-induced reduction in activity; it occurred in 13 recordings (Liu, 2003).
When a warm block was substituted for the cold one, spontaneous activity fell with all three types of response. For example, in recordings showing a type-I response, a warm stimulus reduced activity from 16.7 +/- 2.4 spikes/s to 7.4 +/- 2.9 spikes/s. After removing the warm block, neuronal activity recovered to baseline within 5 s (Liu, 2003).
On exposure to the cold stimulus, temperature will fall, and the closer the stimulus to the terminal organ, the more rapid the fall. In type-I cells, faster cooling elicits a greater increase in neuronal activity, the maximal increase in activity occurs 1 s later, at a time when that terminal organ temperature was calculated to have fallen 0.30°C. With the cold block at position 1, the maximal increase in spike rate occurs at 16 s when a 0.43°C decline was calculated to have occured. At the intermediate position 2, a fall of 0.45°C at 7 s was calculated when activity was maximal. Thus, the response to a cold stimulus seems to be a function of the rate of cooling and the change in temperature (Liu, 2003).
To change temperature by another means, a Peltier-based device was used to cool the solution bathing the larval body. Recordings were studied that showed a type-I response and lowered temperature 0.1-1°C from room temperature. This cooling increased spike frequency by 20%. Thus, similar results were obtained with different methods of lowering temperature and with independent means of measuring the response (Liu, 2003).
Because cooling stimulates terminal organ activity, it was hypothesized that disrupting terminal organ function would blunt the behavioral response to a reduced temperature. Earlier work showed that the GH86 promoter drives expression in the terminal organ; it also drives expression in the dorsal organ, epidermis, enocytes and pharyngeal muscle. The GH86-Gal4 promoter was used to drive a UAS-tetanus toxin light chain transgene (UAS-TNT-C), which specifically degrades synaptobrevin and thereby blocks neurotransmitter release. Because both the GH86-Gal4 and UAS-TNT-C transgenes are located on the X chromosome, only female F1 larvae that contained both transgenes were studied. The GH86 x TNT-C cross showed a reduced preference for 18°C compared with 11°C. These data suggest that larvae use terminal organ thermosensors to sense cool temperatures. Larval terminal organs contain more than 30 neurons and also likely respond to other sensory stimuli. For example, some terminal organ neurons may be involved in the response to low concentrations of salt (Liu, 2003).
To test thermosensitivity at another site, neurons in lateral body segments 5-8 were examined. At this location, there was less interference from fluorescence of the central nervous system and salivary glands than in segments 1-4. Depending on the location of the neurons, it was possible to measure fluorescence from some individual neurons. In other cases, clusters of neurons were studied because single neurons could not be reproducibly identified. However, temperature-dependent changes in the clusters likely arise from single neurons because the fluorescence changes often occur at a single spot within the cluster (Liu, 2003).
Multidendritic neurons show the greatest response to temperature changes, and the amplitude of [Ca2+]i response varies for different neurons and clusters. Different neurons also show distinct thermosensory responses. For example, for neurons in clusters 2 and 3, and neuron ddaB, cooling reduces and heating elevates [Ca2+]i. This response is the opposite of that in the terminal organ. The neurons in cluster 1 and lch5 increase [Ca2+]i when the temperature is either raised or lowered from the preferred temperature of 18°C. The distinct response patterns observed suggest that different neurons carry specific information to the central nervous system (Liu, 2003).
These data provide a functional map of thermosensitive neurons in Drosophila larvae. Neurons with different temperature responses were to be anatomically segregated. Moreover, within different regions there was a striking diversity in the behavior of thermosensitive neurons (Liu, 2003).
Terminal organ cold sensors show activity at room temperature, and this activity increases with cooling and falls with heating. Thus, these thermosensors are poised to respond whenever temperature changes, even slightly. Moreover, terminal organ function is apparently required for a normal response to cool temperature because expressing the tetanus toxin light chain in the terminal organ blunts the behavioral preference for 18°C versus 11°C. The calculations of the temperature shifts induced by a cold block and the measurements of bath temperature indicate that the terminal organ responds to changes of ~1°C. Thus, these data explain how larvae respond to a change in temperature, but how they respond to absolute temperatures remains unclear. The answer probably lies in central integration of output from the complex mixture of thermosensory neurons. In this regard, thermoreceptors with oscillating discharges may be particularly important to sensing absolute temperatures. It has also been suggested that the substantial complexity of thermoreceptive cell types may increase the sensitivity of the system(Liu, 2003).
The data suggest a striking similarity between thermoreceptor physiology in Drosophila and mammals. In both organisms, different types of neurons encode the response to cold and heat stimuli. It was found that in Drosophila, the most common type of terminal organ cold receptive neurons show a characteristic response to cold and heat; they spontaneously discharge at room temperature, cooling reduces the frequency of nerve impulses, and warming decreases activity. This type of cold receptor neuron is also very common in mammals. Additionally, an oscillatory response to cooling was found in 3 of 36 larval terminal organ neurons. The preliminary observations suggest that oscillatory activity may be more common if temperature is reduced more rapidly and to a greater extent. Interestingly, there are several reports of oscillatory activity in mammalian thermoreceptors. Whether this type of activity results from coordinated effects of temperature on multiple channels or on a single type of channel is unknown. Cluster 1 and Ich5 neurons in Drosophila increase activity on both warming and cooling. Mammals also contain these so-called 'paradoxical' temperature receptors. It will be interesting to learn whether this activity is generated by two different temperature-responsive ion channel receptors that are both expressed in a single neuron. Finally, some Drosophila neurons (multidendritic neurons ddaB and clusters 2 and 3) increase their activity during heating and reduce activity during cooling. This pattern of activity also exists in many mammalian warm receptors (Liu, 2003).
