wingless

Transcriptional Regulation Table of contents

Establishment of segmental expression

The response kinetics of known and putative target genes of Fushi tarazu has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (less than 10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely to be important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes have been analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy (Nasiadka, 1999).

In contrast to engrailed, the segment polarity gene wingless has been identified genetically as a negative target of Ftz. This negative interaction has also been demonstrated in HSFtz embryos. Although all wg stripes are repressed in these embryos, the predominant effect is on odd-numbered stripes, which are completely repressed following Ftz induction. Repression of even-numbered stripes is much less efficient. To assess whether this repression is direct, the kinetics of repression were examined. The differential repression of odd- versus even-numbered stripes of wg was a helpful tool and an internal control for recognizing affected embryos. This curve follows very closely that of ftz autoregulation, with the midpoint of both curves occuring at 18 minutes post-heat shock. This indicates that repression of wg by Ftz is also likely to be direct, and that Ftz can act as both an activator and repressor of transcription. Repression is not the only response exhibited by wg in HSFtz embryos. Weak activation within most of each odd-numbered parasegment is also detected. The kinetic curve of this activation response is considerably delayed relative to the kinetics of the other three responses measured thus far. This suggests that wg activation results from an indirect genetic interaction. A likely intermediary gene in this response is the paired gene. prd is genetically required for the proper initiation of all 14 wg stripes; all 14 wg stripes expand rapidly in HSPrd embryos (Nasiadka, 1999).

To test whether the prd gene acts as an intermediate in the positive response of wg to ectopic Ftz, the spatial and temporal responses of prd were determined in HSFtz embryos. If prd does function as an intermediary gene, its expression should be induced in odd-numbered parasegments where wg activation is later observed. Moreover, the induction of prd transcripts should occur with the same rapid kinetics as the ftz, en and early wg responses. The prd expression pattern was examined 20 minutes after ectopic expression of Ftz. Stripes are significantly wider than those in similarly staged wild-type embryos. Using the most posterior stripe of prd as a landmark, it was seen that each of the expanded stripes had broadened at its anterior edge. These regions of expansion comprise most of each odd-numbered parasegment, which is exactly where ectopic expression of wg occurs. The time course of prd mRNA induction was assessed as described for endogenous ftz, en and wg. Although the slopes of the prd and ftz activation curves differ, the initial responses occur at about the same time, suggesting that the interaction between Ftz and prd is also direct. The differences in the slopes of the two curves are likely due to the autoregulatory nature of the ftz response: for a short time, Ftz is expressed from both heat shock and endogenous promoters, and then maximal expression is sustained via autoregulation at the endogenous locus. In contrast, prd activation takes place in regions of the embryo where neither ftz nor prd autoregulates. Hence, prd transcripts do not accumulate as quickly as those of ftz and soon disappear due to degradation of the ectopically expressed Ftz activator (Nasiadka, 1999).

The role of prd as an intermediary factor in wg activation was tested further by examining wg expression in a HSFtz;prd munus background. In the absence of prd, wg stripes should no longer expand. Ectopic Ftz does indeed fail to activate ectopic wg in the absence of prd. The expression pattern in HSFtz;prd minus embryos is essentially identical to the pattern of wg expressed in prd- embryos: odd-numbered stripes are weak and even-numbered stripes are essentially absent. This result is consistent with the proposed role of prd as a direct activator of wg and as a genetic intermediate between Ftz and wg during ectopic stripe broadening (Nasiadka, 1999).

To verify that the prd protein (Prd) is a direct activator of wg, the nature of the temporal delay between prd and wg activation was analysed. Specifically, the temporal accumulation of Prd protein with respect to prd and wg transcripts was examined. If the interaction between prd and wg is direct, then one would expect that much of the interval between accumulation of the two transcripts would be occupied by synthesis and nuclear transport of the Prd protein. The kinetic curve for ectopic Prd induction closely resembles that of WG mRNA activation except that it is shifted by 1-2 minutes to the left (earlier). This indicates that most of the delay observed between the accumulation of prd and wg transcripts (about 8 minutes) is consumed by the synthesis and localization of Prd protein. The time required (~6-7 minutes) may be fairly typical of other segmentation proteins expressed at this stage. Indeed, the delay between detection of en transcript and protein responses is also 6-7 minutes, with curves that are virtually identical to those of prd transcripts and protein. These data do not exclude the possibility that there are genes in addition to prd that are required for ectopic activation of wg. However, if such gene products are required, their rates of synthesis or removal do not appear to supercede the temporal limitations imposed by the synthesis of Prd (Nasiadka, 1999).

