InteractiveFly: GeneBrief

Dysfusion: Biological Overview | Regulation | Developmental Biology | Effects of RNAi | References

BIOLOGICAL OVERVIEW

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).

The insect tracheal system consists of an intricately branched system of tubules that provide oxygen throughout the animal. The formation of the trachea consists of a series of developmental events, and its analysis provides an excellent model system for studying the morphogenesis of other branched structures, such as the vertebrate lung airways, circulatory system, kidney ducts, and excretory epithelia. The trachea is derived from an array of segmentally repeated clusters of precursor cells. After the tracheal precursor cells divide and invaginate, they extend branches, and the branches from neighboring segments fuse to form the mature tracheal tree. The fusion process is mediated by a distinct fusion cell residing on each branch. Branching and fusion are complex cellular processes and pose a number of developmental questions. How are fusion cells specified during tracheal development? What are the short-range and long-range factors that guide tracheal branches to their fusion partners? What is the nature of the adhesive and contact-guidance interactions that mediate fusion and allow the formation of adherens junctions that seal intercellular junctions? How is the cytoskeleton rearranged to allow the tracheal lumen to extend throughout the branch (Jiang, 2003 and references therein)?

The tracheal primordia extend branches in six directions under the guidance of the branchless gene. All of these branches except the visceral branch will fuse with tracheal branches derived from other primordia. The dorsal trunk is formed by fusion of anterior and posterior branches from adjacent segments, as is the lateral trunk, which is formed by fusion of lateral trunk anterior and posterior branches. The dorsal branches travel over the dorsal side of the embryo and fuse along the dorsal midline to their partner from the identical hemisegment. The ganglionic branches migrate ventrally and join at the ventral midline, although only the three anteriormost branches fuse. There is a single fusion cell for each branch, and the fusion cells are characterized by patterns of gene expression distinct from other tracheal cells. The Escargot (Esg) zinc finger transcription factor is prominently expressed during tracheal development in fusion cells and no other tracheal cell. esg mutants show fusion cell defects in the lateral trunk, dorsal branch, and ganglionic branch, but dorsal trunk fusion is relatively normal. Examination of esg mutants indicates that expression of several, but not all, fusion cell-specific genes and markers is absent in the dorsal branch and the ganglionic branch, and there is an excess of branching and gene expression associated with branching. In addition, esg is required for DE-cadherin expression and the ability of fusion cells to form adhesive contacts and adherens junctions. The loss of esg in lateral trunk is the most extreme, resulting in death of the fusion cells. Important issues regarding the tracheal function of esg deal with the identity of genes regulated by esg and why esg is required for tracheal fusion in some branches but not others (Jiang, 2003 and referencest therein).

Three proteins that function prominently in tracheal development are the Trachealess (Trh), Similar (Sima), and Tango (Tgo) basic helix-loop-helix (bHLH)-PAS proteins. Trh and Tgo form a heterodimer that controls transcription and initial formation of the tracheal primordia along with the Drifter (Dfr) POU-homeobox coactivator. Sima and Tgo form a protein dimer that controls the transcriptional response to hypoxia. Since low cellular oxygen conditions induce additional tracheal branching, Sima may autonomously or nonautonomously be required for terminal tracheal branching. Tgo is found in all embryonic cells. In the absence of a bHLH-PAS partner protein, Tgo is found in the cytoplasm, but in the presence of a partner protein, they dimerize, translocate into the nucleus, bind DNA, and activate transcription. Since there may exist multiple bHLH-PAS partners of Tgo in the same cell, it has been proposed that the function and levels of bHLH-PAS proteins may be regulated by competitive interactions. In part for this reason, it is important to identify all Drosophila bHLH-PAS proteins and determine where they are expressed (Jiang, 2003 and references therein).

A novel bHLH-PAS gene, dysfusion, is expressed in all tracheal fusion cells, as well as the epidermal leading edge cells and several other cell types. Tgo accumulates in nuclei of dys-expressing cells, suggesting that it is a partner of Dys in vivo. dys-RNAi experiments reveal tracheal fusion defects in the lateral trunk, dorsal branch, and ganglionic branch but not in the dorsal trunk. The esg gene is expressed in all fusion cells before dys, and esg expression is normal in dys RNAi-injected embryos. However, esg mutant embryos show an absence of dys expression in tracheal fusion cells in most branches, but not the dorsal trunk, further indicating branch-specific function of esg. The appearance of Dys in tracheal fusion cells coincides with a steep drop in Trh levels in fusion cells, and this reduction is dys dependent. This indicates that one function of dys in tracheal fusion cell development is to downregulate Trh protein levels. This provides the first in vivo evidence that bHLH-PAS proteins regulate levels of other bHLH-PAS proteins during development and possibly influence cell fate and morphogenetic decisions (Jiang, 2003).