These findings reveal a diverse pattern of thermosensory response in larval neurons and provide new insight into the physiology of temperature sensing in Drosophila. Moreover, the results demonstrate common thermosensory response patterns between distantly related animal species. Given the potential relationship between temperature sensing and pain, this work may provide a basis for additional insight into nociception. Thus, these studies help pave the way toward a better understanding of the molecular mechanisms of thermoreception in both insects and mammals (Liu, 2003).
The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented in this study, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, it is shown that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, apoptosis is observed in a third larval pharyngeal organ. Hence, the experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. The conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis (Gendre, 2003).
The external gustatory sensilla of the Drosophila larva appear to follow the general holometabolan fate: they degenerate during metamorphosis and are replaced by adult-specific sensilla that derive from the labial imaginal disc. This study examines whether this rule also applies to the internal gustatory system that is located along the pharyngeal tube. Interestingly, the adult pharynx derives essentially from small, densely packed imaginal cells that comprise the clypeolabral bud, which is closely associated with the larval pharyngeal skeleton. Does this imply that adult pharyngeal sensilla are born during metamorphosis, like their external counterparts, or do the anatomical similarities of certain larval and adult pharyngeal organs rather suggest persistence of sensilla through metamorphosis (Gendre, 2003)?
The data prove that most of the neurons of the three major adult pharyngeal sense organs are persisting larval neurons that were born in the embryo. This is unlike other adult sensory neurons, nearly all of which derive from imaginal discs. This interpretation relies on two independent experimental approaches for demonstrating embryonic birth dates (the use of the embryonic lineage tracer HRP and cell labeling by FLPout at late embryonic stages, a novel use of this technique). The experimental data are supported by anatomical observations showing: (1) an almost identical organization of the larval posterior pharyngeal sense organ (pps) and the adult dorsal cibarial sense organ (dcso); (2) the presence of the pps and dorsal pharyngeal sense organ (dps) sensilla continuously through metamorphosis; (3) an uninterrupted expression of the P[GAL4] lines used in these two organs, and (4) the persistence of dendrites and axons in all surviving neurons (Gendre, 2003).
HRP injected at the syncytial blastoderm stage becomes incorporated into every cell upon cellularization. During subsequent development, the marker remains at high levels in cells that divide only a few times but becomes diluted in cells that undergo repeated divisions. Consequently, labeling in the adult is expected in many neurons of the central nervous system known to be persisting larval neurons (e.g. optic lobe pioneers) but should be absent from tissues derived from imaginal discs. This corresponds to what was observed and leads to the postulation of an embryonic origin for the elements containing high HRP levels in adult pharyngeal sense organs (Gendre, 2003).
This interpretation is supported by the FLPout experiments performed at late embryonic stages with the neuron-specific MJ94 line. In adults deriving from this treatment, exclusively single labeled neurons were detected in sensillum 7 of the labral sense organ (lso), containing eight neurons, and in the five multiply innervated sensilla of the vcso and dcso. Although the cell lineage of these sensilla was not studied, they are probably homologous to other multineuronal terminal-pore gustatory sensilla, which derive from a common sensory mother cell. Indeed, apart from its eight neurons, sensillum 7 of the lso corresponds to a typical insect gustatory sensillum in terms of fine structural and cellular organization, containing no more than three accessory cells. Hence, the single labeled neurons in this sensillum and in all sensilla of the vcso and dcso must have been postmitotic during FLPout. This agrees with the observation that formation of head nerves is complete by embryonic stage 15 (Gendre, 2003).
Could these neurons have remained immature during larval life, differentiating only during metamorphosis, similar to subsets of postmitotic cells in the larval central nervous system CNS? This is thought to be rather unlikely because it would require either the entire sensillum or subsets of neurons in multineuronal sensilla to remain immature. Moreover, there is no indication for immature neurons from tracing their development with the marker line mCD8-GFP. Thus, it is suggested that all the neurons of the dcso and vcso, and sensillum 7 of the lso derive from mature, functional larval neurons. Also, continuous reporter expression through metamorphosis suggests that one of the mononeural lso sensilla (perhaps sensillum 3) might be another persisting larval sensillum (Gendre, 2003).
