The Interactive Fly
Genes involved in tissue and organ development
Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).
The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).
This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).
Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).
This view invoking multiple and overlapping modes of regulation by a homeotic selector protein supports and extends the vision from analyses seeking to explain how Ultrabithorax (Ubx) selector function differentiates between the serially homologous wing and haltere appendages. This analysis supports a role for Ubx in fruit flies transforming a dorsal default state (wing) to haltere, by repressing the accumulation of Wg in the posterior part of the haltere, and by regulating a subset of Dpp or Wg activated targets such as vestigial and spalt related. Additionally, clear evidence has been presented for a nonautonomous action of Ubx via its activity in cells of the D-V organizer where wg is expressed. Ubx thus acts to down-regulate wg in the haltere, but also intervenes to modulate the expression of targets of both dpp and wingless signaling pathways. An analysis of mutants for maxillopedia (mxp), the Tribolium pb homolog, revealed augmented transcription of flour beetle wg within the transformed labial segment. This observation, in full accord with the above results for Ubx, and the current results for Drosophila pb, supports a conserved role for homeotic regulation of nonautonomous signaling input in appendage development. At the same time, mxp mutants show a precocious maxilla-to-leg transformation in larvae, demonstrating a prior, embryonic requirement for mxp. This result is of particular interest since it highlights a temporal aspect of pb action in the fly labial disc: the absence of pb function early has no apparent effect on the labial discs in early L3 larvae, which appear normal. It is only subsequently that these diverge toward leg structure. Thus, the globally conserved activity of mxp/pb in equivalent beetle or fly organs is nonetheless employed in temporally different ways among species. Though it is not clear whether this reflects the existence of species-specific co-factors or rather of the effects of expression dosage and timing, such modifications might offer important possibilities for changing form. Variations on all these themes can probably contribute to the diversification of organism form, within and among species (Joulia, 2005)
The roles of diffusible Wg and Dpp morphogens induced by Hh at the A-P boundary, and the transcriptional programs they induce according to their concentrations within a gradient, are considered central to organizing the group of cells constituting a segment. The present work indicates that pb normally acts downstream of Hh within the organizer, where it maintains Wg and Dpp at low levels in labial imaginal tissue. Overexpressing Wg or Dpp in the labial discs results in malformed, overgrown or transformed 'labial' tissue. These observations support the viewpoint that limiting morphogen accumulation is essential to ensuring that the labial program is correctly applied. This study underlines the potential importance of the absolute levels of wg and dpp-encoded signaling molecules deployed for tissue organization. While a gradient may in principle be formed from any source, part of the spectrum of threshold levels necessary for stimulating specific gene responses is likely removed from the repertoire in the labial environment. The absolute level of activation or inhibition of diverse signaling pathways thus may be in itself a tissue-specific property, allowing gradients of related form but with different instructive capacities that can be a distinctive element in guiding tissue formation and specifying ultimate identity. This integration of diverse sorts of information -- the hh organizer linked to the Hox selector -- may confer order to tissue organization and identity (Joulia, 2005).
The fine-tuning of morphogen signals by Hox selectors coupled with the concomitant regulation of downstream targets thus appears to offer a strategic control point for achieving reliable developmental control coupled with evolutionary flexibility. The modulation of different cell signaling pathways by pb activity implies it can regulate both the tissue “context” generated by the signaling pathways activated in a tissue, and the cellular response to this context. This capacity to meld large-scale patterning with cellular identities merits emphasis (Joulia, 2005).
While the logic described above appears to be conserved, its application leads to widely different results according to the species and the tissue. Quite recently, an analysis of vertebrate Hox function has led to the identification of an intimate developmental link between Hox selector function and hedgehog signaling. This analysis reveals a direct physical interaction between the mouse Ci homolog Gli and Hox homeodomain transcription factors. It thus provides a compelling complement to the present work, since the molecular framework of a direct link between Gli and Hox proteins goes far to rationalise the dose-sensitive interplay between Ci and Pb that was observed in Drosophila. If Hox proteins indeed compete for available nuclear Gli/Ci, this molecular mechanism may also help to understand other phenomena including phenotypic suppression in flies or posterior prevalence in mice. Correspondingly, the current data place Pb in antagonism to Ci within the hedgehog organizer, where it modulates output from the wg and dpp genes and the instructive morphogens they encode. These complementary observations from insect and vertebrate models suggest the existence of an evolutionarily conserved machinery with enormous potential for generating morphological diversity. It will be exciting to know more about how the homeotic selector function is integrated in known cascades that make use of conserved molecules both to ensure the fidelity of normal form, as well as to generate new form (Joulia, 2005).
The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. This analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis (Yasunaga, 2006).
Labial Dll is unique in that it represses hth expression only in the L-layer and, consequently, Dll and hth expression domains in the L-layer are mutually antagonistic while, in the M-layer, they can coexist. This layer-dependent asymmetry in Dll function may persist at least until 24 h APF, when pseudotracheal morphology becomes apparent. In very early stages, Dll and hth expression in the leg disc appears mutually antagonistic, while most Dll-expressing cells co-express hth in the antennal discs. Thus, Dll/hth expression in the labial disc M- and L-layers, respectively, might be similar to those of antennal and leg discs. In other words, the labial disc might possess characters of both antennal and leg discs (Yasunaga, 2006).
pb has been shown to direct labial disc development by specifically repressing leg and antennal genes, such as dac and spalt (Abzhanov, 2001) and by controlling a gross morphological organization through attenuation of dpp and wg expression by counteracting Hh signaling (Joulia, 2005, Joulia, 2006). These results indicate that pb is also involved in repression of cll, a distal-region-specific gene in leg and antennal discs. In contrast to cll, al and Bar, two other distal-most homeobox genes in leg and antenna, were expressed in the distal labial-disc region (Yasunaga, 2006).
Preliminary data suggests that distal al expression is dispensable for normal distiproboscis development. Thus, only some regulatory mechanisms for distal-region-specific gene expression appeared conserved in the labial disc while distal-gene function is suppressed by pb (Yasunaga, 2006).
A fate map is presented of late third instar labial disc based on gene expression at larval and pupal stages and mutant adult phenotypes (see Fate map of the labial disc). Gross organization of 24 h-APF labial discs is basically identical to that of adult distiproboscis. For simplicity, the 24 h-APF labial disc is drawn as a thick disc, where pseudotracheal (PT) and taste bristle (TB) regions, respectively, correspond to the bottom face and distal belt surrounding it (Yasunaga, 2006).
Twenty-four-hours-APF PT is marked by the simultaneous expression of wg, Dll, ptc and hth, whereas the TB region is comprised of four regions with (en + Dll), (dpp + ptc + Dll), (ptc + Dll) or (Dll) expression. In the late third instar disc, the distal L-layer consists of (en + Dll), (dpp + ptc + Dll) and (Dll) subregions, while approximately 50% of the distal M-layer is simultaneously positive to wg, Dll, ptc and hth expression. Most combinations of patterning-gene expression are conserved during larval and pupal labial development. It is thus considered that, in the late third instar, the Dll-positive L-layer region is the TB-region progenitor, with the M-layer (wg + Dll + ptc + hth) domain being the progenitor for the PT region. Consistent with the finding that the L- and M-layers of larval discs are assigned to the dorsal and ventral regions at least as far as the anterior compartment is concerned, the PT and TB regions are localized ventrally and dorsally in 24 h-APF discs and adult proboscis. All Dll-expressing regions, that is, PT and TB regions are situated distally in 24 h-APF pupal discs and adult proboscis (Yasunaga, 2006).
The ventral fate of the larval labial disc is considered regulated by wg and consistent with this, high wg activity is required for PT formation in the adult. In contrast, the dorsal fate of the distal portion of the larval labial disc is apparently determined by high dpp expression, since nearly all TBs were eliminated from dpp hypomorphic mutants. Nearly all and only the PT and TB regions were eliminated from Dll mutants (Yasunaga, 2006).
The fate map A indicates that, in the 24 h-APF pupal disc, the D-V boundary separates the outer surface from the inner surface. In the dorsal region, the A-P boundary is almost perpendicular to the D-V boundary but, in the ventral region, the former runs almost parallel to the D-V boundary. A similar situation is also the case in adult proboscis. Thus, as with third-instar labial disc, the posterior compartment of the 24 h-APF disc and adult proboscis may possess little space for the ventral region. The adult-proboscis A-P boundary in the inner surface region, determined by these experiments, strikingly differs from that presumed by Struhl (1981) based on the mosaic analysis of the outer surface region (Yasunaga, 2006).
The proboscis has been considered as a highly modified appendage lacking segmental organization along the P-xD axis (Abzhanov, 2001). The current fate map gives an additional implication that the proboscis is an appendage evolved to effectively take in food by expanding the ventral region of the anterior compartment to develop pseudotracheal rows (Yasunaga, 2006).
The remarkable diversity of form in arthropods reflects flexible genetic programs deploying many conserved genes. In the insect model Drosophila melanogaster, diversity of form can be observed between serially homologous appendages or when a single appendage is transformed by homeotic mutations, such as the adult labial mouthparts that can present alternative antennal, prothoracic, or maxillary identities. This study has examined the roles of the Hox selector genes proboscipedia (pb) and Sex combs reduced (Scr), and the antennal selectors homothorax (hth) and spineless (ss) in labial specification, by tissue-directed mitotic recombination. Whereas loss of pb function transforms labium to prothoracic leg, loss of Scr, hth, or ss functions results in little or no change in labial specification. Results of analysis of single and multiple mutant combinations support a genetic hierarchy in which the homeotic pb gene possesses a primary role. It is surprising to note that while loss of ss activity alone had no detected effect, all mutant combinations lacking both pb and ss yielded the most severe phenotype observed: stunted, apparently tripartite legs that may correspond to a default state. The roles of the four selector genes are functionally linked to a cell nonautonomous mechanism involving the coupled activities of the decapentaplegic (dpp)/TGF-beta and wingless (wg)/Wnt signaling pathways. Accordingly, several mutant combinations impaired in dpp signaling were seen to reorient labial-to-leg transformations toward antennal aristae. A crucial aspect of selector function in development and evolution may be in regulating diffusible signals, including those emitted by dpp and wg (Joulia, 2006).
