Transcriptional Regulation

The expression pattern of trh is altered with alteration of either AP or DV axis. In wingless mutants, the tracheal pits are normal until stage 13, when cells between the pits, normally expressing wg, assume a tracheal fate. When one copy of decapentaplegic is removed, altering the DV axis, tracheal pits become elongated (Wilk, 1996). trachealess expression in the salivary duct is controlled by the homeotic gene Sex combs reduced and forkhead (Isaac, 1996).

Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions, a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells, through the EGF-receptor acts to block forkhead expression in preduct cells, thereby restricting gland identity to more dorsal cells. Forkhead acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

Salivary gland formation in the Drosophila embryo is dependent on Scr. When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, SCR mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that Scr also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Two homeodomain-containing proteins encoded by the extradenticle and homothorax genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. Scr regulates the nuclear localization of Exd in the salivary gland primordia through repression of homothorax expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands (Henderson, 2000).

To determine if Exd cooperates with Scr to control salivary gland gene expression, the accumulation of two early salivary gland proteins, CrebA and Trh, was examined in embryos lacking exd function. Zygotic loss of exd function results in a reduction in the number of salivary gland cells expressing CrebA and Trh, as well as a decrease in the level of protein made in these cells. This reduced level of salivary gland protein expression is not as severe as the one seen in Scr mutant embryos. Unlike SCR, EXD mRNA is supplied maternally and, thus, the maternal contribution may partially compensate for the loss of zygotic function. To test this possibility, the maternal contribution of exd was removed using the FLP-FRT system. In embryos lacking maternal exd function, salivary gland expression of CrebA and Trh is at wild-type levels. However, salivary gland expression of CrebA and Trh is completely absent in embryos lacking both the maternal and the zygotic contributions of exd. Thus, exd is required for embryonic salivary gland gene expression. Moreover, zygotically provided exd can rescue the loss of maternally provided exd and maternally provided exd can partially compensate for zygotic loss of exd (Henderson, 2000).

The posterior spiracle defects of the domeless/mom mutation have led to an examination of functions of the Hop/Stat92E pathway in tracheal formation. Trachea form from 10 tracheal pits, 1 per hemisegment. The trachealess gene selects the tracheal primordia in the embryonic ectoderm and drives the conversion of these planar epithelial regions into tracheal pits. The tracheal pits then sprout successively finer branches and fuse together, forming the tracheal network. The trachea is further connected to the posterior spiracle, forming a functional tracheal system. Tracheal formation was examined in mom, hop, and STAT92E mutants by using an enhancer trap line in the trachealess gene (1-eve-1) and an antibody [(mAb)2A12] that stains tracheal branches and trunks. In hop null embryos, trachealess expression is completely abolished and tracheal formation is completely blocked. In paternally rescued embryos, a defective tracheal system forms, generally with several disruptions in the main trunk and several branches. Because all of the mom and STAT92E mutants examined were enhancer trap lines, trachealess gene expression could not be examined by directly using the 1-eve-1 enhancer trap line. However, in the paternally rescued STAT92E and mom mutant embryos, similar to the hop mutant embryos, a defective tracheal system formed, generally with several disruptions in the main trunk and several branches. These data suggest that Mom and the Hop/Stat92E signal transduction pathway play an indispensable role in tracheal formation (Chen, 2002).

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 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)

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chig320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chig320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92EHJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome367 allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chig320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif (Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT+/Ken+) as well as sites with which Ken cannot associate (STAT+/Ken) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT+/Ken+ wild-type reporter, STAT+/Ken and STAT/Ken variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc167 Drosophila cell line, both STAT+/Ken+ and STAT+/Ken reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT+/Ken+ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT+/Ken+ assay shown is statistically significant, the STAT+/Ken reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT/Ken) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT+/Ken+ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT+/Ken+ and STAT+/Ken reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT+/Ken reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in updOS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the updOS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In updOS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest kenk11035 allele and Df(2R)Chig320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When kenk11035/Df(2R)Chig320 embryos simultaneously mutant for the amorphic updOS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT+/Ken+ and STAT+/Ken binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT+/Ken+ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT+/Ken+ and two STAT+/Ken binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

Targets of Activity

TRH protein regulates itself, perpetuating an autoregulatory loop (Wilk, 1996). Ectopic expression of trachealess results in ectopic breathless expression (Wilk, 1996).

The gene tracheae defective (tdf), now termed apontic, is required for the formation of the tracheal system during Drosophila embryogenesis. It encodes a putative bZIP transcription factor (TDF). Antibodies directed against TDF detect a nuclear protein in all tracheal cells before invagination and throughout tracheal system morphogenesis. Examination of tdf mutants reveals that tdf activity is not necessary for determining tracheal cell identity but for subsequent morphogenetic cell movements. tdf activity is under the control of trachealess, the key regulator gene for tracheal development. In contrast, tdf activity is not dependent on and does not interfere with the fibroblast growth factor- (FGF) and Decapentaplegic- (DPP) mediated signaling that directs guided tracheal cell migration. These results suggest that lack of tdf activity affects tracheal cell migration in general rather than specific aspects of cell migration. tdf activity involves a maternal and zygotic component; its requirement is not limited to tracheal system formation. The complex spatiotemporal patterns of TDF expression in the embryo correspond to defects, suggesting that cell migration is impaired. It is proposed that the bZIP protein TDF functions as a co-regulator of target genes that provide cells with the ability to migrate (Eulenberg, 1997).

The development of Drosophila trachea is under the control of spatially and/or quantitatively regulated activity involving the FGF receptor known as Breathless, which is also essential for midline glial migration. Examination of the proximal promoter region of the breathless promoter reveals three conserved elements (central midline elements or CMEs) resembling previously identified putative binding sites for Sim/Arnt heterodimers (Swanson, 1995) within a 150 base pair region, from -606 to -447 bases, relative to the P2 transcriptional initiation site. These three sites account for breathless expression in midline precursor cells (Ohshiro, 1997).

breathless expression in developing trachea is regulated by direct interactions between Trachealess/Tango heterodimers and three identical central midline elements (TACGTGs) situated in the minimum enhancer region. To test whether the heterodimer of Tango and Trh is capable of binding to CMEs, Trh and Tango fusion proteins were subjected to electrophoretic mobility shift assays of a CME1-containing oligonucleotide. In the absence of either Tango or Trh or both, there is little or nor protein-DNA interaction. In contrast, a retardation band can be seen when the oligonucleotide is incubated with a reaction mixture containing both Tango and Trh, indicating that Trh and Tango are capable of forming a heterodimer which exhibits DNA-binding activity (Ohshiro, 1997).

The Drosophila tracheal system is a network of epithelial tubes that arises from the tracheal placodes, lateral clusters of ectodermal cells in ten embryonic segments. The cells of each cluster invaginate and subsequent formation of the tracheal tree occurs by cell migration and fusion of tracheal branches, without cell division. The combined action of the Decapentaplegic (Dpp), Epidermal growth factor (EGF) and breathless/ branchless pathways are thought to be responsible for the pattern of tracheal branches. It is asked how these transduction pathways regulate cell migration and the consequences on cell behaviour of the Dpp and EGF pathways is examined. rhomboid (rho) mutant embryos display defects not only in tracheal cell migration but also in tracheal cell invagination unveiling a new role for EGF signaling in the formation of the tracheal system. These results indicate that the transduction pathways that control tracheal cell migration are active in different steps of tracheal formation, beginning at invagination (Llimargas, 1999).

Defects in tracheal migration are associated with defects in invagination in rho and vvl mutant embryos, but not in bnl and btl mutant embryos. This is consistent with the observation that EGF-dependent activation of MAP kinase (ERK) in the tracheal placode precedes ERK activation by the Bnl/Btl pathway. Thus the tracheal phenotype of mutations in the EGF pathway, which has been shown to result from impaired activity of the pathway in the trachea, is likely to originate before the onset of migration. It has been proposed that the EGF pathway might be required for tracheal cells in specific branches to follow the leading cell. Tracheal migration defects of rho mutant embryos are due, at least in part, to the failure of some tracheal cells to invaginate (Llimargas, 1999).

Invagination of the tracheal pits is dependent on trh. This process associates with an accumulation of actin in the cell surfaces facing the invagination and both actin accumulation and invagination are dependent on trh activity. Thus, the role of trh as an inducer of tubulogenesis could stem, at least in part, from its potential to reorganize the actin cytoskeleton. These results also indicate that induction of tracheal invagination by trh involves making cells competent to EGF signaling by regulating rho expression. However, there must be other targets of trh because tracheal invagination is only partially affected in rho mutant embryos. Interestingly, an interaction between EGF signaling and trh also occurs in salivary duct determination, suggesting that the coordinated activity of trh and the EGF pathway could be part of a more general mechanism for cell invagination and tubulogenesis (Llimargas, 1999).

The jing zinc-finger transcription factor, identified as a downstream target of slbo required for developmental control of border cell migration also plays an essential role in controlling CNS midline and tracheal cell differentiation. The jing locus ('jing' means 'still' in Chinese) was initially identified in a screen for mutations that cause border cell migration defects in mosaic clones. Embryonic recessive lethal jing mutations display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes sim and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning (Sedaghat, 2002).

jing's involvement in tracheal patterning was assessed. Embryos homozygous for a jing deficiency [Df(2R)ST1] and jing3 mutations are associated with losses of the dorsal trunk, severely disrupted transverse connectives and absences of the visceral branch. Embryos doubly mutant for jing and trh lack all tracheal tubules and display phenotypes identical to trh homozygous mutants. Therefore, trh loss-of-function is epistatic to jing loss-of-function, implying that jing functions downstream of trh (Sedaghat, 2002).

To determine the effects of overexpressing jing in the trachea, flies containing the P[breathless (btl)-GAL4] driver were crossed to those containing P[jing-UAS]. Progeny from this cross were stained with 2A12 antibody and tracheal tubule development was analyzed by light microscopy. Overexpression of jing in the trachea is associated with defects in dorsal trunk fusion, as well as improper formation of the transverse connective, dorsal branch and visceral branch. Therefore, jing overexpression tracheal phenotypes are similar to jing loss-of-function tracheal phenotypes (Sedaghat, 2002).

