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tailless REGULATION Targets of Activity In the anterior domain Tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed. In its posterior domain of action, Tailless is responsible for the establishment ofAbdominal-B expression and demarcating the posterior boundary of the initial domain of expression of Ultrabithorax (Reinitz, 1990). Cis-acting elements for the expression of buttonhead head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter, The four maternal coordinate systems are necessary for correct btd head stripe expression, most likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on bicoid. bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, tailless, a torso dependent repressor of btd, takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity (Wimmer, 1995).The terminal genes of Drosophila specify non-segmented regions of the larval body that are derived from the anterior and posterior regions of the early embryo. Terminal class genes include both maternal-effect loci (typified by the receptor tyrosine kinase Torso) that encode components of a signal transduction cascade and zygotic genes (e.g. Tailless and Huckebein) that are transcribed at the poles of the embryo in response to the local activation of the pathway. A zygotic gene, bowel, has been characterized that is a zinc finger homolog of the pair-rule segmentation gene odd-skipped. bowel transcripts are initially expressed at both poles of the blastoderm embryo and in a single cephalic stripe. This pattern depends upon Torso and Tailless activity, but is not affected in huckebein mutants. Five mutations that affect the Bowel protein were isolated and sequenced, including a nonsense mutation upstream of the zinc fingers and a missense mutation in a putative zinc-chelating residue. bowel mutants die as late embryos with defects in terminal derivatives including the hindgut and proventriculus. These results indicate that the developmental roles of odd-skipped and bowel have diverged substantially, and that bowel represents a new member of the terminal hierarchy that acts downstream of tailless and mediates a subset of tailless functions in the posterior of the embryo (Wang, 1996).The expression of the pair-rule gene hairy (h) in seven evenly spaced stripes along the longitudinal axis of the Drosophila blastoderm embryo ismediated by a modular array of separate stripe enhancer elements. The minimal enhancer element, which generates reporter geneexpression in place of the most posterior h stripe 7 (h7-element), contains a dense array of binding sites for factors providing thetrans-acting control of h stripe 7 expression as revealed by genetic analyses. The stripe seven enhancer is found in a minimal 932 bp region from a 1.5 kb DNA fragment of the h upstream region. The h7-element mediates position-dependent geneexpression by sensing region-specific combinations and concentrations of both the maternal homeodomain transcriptional activators,Caudal and Bicoid, and of transcriptional repressors encoded by locally expressed zygotic gap genes. Zygotic caudal expression is not required for activation. Caudal and Bicoid, which formcomplementing concentration gradients along the longitudinal axis of the embryo, function as redundant activators, indicating that theanterior determinant Bicoid is able to activate gene expression in the most posterior region of the embryo. The spatial limits of the hstripe-7 domain are brought about by the local activities of repressors that prevent activation. The spatial limit of h7 is significantly altered in the gap mutants tailless, knirps and kruppel, but not in embryos lacking either hunchback, giant or huckebein. There are seven binding sites for Bcd, twenty-three for caudal, five for Kruppel, fourteen for Knirps, eight for Hunchback and five for Tailless. In the absence of both cad and bcd, activation still occurs. Thus, a third activator, likely to be Kr, must function in such embryos. It is thought that Kr acts as both a repressor and an activator within the h7 element depending on its concentration. The posterior border is set in response to Tll activity under the control of the terminal maternal organizer system. The anterior border of the expression domain is due to repression in response to Kni. The results suggest that the gradientsof Bicoid and Caudal combine their activities to activate segmentation genes along the entire axis of the embryo (La Rosee, 1997). Drosophila pair-rule gene expression, in an array of seven evenly spaced stripes along theanterior-posterior axis of the blastoderm embryo, is controlled by distinct cis-acting stripe elements. Inthe anterior region, such elements mediate transcriptional activation in response to (1) the maternalconcentration gradient of the anterior determinant Bicoid and (2) repression by spatially distinct activitiesof zygotic gap genes. In the posterior region, activation of hairy stripe 6 has been shown to depend onthe activity of the gap gene knirps, suggesting that posterior stripe expression is exclusively controlledby zygotic regulators. The zygotic activation of hairy stripe 6 expression is precededby activation in response to maternal caudal activity. Thus, transcriptional activation of posterior stripeexpression is likely to be controlled by maternal and zygotic factors as has been observed for anteriorstripes. To establish the potential of Cad and Kni to interact with the cis-acting DNA that mediates hairy stripe 6-like expression in the embryo, in vitro footprinting experiments were performed with the 532 bp hairy stripe 6-element DNA. Cad and Kni bind to thirty six in vitro binding sites, some of which overlap, throughout the element. The sequence of the Cad and Kni binding sites matches the consensus described for each of the two proteins. Most of the potential Cad and Kni binding sites are close to or overlapped by binding sites for Kruppel (eight sites), Hunchback (eight sites), and Tailless (five sites). Tests using fragments of the 532 bp enhancer and of another element, 284-HT, show that sequences mediating activation of reporter expression are not maintained within a minimal activation element but instead are dispersed throughout the enhancer (Hader, 1998). A 500 bpDNA fragment from an ehancer region of Ultrabithorax, approximately 30 kb away from the structural gene,contains one of the distant UBX regulatory elements (bx region enhancer, BRE). Hunchback repressesUBX expression directly by binding to BRE and probably other Ubx regulatory elements. Inaddition, the BRE pattern requires input from other segmentation genes, among them tailless andfushi tarazu but not Krüppel and knirps (Qian, 1993).The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatialexpression of the segment polarity genes, wg and hh, have been analyzed at the late blastoderm stageand during subsequent development. Both wg and hh are normally expressed at blastoderm stagein 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 cephalicregion: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and