Drosophila Dys belongs to a novel conserved subfamily of bHLH-PAS proteins that includes C. elegans C15C8.2 and H-NXF. Dys also likely belongs to an extended family of Drosophila bHLH-PAS proteins, including Sim, Sima, Ss, and Trh, that dimerize with Tgo. The protein structure of Dys is conventional for bHLH-PAS proteins. The bHLH domain is near the N terminus of the protein and is followed by the PAS-1 and PAS-2 domains. One unusual feature compared to other bHLH-PAS proteins is the relatively long (152 aa) region N-terminal to the bHLH domain. This region has a large number of glutamine residues and may act as a transcriptional activation domain. The C-terminal residues after the PAS-2 domain are unconserved with C15C8.2, H-NXF, or any other protein but have histidine-rich, proline-rich, and glutamine-rich regions. These residues may also be transcriptional activation domains. The structure of the Dys protein suggests a DNA-binding transcriptional activator, but this needs to be tested biochemically (Jiang, 2003).

Drosophila dys is expressed in a variety of embryonic cell types, including tracheal fusion, leading edge, foregut atrium, brain or stomagastric nervous system, hindgut, and anal pad cells. Initial expression is observed during mid embryogenesis at stage 12. The function of dys in these cell types is unknown, with the exception of the tracheal fusion cells, in which dys plays a developmental role. The dys-RNAi results show that dys is an essential gene. However, dys-dsRNA-injected embryos do not die as embryos, but as second- and third-instar larvae. Thus, the role of dys, as assayed by RNAi, in the various embryonic cell types is not dramatic enough to cause embryonic lethality. Mutations in esg show fusion defects in the same tracheal branches as dys and are also lethal to larvae. The tracheal fusion defects and resulting putative respiratory deficiencies may be the cause of the larval lethality, since other observations have shown that animals with defective tracheae survive until late larval periods. However, both genes are expressed elsewhere, and defects in the other cell types may contribute to lethality (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)

The trh gene is required for initiation of tracheal formation. trh expression is maintained throughout embryonic development in most tracheal cells, and this continued expression is due to autoregulation. However, the role of trh beyond its role in initiating tracheal formation is not well understood. Trh protein levels fall specifically in all classes of tracheal fusion cells coincident with the rise in Dys levels. The nuclear levels of Tgo, the partner for both Dys and Trh, remain constant in fusion cells. The biological significance of the reduction in Trh remains to be investigated, but it is possible that fusion requires a reduction in Trh. One possibility is that Trh:Tgo is required for the expression or function of the breathless (btl) tyrosine kinase receptor that guides growing tracheal branches and that btl function must be inhibited in fusion cells. Potentially, the only function of dys is to reduce Trh levels (Jiang, 2003)

There are multiple mechanisms in which Dys could regulate Trh levels. These mechanisms include (1) competition between Dys and Trh for dimerization with Tgo, (2) competitive Dys:Tgo binding to Trh:Tgo autoregulatory binding sites in the trh gene, (3) activation of genes by Dys that encode proteins influencing trh RNA or protein stability, and (4) inhibition of protein kinase B that is required for Trh nuclear transport. Conceptually, the first model is the simplest and most attractive. Trh autoregulates its own expression, and reduction in Trh:Tgo complexes by competition for Tgo by Dys would lead to a reduction in trh RNA and protein. In the second model, Dys:Tgo would function as a transcriptional repressor and extinguish trh RNA synthesis by binding Trh:Tgo autoregulatory sequences within the trh gene (Jiang, 2003)

Evidence for the possible roles of these mechanisms has emerged from studies on vertebrate and Drosophila bHLH-PAS proteins. In one study, it was demonstrated that HIF-1alpha outcompetes the Aryl hydrocarbon receptor (Ahr) bHLH-PAS protein for their common dimerization partner, Arnt, which is the vertebrate Tgo ortholog. In another study, Sim2 was shown to compete with HIF-1alpha for Arnt and partially block expression of a HIF-1alpha:Arnt responsive reporter gene. Sim2 can repress transcription and, by binding to HIF-1alpha:Arnt recognition sites on the reporter gene, Sim2 reduced reporter expression. The third model in which the presence of Dys reduces protein levels by activating the transcription of repressive or inhibitory factors is analogous to how Sim:Tgo represses the expression of genes in the central nervous system midline cells by activating transcription of a repressor. One additional issue is whether Dys reduces the levels of other Drosophila bHLH-PAS proteins in addition to Trh. One possibility is Sim. Both Sim and Dys are expressed in anal pad cells, and sim mutants have anal pad defects (Jiang, 2003 and references therein)