Wingless is expressed in a narrow strip of cells at the posterior boundary of each parasegment. Sloppy paired regulates both wg and engrailed. Removal of slp gene function causes embryos to exhibit a severe pair-rule/segment polarity phenotype. slp activity is an absolute requirement for maintenance of wg expression; in a related fashion, wg transcription is dependent on HH. In each parasegment, the SLP proteins are expressed in a broad strip just anterior to the EN-positive cells, overlapping the narrow WG strip. By virtue of their ability to activate wg and repress en expression, the distribution of the SLP proteins define the WG-competent and EN-competent groups. Consistent with this hypothesis, ubiquitous expression of SLP protein throughout the parasegement abolishes en expression; in ptc mutant embryos this results in a near ubiquitous distribution of wg transcripts. In addition to SLP's role in maintaining segment polarity, it has been suggested that SLP works in or parallel with the patched/hedgehog signal transduction pathway to regulate wg transcription (Cardigan, 1994).

wingless expression is also regulated by both odd-skipped and the pair-rule gene paired. odd-skipped represses wg expression, while paired restricts the domain of expression of odd-skipped (Mullen, 1995).

The early bell-shaped gradient of even-skipped expression is sufficient for generating stable parasegment borders. The anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not (Fujioka, 1995).

The gene, hopscotch (hop), coding for the Drosophila JAK serine threonine kinase is required for the establishment of the normal array of embryonic segments. In hop mutant embryos, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. The effect of hop on the expression of these genes is stripe-specific (Binari, 1994).

The pair-rule gene, odd-paired (opa), is essential for parasegmental subdivision of the Drosophila embryo. opa is also required for the timely activation of wg in the remaining parasegments and for the timely activation of engrailed in all parasegments. opa activity is essential for the establishment of alternate parasegments, suggesting opa expression or activity would be spatially restricted like other pair-rule genes. Instead, OPA mRNA and protein are found throughout all segment primordia. Thus, opa does not act in a spatially restricted manner to establish the position of en and wg expression. Rather, OPA must cooperate with other spatially restricted proteins to achieve proper subdivision of the Drosophila embryo (Benedyk, 1994).

There are also clearly segment-specific defects in wingless expression in Dichaete mutants. The loss of wg stipes in the ventral regions of the maxillary and labial segments is independent of corresponding defects in ftz or eve expression (Nambu, 1996).

Single-minded represses wingless, hedgehog and vnd gene expression in developing midline cells. By doing this sim plays a key role in proper patterning of the neuroetoderm by helping to generate the boundary between mesectoderm and ventral ectoderm. This process likely requires simultaneous function of SIM as both a transcriptional activator (of slit and Toll) and transcriptional repressor within the developing midline cells (Xiao, 1996).

The effects of mutations in five anterior gap genes (huckebein, tailless, orthodenticle, empty spiricles and buttonhead) on the spatial expression of segment polarity genes wg and hh has been analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at the 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. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene (Mohler, 1995b).

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. The embryonic head phenotype of col¹ hemizygous mutant embryos indicates a loss of skeletal structures derived from the intercalary, and possibly mandibular, segments without transformation toward another segment identity. A determination was made of whether col mutations affect the expression of wg and En, which mark the anterior and posterior compartments of each segment, respectively. In col¹ hemizygous embryos, both the intercalary stripe of En and the spot of wg expression are missing. Since col expression does not overlap the intercalary Wg spot, the loss of this spot in col mutant embryos suggested that col does not regulate wg expression directly but possibly by an hh-dependent mechanism. It has indeed been found that in col mutant embryos, the intercalary stripe of hh is also absent, or much reduced. Together, these results show that col controls hh, en and wg expression in the intercalary segment and is required for establishing the PS(-1)/PS0 parasegmental border (Crozatier, 1999b).

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted. Previous studies could not establish unambiguously whether Odd acts as a direct or indirect repressor of the en and wg genes. The data presented here show that during gastrulation Odd appears to regulate both genes, not only directly, but indirectly as well. Indirect repression is mediated by selective repression of the en and wg activators: ftz, prd, eve and slp. The result of these interactions in hs-odd embryos is first the loss of all fourteen en and wg stripes due to direct repression and then failure of certain stripes to reinitiate. These results indicate that the activity of Odd is highly dependent on the presence of cofactors and/or overriding inhibitors. Based on these results, and the segmental phenotypes generated by ectopic Odd, a number of new roles for Odd in the patterning of embryonic segments are suggested. These include gap-, pair rule- and segment polarity-type functions (Dréan, 1998).

The two signaling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper it is shown that one component of the Hedgehog pathway, the kinase Fused accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signaling. Direct evidence is presented that Fused is required autonomously in anterior cells neighboring Hedgehog in order to maintain patched and wingless expression, while in turn, Wingless is maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells, it could shown that the Hedgehog signaling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression, whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, the morphogen Hedgehog can pattern the dorsal cuticle independent of Fused. In order to account for these differences in Fused requirements, the existence of position-specific modulators of the Hedgehog response is proposed (Thérond, 1999).