emsregulate hh blastoderm expression. (Mohler, 1995). The tll gene has negative effects on gap genes knirps, Krüppel and giant, setting up their posterior borders of expression (Huelskamp, 1991).The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early duringcellularization in the presumptive abdomen in a broad domain that soon resolves into two stripes.This expression pattern is regulated by the same mechanisms that define gap gene expressiondomains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless,and the gradient morphogen encoded by hunchback. The resolution into two stripes is controlledby the gap gene knirps (Cockerill, 1993). Tailless activates specific domains of expression of caudal and forkhead and Krüppel (Mlodzik, 1987 and Gaul, 1991). Terminal gap genes tailless and huckebein direct theformation of the posterior hunchback stripe. The anterior border of the posterior hb stripeis determined by TLL concentration in a manner analogous to the activation of anterior hb expressionby Bicoid (Margolis, 1995). Tailless protein activates the seventh stripe of expression of at least three pair-rule genes: even-skipped, hairy and fushi tarazu. tailless alleles can be placed in the same order of phenotypic strength on the basis of the deletion of either external structures (A8 and anal pads) or an internal structure (hindugt). Furthermore, different tll alleles result in a gradation in levels of T-related gene expression and these levels of Trg expression are correlated with the size of the differentiated hindgut (references in Diaz, 1996).Tailless is required for the normal pattern of the paired protein in the embryo. Specifically, stripes 6 and 7 are broader and shift posteriorly while in tailless mutants stripe 8 never appears (Gutjahr, 1993).There are several distinct phases of runt expression in the early embryo. Each phase depends on a different set of regulators. The first phase of expression is a broad-field of mRNA accumulation in the central regions of syncytial blastoderm stage embryos. This pattern is due to terminal repression by the anterior and terminal maternalsystems. The effect of the terminal system, even at this early stage, is mediated by two zygotic gapgenes, tailless and huckebein. A 7 stripe pattern of Runt mRNA accumulation emerges during the process of cellularization. The initial formation of this pattern depends on position-specificrepression by zygotic gap genes (Klingler, 1993).Ectopic expression of the pair-rule gene runt in the anterior end of the Drosophila embryo antagonizestranscriptional activation of the head gap gene orthodenticle (otd) by the anterior morphogen bicoid.The relevance of runt's activity as a repressor of otd in normal Drosophilaembryogenesis has been investigated. otd expression is activated in the posterior region of embryos that are mutant for runt.This posterior expression domain of otd depends on the activity of the orphan nuclear receptor proteinTailless. Repression of otd by runt does not require the conserved VVVRPY motif, which mediatesinteraction between Runt and the co-repressor protein Groucho. It is speculated that the genetic interactions between runt and tll involve physical interactions between the two proteins. It is interesting to note that interactions between Runt and another orphan nuclear receptor protein, Ftz-F1 have been invoked to explain runt's regulation of the pair-rule gene fushi tarazu. However, in this case runt functions to activate, rather than repress Ftz-F1 dependent transcription. It will be interesting to determine if there are binding sites for Tll that are essential for the activation of otd in the posterior region and whether these sites respond to the repressive activity of runt. It is noted that the activity of tll is necessary, but not sufficient for otd expression in the posterior region of the embryo.The observed functional interactionsbetween runt and tailless on otd expression may indicate there are other contexts where members ofthese two families of transcriptional regulators interact to regulate gene expression during development (Tsai, 1998).An effect on the early stripe of Goosecoid expression is observed in sloppy-paired, orthodenticle, tailless and decapentaplegic mutants. Both slp and otd affect Gsc in a similar way: the early stripe of Gsc appears normally but at the end of the cellularization stage, there is no reinforcement of its expression and it is prematurely lost. dpp is necessary to bring aboud Gsc repression in the dorsal-most region of the embryo, while tll is required to promote Gsc expression in the lateral region, or to prevent its repression by the dorsoventral patterning system (Goriely, 1996). Tailless activates the Drosophila T-related gene, the homolog of vertebrate Brachyury gene (Kispert, 1994).By examining expression of arc in different mutant embryos,it was determined that transcription factors known tobe required for patterning and maintenance of variousdeveloping epithelia control arc expression in those domains. tll and hkb, whichare required to pattern the posterior 15% of the embryo, control arc expressionin the posterior midgut primordium. fkh, which appears toact as a maintenance, or permissive, transcription factor, isrequired for expression of arc throughout the gut. byn,which is required for hindgut development and specifies itscentral domain (the large intestine),controls expression of arc in the elongating hindgut. Kr andcut, required for evagination and extension of the Malpighiantubule budscontrol expression of arc in the tubule primordia (Liu, 2000).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 tllgain-of-function embryos. A 304bp element in the ems-enhanceris 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 expressionpattern of the first zygotic genes in the anterior region of theembryo. This isthought to be achieved by differences in the affinity of theBcd binding sites within the promotors of these zygoticgenes. Thus, the broadly expressed gap gene hb containsstrong Bcd binding sites and requires only a low level ofBcd 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 itis assumed that these binding sites have a low affinity forBcd. A 304 bp fragment of the ems enhancer that issufficient to generate an ems like expression pattern in thehead primordium contains two Bcd consensus sites thatbind Bcd in vitro. These sites in the ems enhancer elementare medium affinity binding sites. This might reflect the factthat ems is expressed posterior to otd and thus requires alower threshold level of Bcd for its activation compared tootd. Mutations of these Bcd binding sites show that theyare also essential for the in vivo function of this enhancerelement during early head patterning. This suggests thatBcd, or a protein with similar binding specificity, directlyactivates ems expression in the head primordium. The onlyknown 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 reductionand an anterior shift of ems and btd expression at the blastoderm stage. Thus, it has been proposed that head-specific emsexpression at the blastoderm stage requires