Drosophila has four bHLH-PAS proteins that dimerize with Tgo: Sim, Sima, Ss, and Trh. Dys is also likely to dimerize with Tgo. One particularly interesting observation is that Drosophila Dys, Trh, and Sima are all involved in aspects of tracheal development. Mammals have closely related members of all of these proteins. The mammalian proteins dimerize with either Arnt or the closely related Arnt2. C. elegans has four bHLH-PAS partners for AHA-1, the worm Tgo/Arnt ortholog. These partners include (1) C15C8.2, which is related to Dys and mammalian NXF; (2) AHR-1, which is related to Drosophila Ss and vertebrate Ahr; (3) HIF-1, which is related to Drosophila Sima and vertebrate HIF-1alpha, and (4) T01D3.2, which is related to Sim and Trh. Since orthologs of Dys, Ss, Sima, and Sim/Trh are found in vertebrates, insects, and nematodes, these proteins had already diverged in the common ancestor of these species. The C. elegans C15C8.2 gene is expressed in the pharynx, a feeding organ. The mammalian dys gene, NXF, has been detected only in the brain. The evolutionary conservation of the Dys subfamily of proteins suggests a functional relationship, although the sites of expression in Drosophila and the other organisms, as studied to date, are diverse. One possibility based on the tracheal fusion phenotypes is that Dys regulates aspects of cell recognition or cell adhesion events. It will be interesting to determine in future studies what biochemical, developmental, or physiological features of the Dys proteins are conserved, as well as the evolutionary origins regarding the tracheal functions of Dys, Sima, and Trh (Jiang, 2003)

REGULATION

Transcriptional Regulation

Analysis of esg mutants has revealed that expression of fusion cell genes is affected in some branches (dorsal branch and ganglionic branch) but not others (dorsal trunk). In the dorsal branch and the ganglionic branch, several fusion cell genes require esg function, but others do not. Expression of dys follows esg expression in all tracheal branches, raising the possibility that its expression requires esg function. Multiple esg mutants were analyzed, including the deletion strain esg^G66. Analysis of esg^G66 mutant embryos showed a complete absence of dorsal branch and ganglionic branch dys expression in esg mutant fusion cells. The lateral trunk also had no Dys-positive cells but had some esg-lacZ-positive cells, a result consistent with an earlier report that lateral trunk fusion cells die in esg mutant embryos. Thus, it is difficult to assess whether esg controls dys expression in the lateral trunk. In contrast, all dorsal trunk fusion cells were Dys positive. The P-element mutant, esg⁰⁵⁷³⁰, showed a less severe phenotype. The dorsal branch fusion cells were all Dys negative, and dorsal trunk fusion cells were all Dys positive, as with the esg^G66 mutant embryos. However, there were occasionally esg-lacZ-positive cells present in the defective, unfused lateral trunk branches; some were Dys positive, and some were Dys negative. This was also observed in esg^G66, but only rarely. The ganglionic fusion cells of esg⁰⁵⁷³⁰ were also occasionally Dys positive. In summary, dys fusion cell expression requires esg in fusion cells of the dorsal branch and ganglionic branch, and possibly the lateral trunk, but not in the dorsal trunk (Jiang, 2003).

Targets of Activity

trh is expressed in all tracheal cells at the beginning of tracheal development and acts as a master regulator of tracheal development. Trh protein levels remain high during tracheal development in most tracheal cells. Since Tgo is the likely dimerization partner for both Dys and Trh, and these proteins could, in principle, compete for Tgo in the same cells, whether Trh levels remained constant in tracheal fusion cells in the presence of high levels of Dys was examined. Heterozygous esg⁰⁵⁷³⁰ embryos expressing esg-lacZ to mark fusion cells were stained for Dys, ß-Gal, and Trh. Before dys is expressed, Trh is at high levels in fusion cells at equivalent levels to adjacent tracheal cells. As Dys protein accumulates in the fusion cells, Trh levels decline and remain low through the end of tracheal development. This occurs in fusion cells in the dorsal trunk, lateral trunk, dorsal branch, and ganglionic branch. Examination of Dys-positive tracheal fusion cells indicates that 93% show a marked reduction in Trh levels compared to adjacent tracheal cells (Jiang, 2003).

