The Interactive Fly

Genes involved in tissue and organ development

Haltere Development and Function

Flies tune the activity of their multifunctional gyroscope

Members of the order Diptera, the true flies, are among the most maneuverable flying animals. These aerial capabilities are partially attributed to flies' possession of halteres, tiny club-shaped structures that evolved from the hindwings and play a crucial role in flight control. Halteres are renowned for acting as biological gyroscopes that rapidly detect rotational perturbations and help flies maintain a stable flight posture. Additionally, halteres provide rhythmic input to the wing steering system that can be indirectly modulated by the visual system. The multifunctional capacity of the haltere is thought to depend on arrays of embedded mechanosensors called campaniform sensilla that are arranged in distinct groups on the haltere's dorsal and ventral surfaces. Although longstanding hypotheses suggest that each array provides different information relevant to the flight control circuitry, little is known about how the haltere campaniforms are functionally organized. This study used in vivo calcium imaging during tethered flight to obtain population-level recordings of the haltere sensory afferents in specific fields of sensilla. Haltere feedback from both dorsal fields was found to be continuously active, modulated under closed-loop flight conditions, and recruited during saccades to help flies actively maneuver. This study also found that the haltere's multifaceted role may arise from the steering muscles of the haltere itself, regulating haltere stroke amplitude to modulate campaniform activity. Taken together, these results underscore the crucial role of efferent control in regulating sensor activity and provide insight into how the sensory and motor systems of flies coevolved (Verbe, 2024).

Animals navigate unpredictable and challenging three-dimensional environments. This requires the ability to both remain stable, to counteract perturbations, and be maneuverable, to actively pursue prey or escape a predator at short timescales. As a result, neural circuits that detect precise spike timing are essential to survival, and many organisms use specialized organs to detect tiny timing differences. Organs such as the ears of barn owls or the electric organ in knifefish have large arrays of mechanosensory cells that possess specific anatomical and mechanical properties that biomechanically filter the kinetic energy of a stimulus, thereby extracting relevant information while minimizing neural processing time. Although several circuits that detect timing differences have been identified, how the encoding by specialized sensors relates to the functional organization of the motor system is less understood. This is a critical knowledge gap as the timing of neural input has important consequences on the biomechanical properties of the musculoskeletal system, which ultimately executes behavior (Verbe, 2024).

These issues are particularly pronounced for flying insects, which collect and process incoming information to control wing motion at sub-millisecond timescales. Among flying insects, true flies (order Diptera) stand out for their adept aerial maneuvers. Indeed, flies have evolved multiple physiological specializations in both the sensory and motor systems that make them remarkable fliers and one of the most diverse insect orders (Verbe, 2024).

For example, flies possess a unique neural superposition visual system that helps process optic flow at high speeds. Furthermore, in flies (as in many other insect orders) the flight muscles, which reside solely in the thorax, are functionally segregated into two major groups: power muscles that provide the force necessary to flap the wings and generate lift, and steering muscles that control the subtle changes in wing motion needed to accomplish maneuvers. Exoskeletal deformation during flapping activates the power muscles via stretch to achieve the high frequencies needed to produce lift. These muscles are ‘asynchronous:’ a single action potential triggers multiple contraction cycles. By contrast, the wing steering muscles, each innervated by a single motor neuron, are ‘synchronous’; their firing times rely on mechanosensory feedback that arrives during each wingstroke. There are two major sources of mechanosensory feedback that structure the firing times of the steering muscles: the wings themselves, and vestigial hindwings unique to flies called halteres (Verbe, 2024).

Halteres are tiny club-shaped structures found on the metathorax that do not serve an aerodynamic function but instead provide sensory information crucial to flight. Halteres oscillate during flight, providing rhythmic input to the wing steering system via arrays of embedded mechanosensors (campaniform sensilla) that are sensitive to cuticular strain. Both the wings and halteres possess campaniform sensilla, but they have hypertrophied on the haltere such that in the case of Drosophila melanogaster, the haltere has approximately 140 campaniforms compared to less than 50 on the wing (Verbe, 2024).

