Mothers against dpp


Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction

Retrograde signaling is essential for neuronal growth, function and survival; however, little is known about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. This study identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but a significant decrease in the number of presynaptic release sites was found. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Overexpression of the type II TGFβ receptor Wit enhanced presynaptic pMad levels. In Khc-73 mutants, this enhancement was significantly suppressed. Similarly, muscle overexpression of the ligand Gbb enhanced pMAD levels in presynaptic boutons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, loss of Khc-73 was shown to curb the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. These findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons (Liao, 2018).

Khc-73 function plays a supporting role in retrograde BMP signaling under basal conditions. However under conditions of enhanced BMP signaling, this endosomal coordination by Khc-73 becomes critical to transmit the retrograde signal from the synapse to the neuronal cell body (Liao, 2018).

Efficient retrograde signaling from synaptic terminals back to the neuronal soma is critical for appropriate neuronal function and survival. Nevertheless, little is known about the molecular steps that facilitate the routing of synaptic endosomes destined for retrograde axonal pathways. This study describes several lines of evidence for a potential role for Khc-73 in this process. Khc-73 mutant larvae develop grossly normal synaptic structure and function at the Drosophila larval neuromuscular junction (NMJ), but this study finds a reduction in the number of presynaptic release sites. Through genetic interaction experiments, this defect was shown to most likely be the result of abnormal BMP signaling in motoneurons: transheterozygous combinations of Khc-73 and Medea or wit mutants show a significant loss of presynaptic release sites compared to control. Khc-73 becomes even more critical, when higher demand is put on the motoneuron by activating BMP signaling: loss of Khc-73 largely blocks the retrograde enhancement in synaptic release in response to activation of BMP pathway in motor neurons. Consistently it has been shown previously that transgenic knock down of Khc-73 in motoneurons blocks the ability of the NMJ to undergo retrograde synaptic homeostatic compensation (Tsurudome, 2010). The current findings show that when BMP signaling is activated, loss of Khc-73 reduces the accumulation of pMad in motoneuron nuclei, suggesting a role for Khc-73 in the regulation of retrograde signaling. Immunohistochemical assessment and live imaging analysis of Khc-73 mutant larvae provide evidence for involvement of Khc-73 in at least two steps in endosomal dynamics in motoneurons. On the one hand, Khc-73 is required for normal dynamics of internalized endosomes through late endosomal and multivesicular stages, and on the other Khc-73 plays a role in facilitating the routing of endosomes onto the retrograde pathway. These defects have two main consequences: first, an accumulation of BMP receptors was found at the NMJ (possibly in multivesicular bodies) without increased local signaling, suggesting that these receptor containing endosomes might be trapped in a state between late endosomal and lysosomal stage. Second, a dampening was seen of the ability of retrograde bound Rab7:GFP tagged endosomes to join the retrograde pathway, illustrating a defect in retrograde movement of vesicles and possibly providing an underlying explanation for the reduction in pMAD when retrograde BMP signaling is activated in Khc-73 mutants. These results together present Khc-73, a plus-end microtubule motor, in the unexpected role of regulation of endosomal traffic from synapse to the soma in motoneurons with a role for ensuring the efficiency of retrograde BMP signaling (Liao, 2018).

The findings point to a model in which Khc-73 facilitates the routing of retrograde bound vesicles onto the retrograde axonal pathway. This model predicts coordination between endosomes, dynein motors and kinesin Khc-73. The coordinated involvement of dynein and kinesin motor proteins in the transport and sorting of endosomes has been previously proposed and examples supporting this model are mounting. Previously published data for Khc-73 and KIF13B have provided evidence that interaction between early endosomes, dynein motors and microtubules are possible. Khc-73/KIF13B is capable of binding to the GTPase Rab5 (found on early endosomes), thus allowing Khc-73 to localize directly to Rab5 endosomes. As a kinesin motor protein, Khc-73 could then transport these endosomes to the retrograde pathway by moving along the microtubule network in the synapse (Liao, 2018).

Compelling evidence for a dynein interaction with Khc-73 has been previously demonstrated during mitotic spindle formation. The Khc-73/KIF13B stalk domain is phosphorylated by Par1b and this creates a 14-3-3 adapter protein binding motif. It has been proposed that physical interaction between Khc-73 stalk domain and the dynein interacting protein NudE via 14-3-3 ε/ζ might underlie the interaction between Khc-73 and dynein that is necessary for appropriate mitotic spindle formation. Interestingly, transgenic knock down of NudE in Drosophila larval motoneurons leads to a reduction in the number of presynaptic release sites, a phenotype reminiscent of Khc-73 loss of function. Thus, Khc-73 contains domains and protein-protein interactions that are capable of coordinating endosomes, microtubules and dynein. It is proposed that Khc-73 is necessary for the normal endosomal sorting and exit of endosomes from the NMJ to support efficient retrograde BMP signaling (Liao, 2018).

Targets of Activity

The role of Mad in Dpp-mediated signaling was examined by utilizing tkvQ199D, an activated form of the Dpp type I receptor serine-threonine kinase thick veins (tkv). In the midgut, dpp is expressed in the visceral mesoderm of parasegments 3 and 7. In response to Dpp signals, cells expressing dpp in parasegment 3 repress the expression of the homeotic gene Sex combs reduced. Dpp signals are also required to maintain dpp expression in parasegment 3 through an autocrine feedback loop. However, cells in parasegment 4 do not appear to be affected by Dpp signals; Scr is expressed while dpp is not. In ps7, the homeotic gene Ultrabithorax initiates dpp expression. Subsequently, Dpp functions in an autocrine manner to maintain Ubx as well as its own. In ps7, Dpp also signals between germ layers to the underlying endoderm. Within the midgut endoderm, which does not express dpp, expression of the homeotic gene labial is dependent on Dpp signals (Newfeld, 1997).

In the embryonic midgut, tkvQ199D mimics Dpp-mediated inductive interactions. There is an anterior expansion of labial midgut endoderm expression in response to ubiquitously expressed tkvQ199D. In early stage tkvQ199D embryos, dpp expressin is expanded to include ps4, ps5 and ps6. In late stage tkvQ199D embryos, the expanded domain of expression is maintained at very high levels, while in late stage Mad mull tkvQ199D embryos, this is not observed. Analysis of Scr expression in Mad null embryos, combined with tkvQ199D, reveals an anterior expansion of Scr, showing that Mad and dpp are required for repressing Scr. Mad function is epistatic to tkvQ199D in the repression of Scr. Thus homozygous Mad mutations block signaling by tkvQ199D and appropriate responses to signaling by tkvQ199D are restored by expression of MAD protein in Dpp-target cells (Newfeld, 1997).

Endogenous Mad is phosphorylated in a ligand-dependent manner in Drosophila cell culture. Dpp overexpression in the embryonic midgut induces Mad nuclear accumulation; after withdrawal of the overexpressed Dpp signal, Mad is detected only in the cytoplasm. However, in three different tissues and developmental stages actively responding to endogenous Dpp, Mad protein is detected in the cytoplasm but not in the nucleus. To date, in live animals, the nuclear accumulation of Mad in response to Dpp has been observed only with Dpp overexpression. It is possible that even under conditions of maximal endogenous signaling the fraction of Mad translocated to the nucleus is too small to be detected by current techniques. Alternatively Mad's biological role is independent of observed subcellular translocation (Newfeld, 1997).

The function of the Drosophila mef2 gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis, mef2 undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. Evidence is presented that temporally and spatially specific mef2 expression is controlled by a complex array of cis-acting regulatory modules that are responsive to different genetic signals. Functional testing of approximately 12 kb of 5' flanking region of the mef2 gene shows that the initial widespread mesodermal expression is achieved through a 280-bp twist-dependent enhancer. Subsequent dorsal mesoderm-restricted mef2 expression is mediated through a 460-bp dpp-responsive regulatory module, which involves the function of the Smad4 homolog Medea and contains several binding sites for Medea and Mad. Regulated mef2 expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular gut musculatures, respectively, is under the control of distinct enhancer elements. In addition, mef2 expression in the cardioblasts of the heart is dependent on at least two distinct enhancers, which are active at different periods during embryogenesis. Mef2 expressing cells are coincident with those expressing Tinman. Notably, both Mef2 and Tinman expression are in four of six cardioblasts that are present per hemisegment. The complete overlap between the two expression patterns suggests that the activity of this enhancer element could be dependent on tinman function or under similar regulatory controls as is tinman. The cardiac enhancer that functions at later stages also drives mef2 expression in the caudal visceral mesoderm as well as in the somatic mesoderm. Moreover, multiple regulatory elements are differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts, and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of the mef2 gene in myogenic lineages in the Drosophila embryo is the result of multiple genetic inputs that act in an additive manner on distinct enhancers in the 5' flanking region (Nguyen, 1999).

spalt is a target of MAD in the wing. Overexpression of dpp causes an expansion of the wing along the AP axis, the width of the spalt domain expanding relative to the DPP stripe. Cells with reduced MAD fail to express spalt, suggesting that cells must be able to transduce the DPP signal to express spalt (Lecuit, 1996).

Loss of DPP signaling in the Drosophila eye can lead to ectopic expression of wingless, suggesting that MAD negatively regulates wingless transcription. Mutant Mad clones are found to express wingless in eye imaginal discs. Similarly, bifurcations of antennae are associated with mutant Mad clones. Such clones express wg and are overgrown when located in the dorsal region of the antennal disc. Thus the antagonistic effect of DPP signaling in wg expression is also observed in other discs and might be a general mechanism during Drosophila imaginal disc development (Wiersdorff, 1996).

The observation that dpp is not expressed in mutant Mad eye margin clones raises the possibility that DPP signaling is required for the maintenance of dpp expression. Supporting this, mutation in dpp itself reduces expression of a dpp reporter gene in the eye margin (Wiersdorff, 1996).

The spalt and optomotor-blind genes are expressed in a nested pattern in the wing imaginal disc, centered on and extending up to 20 cell diameters away from the stripe of decapentaplegic expression along the anteroposterior compartment boundary. The vestigial gene is even more broadly expressed and is required in all cells of the developing wing. Activation of vg through the quadrant enhancer is responsible for the broad expression of vg in the wing blade. The quadrant enhancer is found in an 806 bp DNA segment in the fourth intron of the vg gene. This enhancer is sufficient to drive expression in all of the developing wing blade. vg expression in developing wing-blade cells, beginning from the wing-blade primordium of the wing imaginal disc, requires a dpp signal from the A/P boundary and another signal from the D/V boundary. The term quadrant enhancer refers to the expression pattern, which is bisected by non-expressing DV boundary cells and becomes less intense along the A/P boundary in the late third instar, producing a quadrant-like pattern. Unlike the D/V boundary enhancer, which is regulated by the Notch pathway and is active in the second instar, the quadrant enhancer is not active until the early third instar. The quadrant enhancer is Suppressor of hairless independent, suggesting that Notch function is not required for formation of this part of the wing during the third instar. The temporal order of D/V boundary and quadrant enhancer activation suggests that D/V boundary cells must produce a signal (or signals) that organize the rest of the wing, including expression from the quadrant enhancer. Activation of the quadrant enhancer requires dpp, since thick veins mutants exhibit reduced Vg protein levels and have smaller wings and wing discs (Kim, 1996).

Because Mad is an intracellular signal transducer of Dpp receptors, the requirement of Mad activity for Vg expression in the wing imaginal disc was examined in mitotic clones with reduced Mad function. Mad has no effect on the Notch-dependent dorsoventral boundary enhancer, but the quadrant enhancer requires Dpp signaling and Mad function. The N-terminal MAD homology region 1 (MH1) plus the central less conserved proline-rich linker region bind DNA and protect a single interval within the quadrant enhancer. A 39 bp double stranded oligonucleotide of the vg quadrant enhancer contains the Mad-protected region. Mutating 12 base pairs within this region prevents Mad-directed expression. The C-terminal MH2 domain of Mad effectively inhibits DNA binding and suggests a mechanism that might contribute to inactivation of Mad in the absence of Dpp signaling. Examination of the Ultrabithorax midgut enhancer, for which the Dpp response element has been localized to the 95 bp DI-DII interval, and the labial endoderm enhancer reveals a Mad binding-site consensus of GCCGnCGC. The two sites of highest affinity match this consensus perfectly, and three lower-affinity sites contain mismatches in one or as many as three positions (Kim, 1997).

Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs

In both Drosophila and Xenopus embryos, gradients of Dpp/BMP activity are established that are responsible for patterning along the dorsoventral axis. Dpp activity has its highest levels along the dorsal midline of the cellular blastoderm embryo and declines toward more lateral regions where it is inhibited by the product of the short gastrulation (sog) gene. The high levels determine the cell fate of the amnioserosa in the dorsal-most cells, whereas lower levels specify aspects of the dorsal epidermis in dorsolateral cells. The absence of Dpp activity in ventrolateral regions permits the formation of the neurogenic ectoderm, which gives rise to both the ventral epidermis and the central nervous system (Rushlow, 2001).

How does Dpp specify cell fate in a concentration-dependent manner? It is thought that Dpp signaling in the early embryo regulates the transcription of downstream target genes that are expressed in nested domains centered around the dorsal midline. High-level Dpp targets such as Race and u-shaped (ush) are expressed in the presumptive amnioserosa. pannier (pnr) is expressed in a broader domain that spans the amnioserosa and part of the dorsal ectoderm. Thus, it requires lower levels of Dpp. Finally, low-level targets such as early zerknullt (zen) and dpp are expressed in an even broader domain that abuts the ventral ectoderm. A possible molecular mechanism to explain the threshold responses of Dpp target genes is that their promoters have different affinities to Smads and therefore can be induced by different levels of nuclear Smads, similar to the mechanism of differential activation by the Drosophila morphogens Dorsal (Dl) and Bicoid (Bcd). The fact that an additional mechanism is involved came from the characterization of the brinker (brk) gene. brk negatively regulates low-level and intermediate-level target genes. Study of the response elements of these target genes can therefore provide clues about the mechanisms of threshold responses to the Dpp morphogen, as well as the interplay of positive and negative inputs in the expression of target genes (Rushlow, 2001 and references therein). zen has a dynamic pattern of expression in the early embryo. During precellular nuclear division cycles 11-13 and during early cellularization (nuclear cycle 14), zen is expressed in a broad dorsal-on/ventral-off pattern. This pattern is thought to be activated by an unknown ubiquitous activator present throughout the embryo and repressed by the Dl morphogen localized in ventral regions. It is Dpp-independent because early zen expression is normal in dpp null mutants. However, slightly later, during early to mid-cellularization, maintenance of the zen pattern becomes dependent on Dpp because zen transcripts fade away suddenly in dpp null mutants. It also becomes dependent on Brk repression because zen transcripts expand into the ventral ectoderm in brk mutants. Thus, the broad pattern of zen is maintained by Dpp in the dorsal region and repressed by Brk in ventral regions. During mid- to late-cellularization, this pattern undergoes a process of refinement in which zen transcripts are lost from the lateral regions and become restricted to a narrow domain of the dorsal-most cells. Brk plays no role in refinement because in brk mutants, although zen expands ventrally, it refines normally (Rushlow, 2001 and references therein).

zen expression is directed by 1.6 kb of 5' flanking DNA sequences referred to as the zen promoter. The distal part of the promoter between 1.2 and 1.4 kb is responsible for Dl-dependent ventral repression. Sequences required for the initiation, maintenance, and refined expression of zen are located in the proximal 0.7 kb of the promoter, but they are not well-characterized (Rushlow, 2001 and references therein).

The regulation of zen during cellular blastoderm formation has been analyzed. Low levels of the Dpp signal transducer p-Mad (phosphorylated Mad), together with Brinker, define the spatial limits of zen transcription in a broad dorsal-on/ventral-off domain. The subsequent refinement of this pattern to the dorsal-most cells, however, correlates with high levels of p-Mad that accumulate in the same region during late blastoderm. Examination of the zen regulatory sequences reveals the presence of multiple Mad and Brk binding sites, and these results indicate that a full occupancy of the Mad sites due to high concentrations of nuclear Mad is the primary mechanism for refinement of zen. Interestingly, several Mad and Brk binding sites overlap, and it has been shown that Mad and Brk cannot bind simultaneously to such sites. A model is proposed whereby competition between Mad and Brk determines spatially restricted domains of expression of Dpp target genes (Rushlow, 2001).