These results clearly support the involvement of the Fu serine/threonine kinase in Hh signal transduction in the embryonic segment within cells producing Wg. Although Fu is present in all embryonic cells, expression of Fu only in the wg-expressing cells of the anterior compartment in a fu mutant context is sufficient to restore a wild-type transcription pattern of both wg and en and a normal cuticular pattern. In contrast, Fu expression in the posterior compartment -- the en-expressing compartment -- has apparently no effect, either on the transcription of wg and en or at the phenotypic level. Together these data show that Fu is not necessary in the majority of cells where it is expressed and suggest that its activity could be induced in wg-expressing cells in response to Hh. Fu protein is evenly distributed in the embryo until stage 9 when it begins to accumulate in the anterior compartment (Thérond, 1999).

What is regulating Fu accumulation? Because FU mRNA distribution is uniform at least until stage 10 it is hypothesized that the localization is due to post-translational regulation. Another component of the Hh pathway, Cos2, also accumulates in the anterior compartment at this stage independent of the level of the Hh signal. As for Cos2, the uniform level of Fu in the anterior compartment seems to be constitutive to anterior cells and independent of Hh signal. Indeed, this regulation is observed during the time when local signaling by Wg and Hh stabilize each other’s expression. At this stage anterior cells at the A/P border are receiving Hh and responding to it. Other anterior cells distal to the A/P border do not receive Hh but have the potential to respond to it. Thus, these results are inconsistent with Hh regulating Fu and Cos2 accumulation in the entire anterior compartment. Fu accumulation could be related to its association with Ci and Cos2 within the same protein complex. Nevertheless, since fu expression is only required in the wg-expressing cells for proper patterning, the higher Fu protein levels in the whole of the anterior compartment do not seem to have any functional significance (Thérond, 1999).

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterized Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity (Janody, 2000).

The results indicate that early anterior expression of a labral determinant, cnc, and of two foregut determinants, wg and hh, is repressed when zygotic expression of hb is allowed to persist at the anterior pole of the Drosophila blastoderm embryo. Expression of cnc, wg and hh is under the positive regulation of bcd and torso but no zygotic gene has yet been implicated in this control. This suggests that the Hb protein is able to repress the three genes cnc, wg and hh, and that torso-induced anterior repression of hb is necessary for their positive control by torso. To determine whether the positive control of cnc, wg and hh by torso could be the result of a double negative control involving hb, expression of these genes was analysed in hb zygotic mutant embryos derived from torso females. If the lack of early anterior expression of cnc, wg and hh was solely due to the absence of repression of hb at the pole, expression of these genes should be recovered in hb minus embryos derived from torso females. Early anterior expression of cnc, wg and hh is not recovered in hb minus embryos derived from torso females whereas it is normal in hb minus embryos. This indicates that, although necessary, the anterior repression of hb is not sufficient to mediate Torso positive control on cnc, wg and hh early anterior expression (Janody, 2000).

The segment polarity gene lines (lin)was identified by Nusslein-Volhard because of its effects the dorsal epidermal pattern. Evidence is provided that Lin is involved in stage specific Wingless signaling activity; it acts in the cell receiving the Wg signal. In addition, Lin can localize to the nuclei of cells signaled by Wg-secreting cells. It is hypothesized that Lin interacts with nuclear Wg signal transducers and confers stage specificity to the pathway. Lin also localizes to the cytoplasm of cells receiving the Hedgehog signal, suggesting that Hh competes with Wg signaling by exporting Lin from the nucleus. Evidence for the suggestion that Lines acts in some way to specify dorsal cell fate has relied on cuticle analysis, which represents the final differentiated state of the cells, first visible at about 13 hr AEL. However, Wg signaling specifies these cell fates between 6 and 9 hr AEL. Thus, to test whether or not Lin acts in concert with Wg, earlier molecular markers for Wg patterning need to be identified and the effects of Lin activity on these markers need to be tested. The first Wg-dependent target gene is wg itself. If Wg function is blocked late by expression of dominant-negative Pan with the Ptc-GAL4 driver, late wg expression is lost from both the dorsal and the ventral epidermis. Reciprocally, if Wg signaling is activated by driving of activated Arm expression with the Ptc-GAL4 driver, an ectopic Wg stripe is induced posterior to the En domain. Thus, wg gene expression depends on Wg input and provides a molecular readout for the pathway. In lin mutants, late wg expression fades from the dorsal epidermis of fully retracted embryos (10 hr AEL). Reciprocally, overexpression of Lin using the Ptc-GAL4 driver activates wg expression posterior to the En domain in the dorsal epidermis. The ectopic expression of wg is identical to that obtained by expression of activated Arm posterior to the En domain. The only distinction is that the effect of Lin is restricted to the dorsal epidermis, while global activation of Wg signaling affects the ventral epidermis as well. Thus, Wg input and Lin are both necessary and sufficient for activation of wg gene expression in dorsal epidermis (Hatini, 2000).

