Interactive Fly, Drosophila



Transcriptional Regulation

Both escargot and snail have similar DNA binding specificity and both are required in the wing imaginal disc. Both escargot and snail are required for for their own expression (autoregulation) and for regulation of expression of each other (Fuse, 1996).

Both escargot and snail are coexpressed in the wing imaginal disc beginning at stage 12 in embryonic development and in the genital disc (Fuse, 1996).

Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains on either side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot (esg), whereas the intermediate column does not. esg expression was examined as a probe to investigate the mechanism of neuroectoderm patterning. The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but is de-repressed when Egfr activity is either elevated or reduced. A neurogenic enhancer of esg was identified, and shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression. It is concluded that decapentaplegic, snail, twist and an activator all act through the distal region to initiate transcription of esg in the neuroectoderm. It is proposed that the combination of opposing gradients of Egfr and its ligand creates a peak of Egfr activity in the intermediate column, where Egfr represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning (Yagi, 1997).

The Drosophila ventral nerve cord derives from a stereotypic population of about 30 neural stem cells (the neuroblasts) per hemineuromere. Previous experiments have provided indications for inductive signals at ventral sites of the neuroectoderm that confer neuroblast identities. Using cell lineage analysis, molecular markers and cell transplantation, it has been shown that Egf receptor (Egfr) signaling plays an instructive role in CNS patterning and exerts differential effects on dorsoventral subpopulations of neuroblasts. The Egfr is capable of cell autonomously specifiying medial and intermediate neuroblast cell fates (referring to neuroblasts arrayed in medial and intermediate columns in the ventral neuroblast proliferative zone). Egfr signalling appears to be most critical for proper development of intermediate neuroblasts and less important for medial neuroblasts. It is not required for the more lateral column of neuroblast lineages or for cells to adopt CNS midline cell fate. Thus, dorsoventral patterning of the CNS involves both Egfr-dependent and -independent regulatory pathways. Furthermore, there appear to be different phases of Egfr activation during neuroectodermal patterning with an early phase independent of midline-derived signals (Udolph, 1998).

The results of isotopic cell transplantation experiments in conjunction with the antibody stainings show that Egfr activity is most crucial for the development of intermediate NBs. Intermediate NB lineages, like NB 4-2 and NB 7-3, are severely affected by the loss of Egfr function. The role of Egfr in specifying intermediate NB cell fates is also reflected by the expression pattern of the escargot (esg) gene (Yagi, 1997). esg, which encodes a zinc finger transcription factor, is expressed in two longitudinal stripes on each side of the wild-type embryo covering the medial and lateral regions of the NE. It is repressed specifically in the intermediate column of the NE. In Egfr mutants, however, esg becomes derepressed and can be found all over the dorsoventral axis of the NE as early as stage 6 (Yagi, 1997). It is interesting to note that, in tissue culture cells, esg interferes with the transcriptional activation function of the proneural Scute protein. Another possible target regulated by the Egfr pathway is the homeobox gene msh (muscle segment homeobox). Activation of the Egfr pathway is necessary to restrict Msh to the dorsal region of the NE, thereby prohibiting the expression within the more ventral regions of the NE. Ectopic expression of msh in the ventral NE severely affects the lineages of NBs derived from that region; thus, the Egfr-mediated repression of msh is crucial to allow normal ventral NE fate development (Udolph, 1998 and references).

Patterning in insect legs is organized along anteroposterior (AP), dorsoventral (DV) and proximodistal (PD) axes. In the case of Drosophila, AP and DV axes of the leg imaginal discs are established along the embryonic AP and DV axes, which are set up based on maternal positional information. The PD axis, however, is zygotically specified by cellular interactions involving the secreted signaling molecules Wingless and Decapentaplegic (Goto, 1999 and references).

PD axis formation in the leg disc first becomes evident when cells expressing either Escargot or Distal-less (Dll) are arranged in a circular pattern. Dll expression defines the central, distal domain. Esg-expressing cells become the proximal domain, which surrounds the distal domain. The Meis family homeodomain protein Homothorax (Hth) is expressed in the proximal domain as well as in the surrounding body wall. Hth regulates nuclear localization of another homeodomain protein, Extradenticle (Exd). Exd is active in the nucleus but inactive in the cytoplasm. The genetic requirements for Dll, Exd and Hth suggest that the distal domain gives rise to the majority of the adult leg including tarsus, tibia, femur and trochanter and that the proximal domain gives rise to the coxa and the ventral thoracic body wall. Initial PD subdivision in the embryonic leg disc becomes elaborated during larval stages by activation of additional genes, such as dachshund (dac), in a circular intermediate domain between the distal and proximal domains. dac is required for specification of the intermediate fate (Goto, 1999 and references).