synergistic activationby 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 determiningits posterior expression border, how is the anterior expressionborder of ems established? This study provides severallines of evidence that indicate that the anterior border ofems expression is set up by repression from another gapgene, namely tll. Thus, in tll mutants, ems expressionexpands anteriorly, which suggests that the absence of tllresults in a derepression of ems transcription in this domain.Moreover, two consensus tll target sites have been identified inthe ems enhancer that bind Tll in vitro and which areessential for the function of this element in vivo. Mutationof these Tll binding sites results in an anterior expansion ofems reporter gene expression in the head primordium in amanner similar to the tll loss-of-function phenotype. Therefore,it is proposed that Tll directly inhibits ems expressionin the head primordium (Hartmann, 2002).The gap genes that are expressed in the trunk region ofthe embryo tightly regulate each others expression domainsand show only little overlap. Most of the head gap genes, on the contrary,were expressed in largely overlapping domains and were sofar presumed not to interact with each other, butcan be regulated by terminal gap genes. Otd is regulated bythe gap gene huckebein and btd is underthe control of tll (Hartmann, 2002 and references therein).This study gives another example of two gap genes in thehead, tll and ems, which behave like gap genes in the trunk,in that they are expressed in nonoverlapping domains anddirectly 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 appropriatelyat the blastoderm stage, is also sufficient to controllater ems expression in neurectoderm and brain domains. Itcould be imagined, however, that Tll plays no direct rolein the regulation of these later ems expression domainsanymore. Thus, a loss of Tll could lead to shifts in theblastoderm fate map of the embryo and all changes in theems expression pattern thereafter would be a consequence ofthese fate map changes. It has been shown, however, that mutationsof the two Tll binding sites in a transgenic reporter lineresult in an expansion of reporter gene expression in thepreantennal neurectoderm and the protocerebral brainanlage at later stages. This suggests that the two Tll bindingsites, which are located within the ems enhancerelement IV, are also necessary for tll mediated inhibitionof ems expression in more anterior neurectoderm regions(the preantennal segment) or in the most anterior neuromereof the brain (the protocerebral brain anlage) (Hartmann, 2002).The fact that such a small enhancer element can driveboth early and late ems expression is remarkable. A900 bp fragment in the otd enhancer, which has been shown tobe responsible for blastoderm expression, can not drivelater expression in the embryo (Hartmann, 2002 and referencs therein).Comparative studies carried out in fish and mouse haveuncovered two conserved short motifs within the enhancerof the Otx2 gene that were found to be required for mesencephalicneural 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).Dynamical analysis of regulatory interactions in the gap gene system of DrosophilaGenetic studies have revealed that segment determination in Drosophilamelanogaster is based on hierarchical regulatory interactions among maternalcoordinate and zygotic segmentation genes. The gap gene system constitutes themost upstream zygotic layer of this regulatory hierarchy, responsible for theinitial interpretation of positional information encoded by maternal gradients.A detailed analysis of regulatory interactions involved in gap generegulation is presented based on gap gene circuits, which are mathematical gene networkmodels used to infer regulatory interactions from quantitative gene expressiondata. The models reproduce gap gene expression at high accuracy and temporalresolution. Regulatory interactions found in gap gene circuits provideconsistent and sufficient mechanisms for gap gene expression, which largelyagree with mechanisms previously inferred from qualitative studies of mutantgene expression patterns. These models predict activation of Kr by Cad andclarify several other regulatory interactions. This analysis suggests a centralrole for repressive feedback loops between complementary gap genes.Repressive interactions among overlapping gap genes show anteroposteriorasymmetry with posterior dominance. Finally, these models suggest a correlationbetween timing of gap domain boundary formation and regulatory contributionsfrom the terminal maternal system (Jaeger, 2004b). Although activating contributions from Bcd and Cad show some degree oflocalization, positioning of gap gene boundariesduring cycle 14A is largely under the control of repressive gap-gapcross-regulatory interactions. Thereby, activation is a prerequisite forrepressive boundary control, which counteracts broad activation of gap genes ina spatially specific manner. In addition, gap genes show a tendency towardautoactivation, which increasingly potentiatesactivation by Bcd and Cad during cycle 14A.Autoactivation is involved in maintenance of gap gene expression within givendomains and sharpening of gap domain boundaries during cycle 14A (Jaeger, 2004b). Regulatory loops of mutual repression create positive regulatory feedbackbetween complementary gap genes, providing a straightforward mechanism for theirmutually exclusive expression patterns. Such a mechanism of 'alternatingcushions' of gap domains has been proposed previously. The resultssuggest that this mechanism is complemented by repression among overlapping gapgenes. Overlap in expression patterns of two repressors imposes a limit on thestrength of repressive interactions between them. Accordingly, repressionbetween neighboring gap genes is generally weaker than that betweencomplementary ones. Moreover, repression amongoverlapping gap genes is asymmetric, centered on the Kr domain. Posterior to this domain, only posterior neighborscontribute functional repressive inputs to gap gene expression, while anteriorneighbors do not. This asymmetry is responsible foranterior shifts of posterior gap gene domains during cycle 14A (Jaeger, 2004b). Repression by Tll mediates regulatory input to gap gene expression by theterminal maternal system. Tll provides the main repressiveinput to early regulation of the posterior boundary of posterior gt, and activation by Tll is required for posteriorhb expression. Note that these two featuresform only during cycle 13 and early cycle 14A,while other gap domain boundaries are already present at the transcript levelduring cycles 10-12 and largelydepend on the anterior and posterior maternal systems for their initialestablishment. The delayed formation of posterior patterning features andtheir distinct mode of regulation are reminiscent of segment determination inprimitive dipterans and intermediate germ-band insects, supporting a conserveddynamical mechanism across different insect taxa (Jaeger, 2004b). The set of regulatory interactions presented here provides a consistent