Based on genetic and phenotypic analyses, a role is proposed for jing downstream of sim and trh during CNS midline and tracheal development, respectively. (1) jing expression is not observed prior to that of either sim or trh in the CNS midline and trachea, respectively. jing expression is detected in the CNS midline during stage 9, which comes after the initiation of sim expression and establishment of midline fates. Jing protein is present in tracheal precursor nuclei, coincident with Trh during stage 10. (2) The CNS axon and tracheal phenotypes of homozygous jing mutations are less severe than those of homozygous sim and trh mutations, respectively. However, it cannot be rule out that maternal Jing may rescue the effects of zygotic jing mutations or that jing functions in a combinatorial fashion and therefore may not display severe phenotypes. (3) jing can be activated by ectopic expression of sim, suggesting that sim may regulate jing. The presence of three E-box ACGTG core sites in the 5' regulatory region of jing suggest that this regulation may be direct. (4) The sim and trh embryonic phenotypes are epistatic to that of jing, as shown by double mutant analysis. (5) jing mutations genetically interact with mutations in bHLH-PAS target genes such as sli and btl. The ventral displacement of midline cells in jing and sli double heterozygotes strongly suggests that jing is required for proper sli regulation (Sedaghat, 2002).

Analysis of genomic DNA sequence (GenBank accession number, AF285778) surrounding two lethal P-element insertions in jing reveals that there are three putative DNA binding sites for Tgo::Sim and Tgo::Trh (CMEs), and one for the HMG SOX protein called Fish-hook (also known as Dichaete, D) (TACAAT) in the 5' regulatory region of jing. This raises the possibility that jing may be a direct transcriptional target of bHLH-PAS heterodimers and SOX proteins including Tgo:Sim, Tgo:Trh or Fish-hook (Sedaghat, 2002).

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. Late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open (Yan), a repressor for late btl expression. Biochemical and genetic analysis has indicated that the minimum btl enhancer includes binding sites of Anterior-open (Ohshiro, 2002).

The minimum btl enhancer consists of B2 and B3 regions, the latter, a late enhancer. lacZ expression driven by B3 enhancer mimics btl late expression. The B3 enhancer possesses two of three CMEs sites for binding of Trh/Tgo complexes. The disruption of three CMEs in the btl enhancer brings about the complete loss of btl expression in tracheal cells at later stages. Thus, Trh/Tgo may also be required for late btl expression. A POU-Homeobox containing protein, Ventral veinless (Vvl)/Drifter is required for maintenance of btl expression in developing trachea. Pnt, Trh/Tgo, and Ventral veinless/Drifter thus quite likely synergistically activate btl expression in DB, VB, and LTa/p whereas Aop and/or Sal activity represses btl to prevent its expression in TC and/or DT (Ohshiro, 2002).

The Drosophila salivary gland is a simple tubular organ derived from a contiguous epithelial primordium, which is established by the activities of the homeodomain-containing proteins Sex combs reduced (Scr), Extradenticle (Exd), and Homothorax (Hth). EGF signaling along the ventral midline specifies the salivary duct fate for cells in the center of the primordium, while cells farther away from the source of EGF signal adopt a secretory cell fate. EGF signaling works, at least in part, by repressing expression of secretory cell genes in the duct primordium, including fork head (fkh), which encodes a winged-helix transcription factor. Fkh, in turn, represses trachealess (trh), a duct-specific gene initially expressed throughout the salivary gland primordium. trh encodes a basic helix-loop-helix PAS-domain containing transcription factor that has been proposed to specify the salivary duct fate. In conflict with this is the idea that trh specifies salivary duct fate: three genes, dead ringer (dri), Serrate (Ser), and trh itself, are expressed in the duct independently of trh. Expression of all three duct genes is repressed in the secretory cells by Fkh. Ser in the duct cells signals to the adjacent secretory cells to specify a third cell type, the imaginal ring cells. Thus, localized EGF- and Notch-signaling transform a uniform epithelial sheet into three distinct cell types. In addition, Ser directs formation of actin rings in the salivary duct (Haberman, 2003).

In trh mutants, salivary duct cells fail to invaginate and remain clustered on the embryo surface. Based on this phenotype and the loss of expression of other duct genes in trh mutant embryos, it has been proposed that trh is required to establish salivary duct identity. Based on this model, expression of all duct genes would be dependent on trh, even trh itself. Indeed, trh activity is required to maintain trh expression in the trachea. It was asked if trh is required to maintain its own expression in the salivary duct as well. In embryos mutant for two EMS trh alleles, trh1 and trh2, trh RNA was absent from the trachea at late stages. However, trh RNA was still observed at approximately wild-type levels in the salivary duct cells, indicating that trh does not autoregulate in the salivary duct as it does in the trachea (Haberman, 2003).

Two other genes, dead ringer (dri; also known as retained) and Serrate (Ser), are expressed to high levels in the salivary duct. dri encodes an ARID-box transcription factor whose role in the salivary duct has not yet been determined. Ser encodes a ligand for the Notch receptor, whose role in this tissue is also unknown. Expression levels of both dri and Ser are unaffected in trh mutants. Dri protein is present in the uninvaginated salivary duct cells that remain on the surface of trh mutants. Similarly, both Ser RNA and ß-galactosidase expressed under the control of a Ser enhancer (Ser-lacZ) are expressed in salivary duct cells in trh mutants. Thus, trh is neither required for its own expression nor for the expression of at least two other salivary duct genes (Haberman, 2003).

Since dri and Ser are expressed independently of trh, it was asked whether there is any regulatory relationship among the three genes. trh expression is not altered in embryos mutant for dri or Ser. Similarly, Ser expression is not altered in dri mutants, and Dri expression is not altered in Ser mutants. Thus, all three genes are expressed in the salivary duct independently of the other two (Haberman, 2003).

trh is initially expressed throughout the salivary gland, in both duct and secretory cell primordia, but becomes restricted to the duct cells by fkh. It has been suggested that Fkh acts through repression of trh to limit expression of all duct genes to only the ventral preduct portion of the salivary gland primordium. Since it has been shown that expression of at least three genes is trh-independent, it is unclear how their expression is limited to the duct. Whether or not expression of the trh-independent duct genes is affected by Fkh was tested. Since salivary gland cells undergo apoptosis in fkh mutants, the experiments were performed in the background of the H99 deficiency, which blocks apoptosis by removing the apoptosis-activating genes hid, grim, and reaper. As in fkh mutants alone, all salivary gland cells remain on the surface of the embryo in fkh H99 embryos. In these embryos, secretory cells express the secretory marker Pasilla (PS) and Trh is expressed in all salivary gland cells. Similarly, expression of both Dri and Ser expanded into the secretory cells of fkh H99 embryos, suggesting that fkh is required to prevent secretory cell expression of multiple duct genes independently. Expression of all three genes is also observed throughout the salivary gland primordium of fkh mutants without the H99 deficiency, demonstrating that the observed expression profiles are not affected by the H99 deficiency. Also, expression of all of these genes is unchanged in H99 homozygous embryos, further indicating that the changes in gene expression are due to fkh (Haberman, 2003).

Given the role of trh in salivary duct morphogenesis, what is the role of the two Trh-independent salivary duct genes? Staining of dri mutants with the duct markers Trh, Ser, or Crb did not reveal any overt morphological changes from wild-type embryos. Staining of Ser mutants with Dri revealed only a subtle, partially penetrant defect, where the distal ends of the individual ducts are slightly enlarged. Differences between Ser and wild-type embryos in the distal ends of the salivary ducts are more apparent with staining for cytoplasmic Ser-lacZ, which reveals that the ends of the individual ducts are splayed in the region where they contacted the secretory cells (Haberman, 2003).

Thus, trh is not the primary determinant of duct cell fate. Instead, these findings support an earlier model in which trh is required for the morphogenesis of the tubes that comprise the salivary duct, in keeping with its role in the trachea and filzkörper. In all three of these tissues, the primordial cells fail to invaginate and form tubular organs, although other tissue-specific markers are still expressed. Thus, it is expected that, in the salivary duct, Trh regulates expression of genes required for tube morphogenesis, as has been shown for the trachea. Indeed, btl, which encodes the FGF-receptor required for tracheal branch migration, is a Trh target in both the trachea and salivary duct, although its role in the salivary duct is unclear, since the loss of btl does not overtly affect salivary gland formation (Haberman, 2003).

fkh has many roles in secretory cell development. Fkh prevents secretory cell apoptosis, mediates apical constriction during invagination, regulates its own expression, maintains expression of dCrebA, and regulates expression of the ecdysone-stimulated glue genes sgs3 and sgs4. Fkh has been found to represses expression of all tested duct genes in the secretory cells. In fkh mutants, trh, Ser, and dri are expressed throughout the salivary gland primordium in both duct and secretory cells. It is unclear whether Fkh directly regulates duct gene expression or regulates expression through some currently unidentified upstream activator(s). The 4-kb Ser salivary duct enhancer used in these studies contains several potential Fkh binding sites, indicating that Fkh repression of Ser could be direct. Fkh repression of duct gene expression suggests a role for Fkh in reinforcing the secretory cell fate. Fkh is required to maintain the distinction between duct and secretory primordia that is initially established by EGF-signaling. First, EGF-signaling initiates the distinctions between duct and secretory cells by blocking expression of secretory-specific genes in the duct primordium. Then, the genes whose duct expression is blocked by EGF-signaling, specifically fkh, maintain this distinction by repressing duct gene expression and maintaining their own expression, thus sharpening the boundaries between duct and secretory primordia by interpreting the gradient of EGF-signaling into a binary cell fate decision (Haberman, 2003).

The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway

Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5'cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. This study shows that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea (Morozova, 2010).

In the developing Drosophila trachea, transcriptional regulation must be precisely coordinated with growth factor signaling to induce the appropriate cellular response. Studies of downstream transcriptional response elements in the transforming growth factor β (TGF-β) signaling pathway show the importance of discrete sequence changes differentiating an activation versus repressive response. Furthermore, such an activating enhancer element in the knirps gene in this pathway requires a cooperative effect with Trh and Tgo to possibly direct tissue specificity in the trachea. Tracheal gene expression is also controlled combinatorially by Trh/Tgo and Dfr/Vvl or either alone. Similarly, this study shows that Trh/Tgo response elements in the jing gene require additional elements to specify embryonic tracheal expression (Morozova, 2010).