The fact that the pps and dps persist through metamorphosis is remarkable given the origin of the adult labrum and cibarium from imaginal cells of the clypeolabral bud. Massive labeling of pharyngeal epithelial cells was observed after early pupal BrdU application. Moreover, the pharyngeal cuticle is shed and regenerates, a process that includes the cuticular part of the sensilla in question. Perhaps the birth of additional accessory cells during metamorphosis (e.g. in the dcso or vcso, containing exclusively persisting neurons) is related to this modification. Formation of new cuticular structures is also known from persisting external sensilla during larval molts, but the survival of pharyngeal sensilla during the extensive remodeling of the pharynx remains stunning. The morphogenetic movements observed in the sensory system certainly reflect these dramatic changes (Gendre, 2003). Why is the larval pharyngeal sensory apparatus largely conserved through metamorphosis? Small subsets of neurons associated with leg imaginal discs or with abdominal segments persist through metamorphosis. In the fly Phormia, such leg-disc-associated neurons remain immature, implying that they are non-functional. Laser ablation studies suggest that persisting neurons might help adult afferents to navigate from the imaginal discs to their central targets. Whether they become truly integrated in the adult nervous system or die after reaching adulthood (having completed their pathway role) remains to be shown (Gendre, 2003).
The data demonstrate for the first time experimentally the integration of larval sensory neurons into the adult nervous system of Drosophila. Particularly striking and novel is the fact that entire, fully differentiated larval sense organs become incorporated. Also, this is the first observation of metamorphic survival in the chemosensory system (Gendre, 2003).
Concerning the persisting neurons of the lso, a pathway function for the newly developing afferents toward and inside the central nervous system is certainly possible. However, the
integration of the surviving pharyngeal neurons into the adult sensory system invites other interpretations. For example, these neurons and/or their central projections might be particularly
precious, allowing, for example, the persistence of specific feeding-associated gustatory tasks through metamorphosis. As an alternative explanation, survival might be due to reasons of economy,
a principle that governs the metamorphosis of the nervous system. Although neuronal reorganization is indispensable owing to the changing demands of larval and adult life, it is kept at a
minimum, as shown by the survival of most larval interneurons and motor neurons. Sophisticated adult sense organs, however, might be easier to build de novo than by the transformation of simple
larval organs, explaining the almost complete replacement of the larval sensory system. Why pharyngeal sense organs do not follow this general rule might relate to their largely conserved
function at the two stages of life (analyzing the quality of ingested food of similar composition). The presence of larva-specific and adult-specific sensilla, however, suggests the existence of
stage-specific gustatory tasks (Gendre, 2003).
Neurogenesis depends on a family of proneural transcriptional activator
proteins, but the 'proneural' function of these factors is poorly understood,
in part because the ensemble of genes they activate, directly or indirectly,
has not been identified systematically. A direct approach to this
problem has been undertaken in Drosophila. Fluorescence-activated cell sorting was used to recover
a purified population of the cells that comprise the 'proneural clusters' from
which sensory organ precursors of the peripheral nervous system (PNS) arise.
Whole-genome microarray analysis and in situ hybridization was then used to
identify and verify a set of genes that are preferentially expressed in
proneural cluster cells. Genes in this set encode proteins with a diverse array
of implied functions, and loss-of-function analysis of two candidate genes
shows that they are indeed required for normal PNS development. Bioinformatic
and reporter gene studies further illuminate the cis-regulatory codes that
direct expression in proneural clusters (Reeves, 2005).
The PNC cells that express the proneural genes achaete (ac)
and scute (sc) comprise only a small fraction of the wing
imaginal disc of the late third-instar Drosophila larva. It is
anticipated that this might frustrate attempts to characterize PNC-specific
gene expression in unfractionated wing discs (e.g., by comparison of wild-type
and ac-sc mutant tissue). Accordingly, PNC cells were purified by
using fluorescence-activated cell sorting (FACS). As a PNC-specific marker,
a GFP reporter was chosen representing the Bearded family gene E(spl)m4.
m4 is strongly and specifically expressed
in PNCs, and a cis-regulatory module has been identified sufficient to recapitulate this activity. Wing imaginal discs were dissected
from late third-instar larvae carrying the m4-GFP transgene and
dissociated in trypsin-EDTA; cells with fluorescence greater than that of
w1118 control cells (GFP-positive cells) and cells with
fluorescence comparable to the control (GFP-negative cells) were recovered separately by FACS (Reeves, 2005).
Transcripts from several genes
known to be expressed in domains of the wing disc outside of PNCs (en,
hh, and twi) were found to be greatly depleted in the
GFP-positive cell population. These negative controls provide further evidence of successful separation of PNC cells from other disc cells (Reeves, 2005).
Since the microarray data clearly associates
expression of known genes preferentially with the expected cell populations,
43 candidates not known to be
expressed in wing imaginal discs were chosen for further analysis. Candidate
genes for which cDNA clones were available from the Drosophila Gene
Collection were favored. The selected genes exhibit a wide variety of GFP+/GFP-
expression ratios in the microarray data, and their products have a broad
spectrum of predicted functions (Reeves, 2005).
In situ hybridization was employed as a
secondary screening method, both to verify that candidate genes selected from
these microarray data are expressed in wing imaginal discs, and to determine
their specific patterns of transcript accumulation. The wing disc expression
patterns observed can be sorted into three major classes: PNC patterns, SOP
patterns, and overlapping patterns.