andsufficient dynamical mechanism for gap gene expression. Insummary, this set of interactions consists of the following five basicregulatory mechanisms: (1) broad activation byBcd and/or Cad, (2) autoactivation, (3) strong repressive feedback betweenmutually exclusive gap genes, (4) asymmetric repression between overlapping gapgenes, and (5) feed-forward repression of posterior domain boundaries by theterminal gap gene tll. In the following subsections, evidence is discussedconcerning specific regulatory interactions involved in each of these basicmechanisms in some detail (Jaeger, 2004b). Activation by Bcd and Cad: Activation of gap gene expression by Bcd and Cad is supported by the following.Bcd binds to the regulatory regions of hb, Kr, and kni. The kni regulatory region also contains binding sitesfor Cad. The anterior domains of gt and hb areabsent in embryos from bcd mothers. The posterior domain of gt ismissing in embryos mutant for both maternal and zygotic cad, while theposterior domain of kni is absent in embryos mutant for maternalbcd plus maternal and zygotic cad. Theseresults suggest partial redundancy of activation of kni by Bcd,consistent with evidence from zygotic cad embryos from bcdmothers, where maternally provided Cad is sufficient to activate kni (Jaeger, 2004b). Kr expression expands anteriorly in embryos from bcd mothers, which is due to the absence of the anterior gt and hbdomains. Bcd has been shown to activate expression ofKr reporter constructs. The fact that Kr is still expressed inembryos from bcd mutant mothers has been attributed to activation bygeneral transcription factors or low levels of Hb. In contrast, themodels predict that this activation is provided by Cad. Although Krexpression is normal in embryos overexpressing cad, repressivecontrol of Kr boundaries could account for the lack of expansion of theKr domain in such embryos (Jaeger, 2004b). The activating effect of Cad on hb found in gap gene circuits is likelyto be spurious. The anterior hb domain is absent in embryos frombcd mutant mothers,which show uniformly high levels of Cad. Moreover, thecomplete absence of the posterior hb domain in tll mutants suggests activation of posteriorhb by Tll rather than by Cad. It is believed that this spurious activation ofhb by Cad is due to the absence of hkb in gap gene circuits. Theposterior hb domain fails to retract from the posterior pole inhkb mutants, suggesting a repressive role of Hkbin regulation of the posterior hb border. Consistent with this, theposterior boundary of the posterior hb domain never fully forms in any ofthe circuits. Moreover, Tll is constrained to avery small or no interaction with hb dueto the absence of the posterior repressor Hkb, since activation of hb byTll would lead to increasing hb expression extending to the posteriorpole (Jaeger, 2004b). Autoactivation:: A role for autoactivation in the late phase of hb regulation is supported by the fact that the posterior border of anteriorhb is shifted anteriorly in a concentration-dependent manner in embryoswith decreasing doses of zygotic Hb.Weakened and narrowed expression of Kr in mutants encoding a functionallydefective Kr protein suggests Kr autoactivation. Similarly, adelay in the expression of gt in mutants encoding a defective Gt proteinindicates gt autoactivation. However, the results suggest thatgt autoactivation is not essential. It is generally weaker thanautoactivation of other gap genes, andcircuits lacking gt autoactivation show no specific defects in gtexpression. Finally, in the case of kni, there is no experimentalevidence for autoactivation, while some authors have even suggested kniautorepression. No such autorepression has been detected in any gap gene circuit (Jaeger, 2004b). Repression between complementary gap genes: Mutual repression of gt and Kr is supported by the following.gt expression expands into the region of the central Kr domain inKr embryos. In contrast, Kr expression is notaltered in gt mutants before germ-band extension. However, Gt binds to the Kr regulatoryregion, and the central domain of Kr is absent in embryos overexpressinggt. Moreover, Kr expression extends furtheranterior in hb gt double mutants than in hb mutants alone. The above is consistent with this analysis, which shows nosignificant derepression of Kr in the absence of Gt even thoughrepression of Kr by Gt is quite strong (Jaeger, 2004b).Hb binds to the kni regulatory region, and the posterior knidomain expands anteriorly in hb mutants. Embryos overexpressing hbshow no kni expression at all, and embryosmisexpressing hb show spatially specific repression of kniexpression.There is no clear posterior expansion of kni in hb mutants. This could be due to the relatively weak and late repressive contribution of Hb on theposterior kni boundary or due to partial redundancy with repression by Gtand Tll. The posterior hb domainexpands anteriorly in kni mutants, but anterior hb expression isnot altered in these embryos. Nevertheless, a role of Kni in positioning the anterior hbdomain is suggested by the fact that misexpression of kni leads tospatially specific repression of both anterior and posterior hb domains. Moreover, onlyslight posterior expansion of anterior hb is observed in Krmutants, while hb is completely derepressed between its anterior andposterior domains in Kr kni double mutants (Jaeger, 2004b). Repression between overlapping gapgenes: gt, kni, and Kr show repression by their immediateposterior neighbors hb, gt, and kni, respectively. Retraction of posterior Gt from the posterior poleduring midcycle 14A fails to occur in hb mutants, and no gt expression is observed in embryos overexpressinghb. The posterior kni boundary isshifted posteriorly in gt mutant embryos, and kniexpression is reduced in embryos overexpressing gt. Note thatthese effects are very subtle and were not reported in similar studies bydifferent authors. A weak but functional interaction of Gt with kniis consistent with these results. This interaction was found to be essential evenin a circuit where it was deemed below significance level. Finally, Kni has beenshown to bind to the Kr regulatory region, and the central Kr domainexpands posteriorly in kni mutants (Jaeger, 2004b). In contrast, no effect of Kr on hb was detected. However, hb expression expandsposteriorly in Kr mutants. This effect islikely to involve repression of hb by Kni. Kni levels are reduced inKr embryos. hb is completely derepressed between its anterior andposterior domains in Kr kni double mutants, whereas anterior hbdoes not expand at all in kni mutants alone. Taken together these results suggests that there is direct repression of hb by Kr in the embryo,but it is at least partially redundant with repression of hb by Kni (Jaeger, 2004b). Unlike repression by posterior neighbors, no or only weakrepression was found of posterior kni, gt, and hb by their anteriorneighbors Kr, kni, and gt, respectively. Most gap gene circuits show weak activation ofhb by Gt. Graphical analysisfailed to reveal any functional role for such activation. Moreover, no functional