The functional organization of central motor circuits underlying feeding behaviors is not well understood. This study combined electrophysiological and genetic approaches to investigate the regulatory networks upstream of the motor program underlying food intake in the Drosophila larval central nervous system. The serotonergic network of the CNS was found to be capable of setting the motor rhythm frequency of pharyngeal pumping. Pharmacological experiments verified that modulation of the feeding motor pattern is based on the release of serotonin. Classical lesion and laser based cell ablation indicated that the serotonergic neurons in the subesophageal zone represent a redundant network for motor control of larval food intake (Schoofs, 2017).
Rhythmic motor behaviors such as feeding are driven by neural networks that can be modulated by external stimuli and internal states. In Drosophila, ingestion is accomplished by a pump that draws fluid into the esophagus. Here we examine how pumping is regulated and characterize motor neurons innervating the pump. Frequency of pumping is not affected by sucrose concentration or hunger but is altered by fluid viscosity. Inactivating motor neurons disrupts pumping and ingestion, whereas activating them elicits arrhythmic pumping. These motor neurons respond to taste stimuli and show prolonged activity to palatable substances. This work describes an important component of the neural circuit for feeding in Drosophila and is a step toward understanding the rhythmic activity producing ingestion (Manzo, 2012).
In many systems, complex motion is controlled by central pattern generators (CPGs), neural circuits that can produce oscillatory activity independent of sensory input. Feeding behaviors such as chewing and sucking require coordinated contraction of different muscle groups in a rhythmic pattern. In Drosophila, ingestion is driven by a pump located in the proboscis. Although the mechanics of fluid ingestion have been examined in other insects, the neural circuits controlling ingestion have not been extensively characterized (Manzo, 2012).
The fruit fly Drosophila melanogaster is an excellent model system for examining neural control of fluid ingestion because both neurons and behavior can be studied using molecular and genetic approaches. In Drosophila, feeding begins with detection of a palatable food source followed by proboscis extension and fluid ingestion. Sensory neurons located in the proboscis, legs, mouthparts, wing margins, and ovipositor allow the fly to detect a variety of compounds, including sugars, bitter substances, carbon dioxide, and water. Many of these neurons send projections to the subesophageal ganglion (SOG) of the fly brain. Also located in the SOG are motor neurons that innervate muscles involved in feeding behaviors. Two muscles (muscles 11 and 12) constitute a pump; the activity of these muscles fills a chamber (the cibarium) with fluid and expels the fluid into the esophagus (see Anatomy of cibarial pump motor neurons and muscle innervation). Previous work has identified motor neurons projecting to muscle 11; when these neurons are inhibited, food consumption on a short timescale decreases. Although neurons comprising a pump CPG have not been identified, there is evidence for a larval feeding CPG in Drosophila and other insects (Manzo, 2012).
How do pump motor neurons control ingestion? Motor neurons may be passive effectors of a pumping CPG or could contribute to its rhythm. Inducible activation and neuronal inhibition experiments were performed to determine how motor neuron activity affects pumping. If motor neurons were passive effectors, activation would lead to prolonged contraction of the target muscles. If motor neuron activity influenced a pumping CPG, activation could produce pumping (Manzo, 2012).
This work examined the regulation of pumping behavior and characterize the role of motor neurons in producing pumping. These studies describe a key component of the feeding circuit in Drosophila and provide insight into the rhythmic activity underlying fluid ingestion (Manzo, 2012).
The experiments suggest that the two muscles constituting the pump have different roles in fluid ingestion. Inactivation of muscle 12 motor neurons inhibits pumping but does not reduce the volume per pump, whereas inactivation of muscle 11 motor neurons does not affect pumping but does affect the volume per pump. Similarly, activation of MN12 neurons produces cibarial expansion, whereas activation of MN11 neurons does not. In addition, anatomical studies have shown that muscle 12 inserts in the roof of the cibarium, whereas muscle 11 is located more posteriorly (3, 4). These data argue that the two muscles have distinct roles in fluid ingestion. Contraction of muscle 12 produces cibarial expansion, whereas contraction of muscle 11 leads to ingestion downstream of pumping, likely allowing fluid to exit the cibarium to the esophagus (Manzo, 2012).
The data suggests a model for how pump motor neurons and muscles produce pumping behavior. Input from sensory neurons signaling the presence of palatable food leads to activation of muscle 11 and muscle 12 motor neurons. Muscle 12 activation produces cibarial opening, whereas muscle 11 activation leads to fluid ingestion downstream of pumping. It is proposed that the activity of these muscles is coordinated such that muscle 12 contraction allows fluid to fill the cibarium, followed by contraction of muscle 11 and relaxation of muscle 12, which propels fluid out of the cibarium and into the esophagus. Muscle 11 then relaxes and the cycle begins again. A CPG upstream of the motor neurons may produce the rhythmic activity that coordinates these muscle movements as well as integrate inputs that signal the continuation and termination of feeding. This study provides a framework for future tests of this model (Manzo, 2012).
In many systems, CPGs produce rhythmic output that drives motor behaviors such as feeding. CPGs are usually composed of interneurons that then synapse onto motor neurons. Motor neurons can be simple passive effectors of the CPG, or they can interact with the CPG to influence its output. For example, excitatory motor neurons that receive input from the leech swimming CPG do not influence its output pattern; by contrast, inhibitory motor neurons synapse onto CPG neurons and can affect their activity. Motor neurons can also form part of the CPG. In the crustacean stomatogastric system, motor neurons innervate stomach muscles and also synapse with each other to form a CPG that controls the chewing and filtering of food (Manzo, 2012).
The data show that motor neurons innervating the cibarial pump in Drosophila influence its output. Inactivating MN11+12 and MN12 neurons produces slower pumping, whereas activating MN11+12 neurons elicits pumping. However, activating MN12 neurons produces prolonged cibarial expansion, whereas activating MN11 neurons produces no obvious effect. Because MN11+12 neuronal activation produces pumping, muscle 11 neurons may indirectly inhibit muscle 12 neurons and cause cibarial emptying. Alternatively, muscle 12 motor neurons in the MN12 and MN11+12 lines may not be identical and could have different effects on the pump (Manzo, 2012).
Motor neurons could influence the pumping CPG in several ways. These motor neurons could form part of the CPG (as in the crustacean stomatogastric system) or could influence the CPG through synaptic connections (similar to the leech swimming system). Consistent with these models, expressing the presynaptic terminal marker UAS-n-synaptobrevin-GFP (25) in MN11+12 neurons leads to labeling in the brain as well as in pump muscles, suggesting they may have postsynaptic partners in the brain. Dissecting the circuit responsible for pumping in Drosophila will require a closer examination of the functional connectivity of motor neurons with each other and with unidentified upstream neurons (Manzo, 2012).
Although CPGs can produce oscillatory activity in isolation, sensory and other modulatory inputs can affect the output pattern. Similarly, pumping is likely to be influenced by feedback from receptors that monitor cibarial expansion or fluid content. The observation that viscous fluids reduce pump frequency supports this idea. Moreover, this reduction is largely due to an increase in the duration of cibarial filling, suggesting that achieving a specific level of cibarial expansion or fluid content may promote emptying of the cibarium. Proprioceptors lining the cibarial walls have been described in the blowfly and if present in Drosophila may provide this input ; chemosensory cells are also present within the cibarium (Manzo, 2012).
How cibarial pump motor neurons interact with the rest of the feeding circuit in Drosophila is an open question. Fluid ingestion must be coordinated with other aspects of the feeding motor program, such as proboscis extension and retraction. Furthermore, proboscis extension response and pumping may be initiated by different chemosensory inputs, because taste neurons are present within the cibarium itself, and stimulation of tarsal sensory neurons can elicit proboscis extension response but not pumping (Manzo, 2012).
Imaging data show that pump neurons can be activated in response to multiple substances, although sucrose elicits greater activity compared with water or caffeine. Similarly, flies prefer sucrose over water or caffeine. Both MN11 and MN12 neurons show longer response durations to sucrose, whereas MN12 neurons also show an increase in peak fluorescence changes. These results suggest that increased activation of acceptance sensory neurons is propagated to the motor neurons through the pump circuit. Electrophysiological experiments that simultaneously monitor sensory neurons, motor neurons, and muscle activity will aid in understanding the sensorimotor transformation that leads to pumping (Manzo, 2012).
Our work paves the way for future studies of the function of pump motor neurons and their role in the feeding circuit. A more precise understanding of these neurons will provide insight into the architecture of neural circuits underlying behavior as well as elucidate mechanisms by which rhythmic motor activity is generated (Manzo, 2012).
Food texture has enormous effects on food preferences. However, the mechanosensory
cells and key molecules responsible for sensing the physical
properties of food are unknown. This study shows that akin to mammals, the
fruit fly, Drosophila melanogaster, prefers food
with a specific hardness or viscosity. This food texture discrimination
depends upon a previously unknown multidendritic (md-L) neuron, which
extends elaborate dendritic arbors innervating the bases of taste hairs.
The md-L neurons exhibit directional selectivity in response to mechanical
stimuli. Moreover, these neurons orchestrate different feeding behaviors
depending on the magnitude of the stimulus. It was demonstrated that the
single Drosophila transmembrane
channel-like (TMC) protein is expressed in md-L neurons, where it is
required for sensing two key textural features of food-hardness and
viscosity. The study proposes that md-L neurons are long sought after
mechanoreceptor cells through which food mechanics are perceived and
encoded by a taste organ, and that this sensation depends on TMC (Zhang, 2016). Food preferences are affected greatly by the qualities of food,
including nutrient value, texture, and the taste valence of sweet,
bitter, salty, and sour qualities. During the last 15 years,
many of the gustatory receptor proteins that participate in the
discrimination of the chemical composition of food have been defined. In sharp contrast, the basis through which food texture is detected is enigmatic, despite the universal
appreciation that the physical properties of food greatly influence
decisions to consume a prospective offering.
There are specific tactile features associated with liquid or
solid food. Viscosity and creaminess are typical textural features
of liquid food, whereas hardness, crispiness, and softness are
the main physical characteristics of solid food. Similar to food tastes, food texture provides important information concerning food quality, including freshness and
wholesomeness. For instance, people prefer freshly baked
bread with relatively soft texture, and tend to reject older bread
with a harder texture, even though the chemical composition
has not changed significantly over the course of a couple of days. Furthermore, while exploring the food landscape, an animal must make assessments of food hardness and viscosity in order to exert the appropriate force to chew or ingest. Insufficient chewing force results in poor food processing, while excessive force can cause injury to the tongue or teeth (Zhang, 2016).