The leg imaginal disc is also divided into a posterior compartment, which expresses the secreted molecule Hedgehog (Hh) and an anterior compartment, which responds to Hh by expressing Wg and Dpp along the AP compartment boundary. Mutual repression between Wg and Dpp limits Wg expression to the ventral side and Dpp expression to the dorsal side. This spatial restriction of Wg and Dpp expression is essential for DV patterning of the leg. In addition, graded activities of Wg and Dpp are required for the expression of Dll and dac and repression of hth in the distal domain. In the proximal domain, target gene activation by Dpp and Wg is inhibited by Hth and Exd, suggesting that the distal and proximal domains have distinct characters to respond to Dpp and Wg (Goto, 1999 and references).

Based on the above observations, it was proposed that the circular patterns of gene expression along the PD axis in the distal domain are organized by the gradient of the combined activity of Dpp and Wg. In the central, distal region, where combined activity of Dpp and Wg would be high, Dll is activated and dac is repressed. An intermediate level of Wg and Dpp activities would allow dac expression in the intermediate domain. Ectopic expression of Dll in the dorsal-proximal region induces wg, which is thought to interact with dpp to specify a new PD axis. These results suggest that the combination of Wg and Dpp constitute a 'distalizing' signal for the PD axis (Goto, 1999 and references).

Although these results suggest that the combination of Wg and Dpp activities centered at the distal tip is essential for PD patterning, it is not known whether Wg and Dpp are sufficient to account for all aspects of PD positional information. In fact, the grafting and regeneration experiments using larval cockroach legs suggest that the reciprocal communication between distal and proximal parts of a leg segment promotes regeneration of the intermediate part. Thus it can be speculated that a proximal to distal cell communication may also be used in PD patterning of the leg during development. Esg is expressed in the proximal domain throughout leg development. Ectopic expression of Esg and its activator Hth in the distal domain induces the intermediate fate in surrounding cells by inducing dac expression. Esg and Hth-expressing cells in the distal domain undergo a change in their adhesive property to sort out from surrounding cells. The proximal to distal inductive communication is unexpected from the model based on the graded activity of Dpp and Wg. Thus an intercalary mechanism that elaborates the PD axis pattern of the leg has been proposed. During the transition from the second to third instar, dac expression in the intermediate domain is induced by (1) a combination of a signal from proximal cells, and (2) Wg and Dpp signaling from the AP compartment boundary. The range of each signaling limits dac expression to the intermediate domain. The proximal to distal signaling dependent on Esg and Hth may provide a molecular basis for the intercalary expression of dac (Goto, 1999 and references).

Thus, it has not been clear whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression in the Drosophila leg. A proximal gene escargot and its activator homothorax have been shown to regulate proximodistal patterning in the distal domain. Clones of cells expressing either escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient the planar cell polarity of those cells. escargot and homothorax-expressing cells also sort out from other cells in the distal domain. Thus, inductive cell communication between the proximodistal domains is the cellular basis for an intercalary mechanism, involving expression of dachshund, during proximodistal axis patterning of the limb (Goto, 1999).

The first sign of proximodistal axis formation in the leg imaginal disc was a circular arrangement of cells expressing either Esg or Dll during embryogenesis. As the disc grows in size and evolves circular folds that separated tarsus, tibia, femur, trochanter and coxa, the pattern of esg expression is maintained. At the late stage of the third instar, more Esg protein is detected in the proximal region corresponding to the coxa and trochanter. The distal most part of the esg-expressing domain partially overlaps with the Dac-expressing domain in the trochanter. The esg expression in the overlapping domain is weaker than that in the more proximal domain, where only esg is detected. The domain of Esg expression appears to overlap with the proximal domain defined by expression of homothorax and teashirt, and nuclear localization of Extradenticle (Goto, 1999 and references).