Jing is implicated in transcriptional regulation in numerous biological processes, but its exact role is not known. This study extend previous observations of a role for jing in the trachea by establishing it as a direct downstream target of Trh/Tgo heterodimers. By analyzing jing 5' cis-regulatory regions, this study shows combinatorial basis to Trh/Tgo-mediated jing activation. A 2.8-kb jing enhancer recapitulates endogenous jing expression in the embryonic trachea, ectodermal stripes, and CNS midline. jing2.8 includes a distal 1.5-kb of genomic DNA that has three CMEs which are known for their involvement in combinatorial transcriptional regulation. The best evidence that Trh/Tgo complexes are able to directly activate the jing1.5 enhancer was gathered from Drosophila S2 cells by Luciferase reporter and ChIP assays. The CMEs in jing1.5-luc were required for activation by Trh/Tgo suggesting a protein-DNA interaction. Furthermore, Trh/Tgo heterodimers associated with and activated the jing1.5 enhancer. However, the combination of DNA-binding sites for bHLH-PAS, POU, and ETS transcription factors in jing1.5 is not capable of driving tracheal β-Gal expression in a pattern similar to that of endogenous jing. The jing1.3 enhancer cannot drive tracheal expression. Evidence is shown, in vitro and in vivo, that trh, pnt, and dfr/vvl regulate jing mRNA and even jing1.5-lacZ fusion cell expression. Given these results, along with the absence of additional CMEs and consensus POU domain-binding sites in jing1.3, it is proposed that trh and dfr/vvl regulate jing tracheal expression in combination with additional elements in jing1.3 (Morozova, 2010).

jing1.5 specifies a fusion cell component of jing expression that may instead be regulated by the bHLH-PAS transcription factors, Dys/Tgo. This is consistent with the presence of preferred and less preferred Dys/Tgo DNA-binding sites in the jing 1.5-lacZ enhancer. Prior to embryonic stage 12, trh is required for dys expression and then Dys and Archipelago downregulate trh specifically in fusion cells during stage 12. Therefore, Trh cannot activate jing1.5-lacZ in fusion cells from stage 12 which is consistent with the presence of fusion cell lacZ expression in embryos carrying CME deletions in jing1.5. The reductions in jing1.5-lacZ expression in the fusion cells of trh mutants may therefore result from subsequent reductions in dys expression (Morozova, 2010).

This study also characterized jing cis-regulatory elements controlling different aspects of jing expression in CNS glia and Engrailed-expressing midline neurons and segmental ectodermal cells. The midline expression of jing enhancers provided an opportunity to compare jing transcriptional regulation in two tissues. The data show that jing1.5 is sufficient to drive expression in MG and neurons where Jing is normally expressed. The CNS midline identity of jing1.5-lacZ-expressing cells was shown in several ways. First, jing1.5-lacZ expression was absent in a homozygous sim mutant background. Second, the jing1.5-lacZ expression domain was expanded by activating the Spitz Egfr ligand thereby forcing midline glial survival. Lastly, MG characteristics, such as oblong shape and dorsal positions, are shown by some jing1.5-lacZ-expressing midline cells. Therefore, this enhancer is differentially activated in the CNS midline and trachea suggesting that there may be differences in the mechanism by which Sim/Tgo and Trh/Tgo heterodimers activate transcription. This is consistent with the differential abilities of Sim/Tgo and Trh/Tgo to associate with Dfr/Vvl in vitro and the inability of trh to induce ectopic CNS midline gene expression (Morozova, 2010).

Strong CNS midline expression was also driven by the jing1.3 enhancer despite the absence of Sim/Tgo or Dfr/Vvl consensus DNA-binding sites. However, upon further characterization, the jing1.3-lacZ-expressing midline cells were found to express the segment polarity gene, engrailed (en). En-expressing CNS midline cells take up the posterior-most position within each VNC segment. Another En-positive midline cell lineage includes four to six MGP which are present at stage 13 but not at stage 17. The round shape of En-positive jing1.3-lacZ-expressing midline cells suggests that they belong to the MNB lineage and its progeny and do not belong to the MGP lineage. The mechanism of midline activation of jing1.3 is not known, but the ability of Jing to function as a repressor suggests that it may function combinatorially with En in segmental patterning. Further studies will be aimed at determining whether jing plays a role in segmental ectodermal patterning and its associated gene expression programs (Morozova, 2010).

An efficient approach to isolate STAT regulated enhancers uncovers STAT92E fundamental role in Drosophila tracheal development

The ventral veinless (vvl) and trachealess (trh) genes are determinants of the Drosophila trachea. Early in development both genes are independently activated in the tracheal primordia by signals that are ill defined. Mutants blocking JAK/STAT signaling at any level do not form a tracheal tree suggesting that STAT92E may be an upstream transcriptional activator of the early trachea determinants. To test this hypothesis STAT92E responsive enhancers activating the expression of vvl and trh in the tracheal primordia were sought. STAT92E regulated enhancers can be rapidly and efficiently isolated by focusing the analysis on genomic regions with clusters of putative STAT binding sites where at least some of them are phylogenetically conserved. Detailed analysis of a vvl early tracheal enhancer shows that non-conserved sites collaborate with conserved sites for enhancer activation. It was found that STAT92E regulated enhancers can be located as far 60 kb from the promoters. The results indicate that vvl and trh are independently activated by STAT92E which is the most important transcription factor required for trachea specification (Sotillos, 2010).

Mutations in the JAK/STAT pathway result in embryos only forming residual trachea fragments. This is caused by the abnormal activation of the early tracheal genes trh and vvl suggesting that the early trachea enhancers may be directly regulated by STAT92E in which case the trachea enhancers would be associated to STAT92E binding sites (Sotillos, 2010).

To test this the putative STAT92E binding sites were first localized in silico in the vvl 144 kb intergenic region. Then in vivo the enhancer activity was tested of regions that either (1) contain putative STAT92E sites that are conserved in several Drosophila species; (2) contain non-conserved putative STAT92E sites; or (3) contain no putative STAT92E sites. Of the 12 reporter lines made 10 have enhancer activity consistent with harboring embryonic vvl cis-regulatory elements. The expression of the enhancers either lacking STAT92E sites or containing non-conserved STAT92E sites is independent of JAK/STAT function. In contrast, of the seven vvl enhancers containing conserved sites, the expression of three of them required JAK/STAT regulation in the embryo. One of these enhancers drives expression in the hindgut and two are expressed in the trachea at stage 10. These results suggested that exclusively looking for conserved STAT92E sites would be sufficient to localize the STAT92E regulated sites, a prediction confirmed by isolating the trh gene tracheal enhancers. Although the presence of cryptic STAT binding sites that diverge from the ideal consensus could not be discarded in this analysis, such elements will probably have a minor contribution as ignoring their existence allowed finding of the main regulatory elements. Analysis of only eight fragments comprising a seventh of the trh locus was sufficient to find the early tracheal STAT92E responsive elements. This analysis revealed that the locus extends at least 40 kb upstream the trh promoter, with some enhancers located beyond the first predicted neighboring gene (Sotillos, 2010).

In both the trh and vvl genes it was found that other late tracheal enhancers are associated to STAT92E conserved sites suggesting that the STAT92E protein may be repeatedly used to control tracheal gene expression during development. This possibility is backed by the fact that upd is transcribed up to stage 13 and phosphorylated STAT92E can be detected in the trachea well after the stage 10 early specification stage. This late trachea specific expression of upd depends on trh suggesting that a feed-back loop maintains JAK/STAT activity during tracheal development (Sotillos, 2010).

It was confirmed that the expression of the vvl1+2 early tracheal enhancer depends on STAT92E by mutating its putative STAT92E binding sites. The results show that mutation of all three putative STAT92E sites in the vvl1+2 enhancer causes a severe loss of expression, indicating that a bona-fide direct enhancer was isolated. However, other possible direct enhancers like the vvlds1.5 hindgut element show an unexpected behavior. While vvlds1.5 perfectly recapitulates the vvl early hindgut activation its expression does not stop at stage 15, but keeps transcribing lacZ up to stage 17 well after the endogenous vvl gut transcription ceased. Despite this abnormal behavior, and pending direct site mutagenesis confirmation, it is believeed that these experiments show convincingly that the analysis of the genomic regions containing conserved STAT binding site clusters is an efficient way to quickly identify direct STAT92E regulated enhancers (Sotillos, 2010).

An important finding of this analysis has been the observation that STAT regulated enhancers can be tens of kilobases away from the transcriptional start of the target gene, even separated by another predicted gene. This indicates that STAT regulated enhancers can be functional at great distances and that search of STAT binding sites should not be restricted to the immediate vicinity of a gene (Sotillos, 2010).

Assuming there was no base content bias, the probability of finding in the genome either a 3n or a 4n STAT92E site by chance is (2 × 1/46) which is close to one site every 2 kb. In the 144 kb vvl genomic region there are 85 putative STAT92E sites, that is, 15 more than the expected 70 sites. Similarly, in the 70 kb trh locus there are 53 putative sites, 19 more than the expected 34 sites. The excess of sites found can be partially explained by selective pressure as there are 20 conserved sites in vvl. However, in the trh locus the 9 conserved STAT92E sites represent only half of the observed excess. An additional explanation for the excess STAT92E sites could be provided by the observation that in the regions were evidence was found for JAK/STAT regulation, there are clusters of conserved and non-conserved sites. It has been suggested that STAT site clustering helps forming tetramers that co-operatively increase STAT transcriptional output. Although STAT92E may form similar tetrameres in Drosophila, the distance of the clustered sites observed in this study is probably too large to allow tetramer formation and their existence must serve another purpose. When the vvl1+2 enhancer was subdivided it was observed that the distal non-conserved STAT92E site is dispensable for the enhancer function. However mutagenesis of the two conserved sites in vvl1+2 without mutating the distal non-conserved site has a mild decrease in the enhancer expression. Only when all three sites were mutated, including the non-conserved site, there is a strong effect on vvl1+2 expression. This shows that non-conserved sites may be functional in vivo even though they are not absolutely necessary. The results indicate that sites appearing distal to a STAT92E regulated enhancer may substitute for the proximal conserved sites, suggesting a way in which novel functional sites could eventually substitute conserved sites during evolution (Sotillos, 2010).