Five of the 43 selected candidate genes exhibit a complete PNC pattern of
expression, while 3 other candidates
are expressed in subsets of PNCs; phyl is expressed in the
SOP and in a subset of non-SOP cells in each PNC. An unexpected 18 candidates are expressed in the
presumptive SOP cells of the wing disc. Fourteen of these SOP genes are
expressed in a complete pattern of SOPs, whereas the remaining four are expressed
either late in SOP development or in subsets of SOPs. The existence of
the latter group suggests that the cell sorting strategy made it possible to
identify genes that are expressed preferentially in just a few cells of the
wing disc. Overall, 27 (63%) of the tested candidates were found to display
PNC- or SOP-specific expression patterns. This is likely to be an underestimate
of the true success rate of the microarray analysis, since 23 genes known to be
expressed in these patterns are not included in the statistic, though they were
reidentified in the screen (Reeves, 2005).
In addition to those expressed specifically
in PNCs and SOPs, a small group of candidate genes was found that is expressed
in patterns that overlap PNCs but appears to be distinct from them.
Detection of this class of genes is an important confirmation
of the efficacy and unbiased nature of the experimental approach (Reeves, 2005).
Patterned expression of the
proneural genes ac and sc defines the PNCs for most external
sensory bristles in adult Drosophila, and ac-sc function is
required for PNC and SOP gene expression, as well as for specification of the
SOP cell fate. Fifteen of the genes identified by the combined cell
sorting/microarray approach also require proneural gene function for their
expression. In an ac− sc− proneural
mutant background, transcript accumulation from members of both the PNC
(CG11798, CG32434/loner, edl, PFE) and SOP
(CG3227, CG30492, CG32150, CG32392, Men,
qua) classes is lost from PNCs that require ac-sc function.
This result is further
evidence that the approach has identified bona fide PNC genes, and it
demonstrates that expression of these ten genes is, directly or indirectly,
downstream of the bHLH activators encoded by ac and sc. The data
further show that the PNC-specific imaginal disc expression of the previously
studied genes mira, phyl, rho, Spn43Aa, and Traf1
is likewise downstream of proneural gene function (Reeves, 2005).
The identification of sets of genes comprising the genetic programs deployed in
PNCs and SOPs by the action of proneural proteins offers a powerful opportunity
to investigate the regulatory organization of these programs. Specifically, it was of interest
to find out (1) which genes are directly activated by proneural
regulators, and which indirectly, and (2) the nature of the
cis-regulatory sequences and their cognate transcription factors that
distinguish PNC- versus SOP-specific target gene expression. This analysis was initiated
by examining potential regulatory sequences of several of the
genes that have been identified for the presence of conserved, high-affinity proneural
protein binding sites of the form RCAGSTG. The initial approach was to ask
whether evolutionarily conserved clusters of these binding sites identify
cis-regulatory modules of the appropriate specificity. To date, this
strategy has proven very successful. Genomic DNA fragments bearing
proneural protein binding site clusters associated with CG11798,
edl, Traf1, CG32434/loner, and rho confer
PNC-specific activity on a heterologous promoter,
while similar modules from CG32150, mira, and PFE drive
SOP-specific expression. In three cases, double
labeling with the SOP marker anti-Hindsight (Hnt) reveals that PNC-specific
expression of the reporter gene includes the SOP as well as the non-SOP cells.
Mutation of the proneural protein binding sites in four of the
enhancer-bearing fragments severely reduces (CG11798) or abolishes (CG32150,
edl, Traf1) reporter gene
expression in PNCs/SOPs. Such results indicate that these genes are indeed
direct targets of activation by proneural proteins in vivo (Reeves, 2005).
Holometabolous insects like Drosophila carry out two major
phases of PNS neurogenesis, one in embryogenesis to form the larval PNS, and a
second in the late larval and early pupal stages to construct the adult PNS.
Many known genes participate in both phases. Accordingly, it was of interest to
determine whether genes identified as being expressed in imaginal disc PNCs
or SOPs are also expressed in the developing larval PNS. In situ hybridization reveals that,
among others, the PNC genes CG11798 and CG32434/loner and the SOP
genes CG3227, CG32150, and CG32392 are indeed expressed in
embryonic PNCs and SOPs, respectively (Reeves, 2005).
To determine whether this combined cell
sorting/microarray/in situ hybridization approach had indeed identified gene
functions required for proper PNS development, loss-of-function
alleles of two loci, CG11798 and CG3227, were generated. These were chosen
because (1) transcript accumulation from both genes was detected in the
primordia of both the larval and adult PNSs; (2) both genes encode proteins
with conserved domains; and (3) mobilizable P element transposon insertions
were available adjacent to these genes (Reeves, 2005).
CG11798 is predicted to encode
a probable transcription factor with four zinc finger domains.
Loss-of-function alleles of the gene were generated
by mobilizing KG03781, a P element located immediately downstream. A
precise excision of the P transposon and two partial deletions of the
CG11798 coding region were recovered and characterized by sequencing.