The haltere is also the only true biological gyroscope: during rotations, it experiences Coriolis forces due to its tendency to resist changes its plane of oscillation, triggering equilibrium reflexes of the head and wings. Underscoring their evolutionary history as a hindwing, halteres are equipped with a single power muscle and a set of seven control muscles that are serially homologous to those of the wings. These muscles receive visual input, allowing flies to modulate the activity of the wing steering system via the halteres even in the absence of body rotations, a process known as the control-loop hypothesis. Through active control of the haltere muscles, flies may execute voluntary movements without triggering counteracting reflexes. Thus, halteres serve as multifunctional sensory organs that provide essential timing information to flight circuitry. However, how this multifunctional role is achieved is poorly understood (Verbe, 2024).

Whereas previous electrophysiological work demonstrated how haltere neurons encode mechanical input using quiescent flies, this study used in vivo calcium imaging during tethered flight to obtain population-level recordings of the haltere afferents in specific fields of sensilla. Further evidence is provided that haltere feedback is continuously modulated by visual stimuli to stabilize flight, and such feedback may trigger saccades and help flies actively maneuver. These different roles for flight control are determined by the haltere motor system, which subtly alters haltere kinematics to modulate campaniform activity. Thus, these results demonstrate how biomechanics can filter sensory stimuli to help produce flexible and robust behavior (Verbe, 2024).

Like other insect campaniform sensilla, the orientation and location of the haltere fields are hypothesized to have functional significance. For example, the fields of sensilla that lie parallel to the haltere’s long axis–i.e., dF1, vF1, and vF2–are predicted to detect the in-plane strains produced by the large vertical oscillations. Similarly, the diagonally orientated sensilla of dF2 should be maximally sensitive to the strains produced either by lateral bending from gyroscopic torques or from active lateral haltere movements from the activity of the haltere steering muscles. Therefore, it was expected hat dF1 alone is continuously active during tethered flight, and that df2 is recruited during active maneuvers (Verbe, 2024).

Instead, the activity of both dF1 and dF2 were found to be continuously regulated during flight. These results are significant in two respects. First, the observation that dF2 activity is modulated by specific directions of wide-field visual motion provides further confirmation of the control-loop hypothesis. The tuning characteristics of dF2, and its alignment with steering responses, suggest that the haltere steering muscles manipulate haltere motion to modulate haltere feedback. Indeed, the experiments provide the first direct evidence that the haltere steering muscles regulate stroke amplitude during flight. This finding is consistent with previous work showing that the haltere steering system is also tuned to visual motion, yet its peak sensitivity is in the opposite direction of wing steering responses. Thus, the haltere motor system may regulate campaniform field activity via tiny changes in flapping amplitude that depend on the wide-field visual environment (Verbe, 2024).

Second, the results suggest that dF1 may mediate flight control. Previous anatomical and physiological work in the blowfly Calliphora showed that haltere afferents provide excitatory input to the wing motor neuron b1, through a mixed electrical and chemical synapse. However, this feedback is provided by a single field of campaniforms, dF2: ablating dF2 and stimulating the remaining campaniform fields, including dF1, failed to generate postsynaptic potentials in the b1 motor neuron. Yet in these experiments, dF1 is recruited in much the same manner as dF2, for both visually mediated reflexes and active maneuvers, indicating that dF1 is not merely a passive sensor for the haltere’s large oscillation. Moreover, anatomical work shows extensive projection patterns of each field within the fly central nervous system, suggesting that all fields play an important part in controlling flight. Future work with new connectomics data will reveal the organizational logic of the haltere campaniform fields with the wing steering motor neurons and other targets (Verbe, 2024).

