empty spiracles

Promoter Structure and Transcriptional regulation

The early expression of ems is controlled by the anterior morphogen Bicoid. A cis-acting element is found in the promoter of ems , itself a target for the bcd gene product (Grossniklaus, 1994).

Homeobox genes encode a class of highly evolutionarily conserved transcription factors that control embryonic development. The Drosophila melanogaster empty spiracles gene is the homolog of the two human homeobox genes EMX1 and EMX2. These genes are necessary for central nervous system development. A regulatory element of the empty spiracles gene was used to study the control of homeobox gene expression. The 1.2-kilobase (kb) cis-regulatory element located 3 kb 5' of the transcription start site of the empty spiracles gene was analyzed by evolutionary sequence comparisons, gel mobility shift assays, DNase footprinting, and the generation of transgenic flies. The corresponding element from a related species, Drosophila hydei, was cloned. Three discrete, approximately 100 base pair (bp) regions of sequence homology were identified. Each had two blocks of 10 to 40 bp of near perfect sequence identity. Fusion proteins were produced containing the Abdominal-B homeodomain or the Empty spiracles homeodomain, known regulators of empty spiracles gene expression. Gel mobility shift assays showed that each of the three regions is bound by both proteins. DNase footprinting revealed closely linked Empty spiracles and Abdominal-B binding sites. Transgenic flies containing a reporter linked to individual conserved regions of the enhancer were prepared. Reporter expression was evident only outside of the usual empty spiracles expression domain. These elements are not sufficient alone; a combinatorial model is proposed. Conserved discrete areas within a homeobox gene regulatory element, which function as homeodomain protein transcription factor binding sites, are used in a combinatorial fashion to regulate these developmentally important genes (Taylor, 1998).

Unlike gap genes in the trunk region of Drosophila embryos, gap genes in the head were presumed not to regulate each other's transcription. However, in tailless loss-of-function mutants the empty spiracles expression domain in the head expands, whereas it retracts in tll gain-of-function embryos. A 304bp element in the ems-enhancer is sufficient to drive expression in the head and brain; it contains two Tll and two Bcd binding sites. Transgenic reporter gene lines containing mutations of the Tll binding sites demonstrate that tll directly inhibits the expression of ems in the early embryonic head and the protocerebral brain anlage. These results are the first demonstration of direct transcriptional regulation between gap genes in the head (Hartmann, 2002).

The protein product of the anterior maternal system gene, bcd, is a morphogen and differentially directs the expression pattern of the first zygotic genes in the anterior region of the embryo. This is thought to be achieved by differences in the affinity of the Bcd binding sites within the promotors of these zygotic genes. Thus, the broadly expressed gap gene hb contains strong Bcd binding sites and requires only a low level of Bcd for its activation. In contrast, the cephalic gap genes ems, orthodenticle (otd), buttonhead (btd) and sloppy paired (slp), whose expression patterns are restricted to anterior regions of the embryo, are presumed to contain low affinity Bcd binding sites requiring high levels of Bcd for their activation. So far, Bcd binding sites have only been mapped for otd, and it is assumed that these binding sites have a low affinity for Bcd. A 304 bp fragment of the ems enhancer that is sufficient to generate an ems like expression pattern in the head primordium contains two Bcd consensus sites that bind Bcd in vitro. These sites in the ems enhancer element are medium affinity binding sites. This might reflect the fact that ems is expressed posterior to otd and thus requires a lower threshold level of Bcd for its activation compared to otd. Mutations of these Bcd binding sites show that they are also essential for the in vivo function of this enhancer element during early head patterning. This suggests that Bcd, or a protein with similar binding specificity, directly activates ems expression in the head primordium. The only known protein with a similar binding specificity as Bcd is Otd. Since ems activation is independent of otd, it is posited that Bcd directly regulates early ems expression (Hartmann, 2002).

Embryos lacking both maternal and zygotic hb display a reduction and an anterior shift of ems and btd expression at the blastoderm stage. Thus, it has been proposed that head-specific ems expression at the blastoderm stage requires synergistic activation by bcd and hb. However, no hb consensus site could be detected within the 304 bp enhancer element. It cannot be excluded that hb binding sites exist in the ems enhancer outside this element. However, the results suggest that hb plays a relatively minor role in ems expression control in the head and brain (Hartmann, 2002).