Deletions 19E and 34E are both homozygous lethal
during early larval stages, and both confer clear defects in the development of
the larval PNS. 19E causes the loss
or misplacement of sensory neurons marked by mAb 22C10 and sensory organ accessory cells marked
by anti-Prospero (αPros). Deletion 34E confers an even more severe PNS phenotype
and removes or misplaces many more 22C10-positive and Pros-positive
sensory organ cells in each hemisegment. The difference in the
severity of the 19E and 34E mutant phenotypes may be due to the fact that the
latter deletes a larger portion of the CG11798 coding region, including
the codons for the four zinc fingers. As a control genotype, use was made of the
precise excision (PE) derivative of the KG03781 transposon insertion. No
PNS mutant phenotype was detected in homozygous PE embryos,
demonstrating that the defects observed in
the 19E and 34E deletion homozygotes do not result from a second-site mutation
on the original KG03781 chromosome. The results of complementation tests
led to the
conclusion that CG11798 corresponds to the previously described
charlatan (chn) locus (Reeves, 2005).
To generate loss-of-function alleles of CG3227,
the P element transposon KG07404, inserted
just upstream of the gene, was mobilized. Imprecise
excision created two deletions, 23B and 23I.
Homozygosity for either results in nearly complete lethality before adulthood.
Mosaic adult flies carrying FLP/FRT-generated mutant clones exhibit a severe
PNS defect in which most mechanosensory bristles within the clonal territory
not only lack shaft structures but also bear multiple socket structures,
suggestive of shaft-to-socket cell fate transformations. The major defects observed in sensory
structures in both the larval and adult PNSs prompted giving CG3227 the new name insensitive (insv) (Reeves, 2005).
insv is predicted to encode a protein containing a conserved C-terminal domain of
unknown function called DUF1172. DUF1172 was
first recognized in the vertebrate NAC1 proteins, transcription factors that
also contain BTB/POZ protein-protein interaction domains. Alignment of
arthropod and vertebrate DUF1172s reveals that the domain is large
(approximately 125 amino acids) and contains a highly conserved central region
of alternating polar/charged residues and nonpolar residues.
This is the first described loss-of-function phenotype for a gene encoding a
DUF1172 domain protein (Reeves, 2005).
Several known or potential components of
signaling pathways were uncovered in this analysis as exhibiting either PNC- or
SOP-specific expression. These include genes encoding a putative G
protein-coupled receptor (CG31660), a receptor tyrosine kinase
(Ror), a regulator of G protein signaling (loco), and a modulator
of Ets protein activity (edl). Earlier studies have linked both G
protein function and Ras/MAPK signaling to the development of Drosophila
sensory bristles, but much remains
to be learned about their roles in this process. These findings suggest functions
in PNS development for both known and previously uncharacterized signaling
pathway components (Reeves, 2005).
Perhaps surprisingly, the data indicate the PNS-specific
expression in imaginal discs of several genes predicted to encode metabolic
enzymes, including a uridine phosphorylase (CG6330), a
maleylacetoacetate isomerase (CG9363), and a malate dehydrogenase
(Men). Exceptional metabolic requirements or signaling activities in
developing sensory organs may underlie these observations (Reeves, 2005).
Loss-of-function analysis of two genes identified by the cell
sorting/microarray/in situ hybridization approach, one expressed in PNCs
(CG11798/chn) and one in SOPs (CG3227), confirms that they are
indeed required for normal PNS development in Drosophila. Deletion
mutations of CG3227 (insensitive)
cause severe defects in the specification and differentiation of
sensory organ cells in the adult PNS, as assayed in mosaic clones. Particularly
prevalent is an apparent transformation of the shaft cell to the fate of its
sister, the socket cell; this is the same phenotype conferred by
loss-of-function mutations in N pathway antagonists such as Hairless and
numb. The definition of a loss-of-function phenotype for a DUF1172 gene
should prove valuable in investigating the in vivo function of this
uncharacterized protein domain (Reeves, 2005).
Certain SOP-specific genes, exemplified by
sens and phyl, are
required for the execution of the SOP fate itself. insv, by contrast,
represents a distinct class of SOP gene, required not for the fate of this
cell, but for the specification and/or differentiation of one or more of its
progeny. Thus, SOP-specific (or, more generally, precursor-specific) gene
expression can serve the same function as maternal gene
expression -- providing gene products essential to the development of
descendants. It is anticipated that a number of the SOP genes identified
will prove to act similarly (Reeves, 2005).
The function of proneural
bHLH proteins in Drosophila PNS development is complex, since they not
only activate in SOPs genes that promote the neural precursor cell fate (e.g.,
ac and sc themselves, sens and
phyl); they also activate in non-SOPs genes involved in inhibiting
this fate (e.g., genes of the Enhancer of split Complex).
The nature of the cis-regulatory
'codes' (combinations of transcription factor binding sites) that
distinguish the PNC versus SOP expression specificities is of particular interest.
One code has been identified for the expression of N-responsive genes in the non-SOP
cells of the PNC that consists of binding sites for the proneural proteins plus
sites for the N-activated transcription factor Suppressor of Hairless (Su(H)). Importantly, none
of the PNC modules identified in this study includes a conserved high-affinity
Su(H) site, yet at least three of them do mediate direct transcriptional
activation by the proneural proteins. Moreover, the expression driven by these
new PNC modules includes the SOP, whereas the 'Su(H) plus
proneural' code directs expression that excludes it.
These findings indicate the existence of at least one novel code
for PNC expression, and of a heretofore hypothetical class of genes -- ones
that are directly regulated by the proneural proteins in PNCs/SOPs but are
evidently not activated in response to N-mediated lateral inhibitory signaling,
perhaps because they are not involved in the inhibitory process (Reeves, 2005).