The crb and dome genes, which have been shown in vivo to be STAT92E targets, also show an accumulation of STAT92E sites in the introns where the enhancers localize. In the case of dome, five STAT92E sites cluster in a 700 bp fragment. Although only two of the five sites are conserved, and dissection of the enhancer showed that the conserved sites are crucial for JAK/STAT regulation, the non-conserved sites were also required for full enhancer expression. Therefore, clustered conserved and non-conserved STAT92E sites contribute to target gene regulation. It will be important to understand how STAT92E proteins binding to these distant sites interact with other transcriptional co-factors that presumably bind to the core enhancer (Sotillos, 2010).

The earliest genes activated specifically in the tracheal primordia are trh and vvl. Both genes have cross-regulatory interactions that help maintain each other's expression in the trachea, but their early activation is independent of each other. This study has localized the early trachea enhancers in trh and vvl and have shown that their activation in both cases depends on the JAK/STAT pathway making STAT92E the most important trachea activator. Stage 10 upd expression is consistent with a model where trh and vvl activation in the tracheae primordia is specified by a competitive interaction between the JAK/STAT and the WNT signaling pathways. STAT92E is probably acting with some other transcription factorS as inactivation of the JAK/STAT pathway does not result in a complete lack of expression of the enhancers. These other transcription factors would not necessarily have a restricted spatial expression as the precise positional activation of vvl could be provided by upd and wg. Although the early requirement of STAT92E for early tracheal specification precludes any studies at later stages, the maintained expression of upd in later tracheal development and the presence of STAT92E conserved sites associated to late tracheal enhancers suggest that the pathway is important for vvl and trh expression maintenance during tracheal development (Sotillos, 2010).

The observation that in crustaceans trh and vvl are co-expressed in the epipods that form the gills suggest that both genes where co-opted early in arthropod evolution to control the formation of the respiratory system (Franch-Marro, 2006). It would be interesting to find out if JAK/STAT is also required for trh and vvl expression in the crustaceans as that would suggest that the pathway had been adopted early in evolution fixing the regulation of both genes in the respiratory system (Sotillos, 2010).

A comparison of midline and tracheal gene regulation during Drosophila development

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, novel enhancers were identified and analyzed that drive midline and/or tracheal expression, and they were compared to previously characterized enhancers. In addition, expression was tested of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea (Long, 2014).

Several families of transcription factors contain members that bind related, but slightly different DNA recognition sequences. Examples include members of the nuclear receptor family and bHLH proteins. Nuclear receptor homodimers and heterodimers bind DNA response elements consisting of two inverted repeats separated by a trinucleotide spacer. Specificity is determined by interactions between protein loops on the second zinc finger of a particular steroid receptor DNA binding domain and the trinucleotide spacer within the DNA recognition site. Similarly, the recognition sequence of bHLH transcription factors is called the E box and consists of the sequence CANNTG. Specific bHLH heterodimers preferentially bind E boxes containing various internal dinucleotides (represented by the NN within the E box). The bHLH-PAS proteins investigated in this study are a subfamily within the bHLH superfamily of transcription factors. The PAS domain helps stabilize protein-protein interactions with other PAS proteins, as well as with additional co-factors, some of which mediate interactions with the environment. The evolutionary relationship of bHLH and bHLH-PAS proteins is also reflected in the similarity of their DNA recognition sequences. The CME is related to the E box and historically has been considered to consist of a five rather than six base pair consensus. Previous results indicated bHLH-PAS heterodimers strongly prefer the internal two nucleotides of the binding site to be 'CG', while the nucleotide immediately 5' to this core helps to discriminate which heterodimer binds the site. The first crystal structure of a bHLH-PAS heterodimer bound to DNA reveals that the recognition sequence of the human Clock/Bmal bHLH-PAS heterodimer actually consists of seven base pairs, rather than five. This is consistent with results reported in this study that suggest Sim/Tgo and Trh/Tgo heterodimers preferentially bind highly related, but slightly different seven base pair sequences. In addition, experiments with fly Sim and human Tgo, called Aryl hydrocarbon receptor nuclear translocator protein (Arnt), using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach, identify the sequence DDRCGTG (D = A, C or T and R = either purine) as the Sim/Tgo binding site. The current results agree with this, although the consensus sequence this study identified by examining known enhancers, is shifted by one nucleotide (DACGTG). In the midline and tracheal enhancers, sixty-six copies of the CME, ACGTG, were found and forty-eight copies of the related sequence, GCGTG, also identified in the SELEX experiments. Half of these GCGTG sites fit the seven bp consensus TGCGTGR and future experiments are needed to determine their importance within the various enhancers. The results indicated that the CME context favored within midline and tracheal enhancers as well as enhancers active in both tissues, was very similar, yet clearly distinct from binding sites of other bHLH and bHLH-PAS heterodimers. Based on the expression pattern of certain reporter genes examined in this study, the same CME may be bound by Sim/Tgo in the midline and Trh/Tgo in the trachea within certain enhancers. Within other contexts, the CME appears to be discriminated by these different heterodimers, because some enhancers drive expression in only one tissue or the other (Long, 2014).

Results from both endogenous enhancers and the synthetic reporter genes confirm the importance of the proximal sequences in limiting expression to either the midline or trachea. While the proximal context of the CME plays a role, additional sequences clearly combine with the CME to ultimately determine if an enhancer is functional in the midline or trachea. Taken together, these results indicate that proximal motifs combine with additional sequences not only to determine whether or not a gene is expressed in the midline or trachea, but also to determine which cellular subtypes express the gene and when it is activated within a tissue. Future experiments will reveal if 1) changing the sequence, AACGTGC, to TACGTGC within a midline enhancer will cause the enhancer to drive expression in trachea as well and 2) if changing the sequence, TACGTGC, to AACGTGC within an enhancer that drives expression in both the midline and trachea, will restrict expression to only the trachea. Sequences proximal to the CME likely affect the affinity of either Sim/Tgo and/or Trh/Tgo heterodimers to the DNA, but binding sites for additional factors that interact cooperatively to stabilize an entire transcription complex are needed for high levels of expression within a particular cell. Moreover, recent experiments indicate that enhancers containing multiple CMEs are activated earlier in the embryonic midline than enhancers containing only one CME. It has been suggested that Sim/Tgo binding sites may be sufficient for activation in the early embryo, but that binding sites for additional transcription factors must combine with the CME to drive expression within the later, more complex embryo (Long, 2014).

Protein Interactions

Trachealess (Trh) and Single-minded (Sim) are highly similar Drosophila bHLH/PAS transcription factors. They activate nonoverlapping target genes and induce diverse cell fates. A single Drosophila gene encoding a bHLH/PAS protein homologous to the vertebrate ARNT protein was isolated and may serve as a partner for both Trh and Sim. The Drosophila ARNT (DARNT, officially termed Tango )protein shows complete identity to the human protein in the basic domain, 95% identity in the HLH region, and 56% identity in the region including PAS A, PAS B and the spacer between them. DARNT is expressed ubiquitously during embryogenesis with elevated levels in the tracheal placodes and pits (Zelzer, 1997).

Trh and Sim complexes recognize similar DNA-binding sites in the embryo. To examine the basis for their distinct target gene specificity, the activity of Trh-Sim chimeric proteins was monitored in embryos. Replacement of the Trh PAS domain by the analogous region of Sim is sufficient to convert it into a functional Sim protein. It is concluded that the basic domains of Trh and Sim bind the same site on the DNA and do not confer specificity. The PAS domain of Sim provides midline specificity. The PAS domain of Trh was replaced with that of SIM. Ubiquitous expression of this protein results in a phenotype identical to the one induced by ectopic Sim expression: wide expansion of midline fates up to the ventral border of the tracheal pits. Expression of genes that are known to be targets of Sim, including breathless, rhomboid and S55 also show an expansion of midline cell fates. The PAS domain thus mediates all the features conferring specificity and the distinct recognition of target genes. The normal expression pattern of additional proteins essential for the activity of the Trh or Sim complexes can be inferred from the induction pattern of target genes and binding-site reporters, triggered by ubiquitous expression of Trh or Sim. It is thought that the capacity of bHLH/PAS heterodimers to associate, through the PAS domain, with additional distinct proteins that bind target-gene DNA, is essential to confer specificity (Zelzer, 1997).

The Drosophila single-minded and trachealess bHLH-PAS genes control transcription and development of the CNS midline cell lineage and tracheal tubules, respectively. Single-minded and Trachealess activate transcription by forming dimers with the Drosophila Tango protein, which is an ortholog of the mammalian Arnt protein. Both cell culture and in vivo studies show that a DNA enhancer element acts as a binding site for both Single-minded::Tango and Trachealess::Tango heterodimers and functions in controlling CNS midline and tracheal transcription. Isolation and analysis of tango mutants reveal CNS midline and tracheal defects. Gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. These experiments support the existence of an evolutionarily conserved, functionally diverse bHLH-PAS protein regulatory system (Sonnenfeld, 1997).

bHLH-PAS proteins represent a class of transcription factors involved in diverse biological activities. Previous experiments have demonstrated that the PAS domain confers target specificity. This suggests an association between the PAS domain and additional DNA-binding proteins, which is essential for the induction of specific target genes. A candidate for interaction with Trh is Drifter/Ventral veinless. A dual requirement for Trh and Drifter has been identified for the autoregulation of Trh and Drifter expression. Furthermore, ectopic expression of both Trh and Dfr (but not each one alone) triggers trh autoregulation in several embryonic tissues. A direct interaction between Drifter and Trh proteins, mediated by the PAS domain of Trh and the POU domain of Drifter, has been demonstrated (Zelzer, 2000).