The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. This study examines the role of the transcription factor Cubitus interruptus (Ci) in this process. So far, Ci has been thought to be the most downstream component of the Hh pathway, capable of activating all Hh functions. However, the study of a null ci allele indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity. Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. Evidence is presented that the zinc-finger protein Teashirt is one candidate for this activity. It is required positively for rhomboid expression and Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk (Gallet, 2000).

Ci is required to transduce Hh signal in order to activate its target genes. In cells that do not receive Hh, Ci is cleaved and represses Hh target genes. However, compelling results point out a more complex role for Ci activity during embryonic development of Drosophila. The embryonic phenotype resulting from the complete loss of Ci function is weaker than the complete loss of Hh function. The phenotypic differences observed between hh and ci null mutations reside in the following observations: in ci⁹⁴ embryos one observes (1) the presence of segmentation due to maintenance of wg expression until stage 11 and (2) the presence of denticle diversities due to an expansion of EGF signaling illustrated by an expansion of rho expression. Ci does not have a maternal contribution, since ci⁹⁴ homozygotes issuing from germ-line clones homozygous for ci⁹⁴, do not show a stronger phenotype than embryos lacking only zygotic Ci product, and also embryos hatching after ci RNA interference experiments show phenotypes similar to ci⁹⁴ embryos. Furthermore, if rho expression present in ci mutants is due to maternal production of Ci, one has to explain how two Ci targets would behave differently in the absence of zygotic Ci contribution; wg expression disappears, whereas rho is expressed in more cells. For all these reasons it can be confidently concluded that Ci has no maternal contribution. Consequently, other factors are substituting for Ci activity in the transduction of Hh signaling (Gallet, 2000).

Loss of ci induces an expansion of rho expression instead of a reduction, as seen in a hh loss of function, showing that Ci is not involved in the activation of rho expression. The fact that rho disappears in tsh mutant embryos strongly suggests that the Tsh zinc-finger protein regulates rho expression or is at least necessary for instructing cells to respond to Hh for rho expression. Nevertheless, one has to explain why rho expression is expanded in ci⁹⁴ . Loss of Ci^rep activity could be responsible for this effect. Indeed, overexpression of Ci^rep in a ci null background or analyses of the ci^Ce2 mutant, which ectopically expresses Ci^rep, reveals a repressive effect of Ci^rep on rho expression. Therefore, Ci^rep could be used as a gatekeeper in order to repress hh target genes tightly where they should not be expressed, and thus to overcome mis-regulation of key genes such as rho or wg. Nevertheless, these observations contradict previous analyses showing that Ci^rep is not required for correct embryogenesis, since loss of ci function is rescued by a ci transgene lacking the Ci⁷⁵ repressor form of Ci. An alternative explanation can be gleaned from the fact that ci⁹⁴ cuticle phenotypes resemble those lacking Wg activity during the cell specification stage. Because it has been shown that Wg exerts a repressive role on rho expression (since absence of Wg activity promotes ectopic expression of rho), rho expansion in ci⁹⁴ could be an indirect consequence of the late disappearance of wg expression during stage 10-11 (Gallet, 2000).

Before stage 11, either Tsh or Ci is sufficient for wg regulation because only the loss of both gene activities results in a downregulation of wg, a situation comparable with that observed in hh mutants. It is interesting to note that Ci seems to display differential requirements for wg maintenance and naked cuticle differentiation in the abdomen versus the thorax. While Ci is dispensable until stage 11 for wg expression and naked cuticle differentiation in the abdomen, its presence in the thorax is required. This specific Ci function in the thorax is currently being studied (Gallet, 2000).

Both Ci and Tsh transcription factors, when overexpressed can induce ectopic wg expression. The two factors do not display the same features: Tsh has three atypical, widely spaced, zinc-finger motifs, whereas Ci has conserved spacer regions between its five zinc fingers; the binding sites identified so far for these two proteins are different. It would be interesting to know whether Tsh can bind directly to the wg promoter and to identify its binding sites. It is also noteworthy that between stage 8 and 10 wg requires, in parallel to Hh, its own activity for the maintenance of its transcription. It has previously been shown that Tsh is a modulator of Wg signaling. Tsh becomes phosphorylated and accumulates at a higher level in the nucleus in Wg-receiving cells compared with cells lacking Wg signal. Hence, in the trunk Tsh could be employed both by Wg and Hh signaling in order to maintain wg transcription (Gallet, 2000).

The redundancy exhibited between Tsh and Ci for wg regulation changes after stage 10, since loss of either Ci or Tsh results in the downregulation of wg transcripts. It is not known if this observation is the result of a cooperation between Tsh and Ci. At least one other gene, gooseberry, is required for the maintenance of wg transcription at this stage, indicating that multiple inputs for the maintenance of wg expression are necessary for normal embryonic development (Gallet, 2000).

