trachealess
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 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)
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).
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).
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).
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).
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).
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).
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: 20 December 2006
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