Transcription of the trh gene is autoregulated, thus maintaining its expression throughout tracheal development, after the initial cues that determine the position of the tracheal placodes have disappeared. However, several experimental results suggest that the Trh/ARNT heterodimer is not sufficient for autoregulation of the trh gene. (1) Examination of Trh-Sim chimeras demonstrates that target gene specificity is determined by the PAS domain, possibly through interactions with other proteins. (2) Ubiquitous Trh can induce ectopic trh expression occasionally, at stage 11, but only at the position of tracheal pits in segments that do not normally form tracheal pits, suggesting that additional protein(s) expressed in this pattern need to cooperate with Trh. A candidate protein that may interact with Trh is the POU-domain protein Drifter/Ventral veinless (Dfr). This protein was previously shown to participate in tracheal morphogenesis. Initially, dfr is expressed in the ten tracheal placodes, as well as in the position of placodes in segments that normally do not produce tracheal pits. dfr mutations show a reduced expression of tracheal-specific genes such as breathless (btl), and accordingly exhibit migration defects that are reminiscent of the btl phenotype (Zelzer, 2000 and references).

An important feature of both Trh and Dfr expression is their capacity to be autoregulated. Once the exogenous cues that direct expression of these genes in the tracheal placodes diminish, expression is maintained by autoregulation. Since the trh and dfr genes themselves can be regarded as targets for Trh or Dfr, respectively, a test was performed to see whether autoregulation of each of the two genes requires both Trh and Dfr. Two phases of Trh expression have been defined; at stage 12, expression induced by exogenous cues is diminished and autoregulation ensues. Staining for the Trh protein in dfr mutant embryos has demonstrated that the initial phase of Trh expression in the placodes is normal. However, starting at stage 12 the levels of Trh are reduced, and are almost undetectable by stage 15. Failure of the cells in the tracheal pits of dfr mutant embryos to express Trh is not due to the death of these cells. Previous examination of the tracheal pits of dfr mutant embryos has shown that the cells are viable and capable of secreting tracheal lumen material, regardless of their failure to migrate properly. It can be concluded that Dfr is required for the autoregulation, and hence the maintenance of trh expression (Zelzer, 2000).

In the case of Dfr, a distinct 514 bp fragment has been defined as the dfr-autoregulatory element, which begins to drive Dfr expression at stage 11/12. This fragment also confers expression in the oenocytes. In trh mutant embryos, lacZ expression driven by this fragment in the oenocytes is retained, but completely abolished in the trachea. Again, the absence of expression in the tracheal placodes, which fail to invaginate in the trh mutant background, is not due to death of these cells. Staining of trh mutant embryos with anti-Dfr antibodies or with a probe detecting dfr RNA, has revealed the early, Trh-independent phase of expression up to stage 11. The uninvaginated placode cells in trh mutant embryos are thus intact, but fail to express the dfr autoregulation reporter. These experiments demonstrate that Trh and Dfr are required simultaneously for the autoregulation of Trh and Dfr themselves (Zelzer, 2000).

Rho (Rhomboid) functions as a regulator for processing the EGF receptor ligand Spitz, and is expressed a embryonic stage 9/10 in the midline glial cells, as well as in cells positioned at the center of the tracheal placodes. The parallel expression of rho in the tissues where Sim and Trh are functional, suggests that it may be a transcriptional target of these two bHLH-PAS proteins. In trh mutant embryos, expression of rho in the tracheal placodes is abolished. Similarly, in sim mutant embryos, expression of rho in the midline is eliminated. To determine if rho expression is regulated by direct binding of Sim and Trh, a 762 bp fragment of the rho 50 regulatory region was dissected: this is sufficient for midline and tracheal expression. The sequence of this fragment contains four sites with the Sim/Trh (ST) binding consensus. Similar sites have previously been shown by in vivo and in vitro analysis to represent the binding sites for Sim/ARNT or Trh/ARNT heterodimers. The 762 bp rho regulatory region was further dissected, and the capacity of smaller fragments to induce midline or tracheal expression in embryos was followed. The following conclusions were reached: Sim/Trh binding sites STc and STd are neither sufficient nor necessary for tracheal or midline expression. In contrast, Sim/Trh binding sites STa and STb are essential for midline and tracheal expression. Distinct cis elements appear to be required to promote midline vs. tracheal expression (Zelzer, 2000).

The paradigm that Trh or Dfr alone are not sufficient to induce their target genes or autoregulation, broadens the scope of activities of the two proteins. Trh is required not only for the induction of tracheal fates, but also for patterning the salivary ducts and posterior spiracles. It is possible that in these tissues, Trh associates with other proteins and induces a different set of tissue-specific target genes. Similarly, Dfr is also expressed in the midline cells. Dfr is not necessary for the induction of Sim-target genes, as can be deduced from the normal expression of rhomboid in the midline of dfr-mutant embryos. However, Dfr could be functioning in conjunction with other midline proteins such as the Sox-domain protein Dichaete (Zelzer, 2000).

Protein kinase B (PKB, also termed Akt) is a phosphatidylinositol 3' kinase (PI3'K)-dependent enzyme implicated in survival signaling and human tumorigenesis. To identify potential targets of this protein kinase, a genetic screen was employed in Drosophila. Among several genes that genetically interacted with PKB is trachealess (trh), which encodes a bHLH-PAS domain transcription factor required for development of the trachea and other tubular organs. Trh activates expression of the fibroblast growth factor receptor Breathless, which, in turn, is required for directed migration of all tracheal branches. Using a combination of biochemical and transgenic approaches, it has been shown that direct phosphorylation of Trh by PKB at serine 665 is essential for nuclear localization and functional activation of this regulator of branching morphogenesis (Jin, 2001).

Trh has a crucial role in the internalization of the primordia to form the tracheal sacs from which the various branches of the trachea derive. Trh controls this and related processes through the transcriptional regulation of downstream target genes. The Trh transcription factor is a direct substrate for PKB/Dakt1 kinase and is selectively phosphorylated at S665. This phosphorylation event is critical for Trh nuclear localization and for its function as a transcriptional coactivator. Further, loss of function of any of Dakt1, dPTEN, or PI3'K (p60A) in Drosophila embryos results in aberrant Trh function. The PI3'K/PTEN/Dakt1 signaling pathway is therefore required for Trh activity and, consequently, tracheal development. This signaling pathway is relevant for Trh function after the initial activation of trh transcription by developmental cues governing the anterior-posterior and dorsal-ventral axes. Since the initial developmental signals regulating trh expression are transient, later stages of tracheal expression are through autoregulation. These results suggest the model whereby PKB activity, as regulated by PI3'K signaling, positively regulates the nuclear localization of Trh via phosphorylation of S665. This leads to the accumulation of Trh within the nucleus, thus promoting an autoregulatory loop, which requires phosphorylation to be maintained. PKB regulation is important for Trh function, since the effects of ectopic Trh expression are suppressed in Dakt1 mutant embryos (Jin, 2001).

To date, a few direct phosphorylation targets of PKB have been identified: Bad, GSK-3ß, and the FKHR transcription factors. Studying these substrates of PKB suggested that PKB may have evolved a substrate selection that is skewed toward motifs also bound by 14-3-3 proteins. These substrates are also negatively regulated by PKB, whereas Trh is positively regulated and does not contain 14-3-3 binding motifs. In the case of FKHR proteins, PKB phosphorylation leads to nuclear exclusion, in contrast to the case for Trh. Thus, Trh may represent another paradigm for regulation by PKB, raising the possibility of other bHLH-PAS domain proteins serving as potential substrates for PKB (Jin, 2001).

In vertebrates, branching morphogenesis is a central component of the development of tubular structures such as lungs, vasculature, kidneys, and mammary glands. Tracheal development in Drosophila has been shown to be a useful model for studying the molecular and morphological aspect of branching morphogenesis. Since PKB is involved in tracheal development through the regulation of Trh, it therefore follows that PKB may have similar role(s) during mammalian branching morphogenesis. During tumorigenesis, branching morphogenesis becomes important during the process of angiogenesis, which is a prerequisite for tumor expansion. A role for PKB/Akt in angiogenesis has been suggested. One provocative study has proposed that the loss of PTEN leads to tumor expansion through ectopic activation of PKB/Akt and hypoxia-inducible factor 1alpha (HIF-1alpha-regulated downstream target genes. Other studies have also linked PI3'K/PKB signaling to the regulation of HIF-1alpha downstream target genes. HIF-1alpha is a bHLH-PAS protein whose levels are elevated in response to hypoxic stress and is structurally similar to Trh. Since human HIF-1alpha expression can induce btl transcription and tracheal structures in Drosophila embryos, it follows that Trh and HIF-1alpha are functionally conserved. This study therefore suggests the mechanism whereby PKB/Akt regulates the expression of genes required for angiogenesis through direct phosphorylation of HIF-1alpha or a related Trh homolog. Several hypoxic response bHLH-PAS factors have been postulated to harbor PKB/Akt consensus phosphorylation sites. Identification of a human bHLH-PAS factor analogous to Trh may provide a valuable target for intervention of the angiogenic response in tumors harboring an activated PI3'K/PTEN/PKB signaling axis (Jin, 2001).

The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

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).



trachealess expression is first detected in tracheal pits and placodes at stage 11. Expression is seen in the salivary placode and expression continues here through stage 15. At stage 15 [Image], staining is detected in the nuclei of posterior spiracles (Wilk, 1996). trachealess expression is also seen in the CNS (Isaac, 1996).

Association of tracheal placodes with leg primordia in Drosophila and implications for the origin of insect tracheal systems

Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. Early events that trigger leg specification have been elucidated and it is shown that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. It is concluded that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. An evolutionary scenario is proposed that accommodates the available data (Franch-Marro, 2006).

The Drosophila tracheal system has a clearly metameric origin, arising from clusters of cells, on either side of each thoracic and abdominal segment, that express the tracheal inducer genes trachealess (trh) and ventral veinless (vvl). Conversely, the leg precursors can be recognized as clusters of cells that express the Distal-less (Dll) gene, on either side of each thoracic segment; these will give rise both to the Keilin's Organs (KOs, the rudimentary legs of the larvae) and to the three pairs of imaginal discs that will give rise to the legs of the adult fly (Franch-Marro, 2006).

To investigate whether there is a direct physical association between the leg and tracheal primordia, Drosophila embryos co-stained for the expression of trh and early markers of leg primordia were examined. Although Dll is one of the most commonly used markers for the leg primordia, it is not the earliest gene required for their specification. Instead, a couple of related and apparently redundant genes, buttonhead (btd) and Sp1, act upstream of Dll in the specification of these primordia (Estella, 2003). Examining the specification of tracheal cells with respect to btd expression, tracheal cells were observed to appear in close apposition to btd-expressing cells, from the earliest stages of their appearance (by stage 9/early stage 10). Interestingly, unlike Dll, btd is initially expressed both in the thoracic and abdominal segments, and its expression is restricted to the thoracic segments later, under the influence of the BX-C. Thus, the cells of the respiratory system in Drosophila always arise in close proximity to the cells that are fated to give rise to the legs (Franch-Marro, 2006).