The requirement of dys in regulating Trh levels was examined by staining esg⁰⁵⁷³⁰ esg-lacZ embryos injected with dys-dsRNA for Trh in fusion cells. Only heterozygous embryos were examined. Tracheal fusion cells were identified by staining for ß-Gal. Levels of ß-Gal in fusion cells were comparable to control-injected embryos, indicating that esg transcription is not substantially affected by the depletion of Dys. Trh levels in Dys-depleted fusion cells were at high levels comparable to the levels observed in adjacent tracheal cells. Quantitatively, 96% of fusion cells examined in dys-dsRNA-injected embryos showed high levels of Trh. This indicates that the reduction in Trh levels in fusion cells is dependent on dys function (Jiang, 2003).

The trh, tgo, and dfr genes encode a transcription factor complex that is required for the development of tracheal cells from dorsal ectoderm and, directly or indirectly, the expression of most genes expressed in the trachea. Appearance of Trh protein precedes Dys by several hours. The expression of dys was examined in a trh mutant background and found to be absent. Thus, trh is required for dys expression, and Dys, in turn, modulates Trh levels (Jiang, 2003).

dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion

The Drosophila dysfusion basic-helix-loop-helix-PAS transcription factor gene is expressed in specialized fusion cells that reside at the tips of migrating tracheal branches. dysfusion mutants were isolated, and genetic analysis of live embryos revealed that mutant tracheal branches migrate to close proximity but fail to recognize and adhere to each other. Misexpression of dysfusion throughout the trachea further indicated that dysfusion has the ability to both inhibit cell migration and promote ectopic tracheal fusion. Nineteen genes whose expression either increases or decreases in fusion cells during development were analyzed in dysfusion mutant embryos. dysfusion upregulates the levels of four genes, including the shotgun cell adhesion protein gene and the zona pellucida family transmembrane protein gene, CG13196. Misexpression experiments with CG13196 result in ectopic tracheal fusion events, suggesting that it also encodes a cell adhesion protein. Another target gene of dysfusion is members only, which inhibits protein nuclear export and influences tracheal fusion. dysfusion also indirectly downregulates protein levels of Trachealess, an important regulator of tracheal development. These results indicate that fusion cells undergo dynamic changes in gene expression as they switch from migratory to fusion modes and that dysfusion regulates a discrete, but important, set of these genes (Jiang, 2006; full text of article).

The isolation of dys mutants allowed detailed phenotypic analysis using time-lapse confocal microscopy of live embryos. Migration and the presence of filopodia during DB branching appeared normal in dys mutant embryos. The two DB fusion cells moved close together, and their filopodia were observed to touch. However, unlike wild-type cells, the dys mutant fusion cells failed to stably adhere. Thus, no fusion occurred, and ultimately the branches retracted from one another. Additional insight into the role of dys emerged from experiments in which dys was misexpressed throughout the trachea. Ectopic fusion events were observed, which is consistent with the dys mutant phenotype and indicates that dys promotes fusion cell recognition/adhesion. In addition, dys misexpression results in a strong reduction in both tracheal branching and formation of MAb 2A12 luminal material at fusion sites. The reduction in branching suggests that another possible function of dys is to inhibit migration in preparation for fusion. The reduction in MAb 2A12 luminal material at sites of normal and ectopic fusion suggests that the fusion process is incomplete. This can be explained for the ectopic fusion results by proposing that Dys activates genes that can mediate fusion but not lumen formation. Since the ectopic cells are not fusion cells, the additional lumen-forming functions would not be present. In contrast, since lumen formation was also defective at normal fusion sites, it is possible that Dys overexpression inhibits lumen formation. In summary, the genetic and misexpression experiments suggest that dys is activated late in the branching process to inhibit migration and promote branch recognition and adhesion. It may also play additional roles after branches join (Jiang, 2006).