interaction was found betweengt and Kni. Although relativelyweak repression of kni by Kr was found in 6 out of 10 circuits, no specific patterning defects could bedetected in the other 4. Consistent with the above, expression of posteriorhb is normal in gt mutants, and both the anterior boundaries ofposterior gt and kni are positioned correctly in kni andKr mutant embryos, respectively (Jaeger, 2004b). Note that activation of kni by Kr, which has been proposed to explaindecreased expression levels of kni in Kr mutants, was never found. The results strongly support the view that this interaction is indirect through Gt, which isfurther corroborated by the fact that kni expression is completelyrestored in Kr gt double mutants compared to that in Kr mutantsalone (Jaeger, 2004b).A significant repressive effect of Hb on Kr was found.Consistent with this, Hb has been shown tobind to the Kr regulatory region, and the central Kr domainexpands anteriorly in hb mutants. However,partial redundancy of this interaction is suggested by correct positioning andshape of the anterior Kr domain in a circuit that does not showrepression of Kr by Hb (Jaeger, 2004b). It has been proposed that Hb plays a dual role as both activator and repressorof Kr. In the framework of the gene circuit model,concentration-dependent switching of regulative action could be implemented byallowing genetic interconnection parameters to switch sign at certain regulatorconcentration thresholds. The current model explicitly does not include such apossibility. Nevertheless, circuits have been obtained that reproduceKr expression faithfully, suggesting thata dual role of Hb is not required for proper Kr expression. Moreover, activation of Kr by Hb was ever observed in any of the circuits. Therefore, the results support a mechanismin which the activation of Kr by Hb is indirect through derepression of kni (Jaeger, 2004b). Repression by Tll: Only a few earliertheoretical approaches have considered terminal gap genes. Gap genecircuits accurately reproduce tll expression. However,in gene circuits, tll is subject to regulation by other gap genes, whichis inconsistent with experimental evidence. In contrast, the correct expression patternof tll in gap gene circuits allows its effect on other gap genes to be studiedin great detail. Strong repressive effects of Tll on Kr,kni, and gt have been found. Tll binding sites havebeen found in the regulatory regions of Kr and kni. Intll mutants, Kr expression is normal, whereas expression of kni expands posteriorly, and theposterior gt domain fails to retract from the posterior pole. No expression of Kr, kni, orgt can be detected in embryos overexpressing tll under aheat-shock promoter (Jaeger, 2004b). Reverse engineering the gap gene network of DrosophilaA fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. A new strategy is described for solving this problem and it is applied to recently published data on early Drosophila development. The method is orders of magnitude faster than current fitting methods and allows fitting of different types of rules for expressing regulatory relationships. Specifically, this approach is sused to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. The technique also allows inference of regulatory relationships de novo or testing network structures suggested by the literature. A series of models is fitted to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on the modeling results and validation against the experimental literature, a revised network structure is proposed for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for, or even inconsistent with, the details of gap gene expression during wild-type development (Perkins, 2006).The regulatory structure of the Combined model is itself sufficient to reproduce all six gap gene domains using either the gene circuit or logical formalisms for production rate functions. Support is cited for the Combined model, and then consider the results of the individual models in light of several outstanding questions about gap gene regulation are discussed (Perkins, 2006).The maternal proteins Bcd and Cad are largely responsible for activating the trunk gap genes, with Bcd being more important for the anterior domains and Cad more important for the posterior domains. Bcd is a primary activator of the anterior hb domain, the anterior gt domain, and the Kr domain. Cad activates posterior gt. The kni domain is present in bcd mutants and in cad mutants, but not in bcd;cad double mutants. This suggests redundant activation by the two maternal factors. Such redundant activation of kni is present in the Unc-GC model. For the other models, the optimization selected one or the other as activators, but not both. Tll is crucial for activating the posterior hb domain, while it represses Kr, kni, and gt, preventing their expression in the extreme posterior. All the regulatory relationships between the gap genes in the Combined model are repressive. The complementary gap gene pairs, hb-kni and Kr-gt are known to be strongly mutually repressive, as was found in nearly all the models. [Repression of hb by Kni is not part of the Rivera-Pomar and Jäckle (RPJ) regulatory relationships (Rivera-Pomar, 1996), but the unconstrained gene circuit (Unc-GC) model and Unc-Logic model (that employs the regulatory structure discovered by the unconstrained gene circuit fit, except that Gt activation of hb and Kni activation of gt were removed) included the link.] The models also suggest that mutual repression between hb and Kr helps to set the boundary between those two domains. A chain of repressive relationships, hb-gt-kni-Kr, causes the shifts in the Kr, kni, and posterior gt domains. Autoactivation by hb is well-established, and there is also some evidence for autoactivation by Kr and gt (Perkins, 2006).Does Hb have a dual regulatory effect on Kr? There is a long-running debate about whether or not low levels of Hb activate Kr. In hb mutants, the Kr domain expands anteriorly, suggesting that Hb represses Kr. However, Kr expression in these mutants is lower than in wild-type and expands posteriorly in embryos overexpressing Hb. Further, in embryos lacking Bcd and Hb, the Kr domain is absent, but can be restored in a dosage-dependent manner by reintroducing Hb. These observations suggest that Hb activates Kr. It has been suggested, therefore, that low levels of Hb activate Kr while high levels repress it. An alternative explanation, however, is that the apparently activating effects of Hb are indirect, via Hb's repression of kni and Kni's repression of Kr. Optimization of the Unc-GC model, which could have resulted in activation or repression of Kr by Hb, but not both, resulted in repression. The RPJ models allow for a dual effect, but activation by Hb was eliminated during optimization of the RPJ-Logic model. The RPJ-GC model retained functional activation and repression of Kr by Hb. However, Kr expression in this model is defective. Kr is not properly repressed in the anterior. Further, Kr is ectopically expressed in a small domain in the posterior of the