If Bcd is responsible for activating ems and determining its posterior expression border, how is the anterior expression border of ems established? This study provides several lines of evidence that indicate that the anterior border of ems expression is set up by repression from another gap gene, namely tll. Thus, in tll mutants, ems expression expands anteriorly, which suggests that the absence of tll results in a derepression of ems transcription in this domain. Moreover, two consensus tll target sites have been identified in the ems enhancer that bind Tll in vitro and which are essential for the function of this element in vivo. Mutation of these Tll binding sites results in an anterior expansion of ems reporter gene expression in the head primordium in a manner similar to the tll loss-of-function phenotype. Therefore, it is proposed that Tll directly inhibits ems expression in the head primordium (Hartmann, 2002).

The gap genes that are expressed in the trunk region of the embryo tightly regulate each others expression domains and show only little overlap. Most of the head gap genes, on the contrary, were expressed in largely overlapping domains and were so far presumed not to interact with each other, but can be regulated by terminal gap genes. Otd is regulated by the gap gene huckebein and btd is under the control of tll (Hartmann, 2002 and references therein).

This study gives another example of two gap genes in the head, tll and ems, which behave like gap genes in the trunk, in that they are expressed in nonoverlapping domains and directly interact with each other. It remains to be seen whether ems in turn acts as repressor of tll transcription (Hartmann, 2002).

Interestingly, the 304 bp region in the ems enhancer, which is necessary and sufficient to drive expression appropriately at the blastoderm stage, is also sufficient to control later ems expression in neurectoderm and brain domains. It could be imagined, however, that Tll plays no direct role in the regulation of these later ems expression domains anymore. Thus, a loss of Tll could lead to shifts in the blastoderm fate map of the embryo and all changes in the ems expression pattern thereafter would be a consequence of these fate map changes. It has been shown, however, that mutations of the two Tll binding sites in a transgenic reporter line result in an expansion of reporter gene expression in the preantennal neurectoderm and the protocerebral brain anlage at later stages. This suggests that the two Tll binding sites, which are located within the ems enhancer element IV, are also necessary for tll mediated inhibition of ems expression in more anterior neurectoderm regions (the preantennal segment) or in the most anterior neuromere of the brain (the protocerebral brain anlage) (Hartmann, 2002).

The fact that such a small enhancer element can drive both early and late ems expression is remarkable. A 900 bp fragment in the otd enhancer, which has been shown to be responsible for blastoderm expression, can not drive later expression in the embryo (Hartmann, 2002 and referencs therein).

Comparative studies carried out in fish and mouse have uncovered two conserved short motifs within the enhancer of the Otx2 gene that were found to be required for mesencephalic neural crest expression. One of these motifs (TAAATCTG) showed similarities to a repeat unit that was identified in a head-specific enhancer element for otd expression in Drosophila (ATCT). Surprisingly, a similar motif is also found repeated three times in the 304 bp ems enhancer element (AATCT). It would be interesting to determine whether a similar control element or binding domains for Tlx, the vertebrate tll homolog, exist in the cis-regulatory control region of Emx genes (Hartmann, 2002).

empty spiracles is required for the development of the Drosophila larval filzkörper, which are structural specializations of the eighth abdominal segment. Filzkörper development is also dependent on the function of the homeotic selector gene Abdominal-B (Abd-B). ems is a downstream gene that is transcriptionally regulated by Abd-B proteins. This regulation is mediated by an Abd-B-dependent ems cis-regulatory element that in early- to mid-stage embryos is activated only in the eighth abdominal segment. Genetic epistasis tests suggest that both ems and Abd-B are required in combination for the specification of the filzkörper primordia (Jones, 1993).

Variations of the ems expression pattern in bicoid mutants suggest that the morphogen protein produced by bicoid has a concentration-dependent regulatory role in the establishment of head-specific ems expression. In contrast, the metameric ems expression pattern is initiated independent of Bicoid protein, and ems becomes expressed at high levels in the primordia of the duplicated filzkörper that develop in the anterior half of bicoid mutant embryos (Dalton, 1989 and Walldorf, 1992).