The proneural genes were first identified by their function
in the ectoderm in specifying neural cell fates, and they have been studied
almost exclusively in that context in both vertebrates and invertebrates.
However, it has become clear that these genes function as well in the other two
germ layers. The Drosophila proneural gene lethal of scute
(l'sc) is required to specify the fates of muscle progenitor
cells in the embryonic mesoderm, and the same gene (and probably also sc) is required
for the adult midgut precursor (AMP) cell fate in the embryonic endoderm. In both
of these nonectodermal settings, a striking parallel with neurogenesis is seen
in the manner in which proneural genes function in close association with the N
pathway to select individual precursor cells. In the mesoderm,
l'sc is deployed in 'pro-muscle clusters' from
which single muscle progenitors emerge by N-mediated 'lateral
inhibition'; in the endoderm, where proneural gene expression is initially
uniform, AMPs are spaced apart from each other by N signaling in a manner very
reminiscent of the spacing of microchaete bristles on the adult thorax.
The mouse proneural protein Atoh1 (Math1) has been shown to be
required for the specification of nonneural secretory cell precursors in the
intestinal epithelium. Thus, proneural transcription factors are not dedicated specifiers
of neural cell fates; rather, they appear to be very effective in first
conferring on a group of cells the potential to adopt a particular cell fate
and then promoting the selection of an individual committed progenitor from
within that group. This suggests the existence of a 'core' set of
genes that function downstream of the proneural proteins in all such contexts,
with other sets of genes contributing to context-dependent (e.g., germ
layer-specific) programs. Further investigation of the genes identified in this
study should permit a test of this intriguing hypothesis (Reeves, 2005).
Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, da neuron axon projections were analyzed in embryonic and larval stages. It was found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. A class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development was identified; this layering allows different classes of da neurons to develop differential connectivity to second-order neurons. The effect of Robo family members on class-specific axon lamination was examined, and a forward genetic approach has also been taken to identify new genes involved in axon and dendrite development. For the latter, the third chromosome was screened at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology (Grueber, 2007).
Drosophila dendritic arborization (da) neurons have been
segregated into four classes (classes I-IV) that reflect arbor complexity,
arbor size and the length of terminal branches. The
cell bodies are distributed in ventral, ventral', lateral and dorsal
clusters between the epidermis and muscles, spreading dendrites across the
body wall, and axons to the ventral nerve cord. It was reasoned that if
morphological classes correspond to at least partially distinct sensory
systems, then their axons may have divergent morphologies and target
non-overlapping regions of the ventral nerve cord, where information will be
relayed to second-order neurons. Previous studies have found that most da
neurons arborize together in a common fascicle in the ventral CNS, with a
subset, including at least some class I da neurons, projecting to more dorsal
neuropil. In light of studies showing distinct morphological types of da neurons, mosaic analysis with a repressible cell marker (MARCM) was used to examine the morphology of da neuron dendrites and axons in third instar larvae. As a MARCM driver Gal4109(2)80 was used; this labels all multidendritic (md) sensory neurons, including those belonging to the da subgroup. Owing to the sparse labeling of central neurons, the 109(2)80 driver combined with MARCM allowed resolution of axon morphology of individual neurons (Grueber, 2007).
Data was collected from wild-type da neuron clones in segments A2-A7 to
identify their axon projections in the CNS. Different da
classes showed distinctive types of central projections. Class I neurons were
unique in their projection to the dorsal neuropil.
Class II axons showed collateral branches (branches exiting from the main
axonal trunk, although the timing of their emergence has not been determined)
that were not observed in class III and IV neurons. The
class I neuron vpda also showed such a branch. Class IV neurons
projected axon branches across the midline, but these were only rarely
observed for the other classes. Dorsal and ventral' class IV
axons crossed the midline, but axons from the ventral neuron did not. Each
class IV neuron also showed a large accumulation of branches medial to the
commissural/longitudinal branch bifurcation. The class III
terminals extended in an anteroposterior (AP) orientation and were relatively
unbranched, showing neither the collateral branches observed in class II
neurons, nor contralateral projections observed among class IV neurons (Grueber, 2007).
The axons of class I and class IV neurons also showed evidence of
somatotopic arrangements in the CNS. The trajectory of class I neurons in the
CNS mirrored the polarity of their dendrites in the periphery. Dorsal class I
neurons have distinct polarity with respect to the AP body axis: dendrites of
ddaD extend anteriorly and dendrites of ddaE extend posteriorly.
Likewise, it was found that the ddaD axons extended anteriorly in the CNS, whereas
the ddaE axons extended posteriorly. Among the class IV neurons, only neurons positioned in the dorsal and ventral regions of the body wall, but not the lateral region,
extended axons across the midline, fitting with principles of somatotopy established for body wall bristle neurons. These data together demonstrate that da neuron classes have
distinguishing axon terminals, and that neurons in the same class show
evidence of somatotopic organization (Grueber, 2007).