Dll induces distal leg development when expressed ectopically in the proximal domain. To determine if any of the proximal genes have an organizing activity analogous to that of Dll, esg was induced ectopically using the flip-out technique. In the adult, Esg-positive clones marked by GFP are found as vesicles inside the leg cuticle and are often associated with malformation. In the region proximal to the clones, the bristles and epidermal hairs, which normally point distally, are often reversed. These bristles and hairs are genetically wild type, suggesting that the polarizing activity of Esg is non-cell-autonomous (Goto, 1999).

In the third instar leg disc, dac is expressed in a partially overlapping manner with the expression of Dll and esg in an intermediate ring that corresponds to the proximal tarsus, tibia, femur and trochanter. When esg expression is induced during the second instar, clones in the distal tarsal region show compact morphology; and many of them are associated with ectopic dac expression in cells within and surrounding the clone. The ectopic dac expression results in a local reversion of the proximal-distal order of the gene expression, which prefigures the change in the cell polarity in the adult leg. The esg-positive clones in the coxa spread normally and do not show induced dac. The non-cell-autonomy of the Esg function could be due to a modulation of known secreted molecules controlling anteroposterior and dorsoventral patterning. However, the expression patterns of the Hh target genes wg and dpp, and optomotor-blind (omb, 1996), a target gene of Dpp, are unaffected by misexpression of Esg (Goto, 1999).

esgG66B null mutant clones were used to assess the requirement of Esg for dac expression. esgG66B is a derivative of an enhancer trap and lacks the coding region of esg but retains the lacZ gene that reproduces the expression pattern of esg. esg mutant cells are marked by the loss of Myc antigen or by the high expression of beta-gal produced from the two copies of the lacZ gene. dac expression is frequently lost in clones induced at the late second instar larval stage. The partial loss of dac expression in large clones may have been due to a non-cell-autonomous rescue by esg+ cells next to the clones. The clones are sometimes associated with ectopic fold formation. Taken together with the gain-of-function analysis, these data suggest that Esg is necessary and sufficient for dac induction (Goto, 1999).

Proximal cell identity is, at least in part, controlled by the homeodomain protein Hth, which regulates nuclear localization of Exd. When expressed ectopically in the tarsal region, Hth causes non-cell-autonomous induction of dac expression and reversal of bristle and cell polarity. These phenotypes are very similar to those caused by Esg. Unlike esg-expressing clones, which secrete a smooth cuticle, hth-expressing clones in the distal part of the leg sometimes form thick socketed bristles without bracts, a characteristic of the bristles in the proximal part of the leg. Hth strongly activates a reporter gene under the control of the esg enhancer in the distal domain, but it does so weakly, if at all, in the proximal domain. This effect is cell-autonomous, suggesting that Hth may directly regulate transcription of esg. In contrast, neither a loss nor a gain of esg expression affects the activity of Hth/Exd as assessed by the expression of Hth and nuclear localization of Exd, nor is esg expression affected by the expression of another proximal gene, teashirt. These results suggest that Esg acts downstream of Hth/Exd to regulate proximodistal patterning (Goto, 1999).

The esg- or hth-expressing clones in the distal region are round in shape with smooth borders and often invaginated basally to form vesicles in the adult legs and in the larval discs. In contrast, control clones expressing non-functional esg, which lacks the zinc-finger domain, and esg-expressing clones located in the coxa and trochanter, have ragged borders. The epithelial-type homophilic cell adhesion molecule DE-cadherin is expressed throughout the leg discs and its apical localization is maintained normally in esg-expressing clones, suggesting that these cells keep their epithelial character. These results of ectopic expression studies, together with the loss of function studies on hth, indicate that Hth and Esg regulate a cell surface property that distinguishes the proximal and distal domains. It is suggested that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb (Goto, 1999).

Tramtrack regulates different morphogenetic events during Drosophila tracheal development

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, a detailed analysis of Ttk function was undertaken. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, it was found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, Ttk was defined as the first identified regulator of intracellular lumen formation and; it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development (Araújo, 2007).

As with the transcription factors Trh and Vvl, which are involved in orchestrating early events of tracheal development, Ttk plays a role in orchestrating several late tracheal events. Ttk69 has been found to act mostly as a repressor. This study identified Ttk targets that appear to be negatively regulated (such as mummy (mmy), encodes a UDP-N-acetylglucosamine pyrophosphorylase enzyme required for the synthesis of the building blocks of chitin, and escargot (esg) whereas others appear to be positively regulated (such as polychaetoid (pyd) and branchless (bnl). In this latter case, Ttk might be converted into a positive regulator, as already described during photoreceptor development (Araújo, 2007).