In Drosophila the Polycomb group genes are required for the long-term maintenance of the repressed state of many developmentally crucial regulatory genes. Their gene products are thought to function in a common multimeric complex that associates with Polycomb group response elements (PREs) in target genes and regulates higher-order chromatin structure. The chromodomain of Polycomb is necessary for protein-protein interactions within a Polycomb-Polyhomeotic complex. Posterior sexcombs protein coimmunoprecipitates Polycomb and Polyhomeotic, indicating that all three are members of a common multimeric protein complex. The B promoter of Abdominal-B is devoid of all three PcG proteins. Ph and Psc are not associated with the peak Pc binding element A (overlapping the gamma promoter). However, other fragments in the vicinity of gamma and C promoters are associated with Ph and/or Psc, and it may be that this regulatory region is unusually complex and contains several PREs that regulate the different Abd-B promoters. Both Ph and Psc are enriched for a restriction fragment in the 3' region of Abd-B, which is relatively poorly enriched by Pc. This element is strongly associated with GAGA factor. In the empty spiracles gene Psc is associated with an upstream fragment, covering a previously identified ems enhancer element. Pc and Ph are not found at this transcribed locus. These results suggest that there may be multiple different Polycomb group protein complexes which function at different target sites. Polyhomeotic and Posterior sexcombs are also associated with expressed genes. Polyhomeotic and Posterior sex combs may participate in a more general transcriptional mechanism that causes modulated gene repression, whereas the inclusion of Polycomb protein in the complex at PREs leads to stable silencing (Strutt, 1997).

Genetic evidence is presented showing that lines, a Drosophila segment polarity gene that has yet to be cloned, is required for the function of the Abdominal-B protein. In lines mutant embryos Abdominal-B protein expression is normal but is incapable of promoting its normal function: formation of the posterior spiracles and specification of an eighth abdominal denticle belt. The tail and A8 segment of lin embryos are highly abnormal. The A8 denticle belt is replaced by naked cuticle that occasionally forms a few denticles less pigmented than the normal ventral denticles. This abnormal A8 cuticle does not resemble the cuticle of any region of the wild-type or of the lin mutant embryo. The absence of anal pads and the abnormal hindgut suggests abnormal development of abdominal segment 11, however, other aspects of the tail development are normal, such as the formation of an anal tuft. In lin embryos the sensory organs are formed at roughly correct positions but have an abnormal shape (Castelli-Gair, 1998).

The Abd-B gene directs the formation of the posterior spiracles by controlling downstream target genes. The defects associated with lines mutation arise because in lines mutant embryos the Abdominal-B protein cannot activate its direct target empty spiracles (ems) or other downstream genes, such as cut(ct) and spalt(sal), while it can still function as a repressor of Ultrabithorax and abdominal-A. ems is one gene required for the formation of posterior spiracles. ems expression in the posterior spiracles is regulated by Abd-B. In lin embryos the transcription of ems is not activated in the posterior spiracles, showing that lin is required for Abd-B to activate its direct downstream target. The other putative Abd-B downstream targets (cut and spalt are also required for the normal development of the posterior spiracles. The activation of ct and sal in the anlage of the posterior spiracles requires Abd-B function but their activation remains independent of one another and of ems, suggesting that all three genes are independently controlled by Abd-B. In lin mutants neither ct nor sal are activated in the anlage of the posterior spiracles. These results show that in lin mutant embryos, Abd-B is incapable of activating some of its targets. The requirement of lines for Abd-B function is not a specific property of the A8 segment. In wild-type embryos, ectopic Abd-B expression using the GAL4 targeting system results in the formation of ectopic posterior spiracles in segments anterior to A8. In contrast, ectopic Abd-B expression in lin mutants does not form ectopic posterior spiracles showing that no matter where the Abd-B protein is expressed in the embryo it requires lines to be fully functional (Castelli-Gair, 1998).