Food texture in mammals is predominantly detected through
poorly understood mechanisms in taste organs. In rodents and
humans, a subset of trigeminal nerves such as the lingual nerve
provides somatosensitive afferents to the tongue. Due to the intrinsic mechanical properties of food, mastication produces compression and shearing forces, which in turn activate mechanosensory neuronsin taste organs. However, the molecular identities of mechanosensory neurons and signaling proteins that enable animals to detect food texture are unknown.
To address the fundamental issue concerning the cellular and
molecular mechanisms that function in the sensation of food
texture, this study turned to the fruit fly, Drosophila melanogaster, as
an animal model. In flies, food quality is evaluated largely through
external sensory hairs (sensilla), which decorate the fly tongue
(the labellum) and several other body parts. These sensilla, which house several sensory neurons, allow the chemical composition of foods, such as sugars and bitter compounds, to be detected prior to entering the mouthparts (Zhang, 2016).
This study found that Drosophila can discriminate between
foods on the basis of hardness and viscosity. A previously unknown type of mechanosensory neuron was identified in the fly tongue that is dedicated to detecting food mechanics. These
multidendritic neurons in the labellum (md-L) extend their projections into the bases of most of the external sensilla and are activated by deflections induced by hard and viscous food.
The ability of md-L neurons to sense food mechanics is virtually
lost due to elimination of the only Drosophila member of the
transmembrane channel-like (TMC) family. Mice and humans
each encode eight TMC proteins, and mutations in the founding member of this family, TMC1, cause
deafness in mammals. This study found that tmc is broadly tuned to detect both soft
and hard food textures. Remarkably, optogenetic stimulation
of the md-L neurons with different light intensities yields
opposing behavioral outcomes-weak light promotes feeding, while strong light represses feeding. It is concluded that md-L neurons and TMC are critical cellular and molecular components
that enable external sensory bristles on the fly tongue to communicate textural features to the brain, and do so through a pre-ingestive mechanism (Zhang, 2016).
This study demonstrates that the attraction of wild-type flies to the same
concentration of sucrose is altered by the viscosity or hardness
of the food. If the sucrose-containing substrate is too sticky, soft,
or hard, the appeal of the food declines. These observations
establish the Drosophila taste system as a model to explore
the cellular and molecular underpinnings that allow an animal
to sense food texture. Moreover, similar to the chemosensory
evaluation of food by external sensilla decorating the labellum,
the textural assessment of foods is pre-ingestive in flies (Zhang, 2016).
This study has identified md-L, a previously undefined neuron in each of
the two bilateral symmetrical labella, which extend a complex
array of dendrites to the bases of many sensilla. Several observations demonstrate that md-L neurons play an indispensable role in food texture sensation. First, selective abolition of
neurotransmission from md-L caused significant impairments in
food texture discrimination. Second, laser ablation of md-L resulted in severe defects in perceiving the viscosity or hardness of foods. Third, low or moderate artificial activation of md-L neurons was sufficient to trigger proboscis extension. Thus, the
loss-of-function and gain-of-function analyses of md-L neurons
has lead to a conclusion that md-L neurons are key mechanoreceptor
cells controlling sensation of food mechanics (Zhang, 2016).
Unexpectedly, while low-intensity optogenetic stimulation of
md-L provoked proboscis extension, high-intensity light induced
contraction of the proboscis. Thus, md-L neurons are tuned to
different levels of mechanical stimuli that give rise to drastically different feeding behaviors. it is proposed that weak or moderate light mimics the response to softer foods that simulates feeding,
while strong light induces a higher level of activity that mimics
hard foods and discourages feeding. When a fly is offered sucrose in combination with optogenetic stimulation of md-L neurons with strong light, this caused the animal to reject the
otherwise appetitive food. It is proposed that this rejection occurred
because the animal perceived the texture of the sucrose as too
hard. Thus, it is suggested that texture sensation is mediated by
md-L neurons through an intensity-dependent rather than a
labeled-line mechanism. While md-L are required, it is not
excluded that other neurons in the labella contribute to food
texture sensation. Ultrastructural studies of taste sensilla led to
the proposal that a neuron positioned at the base of each taste
sensillum is a mechanosensory neuron. However, it currently remains unclear as to whether these neurons contribute to some aspect of food texture detection (Zhang, 2016).
In Drosophila, most taste sensilla point toward the ventral direction. The md-L neuron produced much stronger neuronal activity
in response to forces applied to taste hairs that were deflected
dorsally than those deflected in other directions. Thus, taste sensilla are most
sensitive to force applied opposite to the
direction in which they point. Notably,
this direction-dependent feature of taste
sensilla is reminiscent of the directional
sensitivity of hair in mammals, suggesting that it is a widely used neural
coding strategy for sensation in the animal kingdom (Zhang, 2016).
The directional sensitivity of taste
sensilla differs from the macrochaete bristles in the thorax, since these latter bristles are most sensitive to force applied in the same direction in which
they point. The profound differences inmforce-directional sensitivity reflect the functional divergence between these two types of mechanosensory bristles. The direction-tuning feature of md-L neurons might be an evolutionary
adaptation to help fruit flies sample food. While exploring the
food landscape, a fruit fly normally extends its proboscis in the
ventral direction. As a consequence, the forces arising from
the food will bend taste sensilla in the opposite dorsal direction (Zhang, 2016).
Thus, it is suggested that md-L neurons evolved to become most
sensitive to forces emanating from the dorsal direction It is concluded that Drosophila TMC is required for detecting food hardness. TMC is expressed and required in md-L neurons.
Furthermore, loss of tmc greatly reduced the ability to behaviorally discriminate the preferred softness (1% agarose) or smoothness (sucrose solution only) from harder or stickier food options, respectively. However, the responses to tastants,
such as sucrose, salt, or caffeine, were unaffected in tmc1, indicating that TMC was specifically required for sensing food
texture rather than the chemical composition of food (Zhang, 2016).
An important question concerns the mechanism through which TMC enables md-L neurons to sense food hardness. It is proposed
that deflection of gustatory sensilla by food hardness imposes mechanical force on these neurons. The harder the food, the greater
the stimulation of md-L neurons, which sense force through the
dendrites innervating the bases of many sensilla. Given the expression of TMC in dendrites, an appealing possibility is that TMC is a key component of a mechanically activated channel that endows the fly tongue with the ability to sense food hardness. A TMC
protein (TMC-1) is expressed in worms and is proposed to be
required for salt sensation (Chatzigeorgiou, 2013). Furthermore, TMC-1 plays a critical role in alkali sensation in vivo (Wang, 2016). As such, it appears that the worm TMC-1 controls
multiple aspects of chemosensation. Mammalian TMC1 and
TMC2 are required for hearing and expressed in the inner ear (Kawashima, 2011; Pan, 2013). Currently, it is not known if mammalian TMCs are subunits of a channel, or
whether they are mechanically activated, since problems with
cell-surface expression of these proteins in heterologous expression systems have precluded biophysical characterizations. It is possible that TMCs may depend on additional subunits for trafficking or to form functional ion channels. Drosophila TMC may also be one subunit of a mechanically activated channel, and it is proposed that this feature might allow md-L neurons to be stimulated in response to bending of taste sensilla by hard foods (Zhang, 2016).
In conclusion, this study has elucidated a cellular mechanism through
which food mechanics influence the taste preference of an animal. The md-L neurons define a novel class of mechanosensory neurons that enable flies to detect food hardness and
viscosity. A future question concerns the mapping of the
brain region where mechanical and chemosensory pathways
converge to dictate gustatory decisions. An appealing possibility is that md-L and GRN axons coordinately signal to a pair of command interneurons (Fdg neurons) that have extensive arborizations in the SEZ and control feeding behavior. Finally, the results demonstrate that TMC is essential for food texture sensation. These results raise the possibility
that homologs of fly TMC may be dedicated to the gustatory
discrimination of texture in many other animals, including mammals (Zhang, 2016).
Animals discriminate nutritious food from toxic substances using their sense of taste. Since taste perception requires taste receptor cells to come into contact with water-soluble chemicals, it is a form of contact chemosensation. Concurrent with that contact, mechanosensitive cells detect the texture of food and also contribute to the regulation of feeding. Little is known, however, about the extent to which chemosensitive and mechanosensitive circuits interact. This study shows Drosophila prefers soft food at the expense of sweetness and that this preference requires labellar mechanosensory neurons (MNs) and the mechanosensory channel Nanchung. Activation of these labellar MNs causes GABAergic inhibition of sweet-sensing gustatory receptor neurons, reducing the perceived intensity of a sweet stimulus. These findings expand understanding of the ways different sensory modalities cooperate to shape animal behaviour (Jeong, 2016).
Animals must eat to survive, but not all food sources are equally desirable. Animals use their sense of taste to discriminate nutritious foods and toxic substances. Although a food's taste is a major determinant of its acceptability, animals must assess a food's visual appearance, smell, temperature and texture as well. What humans call 'flavour' is actually a complex multisensory picture of a food's general desirability. In fact, each person has direct experience with the interaction of multiple sensory modalities in the general perception of food quality. Who hasn't noticed a change in a food's flavour on catching a cold severe enough to block their sense of smell (Jeong, 2016)?
Despite its obvious importance, the mechanisms by which multimodal sensory information is incorporated into feeding decisions are not well understood. Psychologists and neuroscientists have begun to explore the ways the individual channels of sensory input affect the perception of flavour, but understanding of cross-modal interactions lags behind. This is partly due to difficulties with parsing the individual components that make up the gestalt of flavour perception, and partly due to technical difficulties associated with the controlled delivery of precisely defined multimodal stimuli. Because of these difficulties, it is suggested that the exploration of simpler model systems can help extend understanding of the multisensory perception of flavour that directs feeding decisions (Jeong, 2016).
In particular, this study concerns itself with the ways neural circuits integrate taste and texture information. Texture is a product of mechanosensation. Animals, of course, use mechanosensory information to help determine their food's precise location, but it is the food's physical properties (for example, its hardness or viscosity) that contribute to determining its palatability. Several studies have demonstrated flavour perception can be altered by a food's hardness or viscosity. In particular, A negative correlation between food viscosity and perceived sweetness has been found in humans; as a food's viscosity increases, it is perceived as being less sweet. Since these sorts of interactions exist, they presumably offer some utility, but the neural mechanisms by which they help coordinate appropriate feeding behaviours are not understood in any system (Jeong, 2016).