embryo. Thus, the current models provide no support for activation of Kr by Hb. The only support found, which is crucial in all models except Unc-Logic and also consistent with the mutant and overexpression studies, is for repression of Kr by Hb (Perkins, 2006).What represses hb between the anterior and posterior domains? Another point of disagreement in the literature is what prevents the expression of hb between its two domains. In the model of Rivera-Pomar and Jäckle (1996), repression by Kr is the explanation. The RPJ models confirm that this mechanism is sufficient. Specifically, in these models Kr repression prevents hb expression just to the posterior of the anterior hb domain. Between the Kr and posterior hb domains, there is no explicit repression of hb. Rather, Hb is not produced simply because of a lack of activating factors. In contrast, the models of Jaeger (2004a and b) detected no effect of Kr and attributed repression solely to Kni. The Unc-GC and Unc-Logic models found repression by Kni, but in addition to repression by Kr, not instead of it. Kr is more responsible for repression near the anterior hb domain and Kni is more responsible for repression near the posterior hb domain. This is consistent with observations of expression in mutant embryos. Embryos mutant for Kr show slight expansion of the anterior hb domain, while kni embryos show expansion of the posterior hb domain. In Kr;kni double mutants, hb is completely derepressed between its two usual domains. This suggests, as seen in the Unc-GC and Unc-Logic models, that Kr and Kni are both repressors of hb, that their activity is redundant in the center of the trunk, and that Kr and Kni are the dominant repressors for setting the boundaries of the anterior and posterior domains, respectively. This interpretation was also favored by Jaeger (2004a and b), on the basis of the mutant data, even though Jaeger's models did not find repression by Kr (Perkins, 2006).The posterior hb domain. In all of the current models, the posterior hb domain is activated by Tll and sustained by Tll and hb autoactivation. Rivera-Pomar (1996) did not consider the posterior hb domain, and did not include activation by Tll in his model. That link was added to the RPJ network structure because otherwise it was not possible to capture the posterior hb domain. The model of Jaeger (2004a and b) captured the domain without Tll activation by substituting activation from cad. However, there is no confirming evidence for such an interaction. The absence of posterior hb in tll mutants and the inability of the models to explain posterior hb by other means, leads to the straightforward hypothesis that Tll activates posterior hb. Posterior hb is unique in that the domain begins to form later than the other five domains modeled. In the RPJ models, this happens simply because high levels of Tll are needed to activate hb -- levels that are reached only at about t = 30 min. The Unc-GC and Unc-Logic models also employ repression by Cad to slightly delay Hb production in the posterior. However, there is no confirming evidence for such repression, and it is omitted from the Combined model (Perkins, 2006).Shifting of the Kr, kni, and posterior gt domains. Domain shifting was first observed by Jaeger (2004a and b) and attributed to a chain of repressive regulatory relationships, hb-gt-kni-Kr. The current models largely support the importance of this regulatory chain, particularly the final two links. Repression of Kr by Kni was significant in all of the current models. Repression of kni by Gt was present in all models except RPJ-Logic, where it would be of little impact anyway, since RPJ-Logic has a defective posterior gt domain. Consistent with these findings, Kni binds to the regulatory region of Kr, and the Kr domain expands towards the posterior in kni mutants. Similarly, the kni domain expands posteriorly in gt mutants, while embryos overexpressing gt show reduced kni expression (Perkins, 2006).Repression of gt by Hb is not as well supported by the current models. The Unc-GC model included the link, though the regulatory weight was the smallest of all those in the model. The link was eliminated from Unc-Logic and, of course, not present in the RPJ network structure. Instead, the models utilized decreasing activation by Cad (Unc-GC, Unc-Logic) and repression by Tll (Unc-GC, RPJ-GC) to shift the posterior gt domain. Even with these links, however, shifting of the domain is not well-captured. RPJ-GC appears to capture the posterior gt shift best (47. The models suggest hb autoactivation also plays a crucial role in sustaining the posterior domain, once it has been initiated by Tll, a role not previously emphasized. Autoactivation for the other genes was found by the Unc-GC model, but is not part of the RPJ network structure. It included autoactivation only for Kr and gt in the Combined model, on the basis of a weakened and narrowed Kr domain in embryos producing defective Kr protein and a delay in gt expression in embryos producing defective gt protein. Interestingly, the gene circuit models of Jaeger (2004a and b) also found autoactivation for all four gap genes, but they considered autoactivation by gt to be the weakest and least certain. In contrast, the Unc-Logic model retained gt autoactivation while eliminating autoactivation for Kr and kni. The RPJ-Logic model was unable to reproduce the posterior gt domain. However, it was found that by adding gt autoactivation to the model, it was able to create and sustain posterior gt correctly, bringing the error of the model down to 15.34. This suggests that, after hb, gt is the most likely candidate for autoactivation. However, even this is not strictly necessary. The RPJ-GC model is able to reproduce and sustain the posterior gt domain without autoactivation by relying on cooperative activation from Bcd and Cad (Perkins, 2006).Comparison of regulatory architectures. The regulatory relationships proposed by Rivera-Pomar and Jäckle (1996) are not fully consistent with the data and require amending. Repression of gt by Kni, which contradicts the mechanism of domain shifts described by Jaeger (2004a and b), was eliminated by the optimization in both of the current models based on the RPJ regulators. Activation of kni by Kr was never observed. No support was found for a dual regulatory effect of Hb on Kr. Activation of Kr at low levels of Hb was eliminated in the RPJ-Logic model. It was retained in the RPJ-GC model, but resulted in serious patterning defects. Inclusion of Tll as an activator of hb was sufficient to produce the posterior hb domain. Based on the current fits and the primary experimental literature, there are likely other regulatory links missing from the model of Rivera-Pomar and Jäckle, though they are not strictly required to reproduce the wild-type gap gene patterns. Foremost is repression of hb by Kni, which appears important for eliminating hb expression anterior of the posterior domain. Fits based on the Sanchez and Thieffry (2001) regulatory relationships also support these conclusions (Perkins, 2006).In