Studies on the developing wing blade show that Ci transduces all Hh-delivered information. However, this study and others on the Hh pathway support the idea that Ci is not always involved in Hh signaling, showing that branchpoints are common for distinct Hh signaling steps for the following five reasons. (1) It has been shown that neither Ci nor Fused (Fu) are involved in the Hh-dependent formation of Bolwig's organ in Drosophila. (2) A Hh-responsive wg reporter gene with no Ci-binding sites does not require Ci activity for its regulation until stage 11. (3) Studies on the talpid 3 gene in chicken suggest that Gli proteins, the vertebrate homologues of Ci, regulate only a subset of Hh target genes, the others being regulated by an unidentified transcription factor. (4) A Sonic hedgehog response element on the COUP-TFII promoter binds to a factor distinct from Gli. (5) Hh signaling does not require Ci activity to regulate rho. Although the authors favor the idea that Tsh regulates rho expression directly in response to Hh signal the hypothesis that Tsh plays a more permissive role allowing Hh to regulate rho via another factor apart from Ci cannot be excluded (Gallet, 2000 and references therein).

In conclusion, Hh requires at least two different transcription factors during Drosophila embryogenesis to regulate its multiple target genes and to instruct cells with precise behaviors. The transcription factors may act independently (e.g. Ci for ptc; Tsh for rho), cooperatively (e.g. Ci and Tsh for wg maintenance during the cell specification phase) or redundantly (e.g. Ci and Tsh for wg maintenance earlier during the stabilization phase). The possibility that other transcription factors like gooseberry might be recruited for Hh signaling cannot be excluded, especially since denticle density is weaker in tsh;ci double mutants as compared with hh single mutants. Furthermore the dorsal phenotypes of the tsh;ci double mutants are weaker than those of hh. (1) wg transcripts are still present in dorsal patches in tsh;ci mutations whereas they are not present in hh embryos. (2) Dorsal cuticle is not as severely perturbed in tsh;ci larvae as compared with hh null ones (Gallet, 2000).

Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. The functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. A model is suggested in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for Extradenticle and Homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins (Kobayashi, 2003).

Exd cooperates with En to repress target genes and to pattern embryos. Loss of exd function has been shown to result in a loss of en expression at later embryonic stages. Because en function is required to maintain its own expression, the loss of en expression could be a downstream effect of a loss of en function, or it could be due to some other consequence of the lack of exd. This ambiguity concerning the role of exd in en function led to an investigation of whether the activities of ectopically expressed En are dependent on exd function. En was ectopically expressed in two ways: from a heat-shock promoter and using a patterned Gal4 'driver' transgene. An advantage of the former approach is that one can often distinguish between immediate and secondary downstream effects based on how rapidly they occur following heat induction. Advantages of the second approach include having normal and altered expression in parts of the same embryo, providing a rigorous internal control. Both of these approaches led to similar conclusions, that exd function is important for the repression by En of its direct target gene slp, that wg also shows a strong dependence on exd function for its repression by En and that the ability of En to alter the pattern of embryonic cuticles is sensitive to the gene dosage of exd. Further, in each set of experiments, the observed dependence of repression on exd was accompanied by a residual repression activity when exd function was removed both maternally and zygotically. This residual exd-independent repression activity might be due to the ability of En to bind to target sites independently of exd but with a reduced affinity, or it could be accounted for by the existence of two classes of binding sites, one exd dependent and the other exd independent. This possibility is paralleled by the relationship of Exd with Ubx, which has been shown to function either co-operatively with Exd or alone on multiple binding sites in target genes. Alternatively, exd might be exerting an indirect effect on repression by En. However, because Exd forms complexes with En in yeast and in vitro, and because it appears to facilitate repression by En directly in cultured cells, it seems likely that the dependence of En on exd function in vivo is due at least in part to the direct action of En-Exd complexes. Confirmation of this model will require the analysis of specific regulatory sites, which have not yet been identified, in target genes such as slp. If this model is correct then these results suggest that the repression activity of Exd-En complexes might come exclusively from En repression domains, because Exd has been shown to act as a cofactor in the activation of target genes in vivo in conjunction with Hox proteins (Kobayashi, 2003).

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. The role of these modifications in regulating subcellular localization of Hedgehog has been investigated in the Drosophila embryonic epithelium. Cholesterol modification of Hedgehog is responsible for Hedgehog assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. Movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog's functional diversity in epithelial cells (Gallet, 2003).

The repeated pattern of the Drosophila larval ectoderm (which secretes cuticle) has been used to follow Hh activity. Each abdominal segment is composed of two types of cuticle: the naked (or smooth) cuticle and the denticle belts, subdivided into six rows of denticles, easily identifiable by their orientation and shape. This cuticle pattern is under the control of several signaling pathways that are indirectly regulated by Hh. Engrailed (En) controls hh expression in the two rows of cells that define the posterior compartment of the segment. Across the parasegmental boundary (in cells anterior to the En/Hh domain), Hh maintains wingless (wg) transcription in one row of cells. The Wg signal then controls the specification of the naked cuticle. Posterior to the En/Hh domain, Hh initiates rhomboid (rho) transcription in one to two rows of cells. rho activation induces EGF signaling, allowing differentiation of denticles 1-4. Finally, Hh and Wg are required for serrate (ser) repression and restrict its expression in three rows of cells posterior to the rho-expressing cells. Ser initiates a third row of rho expression in adjacent cells. The Hh receptor Patched (Ptc) is also transcriptionally upregulated by the Hh pathway in cells on both sides of the En/Hh domain (Gallet, 2003).