Drosophila presents an especially attractive system for exploring interactions between taste and mechanosensation with regard to feeding decisions. Although both taste and olfaction are forms of chemosensation, because odorants are airborne and tastants are water-soluble, only taste requires contact with the stimulus. Indeed, while Drosophila olfactory sensilla lack mechanosensory neurons (MNs), the gustatory receptor neurons (GRNs) of each taste sensillum are accompanied by a MN. Thus, as a fly feeds the sensory sensilla on its labellum (mouthparts) unavoidably receive concurrent taste and mechanical activation. In addition, the molecular genetic tools available in the fly allow examination of the role each type of sensory information plays in directing feeding behaviour via selective activation or inactivation of each class of sensory neuron (Jeong, 2016).
This paper presents an exploration of the circuit-level interactions between the perception of gustatory and mechanical stimuli that help direct feeding decisions in Drosophila. It was discovered that Drosophila prefer soft food at the expense of sweetness and that this preference depends on labellar MNs and their expression of the mechanosensory channel Nanchung. Activation of these labellar MNs attenuates the perceived intensity of a sweet stimulus by suppressing the calcium responses of sweet GRN termini via the inhibitory neurotransmitter GABA. These findings expand understanding of the mechanisms by which the neural circuits responsible for the various modes of sensory perception can cooperate to shape animal behaviour (Jeong, 2016).
This study has uncovered a mechanism by which tactile sensation regulates feeding by controlling the presynaptic gain of phagostimulatory GRNs. Activation of MNs inhibits calcium responses in sweet GRNs via the inhibitory neurotransmitter GABA. This effect likely contributes to Drosophila's preference for ripe or overripe rather than fresh fruits, as both sweetness and hardness change with decay (Jeong, 2016).
The association of MNs with GRNs in labellar taste bristles and taste pegs was first observed several decades ago, but the physiologic significance of this association was never investigated. This study has shown labellar MNs produce GABA in the SEZ to inhibit signalling through the sweet GRNs. The activation and inhibition of R55B01-GAL4-expressing cells show similar effects on presynaptic gain in sweet GRNs as activation and inhibition of R41E11-GAL4-expressing cells and VT2692-GAL4-expressing cells. This implicates the taste bristle MNs labelled by all three of these lines rather than the taste peg MNs in the interaction between sweet sensing and mechanosensation. The projection of taste peg MNs to an area of the SEZ distinct from that innervated by sweet and bitter GRNs project further supports this idea (Jeong, 2016).
In flies, the tarsal segments of the legs also have chemosensory and mechanosensory sensilla that can be activated during food foraging. Two other groups recently explored the role these tarsal MNs play in behavioural regulation. Ramdya (2015) reported that tarsal MNs provide sensory information that drives collective behaviour, and Mann (2013) showed that tarsal MNs inhibit feeding via a population of thoracic ganglion interneurons. The fact that the R41E11-GAL4 and VT2692-GAL4 drivers used in this study are expressed not in the MNs of the legs but in their supporting cells, suggests the tarsal MNs play no role in food hardness detection. In further support of this conclusion, this study found inactivation of the tarsal MNs using Gr68a-GAL4 does not impair hardness-mediated food preference. Thus, it is clear the tarsal and labellar MNs play different roles in controlling animal behaviour (Jeong, 2016).
Although soft food preference is strongly affected by both silencing of the labellar MNs and the loss of Nan, both of these conditions still show a slight residual preference for soft food. This suggests the presence of another mechanosensory system involved in food hardness detection, perhaps the pharyngeal MNs or labellar multidendritic neurons (Jeong, 2016).
Despite being unable to detect any role for NompC in food hardness detection using a preference assay, NompC's expression in the labellar taste bristle MNs makes it a plausible secondary candidate for the labellar MN mechanosensor. In other words, while Nan may act as the mechanosensor in labellar MNs with NompC modulating its function, the reverse may also be true, as is the case in the chordotonal neurons (Jeong, 2016).
In Drosophila, GABABR2 is required in sweet GRN axon termini for the suppression of sweet responses by bitter stimuli when sweet and bitter tastants are mixed together. Knockdown of GABABR2 in sweet GRNs increases the PER to sugar as well as to sugar/bitter mixtures. In this study, knockdown of GABABR2 in sweet GRNs impairs soft food preference at the expense of sweetness, but it does not affect preference for sweetness in the absence of differences in food hardness. These data suggest sweet GRNs receive multiple GABAergic inputs from different sensory circuits (Jeong, 2016).
This study has shown taste-related mechanosensory information can inhibit sweet perception in the primary taste relay centre, the SEZ, but it remains unclear whether mechanosensation modulates the perception of sweet tastants only at the level of the GRNs or whether the tactile information is relayed to higher brain centres for integration. It will be interesting to see which other parts of the brain these MNs innervate and what other behaviours, apart from food hardness perception, they regulate. It will also be interesting to see whether these or any other MNs interact with taste information in any higher brain centres. During feeding, multiple modes of sensory information must be perceived and integrated to produce the perception of 'flavour'. This phenomenon is well-described in humans using mainly a psychophysiological approach, but the molecular mechanisms and neural circuits that produce it remain unclear. Using the Drosophila model system, this study has explored potential circuit motifs underlying multimodal sensory processing and has demonstrated an intriguing interaction between sweet GRNs and MNs that modulates feeding decision-making (Jeong, 2016).
Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. It was recently proposed that all acids are aversive at high concentrations, a response that is mediated by low pH as well as specific anions in Drosophila melanogaster. Particularly, some carboxylic acids such as glycolic acid, citric acid, and lactic acid are highly attractive to Drosophila compared with acetic acid. The present study determined that attractive carboxylic acids were mediated by broadly expressed Ir25a and Ir76b, as demonstrated by a candidate mutant library screen. The mutant deficits were completely recovered via wild-type cDNA expression in sweet-sensing gustatory receptor neurons. Furthermore, sweet gustatory receptors such as Gr5a, Gr61a, and Gr64a-f modulate attractive responses. These genetic defects were confirmed using binary food choice assays as well as electrophysiology in the labellum. Taken together, these findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids (Shrestha, 2021).
The extent of diversity among bitter-sensing neurons is a fundamental issue in the field of taste. Data are limited and conflicting as to whether bitter neurons are broadly tuned and uniform, resulting in indiscriminate avoidance of bitter stimuli, or diverse, allowing a more discerning evaluation of food sources. This study provides a systematic analysis of how bitter taste is encoded by the major taste organ of the Drosophila head, the labellum. Each of 16 bitter compounds is tested physiologically against all 31 taste hairs, revealing responses that are diverse in magnitude and dynamics (see The Drosophila labellum and its physiological responses). Four functional classes of bitter neurons are defined. Four corresponding classes are defined through expression analysis of all 68 gustatory taste receptors. A receptor-to-neuron-to-tastant map is constructed. Misexpression of one receptor confers bitter responses as predicted by the map. These results reveal a degree of complexity that greatly expands the capacity of the system to encode bitter taste (Weiss, 2011).
This study has defined five distinct classes of sensilla in the Drosophila labellum on the basis of their responses to bitter compounds (see Labellar sensilla fall into five expression classes that are similar to the functional classes). Four of these sensillar classes contain bitter-sensing neurons; other sensilla did not respond physiologically to any of the bitter tastants. This analysis, then, has defined four classes of bitter-sensing neurons that are diverse in their response profiles. Some are broadly tuned with respect to a panel of bitter compounds and some are more narrowly tuned. The neurons also vary in the temporal dynamics of their responses. Different neurons respond to the same tastant with different onset kinetics, and an individual neuron responds to distinct tastants with diverse dynamics. The functional diversity of bitter-sensing neurons expands the coding capacity of the system: different tastants elicit responses from different subsets of neurons, and distinct tastants elicit diverse temporal patterns of activity from these neurons (Weiss, 2011).
This systematic analysis does not support previous models that suggest functional uniformity among bitter neurons. A previous physiological study of the labellum did not reveal functionally distinct neuronal classes, but was limited in the number of sensilla and tastants that were examined. There are major technical challenges in recording from I and S sensilla; the S sensilla in particular are small, curved, and difficult to access because of their position on the labellar surface. The finding of functional heterogeneity in labellar sensilla is consistent with the finding that two taste sensilla on the prothoracic leg responded to berberine but not quinine, whereas another sensillum responded to quinine but not berberine. A recent study found that DEET elicited different responses from several labellar sensilla tested. Functionally distinct bitter neurons have also been described in taste organs of caterpillars, and in the case of the Manduca larva, aristolochic acid and salicin activate spike trains that differ in dynamics (Weiss, 2011).
The functional differences among neurons in the Drosophila labellum suggested underlying molecular differences. In particular, it was asked whether the four classes of bitter taste neurons defined by physiological analysis could be distinguished by molecular analysis. A receptor-to-neuron map of the entire Gr repertoire was constructed, and it was found that four classes of bitter taste neurons emerged on the basis of receptor expression, classes that coincided closely with the four functional classes. Moreover, the neuronal classes that were more broadly tuned expressed more receptors (Weiss, 2011).
While the physiological and molecular analyses support each other well, there are limitations to each analysis that raise interesting considerations. The functional analysis is based on a limited number of taste stimuli. Bitter tastants were selected that were structurally diverse, but bitter compounds vary enormously in structure and only a small fraction of them can be sampled. It is possible that by testing more tastants, by testing them over a greater concentration range, or by analyzing temporal dynamics in greater detail, that even more diversity would become apparent among the bitter-sensing neurons (Weiss, 2011).
There are also limitations to the receptor-to-neuron map. First, the map considers exclusively the 68 Grs. There are at least two additional receptors that can mediate bitter taste. DmXR, a G-protein coupled receptor, is expressed in bitter neurons of the labellum and is required for behavioral avoidance of L-canavanine, a naturally occurring insecticide; the TRPA1 cation channel, also expressed in a subset of bitter neurons in the labellum, is required for behavioral and electrophysiological responses to aristolochic acid. Second, Gr-GAL4 drivers may not provide a fully accurate representation of Gr gene expression in every case. Genetic analysis has shown that Gr64a is required for the physiological responses of labellar sensilla to some sugars and is therefore expected to be expressed in labellar sugar neurons. The Gr64a-GAL4 driver, however, is not expressed in these neurons, suggesting the lack of a regulatory element. In light of the limitations to the use of the GAL4 system to assess receptor expression, it was encouraging that drivers representing all 68 Grs were expressed in chemosensory neurons, with very few exceptions, and that the expression patterns in the labellum agreed well with the patterns of physiological responses. In addition, it was possible to integrate the functional and expression data and predict a function for one Gr (Weiss, 2011).