The effect of lin on Abd-B can be explained at the molecular level if lin is required for protein posttranscriptional modification or as a transcriptional cofactor of Abd-B. There is some evidence that the Abd-B protein is posttranslationally modified. If Lin were mediating this process, it would imply that such posttranscriptional modification is functional in vivo. Alternatively if Lines is a transcriptional cofactor of Abd-B, Lines would be interacting with Abd-B in a similar way to that proposed for Extradenticle with Ubx and Abd-A, or Ftz-F1 with Ftz. It is interesting that Exd does not have any effect on Abd-B protein binding or function, and that lin is specific for Abd-B but not for the other Hox genes tested. This suggests that different HOX proteins use different cofactors that contribute to the DNA binding specificity of the HOX proteins (Castelli-Gair, 1998).

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).

To follow the movements of the spiracular chamber cells as they invaginate, constructs were examined that were made with particular enhancers of the cut, ems, and grh genes, each of which drive expression of beta-gal in a subset of cells that express the cut gene at stage 11. These enhancers do not drive the whole spiracular expression of their genes, but are good tools for studying cell specification and the morphogenetic movements of the posterior spiracle cells. The expression of cut in the posterior spiracle is controlled by at least three different enhancers, two of which have been used in this study. From stage 13, the ct-A4.2 enhancer marks the precursors of the four spiracular hairs. The grh-D4 enhancer of the grh gene is expressed in a single group of cells in this area. The expression of ems in the spiracle is driven by at least by one enhancer: ems-1.2. From stage 11 this enhancer marks a group of cells abutting the tracheal pit. Double stainings of the cut-D2.3, ems-1.2, and grh-D4 lacZ constructs show that they are expressed in non-overlapping subsets of cells. The correlation of the expression of these three constructs allows the fate mapping of the spiracular chamber primordium when it is a two dimensional sheet of cells. The different spatial expression of these enhancers at stage 11 shows that the two-dimensional sheet of cells is already patterned and that the cells invaginate to precise positions during development (Hu, 1999 and references therein).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

To maintain cell identity during development and differentiation, mechanisms of cellular memory have evolved that preserve transcription patterns in an epigenetic manner. The proteins of the Polycomb group (PcG) are part of such a mechanism, maintaining gene silencing. They act as repressive multiprotein complexes that may render target genes inaccessible to the transcriptional machinery, inhibit chromatin remodelling, influence chromosome domain topology and recruit histone deacetylases (HDACs). PcG proteins have also been found to bind to core promoter regions, but the mechanism by which they regulate transcription remains unknown. To address this, formaldehyde-crosslinked chromatin immunoprecipitation (X-ChIP) was used to map TATA-binding protein (TBP), transcription initiation factor IIB (TFIIB) and IIF (TFIIF), and dHDAC1 (RPD3) across several Drosophila promoter regions. Binding of PcG proteins to repressed promoters does not exclude general transcription factors (GTFs) and depletion of PcG proteins by double-stranded RNA interference leads to de-repression of developmentally regulated genes. PcG proteins interact in vitro with GTFs. It is suggested that PcG complexes maintain silencing by inhibiting GTF-mediated activation of transcription (Breiling, 2001).

For X-ChIP analysis of promoter regions, the following PcG target genes were chosen: Abdominal-B (Abd-B, B-promoter), iab-4, abdominal-A (abd-A, AI-promoter) and Ultrabithorax (Ubx), all located in the Bithorax complex (BX-C), engrailed (en) and empty spiracles (ems). Also chosen were RpII140 (the subunit of RNA polymerase II with relative molecular mass 140,000 [Mr 140K]) and brown (bw): these last two do not reside in PC binding sites on polytene chromosomes and thus are most probably not PcG regulated. Expression of these genes in Drosophila SL-2 culture cells was assessed by polymerase chain reaction with reverse transcription (RT-PCR) and it was found that Abd-B and RpII140 are transcribed whereas iab-4, abd-A, Ubx, en, ems and bw are inactive (Breiling, 2001).

Acetylation of histones H3 and H4 is considered to be a mark for ongoing transcription. Thus, the promoters of the genes were screened for the presence of amino-terminally acetylated H4 and H3 by X-ChIP. Two antisera were used, one that recognizes H4 acetylated at lysine 12 and one or more other lysines, and one that recognizes H3 acetylated at lysines 9 and/or 18. H4 was found generally acetylated across the promoter regions analysed, in some cases with reduced levels in upstream and downstream regions. H3 is strongly acetylated in the active Abd-B and RpII140 promoters, whereas the inactive loci (iab-4, abd-A, Ubx, en, ems and bw) showed a decrease (5-10 times less than the H3 signal in the active Abd-B and RpII140 promoters) or absence of acetylation both at the core promoters as well as downstream of the initiator. Thus, H3 is acetylated in the active but underacetylated in the inactive promoters, whereas H4 acetylation shows no such changes. Acetylation of histones H3 and H4 seems to be regulated independently across the BX-C, consistent with results in other systems (Breiling, 2001).