To fully endorse this conclusion it is necessary to show that the btd-expressing cells in the abdomen correspond to cryptic leg primordia. This may be a key point because, although many of the genes required for leg development are already known, it has not yet been possible to induce leg development in abdominal segments (except by transforming these segments into thoracic ones). In particular, although the Dll promoter contains BX-C binding sites that repress its expression in the abdominal segments, no ectopic appendage has been reported by misexpressing Dll in the abdomen. These observations have lead to some doubts as to whether a leg developmental program is at all compatible with abdominal segmental identity (Franch-Marro, 2006).

Since the initial expression of btd in the abdominal segments is downregulated by the BX-C genes, it was reasoned that sustained expression of btd might overcome the repressive effect of the BX-C genes and force the induction of leg structures in the abdomen. To test this, a btd-GAL4 driver was used to drive btd expression, expecting that the perdurance of the GAL4/UAS system would ensure a more persistent expression of btd in its endogenous expression domain. No sign was ever obtained of ectopic Dll expression or KOs in the abdominal segments, but the increased expression of btd had an effect on the KOs of the thoracic segments, which had more sensory hairs than the three normally found in wild-type KOs. Thus, on its own, btd seems unable to overcome BX-C repression of leg development (Franch-Marro, 2006).

One possibility would be that the BX-C genes could suppress appendage development in the abdomen by independently repressing both btd and Dll in this region. To assess this possibility, the same btd-GAL4 driver was used to simultaneously induce the expression of both btd and Dll. Under these circumstances, it was observed that KOs develop in otherwise normal abdominal segments; as in the previous experiment, the newly formed KOs have more than three sensory hairs. These results suggest that expression of btd and Dll in the btd-expressing abdominal primordia is sufficient to induce the development of leg structures in the abdomen, overcoming the repressive effect of the BX-C genes. Furthermore, these results demonstrate that these clusters of btd-expressing cells in the abdomen are indeed cryptic leg primordia. These results clearly show that tracheal cells are specified in close proximity to the leg primordia, in both thoracic and abdominal segments (Franch-Marro, 2006).

Previous results have shown that the leg primordia are specified straddling the segmental stripes of wingless (wg) expression in the early embryonic ectoderm, whereas tracheal cells are specified in between these stripes. To investigate whether wg might play a role in determining the fate of these primordia, what happens when the normal pattern of wg expression is disrupted was studied. In wg mutant embryos, trh and vvl from the earliest stages of their expression are no longer restricted to separate clusters of cells; instead larger patches of expression add up to a continuous band of cells running along the anteroposterior axis of the embryo, while btd expression is suppressed in this part of the embryonic ectoderm. Conversely, ubiquitous expression of wg suppresses trh expression, while causing an expansion of btd expression along the embryo. Restricted activation or inactivation of the wg pathway by the expression of a constitutive form of armadillo or a dominant-negative form of dTCF, respectively, are also able to specifically induce or repress trh and btd expression. trh/vvl and btd seem to respond independently to wg signalling and there is no sign of cross-regulation among them, since btd expression is normal in trh vvl double mutants, and trh and vvl expression is normal in mutants for a deficiency uncovering btd and Sp1 (Franch-Marro, 2006).

The role of wg as a repressor of the tracheal fate is further illustrated by looking at the behaviour of transformed cells: the clusters of cells that have lost btd expression and gained trh and vvl expression in wg mutant embryos begin a process of invagination that is characteristic of tracheal cells. Furthermore, these cells also express the dof (stumps) gene, a target gene of both trh and vvl in the tracheal cells. Although further development of these cells is hard to ascertain because of gross abnormalities in wg- embryos, these results indicate that they have been specified as tracheal cells. Thus, wg appears to act as a genetic switch that decides between two mutually exclusive fates in this part of the embryonic ectoderm: the tracheal fate, which is followed in the absence of wg signalling; and the leg fate, which is followed upon activation of the wg pathway. Given that there are no cell lineage restrictions setting apart the cells of the tracheal and leg primordia, these two cell populations could be considered as a single equivalence group, with the differences in their fate controlled by the activation state of the wg signalling pathway (Franch-Marro, 2006).

A link between respiratory organs and appendages is also found in many primitively aquatic arthropods, like crustaceans, where gills typically develop as distinct dorsal branches (or lobes) of appendages called epipods. Following the current observations, which suggest a link between respiratory organs and appendages in Drosophila, whether further similarities could be found between insect tracheal cells and crustacean gills was examined. Specifically, whether homologues of the tracheal inducing genes might have a role in the development of appendage-associated gills in crustaceans was considered (Franch-Marro, 2006).

RT-PCR was used to clone fragments of the vvl and trh homologues from Artemia franciscana and from Parhyale hawaiensis, representing two major divergent groups of crustaceans (members of the branchiopod and malacostracan crustaceans, respectively). In the case of Artemia vvl, a fragment was cloned that corresponds to the APH-1 gene and an antibody was generated for immunochemical staining in developing Artemia larvae. It was observed that Artemia Vvl is initially absent from early limb buds; it becomes weakly and uniformly expressed while the limb is developing its characteristic branching morphology, and becomes strongly upregulated in one of the epipods as its cells begin to differentiate. Uniform weak expression persists in mature limbs, but expression levels in the epipod are always significantly higher. Expression of the trh homologue from Artemia appears to be restricted to the same epipod as Vvl. Similarly, homologues of vvl and trh were cloned from Parhyale hawaiensis and their expression was studied by in situ hybridization. Both genes are specifically expressed in the epipods of developing thoracic appendages. Besides epipods, the Artemia trh and vvl homologues are also expressed in the larval salt gland, an organ with osmoregulatory functions during early larval stages of Artemia development (Franch-Marro, 2006).

What is the significance of the two Drosophila tracheal inducer genes being specifically expressed in crustacean epipods/gills? One possibility is that the expression of these two genes was acquired independently in insect tracheae and in crustacean gills. Alternatively, tracheal systems and gills may have inherited these expression patterns from a common evolutionary precursor, perhaps a respiratory/osmoregulatory structure that was already present in the common ancestors of crustaceans and insects (Franch-Marro, 2006).

The latter possibility is considered unlikely by conventional views, because of the structural differences between gills and tracheae (external versus internal organs, discrete segmental organs versus fused network of tubes), and the difficulty to conceive a smooth transition between these structures. Yet, analogous transformations have occurred during arthropod evolution: tracheae can be organized as large interconnected networks or as isolated entities in each segment (as in some apterygote insects), invagination of external respiratory structures is well documented among groups that have made the transition from aquatic to terrestrial environments (terrestrial crustaceans, spiders and scorpions), and conversely evagination of respiratory surfaces is common in animals that have returned to an aquatic environment (tracheal gills or blood gills in aquatic insect larvae). A very similar (but independent) evolutionary transition is, in fact, thought to have occurred in arachnids, where gills have been internalised to give rise to book lungs, and these in turn have been modified to give rise to tracheae in some groups of spiders. Thus, a relationship between insect tracheae and crustacean gills is plausible (Franch-Marro, 2006).

A particular type of epipod/gill has also been proposed as the origin of insect wings, a hypothesis that has received support from the specific expression in a crustacean epipod of the pdm/nubbin (nub) and apterous (ap) genes - that have wing-specific functions in Drosophila. In fact, the Artemia nub and ap homologues are expressed in the same epipod as trh and vvl, raising questions as to the specific relationship of this epipod with either tracheae or wings. A resolution to this conundrum becomes apparent when one considers the different types of epipods/gills found in aquatic arthropods, and their relative positions with respect to other parts of the appendage (Franch-Marro, 2006).

The primary branches of arthropod appendages, the endopod/leg and exopod, develop straddling the anteroposterior (AP) compartment boundary, which corresponds to a widely conserved patterning landmark in all arthropods. Different types of epipods/gills, however, differ in their position with respect to this boundary. For example, in the thoracic appendages of the crayfish, some epipods develop spanning the AP boundary [visualized by engrailed (en) expression running across the epipod], whereas others develop exclusively from anterior cells (with no en expression). Given that wing primordia comprise cells from both the anterior and posterior compartments, wings probably derived from structures that were straddling the AP boundary. Conversely, given that tracheal primordia arise exclusively from cells of the anterior compartment (anterior to en and even wg-expressing cells), it seems probable that tracheal cells evolved from a population of cells that was located in the anterior compartment. In this respect, it is interesting to note that the former type of epipods express nub, whereas the latter do not (Franch-Marro, 2006).

In summary, it is suggested that the ancestors of arthropods had specific areas on the surface of their body that were specialized for osmoregulation and gas exchange. Homologues of trh and vvl were probably expressed in all of these cells and played a role in their specification, differentiation or function. Some of these structures were probably associated with appendages, in the form of epipods/gills or other types of respiratory surfaces. A particular type of gill, straddling the AP compartment boundary, is likely to have given rise to wings, whereas respiratory surfaces arising from anterior cells only may have given rise to the tracheal system of insects. Confirmation of this hypothetical scenario may ultimately come from the discovery of new fossils, capturing intermediate states in the transition of insects from an aquatic to a terrestrial lifestyle (Franch-Marro, 2006).

Common origin of insect trachea and endocrine organs from a segmentally repeated precursor

Segmented organisms have serially repeated structures that become specialized in some segments. The Drosophila corpora allata, prothoracic glands, and trachea are shown to have a homologous origin and can convert into each other. The tracheal epithelial tubes develop from ten trunk placodes, and homologous ectodermal cells in the maxilla and labium form the corpora allata and the prothoracic glands. The early endocrine and trachea gene networks are similar, with STAT and Hox genes inducing their activation. The initial invagination of the trachea and the endocrine primordia is identical, but activation of Snail in the glands induces an epithelial-mesenchymal transition (EMT), after which the corpora allata and prothoracic gland primordia coalesce and migrate dorsally, joining the corpora cardiaca to form the ring gland. It is proposed that the arthropod ectodermal endocrine glands and respiratory organs arose through an extreme process of divergent evolution from a metameric repeated structure (Sanchez-Higueras, 2013).