Examination of p-Mad staining in wild-type embryos indicates that maintenance and refinement require different levels of signaling. Only the highest p-Mad levels in the dorsal-most five to six nuclei are capable of driving zen transcription during late cellularization. The lower levels present in the three to four lateral nuclei to either side are not sufficient to activate zen, although earlier they were sufficient for its maintenance. This indicates that maintenance may involve the contribution of an additional activator, perhaps the same ubiquitous activator that initiates zen earlier (Rushlow, 2001).

Later during refinement, p-Mad at peak levels is sufficient to up-regulate zen. Interestingly, the Dpp target gene ush is expressed in a broader domain than refined zen that includes the three to four lateral nuclei. This indicates that ush can be activated by a lower level of signaling than refined zen and that the high-level class of Dpp target genes can be further subdivided (Rushlow, 2001).

Because the amount of p-Mad depends on the amount of Dpp activity, the simplest explanation is that the zen promoter responds to differences in Dpp activity by measuring the level of nuclear Smads. Such a conclusion is consistent with the presence of multiple Mad/Medea binding sites and mutagenesis analysis. Deletion of only two Mad/Medea sites results in the loss of refined expression; therefore, most if not all of the Smad binding sites are required for this function, as are the peak levels of p-Mad activity (because weak dpp mutants do not refine). However, maintenance is not affected, possibly because several sites remain intact and this function does not require full p-Mad activity. That the zen promoter measures the level of nuclear Smads also explains the broad dorsolateral pattern of both p-Mad immunostaining and zen expression in sog embryos. In the absence of inhibition by Sog, Dpp continues to signal, and p-Mad can accumulate in the dorsolateral region of the embryo and induce zen expression (Rushlow, 2001).

The experiments presented here show that Mad/Medea and Brk regulate zen by binding to separated and overlapping DNA binding sites. There are 10 Mad/Medea and 6 Brk binding sites in the zen promoter, 5 of which are shared, indicating duality in their function. Indeed, the results from mutagenesis of the zen promoter show that the shared sites mediate both Brk and Mad/Medea functions. Five Brk and nine Smad binding sites are clustered in the zen proximal regulatory element over about 600 bp with spacing not exceeding 120 bp. This organization is similar to that of several well-studied enhancers from Drosophila. These enhancers are activated by a variety of transcriptional activators and repressed by short-range repressors such as Snail (Sna), Knirps (Kni), and Krüppel (Kr). All three of these repressors are DNA-binding proteins that can inhibit activator function when they are bound not further than 150 bp away from the activator binding site. It has been shown that they all contain a short stretch of amino acids, P-DLS-K, that is required for recruitment of the corepressor dCtBP. Analysis of zen regulation indicates that Brk also may be a short-range repressor. It is a DNA-binding protein and contains a PMDLSG domain. Preliminary in vitro experiments showed that Brk interacts with dCtBP; however, embryos devoid of dCtBP activity do not ectopically express zen and dpp, indicating that dCtBP is dispensable for Brk repression and other corepressors interact with Brk, or that Brk repression of these targets does not require additional factors (Rushlow, 2001).

What remains illusive is the identity of the ubiquitous transcriptional activator that activates zen in the dorsal ectoderm during precellular stages and early cellularization. It is possible that this activator interacts with Smads to enhance transcription of zen at a time when p-Mad levels are low. Also, Brk represses the ubiquitous activator, because zen becomes ectopically expressed in brk mutants. Thus far, deletion analysis of the zen promoter has not uncovered any sequences that might interact with this putative activator. It is possible that these sequences are redundant and scattered over the entire promoter and may in fact overlap with Smad and/or Brk binding sites (Rushlow, 2001).

In the cellularizing embryo, Dpp and Brk activities overlap in the lateral-most region. Here Dpp and Brk function to set thresholds of response for target genes such as zen and pnr. In this same region, Dpp signaling negatively regulates brk expression. Similarly, in the wing disc, the Brk expression domain overlaps with that of the Dpp target gene omb in the region where activated p-Mad is present. It has been proposed that a dual mechanism whereby Dpp can simultaneously down-regulate Brk repressor levels and antagonize its function on target gene promoters would be very efficient in establishing sharp threshold responses. Based on the experiments described here, a molecular model is proposed to explain mechanistically the antagonizing activities of Brk and Smads. It is proposed that they are involved in direct competition for binding to shared binding sites on target promoters. Thus, it is the balance of their opposing activities that determines the transcriptional state of the target genes. Two sets of experiments support this model: (1) ectopic expression of Brk in eve-stripe 2 abolishes zen expression in those cells. The elevated level of Brk in the stripe was therefore sufficient to repress the zen promoter even in the presence of activated Smads. The possibility that zen is repressed indirectly through Brk-mediated repression of dpp is highly unlikely because there was no delay in zen repression. (2) In vitro competition experiments also support the model. Especially revealing is the fact that the outcome of competition depends on the relative concentrations of both proteins and their binding affinities. Competitive mechanisms have been proposed to operate on many promoters where mutually exclusive DNA-binding factors are involved, and, in some instances, DNA-binding assays similar to the ones used in this study were used to show competition for binding between activator and repressor proteins. For example, bHLH proteins compete with a zinc-finger repressor for E-box binding in the immunoglobulin heavy chain enhancer (Rushlow, 2001 and references therein).

The findings presented here provide a framework for further study of the mechanisms of regulation of Dpp morphogen targets. zen is the only one of the known Dpp target genes that responds to two threshold activities: low (during early to mid-cellularization) and high (during late cellularization). Based on the results presented here and the proposed competition mechanism for activation and repression of the zen promoter, predictions can be made about the organization of the regulatory elements of the other Dpp target genes. High-level targets such as ush strongly depend on high levels of Smads, and their regulatory elements may have many, and possibly closely packed, Smad binding sites. Low-level targets such as omb in the wing imaginal disc may be repressed by Brk binding to their regulatory sequences. The spatial domains of expression of the intermediate targets such as pnr in the embryo and sal in the wing disc, which are dependent on both Dpp signaling and Brk repression, might be determined by the net balance of positive and negative inputs. Interestingly, this type of mechanism can result in expression domains that vary largely in size and may result in even broader domains than the low-level targets. An example is the vg gene. In third-instar imaginal wing discs, vg is expressed in a broader domain than omb. Its expression along the anterior-posterior boundary in the wing pouch is activated by the quadrant enhancer that contains Mad binding sites essential for activation. At the same time, vg is repressed by Brk. However, the essential Smad binding sites do not match the Brk binding sites, like many of the Smad sites in the zen promoter, suggesting that they will have no or low affinity for the Brk protein. Neither are there strong zen-like Brk binding sites in the quadrant enhancer. Its broad expression domain could then be explained if the positive inputs from Smads, enhanced by signals from the dorsoventral boundary, are able to overcome Brk repression far from the Dpp source (Rushlow, 2001).

Further studies of the arrangement, affinities, and numbers of repressor and activator sites in Dpp target promoters will determine to what extent the different thresholds of responses to the Dpp morphogen activity are shaped by a simple balance of positive and negative transcriptional inputs (Rushlow, 2001).

The zinc finger protein Schnurri acts as a Smad partner in mediating the transcriptional response to Decapentaplegic

Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study it is shown that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes a cell culture assay was developed and it has been shown that Shn and Mad act synergistically to induce transcription. These results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor (Dai, 2000).

Although mutations in the binding sites for Mad result in more severe loss of Ubx B expression compared to that caused by mutations in the Shn sites, in neither case is the expression abolished, raising the possibility that inputs from both proteins contribute to the regulation of Ubx B. There is increasing evidence that protein-protein interactions between Smads and accessory transcriptional factors can result in cooperative binding and synergistic transcription of reporter genes. The fact that a Ubx B reporter that lacks Shn-binding sites (BS1S2) shows residual staining, while Ubx B expression is completely absent in shn mutant embryos, suggests that loss of Shn protein has more severe consequences than loss of Shn-binding sites. In order to determine whether protein-protein interactions as well as DNA-binding contribute to activation of Ubx B by Shn and Mad, an assay was developed to study the nuclear response to Dpp signaling. The B enhancer was cloned upstream of a minimal promoter driving expression of the luciferase gene (Ubx B-Luc) and its activity was examined in cultured cells. This reporter shows very low levels of basal expression in the BMP-responsive C3H10T1/2 cells. Cotransfection with Mad/ Medea results in only a slight elevation of luciferase activity. However coexpression of Mad/Medea with constitutively activated TkvA results in a dramatic 25-fold increase in promoter activity relative to the basal response. In other words, coexpression of all three components causes a 5-fold stronger stimulation than expression of either Mad/Medea or TkvA alone. The response to TkvA is dependent on Mad and Medea since transfection with the receptor alone leads to only a small increase in transcription over basal levels, perhaps due to phosphorylation of endogenous BMP-specific Smads (Dai, 2000).

Whether coexpression of Shn with Mad and Medea could enhance transcriptional activation of Ubx B-Luc was examined. Expression of Shn or Mad/Medea alone elicits a weak transcriptional response. However coexpression of all three proteins results in a 32-fold induction of reporter gene activity relative to the basal response. This is a 6-fold increase over the response to either Shn or Mad/Medea alone. More strikingly this induction is 3-fold greater than the expected additive response to expression of the individual proteins. To test the importance of Mad and Shn DNA-binding to synergistic activation, a luciferase reporter construct was generated lacking both Mad sites known to be required for expression in the embryo (Ubx BM2). As anticipated, it was found that the response of Ubx BM2-Luc to stimulation by TkvA and Mad is significantly reduced when compared to wild-type Ubx B. Interestingly, however, deletion of the Mad-binding sites in BM2 does not affect the induction of reporter activity by Mad/Medea in the presence of Shn. In analogous experiments using a Ubx BS1S2-Luc reporter, loss of the Shn-binding sites only marginally affects the cooperative response to Shn and Mad/Medea. These results could indicate that synergistic transcriptional activation by overexpression of Shn and Mad/Medea does not depend entirely on their ability to bind DNA, but involves cooperative protein-protein interactions (Dai, 2000).

To test this, a reporter was constructed that lacks both Mad as well as Shn-binding sites (Ubx BM2S1S2-luc). The response of Ubx B to overexpression of Mad/Medea and Shn is strongly reduced in the double mutant. It is concluded that binding sites for either Mad or Shn are sufficient to mediate synergistic activation of the Ubx B reporter. However, when neither protein can bind the enhancer, it is no longer possible to elicit a transcriptional response. While the data may be interpreted as redundancy for Mad/Medea and Shn in stimulating UbxB transcription, this view is contradicted by the fact that expression of either protein alone clearly does not stimulate maximal response of the UbxB reporter. Taken together these data indicate that Shn can act as a transcriptional coactivator with Mad to regulate the expression of the Ubx B enhancer (Dai, 2000).

Repression of Dpp targets by binding of Brinker to Mad sites

Signaling by Dpp activates targets such as vestigial indirectly through negative regulation of brinker. The Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity is found in an amino-terminal region containing a putative homeodomain. Brk binds to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for Mad. Furthermore, Brk is able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs from the Smad3/Smad4 binding site consensus. The Dpp response element from Ubx is more sensitive to repression by Brk than is the vg quadrant enhancer. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Evidence suggests that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad (Kirkpatrick, 2001).

Binding of Brk to the Ubx and vg probes generates multiple bands, possibly indicating that Brk binds to more than one site. The Ubx element contains an inverted repeat of GGCGCT that overlaps a previously identified Mad binding site. Whereas the Mad site embedded in this repeat resembles the vg Mad site, the repeat as a whole is only matched at 7 of 12 positions in vg. Brk was tested for the ability to bind one copy of this sequence in a DNA probe that was otherwise divergent in sequence from the Ubx element. Brk binds to the GGCGCT probe with affinity that is similar to its affinity for the Ubx probe and yields a single major shifted band at about the same position as the lower most band observed with the Ubx probe. Although two weak upper bands are also observed with the GGCGCT probe, overall, these results are consistent with high affinity interaction of Brk with just one site in the GGCGCT probe (Kirkpatrick, 2001).

To investigate the specificity of Brk for the GGCGCT sequence, the effects of single base pair substitutions were determined. This was done measuring the ability of unlabeled 'wildtype' (GGCGCT) and mutant DNAs to compete with the labeled GGCGCT probe. In all, five mutants exhibited an ~20-fold reduction in the binding affinity, whereas the least critical position contributed as much as 3-fold to binding affinity. These results indicate that Brk makes base-specific contacts across the entire GGCGCT sequence (Kirkpatrick, 2001).

The GGCGCT repeat in the Ubx element overlaps a Mad binding site that can be modeled to consist of two degenerate Smad boxes, suggesting that Brk may compete with Mad for binding. This could not be determined unequivocally using the Ubx probe because Mad and Brk complexes have nearly identical mobilities in the band shift assay. However, the GGCGCT probe forms a complex with Mad that is easily resolved from the main complex formed with Brk; this probe makes clear that formation of Brk complexes correlates with reduced binding of Mad. In contrast, the same amount of Brk did not reduce binding of Mad to the M7 probe, evidence that Brk reduces the level of Mad binding by competition rather than by sequence-independent inhibition (Kirkpatrick, 2001).

To determine whether the Brk binding sites identified using the band shift assay are actually required for repression, the Ubx element was mutated to disrupt Brk binding. Each of three GGCG(C/T) sequences was changed to GTCG or to GGCGA, both of which dramatically reduce Brk binding but still allow Mad to bind. Introduction of the same triple-substitutions into the 2×Ubx-lacZ reporter result in an ~100-fold decrease in sensitivity to repression by cotransfected Brk. These results demonstrate that Brk binding sites are required for repression and confirm that the sequence specificity characterized in band shift experiments is also observed in cells (Kirkpatrick, 2001).

The overlap of Mad and Brk binding sites in the Ubx midgut element suggests that Brk might repress Dpp targets by simply competing with Mad for occupancy of an enhancer element. However, repressors generally function by quenching the activating potential of transcription factors bound nearby or by means of long range interfering effects on the general transcription machinery. To determine whether Brk is capable of functioning as an active repressor, Brk binding sites were positioned adjacent to sites for the unrelated Notch-responsive activator, Suppressor of Hairless [Su(H)] and reporter expression was monitored in response to cotransfected Brk, Su(H), and activated Notch effector plasmids. Brk completely prevents activation by Su(H), whereas a control reporter containing only Su(H) sites was repressed only 2-4-fold, an effect that may have been caused by the presence of a single Brk binding site adjacent to the hsp70 TATA box. Given this ability of Brk to function as a generic active repressor, it is reasonable to speculate that Brk might control a subset of Dpp targets without the direct involvement of Mad (Kirkpatrick, 2001).

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Ultrabithorax (Ubx) is a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. Ubx expression is negatively regulated by Dpp. Brinker represses Ubx in the embryonic midgut. The functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp (Saller, 2001).

The control of Ubx by Dpp and Wg signalling has been studied by functional dissection of a minimal midgut enhancer called Ubx B. This enhancer directs expression of a linked ß-galactosidase (lacZ) gene in parasegments (ps) 6-9, and also in ps3, of the midgut mesoderm as a result of stimulation by Dpp and Wg, which are expressed in or near these regions. This stimulation requires distinct Dpp and Wg response sequences (DRS and WRS) within Ubx B. In addition, Ubx B is repressible by high Wg levels near the Wg signalling source, and is also repressed in the absence of Wg signalling in cells remote from the source. The former repression is mediated by the WRS-R, a sequence coinciding with the Mad binding sites within the DRS, the latter by the WRS, a binding site for Pangolin, the Drosophila T cell factor (Saller, 2001).

Since Ubx is a Dpp target gene in the embryonic midgut, it was asked whether this HOX gene might be under brk control. Thus, brk mutant embryos were stained with an antibody against Ubx and weak ectopic Ubx staining was found in the posterior midgut mesoderm of these mutants. Normally, the HOX protein Abdominal-A represses Ubx in the posterior midgut, but evidently this is not sufficient to keep Ubx repressed in the absence of brk. However, no Ubx derepression was observed in the anterior midgut of brk mutants, probably because of the silencing of Ubx in this region by Polycomb. But derepression was found in the anterior and posterior midgut of brk mutant embryos when examining lacZ expression conferred by an extensive Ubx midgut enhancer called RP9, the expression of which closely resembles Ubx expression in the midgut. These stainings show that brk represses Ubx in the embryonic midgut (Saller, 2001).