The same promoter regions were analyzed by X-ChIP using antibodies against the PcG proteins Polycomb (PC) and Polyhomeotic (PH), dHDAC1, TBP, TFIIB and TFIIF (RAP 30 subunit, associated with RNA polymerase II). All six proteins were found in the core promoter regions (200 base pairs [bp] around the initiator) of the Abd-B, iab-4, abd-A, Ubx, en and ems transcription units. PC was found in most regions both upstream and downstream of the transcription start site (Breiling, 2001).

The major conclusion from this work is that promoters constitute a key target of PcG function. Evidence is provided that, unexpectedly, GTFs are retained at PcG-repressed promoters and that PcG proteins may function through direct physical interactions with GTFs. This mechanism of transcriptional regulation may provide both transcriptional competence and the flexibility necessary for the rapid re-arrangement of patterns of gene expression in response to developmental signals. Thus, the presence of GTFs and some trxG proteins at PcG-repressed promoters would allow a relatively fast re-activation of these genes, as differentiation processes require. In this context, PcG proteins would need to be continuously present at target gene promoters to constitutively inhibit transcription, a prediction supported by the finding that PcG-repressed genes are re-expressed in cells depleted of PcG proteins by dsRNA interference (Breiling, 2001).

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. This study describes a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. Fifteen deficiencies were found that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways; this suggests an extensive use of signaling in the control of Hox gene function (Marabet, 2002).

This study surveyed 60% of the genome and 11 genomic regions were found acting as recessive activators of ems HRE; 4 were found acting as recessive repressors of ems HRE, and 18 were found acting as recessive repressors of wg HRE. So far, the only known gene in addition to AbdB required for ems activation is lines. Df(2R)H3E1, which uncovers lines, has been recovered from the screen for AbdB modifiers. A search for discrete mutations that reproduce the deficiency phenotypes allowed identification of four ems HRE modifier genes: dally, ds, scw, and ttk. Although ttk and scw have already been linked to filzkörper development, none of the four genes had previously been involved in the control of ems expression in posterior spiracles. The screen for AbdA modifiers was restricted to genomic regions leading to ectopic activation of the wg HRE; these response elements relate to functions that repress the enhancer. Accordingly, genomic regions or genes already known to play a role in wg activation, such as abdA, exd, hth, or genes coding for components of the Dpp signaling pathway, were not recovered. Five mutations at specific loci reproduce the phenotypes caused by original deficiencies. Four of these mutations identify tsl, ttk, and genes encoding a putative MPK and a putative CBP as candidate modifiers of wg HRE. None of these genes has so far been involved in the regulation of wg in the visceral mesoderm (Marabet, 2002).

Three of the four candidate genes identified from the ems screen encode molecules acting in or acting in connection with signal transduction pathways. The Scw protein is a secreted factor of the TGF-ß family. The loss of ems expression induced by Brk, a potent repressor of the Dpp/TGF-ß target gene, strongly supports this hypothesis. The involvement of additional signaling pathways in the regulation of ems is more indirectly suggested by the identification of ds and dally that act in connection with several signaling pathways. ds codes for a calcium-dependent cell adhesion molecule of the cadherin superfamily and genetically interacts with shotgun and rhomboid, two genes involved in epidermal growth factor (EGF) signaling, as well as with armadillo (arm), which produces a nuclear effector of the Wg transduction pathway. dally encodes a heparin sulfate proteoglycan involved in the reception of Wg. Although additional experiments are required to firmly establish the involvement of the Wg and EGF pathways, the integration of multiple signals seems to be required for accurate ems regulation by AbdB (Marabet, 2002).