This study identified multiple tracheal requirements for Ttk. Interestingly, most of them depend on Ttk regulating events downstream of cell fate specification, at the level of cellular responses. Additionally, a few other requirements depend on cell fate specification, as has been described for most other functions of Ttk in other developmental situations. For instance, Ttk regulates fusion cell specification by acting as a target and mediator of Notch, as occurs during sensory organ development and oogenesis. Such regulation of Ttk by N might be post-transcriptional, as occurs during sensory organ development. Remarkably, it was found that, although Ttk is sufficient to repress esg expression in fusion cells, it might not be the only esg- and fusion fate-repressor, because absence of Ttk does not increase the number of Esg-positive cells, as does downregulating N. Other N targets might be redundant with Ttk, and such redundancy could reinforce N-mediated repression of fusion fate in positions in which inductive signals (such as Bnl, Dpp and Wg) are very high, particularly near the branch tips (Araújo, 2007).

Cell rearrangements during development are common to most animals and ensure proper morphogenesis. During tracheal development, many branches grow and extend by cell intercalation. Several cellular and genetic aspects of tracheal intercalation have been well described. However, targets of Sal (which inhibits intercalation) are currently unknown (Araújo, 2007).

This study identified Ttk as a new and positive regulator of intercalation. Ttk is involved in cell junction modulation by transcriptionally regulating pyd, the only junctional protein shown, so far, to affect intercalation. In fact, modulation of AJs has been proposed to play a role during intercalation. However, Pyd cannot be the only Ttk effector of intercalation, because the pyd mutant phenotype is much weaker than that of ttk mutants. Accordingly, it was found that, in ttk mutants, cells in branches that usually intercalate remain paired and cuboidal, and appear unable to change shape and elongate. Although other explanations could account for the impaired intercalation detected in ttk mutants, it is proposed that inefficient cell shape changes represent the main cause, and might prevent the proper accomplishment of several events, such as the sliding of cells, formation of a first autocellular contact and zipping up, thereby blocking intercalation. Hence, it is proposed that cell shape changes, particularly cell elongation, are an obligate requisite for different steps of intercalation. Other targets of Ttk might presumably be regulators or components of the cytoskeleton involved in cell shape changes. It is relevant to point out here that Ttk has also been proposed to regulate morphogenetic changes required for dorsal appendage elongation (Araújo, 2007).

How does Ttk relate to the known genetic circuit (Sal-dependent) involved in intercalation? Being a transcription factor, Ttk initially appeared as an excellent candidate to participate in this genetic network by regulating sal and/or kni expression. However, both these genes to be normally expressed in ttk mutants, and several differences were detected in the intercalation phenotype of ttk loss versus sal upregulation. For instance, although both situations block intercalation, cells expressing sal, unlike those lacking ttk, are still able to undergo a certain change in shape, from cuboidal to elongated. Therefore, the results fit a model in which Ttk acts in a different and parallel pathway to Sal during intercalation. Consistent with this model, it was found that Ttk is not sufficient to promote intercalation on its own, because its overexpression cannot overcome the inhibition of intercalation imposed by Sal in the DT. Finally, genetic interactions also favour this model, because it was found that: (1) ttk overexpression did not rescue lack of intercalation produced by sal overexpression (even though it rescued the intercalation defects of ttk mutants), and (2) absence of sal (by means of the constitutive activation of the Dpp pathway) does not overcome the intercalation defects of ttk mutants. Therefore, it is proposed that Ttk promotes intercalation by endorsing changes in cell shape, but absence of Sal is still required to allow other aspects of intercalation to occur (Araújo, 2007).

Tube size regulation is essential for functionality. It was found that Ttk is involved in such regulation. Tube expansion and extension relies on a luminal chitin filament that assembles transiently in the tracheal tubes. The metabolic pathway that leads to chitin synthesis involves several enzymes, among which are Mmy and krotzkopf verkehrt (Kkv, a Chitin synthase). In addition, other proteins are known to participate in the proper assembly and/or modification of the chitin filament, such as Knk, Rtv, Verm and Serp. SJs are also required to regulate tube size and it was proposed that they exert this activity, at least partly, via the control of the apical secretion of chitin modifiers. The current results revealed that ttk acts as a key gene in tube size control, playing at least two roles: it regulates chitin filament synthesis and septate junction (SJ) activity (Araújo, 2007).