The position of sensory axons defines the population of possible
second-order targets and thus contributes strongly to sensory information
processing in the CNS. Axons of tactile receptors typically project to ventral
areas of the neuropil, whereas strain-sensing or proprioceptive neurons
usually project to more dorsal regions. Fasciclin II-labeled axon tracts provide a frame of reference for assessing dorsoventral (DV) position in the CNS. The DV
positions of axons was studied in 42 ventral nerve cords (VNCs) using MARCM, and 18 VNCs
using the FLP-out system. Both techniques revealed that each class I neuron extended
axons to dorsal regions of the neuropil, terminating just lateral to the
dorsomedial (DM) fascicle. The position of class I axons was therefore indistinguishable
at this level of resolution from the position of the dbd terminal arbor,
implying that information from class I neurons and the putative
stretch-sensing dbd neuron might be processed similarly in the CNS. Class II,
III and IV axons targeted the ventral CNS without obvious class-specific
dorsoventral lamination of terminal position. The positions of
the class II collateral branches were somewhat variable, either terminating on
the ventrolateral (VL) fascicle, or slightly lateral to VL (vdaA often had a more lateral termination). These data together provided anatomical support
for distinct functions among different da neurons, fitting with their distinct
dendritic arbor morphologies. Class II, III and IV axons project similarly to
known tactile afferents, while class I neurons have projections like known
proprioceptive or strain-sensing neurons (Grueber, 2007).
Whether the terminal positions of the ventral-projecting
class II, III and IV neurons could be further distinguished by their position was examined. Short pickpocket (ppk) enhancer sequences can drive gene
expression strongly in all class IV neurons and weakly in class III neurons.
Viewing all class IV neurons together revealed that they crossed the midline
in a single fascicle, that the stereotyped branching at the
commissural-longitudinal junction overlapped for all neurons, and that
longitudinal projections were not always tightly fasciculated. In ppk-eGFP
and ppk-Gal4, UAS-CD8::GFP animals, a strongly labeled
set of medial axons and a weakly labeled, slightly more lateral, layer of
terminals were observed. It is suspected that the weakly labeled axons were class III axons, which may form a layer next to class IV axons. To test this idea,
ppk-Gal4 was introduced into the FLP-out mosaic system. The relative
locations were observed of all class III axons except ddaF (whose axon was labeled too
weakly) and it was found that their major longitudinal projections terminated
immediately lateral to the scaffold of class IV axons (Grueber, 2007).
The ppk reporter lines alone do not label the class II axons, and
thus did not allow determination of whether all da classes form a laminar
organization or only the class III and IV neurons. However, examination of
FLP-out clones produced with Gal4109(2)80, with or without
ppk-eGFP to label class IV neurons, permitted labeling of different
axon groups. It was found that class II neurons with a significant longitudinal
projection formed a third layer of sensory axons that was lateral to both
class III and class IV axons, with class II collateral branches terminating in a distinct, even more lateral, position (Grueber, 2007).
The FLP-out data was confirmed by mapping the relationships of individual
pairs of sensory afferents using the MARCM technique. Within hemisegments, or
in adjacent hemisegments, having two or more da neuron clones
axons were organized (medial>lateral) class IV>class III>class II. Laminar patterning was independent of peripheral cell body position. These data together
indicate the presence of a laminar arrangement of somatosensory axons in the
Drosophila CNS. These data also suggest that somatosensory
information carried by different classes of da neurons might be distinguished
by sensory axon connectivity to second-order targets (Grueber, 2007).
The above FLP-out and MARCM data were collected from third instar larval
stages, so when during development layering of the different classes of axons could be observed was examined. To achieve live two-color discrimination of
different neuronal classes in embryonic and early larval stages
transgenic flies were generated expressing a photoconvertible fluorescent protein, Kaede, and
expression was placed under the control of Gal4109(2)80 in the
presence of ppk-eGFP. The Kaede protein was converted from green to red
fluorescence using a 10-30 second UV pulse and the position of all da
axons was examined relative to ppk-eGFP-labeled class IV axons. As early as the
sensory axon scaffold could be visualized (stage 17), class IV axons occupied
a medialmost layer with respect to other classes. These data
indicate that a laminar pattern develops at least by late embryonic stages and
is maintained without qualitative change in larvae (Grueber, 2007).
Much of the knowledge about somatotopic maps in insect mechanosensory
systems derives from studies of bristle afferents with peripheral receptive
fields that approximate a point source. Drosophila da neurons have
largely overlapping peripheral sensory fields and may, as a group, respond to
several distinct stimuli. How is information from this predominant body wall sensory
system represented in the CNS and what might this organization reveal about
the possible functions of da neurons? Neurons with different
dendritic branching morphologies target distinct regions of the CNS,
supporting the existence of a modality map of da neuron axons. Evidence is provided for nested somatotopic mapping in class I and class IV da
neurons. Individual class I neurons extend their dendritic and axonal arbors
in the same preferred direction along the AP body axis. Class IV axons
project across the midline according to the cell body position along the DV
axis of the body wall, with dorsal and ventral cells, but not more lateral
cells, crossing the midline. It is possible that class II and III neurons also
project in a somatotopic pattern that was not uncovered by this analysis. Thus, da neuron connectivity appears to incorporate both class and position-specific components, with some apparent correlations with dendritic field orientation (Grueber, 2007).