While the data support the hypothesis that Gr59c encodes a bitter receptor for berberine, denatonium and lobeline, Gr59c is not sufficient for responses to these compounds in sugar neurons. It is also apparently not necessary, in the sense that physiological responses to these tastants were observed in S-a sensilla that do not express the Gr59c driver. These observations suggest that there is another receptor for berberine, denatonium and lobeline that may recognize a different moiety of these tastants, providing multiple means of detecting some of the most behaviorally aversive bitter tastants in the panel (Weiss, 2011).
It is noted that 38 of the Gr-GAL4 drivers, slightly more than half, showed expression in the labellum. The other Grs are likely expressed in other chemosensory neurons of the adult and larva. Of the 38 labellar Gr-GAL4 drivers, 33 are expressed in bitter neurons, and only a few in sugar neurons. It seems likely that a high fraction of Grs are devoted to bitter perception because of the number and structural complexity of bitter compounds. Sugars are simpler and more similar in structure. In order to detect the wide diversity of noxious bitter substances that an animal may encounter, a larger and more versatile repertoire of receptors is likely needed. It is noted that in mice and rats, 36 bitter receptors have been identified (Weiss, 2011).
Among the Grs mapped to bitter neurons, five map to all bitter neurons: Gr32a, Gr33a, Gr39a.a, Gr66a, and Gr89a. Some or all of these 'core bitter Grs' may function as coreceptors, perhaps forming multimers with other Grs. These core Grs might play a role analogous to Or83b, an Or that is broadly expressed in olfactory receptor neurons and that functions in the transport of other Ors and as a channel, rather than conferring odor-specificity per se. If so, the core Grs may be useful in deorphanizing other Grs in heterologous expression systems. It is noted that in mammals, T1R3 functions as a common coreceptor with either T1R1 or T1R2 to mediate gustatory responses to amino acids or sugars, respectively (Weiss, 2011).
Finally it is noted that the receptor-to-neuron map defines intriguing developmental problems. How do the five classes of sensilla acquire their diverse functional identities? How does an individual taste neuron select, from among a large Gr repertoire, which receptor genes to express? In the olfactory system of the fly, the expression of each receptor gene is dictated by a combinatorial code of cis-regulatory elements and by a combinatorial code of transcription factors. Mechanisms of receptor gene choice were elucidated in part by identifying upstream regulatory elements that were common to coexpressed Or genes. The receptor-to-neuron map that this study has established for the taste system lays a foundation for identifying regulatory elements shared by coexpressed Gr genes, which in turn may elucidate mechanisms of receptor gene choice in the taste system. It will be interesting to determine whether the mechanisms used in the olfactory and taste systems are similar (Weiss, 2011).
In principle the design of the Drosophila taste system could have been extremely simple. Every sensillum could be identical, and all sensilla could report uniformly the valence of each tastant, e.g. positive for most sugars and negative for bitter compounds. Such a design would be economical to encode in the genome and to execute during development (Weiss, 2011).
The design of the Drosophila olfactory system is not so simple. Physiological analysis of the fly has identified ~17 functionally distinct types of olfactory sensilla. This design allows for the combinatorial coding of odors. A recent study of the Drosophila larva defined an odor space in which each dimension represents the response of each component of olfactory input . The distance between two odors in this space was proportional to the perceptual relationship between them. In principle, a coding space of high dimension may enhance sensory discrimination and allow for a more adaptive behavioral response to a sensory stimulus (Weiss, 2011).
This study has found that the fly's taste system is similar to its olfactory system in that its sensilla fall into at least five functionally distinct types, four of which respond to bitter stimuli. This heterogeneity provides the basis for a combinatorial code for tastes and for a multidimensional taste space. A recent report has suggested that flies can not discriminate between pairs of bitter stimuli when applied to leg sensilla (Masek, 2010); it will be interesting to extend such analysis to the labellum, and especially to examine pairs of stimuli that have been shown to activate distinct populations of neurons. Physiological analysis thus invites an extensive behavioral analysis, beyond the scope of the current study, which explores the extent to which such a taste space supports taste discrimination in the fly (Weiss, 2011).
Why might there be selective pressure to enhance the coding of bitter taste? Why not simply coexpress all bitter receptors in one type of neuron that activates a single circuit, thereby triggering equivalent avoidance of all bitter compounds? Not all bitter compounds are equally toxic, and it is not clear that there is a direct correlation between bitterness and toxicity. It is even possible that in certain contexts, such as the selection of egg-laying sites or self-medication, some bitter tastants may have a positive valence. It is noted that in the behavioral analysis carried out in this study, flies tended to be more sensitive to bitter compounds that activate I-a than I-b neurons, suggesting that I-a ligands are perceived to be more bitter than those of I-b ligand, as if I-a ligands were more toxic. A more nuanced behavioral decision based on the intensities of bitter compounds may exist within the complex milieu of rotting fruit (Weiss, 2011).
The olfactory and taste systems of the fly differ in the anatomy of their projections to the brain. Olfactory receptor neurons (ORNs) project to the antennal lobe, which consists of spherical modules called glomeruli. ORNs of a particular functional specificity converge upon a common glomerulus, and there is a distinct glomerulus for each type of ORN. Taste neurons project from the labellum to a region of the ventral brain called the subesophageal ganglion (SOG) that does not have such an obviously modular structure. A study using Gr66a-GAL4, which marks all or almost all bitter cells in the labellum, and Gr5a-GAL4, which marks all or almost all sugar cells, revealed that the two classes of cells project to spatially segregated regions of the SOG (Thorne, 2004; Wang, 2004). However, subsets of bitter cells labeled by Gr-GAL4 drivers did not show obvious spatial segregation within the region of the SOG labeled by Gr66a-GAL4. Markers of different subsets of sugar cells also showed overlapping projections in the SOG. These studies did not, then, reveal at a gross level the kind of spatially discrete projections that are characteristic of the olfactory system (Weiss, 2011).
However, analysis of the SOG at higher resolution has recently revealed more detailed substructure (Miyazaki, 2010). Different sets of Gr66a-expressing neurons, such as those expressing Gr47a, an I-b-specific receptor, showed distinguishable projection patterns, leading to the suggestion that different subregions process different subsets of bitter compounds. Moreover, similarity in projection patterns does not imply identity of function. For example, in the antennal lobe, ORNs that express the odor receptor Or67d converge on the DA1 glomerulus in both males and females, but the projections from DA1 to the protocerebrum are sexually dimorphic. Activation of these ORNs elicits different behaviors in males and females. Taste neurons that project to similar locations in the SOG could also activate different circuits, with distinguishable behavioral consequences. Like the fly taste system, the C. elegans olfactory system does not contain glomeruli and its sensory neurons coexpress many receptors, yet the worm is able to discriminate odors. Finally, it is noted that different sensory neurons that project to similar positions may carry distinguishable information by virtue of differences in the temporal dynamics of their firing. Differences have been identified in the temporal dynamics elicited by different tastants. In summary, it is difficult to draw definitive conclusions about the functional roles of taste neurons from the currently available anatomical analysis (Weiss, 2011).
A final consideration raised by this analysis is how the responses of the different functional classes of taste sensilla are temporally integrated to control feeding behavior. The different functional classes of sensilla differ in length and are located in different regions of the labellar surface. Moreover, during the course of feeding the labellum expands, changing the positions of the various sensilla with respect to the food source. It seems likely that there is a temporal order in which labellar taste sensilla send information to the CNS (Weiss, 2011).
In summary, this study has provided a systematic behavioral, physiological, and molecular analysis of the primary representation of bitter compounds in a major taste organ. This study has defined the molecular and cellular organization of the bitter-sensitive neurons, and extensive functional diversity was found in their responses. The results provide a foundation for investigating how this primary tastant representation is transformed into successive representations in the CNS and ultimately into behavior (Weiss, 2011).
Diversification of Drosophila segmental and cellular identities requires the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that was used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated Ey proteins. Apart from eye loss, these new eyeless alleles led to defects in the adult olfactory appendages -- the maxillary palps and antennae. In support of these observations, both ey and pb were seen to be expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either Pb or Ey proteins. A combination of in vivo and in vitro approaches indicates that Pb suppresses Ey transactivation activity via protein-protein contacts of the Pb homeodomain and Ey Paired domain. The direct functional antagonism between Pb and Ey proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis (Benassayag, 2003).
To better understand the relationship between Pb and Ey in normal
development, the phenotypic effects of ey mutations were studied in
the sensitized HSPbsy genetic context (ectopic expression of pb). Two copies of the HSPbsy transgene (the sensitizing condition) showed no marked
effect. In contrast, pharate adult females with 2x HSPbsy and homozygous for eyJD showed strong maxillary palp and antennal defects. In some cases, the maxillary palp, whose identity is indicated by the distinctive distal bristles, remains adjoined to the antennal appendage. However,
differentiation of the proboscis (which likewise depends on pb
function) is not affected. Thus in the sensitized context, ey mutations
can provoke strong defects of the maxillary and antennal appendages. The
phenotype suggests that ey+ may
participate in partitioning imaginal disc cells into antenna and maxillary
palp during morphogenesis (Benassayag, 2003).
To confirm a role of eyeless in this process,
HSPbsy was removed from the genetic background to examine the effects of the
new eyeless mutations alone. All four ey alleles appear
recessive in a non-sensitized background as shown for eyJD, and can be
interpreted as loss-of-function mutations in accord with their molecular
lesions. All give homozygous escapers with visible defects, allowing for the
composition of an allelic series, from weakest to strongest:
ey11>eyD1Da>eyEH>eyJD.
Analysis of the phenotypes of hemizygotes with Df(4)BA led to the same
conclusion (Benassayag, 2003).
eyJD homozygotes display eye reduction or loss, low viability and
strong brain defects associated with abnormal behavior.
Furthermore, a minority of surviving eyJD homozygotes
(10%-20%, after outcrossing) show alterations in the size and/or shape of
maxillary palps and antennae. The altered maxillary palps of eyJD
homozygotes still harbor the two characteristic sensilla trichodea,
suggesting that maxillary identity per se is not affected. Reduced, malformed
maxillary palps are often accompanied by enlarged, misshapen antennae. Similar although weaker defects are likewise detected for
eyEH homozygotes, as well as for certain
trans-heterozygous combinations with other Enhancer alleles in the sensitized
background. The reciprocal effect of eyJD on
appendages that derive from the same antennal imaginal disc constitutes
evidence of a potential role for ey in apportioning the maxillary
portion of this disc. The defects observed in eyJD
homozygotes appeared stronger than in the hemizygous combination,
eyJD/Df(4)BA. Thus, the truncated protein may have a
limited antimorphic character not detected in the presence of wild-type
protein (Benassayag, 2003).