Since dys encodes a transcription factor, it is expected that it functions by regulating gene expression. Previous work had identified several genes prominently expressed in fusion cells, as well as additional trachea-expressed genes whose fusion cell expression was low or absent. This paper further shows that a number of prominent trachea-expressed genes are also downregulated in fusion cells, indicating that this is a common occurrence. Expression of 19 genes was assayed in dys mutant embryos to identify Dys target genes. RNA levels of four genes (CG13196, CG15252, mbo, and shg) were reduced. In contrast, Trh protein levels, which normally decline in fusion cells, increased in dys mutants. These results were confirmed by dys misexpression experiments, in which CG13196 and CG15252 were increased and Trh protein levels declined. Despite dys expression in all tracheal fusion cells, there exist branch-specific differences in Dys-regulated gene expression. CG13196 is expressed in all fusion cells, and dys is required for its expression. In contrast, shg is upregulated in DB and DT fusion cells, but only DB upregulation requires Dys, an effect also seen for Esg. CG15252 is expressed only in DT fusion cells, and this restriction may be due to the positive or negative action of branch-specific transcription factors, such as Spalt major (DT specific, positively acting) or Kni and Knrl (non-DT branches, negatively acting) (Jiang, 2006). All of the dys misexpression defects (and thus probably the mutant defects) require Dys DNA binding, since deletion of the dys basic region, and presumably its ability to bind DNA, abolishes the tracheal defects. Although trh RNA levels decline in fusion cells along with protein levels, the RNA reduction is not dys dependent. Thus, dys likely regulates transcription of a gene that regulates Trh protein levels. Similarly, the requirement of Dys DNA binding to regulate Trh protein levels does not support a model in which Trh levels are reduced as a consequence of Dys competing for their common dimerization partner, Tgo, since this is unlikely to require DNA binding (Jiang, 2006).

The recognition/adhesive properties promoted by dys may be mediated by two Dys target genes, shg and CG13196. Shg is a well-studied adhesion protein, and CG13196 encodes a ZP transmembrane protein, although its function and subcellular localization are unknown. Misexpression of CG13196 results in ectopic fusion events consistent with it playing a role in cell adhesion. Thus, one key role of dys may be to promote tracheal fusion by controlling expression of two or more cell adhesion protein genes. They could work together in the same cellular process or in different aspects of tracheal fusion, lumen formation, or function. The identification of members only (mbo) as a transcriptional target of Dys is intriguing, since mbo mutants have a tracheal fusion defect and it attenuates protein nuclear export. Although the fusion cell protein cargo regulated by mbo is unknown, it presumably includes proteins that are localized to nuclei in fusion cells (Jiang, 2006).

The two major transcription factors studied to date that control fusion cell transcription and development are esg and dys. esg precedes dys during fusion cell development and controls expression of dys in DBs and GBs but not DTs (the case for LTs is unknown, since fusion cells die in esg mutants. dys itself does not affect esg expression. The tracheal fusion phenotypes of both genes are similar. The DT is the one branch that still fuses in both esg and dys mutants, although both show constrictions at the sites of DT fusion. Previous work on esg revealed that, genetically, it is required for both activation of fusion cell gene expression and repression of terminal cell gene expression in fusion cells. In this study, it was found that dys constitutes a transcriptional pathway that carries out a subset of esg functions, focused on upregulating expression of genes involved in cell adhesion and protein localization, although future work may uncover additional target genes. Since a large number of genes are either activated or downregulated in tracheal fusion cells, it will be important to continue genetic and molecular studies to determine which genes are targets of Esg and Dys and whether their control is direct or indirect (Jiang, 2006).

One model of Drosophila dys function is that dys acts as a developmental timer near the end of tracheal branching to inhibit migration and promote cell adhesion and fusion. The adhesion component works, in part, through activation of shg and (possibly) CG13196. The inhibition of migration has only been postulated from misexpression experiments and needs to be confirmed by alternative approaches. It is also important to note that the switch from migration to fusion can also include changes in gene expression that are independent of dys. For example, it is shown here that RNA levels of btl, a gene required for tracheal migration, are downregulated in fusion cells in a dys-independent mode. dys is expressed in a variety of Drosophila embryonic cell types, including leading edge, brain, gut, and anal pad, and the mammalian ortholog is prominently expressed in the brain. However, the function of dys in these cell types is unknown, although a potential connection between tracheal fusion cells and both migrating neuronal axon growth cones and leading-edge cells is worth investigating. The role of dys in controlling fusion cell behavior suggests that it is worthwhile to look in tissues that undergo branching morphogenesis, such as the vertebrate lung and vascular system, for regulatory proteins expressed in tip/fusion cells that control the migration, recognition, and fusion properties of their branches (Jiang, 2006).

The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system: Ago protein is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion

The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. This study shows that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion (Dys) protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease (Mortimer, 2007).

The biological properties of individual F-box proteins are to a large degree determined by their repertoire of target proteins. In the case of the Drosophila Ago F-box protein, failure to degrade these targets promotes excess proliferation of imaginal disc cells. This observation has led to the identification of Cyclin E and Myc proteins as ago targets. However the broad pattern of Ago expression in the embryo suggests that it might regulate distinct processes and targets in other cell types. In view of the rapidly growing body of work showing that inactivation of human ago/Fbw7 is a common event in a variety of cancers (e.g., Malyukova, 2007; O'Neil, 2007; Thompson, 2007), identification of these targets may provide important insight into the biology of cancers lacking ago function (Mortimer, 2007).