In the context of sensory processing, these data suggest distinct functions
for different morphological classes of da neurons. The class II-IV neurons
target a ventral region of the neuropil; thus information from these neurons
might be processed similarly to ventral-projecting tactile sensory bristle
neurons. Within this ventral region, the class-specific laminar
pattern could allow differential connectivity with second-order interneurons.
Additionally, class II neurons, with their collateral branches, might provide
information to distinct central circuits. The class I neurons targeted a more
dorsal region of the neuropil, which is generally a characteristic of
proprioceptive afferents in insects. Indeed, a class of da neurons in Manduca has been shown to target dorsal neuropil, and to respond to stretch of the cuticle. Many
insect proprioceptors, including chordotonal organs and the bipolar dendrite
neurons, have dendrites oriented in a preferential direction relative to the
body axis. Notably, the primary dendrites of each class I neuron are oriented
dorsally and secondary dendrites are oriented anteriorly and posteriorly. This
arrangement could allow larvae to compare distension along major body axes.
While the anatomical arrangement of their axons suggests distinct functions
for different da neurons, dissecting these different functions will ultimately
require behavioral and physiological studies (Grueber, 2007).
A notable feature of the mapping of da sensory afferents is their
predominant organization into class-correlated mediolateral layers, with class IV neurons in a medial layer, class III neurons intermediate, and class II neurons most
lateral. These layers do not correspond to the medial, intermediate and
lateral fascicles that have their position specified by a Robo combinatorial
code; thus novel molecular cues may contribute to this laminar
organization. Indeed, it has been postulated that the Robo code provides
information about the broad zone that a growth cone targets, while a
complementary fasciculation code fine-tunes pathway choice within that zone. It
is conceivable that Robo proteins could participate in specifying the lateral
position of collateral branches, since Robo3 overexpression in individual neurons
induced ectopic branching from the axon shaft. Although no cell-type-specific expression of Robo3 was detected in neurons that normally show such
branching, it is notable that Robo3 has been implicated in cell-type-specific
patterning decisions of PNS axons, and that Slit2 has been proposed as a positive
regulator of collateral branching of dorsal root ganglion sensory axons (Grueber, 2007).
The mechanisms for targeting of somatosensory afferents should act to
properly position axons of different classes relative to one another. Several
alternative mechanisms could contribute to this positioning. One potentially
important component of axon sorting could involve interactions between
homotypic or heterotypic axons. Heterotypic axons could repel each other to
sort to discrete bundles. Likewise, homotypic axons could adhere to one
another to ensure that they terminate together. Olfactory receptor neuron
axons engage in extensive hierarchical interactions to establish precise
targeting in olfactory glomeruli, and dendrites of da neurons engage in
class-selective interactions during development to ensure their proper spacing. It is
therefore conceivable that intraclass and interclass interactions could
participate in the sorting of somatosensory axons. Axons from different
classes could also project to specific layers that are prepatterned by the
processes of target interneurons or by other axons. Finally, the projection of
da axons to different layers could conceivably reflect a temporal order of
sensory axon arrival in the CNS, similar to the three-way correlation between
mediolateral axon position, physiological function and time of differentiation
among Drosophila wing campaniform sensilla. Among
the ventral cluster da neurons, a group with differentiation that has been
examined in the greatest detail, the class II neurons appear to be the
first-born, followed by class III neurons and class IV neurons. These
data suggest a possible correlation between birth order and axon position in the neuropil, although additional early markers of da neurons and further high-resolution imaging studies are required to further test this scenario (Grueber, 2007).
The molecular basis for insect sensory neuron differentiation, as well as
anatomical studies of somatosensory axon mapping and VNC circuitry, have been
subjects of considerable study, and principles are emerging that link the two
areas into molecular models of connectivity and synaptic specificity. Among
embryonic sensory neurons in Drosophila, there is a three-way
correlation between soma position, proneural transcription factor expression
and axon projection pattern, suggesting that these transcription factors may
endow aspects of modality-specific axonal projections. Such a link was recently established between chordotonal-organ-specific expression of the atonal proneural gene and expression of the Robo3 axon guidance molecule in these same organs.
Misexpression experiments with atonal, robo3 and comm
suggest a model whereby Atonal activates expression of Robo3, which in turn
specifies mediolateral positioning in chordotonal versus bipolar dendrite-type
axon projections. These studies provide an important basis for understanding
the establishment of sensory circuitry in the VNC (Grueber, 2007).
To begin to address the molecular basis of axon and dendrite patterning
using the anatomical framework established for the da neurons, a
forward genetic approach, which has proven a successful means to identify regulators of neuronal morphogenesis, was undertaken. A strength of this screen was the ability to simultaneously assess phenotypes in dendrites and axons at the level of single identifiable neurons. Numerous complementation groups were identified that affect axon patterning, including several mutations with a molecular nature as yet unknown. These mutations should allow identification of new genes involved in axon morphogenesis and place these into the context of their effects on somatosensory axon patterning and circuitry. Given that many mutations affecting dendrite morphogenesis have been identified, it is expected that studies of the mutations identified from the screen will also allow addressing of the similarities and distinctions between axon and dendrite development (Grueber, 2007).
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Genes involved in organ development
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