Next, a series of mutant versions of Ubx B was tested that carry nested point mutations. Most of these are still derepressed in brk mutants, e.g. BM1, which has a mutated MadB site. However, three mutant enhancers were no longer derepressed: B4, which has a mutated Pangolin binding site; B4R8, which carries a mutation in a conserved sequence motif; and BM2, in which both Mad binding sites are mutated. Formally, each of these mutations could define a target for Brinker repression. Alternatively, they define sequences that are essential for enhancer activation, in particular for ectopic activation at the midgut ends. This is a clear possibility since B4, BM2 and B4R8 are each considerably less active than Ubx B and other mutant enhancers such as BM1 (Saller, 2001).

Full-length and various fragments of Brinker were expressed as glutathione S-transferase (GST) fusion proteins in bacteria, in order to test whether these fusion proteins can bind to the signal-responsive sequence from Ubx B in gel shift assays. This revealed that full-length Brinker, or its N-terminus alone, can bind to this sequence, whereas the C-terminus cannot. This is consistent with the suggestion that the N-terminus contains a putative DNA binding domain similar to the homeodomain. Indeed, a minimal fragment spanning this domain (BRK44-99) binds to the probe as well as full-length Brinker (Saller, 2001).

Next, Brinker binding to mutant DNA probes was tested. Of these, BM2 and BM0 are the only mutants that no longer show any binding to Brinker. Likewise, Brinker binding to DNA can be competed with an excess of unlabelled wild-type probe, but not with mutant BM2 probe. This shows that Brinker binds to Ubx B in a sequence-specific manner, and that the residues mutated in BM2 and BM0 are critical for Brinker binding (Saller, 2001).

Three perfect matches to a consensus site for Brinker binding, GGCG C/T C/T, are found in Ubx B. These are adjacent to one another, and each of them is mutated in BM2. The results with BM1 indicate that the first of these matches (Brk bs1) is sufficient for Brinker function in vivo and in vitro. However, Brk bs3 alone is unlikely to be sufficient for function, given that Brinker cannot bind to the mutant probe BM0. Finally, the results indicate that Brk bs2 (perhaps together with bs3) can substitute for Brk bs1 and provide full function: BC2 is repressible by brk in vivo, and Brinker binds to BC2, BC and BM01 mutant probes, all of which lack Brk bs1 (Saller, 2001).

Interestingly, the three Brinker binding sites completely overlap the two Mad binding sites within the DRS. Indeed, the Dpp response critically depends on MadA; MadA fully overlaps Brk bs1, which is sufficient for Brinker function in vitro and in vivo. It was thus asked whether Brinker might be able to compete with Mad for DNA binding. Competitive DNA binding experiments were performed using bacterially expressed DNA binding domains of Brinker and Mad. This revealed that the former is capable of competing successfully with the latter for DNA binding at a molar ratio of 1:150, and Brinker almost completely blocks Mad binding at a ratio of 1:15. Note that full-length Mad binds to DNA less efficiently than its isolated DNA binding domain, indicating that Brinker would be able to compete even more successfully with full-length Mad. Thus, Brinker can block Mad binding to DNA in vitro in the presence of a considerable molar excess of Mad (Saller, 2001).

To confirm that the above Brinker binding sites within Ubx are functional targets in vivo, Brinker was expressed throughout the midgut mesoderm with the GAL4 system. This revealed that expression of Ubx in the middle midgut is nearly eliminated in Brinker-overexpressing embryos. Instead, many of these embryos show an endodermal bulge in the middle midgut that is also observed in Ubx mutants. Furthermore, the first and second midgut constrictions are rudimentary at best, and often missing altogether. Again, loss of the second constriction is indicative of mutations of Ubx and dpp, while loss of the first may reflect mutation of the dpp-related gene gbb. Finally, ectopic Brinker also drastically reduces dpp and wg expression in the middle midgut, which is expected since their expression depends on Ubx. This indicates that Brinker, by virtue of repressing Ubx, is capable of blocking the whole process of endoderm induction that depends on this HOX gene (Saller, 2001).

These results indicate that Brinker is a direct repressor of Ubx, and thus a potent antagonist of the Dpp-dependent process of endoderm induction. It is noted that Brinker is expressed in 'signal-free' zones bordering the anterior and posterior limits of the midgut. Its presence in these zones may have a barrier function, helping to block the spread of the Dpp response beyond the midgut limits (Saller, 2001).

Interestingly, the critical Brinker target site within Ubx B overlaps MadA, a functional Mad binding site that is required for the stimulation of this enhancer by Dpp signalling. Furthermore, Brinker competes effectively with Mad in binding to this site in vitro, and blocks activated Mad from stimulating Ubx B in vivo. This indicates that the mechanism by which Brinker repression dominates over stimulatory Dpp inputs is based on direct competition for binding to Dpp target enhancers. Given that most, if not all, Dpp signalling is mediated by Mad, it seems likely that this competition-based mechanism of Brinker repression is widespread and extends to genes that are Dpp targets in other developmental contexts (Saller, 2001).

Notably, MadA is also the target sequence for repression of Ubx B in response to high Wg levels in the middle midgut. MadA is thus a pivotal enhancer sequence that gauges and integrates positive inputs from Dpp and negative inputs from Brinker and Wg. Wg-mediated repression in the middle midgut is mediated by the zinc finger protein Teashirt and can be overriden by simultaneous Dpp stimulation. In contrast, Brinker-mediated repression dominates over simultaneous Dpp stimulation. It thus appears that Brinker is a more potent repressor than Teashirt, and is designed to function as a signal-antagonist even in the presence of high levels of Dpp signalling (Saller, 2001).

Brinker contains the sequence PMDLS, which resembles the P-DLS motif through which a number of transcription factors recruit the co-repressor dCtBP. Indeed, using in vitro pull-down assays, it was found that dCtBP binds to full-length Brinker as well as to an N-terminal Brinker fragment that contains the PMDLS motif. This suggests that Brinker may recruit dCtBP to repress Dpp target genes in the embryo. Interestingly, dCtBP assists various transcription factors, such as Knirps, Snail and Krüppel, that act at short-range to repress their target genes. These short-range repressors bind to autonomous enhancers to quench nearby bound transcriptional activators, which has prompted the suggestion that dCtBP may be specifically designed to quench. Therefore, this quenching ability of dCtBP could enable Brinker to not only compete efficiently with activated Mad in the binding of DNA, but also out-compete the activity of nearby transcription factors such as activated dTCF (Saller, 2001).

Mad and Scalloped: Control of a genetic regulatory network by a selector gene

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped (Sd), the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]2 [Su(H)]2 element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]2 [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).

Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient

Morphogen gradients control body pattern by differentially regulating cellular behavior. Molecular events underlying the primary response to the Dpp/BMP morphogen have been analyzed in Drosophila. Throughout development, Dpp transduction causes the graded transcriptional downregulation of the brinker (brk) gene. Significance for the brk expression gradient is provided by showing that different Brk levels repress distinct combinations of wing genes expressed at different distances from Dpp-secreting cells. The brk regulatory region has been dissected and two separable elements have been identified with opposite properties, a constitutive enhancer and a Dpp morphogen-regulated silencer. Furthermore, genetic and biochemical evidence is presented that the brk silencer serves as a direct target for a protein complex consisting of the Smad homologs Mad/Medea and the zinc finger protein Schnurri. Together, these results provide the molecular framework for a mechanism by which the extracellular Dpp/BMP morphogen establishes a finely tuned, graded read-out of transcriptional repression (Müller, 2003).

The Dpp signaling system shapes an inverse profile of Brk expression, which serves as a mold for casting the spatial domains of Dpp target genes. Thus, the question of how the Dpp morphogen gradient is converted into transcriptional outputs can be largely reduced to the question of how Dpp generates an inverse transcriptional gradient of brk expression. An unbiased approach was applied to this problem by isolating the regulatory elements of brk. A protein complex has been identified and characterized that binds to and regulates the activity of these elements in a Dpp dose-dependent manner (Müller, 2003).

Dissection of the brk locus reveals two separable elements with opposite properties: a constitutive enhancer and a morphogen-regulated silencer. Both elements have a direct effect on the level of brk expression, and it is the net sum of their opposing forces that dictates the transcriptional activity of brk in any given cell. In this sense, expression of the brk gene behaves like a spring that is compressed by Dpp signaling. Its silencer and enhancer embody the variable compressing and constant restoring forces, respectively. As stated by Hooke's law, an increased elastic constant (e.g., two copies of the constitutive enhancer) either shifts the brk levels toward those normally present at more lateral positions or necessitates a correspondingly higher compressing force (e.g., more silencer elements or higher levels of Dpp signaling). Given the central role Brk plays in controlling growth and pattern together with the direct impact of the two regulatory elements on brk levels, it appears inevitable that their quantitative properties must exhibit a fine-tuned evolutionary relationship with each other and with those of the Dpp transduction system. It appears, furthermore, that both the brk enhancer as well as the brk silencer elements represent ideal substrates for evolutionary changes in morphology (Müller, 2003).

Based on combined genetic and biochemical analysis, it is proposed that upon Dpp signaling the following key players meet at the brk silencer elements to execute repression: the Smad proteins Mad and Med and the zinc finger protein Shn. The role of Shn must be to direct the signaling input provided by Mad and Med into transcriptional silencing. In principle, two scenarios can be envisaged by which Shn fulfills this task. Shn could possess repressor activity (presumably via recruitment of corepressors) but lack the ability to bind the brk silencer and, hence, depend on Mad/Med for being targeted to its site of action. Alternatively, Shn could be prebound to the silencer, but only be capable of recruiting corepressors upon interaction with Mad/Med. Based on the observation that a Shn/DNA complex cannot be detected in the absence of Mad/Med, the first of these two possibilities is favored. The molecular architecture of the protein complex binding to the brk silencer as well as the DNA sequences providing the specificity for the local setup of this complex remain to be determined in detail (Müller, 2003).

An additional protein, which appears to influence the events at the brk silencer, is Brk itself. Genetic experiments indicate that Brk negatively modulates its own expression, forming a short regulatory loop that contributes to the final shape of the Brk gradient. This autoregulatory action occurs also via the brk silencer element, suggesting that Brk directly participates in the protein-protein or protein-DNA interactions at this site (Müller, 2003).

Most regulatory events ascribed to Smad proteins to date concern signaling-induced activation of target gene transcription. In the case of the brk silencer Shn could be regarded as a 'switch factor' that converts an inherently activating property of Smad proteins into transcriptional repression activity. Indeed, it has been shown that Smad proteins have the ability to recruit general coactivators with histone acetyl transferase activity. However, in an alternative and more general view, Smad proteins per se may provide no bias toward activation or repression. Their main function may be to assemble transcriptional regulatory complexes involving other DNA binding proteins and endow these complexes with additional DNA binding capacity. Such associated DNA binding factors would not only determine target site specificity, but, by their recruitment of either coactivator or corepressor proteins, also define the kind of regulatory influence exerted on nearby promoters. Since Shn directs Mad/Med activity toward repression, the existence of at least one other such Mad/Med partner in Drosophila is hypothesized to account for Mad/Med-mediated activation of gene expression. Such Mad/Med-mediated activation appears to be required for peak levels of sal and vg transcription, as well as for defining gene expression patterns in domains where brk expression is completely repressed, e.g., close to the Dpp source of the dorsal embryonic ectoderm (Müller, 2003).

At the heart of the model is the direct causal relationship between the formation of a Shn/Mad/Med/brk-silencer complex and the silencing of brk gene transcription. Although the two observations have been derived from different experimental data sets (biochemical versus genetic, respectively), there is a firm correlation between the requirements for either event to occur. brk is not repressed when either (1) the brk silencer elements are lacking or mutated; (2) or when Dpp input is prevented (and hence Mad is neither phosphorylated, nor nuclearly localized, nor associated with Med), or when (3) Shn is not present or is deprived of its C-terminal zinc fingers. The same set of requirements was observed for the formation of the Shn/Mad/Med/brk complex. Moreover, it is the concurrence of all three of these conditions that appears to provide the exquisite specificity to the Dpp-regulated silencing of gene transcription. (1) It only occurs in conjunction with a functional brk silencer, or an equivalent element. (2) There is an absolute requirement for Dpp input in Shn-mediated silencing. Not even a partial repressor activity of Shn was observed in cells that do not receive Dpp signal (e.g., loss of shn function in cells situated in lateral-most positions of the wing disc does not cause a further upregulation of brk transcription). (3) Shn represents only one of several zinc finger proteins expressed in Dpp receiving cells, yet none of the other proteins is able to substitute for Dpp-mediated repression. A major determinant for the specificity with which Shn engages in the signaling-dependent protein/DNA complex appears to be the triple zinc-finger motif. Although it is likely that this structural feature is required for contacting specific nucleotides on the brk silencer, the possibility cannot be not excluded that some of the zinc fingers mediate protein-protein interactions between Shn and Mad, Med or other cofactors (Müller, 2003).

Mad acts on a Hox response element to confer specificity and diversity to Hox protein function

Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).

AbdA is expressed and is active in the third and fourth compartments of the midgut (PS8-PS12), and yet it activates the wg target gene only in PS8. Dpp secreted from PS7 is shown to provide the spatial information required for PS8-localized wg activation and, acting through a newly identified 546 bp enhancer, AbdA and Mad, a transcriptional effector of the Dpp pathway, directly control wg transcription. The convergence of Hox function and Dpp signaling therefore occurs at the levels of DNA and transcription, and endows AbdA with PS8-specific regulatory properties (Grienenberger, 2003).

To identify the enhancer responsible for wg expression in the VM, subfragments of a 9kb genomic region known to drive wg embryonic expression were analyzed in transgenic lines transformed with lacZ reporter constructs. The smallest fragment that drives accurate expression in the VM is a 546 bp XhoI/ClaI (XC) restriction fragment. Its activity is first detected during germ-band retraction, when wg transcripts are visualized in the VM by in situ hybridization, and only in PS8 VM cells. During subsequent development, XC enhancer activity still mimics wg expression, and is associated with the site of central midgut constriction formation. Thus, from early on to the end of embryogenesis, the XC enhancer exclusively and accurately recapitulates wg spatiotemporal expression in the VM (Grienenberger, 2003).

To address whether AbdA and Dpp signaling could directly regulate wg, the sequence of the XC enhancer was examined for the presence of putative binding sites for AbdA and for Mad/Medea (referred to as DRS, for Dpp response sequence), the canonical transcriptional effectors of the Dpp signaling pathway known to recognize identical target sequences. Since genetic and molecular data led to the proposal that, in Drosophila, the CRE sequences to which Creb proteins bind are required to respond to Dpp in addition to DRSs, potential Creb binding sites were sought. Six TAAT core sequences and four sequences resembling the consensual Hox/Pbx binding sites (TGATNNATG/TG/A) were identified as potentially mediating AbdA function. The Hox/Pbx 3 and 2 sequences strongly match the consensus, with seven or six of the eight consensus nucleotides conserved, respectively. Hox/Pbx sequences 1 and 4 only have five of the eight consensus nucleotides conserved. The XC fragment contains three sequences matching DRSs and two potential CRE sites (Grienenberger, 2003).

To assess the evolutionary conservation of the XC enhancer, an homologous fragment from Drosophila virilis was isolated and analyzed for its in vivo activity by transgenesis in Drosophila melanogaster. The D. virilis fragment drives expression in a pattern very similar to that of the XC enhancer, suggesting that sequences conserved between these two enhancers may be important for wg regulation in the midgut. Sequence comparison, including sequences from D. pseudoobscura, revealed that a majority of the TAAT core motifs, the DRSs and the putative Creb-binding sequences are evolutionarily conserved, whereas sequences that match heterodimeric Hox/Pbx consensus binding sites are not. The existence of two large conserved sequences, Box 1 and 2, is noted. Since Box1 lies in a fragment that does not drive reporter gene expression in transgenic flies, particular attention was paid to Box2 (Grienenberger, 2003).