Loss of hh results in loss of both naked cuticle and denticle diversity. This cuticle phenotype correlates with Hh target gene expression: loss of wg, extension of the ser expression domain (which now covers most of the segment) and absence of ptc upregulation. rho expression is strongly reduced, though some remains under the control of Ser. Conversely, ubiquitous expression of full-length hh (Hh^FL) in the ectoderm with the GAL4-UAS system induces an expansion over four to five cells of both wg and rho expression in the anterior and posterior directions, respectively, while ser expression is completely repressed. Accordingly, the denticle belts of these embryos contain several rows of type 2 denticles, reflecting a uniform level of rho expression in response to a uniform level of Hh. Thus, wg, rho, ser, and ptc expressions reflect direct Hh activity in cells anterior and posterior to en/hh-expressing cells (Gallet, 2003).

Two endogenous Hh isoforms are present in vivo: one bearing both posttranslational lipid modifications and another modified only by a cholesterol adduct. To address the role of these different modifications in Hh signaling, the biological activity of different Hh constructs that do not undergo all modifications was assessed (Gallet, 2003).

It is hypothesized that the differences observed could be accounted for by differential activation mechanisms. These results outline the important role of the Hh cholesterol modification in stimulating the anterior target genes wg and ptc across the parasegmental boundary and, subsequently, naked cuticle differentiation, while cholesterol appears dispensable for posterior induction of ptc and rho and, thus, denticle diversity. Because some wg expression can still be activated by Hh-N, the presence of cholesterol modification on Hh might not be the only requirement for anterior target gene regulation. Hh-N-CD2 and Hh-N-GPI activities suggest that the differences observed could be a consequence of Hh differential sorting in the producing cells and/or access and presentation to the target cell surface (Gallet, 2003).

Differential activation of wg and ptc in anterior cells and of rho and ptc in posterior cells is related to the membrane localization of Hh. Cholesterol-dependent LPS formation and apical targeting are shown to be necessary for proper anterior wg activation but dispensable for rho expression in posterior cells. Conversely, basolateral targeting of Hh in cells producing Hh-N-CD2 and Hh-N-GPI is sufficient to activate the posterior rho expression, independent of the presence of cholesterol (Gallet, 2003).

Interestingly, wg is expressed in adjacent cells located just anterior to the Hh-sending cells. Hence, long-range diffusion of Hh should not be required for wg activation. However, in the absence of Ttv function, Hh-Np LPSs are blocked apically in producing cells, and wg is not activated. Ttv-dependent heparan sulfate proteoglycans are required for long-range Hh-Np movement in the wing disc. Thus, these results suggest that, in the embryonic ectoderm, two different mechanisms of Hh pathway activation are present. wg activation requires all the events previously associated with long-range Hh target gene activation and thus depends on Hh secretion and transport mechanisms. However, rho does not require secretion of Hh and can be activated in a cell-cell contact-dependent manner, like a short-range target. This difference could be due to differential accessibility of Hh to anterior versus posterior cells caused by the presence of the parasegmental boundary between en and wg cells. Indeed, when Ttv is expressed exclusively in cells anterior to En cells, both wg- and rho-dependent cell differentiation are rescued. This indicates that a differential transport and/or presentation of Hh-Np could be responsible for the asymmetric cellular response to Hh (Gallet, 2003).

Signals from the BMP family member Decapentaplegic (Dpp) play a role in establishing a variety of positional cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although a reasonably clear picture is available of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. Three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, referred to collectively as Dorsocross, are described that are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. These genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, it has been demonstrated that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend the knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals (Reim, 2003).

In addition to the inputs from dpp, metameric Doc expression in dorsolateral areas of the germ band must depend on the activity of segmental regulators. A direct comparison with the expression of engrailed (en) shows that the clusters of Doc expression straddle the compartmental borders. Although Doc expression overlaps with en in the P compartments, about two-thirds of the Doc expressing cells of each cluster are located in posterior areas of the A compartments. In agreement with this allocation, it has been found that the metameric Doc domains are exactly centered on the stripes of Wingless (Wg) expression. The observed correlation of the segmental registers of Wg and Doc makes wg a good candidate for an upstream regulator of Doc. Dorsolateral Doc expression in the ectoderm and mesoderm is shown to be completely absent if wg is inactive. By contrast, deletion of sloppy paired (slp), a known target of wg in the mesoderm and a wg feedback regulator in the ectoderm, results in a reduction, but not a complete loss of metameric DOC expression. Hence, slp probably affects Doc indirectly through its effect on ectodermal wg expression. Altogether, the data suggest that metameric Doc expression in the ectoderm and mesoderm is triggered by the intersecting activities of Wg and Dpp (Reim, 2003).