SJ regulation by Ttk appears functional rather than structural: mild defects were detected in the accumulation of only some SJ markers and there was a loss of the transepithelial diffusion barrier, whereas accumulation of other markers and SJ localisation remained apparently unaffected. It is speculated that Ttk transcriptionally controls one or several SJ components that contribute to maintain the paracellular barrier and to control a specialised apical secretory pathway. As a result, chitin binding proteins such as Verm or Serp are not properly secreted (Araújo, 2007).

It was also found that mmy is transcriptionally regulated by Ttk. mmy tracheal expression positively depends on a mid-embryonic peak of the insect hormone 20-hydroxyecdysone. Therefore, it is proposed that Ttk and ecdysone exert opposing effects on chitin synthesis. Excess of mmy mRNA results in the abnormal deposition of the chitin filament, as occurs in ttk mutants. Defects in chitin deposition might lead to the irregular organisation of taenidia and the faint larval cuticle observed in ttk mutants. Strikingly, Ttk is also required for normal chorion production, which represents another specialised secreted layer (Araújo, 2007).

ttk mutants are defective in the formation of terminal and fusion branches. These defects are due, in part, to non-autonomous, secondary and/or pleiotropic effects of ttk. For instance, ttk mutants exhibit a dorsal closure defect, which prevents the approach and fusion of contralateral dorsal branches. Additionally, terminal and fusion branches depend on correct cell type specification, which did not reliably occur in ttk mutants. For instance, DSRF (Blistered) was missing in some presumptive terminal cells of ttk mutants, impairing terminal branch formation. These tracheal cell identity specification defects might be related to non-autonomous requirements of ttk. For instance, DSRF is not properly expressed in ttk mutants because of an abnormal expression of its regulator, Bnl (Araújo, 2007).

It is important to note that, in spite of these non-autonomous and cell fate specification defects, two pieces of evidence indicate that ttk also plays a specific and autonomous role in the formation of terminal and fusion tubes. First, markers for fusion and terminal cell specification were expressed in many tracheal cells of ttk mutants, but yet most of these cells did not form terminal or fusion branches. Second, only the tracheal expression of ttk in ttk mutants (but not the constitutive activation of the btl pathway, which regulates the terminal and fusion identity) was able to restore the formation of terminal branches (Araújo, 2007).

A common feature of terminal and fusion branches is that they both display intracellular lumina that lack detectable junctions. The cellular events that precede the formation of fusion and terminal branches differ, but the mechanisms by which their intracellular lumina form has been proposed to be comparable. It was found that, in ttk mutants, terminal and fusion cells engage in the correct cellular changes before intracellular lumen formation. However, neither of these two cell types finalised the cellular events leading to tube formation. It has been proposed that the lumen of terminal and fusion branches forms by the coalescence of intracellular vesicles that use a 'finger' tip provided by the neighbouring stalk cell as a nucleation point. Interestingly, it was found that vesicles containing luminal material are less abundant in ttk mutants. These observations suggest a new role for Ttk in the formation of intracellular lumina in distinct cell types. Intracellular lumen formation also occurs in other branched tubular structures, such as in vertebrate endothelial cells and in the excretory cell of Caenorhabditis elegans, presumably by the coalescence of vesicles. Importantly, a crucial role for vesicle formation and their fusion during intracellular tube formation has been demonstrated (Araújo, 2007).

ttk is the first gene described to be involved in intracellular lumen formation during tracheal development. Possible targets of Ttk might be genes related to the apical surface and the underlying cytoskeleton, because several of these genes are involved in C. elegans excretory canal formation. Additionally, genes involved in intracellular vesicle trafficking might also be good candidates. In this respect, several abnormalities have been detected in ttk mutants that might reflect defects in vesicle trafficking (Araújo, 2007).