Hox signaling integration was examined to determine whether signaling pathways contribute towards specifying how AbdA, a widely expressed Hox selector protein, controls the development of distinct pattern elements at different locations. Dpp signal secreted from PS7 provides the positional cue responsible for localized activation of wg by AbdA. Biochemical and reverse genetics experiments have established that AbdA and Mad directly regulate wg transcription through the XC enhancer, which thus serves as an integrator of Hox and Dpp input. AbdA is impotent with respect to this enhancer in the absence of the Dpp signal, though it can function perfectly well on other genes without Dpp. Therefore, functional interactions between selector proteins and signaling pathways confer specificity to signaling pathways, and reciprocally confer functional diversity to selector proteins (Grienenberger, 2003).

This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).

Further studies are required to understand how Hox selector proteins functionally interact with nuclear effectors of signaling pathways to generate specific transcriptional patterns. In the control of wg by AbdA, several scenarios can be envisioned. In one, the effect of the Dpp transcriptional effector Mad on AbdA activity would be indirect, by antagonizing the function of a repressor that would otherwise act on the XC enhancer to prevent wg expression. The absence of a binding site for this hypothetical repressor in Box2 could explain how Box2 drives AbdA-dependent transcription even without Dpp transcriptional effector binding sites. In a second scenario, Dpp transcriptional effectors would more directly control the activity of AbdA by influencing its DNA binding or transregulatory properties. A direct interaction of HoxC8 and Smad1 has been reported to induce osteoblast differentiation in mammals, suggesting that the coordinate action of AbdA and Dpp signaling might rely on direct AbdA-Mad interaction. In wg regulation, the situation may be different, as additional regulatory inputs are involved. bin and hth are essential, and Wg signaling is required for accurate levels of wg expression. The contribution of Creb might indicate that the Ras/Mapk signaling pathway is involved as well. Ras signaling has been proposed to play a permissive role by acting on CRE sequences of the Ubx and lab enhancers. These observations suggest that AbdA and Hox proteins in general attain specificity and diversity by participating in a variety of protein interactions in enhancer-binding complexes (Grienenberger, 2003).

Function and regulation of homothorax in the wing imaginal disc of Drosophila: hth is latently active in the wing cells and has to be repressed by the continuous activity of the Dpp and Wg signals

The gene homothorax is required for the nuclear import of Extradenticle, The functions of exd/hth and of the Hh/Wg/Dpp pathway are mutually antagonistic: exd blocks the response of Hh/Wg/Dpp target genes such as optomotor-blind and dachshund; high levels of Wg and Dpp eliminate exd function by repressing hth. This repression is mediated by the activity of Dll and dac. One prerequisite for appendage development is the inactivation of the exd/hth genes (Azpiazu, 2000 and references therein).

htx is originally expressed uniformly in the wing imaginal disc but, during development, its activity is restricted to the cells that form the thorax and the hinge, where the wing blade attaches to the thorax, and it is eliminated in the wing pouch, which forms the wing blade. Repression of hth in the wing pouch is a prerequisite for wing development; forcing hth expression prevents growth of the wing blade. Both the Dpp and the Wg pathways are involved in hth repression. Cells unable to process the Dpp signal (lacking thick veins or Mothers against Dpp activity) or the Wg signal (lacking dishevelled function) express hth in the wing pouch. vestigial has been identified as a Wg and Dpp response factor that is involved in hth control. In contrast to its repressing role in the wing pouch, wg upregulates hth expression in the hinge; teashirt is a positive regulator of hth in the hinge. tsh plays a role specifying hinge structures, possibly in co-operation with hth (Azpiazu, 2000).

In the second instar wing disc, the Hth product accumulates uniformly in the thoracic and appendage regions of the disc, but throughout the third larval period hth expression is downregulated and, by the late third instar, Hth only appears in the presumptive regions of the thorax and the wing hinge. The central part of the disc, which gives rise to the wing pouch, shows no hth expression. The repression of hth function is important for wing development, because if hth activity is forced in the wing pouch, the wing does not form. A similar observation has been made in the leg disc; hth or exd expression in the distal part results in a truncated appendage in which all the distal components are missing. In the leg, the subdivision between distal and proximal regions results from the antagonism between Hh signaling and exd/hth function. Hh response genes such as Dll and dac are instrumental in repressing hth (Azpiazu, 2000 and references therein).

The downregulation of hth in the wing pouch is a consequence of the activity of the Dpp and the Wg signaling pathways. In cells in which the response to the Dpp signal is prevented, as in tkv or Mad mutant cells, hth is expressed at high levels. Similarly, dsh minus cells, in which the transduction of Wg is blocked, show ectopic hth activity and consequently nuclear exd expression. These results also indicate that hth is latently active in the wing cells and has to be repressed by the continuous activity of the Dpp and Wg signals. The inability of cell clones to proliferate, cells in which the Dpp or the Wg pathways have been totally eliminated, may be due to high levels of hth expression. The Dpp and Wg pathways repress hth expression independently. This is illustrated by the experiments inducing dsh mutant clones: ectopic hth expression is only observed in clones located away from the AP border. This suggests that the high levels of Dpp expression near the AP border are sufficient to impede hth expression despite the removal of the control by Wg (Azpiazu, 2000).

Transcriptional repression due to high levels of Wingless signalling: Transcriptional activation of Ubx depends Mad and Wg signalling

Extracellular signals can act at different threshold levels to elicit distinct transcriptional and cellular responses. The transcriptional regulation of the Wingless target gene Ultrabithorax has been examined in the embryonic midgut of Drosophila. Ubx transcription is stimulated in this tissue by Dpp and by low levels of Wingless signaling. High levels of Wingless signaling can repress Ubx transcription. The response sequence within the Ubx midgut enhancer required for this repression coincides with a motif required for transcriptional stimulation by Dpp, namely a tandem array of binding sites for the Dpp-tranducing protein, Mad. Indeed, Wingless-mediated repression depends on low levels of Dpp, although apparently not on Mad itself. In contrast, high levels of Dpp signaling antagonize Wingless-mediated repression. This suggests that transcriptional activation of Ubx is subject to competition between Dpp-activated Mad and another Smad whose function as a transcriptional repressor depends on high Wg signaling. Wingless can repress its own expression via an autorepressive feedback loop that results in a change of the Wingless signaling profile during development (Yu, 1998).

Dpp and Wg signaling synergize in the visceral mesoderm to stimulate Ubx transcription, targeting distinct, albeit adjacent, response sequences in the Ubx midgut enhancer. Therefore, efficient stimulation of Ubx transcription by Wg depends on dpp. Wg-mediated repression also depends on dpp, but, remarkably in this case, the response sequence for Wg-mediated repression within the Ubx enhancer coincides with that for Dpp-mediated stimulation. Indeed, the WRS-R/DRS (Wingless response sequence mediating repression and Dpp response sequence) functions in two antipodal responses: it mediates efficient transcriptional stimulation when the signaling levels of Dpp are high and those of Wg are low, but it is also required for transcriptional repression when the Wg signaling levels are high and those of Dpp are low. This raises the possibility that the same factor may confer the two antipodal responses. However, this is unlikely to be the case since Mad itself, which binds to the DRS to mediate the positive response to Dpp, is apparently not required for the Wg-mediated repression (Yu, 1998).

Thus it is proposed that the two antipodal responses are conferred by two distinct factors: by Mad and by a hypothetical protein WR. It is further proposed that WR is a Mad-related protein, i.e. a Smad, since WR acts through Mad-binding sites and since its function as a repressor depends on dpp. It is envisaged that WR, like Mad itself and other Smads, is activated by Dpp signaling through phosphorylation by ligand-bound membrane receptors, an event that promotes their subsequent translocation to the nucleus. In this scenario, Dpp enables WR (which also needs to be activated by high Wg signaling) to occupy the Mad-binding sites within the Ubx enhancer. Once bound to this enhancer, WR dominantly represses Ubx transcription, overriding the activating function of Arm-Pangolin and other transcriptional activators bound to the same enhancer (Yu, 1998).

How is WR's repressor function activated by high Wg levels? It is presumed that high Wg signaling regulates, directly or indirectly, the availability of WR as an enhancer-binding protein: either high Wg signaling controls a post-transcriptional event (e.g. it may promote WR's association with Armadillo, or WR's translocation into the nucleus), or it simply activates transcription of the WR gene. The latter possibility of indirect regulation, which involves transcriptional coupling, is favored because it accomodates readily the dependence of Wg-mediated repression on arm and Pangolin. Whatever the case, it is emphasized that high Wg signaling controls the activity of the protein WR (possibly a Smad), which also requires Dpp signaling. Thus, WR is a common target for two signaling pathways and represents a point of convergence between them (Yu, 1998).

This model readily explains how high Dpp levels antagonize WR, namely by promoting maximal levels of nuclear Mad which now competes with WR for binding to the Ubx enhancer. The outcome of this competition is the transcriptional activation or repression of target genes, depending on the prevalence of Mad or WR. This may illustrate a general principle, namely that the response sequence for the positive effect of one signal is also the response sequence for the negative effect of an antagonistic signal. Such a layout provides a sharp flipping of the response from positive to negative in an area where cells are experiencing increasingly more of one signal and increasingly less of the antagonizing one (Yu, 1998).

Medea is the Smad4 homolog that is known to be the common oligomerization partner for pathway-specific Smads. Furthermore, Medea binds to the same DNA sequences as Mad. This raises the possibility that Medea is an oligomerization partner of WR: while Medea, together with Mad, is expected to activate transcription, together with WR it may repress transcription. A precedent for this scenario is the Myc/Mad/Max system, in which Mad (a bHLH protein that happens to have the same name as the Dpp transducer Mad) is a common dimerization partner for either Myc, a transcriptional activator, or Max, a transcriptional repressor. In addition to antagonism, there is also synergy between Wg and Dpp in the embryonic midgut. This synergy apparently results from cooperation between the nuclear target factors activated by the two signals, i.e. between Arm-Pangolin and Mad/CRE-binding proteins. Other examples of apparent synergy between Wg and Dpp are the leg and wing imaginal discs, where these signals act together in central disc regions to stimulate expression of homeobox genes. But the two signals also antagonize each other in leg discs, as well as in eye discs. Although it is conceivable that the developmental context determines the synergy or antagonism between Dpp and Wg, the situation in the midgut suggests that the decisive factor in each case may be the levels of signaling (Yu, 1998 and references).

It is interesting that Wg signaling can repress its own expression when signaling levels reach a critically high level. This indicates a negative feedback loop, which could account for two observations: (1) Wg signaling shifts its own expression towards the anterior over time. It is not known at present whether this shift has any biological significance. (2) Wg has the potential for switching itself off over time. This is actually observed, since Wg expression becomes undetectable by the end of embryogenesis. Clearly, Wg's negative feedback loop is capable of changing the Wg signaling profile as development procedes. There are negative feedback loops for other signaling pathways in Drosophila. For example, the epidermal growth factor (EGF) receptor inhibits itself eventually, after signaling has reached a critical level, by switching on expression of an inhibitory ligand, Argos. In the ovary, this negative feedback loop causes splitting of a single signaling peak into twin peaks. Furthermore, Hedgehog signaling in the eye imaginal disc is repressive at high Hedgehog levels, but stimulatory in cells, further away from the signaling source, which experience lower Hedgehog levels. Perhaps such 'hard-wired' negative feedback loops in signaling pathways are fairly universal, and serve to stop these pathways from escalating out of control. If so, this would be akin to feedback inhibition of metabolic pathways, which provides homeostatic control (Yu, 1998 and references).

Restricted patterning of vestigial expression in Drosophila wing imaginal discs requires synergistic activation by both Mad and the Drifter POU domain transcription factor

The Drosophila Vestigial protein has been shown to play an essential role in the regulation of cell proliferation and differentiation within the developing wing imaginal disc. Cell-specific expression of vg is controlled by two separate transcriptional enhancers. The boundary enhancer controls expression in cells near the dorsoventral (DV) boundary and is regulated by the Notch signal transduction pathway, while the quadrant enhancer responds to the Decapentaplegic and Wingless morphogen gradients emanating from cells near the anteroposterior (AP) and DV boundaries, respectively. MAD-dependent activation of the vestigial quadrant enhancer results in broad expression throughout the wing pouch but is excluded from cells near the DV boundary. This has previously been thought to be due to direct repression by a signal from the DV boundary; however, this exclusion of quadrant enhancer-dependent expression from the DV boundary has been shown to be due to the absence of an additional essential activator in those cells. The Drosophila POU domain transcriptional regulator, Drifter, is expressed in all cells within the wing pouch expressing a vgQ-lacZ transgene and is also excluded from the DV boundary. Viable drifter hypomorphic mutations cause defects in cell proliferation and wing vein patterning correlated with decreased quadrant enhancer-dependent expression. Drifter misexpression at the DV boundary using the GAL4/UAS system causes ectopic outgrowths at the distal wing tip due to induction of aberrant Vestigial expression, while a dominant-negative Drifter isoform represses expression of vgQ-lacZ and causes severe notching of the adult wing. In addition, an essential evolutionarily conserved sequence element bound by the Drifter protein with high affinity has been identified and it has been located adjacent to the MAD binding site within the quadrant enhancer. These results demonstrate that Drifter functions along with MAD as a direct activator of Vestigial expression in the wing pouch (Certel, 2000).

The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated transcription factors (Pangolin/dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).

Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).

Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by Notch then selects a single progenitor from each equivalence group (Halfon, 2000).

The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their own respective downstream transcriptional effectors, dTCF and Mothers against dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2 (Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).

The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the Egfr and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).

Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the Egfr and Htl signal transduction pathways (Halfon, 2000).

One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2EPC and F2DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).

Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15DA1 and F2DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).

Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).

Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).

To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).

It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).

Smads silence bam in ovarian germ-line stem cells

Stem cells execute self-renewing and asymmetric cell divisions in close association with stromal cells that form a niche. The mechanisms that link stromal cell signaling to self-renewal and asymmetry are only beginning to be identified, but Drosophila oogenic germline stem cells (GSCs) have emerged as an important model for studying stem cell niches. Decapentaplegic sustains ovarian GSCs by suppressing differentiation in the stem cell niche. Dpp overexpression expands the niche, blocks germ cell differentiation, and causes GSC hyperplasty. The bag-of-marbles (bam) differentiation factor is the principal target of Dpp signaling in GSCs; ectopic bam expression restores differentiation even when Dpp is overexpressed. The transcriptional silencer element in the bam gene integrates Dpp control of bam expression. Finally and most significantly, this study demonstrates that Dpp signaling regulates bam expression directly since the bam silencer element (SE) is a strong binding site for the Drosophila Smads, Mad and Medea. These studies provide a simple mechanistic explanation for how stromal cell signals regulate both the self-renewal and asymmetric fates of the products of stem cell division (Chen, 2003).

GSCs divide in the anterior/posterior axis, and this division produces daughters with different fates. The anterior cell of a GSC division retains contact with the stromal cap cells, maintains high levels of Dpp signaling, and continues as a stem cell. The posterior stem cell daughter dissociates from the cap cells and becomes a cystoblast (CB). The CB divides precisely four times with incomplete cytokinesis, giving rise to a cyst of 16 interconnected cells that differentiate further into 1 oocyte and 15 nurse cells. The progress of cyst formation can be followed by monitoring the morphogenesis of a dynamic organelle, termed the fusome, that grows and branches with each cyst cell mitosis (Chen, 2003).

Cystoblasts require the product of the bag-of-marbles (bam) gene, which is both necessary and sufficient for differentiation. Germ cells lacking bam fail to differentiate into cystoblasts and continue to divide with full cytokinesis, producing germ cell hyperplasia. The failure of these proliferating germ cells to differentiate can be recognized by following the fusome since it remains spherical instead of growing into a branched structure. Superficially, therefore, bam mutant cells behave like GSCs, but molecular markers to determine the stage of arrest have been lacking (Chen, 2003).

Studies characterizing bam transcription demonstrate that bam is tightly regulated such that it is off in GSCs and on in CBs. Thus, it was possible to determine if bam mutant cells are 'stem cell-like' since the activity of a bam reporter transgene would distinguish GSCs from CBs precisely. GFP expression was examined in bam mutant animals carrying a transgene with the bam promoter fused to GFP; most germ cells were observed to be GFP positive. Thus, unlike GSCs, germ cells lacking bam advanced to a state of differentiation sufficient to activate bam transcription (Chen, 2003).