More broadly, the observation that both dorsal fields are continuously active is not surprising as the haltere undergoes large oscillations at a high frequency, creating complex patterns of strain across the surface. These strain patterns are transduced by the embedded campaniform sensilla, which are exquisitely sensitive to the slightest deformations of the cuticle. Use of a calcium indicator to monitor haltere activity allowed obtaining population recordings from the two dorsal fields. However, the kinetics of the calcium indicator are too slow to capture crucial information about spike timing from individual sensilla. Intracellular recordings from sensilla axons demonstrate that changes in haltere motion are reported via either shifts in the preferred firing phase of active campaniforms or recruitment of additional campaniforms that have unique preferred firing phases. By adjusting haltere stroke amplitude via modulation of the haltere steering muscles, flies may modulate the strength of this feedback by subtle changes in stroke amplitude to rapidly adjust wing kinematics with precisely timed mechanosensory feedback (Verbe, 2024).

In addition to suggesting that visual information modifies haltere mechanosensory feedback and consequently wing motor output, the control-loop hypothesis predicts that the haltere control muscles and sensilla are active during voluntary maneuvers, e.g., body saccades. Consistent with their role in sensing the Coriolis forces that result from body rotations, feedback from the halteres terminates saccades. However, whether haltere feedback is modulated during the initiation of saccades remains an open question (Verbe, 2024).

Both the haltere motor system, as well as the campaniform fields, were found to be recruited during spontaneous saccades. Moreover, these changes in muscle and campaniform field activity occur prior to the changes in wing kinematics. This suggests that haltere motion can be controlled independently of the wings to initiate a voluntary maneuver. Recent work shows that flies tune the magnitude of a saccade according to the angular velocity of a visual stimulus. Similarly, this study found that flies can actively tune the strength of haltere mechanosensory feedback depending on the magnitude of a saccade (Verbe, 2024).

Recent work in insects suggests a role for efference copy in suppressing visually mediated reflexes during voluntary flight maneuvers. For example, electrophysiological recordings of lobula plate tangential cells (LPTCs) in Drosophila show predictive scalable inhibition or excitation, correlated with spontaneous saccades. Yet in all cases, the source of these efference copies remains unidentified. By contrast, the results suggest that rather than cancel out expected haltere feedback during a turn, flies co-opt existing haltere reflex loops to actively maneuver. An efference copy signal would render a fly susceptible to mechanical perturbations. Instead, by actively manipulating the haltere during a turn, flies can remain sensitive to gyroscopic forces during voluntary maneuvers (Verbe, 2024).

The multifunctional capability of the haltere system suggests that flies can be maneuverable at low cost to their stability. However, how flies can distinguish self-motion mechanosensory feedback from external whole-body mechanical perturbations remains unclear. Campaniform sensilla function through a generic encoding mechanism, termed Derivative Pair Feature Detection (DPFD). DPFD may be an inherent property of neurons with non-specialized Hodgkin-Huxley dynamics, like many mechanosensors. As a result, the anatomical placement and mechanics of a DPFD neuron, rather than the specialized neural computation and membrane dynamics, act as a biomechanical filter and may confer specialized encoding. It is hypothesized that the orientation of the different campaniform fields on the haltere allows for the control-loop and gyroscopic sensing to work synergistically to control flight maneuvers. In this scheme, descending visual commands to the haltere steering system can initiate a turn by causing small changes in haltere kinematics and by recruiting campaniforms from all fields. Then, as the fly begins to rotate and the haltere undergoes changes in its trajectory due to Coriolis forces, the activity of dF2–which should be maximally sensitive to the resulting shear strains–is increased, triggering a corrective maneuver. This hypothesis is consistent with both the observation that haltere feedback is constant and the haltere’s well-established role as a gyroscopic sensor (Verbe, 2024).