This study shows that Drosophila Ago is required for the post-mitotic morphogenesis of the embryonic tracheal system and that this requirement is due, at least in part, to the ability of Ago to bind directly to a previously unrecognized target, the Trh transcription factor, and stimulate its proteasomal degradation. This ago degradation mechanism appears to fulfill different regulatory roles in different populations of tracheal cells. In non-fusion tracheal cells, ago is required to limit overall levels of Trh, which is normally expressed at moderate levels throughout the tracheal system. In tracheal fusion cells the ago degradation mechanism appears to be strongly potentiated by an unidentified signal generated by Dys, such that Trh is completely eliminated from Dys-expressing cells. At a genetic level, the dependence of ago tracheal phenotypes on trh gene dosage argues that that elevated Trh levels are primarily responsible for branching defects that occur in ago zygotic mutant embryos. In support of this, persistent Trh expression is also observed in ago mutant fusion cells in other tracheal branches. This novel role for Ago in tracheal development is supported by the independent finding that homozygosity for a genomic deletion containing the ago locus is associated with cell migration defects in embryonic tracheal metameres (Mortimer, 2007).

Many important developmental events are controlled by multiple mechanisms that collaborate to regulate a key step in the process. This somewhat redundant control insulates the process from defects in any single pathway, such that major defects only occur when multiple control mechanisms are blocked. The observation that the effect of ago mutations on Trh and btl levels is completely penetrant, but the resulting morphological defects are not, suggests that another pathway acts redundantly to ago to control tracheal development. The strong, dominant enhancement of the ago phenotype by a mutation in the abnormal wing disc (awd) gene (Dammai, 2003) fits very well into a model in which multiple pathways are responsible for the precisely timed down-regulation of the Breathless/FGF pathway: ago attenuates btl transcription by degrading Trh, awd lowers levels of Btl protein on the cell surface by promoting its endocytic internalization (Dammai, 2003), and other pathways act independently to control expression of the FGF ligand branchless in non-tracheal cells. Thus, the incomplete penetrance of the ago phenotype is not indicative of an insignificant role for the gene in tracheal development, but rather may indicate that the tracheal system uses multiple mechanisms to redundantly control a key step in its development (Mortimer, 2007).

The Ago WD repeat region binds Cyclin E and dMyc, and the current work demonstrates that it also binds Trh. Broadly, the Ago-Trh interaction is quite similar to interactions with Cyclin E and dMyc: it is required for the down-regulation of substrate levels in vivo, and its disruption elevates levels of substrate that then drive downstream phenotypes. For substrates like Myc, site-specific phosphorylation generates a motif that binds to the Ago WD-region and stimulates rapid, SCF-mediated protein turnover of the target protein (reviewed in Minella, 2005). In contrast, the data in this study suggest that Trh can physically interact with Ago in two distinct configurations: one that does not require an intact WD-domain and a second WD-dependent mode of binding. The observation that the Ago¹ allele can participate in the first complex but not the second and is defective in Trh regulation in vivo, suggests that like other Ago targets, WD-dependent binding is associated with rapid Trh turnover. Expression of Dys appears to shift the balance in favor of this second mode of binding. Combined with the genetic and phenotypic data implicating ago as an in vivo regulator of Trh activity, these molecular data support a model in which Ago can bind to Trh in the absence of Dys and inefficiently stimulate Trh turnover by a WD-dependent mechanism. This inefficient mechanism may be responsible for the fairly mild increase in Trh levels observed in all ago mutant dorsal trunk cells. However, in the presence of Dys, the efficiency of Trh turnover in dorsal trunk fusion cells is enhanced to the degree that the entire pool of Trh is rapidly eliminated. Interestingly, the correlate of this hypothesis - that ectopic expression of dys in non-fusion cells should be sufficient to trigger down-regulation of endogenous Trh - was confirmed in a recent study (Jiang, 2006; Mortimer, 2007).

The nature of the Dys-generated signal responsible for this effect is not currently known. Precedent with other Ago targets suggests that it may involve Trh phosphorylation. Recent work on the mammalian ago ortholog Fbw7 has shown that interactions with substrates can also be modulated by interaction with accessory factors (Punga, 2006), or by conformational changes in the substrate driven by the isomerization of proline residues within the Ago/Fbw7 binding motif (van Drogen, 2006). Proline isomerization has been implicated in the degradation of mammalian c-Myc, but such mechanisms are not currently known to play a role in the degradation of either Myc or bHLH-PAS proteins in Drosophila . An important goal of future studies will be to determine if any of these types of mechanisms are involved in Dys-induced Trh degradation in tracheal cells (Mortimer, 2007).