Two features of GSC divisions demand molecular explanation: how is the anterior daughter of the GSC division retained as a stem cell (self-renewal) and what causes the posterior daughter to differentiate into a CB (asymmetric division)? Stromal cells, including the cap and inner sheath cells, at the germarial tip express several signaling molecules and are a likely source of dpp. dpp signaling has been shown to be required to maintain GSCs, and transcriptional control over Bam has been shown to distinguish GSCs from CBs. These two phenomena can now be linked directly (Chen, 2003).

In GSCs, in which dpp signaling and pMad levels are highest, the Mad:Med complex binds to the bam SE and prevents bam transcription. GSCs self-renew because association of the anterior daughter with stromal cells permits sufficient dpp signaling to block CB differentiation by assembling a repressor complex on the bam SE element. This complex is likely to include other factors required for transcriptional antagonism, such as TGIF, a homeodomain-containing transcriptional corepressor of TGF-beta-dependent gene expression, or Ski/Sno factors, which can recruit histone-modifying enzymes. The complex may also contain Schnurri, a negatively acting Mad cofactor, since shn mutant GSCs also differentiate precociously (Chen, 2003).

During division, a GSC daughter cell is displaced away from the cap cells and into a region of diminished dpp signaling. This cell, the CB precursor (pre-CB), expresses lower pMad levels that would cause the concentration of Mad:Med complexes to fall. Declining occupancy levels of the bam SE would produce derepression of bam transcription and concomittant activation of the CB differentiation program (Chen, 2003).

Embryonic stem cells are considered totipotent because they can populate any of the adult niches. Although the degree of adult stem cell plasticity is currently receiving much attention, assembly of stem cells into signaling niches during postembryonic development might impose differentiation limits. What are the specific effects of stromal cell niches on captured stem cells? In the case of the Drosophila ovarian niche, GSCs are maintained as 'CBs-in-waiting' because a stromal cell signal represses the expression of one key factor (i.e., Bam). Perhaps other types of stem cells are similarly differentiated but blocked by stromal cell signals and require the expression of only one or a few key molecules to resume development (Chen, 2003).

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters (cystoblasts). Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal (Song, 2004).

This study reveals a new function for gbb in the regulation of GSCs in the Drosophila ovary. Loss of gbb function leads to GSC differentiation and stem cell loss, similar to dpp mutants. gbb is expressed in somatic cells but not in germ cells, suggesting that gbb is another niche signal that controls GSC maintenance. Like dpp, gbb contributes to the production of pMad in GSCs and also functions to repress bam expression in GSCs. As in the wing imaginal disc, gbb also probably functions to augment the dpp signal in the regulation of GSCs through common receptors in the Drosophila ovary. In both dpp and gbb mutants, pMad accumulation in GSCs is severely reduced but not completely diminished. Since the dpp or gbb mutants used in this study do not carry complete loss-of-function mutations, it remains possible that complete elimination of either dpp or gbb function is sufficient for eradicating pMad accumulation in GSCs. Alternatively, both dpp and gbb signaling are required independently for full pMad accumulation in GSCs, and thus disrupting either one of them only partially diminishes pMad accumulation in GSCs. The lethality of null dpp and gbb mutants, and the difficulty in completely removing their function in the adult ovary, prevent these possibilities from being tested directly (Song, 2004).

Interestingly, dpp overexpression results in complete suppression of cystoblast differentiation and complete repression of bam transcription in the germ cells, whereas gbb overexpression does not have obvious effects on cystoblast differentiation or bam transcription. Even though the UAS-gbb transgene and the c587 driver for gbb overexpression have been demonstrated to function properly, it is possible that active Gbb proteins are not produced in inner sheath cells and somatic follicle cells because of a lack of proper factors that are required for Gbb translation and processing in those cells, which could explain why the assumed gbb overexpression does not have any effect on cystoblast differentiation. However, since active Dpp proteins can be successfully achieved using the same expression method, and Dpp and Gbb are closely related Bmps, it is unlikely that active Gbb proteins are not produced in inner sheath cells and follicle cells. Alternatively, dpp and gbb signals could have distinct signaling properties, and dpp may play a greater role in regulating GSCs and cystoblasts. Recent studies have indicated that Dpp and Gbb have context-dependent relationships in wing development. In the wing disc, duplications of dpp are able to rescue many but not all of the phenotypes associated with gbb mutants, suggesting that dpp and gbb have not only partly redundant functions but also distinct signaling properties. In the wing and ovary, gbb and dpp function through two Bmp type I receptors, sax and tkv. The puzzling difference between gbb and dpp could be explained by context-dependent modifications of Bmp proteins, which render different signaling properties in different cell types. It will be of great future interest to better understand what causes Bmps to have distinct signaling properties (Song, 2004).

All the defined niches share a commonality, structural asymmetry, which ensures stem cells and their differentiated daughters receive different levels of niche signals. In order for a niche signal to function differently in a stem cell and its immediately differentiating daughter cell that is just one cell away, it has to be short-ranged and localized. This study shows that Bmp signaling mediated by Dpp and Gbb results in preferential expression of pMad and Dad in GSCs. Bmp signaling appears to elicit different levels of responses in GSCs and cystoblasts, suggesting that the cap cells are likely to be a source for active short-ranged Bmp signals. These observations support the idea that Bmp signals are active only around cap cells. Consistently, when GSCs lose contact with the cap cells following the removal of adherens junctions they move away from the niche and then are lost. As gbb and dpp mRNAs are broadly expressed in the other somatic cells of the germarium besides cap cells, localized active Bmp proteins around cap cells could be generated by localized translation and/or activation of Bmp proteins. As they can function as long-range signals, it remains unclear how Dpp and Gbb act as short-range signals in the GSC niche (Song, 2004).

Bmp signaling and bam expression are in direct opposition in Drosophila ovarian GSCs. bam is actively repressed in GSCs through a defined transcriptional silencer. These observations lead to a model in which Bmp signals from the niche maintain adjacent germ cells as GSCs by actively suppressing bam transcription and thus preventing differentiation into cystoblasts. The levels of pMad are correlated with the amount of bam transcriptional repression in GSCs and cystoblasts. In the wild-type germarium, pMad is highly expressed in GSCs and some cystoblasts where bam is repressed. In other cystoblasts and differentiated germline cysts, pMad is reduced to very low levels, and thus bam transcriptional repression is relieved. In the GSCs mutant for dpp, gbb or punt, pMad levels are severely reduced, and bam begins to be expressed. The repression of bam transcription as a result of dpp overexpression seems to be a rapid process; bam mRNA is reduced to below detectable levels two hours after dpp is overexpressed. This suggests that repression of bam transcription by Bmp signaling could be direct. Furthermore, Med and Mad can bind to the defined bam silencer in vitro, which also supports the idea that Bmp signaling acts directly to repress bam transcription. Dpp signaling has also been shown to repress brinker (brk) expression in the wing imaginal disc and in the embryo. The repression of brk expression by Dpp signaling is mediated by the direct binding of Mad and Med to a silencer element in the brk promoter. Since the brk silencer is very similar to the bam silencer, the results suggest that bam repression in GSCs is also mediated directly by Dpp and Gbb in a similar manner (Song, 2004).

It remains unclear how the binding of Med and Mad to the bam silencer results in bam transcriptional repression in GSCs. For the brk silencer, Dpp signaling and Shn are both required to repress brk expression in the Drosophila wing disc and embryo. Mad and Med belong to the Smad protein family, which are known to function as transcriptional activators by recruiting co-activators with histone acetyltransferase activity. In the wing disc, Shn is proposed to function as a switch factor that converts the activating property of Mad and Med proteins into a transcriptional repressor property. Possibly, the Mad-Med complex could also recruit Shn to the bam repressor element. Consistent with the possible role of Shn in repressing bam expression in GSCs is the observation that GSCs that lose shn function differentiate, and thus are lost. Also, it remains possible that Mad and Med could recruit a repressor other than Shn when binding to the bam repressor element. In the future, it will be very important to determine whether Shn itself is a co-repressor for Mad/Med proteins or whether it directly recruits a co-repressor to repress bam transcription in GSCs (Song, 2004).

In Drosophila, primordial germ cells (PGCs) are set aside from somatic cells and subsequently migrate through the embryo and associate with somatic gonadal cells to form the embryonic gonad. During larval stages, PGCs proliferate in the female gonad, and a subset of PGCs are selected at late larval stages to become germ line stem cells (GSCs), the source of continuous egg production throughout adulthood. However, the degree of similarity between PGCs and the self-renewing GSCs is unclear. Many of the genes that are required for GSC maintenance in adults are also required to prevent precocious differentiation of PGCs within the larval ovary. Following overexpression of the GSC-differentiation gene bag of marbles (bam), PGCs differentiate to form cysts without becoming GSCs. Furthermore, PGCs that are mutant for nanos (nos), pumilio (pum) or for signaling components of the decapentaplegic (dpp) pathway also differentiate. The similarity in the genes necessary for GSC maintenance and the repression of PGC differentiation suggest that PGCs and GSCs may be functionally equivalent and that the larval gonad functions as a 'PGC niche' (Gilboa, 2004).

The embryonic gonad in Drosophila forms when somatic gonadal cells encapsulate about 12 primordial germ cells on each side of the embryo. PGCs proliferate in the female gonad during the subsequent three larval stages until, at the late-third larval stage, the gonad carries over 100 germ cells. At the larval/pupal transition, PGCs at the posterior of the gonad differentiate. Similar to differentiating germ line stem cells, differentiating PGCs undergo several rounds of incomplete mitotic divisions to form cysts and subsequently egg chambers with one oocyte and 15 nurse cells. Differentiation of posterior PGCs at the larval/pupal transition was attributed to the hormonal changes that control pupa formation. At the anterior part of the gonad, the newly formed somatic niche prevents the differentiation of PGCs and these are maintained as self-renewing GSCs throughout adult life. It has therefore been proposed that the transition from PGCs to GSCs coincides with the larval/pupal transition and the formation of the somatic niche. This study examines the genetic mechanisms that control PGCs during the proliferative larval stages, to better understand the PGC to GSC transition (Gilboa, 2004).

To explore how PGC differentiation is inhibited in the larval ovary, tests were performed to see whether any of the genes that are needed for either GSC maintenance or differentiation in the adult are likewise required in PGCs. Bag of marbles (Bam) has been shown to be a critical differentiation factor, because it is both necessary and sufficient to induce adult GSCs to differentiate. Therefore whether overexpression of Bam in the larval ovary is sufficient to drive PGCs to differentiate and form cysts was tested. As a marker for cyst formation, an antibody against an adducin-like molecule (1B1) was used, that stains the fusome, a sub-cellular organelle that is spherical in PGCs and GSCs but extends and branches as the single germ cell forms a multicellular cyst. Larvae carrying the bam gene under control of a heat-shock promoter (hs-bam) were heat-shocked at various time points during larval development and their gonads were examined one or two days later. Control wild-type gonads at the larval/pupal transition (LL3) exhibited either single PGCs carrying a spherical fusome or 2-cell cysts, which shared a bar-like fusome. The latter could be PGCs undergoing division, prior to full dissociation, or PGCs that initiated differentiation at the larval/pupal transition. In contrast, in hs-bam gonads, PGCs differentiate, as shown by the many cysts with branched fusome. Differentiation in hs-bam ovaries depended on the time of heat-shock. No cysts were observed in ovaries derived from larvae that were heat-shocked at the end of embryogenesis. Heat-shocking at the end of the first-larval instar (LL1) led to a high fraction (75%) of gonads that carried many cysts, whereas heat-shocking at the end of the second-larval instar (LL2) led to differentiation of all PGCs in all gonads tested. Thus, PGCs are able to differentiate prior to the larval/pupal transition. The time-dependent response to hs-bam could indicate either that PGCs are more capable of differentiation as the animal matures or that transcription from the hs promoter may be more active in the later larval stages. In support of the latter hypothesis, PGCs are transcriptionally quiescent during early embryogenesis and acquire transcriptional competence as they start to migrate. Indeed, the quantity of bam transcript seems limiting because a less rigorous heat-shock regime induces fewer cysts. Furthermore, with a different expression system, PGCs could be induced to differentiate as early as the end of embryogenesis. In support of the notion that differentiating PGCs follow the normal differentiation program, it was found that the time course of mitotic divisions in cysts that were precociously induced at LL2 was similar to that observed in cysts during normal development in either the pupal or the adult ovary (Gilboa, 2004).

These results suggest that all PGCs in the larval ovary are capable of differentiating following overexpression of Bam. Therefore whether active repression is required to keep PGCs in a proliferative state was tested. In adult GSCs, the Decapentaplegic (Dpp) pathway plays a major role in GSC maintenance. Dpp is produced by niche cells and is perceived directly by GSCs. Dpp signaling activates the downstream components Mothers against dpp (Mad) and Medea (Med), which directly bind to the bam promoter and repress the transcription of bam. In the larval gonad, overexpression of Dpp induces overproliferation of PGCs, suggesting that PGCs can respond to a Dpp signal. However, PGCs have not been shown to require Dpp in larval gonads. It was found that abolishing Dpp signaling in PGCs by overexpression of the negative regulator Daughters against Dpp (Dad) or mutations in thickveins (tkv), the Dpp type I receptor, induced differentiation of PGCs. 16-cell cysts were observed already at LL2 (48 hr after hatching), suggesting that PGCs begin differentiation shortly after the end of embryogenesis. Oocyte determination was detected in LL3 gonads, as indicated by accumulation of Orb, an oocyte marker, in one cell of the cyst (Gilboa, 2004).

To further explore the requirement for Dpp within GSCs, it was asked whether Dpp signaling could be detected directly in larval PGCs by monitoring the accumulation of its target, phosphorylated Mad (pMad) in PGCs. In larval gonads all PGCs accumulate pMad in the nucleus, suggesting that during larval development all PGCs receive a Dpp signal that actively represses their differentiation. In the adult, only germ line cells close to the niche contain significant levels of nuclear pMAD. Thus, the larval ovary may function in a similar manner to the adult niche in the prevention of PGCs from differentiation (Gilboa, 2004).

GSC differentiation is repressed by extrinsic factors, such as Dpp, and also by intrinsic factors. To further test whether PGCs employ the same mechanisms as GSCs to repress differentiation, larval ovaries were examined that were mutant for the translational repressors Nanos (Nos) and Pumilio (Pum), which function within GSCs to repress their differentiation. Indeed, nos mutant LL3 gonads contained many developed cysts. pumilio (pum) mutant gonads also contained cysts, although less so than nos mutants. Gonads that were mutant for both nos and pum did not contain more cysts than gonads that were mutant for nos alone. Because the alleles that were used were very strong, this suggests that nos and pum function together in the repression of PGC differentiation (Gilboa, 2004).

In adult ovaries, the differentiation of cysts requires Bam, and increasing amounts of Bam are present during each subsequent mitotic division. A reporter construct of GFP under control of the bam promoter was used to follow bam expression in the larval cysts. Cysts found in nos ML3 larval gonads also expressed higher amounts of GFP as compared to single PGCs. As in adults, the intensity of GFP labeling corresponds to the developmental state of the cyst. In addition to precocious differentiation, nos mutant germ cells displayed aberrations in the shape of the branched fusome and increased amount of small fusomal material as compared with wild-type. It is concluded that both Nos and Pum, which are required for GSC maintenance, are also required to repress PGC differentiation (Gilboa, 2004).

To further test for a possible partnership between nos and pum in GSC maintenance, the time at which nos or pum mutant germ line clones, generated by the FLP-FRT method, were eliminated from the adult ovary was examined. In wild-type, clones of unmarked GSCs were induced in about 25% of the ovarioles and that percent decreased only slightly during the course of the experiment, probably due to the natural rate of GSC loss. nos and pum mutant GSCs, in contrast, were lost rapidly. GSC loss was observed as early as 4 days after clone induction, and by the 6th or 7th day, most ovarioles did not contain a mutant GSC. The striking similarity in the profiles of nos and pum GSC loss therefore suggests that these genes also function together within GSCs (Gilboa, 2004).