A new Prospero and microRNA-279 pathway restricts CO2 receptor neuron formation

CO2 sensation represents an interesting example of nervous system and behavioral evolutionary divergence. The underlying molecular mechanisms, however, are not understood. Loss of microRNA-279 in Drosophila leads to the formation of a CO2 sensory system partly similar to the one of mosquitoes. This study shows that a novel allele of the pleiotropic transcription factor Prospero resembles the miR-279 phenotype. A combination of genetics and in vitro and in vivo analysis was used to demonstrate that Pros participates in the regulation of miR-279 expression, and that reexpression of miR-279 rescues the pros CO2 neuron phenotype. Common target molecules of miR-279 and Pros were identified in bioinformatics analysis, and it was shown that overexpression of the transcription factors Nerfin-1 and Escargot (Esg) is sufficient to induce formation of CO2 neurons on maxillary palps. These results suggest that Prospero restricts CO2 neuron formation indirectly via miR-279 and directly by repressing the shared target molecules, Nerfin-1 and Esg, during olfactory system development. Given the important role of Pros in differentiation of the nervous system, it is anticipated that miR-mediated signal tuning represents a powerful method for olfactory sensory system diversification during evolution (Hartl, 2011).

Targets of Activity

ESG binds as a monomer with high affinity to a specific sequence E2-box consisting of G/ACAGGTG. This is the consensus binding sequence for the basic HLH family of transcription factors. A heterodimer composed of proneural proteins Scute and Daughterless activates transcription from promoters containing E2-boxes. The ESG protein strongly inhibits this activation by competitively binding to E2-boxes (Fuse, 1994).

escargot and snail are both required for the expression of vestigial in the wing disc (Fuse, 1996).

In escargot mutants, diploid imaginal cells arrested in G2 lose Cyclin A and enter an endocycle, the duplication of DNA without subsequent mitosis, creating a state of polyploidy. Mutants in Cdc2, the cyclin dependent kinase (see cell cycle), give similar results. Therefore Escargot acts to maintain high levels of both Cdc2 and Cyclin A in the active form that inhibits entry into S (the DNA synthetic phase) (Hayashi, 1996).

During tracheal development, the tip cells, located at the end of each branch that is going to fuse, extend filopodia to search for targets; later they change their cell shape. The cell adhesion molecule E-cadherin accumulates at the site is essential for the fusion of the tip cell with its target. DE-cadherin expression in tip cells of a subset of branches is dependent on escargot, a zinc finger gene expressed in all tip cells. Such escargot mutant tip cells failed to adhere to one another and continue to search for alternative targets by extending long filopodia. These cells also develop an abnormal cuticle in their blind-ended structure and their terminus floats freely in the body. escargot positively regulates transcription of the DE-cadherin gene, shotgun. Overexpression of DE-cadherin rescues the defect in one of the fusion points in escargot mutants, demonstrating an essential role of DE-cadherin in target recognition and identifying escargot as a key regulator of cell adhesion and motility in tracheal morphogenesis (Tanaka-Matakatsu, 1996). headcase expression in the trachea is regulated by Escargot. Branch fusion in the Drosophila trachea is a complex process involving two specialized cells at the tip of each fusing branch; they undergo a series of morphological changes to generate a bicellular anastomosis and connect the two tracheal branches. In esg mutants, the fusion cells of the dorsal branches fail to undergo the fusion process and express later fusion markers; instead, they express terminal markers and ramify into tracheoles during larval life. Ectopic expression of esg in all tracheal cells is sufficient to induce ectopic branch fusions and suppress terminal branching and expression of terminal genes. hdc expression is absent in the fusion cells of esg mutants. esg was misexpressed in all tracheal cells using the UAS-GAL4 system. This was found to be sufficient to induce hdc expression in one to two additional tracheal cells at the tips of the dorsal and lateral trunk branches. Thus, esg is not only necessary for hdc expression in the fusion cells, but it is also sufficient to induce hdc expression in the cells of the pantip group of the dorsal and lateral branches. Because hdc acts nonautonomously as a branching inhibitor, the above results predict that in esg mutants not only the fusion cell, but also one of the stalk cells may acquire the terminal cell fate. The extra terminal branching phenotype of esg strong loss-of-function mutants was analyzed. An additional cell extends a unicellular branch and expresses the terminal marker blistered (coding for the Drosophila Serum response factor) in ~16% of the 176 dorsal branches analyzed. These results show that esg can suppress terminal cell fate in the fusion cells by repressing terminal genes like blistered, and in neighboring cells by the activation of hdc and perhaps other fusion genes that act non-cell autonomously as branching inhibitors (Steneberg, 1998).