The requirement for ago in tracheal cells suggests that the consequences of ago loss vary considerably depending on the proliferative state of the cells, their location within the organism, and their developmental stage. ago mutant clones in the mitotically active larval eye disc show no evidence of excessive Trh levels or deregulated Btl/FGF signaling and conversely, ago zygotic mutant trachea do not display 'extra cell' defects similar to those observed in the eye. The origins of this tissue specificity are currently not clear, although it might simply reflect the differential expression patterns of Ago targets in various mitotic and post-mitotic cell populations. There is currently no evidence that the mammalian Trh homologs NPAS-1 and NPAS-3 are degraded by an Ago/Fbw7 dependent mechanism in mammalian cells. However the finding that Fbw7 knock-out mouse embryos display defects in vascular development (Tetzlaff, 2004; Tsunematsu, 2004) seems to indicate that the Fbw7 ligase may also target proteins involved in tubular morphogenesis, and intriguingly both NPAS1 and NPAS3 have been linked either to this process (Levesque, 2007) or to the transcriptional control of FGFR genes (Pieper, 2005). It has been suggested that the Fbw7 vascular defects arise due to Notch misregulation. However since FGF signaling is known to control branching morphogenesis of the mammalian vasculature and lung, the data presented in this study raise the possibility that vascular phenotypes in Fbw7 knock-out mouse embryos may also reflect the deregulation of developmental pathways that control branching morphogenesis via mammalian homologs of trh and btl (Mortimer, 2007).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

DEVELOPMENTAL BIOLOGY

Embryonic

Staining with Dys antisera is highly restricted temporally and spatially. Little, if any, expression is observed before stage 12, but at stage 12 expression, begins in many of the Dys-positive embryonic cell types. At stage 14, Dys protein is observed in (1) precursors to the foregut atrium, (2) a subset of nervous system cells that are either part of the medial brain or the frontal ganglion, (3) four rows of tracheal fusion cells, (4) cells at the position of the epidermal leading edge, (5) the hindgut, and (6) the anal pad. The frontal ganglion consists of cells of the stomagastric nervous system located between the brain and roof of the pharynx. Expression remains in all of these cell types through the remainder of embryonic development. All Dys-positive cells show nuclear localization. The leading-edge and tracheal cells have been conclusively identified in this study (Jiang, 2003).

During the process of dorsal closure, the dorsalmost rows of cells on each side are referred to as the leading edge. These cells (1) are required for the process of dorsal closure, (2) respond to cellular signals regulating closure, and (3) form the dorsal zipper after closure. These cells are characterized by changes in cell shape during the closure process, and they interdigitate and adhere to each other after meeting at the dorsal midline. dys was examined for expression in the leading-edge cells by examining for colocalization with Puckered (Puc). The puc gene is expressed in the leading edge, and puc^A251.1F3, an enhancer trap line with a P[lacZ] insertion in the puc gene expresses lacZ in the leading edge cells. Double staining embryos with anti-Dys and anti-ß-Gal reveals colocalization of the two proteins (Jiang, 2003).

Dys protein was observed in four distinct longitudinal, segmentally repeated sets of cells along the ectoderm. The locations of these cells resembles the pattern of genes expressed exclusively in tracheal fusion cells. Embryos were double stained with MAb 2A12, which stains the tracheal lumen, and anti-Dys to determine the relationship between Dys-positive cells and the trachea. Before fusion, each branch contained a single Dys-positive cell, whereas after fusion there were two Dys-positive cells at the fusion site of each branch. This was observed at all sites of tracheal fusion, including the dorsal trunk, lateral trunk, dorsal branch, and ganglionic branch, but in no other tracheal cells. Only the three most anterior (G0, G1, and G2) of the 10 ganglionic branches fuse at the ventral midline. These three branches possess Dys-positive cells, but the others do not. Confirmation that the Dys-positive cells are tracheal fusion cells was shown by double staining an escargot-lacZ (esg-lacZ) enhancer trap line with anti-ß-Gal and anti-Dys. Colocalization was observed in all Esg-positive tracheal fusion cells (Jiang, 2003).