contrast, the regulatory relationships in the Combined model and both the Unc-GC and Unc-Logic models are able to capture the wild-type gap patterns without gross defects. The relationships in the Unc-GC model are very similar to those obtained by Jaeger (2004a and b). For example, the regulation of Kr and kni is qualitatively equivalent in both models, and there is a single minor difference in the regulation of gt. The optimizations correctly identified activation of hb by Tll, which was missed by Jaeger (2004a and b), though the current models did less well at capturing shifting of the posterior gt domain. These regulatory relationships are also similar to those found by Gursky (2004), though that study was based on gap gene expression data with much lower accuracy and temporal resolution than the data used in this study. These similarities show that differences in the mathematical formulations of these models-as ordinary versus partial differential equations, how diffusion and nuclei doubling are modeled, and choice of boundary conditions and other simulation parameters-are not important for the reproduction of the gap gene patterns nor for the inference of regulatory relationships from the data (Perkins, 2006). Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryoPattern formation in the developing embryo relies on key regulatory molecules, many of which are distributed in concentration gradients. For example, a gradient of BMP specifies cell fates along the dorsoventral axis in species ranging from flies to mammals. In Drosophila, a gradient of the BMP molecule Dpp gives rise to nested domains of target gene expression in the dorsal region of the embryo; however, the mechanisms underlying the differential response are not well understood, partly owing to an insufficient number of well-studied targets. This study analyzed how the Dpp gradient regulates expression of pannier (pnr), a candidate low-level Dpp target gene. It was predicted that the pnr enhancer would contain high-affinity binding sites for the Dpp effector Smad transcription factors, which would be occupied in the presence of low-level Dpp. Unexpectedly, the affinity of Smad sites in the pnr enhancer was similar to those in the Race enhancer, a high-level Dpp target gene, suggesting that the affinity threshold mechanism plays a minimal role in the regulation of pnr. The results indicate that a mechanism involving a conserved bipartite motif that is predicted to bind a homeodomain factor in addition to Smads and the Brinker repressor, establishes the pnr expression domain. Furthermore, the pnr enhancer has a highly complex structure that integrates cues not only from the dorsoventral axis, but also from the anteroposterior and terminal patterning systems in the blastoderm embryo (Liang, 2012). Most blastoderm genes are regulated primarily on either the DV or AP axis. For example, the gap genes are expressed in one or two domains of expression along the AP axis and, although some of them may exhibit regulation along the DV axis, they are nonetheless considered AP genes. pnr represents an interesting case because although it was originally reported as a DV gene, closer inspection of its expression pattern in wild-type and mutant embryos and detailed dissection of its cis-regulatory enhancers revealed that pnr is highly regulated by both AP and DV genes. Its pattern is a composite of two superimposed patterns that each exhibit AP and DV spatial regulation: a dorsal patch and six AP stripes, which are limited to the dorsal 30% of the embryo. The patch domain, but not the stripes, disappeared in dpp mutants, whereas both the patch and stripes expand ventrally in the absence of Brk. The stripes are more sensitive to Brk repression because activation of the patch domain is limited to the region where Dpp is present dorsally, whereas the stripes can be activated along the entire DV axis. Brk in the ventrolateral region and Sna in the ventral-most region repress stripe expression. Since pnr specifies dorsomedial fates, restricting its expression to the dorsal 30% of the circumference is crucial. Ectopic expression of pnr ventrally causes transformation of ventral epidermis into dorsomedial epidermis (Liang, 2012). Competition between Brk and Smads for binding to overlapping DNA sequences is likely to set the border of the patch domain. Two Smad sites are particularly important for patch expression, and one of these, the M3 site, is a composite site that binds both Brk and Smads, raising the possibility that the patch border is established by competition between activating inputs from Smads in the dorsal region and repressive inputs from Brk emanating from the ventral region. Competition between Brk and Smads for overlapping binding sites has been observed for several Dpp target enhancers (Liang, 2012). Repression of the AP stripes ventrally requires both Brk sites B1 and B2. The two posterior stripes driven by P3 expand to a lesser degree than the four anterior stripes driven by P4. This can be explained by the fact that P4 lacks Brk site B1, which is a stronger Brk site. Loss of both Brk sites would likely result in expansion to the edge of the mesoderm, as seen in embryos that lack Brk protein. Repression by Sna is likely to involve the Sna binding sites in the pnr enhancer, as genome-wide binding studies have shown that the pnr enhancer is bound by Sna (Liang, 2012). The positioning of the stripes, as well as of the patch, along the AP axis is regulated by the gap genes. The results suggest that Hb, Gt and Tll set the anterior edge of the pnr domain, whereas Tll sets the posterior, and that direct and indirect interactions among the gap proteins establish the stripe borders relative to one another, as has been observed for eve. For example, the broad central stripe seen in kni- could be explained by the lack of direct Kni repression. However, owing to the complex cross-regulatory interactions among the gap genes, it is difficult to predict which gap proteins regulate the pnr stripes directly, although genome-wide binding data of the gap factors support their direct binding to the pnr enhancer. Although Bcd does not appear to bind directly to the pnr enhancer, its effects are mediated through its targets Gt and Hb (Liang, 2012). In depth studies of three genes with different boundary positions in the dorsal region, Race, C15 and pnr, indicate that complex combinatorial mechanisms are employed to establish their expression domains, with each gene having a unique regulatory network of its own. Although they all respond to Dpp signaling, their borders of expression are not set by a simple threshold response to the Dpp gradient that depends on differential binding site affinity (Liang, 2012). The feature that has been shown to be important for high-level Dpp target expression is the feed-forward motif involving Dpp and Zen. High levels of Dpp/Smads first activate zen expression in the dorsal-most region, the presumptive amnioserosa, and then both Zen and Smads bind and activate the Race enhancer. The intermediate-level target C15 has a different