As of the fifth and sixth day after clone induction, it was found that many nos mutant cysts were eliminated from the ovary. These results agree with the death of cysts observed in nos and pum mutants and with the death of nos cysts in pupal ovaries, which may be the cause of the empty ovarioles observed in adult nos females. These results and the previously reported phenotypes of nos and pum suggest that these genes are continually required throughout germ cell life. In the embryo, nos and pum are required for correct migration, transcription, and viability. During larval stages, they are required for the repression of PGC differentiation and, in the adult, for the maintenance and viability of GSCs as well as for the viability of differentiating cysts (Gilboa, 2004).

The targets of Nos and Pum within GSCs remain elusive, and the relationship of these 'intrinsic' GSC maintenance factors to the 'extrinsic' Dpp signal is unclear. To test if Dpp could function partly through Nos, the Nos expression pattern was examined in wild-type and in tkv-mutant GSCs. In wild-type germaria Nos is expressed at intermediate levels in GSCs and their immediate daughters, at very low levels during mitotic divisions of the cyst, and at very high levels in a fraction of the 16-cell cysts. This expression pattern was unchanged in tkv-mutant germ cells. Similar results were obtained for larval PGCs; Nos was expressed at intermediate levels in wild-type and tkv mutant PGCs, at lower levels in cysts undergoing mitosis, and at very high levels in 16-cell cysts. This suggests that Nos expression is independent of Dpp signaling (Gilboa, 2004).

Next, whether nos is required for Dpp function was tested, by analyzing nos mutant PGCs that were overexpressing either Dpp or TkvQD, a constitutively activated form of Tkv. In nos mutant control gonads, fragmented fusomal material as well as branched cysts could be observed. The spherical fusome within nos mutant germ cells remained small or fragmented in nos gonads overexpressing Dpp. Most strikingly, single PGC/GSC like germ cells accumulated in these gonads, and no cysts could be found. Thus, although increased Dpp signaling cannot fully counteract the nos phenotype, it does prevent precocious differentiation of nos mutant PGCs. Similar results were obtained with PGCs expressing TkvQD. In most gonads no cysts could be observed, although occasionally a small branched fusome could be detected, suggesting that Dpp signaling acts directly on PGCs, rather than via a secondary signal. The genetic data show that PGCs that are mutant for nos, can still respond to a Dpp signal, which keeps them in an undifferentiated state (Gilboa, 2004).

During larval stages, PGCs proliferate rather than differentiate. The translational repressors Nos and Pum are required to repress PGCs differentiation during larval stages. It has also been show that the Dpp pathway functions in a similar manner. Both pathways are also required for GSC maintenance. The fact that the spherical fusome remains abnormal in nos mutant gonads even when Dpp is overexpressed may suggest that some of Nos function is downstream of Dpp. However, the Nos expression data and the fact that Dpp signaling can prevent nos mutant PGCs from differentiation are more compatible with the Nos pathway playing a role upstream or in parallel to the Dpp pathway. It remains unclear how these pathways converge within germ cells (Gilboa, 2004).

Germ cells may perceive a Dpp signal from the moment they form at the posterior pole of the embryo until they differentiate to form cysts. Indeed, pMad is present in embryonic pole cells, larval PGCs and adult GSCs. Dpp signaling is not only necessary for GSC maintenance but also required continually through larval stages to actively repress PGC differentiation. Thus, the larval ovary functions in a similar manner to the adult niche with regard to Dpp-mediated repression of differentiation. During the third-larval instar, the adult somatic niche forms, and repression of PGC differentiation may then become limited to the small area of the adult ovary, allowing PGCs outside the confinement of the niche to differentiate (Gilboa, 2004).

Repression of PGC differentiation is required for about 4 days, from the end of embryogenesis to the beginning of pupa formation, whereas GSCs are maintained in the adult for many days. Differences between the 'short-term' and the 'long-term' repression of differentiation may yet be found. However, all the genes tested, dpp, bam, nos, and pum, function similarly in GSCs and PGCs. This similarity suggests that there may not be a clear transition from a 'dividing' PGC to a 'self-renewing' GSC (Gilboa, 2004).

Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc: A potential competition between Mad and Brinker

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene and the proneural genes achaete and scute. Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe) has been examined. These studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).

The D-h and V-h stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit. ac stripes are not expressed until 6 hours after puparium formation (APF). The flanking narrow D-h stripe is positioned a few cells anterior to the compartment boundary, allowing expression of two dorsal ac stripes in the anterior compartment. V-h, however, is expressed directly adjacent to the AP boundary so that there is only one ventral ac stripe in the anterior compartment. Expression of each h stripe in its proper register is essential for positioning of the ac stripes and consequently for sensory bristle patterning in the adult leg. Focus was placed on the mechanisms that lead to expression of the D-h stripe in its precise register near the AP boundary (Kwon, 2004).

Genetic data support a hypothesis in which Brk acts through the REPE of the D-h enhancer to modulate activity of the HHRE. If so, it might be expected that the REPE would contain one or more functional Brk-binding sites. Hence, the REPE was examined for the Brk consensus binding site, GGCG(C/T)(C/T), and a potential Brk binding site was identified that overlaps two sequences similar to a consensus binding sites for Mad: GCCGNCGC, and a sequence similar to a cAMP response element (CRE), TGACGTCA. The sequence of overlapping CRE, Brk and Mad sites was designated the CMB element. Site directed mutational studies are consistent with the hypotheses that Brk acts through the CMB to repress D-h expression (Kwon, 2004).

A short sequence in the REPE, the CMB, has been identified that functions to restrict HHRE expression to a narrow dorsal domain. In this study, evidence is provided for the hypothesis that the transcriptional repressor Brk acts through the CMB to repress D-h expression. Although previous studies have shown that brk expression is very low or undetectable in cells near the Dpp source, a genetic requirement has been demonstrated for brk in repression of D-h in this region. In addition, overexpression of brk results in a dramatic reduction of D-h-GFP expression, but only mildly affects expression from a D-h-GFP transgene with a compromised Brk binding site (Kwon, 2004).

Dpp acts through the REPE to block Brk-mediated repression. It is proposed that high-level Dpp signaling defines the domain of D-h expression within the HHRE-response zone. This idea is supported by the observations that D-h-GFP but not HHRE-GFP expression is dependent on Dpp, indicating that Dpp signals through the REPE, and that elevation of Dpp signaling results in expansion of D-h expression along the AP and DV axes, within the domain of HHRE activity. Current studies suggest that the function of Dpp in regulation of D-h expression may be limited to repression of brk. Yet, the presence of Mad-binding sites in the CMB suggests a potentially more direct role for activated Mad (act-Mad), the transcriptional mediator of Dpp signaling. Brk has been shown to be a potent competitor of Mad in vitro for binding to overlapping binding sites in Dpp target enhancers. Hence, a potential role for Mad would be to prevent Brk from binding the CMB, thereby blocking Brk repression in cells receiving high-level Dpp signaling. If this model is correct, one might have expected the Mad1/Mad2 (MM) mutation to compromise D-h expression, which was not the case. However, the destabilization of Brk binding to the MM mutant might have masked a requirement for the Mad sites in blocking Brk repression (Kwon, 2004).

It has recently been shown that an act-Mad/Shn complex represses brk expression by binding a silencer element. Therefore, since mutation of the Mad sites expands D-h expression, it is possible that Mad acts in concert with Brk through the CMB to repress D-h expression. This notion is not inconsistent with genetic evidence, indicating a requirement for Mad in D-h expression, since loss of Mad function elevates Brk levels, which can overcome the requirement for CMB-sequences other than the Brk site. However, if this were the case, a more severe expansion phenotype might be expected with the MM mutant, in which both Brk and Mad binding are compromised. Further analysis is required to determine the role, if any, of the CMB-Mad-binding sites in D-h expression (Kwon, 2004).

This study has identified a novel function for Brk as repressor of Hh-target gene expression. Brk was originally identified as a repressor of Dpp-target genes and a recent study indicates that Brk can block Wg-mediated transcription as well. Brk was shown to antagonize function of a Wg-responsive element in the midgut enhancer of the Ultrabithorax (Ubx). The Ubx midgut enhancer drives Ubx expression in parasegment (ps) 7 of the embryonic midgut. Two elements, one of which is Wg responsive (the WRS) and another Dpp responsive (the DRS), function synergistically to activate Ubx expression in ps 7 expression. In the adjacent ps8, however, Brk binds to the DRS and blocks the activity of the WRS. Curiously, the D-h-CMB and the Ubx-DRS are similarly organized in that each consists of overlapping CRE/Mad and Brk sites. The Ubx-DRS appears to mediate two modes of signal integration which involve: (1) synergistic activation, in which Mad/Med and dTCF act together to activate expression; and (2) activation and refinement, in which there is Wg mediated activation combined with Brk repression, which is blocked by Dpp. In the D-h enhancer, however, the CMB appears to be a component of a dedicated repression element, which appears to mediate only the second mode of signal integration: activation and refinement. The similar organization of the CMB and DRS suggests that it may be possible to predict the structure of enhancers known to be Brk responsive and which integrate Dpp and a second signal (Kwon, 2004).

Despite the similarities, there are important distinctions between the D-h and Ubx-midgut enhancers, suggesting that the mechanisms of Brk-mediated repression might differ in each case. In the Ubx-midgut enhancer, the DRS and WRS are separated by 10 bp, suggesting that Brk acts at short range to inhibit WRS activity. In the D-h enhancer, however, the CMB is positioned at least 1 kb from the HHRE, implying a long-range effect for this element. Furthermore, Brk repression of the WRS depends on Teashirt (Tsh), which binds Brk and acts as a co-repressor. Tsh is unlikely to be required for D-h repression because it is only expressed in proximal leg segments. The current studies suggest the requirement for a second DNA-bound factor, which binds the CRE, in addition to Brk for repression. The DRS-CRE, however, is required in addition to the Mad-binding sites for activation of Ubx in ps 7 (Kwon, 2004).

Together, these observations are consistent with a model in which Ci, acting through the HHRE, activates D-h expression. The domain of HHRE activity can be divided into two zones, 1 and 2. The HHRE has the potential to direct expression in both zones 1 and 2, but its activity is restricted to zone 1 by Brk and perhaps a second factor, X, which binds the CRE. In zone 2 cells, Brk would bind to the CMB and repress HHRE-directed expression. It is proposed that zone 1 is defined by the overlap of Hh and high-level Dpp signaling. Dpp promotes D-h expression by repressing brk expression in zone 1. However, the presence of Mad-binding sites in the CMB suggests the potential for a more direct role for Mad in D-h regulation, perhaps in competing with Brk for binding to the CMB, or in directly mediating repression. Confirmation of a role for the Mad sites awaits further analysis of the D-h enhancer (Kwon, 2004).

Smad affinity can direct distinct readouts of the embryonic extracellular Dpp gradient in Drosophila

The TGF-β signaling molecule Dpp is an essential morphogen that patterns many tissues during Drosophila development, including the embryonic dorsal ectoderm and larval wing imaginal disc. An activity gradient of Dpp specifies distinct cell fates in the dorsal ectoderm of the embryo through the activation of different transcriptional threshold responses. The gene Race, which is expressed in response to peak levels of Dpp signaling in gastrulating embryos, was analyzed. The Smad transcription factors, which are intracellular transducers of Dpp signaling, are essential activators of Race in vivo. Furthermore, increasing the affinity of the Smad binding sites in the Race enhancer broadens the expression pattern of a linked reporter gene and alters its behavior in mutant embryos to that characteristic of a distinct threshold response. It is concluded that Smad activator affinity is a critical determinant of the threshold response to the extracellular Dpp gradient in the embryo. These results identify a mechanism for interpreting the Dpp gradient in the embryo which is different from the reciprocal repressor gradient model proposed for the wing disc. It is suggested that transcription factor binding site affinity will be a general strategy used in the interpretation of other extracellular morphogen gradients (Wharton, 2004).

Three distinct thresholds of gene expression in response to the Dpp/Scw gradient have been defined in the dorsal ectoderm of gastrulating embryos. Expression of Race, a type I threshold response, is dependent on peak Dpp/Scw signaling and is restricted to a narrow strip of cells in the presumptive amnioserosa. In sog mutant embryos, there is no Dpp/Scw gradient but instead a uniform low level of Dpp/Scw signaling throughout the dorsal epidermis. In these sog- embryos, Race expression is lost from the presumptive amnioserosa, since the level of Dpp/Scw signaling is insufficient to activate Race (Wharton, 2004).

A 533 bp enhancer has been identified that directs expression of a linked lacZ reporter gene in transgenic embryos in a similar pattern to that of endogenous Race. Race expression is dependent on peak Dpp signaling, and the Race enhancer contains three binding sites for the Zerknüllt (Zen) transcription factor. Zen, which is itself activated by Dpp signaling in the presumptive amnioserosa of gastrulating embryos, is essential for Race activation but not sufficient in the absence of Dpp signaling. This raised the possiblity that the Mad and Medea transcription factors, which are the intracellular transducers of Dpp signaling, may function with Zen to directly activate Race. Therefore, whether Mad and Medea could bind directly to the Race enhancer was tested in vitro. DNase I footprinting assays with bacterially expressed GST fusion proteins were used to characterize the binding of Mad and Medea to three overlapping fragments of the Race enhancer. Mad and Medea bind to two of the three enhancer fragments tested. Three areas are protected from DNase digestion in the presence of Mad/Medea fusion proteins. One of these footprints (site A) encompasses nucleotides 28-41, and the other two sites, B and C, are adjacent to each other (nucleotides 464-483 and 484-502, respectively). These adjacent binding sites are flanked by the previously identified Zen binding sites. Mad and Medea recognize the same sites within the Race enhancer, consistent with previous observations that Mad and Medea have overlapping binding specificities (Wharton, 2004).

These binding sites were confirmed by gel retardation assays using oligonucleotide probes containing the protected regions and a GST-Mad fusion protein. It appears that sites A and B are low-affinity binding sites, whereas site C is higher affinity; an increased amount of the retarded complex is observed with probe C and Mad, even though equimolar amounts of probe and the same amount of Mad protein were used. Since similar binding data were obtained for Medea, and given the overlapping binding specificities documented for Mad and Medea, subsequent in vitro binding assays were only performed using Mad (Wharton, 2004).

Two previous studies have identified Mad/Medea consensus binding sites, which together can be represented by the Drosophila Smad consensus (DSC) GCCGC[C/G]G[C/A]. In addition, some Drosophila Smad binding sites contain the Smad binding element (SBE), AGAC, which was identified for human Smad4. Of the three binding sites identified in the Race enhancer, only site C, the higher-affinity site, has a motif close to the consensus with six out of eight matches, although B and C both contain perfect matches to the SBE. Site A contains the sequence CGAC, which differs from the SBE by one nucleotide, and a mutation at this position in the SBE reduces but does not abolish binding of Smad4 (Wharton, 2004).

It is suggested that the high nuclear Smad concentration at the dorsal midline is sufficient to occupy the Smad binding sites in the Race enhancer, two of which are low affinity, whereas the lower levels of nuclear Smads detected in the dorsomedial ectoderm are not. Increasing the affinity of the Smad sites in the Racee enhancer allows occupancy at lower Smad concentrations and hence a wider expression pattern characteristic of a type II threshold. Therefore, expression of Race is restricted to the presumptive amnioserosa due to low Smad affinity in its enhancer, and it is speculateed that this will also be true for at least some other type I thresholds (Wharton, 2004).

Studies in vertebrates have indicated that Smads have low specificity and affinity for DNA, which can be increased in the presence of a Smad binding partner. The results presented here demonstrate that alterations in Smad affinity alone can change the threshold response, but cooperative DNA binding with a Smad binding partner may also be important. It is possible that genes with broader expression patterns have binding sites for different Smad binding partners in their enhancers. It is noted that two of the three Smad binding sites identified in the Race enhancer are flanked by Zen binding sites. Since Mad and Medea interact with Zen in the yeast 2-hybrid assay, Zen may function as a Smad binding partner for Race activation. However, misexpression of Zen using the tolloid or Krüppel enhancers does not expand wild-type Race expression or the lacZ pattern directed by the Race or Racee enhancers (Wharton, 2004).