Although mutant phenotypes implicated both pb and now ey
in maxillary development, no gene expression had been detected in the
maxillary portion of the antennal disc in the third instar larvae.
Pb and Ey expression were examined later, during the prepupal stage when maxillary and
antennal structures evaginate from the eye-antenna imaginal disc. Using a rabbit
anti-Pb serum directed against the C-terminal region (anti-E9), Pb
accumulation was detected in the central part of the maxillary primordium
beginning approximately eight hours after puparium formation, during
evagination of the antennal and maxillary appendages from the composite disc. Ey protein as
visualized by a rabbit anti-Ey serum accumulates in the same primordium and,
within discrimination, at the same time. This expression
appears to be limited to the borders of the primordium, rather than the center as for Pb. Results of tests for co-expression of the two proteins were
mitigated: in situ hybridization or immunostaining experiments were
inconclusive, whereas available antibodies that gave acceptable signals in
this tissue were both rabbit polyclonal antisera. To address whether
endogenous pb and ey patterns in the maxillary palps may
overlap, a pb-GAL4 mini-gene was used based on
descriptions of the pb-promoter region.
Using a pb-GAL4 driver insertion to direct ß-galactosidase
expression (pb-GAL4>UAS-lacZ), the patterns
of pb>lacZ and ey expression were examined by double-immunofluorescence
labeling and confocal microscopy. Early Pb expression is limited to a small
number of cells in the distal maxillary primordium. At
this stage, Ey expression can likewise be detected in a small group of cells
partially overlapping those expressing Pb.
Co-expression appears to be very limited in the progression of a dynamic
pattern. In later prepupae, the expression patterns of
pb>lacZ and ey in the maxillary primordium are adjacent
but exclusive. Taken
together, these data show a previously undisclosed co-temporal expression of
both pb and ey in the maxillary primordium of prepupae, and
support an ephemeral co-expression of these genes in a small number of cells.
This is in agreement with the known function of pb in maxillary
determination, and with the newly established function of ey in this
tissue (Benassayag, 2003).
Thus, etopically expressed homeotic Pb protein, even
a form bereft of DNA-binding capacity, can suppress eye development in a
dose-sensitive manner. Genetic and molecular results indicate a
central role for direct contacts between conserved domains of the Hox selector
protein Pb and the eye selector Ey. One physiological situation where the
interaction between pb and ey is likely to be relevant was
identified, based on their genetic interaction, mutant phenotypes and
expression patterns in forming the adult antennae and maxillary palps (Benassayag, 2003).
This work has identified a previously unrecognized role for
ey in the development of the maxillary palps and antennae. The
mutation employed for most of these experiments, eyJD,
behaves as a strong allele affecting viability, formation of the adult eyes
and brain mushroom bodies, but also of antennal and maxillary differentiation.
Consistent with a late requirement for ey in the antennal disc,
ey expression in the maxillary primordium appears in early stages of
metamorphosis when both eye-antennal discs have fused, and the antennal and
maxillary appendages start to evaginate. After evagination, the maxillary
primordium migrates to join the labial disc in forming the adult mouthparts,
whereas the antennal primordium remains near the eye. When a contiguous
epidermal cell layer has been completed, the head sac is abruptly evaginated
under the internal pressure. Maxillary ey expression in early stages of
prepupal metamorphosis is limited to the boundary between the maxillary
primordium and the antenna, and ey mutant phenotypes often involved
simultaneously reduced palps and enlarged antennae. These reciprocal effects
are consistent with communicating cell populations, suggesting that
ey may contribute to a partitioning of the antennal disc permitting
the establishment of two separate appendages. Further analysis of this process
will require new maxillary-specific markers permitting the fates of these
cells to be followed (Benassayag, 2003).
Starting from a dose-sensitive eye loss provoked by
ectopic Pb, new ey alleles isolated as eye loss
Enhancers were identified. These mutations reveal a role for ey, and a potential
biological relevance for this Hox-PAX6 interaction, in the development of the
antennal and maxillary sensory palps. ey loss-of-function defects in
the sensory palps are exacerbated by Pbsy. The most direct
interpretation of the enhanced ey loss-of-function phenotype with
HSPbsy is that the newly isolated alleles retain a partial function
that can be negated by adequate Pb levels. The molecular characterization of
these alleles is consistent with this hypothesis, because all four alleles
should encode truncated proteins that contain most or all of the interacting
PD (Benassayag, 2003).
To better understand the in vivo relationship between these two selector
genes, attempts were made to examine the effects of double mutants for pb and
ey. Although homozygotes for pb- or for the new
ey mutants showed viabilities of up to 50% compared with
heterozygotes, a double mutant adult was never obtained for any of the
four ey alleles. This result, although suggesting that the double
mutant is synthetic lethal, does not offer insight into the tissue(s)
implicated in this lethality (Benassayag, 2003).
One tissue in which an interaction is clearly indicated from this analysis
is the maxillary palp primordium, where a dynamic expression was detected of Ey
and of Pb (directly or via the pbGAL4 driver) during pre-pupal development.
Transient early co-expression of Pb and Ey in pre-pupae is limited to a small
number of cells, whereas later expression appears exclusive. This result can
be rationalized in two ways: first, co-expressing cells might be rapidly
eliminated by apoptosis, through a coordinate gene-activation process
triggered by a Hox-Pax dimer; second, co-expression of the Ey and Pb
transcription factors could induce a developmental pathway interference
resulting in a G1 cell-cycle arrest. These
possibilities are not fully exclusive. Indeed, one or both mechanisms could
serve to refine the boundaries between antennal and maxillary cell populations
within the antennal disc (Benassayag, 2003).
In vertebrates, Pax6 has multiple known or inferred roles in eye,
brain and nasal development. Apart from the fly eye, several groups have identified an
eyeless function required for development of the mushroom bodies,
neural structures important for olfactory perception and learning. This
study describes a specific role for Ey in concert with Pb in the maxillary and
antennal appendages; both of these are derived from the antennal disc and constitute the
adult olfactory system. An analysis of mutations producing headless
flies has revealed a role for Drosophila Pax6 in head morphogenesis
and thereby suggests a requirement of ey for the development of all
structures derived from eye-antennal discs. These studies involve mutations truncating the Ey protein, which induces head defects. Interestingly,
because these truncated Ey proteins still contain the PD, the fact that the phenotypes obtained reflect an allele-specific antimorphic
effect of the PD cannot be excluded. Taken together, these results strongly suggest that
eyeless, apart from its known role in eye morphogenesis, may also
play multiple other roles in head formation (notably for brain and olfactory
sensory systems) (Benassayag, 2003).
The development of olfactory and visual systems has several common features
in Drosophila. Both systems are derived from the composite
eye-antennal imaginal disc. Moreover, both have similar signal transduction
pathways and appear to share regulatory networks. However, when the expression of ey, so, eya and dac was examined in the
pre-pupal maxillary primordium, only ey expression was detected. This
observation suggests that ey acts there via a distinct combinatorial
code of regulatory genes compared with eye development. One possibility
proposed in this study is that ey activity is modulated by other
co-factors or transcription factors whose activity is likely to be sensitive to Pb.
In this light, it is worth noting that other Enhancer mutations isolated also similarly affect maxillary palps, either singly or in combination with ey. It will be of fundamental interest to better understand the molecular basis for how a single protein might function in multiple, distinct networks (Benassayag, 2003).
The ey mutants studied here were identified as dominant enhancers
of pb-induced eye reduction. Consistent with the antagonism observed
in vivo, Ey and Pb proteins interact directly in vitro, via the Ey Paired
domain and the Pb homeodomain. This interaction with Pb that renders Ey unable
to activate its downstream target genes can be extended to other homeotic
genes because Antp, Scr, Ubx, abdA and AbdB repress eye
development while increasing apoptosis in the eye disc, and their protein
products likewise interact in vitro with Ey protein. This
suggests a combinatorial interaction of homeodomain-containing proteins (Hox
and Pax) to specify a given body segment. An
inhibition through physical association has been proposed between Pax6 and
En-1 during eye development in quail, and
between Pax3 and Msx1 for muscle development in chicken.
Moreover, a similar inhibitory mechanism involving a Hox protein HD has
been reported in vertebrates; in
contrast, physical interaction with Hox-B1 protein leads to increased Pax6
activity in Hela cells, raising the possibility that additional context-dependent
partners modulate the action of Hox-Pax combinations to generate functional
diversity. Based upon genetic and molecular data, it is proposed that variations
on a PD-HD interface can serve to mediate combinatorial or hierarchical
functional relationships among Hox and Pax genes in normal development (Benassayag, 2003).
The results presented here appear to favor a specific role for discrete protein-protein interactions rather than an indirect interference mechanism. Indeed, (1) by
analysing the residues of Pb protein involved in its homeotic function, a Pbsy protein was identified with diminished DNA binding but still
able to inhibit eye development; (2) using this mutant in a genetic screen to
isolate Pb functional partners, four independent eyeless
mutations were isolated, all of them leading to a shortened Ey protein; (3) genetic
interaction tests showed that Pbsy-induced eye loss is highly
sensitive to levels of ey function but independent of several other
eye-determining genes including eyg, eya or so, and (4)
ectopically expressed Pb interferes with ey activity in the eye
imaginal disc by inhibiting so and eya activation without
affecting ey transcription or Ey accumulation (Benassayag, 2003).
In conclusion, these results suggest that a specific Hox/Pax interaction
between Pb and Ey is involved in a normal developmental process defining the
boundary between the antenna and maxillary palp. More generally, the formation
of such protein couples could afford a sensitive and delicate measure of the
balance of Pax6 level, permitting a finely tuned integration to generate
distinct transcriptional outputs during development (Benassayag, 2003).
Mechanoreception detects physical forces in the senses of hearing, touch, and proprioception. This study shows that labellar mechanoreception wires two motor circuits to facilitate and terminate Drosophila feeding. Using patch-clamp recordings, Mechanosensory neurons (MSNs) in taste pegs of the inner labella and taste bristles of the outer labella were identified, both of which rely on the same mechanoreceptor, NOMPC (no mechanoreceptor potential C), to transduce mechanical deflection. Connecting with distinct brain motor circuits, bristle MSNs drive labellar spread to facilitate feeding and peg MSNs elicit proboscis retraction to terminate feeding. Bitter sense modulates these two mechanosensory circuits in opposing manners, preventing labellar spread by bristle MSNs and promoting proboscis retraction by peg MSNs. Together, these labeled-line circuits enable labellar peg and bristle MSNs to use the same mechanoreceptors to direct opposing feeding actions and differentially integrate gustatory information in shaping feeding decisions (Zhao, 2019).