The segmental stripes of wg expression in the embryonic trunk segments initially span the entire dorsoventral extent of the ectoderm, but at stage 11 they become interrupted in dorsolateral areas. A comparison of Wg and Doc expression at this stage shows that the positions of the metameric ectodermal domains of Doc expression correspond to the areas in which the Wg stripes become interrupted. Temporally, there is a brief overlap of ectodermal Wg and Doc expression during stage 10 until Wg expression is downregulated within the Doc domains. In contrast to the wild-type situation, the Wg stripes remain continuous in DocA mutant embryos. Similar observations were made with the homeobox gene product Ladybird (Lb=Lbe + Lbl) as a marker. In wild-type embryos after stage 11, Lb is also expressed in striped domains that are interrupted at the positions of the ectodermal Doc domains, whereas in DocA mutant embryos there is ectopic expression in a pattern of continuous stripes. These data show that Doc activity is required for patterning events in the dorsolateral ectoderm, which include the repression of wg and lb expression in these areas (Reim, 2003).

Ectopic expression experiments with Doc genes provide additional evidence for a repressive activity of Doc on wg expression. Upon ectopic expression of Doc2 in all cells of the ectoderm of wild type embryos, the ventral portions of the Wg stripes are lost. However, the dorsal regions of the Wg stripes appear to be under different regulation, because ectopic Doc results in a uniform domain of dorsal Wg along the anteroposterior axis, albeit at lower levels than in wild-type embryos (Reim, 2003).

Ectopic expression experiments with Doc genes in imaginal discs further confirm their ability to repress wg. In third instar larval wing discs, Doc genes are expressed in four distinct areas that do not overlap with the wg expression domains. Specifically, two large Doc expression domains are located in the centers of the dorsal and ventral regions of the prospective wing blades and two smaller domains in prospective dorsal hinge and posterior notal regions, respectively. In leg discs, low levels of Doc expression can be detected in regions of the prospective body wall and proximal leg segments, which also do not express wg. Importantly, ectopic expression of Doc2 within the Dpp domains of imaginal discs causes wg expression to disappear in the corresponding areas. In agreement with the known role of wg in limb development, its repression by ectopic Doc results in the loss of distal structures of wings, legs and antenna of adult animals. Analogous ectopic expression experiments with Doc1 and Doc3 in embryos and discs produce qualitatively similar (although weaker) effects to those of Doc2 (Reim, 2003).

The maintenance of wg after stage 10 has been shown to depend on two different positive feedback loops, one being active in the dorsal and the other in the ventral ectoderm. The dorsal feedback loop is mediated by the ladybird homeobox genes (lb=lbe and lbl), whereas the ventral loop is mediated by the Pax gene gooseberry (gsb). The Doc genes must interrupt one or both of these feedback loops, although it is not clear whether the primary block is at the level of the wg gene or at the level of the transcription factor-encoding genes lb and/or gsb. Another target for repression by the Doc genes in this pathway could be slp, which is required both dorsally and ventrally in wg feedback regulation. It is thought that lb is unlikely to be the primary target of Doc repression since the failure of wg repression temporally precedes the expansion of the lb stripes in Doc mutant embryos. Furthermore, the observation that the Doc genes can also repress wg in other tissue contexts such as the imaginal discs, where gsb, lb and slp are not components of a wg feedback loop, seems to favor the mechanism of a direct repression of the wg gene by Doc (Reim, 2003).

A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. An ectopic expression system that allows for quantitative manipulation of expression levels was used to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1), most appropriately classified as segment polarity genes, as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. Furthermore Ftz is found to modulate the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation (Swantek, 2004).