The importance of Smad affinity in establishment of the dorsal ectoderm Dpp threshold responses contrasts with the mechanism for setting the limits of the expression patterns of Dpp target genes in the wing disc. In the wing disc, a localized source of Dpp diffuses to form a gradient which activates target genes, eg spalt, optomotor-blind and vg, in nested domains with different widths of expression. Dpp signaling directly represses Brk to create an opposing Brk gradient and it appears that sensitivity to Brk repression is the major determinant of the Dpp threshold responses. Since Brk and Mad can compete for overlapping binding sites, Smad affinity may play a role in the generation of a subset of wing Dpp thresholds by restricting Brk binding to common sites depending on their relative affinities for Brk and the Smads. However, Brk can also mediate repression by binding to sites in an enhancer that are separate from the Smad activator sites and by recruiting specific corepressors, suggesting that for other wing Dpp threshold responses, Brk affinity alone will be the critical factor. In contrast to the wing disc, many Dpp threshold responses in the early embryo are insensitive to Brk repression; therefore, Dpp target genes must use an alternative mechanism for interpreting the extracellular morphogen concentration. The results suggest that specification of at least some type I and II thresholds in the embryo depends on variations in Smad affinity at target gene enhancers (Wharton, 2004).

Bicoid (Bcd) and Dorsal (Dl) are maternally expressed transcription factors that are determinants of the anterior-posterior and dorsal-ventral axes, respectively, and have many of the properties of morphogens. In the precellular embryo, the Bcd and Dl transcription factor gradients are interpreted by binding site affinity. The current results demonstrate that transcriptional activator affinity can also be used to measure the extracellular concentration of a morphogen postcellularization so that dose-dependent gene expression patterns can be generated. These results, which describe how different readouts can be obtained from a Dpp/BMP gradient solely by changes in Smad affinity, may be especially relevant to TGF-β signaling readouts in vertebrates. Moreover, they identify a possible strategy for interpretation of extracellular gradients of different classes of signaling molecules (Wharton, 2004).

Peak levels of BMP in the Drosophila embryo control target genes by a feed-forward mechanism involving Zen and Mad

Gradients of morphogens determine cell fates by specifying discrete thresholds of gene activities. In the Drosophila embryo, a BMP gradient subdivides the dorsal ectoderm into amnioserosa and dorsal epidermis, and also inhibits neuroectoderm formation. A number of genes are differentially expressed in response to the gradient, but how their borders of expression are established is not well understood. Evidence is presented that the BMP gradient, via the Smads, provides a two-fold input in regulating the amnioserosa-specific target genes such as Race. Peak levels of Smads in the presumptive amnioserosa set the expression domain of zen, and then Smads act in combination with Zen to directly activate Race. This situation resembles a feed-forward mechanism of transcriptional regulation. In addition, ectopically expressed Zen can activate targets like Race in the presence of low level Smads, indicating that the role of the highest activity of the BMP gradient is to activate zen (Xu, 2005).

Specific activation or repression of transcription by a combination of transcription factors is a common theme in the regulation of developmentally important genes. The results from the genetic analysis and the molecular dissection of the Race enhancer clearly show that Race is activated by the combined action of Smads and Zen. Although Smads can single handedly activate Race when overexpressed, under normal circumstances concurrent Zen activity is required. Why are both Smads and Zen necessary (Xu, 2005)?

Zen may act to restrict target gene expression specifically to the presumptive amnioserosa. Since the Dpp pathway is used repeatedly during development, other factors must function in combination with Dpp to ensure tissue specificity. Ectopic expression studies support this idea. In normal embryos, Race is activated only in regions where there are peak levels of PMad and Zen. In embryos where Zen is ubiquitously expressed, Race can now be activated in regions where there are lower levels of PMad, indicating that high level PMad is not the determining factor for amnioserosa tissue specificity. Rather PMad allows expression, and Zen determines the border of expression. The overexpression studies where Dpp can activate Race alone are interpreted to be situations where there are such high levels of Smads that Race and hindsight (hnt/pebbled) become activated promiscuously, and hence differential regulation is lost. In normal embryos, the combination of Smads and Zen ensures that the high level target genes are activated only in the presumptive amnioserosa (Xu, 2005).

In contrast, why the need for Smads? One role for Smads is suggested from the observation that Smads facilitate the binding of Zen to the Race enhancer. It is well established that Hox proteins often require co-factors for DNA binding to target enhancers. For example, composite sites that also bind the co-factor Extradenticle (Exd) ensure a greater selectivity for binding over the higher frequency Hox core site such as TAAT. In other examples, binding sites for signaling pathway effectors lie close to Hox/Selector-binding sites. The closely apposed Zen- and Smad-binding sites in the Race enhancer is one such scenario, since Zen can be thought of as a Selector gene. The current studies add to this idea of Smads and Selector cooperativity by demonstrating enhanced binding of Zen in the presence of Smads. Though the observed enhancement observed in in vitro assays is not dramatic, it is possible that in the embryo a moderate enhancement is functionally significant as is the twofold doubling of the dpp dose (Xu, 2005).

Another potential role of Smads was suggested from previous overexpression studies. Zen was able to activate Race only in the absence of Dpp if fused to a strong activation domain derived from VP16. This suggests that Smads provide a transactivation function different from that of Zen. The Smad MH2 domain has been shown to interact with the transcriptional co-activators CBP and p300. Zen has not yet been analyzed for interaction with transcriptional co-activators, however, the activation domain of Zen lies within the C-terminal 119 amino acids, and does not overlap with the homeodomain or the Mad interaction domain. Mechanistically, the difference in the activation potential between Zen and Smads could be due to their ability to recruit different co-activators to the transcriptional machinery (Xu, 2005).

Gradients of morphogens provide positional information to the cells by activating different genes at different threshold concentrations. In early Drosophila embryos, the transcriptional threshold responses to the Bicoid and Dorsal (Dl) morphogens have been extensively studied. The major mechanisms by which thresholds are established exploits the DNA-binding affinities of Bcd and Dl to their operator sites, as well as synergistic interactions with other transcription factors bound to the cis-regulatory sequences (Xu, 2005).

The BMP morphogen gradient also elicits different threshold responses from its targets, and a combinatorial mechanism is used to activate Race, a high level Dpp target. The genetic results indicate that Race, and also another high level target hnt, are activated only when a specific threshold of Zen and Smad activities are reached. In sog mutant embryos, Zen and Smad concentrations are relatively high, though below peak levels, and there is just enough of their combined activity to weakly activate Race. By contrast, in the double heterozygous embryos dpphr4/+; zenw36/+, Race is not activated because Zen and Smad concentrations are below the threshold levels required for activation (Xu, 2005).

A simple way to explain these results is if the Race enhancer has low affinity to Zen and Smad proteins in vivo. To transcribe Race effectively would then require relatively high concentrations of the proteins, which are indeed reached in the dorsalmost cells. It has been known for some time that the enhancers of the high level Dl targets contain binding sites with lower affinity for Dl compared with genes responding to lower levels of Dl. Recently, it has been shown that increasing the affinities of Smad-binding sites in the Race enhancer broadens the Race expression domain, which argues that the affinities of the Smad-binding sites in this high level Dpp target gene enhancer are low. The results suggest that cooperative binding between Smads and Zen, which is dependent on their physical interaction, should increase their binding to the Race enhancer. It is possible that interacting with Smads at the protein level either increases the binding affinity of Zen or effectively increases the local concentration of Zen when Smads bind the adjacent sites. This in turn leads to a robust transcriptional response of Race. The overexpression results are consistent with such a model. Ectopic Zen can only activate Race if some detectable level of PMad is present, and in addition Zen must contain the Smad interaction domain (Xu, 2005).

How are the lower level target genes activated? u-shaped (ush) and rhomboid are expressed in a broader domain, the border coinciding exactly with that of low level PMad staining. The Zen domains, however, do not; refined zen is not broad enough, while early Zen is too broad encompassing the entire dorsal domain, though Zen could possibly be graded in this region. Thus, it is possible that this class of target genes relies on a mechanism that uses numerous high-affinity Smad sites, and/or synergistic action of Smads with other co-factor(s) besides Zen. Such a mechanism resembles the activation of target genes in the neurogenic ectoderm of the embryo by Dorsal. It has been shown that the threshold responses from these genes depend on high-affinity Dl-binding sites, as well as synergistic interactions of Dl with bHLH transcription factors (i.e., dorsal/twist interactions) (Xu, 2005).

The pnr expression domain, which is about three times broader than ush, may represent a third threshold of Dpp activity. However, pnr is a different type of target gene compared with the prior classes, in that it is repressed by Brk, which is present in a reverse gradient to Dpp. In brk mutants, pnr expands into the ventral region, while Race and ush, for example, are unchanged. It is expected that the pnr gene enhancer contains Brk-binding sites, whereas the Race enhancer does not. Brk binding sites often overlap with GNCN sites, and it is possible that in the embryo, as in the wing disc, a concentration-dependent competition between Smads and Brk establishes the expression domains of the target genes regulated by both inputs. However, whether direct competition for binding can generate threshold responses remains to be seen. In summary, it appears that different classes of Dpp target genes are regulated by different combinations of transcription factors (Xu, 2005).

One of the simple regulatory motifs used in transcriptional networks is the feed-forward or self-enabling mechanism, whereby one regulator controls a second regulator and then both bind a common target gene. It has been shown both in prokaryotes and yeasts that this mode of regulation appears relatively frequently and is favored over others, e.g., autoregulation motifs, single input motifs in which one regulator controls several genes, or regulator chain motifs whereby one gene regulates a second which regulates a third, and so on. Such an over-representation of the feed-forward motif is probably due to its potential to provide enhanced sensitivity and temporal control to the transcriptional response. The feed-forward loop is especially suitable for eliciting precise threshold responses of morphogen targets because it allows a strong response of the target gene to small changes in the activity of the regulator that initiates the loop (Dpp), due to the combined action with the second regulator (Zen). In fact Bcd and Dl use mechanisms that are reminiscent of the feed-forward loop to activate their high level targets. Bcd regulates zygotic hunchback (hb) and together Bcd and Hb activate the downstream target even-skipped (eve) stripe 2, and Dl activates sna with the help of Twi. It is striking that the three morphogen gradients involved in specifying the Drosophila embryonic axes use the feed-forward strategy to regulate downstream target genes (Xu, 2005).

An unexpected implication from these results concerns the role of the high concentration end of the BMP morphogen gradient. In Drosophila embryos, the refined zen domain depends on peak levels of BMP activity, and Zen can activate high level targets as long as there is some level of PMad present to facilitate DNA binding. It can be then concluded that, for the high level targets, the role of Dpp is twofold: to set the domain of zen, which can be referred to as a primary target gene; and then to act in combination with Zen to activate the other, secondary, target genes such as Race and hnt. In addition, with respect to the BMP gradient in the Drosophila embryo, it is further proposed that the sole purpose of the peak of the gradient is to set up the zen domain (Xu, 2005).

Integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors and thir impact of Bagpipe expression during Drosophila visceral mesoderm induction

Tissue induction during embryonic development relies to a significant degree on the integration of combinatorial regulatory inputs at the enhancer level of target genes. During mesodermal tissue induction in Drosophila, various combinations of inductive signals and mesoderm-intrinsic transcription factors cooperate to induce the progenitors of different types of muscle and heart precursors at precisely defined positions within the mesoderm layer. Dpp signals are required in cooperation with the mesoderm-specific NK homeodomain transcription factor Tinman (Tin) to induce all dorsal mesodermal tissue derivatives, which include dorsal somatic muscles (the dorsal vessel and visceral muscles of the midgut). Wingless (Wg) signals modulate the responses to Dpp/Tin along anteroposterior positions by cooperating with Dpp/Tin during dorsal vessel and somatic muscle induction while antagonizing Dpp/Tin during visceral mesoderm induction. As a result, dorsal muscle and cardiac progenitors form in a pattern that is reciprocal to that of visceral muscle precursors along the anteroposterior axis. The present study addresses how positive Dpp signals and antagonistic Wg inputs are integrated at the enhancer level of bagpipe (bap), a NK homeobox gene that serves as an early regulator of visceral mesoderm development. An evolutionarily conserved bap enhancer element requires combinatorial binding sites for Tin and Dpp-activated Smad proteins for its activity. Adjacent binding sites for the FoxG transcription factors encoded by the Sloppy paired genes (slp1 and slp2), which are direct targets of the Wg signaling cascade, serve to block the synergistic activity of Tin and activated Smads during bap induction. In addition, binding sites for yet unknown repressors are essential to prevent the induction of the bap enhancer by Dpp in the dorsal ectoderm. These data illustrate how the same signal combinations can have opposite effects on different targets in the same cells during tissue induction (Lee, 2005).

To investigate whether the bap regulators identified genetically, including tin, dpp, slp (downstream of wg) and biniou (bin), can act directly on the early TVM regulatory element of bap, DNaseI protection experiments with recombinant Tin, Bap, Smad (Mad and Medea), Slp and Bin proteins were performed on the 180 bp bap3.2.1 DNA sequence from D. melanogaster. The DNA footprinting results demonstrate that both Tin and Bap proteins can bind to the predicted Tin-binding site, which includes a perfect match to the canonical Tin-binding motif TCAAGTG. In addition to the Tin-binding site, a site with a TAAG core motif can strongly bind Bap but not Tin (CTTA in opposite strand; note that the same core motif is found in binding sites of a Bap ortholog, Nkx3.2). With regard to Dpp signaling mediators, there are five Mad-protected regions, three of which are also protected by recombinant Medea (Mad/Medea-1 to -3). Site 1 includes an AGAC motif that was initially identified as a Smad binding motif in vertebrates whereas sites 3-5 contain GC-rich sequences with CGGC motifs that were first shown to bind Smad proteins in Drosophila. Site 2 may be a combination of the two types (TGAC motif and CG-rich sequences). No clear correlation of either type of site was observed with the binding of Mad versus Medea. Finally, recombinant Slp proteins protect a wide stretch that includes an inverted repeat of core binding motifs for forkhead transcription factors (TAAACA), but extends further downstream. Slp can bind to tandem repeats of CAAA sequences, which are present in three copies in the 3' region of the protected region. Gel mobility shift and competition assays with Slp using wild-type oligonucleotides and a version in which the TAAACA motifs were mutated indicate that Slp can bind to both the TAAACA and the CAAA motifs with roughly equal affinity. In addition, the FoxF family protein Bin binds to the TAAACA inverted repeat region, but less well to the CAAA repeat region when compared with Slp. Taken altogether, these binding data are consistent with the hypothesis that the known mesodermal regulators of bap, namely Tin and Bin (and possibly autoregulatory Bap), as well as the signaling inputs from Dpp and Wg (through Smads and Slp, respectively) are integrated via direct binding to the early TVM enhancer of bap (Lee, 2005).

The present study describes an example of an enhancer whose response to Dpp is suppressed by Wg signals. A comparison of the functional organization of these enhancers provides new insight into molecular strategies of nuclear signal integration to produce differential developmental responses. The data show that bap is a direct target of Dpp signals. Thus, an indirect pathway of bap being activated solely by tin, whose mRNA expression is known to depend on Dpp inputs during the time of bap activation, can be ruled out. Rather, tin acts simultaneously and synergistically with Dpp. In fact, recent data with tin alleles lacking the Dpp-responsive enhancer show that bap can be induced in the absence of Dpp-induced tin products, as long as the twist-activated tin products are present. The molecular basis for this observed synergism of tin and dpp relies on the combinatorial binding of Tin and Dpp-activated Smad proteins to the bap enhancer. Several possible molecular mechanisms could underlie the strict requirement for combinatorial binding of Tin and Smads. For example, the relatively low binding affinity and specificity of Smads might be enhanced by bound Tin, which can engage in protein interactions with Mad and Medea. The combined presence of Tin and Smads in close vicinity or in complexes may also be a prerequisite for the assembly of higher order complexes with transcriptional co-activators such as CBP/p300. In addition, Tin may counteract the function of yet unknown repressors of nuclear Dpp signaling activity so that they can only repress in the ectoderm (Lee, 2005).