In free flight, both stabilization and active maneuvers consist of banked turns that involve coordinated changes about all three cardinal axes. Modeling and behavioral evidence show that the haltere can detect any combination of body rotations. In this regard, it is surprising that visual modulation of campaniform sensilla activity seems to be present only in the yaw-roll plane and nonexistent for rotations about the pitch-roll plane. This is of particular interest as the direction of motion that elicited the strongest response, for both dF1 and dF2, is pure yaw, which is the weakest axis for gyroscopic responses36. It is possible that, in addition to helping initiate active turns, one major role for the control-loop is to help mediate straight flight about the azimuth by instituting rapid corrections. Indeed, flies dedicate a great deal of neural circuitry to descending interneurons that are hypothesized to maintain a straight flight trajectory. Presumably some of those commands are directed to the haltere motor system (Verbe, 2024).

Although the haltere is a unique sensory structure, its core function–regulating the timing of the wing steering system–and control are much like the aerodynamically functional forewings. Indeed, haltere evolved from the hindwing, and past genetic work confirms that it is a serially homologous structure. Moreover, in other flying insects, this homology even extends to detecting perturbations. Fields dF2 and vF1 in flies are serial homologues of campaniform fields on the radial vein of the wing (dorsal Radius A and ventral Radius A, respectively). This study found that this homology extends to the organization of the motor system for each structure, as the haltere motor system is functionally segregated like the wing steering muscles. Interestingly, along with their distinct morphologies, the two fields dF1 and vF2 have no clear homolog on the wing. It is possible that the development of these two fields is an evolutionary novelty that – combined with classic approaches drawn from biomechanics and biophysics will enable for a fuller appreciation of this enigmatic sensory structure (Verbe, 2024).

Specific expression and function of the Six3 optix in Drosophila serially homologous organs

Organ size and pattern results from the integration of two positional information systems. One global, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. Still, the mechanisms of the Hox-signal integration even in this well-studied system are incomplete. This study has investigated this issue by studying the expression and function of the Six3 transcription factor optix during the development of the Drosophila wing and haltere development. In both organs Dpp defines the expression domain of optix through repression, and the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere: In the wing, optix is required for the growth of the most anterior/proximal region (the 'marginal cell') and for the correct formation of sensory structures along the proximal anterior wing margin, and the halteres of optix mutants are also significantly reduced. In addition, in the haltere optix is necessary for the suppression of sensory bristles (Al Khatib, 2017).

In the haltere, Ubx modifies the wing developmental program in two ways. First, as a transcription factor, Ubx regulates the expression of some targets. For example, Ubx represses sal expression. Second, Ubx modifies the shape of the Dpp-generated signaling gradient indirectly, by controlling the expression of proteoglycans required for Dpp dispersion. Globally, these modifications of Dpp signaling and target gene activation by Ubx have been related to the size and patterning differences between halteres and wings (Al Khatib, 2017).

Since Dpp signaling generates a signaling gradient that spans the whole wing pouch and its activity is required throughout the wing, it is expected to control the expression of target genes not only in central region of the pouch, but also in more lateral ones. The Six3-type transcription factor optix has been reported to be expressed in the lateral region of the wing pouch, as well as in the haltere). Functional studies show that optix is required for the normal patterning of the anterior portion of the wing and that its expression is negatively regulated by sal genes. The fact that sal genes are Dpp signaling targets in the wing, places optix downstream of Dpp regulation. However, since sal genes are not expressed in haltere discs, the mechanism of optix regulation in this organ is still unknown. This study analyzed comparatively the expression, function and regulation of optix in wing and haltere discs. In both discs, optix expression is anteriorly restricted by Dpp signaling, although in the wing the precise expression boundary may be set with the collaboration of wing specific Dpp targets, such as sal. optix shows organ-specific functions: in the wing, previous results were confirmed showing it is necessary for the growth of the anterior/proximal wing ('marginal cell') and the development of wing margin sensory bristles. However, in the haltere optix is required for the suppression of sensory bristle formation. Overexpression of optix in the entire wing pouch affects only anterior wing development, suggesting that other parts of the wing cannot integrate ectopic Optix input. This observation may provide a mechanistic explanation for a widespread re-deployment of optix expression in wing spot formation in various butterfly species (Al Khatib, 2017).