Octopamine regulates feeding behavioral responses in Drosophila melanogaster, however the molecular and circuit mechanisms have not been fully elucidated. This study investigated the role of a subset of octopaminergic neurons, the OA-VPM4 cluster, in sucrose acceptance behavior. Thermogenetic activation of Gal4 lines containing OA-VPM4 promoted proboscis extension to sucrose, while optogenetic inactivation reduced extension. Anatomically, the presynaptic terminals of OA-VPM4 are in close proximity to the axons of sugar-responsive gustatory sensory neurons. Moreover, RNAi knockdown of a specific class of octopamine receptor, OAMB, selectively in sugar-sensing gustatory neurons decreased the behavioral response to sucrose. By calcium imaging experiments, this study found that application of octopamine potentiates sensory responses to sucrose in satiated flies. Taken together, these findings suggest a model by which OA-VPM4 promotes feeding behavior by modulating the activity of sensory neurons (Youn, 2018).
Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. This study functionally characterized JHEdup in vivo. The jhedup gene is highly expressed in large basiconic sensilla, housed in the the maxillary palps, that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. These results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids (Steiner, 2017).
The Drosophila maxillary palpus that develops during metamorphosis is composed of two elements: the proximal maxillary socket and distal maxillary palp. The HOX protein, Proboscipedia (PB), was required for development of the proximal maxillary socket and distal maxillary palp. For growth and differentiation of the distal maxillary palp, PB was required in the cells of, or close to, the maxillary socket, as well as the cells of the distal maxillary palp. Therefore, PB is required in cells outside the distal maxillary palp for the expression, by some mechanism, of a growth factor or factors that promote the growth of the distal maxillary palp. Both wingless (wg) and hedgehog (hh) genes were expressed in cells outside the distal maxillary palp in the lancinia and maxillary socket, respectively. Both wg and hh were required for distal maxillary palp growth, and hh was required noncell autonomously for distal maxillary palp growth. However, expression of wg-GAL4 and hh-GAL4 during maxillary palp differentiation did not require PB, ruling out a direct role for PB in the regulation of transcription of these growth factors (Percival-Smith, 2017).
Identification of nutritious compounds is dependent on expression of specific taste receptors in appropriate taste-cell types. In contrast to mammals, which rely on a single, broadly tuned heterodimeric sugar receptor, the Drosophila genome harbors a small subfamily of eight, closely related gustatory receptor (Gr) genes, Gr5a, Gr61a, and Gr64a-Gr64f, of which three have been proposed to mediate sweet taste. However, expression and function of several of these putative sugar Gr genes are not known. This study presents a comprehensive expression and functional analysis using Gr(LEXA/GAL4) alleles that were generated through homologous recombination. Sugar Gr genes are shown to be expressed in a combinatorial manner to yield at least eight sets of sweet-sensing neurons. Behavioral investigations show that most sugar Gr mutations affect taste responses to only a small number of sugars and that effective detection of most sugars is dependent on more than one Gr gene. Surprisingly, Gr64a, one of three Gr genes previously proposed to play a major role in sweet taste, is not expressed in labellar taste neurons, and Gr64a mutant flies exhibit normal sugar responses elicited from the labellum. This analysis provides a molecular rationale for distinct tuning profiles of sweet taste neurons, and it favors a model whereby all sugar Grs contribute to sweet taste. Furthermore, expression in olfactory organs and the brain implies novel roles for sugar Gr genes in olfaction and internal nutrient sensing, respectively. Thus, sugar receptors may contribute to feeding behavior via multiple sensory systems (Fujii, 2015).
Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. This study found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla (Kawano, 2015).
Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, feeding suppression by Na(+)-rich food was found to require Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent Na(+) detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent Na(+)-sensing GRNs in both L- and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-Na(+) attraction. This work extends the physiological implications of Ir76b from low-Na(+) attraction to high-Na(+) aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of Na(+)-evoked gustation coded in heterogeneous Ir76b-positive GRNs (Lee, 2017).
The proboscis is one of the most highly modified appendages in Drosophila melanogaster. However, the phenotypes of proboscipedia (pb) mutants, which transform the proboscis into leg or antenna, indicate a basic homology among these limbs. Recent genetic studies have revealed a developmental system for patterning appendages and identified several genes required for limb development. Among these are: extradenticle (exd), homothorax (hth), dachshund (dac), Distal-less (Dll) and spalt (sal). These limb genes have not been well studied in wild-type mouthparts and their role if any in this appendage is not well understood. This study demonstrates that the homeotic gene products Proboscipedia (Pb) and Sex combs reduced (Scr) regulate the limb genes in the labial disc to give rise to a unique type of appendage, the proboscis. Pb inhibits exd, dac and sal expression and downregulates DLL: This observation explains the ability of Pb to inhibit the effects of ectopically expressed trunk Hox genes in the proboscis, to suppress leg identity in the trunk and to transform antenna to maxillary palp. Scr suppresses sal expression and also downregulates Dll in the labial discs; discs mutant for both pb and Scr give rise to complete antennae, further demonstrating appendage homology. In the labial disc, Pb positively regulates transcription of Scr, whereas in the embryo, Scr positively regulates pb. Additionally, the results suggests a revised fate map of the labial disc. It is concluded that the proboscis constitutes a genetically distinct type of appendage whose morphogenesis does not require several important components of leg and/or antennal patterning systems, but retains distal segmental homology with these appendages (Abzhanov, 2001).
Proboscipedia is a homeotic protein required for the formation of labial and maxillary palps. It is a member of the Antennapedia Complex (ANTP-C), a linked array of homeodomain proteins. Both Proboscipedia (Pb) and Sex combs reduced (Scr) activities are required for determination of
proboscis identity, while Scr determines tarsus identity. Simultaneous removal of Pb and Scr activity results in a proboscis-to-antenna transformation. Previous genetic observations suggest that Pb and Scr activity may interact. Five pieces of evidence support an interaction between Pb and Scr: (1) the proboscis of a null pb mutant is transformed into a pair of tarsi (the terminal segments of the leg), and (2) these alleles also result in reduced maxillary palps, which some investigators have interpreted as a transformation of the maxillary palps into antennae. (3) Ectopic expression of Pb from a heat-shock promoter/pb fusion gene, or in a small clone of cells from a Tubulin a1 (Tub a1) promoter/ pb fusion gene result in the transformation of the antennae into maxillary palps. (4) Ectopic expression of Scr from a heat-shock promoter/Scr fusion gene results in the transformation of the aristae into tarsi. (5) The proboscis of semilethal loss-of-function Scr alleles, and clones of Scr null mutant cells in the proboscis adopt maxillary palp identity (Percival-Smith, 1997 and references).
That both Pb and Scr activities are required for determination of proboscis identity, and that individual expression of Pb and Scr activities determines maxillary palp and tarsus identities, respectively, suggests a simple model for determination of four developmental identities. It is proposed that the expression patterns of Pb and Scr determine antenna, maxillary palp, tarsus and proboscis identities. Specifically, the absence of Pb and Scr expression, the default state, leads to antennal identity, expression of only Pb activity leads to maxillary palp identity, expression of only Scr activity leads to tarsus identity, and expression of both Pb and Scr activities leads to proboscis identity. A prediction of this simple model is that a proboscis primordial cell that is unable to express either Pb or Scr will adopt antennal identity (Percival-Smith, 1997).
Two mechanisms for the role of Pb and Scr in proboscis determination may be proposed. In both models,
Pb regulates a set of Pb-regulated genes which, when expressed in isolation, determine maxillary palp identity. Similarly, Scr regulates a set of Scr-regulated genes that, when expressed in
isolation, determine tarsal identity. In one model, expression of
both sets of Pb-regulated genes and Scr-regulated genes in the same cell determines proboscis identity. In a second model, expression of Pb and Scr proteins in the same cell leads to formation of a Pb-Scr-containing, heteromeric, protein complex that regulates a novel set of genes that determines proboscis identity, the Pb-Scr-regulated genes. If the second model is correct, it should be possible to design dominant
negative Pb and Scr molecules that will inhibit one another's activity (Percival-Smith, 1997).
In choosing the mutations used for the designed dominant negative Pb and Scr molecules, the properties of previously described change of DNA-binding specificity mutants made them ideal candidates. Both Pb and Scr have a glutamine at position 50 of the homeodomain (HD): pb and Scr genes have been created where this glutamine has been substituted for a lysine. This change is expected to change the DNA-binding specificity of Pb and Scr from Antennapedia class DNA-binding sites to Bicoid
class DNA-binding sites, as has been extensively documented for other HDs. The result of this change would be that the Pb Q50K and Scr Q50K molecules, as well the Pb Q50K Scr and Pb-Scr
Q50K -containing complexes, would not only have diminished
affinity for their normal interaction site, but would also have an increased affinity for another set of sites, dragging away from their normal site of interaction the Pb Q50K and Scr Q50K molecules, as well as the Pb Q50K Scr and Pb-Scr Q50K -containing complexes (Percival-Smith, 1997).
Dominant negative Pb molecules inhibit the activity of Scr indicating that Pb and Scr interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of Pb and Scr and the ability of Pb and Scr to interact in a multimeric complex control the determination of four adult structures (see above: antenna, maxillary palp, tarsus and proboscis). However, the Pb-Scr interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the Pb-Scr interaction may be regulated and indirect (for example, an additional factor binding to both proteins). This regulation may also explain why ectopic expression of Scr(Q50K) and Scr does not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of Scr activity for adult pattern formation has shown that ectopic expression of Scr results in an antenna-to-tarsus transformation, but removal of Scr activity in a clone of cells does not result in a tarsus-to-arista transformation. In five independent assays the reason for this apparent contradictory requirement of Scr activity in tarsus determination is shown. Scr activity is required cell nonautonomously for tarsus determination. Specifically, it is proposed that Scr activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity (Percival-Smith, 1997).