The role of Runt as a primary pair-rule gene complicates interpreting the alterations in segment-polarity gene expression that are observed in run mutants. Recent experiments utilizing a GAL4-based NGT-expression system [the transgene construct used to express GAL4 maternally contains the nanos promoter and the 3' untranslated region of an alpha-tubulin mRNA and is thus referred to as an NGT transgene (nanos-GAL4-tubulin)] to manipulate expression in the blastoderm embryo have demonstrated that low levels of Runt repress en in odd-numbered parasegments without altering expression of the pair-rule genes eve and ftz. This observation suggested that this approach might provide a useful tool for defining the role of Runt in regulating other segment-polarity genes. A systematic survey was undertaken of the response of other segmentation genes to increasing levels of NGT-driven Runt expression. These experiments revealed significant differences in sensitivity as well as interesting differences in the nature of the response of different genes to ectopic Runt. The odd-numbered en stripes are repressed at both intermediate and high levels of ectopic runt. After en, the second most sensitive target is slp1. This gene shows a partially penetrant and subtle defect in the spacing of the segmentally repeated stripes in embryos with low levels of NGT-driven Runt. A more pronounced alteration is obtained in embryos with intermediate levels of Runt. In these embryos the slp1 pattern is converted from a segment-polarity-like, 14-stripe pattern to a pair-rule-like, seven-stripe pattern. At this level, expression of other segmentation genes is normal although there are subtle changes in the spacing of the wg stripes and a partial loss of the odd-numbered hh stripes. All three of these genes show clearer alterations at higher levels of NGT-driven Runt, with wg responding in a manner similar to slp1 and hh responding in a manner similar to en. High Runt levels also produce spacing defects in the expression of odd and gsb, as well as a more subtle effect on prd. Several of the changes observed at high levels of ectopic Runt are likely to be indirect and due to alterations in the expression of eve, ftz and hairy. The response of slp1 to ectopic Runt is notable both because of its sensitivity and apparent simplicity, thus suggesting that Runt plays a pivotal role in regulating slp1 transcription (Swantek, 2004).

The differential combinatorial effects of Runt and Ftz on segment-polarity gene regulation emerged as a result of analyzing the sensitive and relatively simple response of slp1 to ectopic Runt. The slp1 transcription unit is one of two redundant genes that comprise the slp locus. This locus was initially characterized as having a pair-rule function in the segmentation gene hierarchy based on a weak pair-rule phenotype associated with loss of slp1 function. The slp1 and slp2 genes are expressed in similar patterns during early embryogenesis. Embryos deficient for both slp1 and slp2 have an unsegmented lawn cuticle phenotype similar to that produced by wg mutations. This raises the question of whether it is most appropriate to consider slp as a pair-rule or segment-polarity locus. In the most straightforward interpretation of the segmentation hierarchy, the role of the pair-rule genes is to establish the initial metameric expression patterns of the segment-polarity genes. The initial expression of the key segment polarity genes en and wg is normal in gastrula stage embryos that are deleted for both slp1 and slp2. The expression of wg begins to become abnormal and is lost during early germband extension. These observations are consistent with the proposal that slp expression identifies cells that are competent to maintain wg expression subsequent to the blastoderm stage. Based on these observations, it is concluded that slp1 and slp2 are most appropriately classified as segment polarity genes, not pair-rule genes (Swantek, 2004).

Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless

Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. This study reports that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, it has been shown that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. The data presented indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain (Buescher, 2004).

Previous work has suggested that Slp permits the Hh-dependent activation of Wg anterior to the En/Hh stripe by antagonizing a repressor of Wg. Based on the data presented above, Mid/H15 appear to be such repressors. To determine if Slp is a negative regulator of mid expression, the effect of slp loss-of-function and slp misexpression was studied on the distribution of mid RNA. In slp mutant embryos the early mid expression is normal. However from early stage 9 onward the mid stripes broaden to approximately twice their normal width. Using mid-positive neuroblasts as a landmark (these remain unchanged in slp mutant embryos), it was possible to characterize the increase in mid expression as an anterior expansion. This aberrant mid expression pattern is unstable; from stage 11 onward mid decays in odd-numbered segments. Conversely, misexpression of slp in the ventral ectoderm from early stage 9 onward led to a complete loss of ectodermal mid expression. These data show that Slp functions as a repressor of mid expression. Taken together with the observation that misexpression of mid in otherwise wild-type embryos results in the loss of Wg expression, these results lead to the conclusion that the Slp-mediated repression of mid anterior to the En/Hh stripe is an important component of wg competence (Buescher, 2004).

As a further test of the relationship between slp and mid, the effect was compared of expressing mid and slp, alone or in combination, on Wg expression. Ectopic expression of mid results in a rapid and almost complete loss of Wg expression, whereas ectopic expression of slp results in weak ectopic expression of Wg posterior to the En/Hh stripe. This slp-induced phenotype resembles that of the loss of mid, except that ectopic Wg expression is weaker and appears randomly in even- and odd-numbered segments. The ectopic Wg expression is blocked when mid and slp are expressed together, suggesting that in this context mid acts downstream of slp. The Wg expression anterior to the En/Hh stripe still decays in UAS-mid/UAS-slp embryos, albeit more slowly and variably than in UAS-mid alone. This result may reflect that Wg expression is sensitive to the amounts of available Mid and Slp. It may also indicate that anterior to the En/Hh stripe, Slp function is required for more than just repression of mid and may possibly have independent activating functions. An analysis of slp1,slp2;mid/H15 quadruple mutants would be highly helpful in clarifying the relationship between slp genes and mid/H15. Unfortunately, the generation of such a quadruple mutant by genetic recombination is impossible because the slp deletion that removes these genes (Δ34B) is on a balancer chromosome that precludes recombination (Buescher, 2004).

Transcriptional Regulation Table of contents

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