The Dpp signaling gradient is required for the patterning of the whole wing, from the center to its margin. This gradient is translated into a series of contiguous domains expressing distinct transcription factors, each required for the specification of specific features in the adult organ. However, while the transcription factors acting in the central wing were known, the most anterior region of the wing -- the region comprised between the longitudinal vein 2 (L2) and the anterior margin (L1) -- lacked a specific transcription factor. This paper shows that this transcription factor, or at least one of them, is Optix (Al Khatib, 2017).

The results confirm previous findings that optix is expressed in, and required for the growth of this most anterior sector of the wing, the so-called margin cell. This study now shows that optix is also required for the growth of the wing's serially homologous organ: the haltere. This role is in agreement with previous results showing that Six3 regulates cell proliferation in vertebrate systems. This study further shows that Dpp signaling plays a major role in setting the optix expression domain. Although it has been reported before that sal genes are required to set the central limit of this domain, in discs lacking sal function optix does not extend all the way to the AP border, suggesting additional mechanisms involved in optix repression. The fact that sal is not expressed in the haltere pouch and still optix does not extend all the way to the AP border, the exclusion of optix expression from intermediate/high Dpp signaling in both wing and haltere, and the requirement of Dpp signaling to repress optix in any position of the anterior wing compartment globally suggested that either Dpp activates a different repressor closer to the AP border, or that Dpp signaling represses directly optix transcription. The current work cannot distinguish between these possibilities. Regarding another well characterized Dpp target, omb, the extensive coexpression of omb and optix in the haltere also seems to exclude omb as a repressor. Therefore, either another unknown repressor exists, or Dpp signaling acts as a direct optix repressor. While in the haltere, the domain of optix would be set directly by Dpp, in the wing sal would be an additional repressor. By intercalating sal, the Dpp positioning system may be able to push the limit of optix expression farther away from the AP border of the wing. The Sal proteins have been previously shown to act as transcriptional repressors of knirps (kni) to position vein L2. Thus, adding sal repression may help to align the optix domain with L2. This additional repression would not be operating in the haltere, which lacks venation (Al Khatib, 2017).

Interestingly, the logic of optix regulation by Dpp is different from that of other Dpp targets. The activation of the sal paralogs (sal-m and sal-r) and aristaless (al), another target required for vein L2 formation, proceeds through a double repression mechanism: In the absence of signal, the Brinker repressor keeps sal and al off. Activation of the pathway leads to the phosphorylation of the nuclear transducer Mad (pMad) which, in turn, represses brk, thus relieving the repression on sal and al. Therefore, optix regulation by Dpp signaling could be more direct similarly to that of brk (Al Khatib, 2017).

One interesting aspect of optix function is that it plays an additional specific role in the haltere. While in the wing optix is required for the development of the anterior-most portion of the wing (including the margin bristles), in the haltere optix serves to suppress the development of sensory bristles, a task known to be carried out by the Hox gene Ubx. A role for optix in regulating Ubx expression has been ruled out, at least when judged from Ubx protein levels. Therefore, optix is required for a subset of Ubx's normal functions. Since optix encodes a Six3-type transcription factor, this interaction could be happening at the level of target enhancers, where the combination of Ubx and Optix would allow the activation or repression of specific sets of genes (Al Khatib, 2017).

Finally, it was observed that the expression of optix in wing and haltere primordia is conserved across higher Diptera. Interestingly, optix is expressed in the developing wings of passion vine butterflies (genus Heliconius). In Heliconius species, optix has been co-opted for red color patterning in wings. However, the ancestral pattern found in basal Heliconiini is in the proximal complex, a region that runs along the base of the forewing costa, the most anterior region of the forewing. This similarity between optix expression patterns in forewings of Diptera and Lepidoptera leads to the hypothesis that an ancestral role of optix might have been 'structural', being required for the development of the anterior wing. Once expressed in the wings, recruitment of red pigmentation genes allowed optix co-option for color pattern diversification through regulatory evolution. It is noted that a pre-requisite for this co-option in wing pigmentation patterning must have been that optix would not interfere with the developmental pathway leading to the formation of a normal wing in the first place. The fact that the effects of optix overexpression throughout the wing primordium in Drosophila are restricted to the anterior/proximal wing -its normal expression domain- indicates that optix cannot engage in promiscuous gene regulation, and that its function depends on other competence factors, which would limit its gene expression regulatory potential (Al Khatib, 2017).

Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells. Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

However, cells of the third instar larval wing and haltere discs are similar in size and shape. The difference between cell size and shape becomes evident at late pupal stages. Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Singh, 2015).

In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous. For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx^6.28) show similar sized twin spot in small clones. Only when very large clones of Ubx^6.28Ubx^6.28 are generated, one can see increased growth compared to their twin spots. This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (Singh, 2015).

Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere. However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling. EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP. However, EGFR negatively regulates events downstream of Yki. The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

(1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

(2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

(3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

(4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).

Control of tissue morphogenesis by the HOX gene Ultrabithorax

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. This study shows that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. These results provide a long-awaited explanation for how Ubx controls morphogenetic transformation (Diaz-de-la-Loza, 2020).

The results reveal how Ubx – a homeotic gene that encodes the founding member of the HOX-family of transcription factors – regulates apical and basal matrix remodelling to control epithelial morphogenesis (see Ubx controls apical and basal ECM degradation to regulate morphogenesis). Ubx strongly represses two genes encoding apical matrix proteases (Np and Sb), as well as partially repressing two genes encoding basal matrix metalloproteases (Mmp1 and Mmp2), while inducing an inhibitor of Mmp1/2 (Timp) in the haltere. In this way, Ubx prevents both apical and basal matrix remodelling in the haltere, a key event in the homeotic wing-to-haltere transformation. In addition to regulating morphogenesis, Ubx controls many other genes affecting wing growth and pattern. Together, the combined repression of morphogenesis, growth and patterning by Ubx is responsible for the full transformation of wing to haltere (Diaz-de-la-Loza, 2020).

Ubx controls apical and basal ECM degradation to regulate morphogenesis. Schematic of Ubx expression and function in Drosophila and a hypothetical four-winged ancestor. Ubx controls organ shape via regulation of aECM and bECM proteases, in addition to its known functions in regulating organ growth and patterning. These target genes have presumably evolved to be specifically regulated in the Drosophila wing and/or haltere, and must be insensitive to Ubx in four-winged ancestors (Diaz-de-la-Loza, 2020).

These findings also support the general view that transcriptional control of matrix synthesis and degradation is a conserved mechanism by which information encoded in the genome is deployed to govern the shape of tissues and organs in animals. Although this concept is broadly appreciated for the regulation of the bECM, the notion that the aECM is also developmentally regulated during tissue morphogenesis needs further investigation, particularly in mammals. Beyond animals, morphogenesis of plants, fungi and bacteria is also known to be fundamentally dependent on patterned synthesis and degradation of the cell wall, a type of ECM. Thus, genetic control of the matrix appears to be a general principle that shapes all life forms (Diaz-de-la-Loza, 2020).

Cross-modal influence of mechanosensory input on gaze responses to visual motion in Drosophila

Animals typically combine inertial and visual information to stabilize their gaze against confounding self-generated visual motion, and to maintain a level gaze when the body is perturbed by external forces. In vertebrates, an inner ear vestibular system provides information about body rotations and accelerations, but gaze stabilization is less understood in insects, which lack a vestibular organ. In flies, the halteres, reduced hindwings imbued with hundreds of mechanosensory cells, sense inertial forces and provide input to neck motoneurons that control gaze. These neck motoneurons also receive input from the visual system. Head movement responses to visual motion and physical rotations of the body have been measured independently, but how inertial information might influence gaze responses to visual motion has not been fully explored. In this study, the head movement responses to visual motion were measured in intact and haltere-ablated tethered flies to explore the haltere's role in modulating visually-guided head movements in the absence of rotation. It is noted that visually-guided head movements occur only during flight. Although halteres are not necessary for head movements, the amplitude of the response is smaller in haltereless flies at higher speeds of visual motion. This modulation occurred in the absence of rotational body movements, demonstrating that the inertial forces associated with straight tethered flight are important for gaze-control behavior. The cross-modal influence of halteres on the fly's responses to fast visual motion indicates that the haltere's role in gaze stabilization extends beyond its canonical function as a sensor of angular rotations of the thorax (Mureli, 2017). <

Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development in Drosophila

Suppression of wing fate and specification of haltere fate in Drosophila by the homeotic gene Ultrabithorax is a classical example of Hox regulation of serial homology (Lewis, E.B. 1978. Nature 276, 565-570) and has served as a paradigm for understanding homeotic gene function. We have used DNA microarray analyses to identify potential targets of Ultrabithorax function during haltere specification. Expression patterns of 18 validated target genes and functional analyses of a subset of these genes suggest that down-regulation of both anterior-posterior and dorso-ventral signaling is critical for haltere fate specification. This is further confirmed by the observation that combined over-expression of Decapentaplegic and Vestigial is sufficient to override the effect of Ubx and cause dramatic haltere-to-wing transformations. Our results also demonstrate that analysis of the differential development of wing and haltere is a good assay system to identify novel regulators of key signaling pathways (Mohit, 2006). <

Negative regulation of Egfr/Ras pathway by Ultrabithorax during haltere development in Drosophila

In Drosophila, wings and halteres are the dorsal appendages of the second and third thoracic segments, respectively. In the third thoracic segment, homeotic selector gene Ultrabithorax (Ubx) suppresses wing development to mediate haltere development (E.B. Lewis, 1978. A gene complex controlling segmentation in Drosophila. Nature 276, 565-570). Halteres lack stout sensory bristles of the wing margin and veins that reticulate the wing blade. Furthermore, wing and haltere epithelia differ in the size, shape, spacing and number of cuticular hairs. The differential development of wing and haltere, thus, constitutes a good genetic system to study cell fate determination. Here, we report that down-regulation of Egfr/Ras pathway is critical for haltere fate specification: over-expression of positive components of this pathway causes significant haltere-to-wing transformations. RNA in situ, immunohistochemistry, and epistasis genetic experiments suggest that Ubx negatively regulates the expression of the ligand vein as well as the receptor Egf-r to down-regulate the signaling pathway. Electromobility shift assays further suggest that Egf-r is a potential direct target of Ubx. These results and other recent findings suggest that homeotic genes may regulate cell fate determination by directly regulating few steps at the top of the hierarchy of selected signal transduction pathways (Pallavi, 2006).

Modulation of Decapentaplegic gradient during haltere specification in Drosophila

Suppression of wing fate and specification of haltere fate in Drosophila by Ultrabithorax is a classical example of Hox regulation of serial homology. However, the mechanism of Ultrabithorax function in specifying haltere size and shape is not well understood. Here we show that Decapentaplegic signaling, which controls wing growth and shape, is a target of Ultrabithorax function during haltere specification. The Decapentaplegic signaling is down-regulated in haltere discs due to a combination of reduced levels of the Dpp, its trapping at the A/P boundary by increased levels of its receptor Thick-vein and its inability to diffuse in the absence of Dally. Results presented here suggest a complex mechanism adopted by Ultrabithorax to modulate Decapentaplegic signaling. We discuss how this complexity may regulate the final form of the adult haltere in the fly, without compromising features such as cell survival, which is also dependent on Decapentaplegic signaling (Makhijani, 2007).