dysfusion functions downstream of escargot

The development of the mature insect trachea requires a complex series of cellular events, including tracheal cell specification, cell migration, tubule branching, and tubule fusion. The Drosophila dysfusion gene encodes a basic helix-loop-helix (bHLH)-PAS protein conserved between Caenorhabditis elegans, insects, and humans; dysfusion controls tracheal fusion events. The Dysfusion protein functions as a heterodimer with the Tango bHLH-PAS protein in vivo to form a putative DNA-binding complex. The dysfusion gene is expressed in a variety of embryonic cell types, including tracheal-fusion, leading-edge, foregut atrium cells, nervous system, hindgut, and anal pad cells. RNAi experiments indicate that dysfusion is required for dorsal branch, lateral trunk, and ganglionic branch fusion but not for fusion of the dorsal trunk. The escargot gene, which is also expressed in fusion cells and is required for tracheal fusion, precedes dysfusion expression. Analysis of escargot mutants indicates a complex pattern of dysfusion regulation, such that dysfusion expression is dependent on escargot in the dorsal and ganglionic branches but not the dorsal trunk. Early in tracheal development, the Trachealess bHLH-PAS protein is present at uniformly high levels in all tracheal cells, but when the levels of Dysfusion rise in wild-type fusion cells, the levels of Trachealess in fusion cells decline. The downregulation of Trachealess is dependent on dysfusion function. These results suggest the possibility that competitive interactions between basic helix-loop-helix-PAS proteins (Dysfusion, Trachealess, and possibly Similar) may be important for the proper development of the trachea (Jiang, 2003).

dys is expressed in tracheal fusion cells, and no other tracheal cells. This was shown by coexpression of dys with esg, a gene that is expressed in fusion cells and regulates tracheal fusion. dys-RNAi experiments were carried out to examine whether dys is involved in tracheal fusion. The results demonstrate that dys is required for fusion of the dorsal branch, lateral trunk, and ganglionic branch but not of the dorsal trunk. This phenotype is similar to the esg mutant phenotype that also affects the dorsal branch, lateral trunk, and ganglionic branch but not the dorsal trunk. Although branches differ in the details of the fusion process, tracheal fusion generally requires migration, recognition, and adhesion of fusion cells. dys-RNAi embryos show relatively normal tracheal branches and migration. The occurrence of a single esg-lacZ cell in each dys-RNAi branch indicates that esg-positive tracheal fusion cells are present, and thus survival and gross cell fate is not controlled by dys. It is possible that dys controls aspects of fusion cell recognition, cell adhesion, or inhibition of nonfusion tracheal functions, such as branching (Jiang, 2003)

Since dys, as well as esg, is expressed in dorsal trunk fusion cells, why is dorsal trunk fusion apparently unaffected in dys-RNAi-injected embryos? It is not likely due to incomplete expressivity of the dys-RNAi, since Dys protein was not detected in dys-RNAi-injected embryos, including dorsal trunk fusion cells, and ~100% of lateral trunk, dorsal branch, and ganglionic branch branches in dys-dsRNA-injected embryos failed to fuse. There are a number of differences between the dorsal trunk and the other branches that could contribute to differences in fusion behavior. The larger diameter dorsal trunk has multiple cells comprising its circumference, unlike most of the other branches, which are thinner and have a single cell comprising the circumference. Dorsal trunk branches are in close proximity to their fusion partner and lack the filopodial extensions that help guide the other branches to their targets. The dorsal trunk also utilizes a mesodermal guidepost cell that mediates fusion. Similar guidepost cells have not been described for the other branches. Finally, breathless RNA levels begin to decline by stage 12 in the dorsal trunk due to spalt repression, which may eliminate the potential need to reduce breathless levels by decreasing Trh levels. These and other possible differences suggest why dys and esg may have different functions in different branches (Jiang, 2003)

The esg gene is required in dorsal branch and ganglionic branch tracheal fusion cells for expression of several genes, including shotgun (DE-cadherin) and three late-expressing fusion cell genes (fusion-4 to fusion-6), as well as repression of terminal branching genes (DSRF and terminal-1). Expression of two early-expressing fusion cell genes (fusion-2 and fusion-3) are not dependent on esg. dys expression is also dependent on esg, in keeping with the role of esg in regulating early-expressing fusion cell gene expression. As with other genes expressed in fusion cells, dys expression is not dependent on esg in dorsal trunk cells. This implies that the ability of esg to activate transcription is fusion cell dependent and is due to the presence of different coregulatory proteins or modifier proteins in the different branches (Jiang, 2003)

escargot: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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