The endocrine control of molting and metamorphosis in insects is regulated by the corpora allata (ca) and the prothoracic glands (pg), which secrete juvenile hormone and ecdysone, respectively. In Diptera, these glands and the corpora cardiaca (cc) fuse during development to form a tripartite endocrine organ called the ring gland. While the corpora cardiaca is known to originate from the migration of anterior mesodermal cells, the origin of the other two ring gland components is unclear (Sanchez-Higueras, 2013).

The tracheae have a completely different structure consisting of a tubular network of polarized cells. The tracheae are specified in the second thoracic to the eighth abdominal segments (T2-A8) by the activation of trachealess (trh) and ventral veinless (vvl) (Sanchez-Higueras, 2013).

The enhancers controlling trh and vvl in the tracheal primordia have been isolated and shown to be activated by JAK/ STAT signaling. While the trh enhancers are restricted to the tracheal primordia in the T2-A8 segments, the vvl1+2 enhancer is also expressed in cells at homologous positions in the maxilla (Mx), labium (Lb), T1, and A9 segments in a pattern reproducing the early transcription of vvl. The fate of these nontracheal vvl-expressing cells was unknown, but it was shown that ectopic trh expression transforms these cells into tracheae. To identify their fate, vvl1+2-EGFP and mCherry constructs were made (Sanchez-Higueras, 2013).

Although the vvl1+2 enhancer drives expression transiently, the stability of the EGFP and mCherry proteins labels these cells during development. It was observed that while the T1 and A9 patches remained in the surface and integrated with the embryonic epidermis, the patches in the Mx and Lb invaginated just as the tracheal primordia did. Next, the Mx and Lb patches fused, and a group of them underwent an epithelial-mesenchymal transition (EMT) initiating a dorsal migration toward the anterior of the aorta, where they integrate into the ring gland. To find out what controls the EMT, the expression of the snail (sna) gene, a key EMT regulator, was studied. Besides its expression in the mesoderm primordium, it was found that sna is also transcribed in two patches of cells that become the migrating primordium. Using sna bacterial artificial chromosomes (BACs) with different cis-regulatory regions, the enhancer activating sna in the ring gland primordium (sna-rg). A sna-rg-GFP construct labels the subset of Mx and Lb vvl1+2-expressing cells that experience EMT and migrate to form the ring gland. Staining with seven-up (svp) and spalt (sal) (also known as salm) markers, which label the ca and the pg, respectively, showed that the sna-rg-GFP cells form these two endocrine glands. The sna-rg-GFP-expressing cells in the Mx activated svp, and those in the Lb activated sal before they coalesced, indicating that the ca and pg are specified in different segments before they migrate (Sanchez-Higueras, 2013).

To test whether Hox genes, the major regulators of anteroposterior segment differentiation, participate in gland morphogenesis, vvl1+2-GFP embryos were stained, and it was found that the Mx vvl1+2 primordium expressed Deformed (Dfd) and the Lb primordium Sex combs reduced (Scr), while the T1 primordium expressed very low levels of Scr. Dfd mutant embryos lacked the ca, while Scr mutant embryos lacked the pg. Dfd and Scr expression in the gland primordia was transient, suggesting that they control their specification. Consistently, in Dfd, Scr double-mutant embryos, vvl1+2 was not activated in the Mx and Lb patches, and the same was true for vvl transcription. In these mutants, the sna-rg-GFP expression was almost absent, and the ca and pg did not form. In each case, Dfd controlled the expression of the Mx patch and Scr of the Lb patch (Sanchez-Higueras, 2013).

The capacity of different Hox genes to rescue the ring gland defects of Scr, Dfd double mutants was tested. Induction of Dfd with the sal-Gal4 line in these mutants restored the expression of vvl1+2 and sna-rg-GFP in the Mx and the Lb. However, in contrast to the wild-type, both segments formed a ca as all cells express Svp. Similarly, induction of Scr also restored the vvl1+2 and sna-rg-GFP expression, but both primordia formed a pg as they activate Sal and Phantom, an enzyme required for ecdysone synthesis. The capacity of both Dfd and Scr to restore vvl expression, regardless of the segment, led to a test of whether other Hox proteins could have the same function. Induction of Antennapaedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), or Abdominal-B (Abd-B) restored vvl1+2 expression in the Mx and Lb, but these cells formed tubes instead of migratory gland primordia. These cephalic tubes are trachea, as they do not activate sna-rg, they express Trh, and their nuclei accumulate Tango (Tgo), a maternal protein that is only translocated to the nucleus in salivary glands and tracheal cells, indicating that the trunk Hox proteins can restore vvl expression in the Mx and Lb but induce their transformation to trachea (Sanchez-Higueras, 2013).

To investigate whether vvl and trh expression is normally under Hox control in the trunk, focus was placed on Antp, which is expressed at high levels in the tracheal pits. In double-mutant Dfd, Antp embryos, vvl1+2 was maintained in the Lb where Scr was present, while the Mx, T1, and T2 patches were missing. In T3-A8, vvl1+2 expression, although reduced, was present, probably due to the expression of Ubx, Abd-A, and Abd-B in the posterior thorax and abdomen. Thus, Antp regulates vvl expression in the tracheal T2 primordium. Surprisingly, in Dfd, Antp double mutants, Trh and Tgo were maintained in the T2 tracheal pit, indicating that although Hox genes can activate ectopic trh expression, in the tracheal primordia they may be acting redundantly with some other unidentified factor, explaining why the capacity of Hox proteins to specify trachea had not been reported previously (Sanchez-Higueras, 2013).

sna null mutants were studied to determine sna's requirement for ring gland development, but their aberrant gastrulation precluded analyzing specific ring gland defects. To investigate sna function in the gland primordia, the sna mutants were rescued with the sna-squish BAC, which drives normal Sna expression except in the ring gland. These embryos have a normal gastrulation and activate the sna-rg- GFP; however, the gland primordia degenerate and disappear. To block apoptosis, these embryos were made homozygous for the H99 deficiency, which removes three apoptotic inducers. In this situation, the ca and pg primordia invaginated and survived, but they did not undergo EMT. As a result, the gland primordia maintain epithelial polarity, do not migrate, and form small pouches that remain attached to the epidermis. Vvl is required for tracheal migration. In vvl mutant embryos, sna-rg-GFP expression was activated, but the cells degenerated. In vvl mutant embryos also mutant for H99, the primordia underwent EMT and migrated up to the primordia coalescence; however, the later dorsal migration did not progress (Sanchez-Higueras, 2013).

This study has shown that the ca and pg develop from vvl-expressing cephalic cells at positions where other segments form trachea, suggesting that they could be part of a segmentally repeated structure that is modified in each segment by the activity of different Hox proteins. As the cephalic primordia are transformed into trachea by ectopic expression of trunk Hox, tests were performed to see whether the trachea primordia could form gland cells. Ectopic expression of Dfd with arm- Gal4 resulted in the activation of sna-rg-GFP on the ventral side of the tracheal pits. These sna-rg-GFP0-expressing cells also expressed vvl1+2 and Trh and had nuclear Tgo, showing that they conserve tracheal characteristics. These sna-rg-GFP-positive cells did not show EMT and remained associated to the ventral anterior tracheal branch. The strength of ectopic sna-rg-GFP expression increased when ectopic Dfd was induced in trh mutant embryos. However, migratory behaviors in the sna-rg-GFP cells were only observed if Dfd was coexpressed with Sal. Thus, sal is expressed several times in the gland primordia, first at st9-10 repressing trunk Hox expression in the cephalic segments and second from st11 in the prothoracic gland. It is uncertain whether the sal requirement for migration is linked to the first function or whether it represents an additional role (Sanchez-Higueras, 2013).

These results show that the endocrine ectodermal glands and the respiratory trachea develop as serially homologous organs in Drosophila. The identical regulation of vvl in the primordia of trachea and gland by the combined action of the JAK/STAT pathway and Hox proteins could represent the vestiges of an ancestral regulatory network retained to specify these serially repeated structures, while the activation of Sna for gland development and Trh and Tgo for trachea formation could represent network modifications recruited later by specific Hox proteins during the functional specialization of each primordium. This hypothesis or alternative possibilities should be confirmed by analyzing the expression of these gene networks in various arthropod species. The diversification of glands and respiratory organs must have occurred before the split of insects and crustaceans, as there is a correspondence between the endocrine glands in both classes, with the corpora cardiaca corresponding to the pericardial organ, the corpora allata to the mandibular organ, and the prothoracic gland to the Y gland. Despite their divergent morphology, a correspondence between the insect trachea and the crustacean gills can also be made, as both respiratory organs coexpress vvl and trh during their organogenesis. Divergence between endocrine glands and respiratory organs may have occurred when the evolution of the arthropod exoskeleton required solving two simultaneous problems: the need to molt to allow growth, and the need for specialized organs for gas exchange (Sanchez-Higueras, 2013).

Effects of Mutation or Deletion

In trachealess mutant embryos, tracheal pits do not form (Isaac, 1996). A dominant negative form of trh does not perturb the formation of tracheal pits, but more specific later processes fail to take place (Wilk, 1996).

Developing sensory axons have been studied in Drosophila embryos mutant for trachealess and/or the twist gene. These embryos manifest an absence of the trachea and/or somatic muscles, a part of the substrate on which sensory axons normally grow. In each of these three mutant backgrounds, the majority of sensory nerves form normally. This indicates that neither the tracheae nor the somatic musculature is absolutely required for pathfinding of the embryonic sensory axons. However, the incidence of misrouted axons is significantly increased, most strongly in the trh, twi double mutant. Furthermore, axonal elongation is considerably slowed down, and sensory neurons which fail to send out an axon are frequent (Younossi-Hartenstein, 1993).