enhancer structure than high-level targets, containing many Smad sites that act in a cumulative manner to drive expression in regions of intermediate Dpp levels. Mutation analysis has shown that the number of intact Smad binding sites, rather than their affinity, is important for the C15 response. Nevertheless, the enhancer structure of C15 might promote high levels of Smad binding in vivo, and this may increase the response to Dpp. Do all intermediate-level Dpp targets have a similar enhancer structure? The enhancer that drives expression of the intermediate-level Dpp target gene tup was examined for putative Smad binding sites (SBEs and GC-rich regions), and observed multiple Smad sites across the enhancer, similar to that seen with C15. Thus, the multiple Smad site signature might be necessary for response to lower than peak levels of Dpp. In addition, intermediate-leveltargets may utilize repression mechanisms to help establish their borders of expression, as was shown for C15 (Liang, 2012). These studies have revealed that the pnr enhancer resembles that of a high-level target in Smad site organization and Smad binding site affinity. In fact, it was surprisingly easy to convert the low-level target enhancer into a high-level target by mutating a single Smad site. This result could be easily explained if the M3 site had a higher affinity for Smads than those in Race; however, comparison of the binding sites by gel shift showed they have similar affinities. Furthermore, replacing the M3 Smad site with a Race Smad site had little effect on the expression pattern. These results suggest that activation of pnr in its broad domain has little to do with Smad binding affinity. How then does pnr respond to low levels of Dpp? One possible mechanism involves the highly conserved AGCAATTAA site that lies adjacent to the Smad sites. In the absence of this site, the P3 enhancer could not respond to low-level Dpp. It is possible that this site, when bound, leads to greater Smad binding, which would then promote pnr activation (Liang, 2012). What factor(s) might bind to the AGCAATTAA site? ATTA is the core binding site for Antp class HD proteins. Although Zen binds to the ATTA site in vitro, neither the endogenous pnr pattern nor P3-lacZ expression is significantly affected in zen mutants. To identify candidate factors, the TOMTOM tool at FlyFactor Survey was used, and the best match was to the HD protein Hmx, which binds CAATTAA. However, Drosophila Hmx is expressed only in an anterior region that does not overlap with pnr (see FlyBase). Likewise, although several Antp class HD proteins were predicted to bind to the ATTA core sequence, their timing or domains of expression do not overlap ideally with those of pnr (Liang, 2012). It has been proposed that the AGCAATTAA site in the Msx2 enhancer might bind a factor in addition to an HD protein via the 5' half of the site, perhaps a transcriptional partner such as FAST1, which was previously shown to function with Smads. Although the search did not reveal any candidates, if this is the case for pnr then the bipartite motif could potentially bind four proteins: Smads, Brk, HD and 'partner X', The combination of these proteins in a given cell along the DV axis would determine pnr transcriptional activity. The fact that the bipartite motif is not present in the enhancers of Race or C15, or in the other pnr enhancers identified, demonstrates the versatility of how Dpp uses different partners to establish multiple target gene domains (Liang, 2012). Is the structure of the pnr enhancer typical for low-level Dpp targets? This is difficult to address owing to the lack of candidate low-level Dpp targets. Brk is considered a low-level Dpp target in imaginal disc development; however, Dpp represses brk, giving rise to a reciprocal gradient of the Brk repressor. Target gene borders are thus established by competition between Smad and Brk for overlapping binding sites, as mentioned above for pnr. The brk enhancer contains multiple enhancer/silencer modules consisting of activator and repressor (Mad/Medea/Schnurri) binding sites, which contribute to threshold responses to the Dpp gradient, and thus it does not resemble the pnr enhancer. Although good progress has been made in understanding how pnr is expressed in regions with low levels of Dpp, learning the general rules that control broad dorsal patterns will require the analysis of more enhancer elements (Liang, 2012). What rules do target genes for other morphogens follow? Long before the 'feed-forward' term was it was shown that both the Dl and Bcd morphogens interact with their high-level targets, Twi and Hb, respectively, to activate downstream; thus, combinatorial motifs are generally utilized. Moreover, as more target genes of Dl and Bcd were identified and studied, it became apparent that the affinity threshold model could not explain all cases of differential response to the gradient. For example, analysis of several enhancers that drive Bcd-dependent expression in anterior regions of the embryo revealed a poor correlation between Bcd binding site affinity and the AP limits of the pattern. Also, although Dl targets remain archetypal examples of genes that utilize the affinity threshold mechanism, it was found that genes expressed in the lateral region also require input from the Zelda (Vielfaltig - FlyBase) transcription factor for expression in regions of low-level Dl. Zelda binding sites are present in target enhancers, and it was proposed that Zelda boosts Dl binding to help activate the neuroectodermal genes (Liang, 2012). Downstream target gene interactions also shape domains of expression, in particular cross-repression among the targets. In both the Drosophila neuroectoderm and the vertebrate neural tube, morphogen targets are expressed in discrete domains rather than nested overlapping domains due to the repression of one target by another. This mechanism establishes sharp boundaries among the target genes (Liang, 2012). Thus, it is clear that additional factors help morphogens set threshold responses. Given that the pnr enhancer could potentially interact with four different factors along the DV axis and at least four factors along the AP axis, several combinations of inputs could regulate other Dpp target genes. More generally, depending on the number of different factors that interact with the cis-regulatory regions of target genes, morphogen gradients could elicit multiple threshold responses, as has been seen for morphogens such as Dl in Drosophila, Activin in the Xenopus blastula and Shh in the vertebrate neural tube, where up to seven threshold responses have been described. Only by dissecting enhancers can it be fully understood how target genes integrate diverse inputs (Liang, 2012). tailless: Biological Overview Evolutionary Homologs Regulation Developmental Biology Effects of Mutation ReferencesHome page: The Interactive Fly 1997 Thomas B. Brody, Ph.D.The Interactive Fly resides on theSociety for Developmental Biology's Web server. 2ff7e9595c
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