It is suggested that the Drosophila leg is made up of two developmental fields: the tarsus and the proximal leg. These two developmental fields may correlate with the nuclear (proximal) versus cytoplasmic (distal) intracellular localization of Extradenticle, and the distal expression of Distalless. It is also proposed that there are four genetic pathways working in leg determination. The first pathway is the cell nonautonomous Scr-dependent, tarsus-inducing, signal pathway, and this lays down the plan for the basic unmodified tarsus. The second pathway is the relatively cell autonomous proximal leg
pathway, which can be activated by the expression of Scr, Antp or Ubx and which lays out the basic plan for the proximal leg. The third and fourth pathways are cell autonomous pathways that Scr and Ubx control. A basic leg plan results in second leg identity, but expression of Scr or Ubx in both the proximal and distal portions of this basic plan brings about modifications resulting in first or third leg
identity, respectively (Percival-Smith, 1997 and references).
The Drosophila homeotic gene proboscipedia (a HoxA2/B2 homolog) is required for the development of adult mouthparts.
Ectopic Pb protein expression from a transgenic heat shock promoter (HSPB) results in
transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to
Pb-induced effects. To study the basis of homeotic tissue specificity,
mutations that modify dominant HSPB-induced phenotypes have been characterised. One HSPB point mutation (Arg5 of the
homeodomain mutated to His) removes homeotic activity in the mouthparts and antennae, but provokes a
dose-sensitive eye loss. Eye loss can be induced by Pbproteins that no longer
effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific,
context-dependent protein-protein interactions. Dominant eye loss may reflect the titration of limiting proteins factor(s) through specific interactions with the altered heat shock induced protein (Benassayag, 1997a).
A transgenic Hsp70-proboscipedia (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary
palps. To identify sequences essential to PB protein function, EMS-induced HSPB
mutations were sought that lead to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified
point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes
in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved
regions outside the homeodomain. Two independent homeodomain mutations that change the highly
conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a
previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations
outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that
contribute quantitatively to pb function. One of the three is in the N-terminus of the protein, a second is 25 residues downstream of the homeodomain, and a third mutations deletes the C-terminal 123 amino acids. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function (Benassayag, 1997b).
Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homolog) provoke
dose-sensitive defects. These effects were used to search for dose-sensitive dominant modifiers of pb
function. Two identified interacting genes are the proto-oncogene Ras1 and its functional antagonist
Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of
pb activity both in normal and ectopic cell contexts, while Gap1, the Ras1-antagonist, has an opposite
effect. Ras1-modulated changes were observed in homeotic effects on cell identity (bristle to distal sex combs, wing trichomes to veins, veins to trichomes or veins to bristles). Only a small number of cell identities in precise contexts are changed by HSPB activity. This suggests that most cells are aware of their positions and their correctly associated fates, perhaps as a consequence of cell-cell communication. Ras1-dependent modifications of segmental identity are also observed. These occur in a concerted fashion on groups of adjacent cells, again suggesting cell communication. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates
functions of the homeotic loci Sex combs reduced and Ultrabithorax. These data suggest that the
modulation occurs by an independent mechanism for the transcriptional control of the homeotic loci
themselves, or of the Ras1/Gap1 genes. Taken together the data support a role for Ras1-mediated cell
signaling in the homeotic control of segmental differentiation (Boube, 1997).
The Drosophila homeotic gene proboscipedia specifies labial identity and directs
formation of the adult distiproboscis from the labial imaginal discs. pb null alleles result
in the homeotic transformation of the distiproboscis into prothoracic (T1) legs.
Homology with other transcription factors, localization to the nucleus, and restricted
embryonic and imaginal expression implicate the PB protein as a transcription
factor. In order to examine the possible roles that PB may play in the specification of
adult mouthparts, PB was expressed in cells of wing, leg and eye-antennal
imaginal discs, and the effects on the development of adult structures were observed. The ectopic expression of PB in the imaginal discs under the control of
the inducible GAL4 system under control of a dpp imaginal disc enhancer
alters the developmental program of adult legs into maxillary or labial palps. Labial-like structures observed include pseudotrachea, shot hairs resembling basiconica, and patches of smooth cuticle usually associated with the labellar bolster at the distal-most end of the labial palps. Leg patterning defects resulting from ectopic PB expression do not include a replacement of the entire leg by labial palps. Instead, an appendage of mixed identity is produced, containing both leg- and mouth-specific structures. These
homeotic transformations have an equal effect on all three sets of legs, indicating an
activity that is not solely dependent upon the unique combinations of other homeotic
genes present in each of the leg discs. Wings expressing PB do not exhibit a homeotic transformation, but are smaller in size than wild type, are missing veins, have ectopic socketed bristles growing from the wing blade surface, and display a generalized crumpled appearance. Segment polarity genes required for
establishing the AP compartment boundary are found to be undisturbed by ectopic
PB. Furthermore, normal patterns of apoptosis are observed in animals expressing
ectopic PB, indicating that PB does not alter or affect cell death. The normal domain of activity of pb is in the labial imaginal discs, tissues that are derived from the embryonic labial segments. The fact that pb can alter the segmental identity of the thoracic imaginal discs, derived from segments more posteriorly located than the labial segment, indicates that pb does not follow the general rule of "posterior dominance" of the HOM-C genes. These results
suggest that molecular events occurring downstream of the establishment of the
compartment boundary are affected by ectopic PB expression in imaginal discs and
point to a general role in "palp" formation, in addition to the specification of labial
identity (Aplin, 1997).
References
Abzhanov, A., Holtzman, S. and Kaufman, T. C. (2001). The Drosophila proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes. Development 128(14): 2803-2814. PubMed ID: 11526085
Aplin, A. C. and Kaufman, T. C. (1997). Homeotic transformation of legs to mouthparts by proboscipedia expression in Drosophila imaginal discs. Mech. Dev. 62 (1): 51-60.
Benassayag, C., et al. (1997a). A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function. Mech. Dev. 63(2): 187-198.
Benassayag, C., et al. (1997b). Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila. Genetics 146(3): 939-949.
Benassayag, C., et al. (2003). Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development. Development 130: 575-586. 12490563
Fujii, S., Yavuz, A., Slone, J., Jagge, C., Song, X. and Amrein, H. (2015). Drosophila sugar receptors in sweet taste perception, olfaction, and internal nutrient sensing. Curr Biol 25: 621-627. PubMed ID: 25702577
Jeong, Y. T., Oh, S. M., Shim, J., Seo, J. T., Kwon, J. Y. and Moon, S. J. (2016). Mechanosensory neurons control sweet sensing in Drosophila. Nat Commun 7: 12872. PubMed ID: 27641708
Joulia, L., Bourbon, H. M. and Cribbs, D. L. (2005). Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts. Dev. Biol. 278(2): 496-510. 15680366
Joulia, L., Deutsch, J., Bourbon, H. M. and Cribbs, D. L. (2006). The specification of a highly derived arthropod appendage, the Drosophila labial palps, requires the joint action of selectors and signaling pathways. Dev Genes Evol 216(7-8): 431-442. PubMed ID: 16773339
Kawano, T., Ryuda, M., Matsumoto, H., Ochiai, M., Oda, Y., Tanimura, T., Csikos, G., Moriya, M. and Hayakawa, Y. (2015). Function of desiccate in gustatory sensilla of Drosophila melanogaster. Sci Rep 5: 17195. PubMed ID: 26610608
Lee, M. J., Sung, H. Y., Jo, H., Kim, H. W., Choi, M. S., Kwon, J. Y. and Kang, K. (2017). Ionotropic Receptor 76b is required for gustatory aversion to excessive Na+ in Drosophila. Mol Cells 40(10): 787-795. PubMed ID: 29081083
Manzo, A., Silies, M., Gohl, D. M. and Scott, K. (2012). Motor neurons controlling fluid ingestion in Drosophila. Proc Natl Acad Sci U S A 109(16): 6307-6312. PubMed ID: 22474379
Percival-Smith, A., et al. (1997). Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster. Development 124(24): 5049-5062
Percival-Smith, A., Ponce, G. and Pelling, J. J. (2017). The non-cell autonomous requirement of Proboscipedia for growth and differentiation of the distal maxillary palp during metamorphosis of Drosophila melanogaster. Genet Res Int 2017: 2624170. PubMed ID: 28357140
Park, J. and Carlson, J. R. (2017). Physiological responses of the Drosophila labellum to amino acids. J Neurogenet: 1-10. PubMed ID: 29191065
Rass, M., Oestreich, S., Guetter, S., Fischer, S. and Schneuwly, S. (2019). The Drosophila fussel gene is required for bitter gustatory neuron differentiation acting within an Rpd3 dependent chromatin modifying complex. PLoS Genet 15(2): e1007940. PubMed ID: 30730884
Schoofs, A., Huckesfeld, S. and Pankratz, M. J. (2018). Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila. J Insect Physiol 106(Pt 1): 36-46. PubMed ID: 28735009
Shrestha, B. and Lee, Y. (2021). Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster. Mol Cells. PubMed ID: 34711686
Steiner, C., Bozzolan, F., Montagne, N., Maibeche, M. and Chertemps, T. (2017). Neofunctionalization of "Juvenile Hormone Esterase Duplication" in Drosophila as an odorant-degrading enzyme towards food odorants. Sci Rep 7(1): 12629. PubMed ID: 28974761
Struhl, G. (1997). Developmental compartments in the proboscis of Drosophila Nature 270: 723-725
Uchizono, S., Itoh, T. Q., Kim, H., Hamada, N., Kwon, J. Y. and Tanimura, T. (2017). Deciphering the genes for taste receptors for fructose in Drosophila. Mol Cells 40(10): 731-736. PubMed ID: 29047261
Weiss, L. A., Dahanukar, A., Kwon, J. Y., Banerjee, D., Carlson J. R. (2011). The molecular and cellular basis of bitter taste in Drosophila. Neuron 69(2): 258-272. PubMed ID: 21262465
Youn, H., Kirkhart, C., Chia, J. and Scott, K. (2018). A subset of octopaminergic neurons that promotes feeding initiation in Drosophila melanogaster. PLoS One 13(6): e0198362. PubMed ID: 29949586
Yasunaga, K., Saigo, K. and Kojima, T. (2006). Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes. Mech Dev 123(12): 893-906. PubMed ID: 17027238
Zhang, Y.V., Aikin, T.J., Li, Z. and Montell, C. (2016). The basis of food texture sensation in Drosophila. Neuron 91(4):863-877. PubMed ID: 27478019
Zhou, Y., Cao, L. H., Sui, X. W., Guo, X. Q. and Luo, D. G. (2019). Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding. Sci Adv 5(5): eaaw5141. PubMed ID: 31131327
Genes involved in organ development
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