Isolation and analysis of tango mutants reveal CNS midline and tracheal defects, and gene dosage studies demonstrate in vivo interactions between single-minded::tango and trachealess::tango. Defects in CNS midline neurons and glia were examined using enhancer trap reporters. In wild-type embryos, the AA142 enhancer trap is expressed in an average of 3.5 midline glia per segment by stage 14 of embryogenesis. In tango mutant embryos, there is a reduction in the number of stained midline glia to approximately one cell per segment. The X55 enhancer trap gene stains the ventral unpaired median neurons (VUMs) and the median neuroblast (MNB) and its progeny in the ventral region of the CNS. In tango mutant embryos, the number of VUM neurons and MNB progeny are reduced in number (60% of wild-type) and do not migrate into the ventral regions of the ventral cord. The role of tango in tracheal development was examined by staining tango mutant embryos with monoclonal antibody 2A12, which stains the lumen of the tracheal tubes. tango mutant embryos are shown to have a variety of tracheal defects. Experiments with heterozygotes show that tango interacts genetically with both trachealess and single minded (Sonnenfeld, 1997).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

Among the most striking effects on tubule development observed is the presence, in trachealess (trh) and krotzkopf verkehrt (kkv) mutant embryos, of a single sack from which the tubules emanate, instead of the two ureters that normally join the anterior and posterior pairs of tubules. Although the grossly abnormal arrangement of the proximal tubules including the ureters is different from the failure of the salivary gland duct cells to invaginate, in both cases trh is required for the proper formation of a tube that joins two other tubular structures. However, although trh expression has been reported in the tracheae and salivary glands, expression has not been reported in the Malpighian tubules or the ureters (Jack, 1999).

In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed. dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).

The pattern of stg expression anticipates and determines the embryonic cell division pattern. stg expression in the embryonic epidermis before mitosis 16, therefore, is highly similar to the pattern of dap expression, which also precedes this terminal mitosis 16. To analyze whether stg and dap expression before mitosis 16 are mechanistically coupled, the distribution of stg transcripts in ventral veins lacking (vvl) and trachealess (trh) embryos was examined. vvl and trh are expressed within the prospective tracheal pit regions and are known to co-operate for the specification of tracheal cell fate. The characteristic early dap expression in tracheal pits is not detected in vvl embryos and it is severely decreased in trh embryos. Interestingly, while the characteristic early expression of stg is not observed in trh embryos, it is normal in vvl mutants. As expected, progression through mitosis 16 also occurred in the normal pattern in these vvl mutants. The absence of the characteristic early dap expression in tracheal pits of vvl embryos, therefore, is not preceded by a change in the proliferation program. These findings indicate that the control of stg and dap expression is mechanistically distinct. Moreover, they suggest that regulators of developmental fates control dap expression directly and not indirectly by controlling the cell proliferation program via other cell cycle regulators (Meyer, 2002).

The RhoGAP crossveinless-c links trachealess and EGFR signaling to cell shape remodeling in Drosophila tracheal invagination

A major issue in morphogenesis is to understand how the activity of genes specifying cell fate affects cytoskeletal components that modify cell shape and induce cell movements. This study approaches this question by investigating how a group of cells from an epithelial sheet initiate invagination to ultimately form the Drosophila tracheal tubes. Tracheal cell behavior is described at invagination; it is show to be associated with, and requires, a distinct recruitment of Myosin II to the apical surface of cells at the invaginating edge. This process is achieved by the activity of crossveinless-c, a gene coding for a RhoGAP and whose specific transcriptional activation in the tracheal cells is triggered by both the trachealess patterning gene and the EGF Receptor (EGFR) signaling pathway. These results identify a developmental pathway linking cell fate genes and cell signaling pathways to intracellular modifications during tracheal cell invagination (Brodu, 2006).

Tracheal cells are singled out as cell clusters in the ectodermal unicellular layer, one at each side of 10 central embryonic segments. This study focused on the central tracheal placodes because the first and last one have distinct features. By stage 10, tracheal cells form a flat epithelium with their neighboring ectodermal cells. Longitudinal optical sections (1 microm apart) show the apical cell membrane, visualized by PKC, in a more exterior plane and the tracheal nuclei in a deeper one. A transverse optical section across the middle of the placode reveals its straight surface. By early stage 11, a group of around six cells reduces its apical cellular perimeter; this is the earliest indication of tracheal invagination since the constricted apical surface of those cells can be detected deeper inside. Local constriction is associated with cell shape changes; those cells pinch at their apical surface while their basal surface and nuclei appear deeper than those of the other tracheal cells. By middle stage 11, the invagination proceeds further; now the apical marker of the cells can be detected in an even deeper position. In addition, at this stage a significant change is observed in the invagination behavior of these cells. On the dorsal side, cells begin a rotation-like movement folding to form a new layer of cells below the epidermal surface. On the ventral side, cells slide below the invaginating dorsal cells. As a result, a finger-like structure originates in a process that has evolved from a cell monolayer to a 'three-layer organization' (two cell layers initiating a tube below the epidermis layer). As development proceeds, this finger-like structure elongates dorsally incorporating more tracheal cells from the embryonic surface toward the inside (Brodu, 2006).

The results suggest a two-step model by which trh induces and organizes tracheal invagination. First, trh activity appears to outline an invagination field, a region of cells that acquire the competence to invaginate. This effect can be clearly observed in mutants that impair EGFR signaling; in those embryos, trh activity is still able to promote a broad depression of the trh-expressing cells that will only further reorganize due to their ability to migrate in response to FGFR signaling. In this regard, there are clearly some consequences of trh that are independent of EGFR signaling and could be connected with the potential of trh to induce a general depression. For instance, it was found that the microtubule network is highly enriched and polarized apically at the site of invagination; while this arrangement is absent in trh mutants, it remains present in the abnormal invaginating tracheal placodes in the absence of both FGF and EGFR signaling (Brodu, 2006).

A second outcome of trh is accomplished by the triggering of EGFR signaling, which leads to the spatial and temporal organization of tracheal invagination. It is the activity of the EGFR pathway that converts the tracheal cell potential to invaginate into the organized process, resulting in a 'three-layer organization' and initiation of tube formation. A partner required for the organization of tracheal invagination is sal, which is expressed in the dorsal half of the tracheal placode and is responsible for the different morphology and behavior of the cells between the two sides of the placodes. The role of sal is, at least in part, achieved through down-regulation of EGFR signaling activity. However, it is not clear how this modulation is translated into differences in invaginating behavior. For example, no differences have been detected in level or distribution of cytoskeletal components along the sal expression border. An intriguing possibility would be that down-regulation of EGFR signaling gives rise to cells with different forces or stiffness (perhaps due to different levels of actin–myosin contractility), and the resulting apposition of two invaginating cell populations with different properties could force one of them to fold and initiate dorsal-oriented rotation, while the other would slide down under the former (Brodu, 2006).

It is worth noting that a well-organized invagination is an absolute requirement for tracheal morphogenesis. All the mutants that cause an abnormal invagination give rise to an impaired tracheal system in which some branches do not develop or develop deficiently. Thus, for example, rho mutants, which were originally thought to affect specifically the formation of two branches, have a general defect in invagination, and many tracheal cells remain clustered at the embryonic surface. In this regard, an important outcome of proper tracheal invagination appears to be that the tracheal cells reach the appropriate position with respect to the cues that will direct their subsequent migration. It has been suggested that the wild-type organization of the tracheal tree depends on having the appropriate number of cells at the correct position facing those signals, such that a specific number of cells contributes to the formation of the different branches (Brodu, 2006).

In many cases, cell fate commitment leads to cell shape modifications and rearrangements. The results of this study depict a developmental pathway that is initiated by the activity of a gene specifying cell fate (trh), which triggers a cell signaling pathway (EGFR) that, in turn, organizes cell invagination. A key step in this pathway is the transcriptional activation of a gene coding for a RhoGAP enzyme, cv-c, that affects actin–myosin apical distribution, likely by regulation of Rho1 activity (Brodu, 2006).

Regulation of RhoGTPases, either by RhoGAPs or RhoGEFs, appears to be a common trait in the control of morphogenesis. Indeed, RhoGAPs and RhoGEFs have been shown to act in different manners to affect actin and myosin. In this regard, some parallelisms can be found between tracheal cell invagination and other morphogenetic events such as gastrulation and neurulation. In particular, clear similarities can be seen with the mechanism of myosin regulation in Drosophila gastrulation. In this case, it is also the activity of a patterning gene (twist) that gives rise to the expression of a signaling molecule (folded gastrulation) that is thought to elicit a signaling pathway requiring a G-protein alpha subunit (concertina) and a RhoGEF (RhoGEF2). Then, RhoGEF2 ultimately leads to phosphorylation of myosin, which then activates actin binding by myosin and increases actomyosin contractility. However, in tracheal invagination, the remaining colocalization of myosin and actin in cv-c mutants suggest that cv-c is not necessary for the interaction between actin and myosin but instead for the proper localization of the actin–myosin complex. This observation fits well with a recent report that indicates that the cv-c RhoGAP acts on the actin apical accumulation in Malpighian tube morphogenesis and during epithelial dorsal closure (Brodu, 2006).

Different RhoGTPases act as substrates of the cv-c RhoGAP enzyme in different tissues. The results indicate that Rho1 is the substrate for cv-c in tracheal invagination. Notably, there appear to be more RhoGAPs and RhoGEFs molecules than RhoGTPases, which has been interpreted as an indication of the importance of a precise regulation of the transition between active and inactive states of RhoGTPases for different cell processes. Additionally, the fact that mutants for cv-c, a negative regulator of Rho1 activity, and Rho1 both impair actin apical organization and cell invagination in the tracheal placodes illustrates the importance of an appropriate regulation of RhoGTPase activity to achieve proper actin organization and cell behavior. In this regard, the fact that the cv-c RhoGAP has a pivotal role in tracheal invagination does not rule out that additional regulatory mechanisms that act on RhoGTPases could also be in place in tracheal invagination. The variable penetrance of null cv-c RhoGAP phenotypes suggests the possible existence of other invagination-regulating molecules under the control of trh. Additionally, EGFR signaling is only one of the programs elicited by the activity of trh. Altogether, these observations indicate that the developmental pathway that induces and organizes tracheal invagination must have diverse branches with additional target outcomes. It is suggested that many morphogenetic events share the same basic operational logic; leading from patterning genes and cell signaling pathways to cell shape changes, although each case may involve diverse target molecules acting at different steps in the regulation of the actin–myosin complex (Brodu, 2006).


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trachealess: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 July 2014

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