Time course experiments were carried-out by double staining P[esg-lacZ] embryos with anti-Dys, anti-ß-Gal, and anti-Trh. Anti-Trh staining visualizes tracheal development at all stages of development. Primary branches originate from tracheal precursor cells at stage 11. At this time, Dys protein is undetectable. At stage 12, before the tracheae fuse in the dorsal trunk, dys expression is first detected. It remains on at high levels throughout the remainder of embryonic development (to stage 17) in the dorsal trunk. Lateral trunk cells fuse later (stage 15) than the dorsal trunk, and dys expression correspondingly also appears later in the lateral trunk (stage 13) than in the dorsal trunk (stage 12). However, dys expression in the lateral trunk still precedes its fusion. The dorsal branches and ganglionic branches fuse later (stages 16 to 17) than the lateral trunk and dorsal trunk, and dys expression also appears later (stage 14) than in the dorsal trunk and lateral trunk. Thus, Dys protein appears before fusion of each branch, and its appearance in each branch type correlates with the timing of branch fusion (Jiang, 2003).

Since Tgo is nuclear only in the presence of a partner bHLH-PAS protein, if Dys and Tgo dimerize in vivo, Tgo should be found in nuclei of Dys-positive cells. Embryos were double stained for Dys and Tgo. Most relevant are the leading-edge cells, since no other bHLH-PAS proteins are known to be expressed in these cells. Strong Tgo nuclear localization was observed that overlaps exactly with Dys. Tracheal fusion cells were also examined for nuclear Tgo levels. Examination of fusion cells of all four types of branches indicated high levels of nuclear Tgo accumulation colocalizing with Dys nuclear localization. These results are consistent with Tgo being a partner for Dys in vivo, although the presence of Trh in tracheal fusion cells could also contribute to Tgo nuclear localization. Of particular note is the finding that, qualitatively, the levels of nuclear Tgo appear constant in fusion cells throughout development, both before and after the appearance of Dys (Jiang, 2003).

REFERENCES

Search PubMed for articles about Drosophila Dysfusion

Cordoba, S. and Estella, C. (2014). The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development. PLoS Genet 10: e1004621. PubMed ID: 25329825

Dammai, V., Adryan, B., Lavenburg, K. R. and Hsu, T. (2003). Drosophila awd, the homolog of human nm23, regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development. Genes Dev. 17(22): 2812-24. PubMed Citation: 14630942

Jiang, L. and Crews, S. T. (2003). The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the Trachealess bHLH-PAS protein. Molec. Cell. Biol. 23: 5625-5637. 12897136

Jiang, L. and Crews, S. T. (2006). dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion. Mol. Cell. Biol. 26: 6547-6556. Medline abstract: 16914738

Levesque, B. M., et al. (2007). NPAS1 regulates branching morphogenesis in embryonic lung. Am. J. Respir. Cell Mol. Biol. 36: 427-34. PubMed Citation: 17110583

Malyukova, A., et al. (2007). The tumor suppressor gene hCDC4 is frequently mutated in human T-cell acute lymphoblastic leukemia with functional consequences for Notch signaling. Cancer Res. 67: 5611-6. PubMed Citation: 17575125

Minella, A. C. and Clurman, B. E. (2005). Mechanisms of tumor suppression by the SCF(Fbw7). Cell Cycle 4: 1356-9. PubMed Citation: 16131838

Mortimer, N. T. and Moberg, K. H. (2007). The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system. Dev. Biol. 312(2): 560-71. PubMed Citation: 17976568

O’Neil, J., et al. (2007). FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to {gamma}-secretase inhibitors. J. Exp. Med. 204(8): 1813-24. PubMed Citation: 17646409

Pieper, A. A., et al. (2005). The neuronal PAS domain protein 3 transcription factor controls FGF-mediated adult hippocampal neurogenesis in mice. Proc. Natl. Acad. Sci. 102: 14052-7. PubMed Citation: 16172381

Punga, T., Bengoechea-Alonso, M. T. and Ericsson, J. (2006). Phosphorylation and ubiquitination of the transcription factor sterol regulatory element-binding protein-1 in response to DNA binding. J. Biol. Chem. 281: 25278-86. PubMed Citation: 16825193

Tetzlaff, M. T., et al. (2004). Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein. Proc. Natl. Acad. Sci. 101: 3338-45. PubMed Citation: 14766969

Thompson, B. J., et al. (2007). The SCFFBW7 ubiquitin ligase complex as a tumor suppressor in T cell leukemia. J Exp Med. 204(8): 1825-35. PubMed Citation: 17646408

Tsunematsu, R. (2004). Mouse Fbw7/Sel-10/Cdc4 is required for notch degradation during vascular development. J. Biol. Chem. 279: 9417-23. PubMed Citation: 14672936

van Drogen, F., et al. (2006). Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms. Mol. Cell 23: 37-48. PubMed Citation: 16818231

date revised: 2 December 2018

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