Two modifier genes obtained from the wg screen are presumably involved in the signal transduction cascade. The first, tsl, encodes a ligand for the RTK Torso receptor and the second encodes a putative MKP. Signaling by Ras/MAPK could thus be part of the genetic network that controls wg expression in the midgut, which has been confirmed by showing that wg transcription is impaired by a constitutive active form of Ras. Interestingly, the Ras/MAPK pathway has been implicated in regulation of the Ubx and lab enhancer in the central midgut, and the ETS-domain-containing transcription factor Pointed, which acts as a nuclear effector of the Ras/MAPK pathway, is expressed in the third midgut chamber (Marabet, 2002).

Several modifiers of wg and ems HRE activities identified in this study encode molecules acting in signal transduction cascades. This indicates that signaling processes play important roles in the control of Hox gene function and extends previous observations from a screen for modifiers of a dominant Pb phenotype. Understanding how cell signaling and transcriptional control by Hox protein are mechanistically integrated requires further study (Marabet, 2002).

Targets of Activity

sloppy paired, in addition to its roles as a segment polarity gene and as a pair rule gene, acts like a gap gene in the head. All three maternal systems active in the cephalic region are required for proper slp expression. Dorsal the morphogen of the dorsoventral system, and Empty spiracles act as repressor and corepressor in the regulation of slp (Grossnicklaus, 1994).

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wingless and hedgehog, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression (Mohler, 1995).

There is no collier expression in embryos from bicoid mothers or in buttonhead homozygous mutant embryos. In empty spiracles mutant embryos, col expression expands ventrally to include mesodermal precursor cells. These results indicate that col acts downstream of the head gap genes in the transcription regulatory cascade that patterns the anterior part of the head, with buttonhead being absolutely required for col activation. In string mutant embryos, col expression during gastrulation is the same as that in wild-type embryos, indicating that the expression of col is independent of the mitotic program of cells in mitotic domain 2 (Crozatier, 1996).

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. The phenotypic analysis is reported of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. col expression is first detected during the interphase of mitotic cycle 14, when expression of head-gap genes has already resolved from initial broad domains into defined stripes. The stripe of col expression is included in that of btd, overlaps that of ems, and is restricted both dorsally and ventrally to neuroectodermal cells. Examination of dorsal (dl) mutant embryos shows that Dl is required for col repression in the mesodermal plate. The ectopic expression of col observed in twist (twi) and snail (sna) mutant embryos suggests that Dl target genes, rather than Dl itself, are involved. Embryos lacking ems function also show a ventral derepression of col expression. Further, at stage 10, ems mutant embryos show an abnormal pattern of col mRNA accumulation, with a mandibular stripe in addition to intercalary stripe of col-expressing cells. This suggests a second role for ems in regulating col. In btd mutant embryos, there is a complete loss of col expression, whereas there is no change in embryos lacking both slp (slp1 and slp2) genes, consistent with previous data establishing that btd but not slp is required for intercalary en and wg expression (Crozatier, 1999).

Phenotypic suppression

In the absence of the eight genes of the homeotic cluster (HOM-C), which specify the identity of head, thoracic and abdominal segments, thoracic and abdominal structures develop a 'ground' pattern which includes cephalic structures called sclerotic plates. These plates are specified by empty spiracles. EMS has the potential to induce sclerotic plates, but this potential is suppressed by the HOM-C genes, including labial, the most anteriorly expressed homeotic gene.

This suppression does not occur at the transcriptional or translational level, since EMS protein reaches normal levels in the cells exhibiting suppression; consequently the phenomenon is termed 'phenotypic suppression.' The phenomenon is used to explain posterior dominance, the observation that a homeotic gene product will have an effect only in the body region anterior to the normal domain of the gene.

Posterior dominance is given an evolutionary context by the argument that the primordial segment pattern is thoracic-like and that head structures are formed by modifying an archetypal thoracic-like pattern. Thus, it is argued that ems thoracic pattern represents the primitive function. In Drosophila and other dipterans, the homeotic complex may be falling apart: the ANTP and BX subcomplexes have already separated, whereas in other insects they are linked. In mammals the original linkage is conserved. It is also suggested that perhaps ems may have been a part of the earlier intact complex (Macías, 1996).

Protein Interactions

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).

The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).

Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).

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