Modulation of Decapentaplegic gradient during haltere specification in Drosophila

The halteres and wings of Drosophila are homologous thoracic appendages, which share common positional information provided by signaling pathways. The activity in the haltere discs of the Ultrabithorax (Ubx) Hox gene establishes the differences between these structures, their different size being an obvious one. We show here that Ubx regulates the activity of the Decapentaplegic (Dpp) signaling pathway at different levels, and that this regulation is instrumental in establishing the size difference. Ubx downregulates dpp transcription and reduces Dpp diffusion by repressing the expression of master of thick veins and division abnormally delayed and by increasing the levels of thick veins, one of the Dpp receptors. Our results suggest that modulation in Dpp expression and spread accounts, in part, for the different size of halteres and wings (de Navas, 2006). <

Regulation of Wingless and Vestigial expression in wing and haltere discs of Drosophila

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Previously, we have shown that Ubx represses dorsoventral (DV) signaling to specify haltere fate. Here we examine the mechanism of Ubx-mediated downregulation of DV signaling. We show that Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on DV boundary function of Wg, it maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions, at least, at two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3 beta to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations (Prasad, 2003). <

Ultrabithorax and the control of cell morphology in Drosophila halteres

The Drosophila haltere is a much reduced and specialised hind wing, which functions as a balance organ. Ultrabithorax (Ubx) is the sole Hox gene responsible for the differential development of the fore-wing and haltere in Drosophila. Previous work on the downstream effects of Ubx has focused on the control of pattern formation. Here we provide the first detailed description of cell differentiation in the haltere epidermis, and of the developmental processes that distinguish wing and haltere cells. By the end of pupal development, haltere cells are 8-fold smaller in apical surface area than wing cells; they differ in cell outline, and in the size and number of cuticular hairs secreted by each cell. Wing cells secrete only a thin cuticle, and undergo apoptosis within 2 hours of eclosion. Haltere cells continue to secrete cuticle after eclosion. Differences in the shape of wing and haltere cells reflect differences in the architecture of the actin cytoskeleton that become apparent between 24 and 48 hours after puparium formation. We show that Ubx protein is not needed later than 6 hours after puparium formation to specify these differences, though it is required at later stages for the correct development of campaniform sensilla on the haltere. We conclude that, during normal development, Ubx protein expressed before pupation controls a cascade of downstream effects that control changes in cell morphology 24-48 hours later. Ectopic expression of Ubx in the pupal wing, up to 30 hours after puparium formation, can still elicit many aspects of haltere cell morphology. The response of wing cells to Ubx at this time is sensitive to both the duration and level of Ubx exposure (Roch, 2000). <

Negative regulation of dorsoventral signaling by the homeotic gene Ultrabithorax during haltere development in Drosophila

Growth and patterning during Drosophila wing development are mediated by signaling from its dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ultrabithorax (Ubx) to mediate development of a pair of tiny balancing organs, the halteres. Here we show that expression of Ubx in the haltere D/V boundary down-regulates its D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx(-) clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhanced the homeotic transformation of Ubx(-) cells in the capitellum but also caused homeotic transformation of even Ubx(+) cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles of Ubx during maintenance of cell fate and patterning in the halteres (Shashidhara, 1999). <

Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of the Drosophila haltere

Arthropods and vertebrates are constructed of many serially homologous structures whose individual patterns are regulated by Hox genes. The Hox-regulated target genes and developmental pathways that determine the morphological differences between any homologous structures are not known. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology, although no Ubx-regulated genes in the haltere have been identified previously. Here, we show that Ubx represses the expression of the Wingless (Wg) signaling protein and a subset of Wg- and Decapentaplegic-activated genes such as spalt-related, vestigial, Serum Response Factor, and achaete-scute, whose products regulate morphological features that differ between the wing and haltere. In addition, we found that some genes in the same developmental pathway are independently regulated by Ubx. Our results suggest that Ubx, and Hox genes in general, independently and selectively regulate genes that act at many levels of regulatory hierarchies to shape the differential development of serially homologous structures (Weatherbee, 1998).

References

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de Navas, L. F., Garaulet, D. L. and Sanchez-Herrero, E. (2006). The Ultrabithorax Hox gene of Drosophila controls haltere size by regulating the Dpp pathway. Development 133: 4495-4506. PubMed ID: 17050628

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Genes involved in tissue development

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