Unlike Dpp, Wg signals act indirectly upon the early bap enhancer. Previous genetic and molecular data have shown that Wg induces the expression of the forkhead domain-encoding gene slp via crucial dTCF/Lef-1 binding sites in both mesoderm and ectoderm. slp, in turn, functions as a repressor of bap. The present data show that slp products exert this function by direct binding to the Dpp-responsive bap enhancer, which obviously results in a suppression of the synergistic activity of bound Tin and Smad complexes. Slp proteins contain eh1 motifs that can potentially bind the Groucho co-repressor and Slp has known repressor activities in other contexts. In addition, the vertebrate counterpart of Slp, FoxG (BF-1), is known to interact with Groucho and histone deacetylases (Yao, 2001). Thus, it is proposed that Slp overrides nuclear Dpp signaling activities by dominantly establishing an inactive state of the chromatin at the bap locus (Lee, 2005).

Why would induction of tin and bap in the mesoderm require Tin as a co-factor of Smads, whereas in the ectoderm, which lacks Tin, the induction of tin and bap needs to be actively repressed? In the case of the tin enhancer, the ectodermal repressor elements are overlapping with the Tin-binding sites. Based upon this situation, a model is proposed in which the repressor would be present in both germ layers, but in cells of the mesoderm it is competed away from binding to the enhancer by Tin. This model is compatible with data showing that ectopic expression of Tin in the ectoderm is able to activate the Dpp-responsive enhancer of tin, even in the presence of the putative repressor binding elements. However unlike full-length Tin, an N-terminally truncated version with an intact homeodomain is not able to allow induction of the tin enhancer in the ectoderm. Furthermore, the putative repressor binding sites in the bap enhancer are separate from the Tin site. Hence, Tin does not compete for binding but may rather block or override the repressor factor(s) functionally. Thus, the positive activity of Tin would dominate over the negative action of this repressor in the mesoderm. By contrast, the repressing activity of Slp dominates over the positive action of Tin. Through this intricate balance of positive and negative switches, Tin could ensure that bap is induced by Dpp only in the mesoderm, while bound Slp prevents Tin from promoting Dpp inputs towards bap in striped domains within this germ layer. However, it can still not be fully explained why the absence of both the functional Tin and ectodermal repressor sites allows enhancer induction in the ectoderm, while preventing it in the mesoderm. The additional positive and negative binding factors involved will need to be identified to gain a full understanding of the germ layer-specific induction of these Dpp-responsive enhancers (Lee, 2005).

The bap enhancer described in this study represents the third example of well-characterized Dpp-responsive enhancers from mesodermal control genes. The other two are from tin, which is induced in the entire dorsal mesoderm, and eve, which is active in a small number of somatic muscle founder cells and pericardial progenitors in the dorsal mesoderm. The activities of the bap and eve enhancers along the anteroposterior axis are reciprocal, which is due to the fact that the eve enhancer requires inputs from Wg, whereas bap enhancer activity is suppressed by Wg. A comparison of the molecular architecture of these three enhancers reveals that they all share a number of important features. Most notably, all three enhancers feature several Tin- and Smad-binding sites in close vicinity that are essential for the activation of the enhancer in the mesoderm. Each enhancer includes both types of known Smad-binding motifs, which have 'AGAC' and 'CG'-rich cores, respectively. Hence, the basic activation mechanisms of each of the three enhancers downstream of Dpp are likely to be closely related. In the enhancers of both tin and bap, binding sites for a nuclear repressor of Dpp signals are key for the germ layer specificity of the inductive response. Although it is not known whether the same repressive mechanism operates at the eve enhancer, it is noted that motifs related to the presumed repressor binding motifs are present and their function can now be tested in vivo. As in the case of bap, the tin enhancer includes also additional sites that are required for Dpp-inducible enhancer activity, which may bind essential Smad co-factors. However, based upon the divergent sequences of these sites (C1 site in the bap and 'CAATGT' motifs in the tin enhancer), they appear to bind different types of factors in each case (Lee, 2005).

On top of this basic arrangement that allows the enhancer to be active in the dorsal mesoderm, the enhancers from bap and eve, but not tin, include binding sites that make them respond to Wg inputs in an opposite fashion. In the case of bap, Wg-induced Slp binds and dominantly suppresses the activity of bound Smad effectors. For the eve enhancer it has been proposed that there is an analogous repressive activity; however, in this case, it is exerted by bound Wg signal effectors, i.e., dTCF/Lef-1, in the absence of Wg signals. In the domains with active Wg signaling, the repressive activity of dTCF/Lef-1 is neutralized by the Wg signaling cascade, which allows the Dpp effectors to be active at the eve enhancer (since it lacks Slp binding sites). Through these switches, the bap and eve enhancers become induced in reciprocal AP patterns. In addition, the eve enhancer includes binding sites for activators and repressors downstream of receptor tyrosine kinases and Notch, respectively, which serve to restrict eve activity to specific subsets of cells within the domains of overlapping Dpp and Wg activities. Clearly, many of the molecular details still need to be clarified. Nevertheless, the basic principles of how differential inputs from inductive signals and tissue-specific activities can be integrated at the enhancer level to achieve distinct patterns of target gene expression during early tissue induction in the Drosophila mesoderm are now beginning to be understood (Lee, 2005).

Patterning function of homothorax/extradenticle in the thorax of Drosophila: Mad represses homothorax

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. This study investigates the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single Hox independent functional unit. Their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and ß subdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. Evidence is presented for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain (Aldaz, 2005).

The role of the Dpp pathway as a negative regulator of hth is based on results showing that Mad - mutant clones in the inter-subdomains region show activation of hth. This is in contrast to the behaviour of those clones in the alpha subdomain, where they have no effect, or in the ß subdomain, where they show suppression of hth. It is believed that the reason for the latter effect is that eyg is up regulated in those clones, and in turn Eyg suppresses hth. The lack of effect of Mad - clones in the alpha subdomain is probably due to the low activity of Dpp in that region. In principle, the observation that the high activity levels generated in the TkvQD clones suppress hth in this subdomain supports this view. Expectedly, TkvQD clones do not affect hth expression in the ß subdomain, because it normally possesses high Dpp activity levels (Aldaz, 2005).

Taking all the results together, the following model of hth regulation is proposed. Since hth is originally expressed in all trunk embryonic cells and in all the notum cells in the early disc, the regulation of hth during wing disc development essentially reflects local repression in specific parts of the disc. The basic idea is that hth is repressed by the joint contribution of eyg and high/moderate levels of the Dpp pathway. Neither of these elements can repress hth individually. Although eyg appears to act uniformly in its domain, the repressing activity of Dpp is concentration dependent. Within the eyg domain, the hth alpha subdomain is located in the anterior region, in which the Dpp levels are too low to be effective and Eyg alone cannot repress hth/exd. In the inter-subdomains region the Dpp levels are high enough to repress hth, since here it acts together with Eyg. The ß subdomain is outside the eyg domain and therefore in the absence of Eyg even the high Dpp levels are not capable of repressing hth/exd. The model is also supported by the experiments of overexpressing eyg. The eyg-expressing clones in the ß subdomain suppress hth because the two repressors are active in the clones, while they have no effect in the alpha subdomain because it normally contains high eyg levels. In principle the experiments overexpressing the Dpp pathway (TkvQD clones) appear to support the model. These clones have no effect in the ß subdomain, which normally possesses high Dpp activity levels, but they suppress hth in the alpha subdomain. However, these clones are known to suppress eyg and therefore hth should not be repressed according to this model. It is possible that in certain circumstances the very high Dpp activity levels induced by these clones may be sufficient to down regulate hth, even in the absence of eyg (Aldaz, 2005).

The presence of two distinct repressors may suggest that the hth promoter region contains binding sites for Eyg and for Mad/Medea that would be responsible for the transcriptional repression. The ubiquitous expression in the absence of these two repressors may be due to a constitutive promoter (Aldaz, 2005).

Mad/Med regulate decapentaplegic expression during Drosophila wing veins pupal development

The differentiation of veins in the Drosophila wing relies on localised expression of decapentaplegic in pro-vein territories during pupal development. The expression of dpp in the pupal veins requires the integrity of the shortvein region (shv), localised 5' to the coding region. It is likely that this DNA integrates positive and negative regulatory signals directing dpp transcription during pupal development. A minimal 0.9 kb fragment has been identified giving localised expression in the vein L5 and a 0.5 kb fragment giving expression in all longitudinal veins. Using a combination of in vivo expression of reporter genes regulated by shv sequences, in vitro binding assays, and sequence comparisons between the shv region of different Drosophila species, binding sites were found for the vein-specific transciption factors Araucan, Knirps and Ventral veinless, as well as binding sites for the Dpp pathway effectors Mad and Med. It is concluded that conserved vein-specific enhancers regulated by transcription factors expressed in individual veins collaborate with general vein and intervein regulators to establish and maintain the expression of dpp confined to the veins during pupal development (Sotillos, 2006).

The expression of dpp in the wing disc is restricted to a narrow stripe of anterior cells localised at the anterior/posterior compartment boundary. This expression is regulated by sequences localised 3′ to the dpp coding region, and the function of the gene at this stage is required for the growth and patterning of the wing. The expression of dpp is still detected at the A/P boundary during the 8 h of pupal development. Later, at 14 h APF novel domains of dpp expression appear corresponding to the developing wing veins. The function of dpp during pupal development requires the integrity of the shv region, which is localised 5′ to the dpp coding region. There are two different transcripts expressed during pupal development, transcripts dpp-RA and dpp-RC, whose promoters (P5 and P3, respectively) are separated by approximately 20 kb of DNA. This DNA includes the first exon of transcript dpp-RC and corresponds to the place where all dpps alleles map. Because the strength of dpps alleles correlates with their distance to the P3 promoter, it is likely that dpp function in pupal development is mediated mainly by transcript dpp-RC. This suggests that dpps mutations affect regulatory sequences necessary to drive dpp expression in presumptive vein territories during pupal development. This possibility was confirmed by analysing the expression of a 8.5 kb construct containing most of the shv region fused to the reporter gene lacZ (shv8.5lacZ). The expression of βGal in shv8.5lacZ is detected exclusively in the pupal veins, indicating that this region includes all dpp wing veins regulatory regions (Sotillos, 2006).

Several constructs were made using different sub-fragments from the original 8.5 kb dpps DNA to identify with more precision the sequences that regulate dpp expression during pupal development. These fragments were cloned in front of a dicistronic lacZGal4 reporter gene and the activity of these constructs was analysed by looking at the expression of βGal in pupal wings from transgenic flies. In addition, to amplify the signal of the dicistronic lacZGal4 gene, the expression was monitored of a reporter gene regulated by UAS sequences. This expression should reveal all places where the Gal4 protein is present. Several regulatory regions were detected that control dpp expression in the veins during pupal development. One regulatory sequence is localised in a 1.1 kb fragment localised 6.5 kb from P3, and drives high levels of expression in most pupal veins and low levels of expression in some interveins. Additional regulatory sequences that efficiently drive expression in most veins are localised in an adjacent 0.5 kb fragment, and further vein-specific regulatory sequences for L5 are localised in the 0.9 kb SalI/KpnI fragment (Sotillos, 2006).

The expression of dpp during embryogenesis is highly dynamic and several independent regulatory regions controlling embryonic dpp expression have already been identified. The shv constructs included in the 8.5 kb EcoRI fragment drive reporter expression during embryonic development from stage 12/13 mainly in three regions of the mesoderm: the oesophagus, gastric caeca and midgut. Regulatory regions controlling dpp expression in the oesophagus appear to be duplicated, because they are localised in the 2.7 kb EcoRI/SalI fragment and also in the 3 kb KpnI/KpnI fragment. Similarly, regions controlling dpp expression in the gastric caeca are also present in the two adjacent fragments 0.9 kb SalI/KpnI and 3 kb KpnI/KpnI. The regions driving reporter expression in the gut are localised in the 3′ end of the shv region (Sotillos, 2006).

To better understand the regulation of dpp expression during vein development, the interactions were analyzed between a 2.5 kb region including wing veins pupal enhancers and several proteins involved in the regulatory network controlling the formation of veins. For this purpose, the 2.5 kb region was subdivided into overlapping fragments of 250-300 bp used as probes to detect the binding of different transcription factors by Electrophoretic Mobility-Shift Assays (EMSAs). Both prepattern specific genes that control vein development, such as Ventral veinless (Vvl) and the Araucan protein (Ara), and transcription factors belonging to the Dpp pathway (Mad and Medea) were analyzed (Sotillos, 2006).

The DNA-binding activity of Drosophila Smad proteins, Mad and Medea, is crucial for the expression of Dpp target genes. The expression of phosphorylated Mad (p-Mad), the activated form of the Mad protein, is restricted to the developing veins during pupal development. The efficiency of ectopic dpp expression to direct vein differentiation depends on the integrity of the shv region, suggesting that Dpp signalling is sufficient, directly or indirectly, for driving the expression of additional dpp transcription via the shv enhancer. Therefore, whether the Smads proteins can bind to the dpps enhancer was studied. Specific binding was obtained using all probes as shown by competition both with cold DNA and with specific oligonucleotides containing consensus binding sequences for Medea and for Mad and Medea. Other oligonucleotides with consensus for the transcription factor Nubbin did not compete the binding. The three main regions of competition with Mad and Medea binding included in the S9 and S10 probes correspond to GC rich sequences characteristic of Smad-response elements. However, only a single consensus sequence for Mad/Med (GCGGCTGT) in S10 is localised in a highly conserved region of different Drosophilids (see below) (Sotillos, 2006).

The pattern of four longitudinal veins is very similar in all Drosophilids despite the differences in wing size and pigmentation that exist between species. This conservation suggests that the mechanisms underlying vein pattern formation are conserved. The availability of the genomic sequence for different Drosophila species allows a direct comparison between their dpps regions. Two Drosophila species from the melanogaster group (D. melanogaster and D. ananassae), one Drosophila from the obscura group (D. pseudoobscura) and D. virilis from the virilis group were compared. It is expected that sequence similarity in non-coding regions corresponds to functional regulatory DNA. In the 2.5 kb region analysed several clusters of sequence conservation were found including most of the binding sites identified by in vitro analysis. Thus, there are four highly conserved regions corresponding to the S1, S4-5, S7-8 and S9-S10 probes containing conserved binding sequences for Vvl, Mad, Med and Ara. This conservation reinforces the importance of these regions to regulate the expression of dpp in the pupal veins. In the case of Vvl specific DNA binding to all probes was shown. However, the putative Vvl binding sequences localised in the conserved regions are only in S1, S3, S7, S8 and S10. In the case of the Dpp pathway transcription factors Mad and Med, putative binding sites are present throughout the enhancer, and accordingly binding of them to all probes was shown. However, only the S5 and S10 probes contain putative binding sites in regions of high sequence conservation. Interestingly, these conserved Mad/Med binding regions contain overlapping binding consensus for the Brinker repressor. This suggests a competition mechanism between Mad/Med and Brinker for binding to the shv enhancer. Competition mechanisms between activator and repressor also occur in several Dpp-downstream genes such as zen and omb. Four consensus binding sequences were found for the transcription factors of the Knirps-complex. The kni genes are expressed in the L2 vein, where they are required for its formation. Three Kni-binding sites were found in the 1.1 kb KpnI/SacII enhancer and one in the 0.5 SacII regions. Only two of the sequences located in the 1.1 kb KpnI/SacII enhancer present some conservation between Drosophilids. Interestingly, the 0.9 kb SalI/KpnI enhancer responsible of dpp expression in the L5 veins does not contain any putative Knirps binding sequence. Although whether Kni binds directly to the shv enhancer has not been analyzed, the presence of Kni-binding sites in conserved regions of the enhancer suggests that, in addition to its role during imaginal development, Kni might also activate dpp transcription during pupal development (Sotillos, 2006).

Therefore, regulatory sequences that drive dpp expression in the pupal veins in 2.5 kb of the dpps region have been found. This regulatory DNA can be subdivided into three fragments, a 1.1 kb fragment that recapitulates almost completely the pupal expression of dpp, a 0.9 kb upstream fragment, which drives expression in the proximal part of L5, and a 0.5 kb fragment that directs expression in all veins. Binding sites were found in these fragments for general transcription factors involved in the development of all veins (Vvl) and for the downstream activators of the dpp pathway, Mad and Medea. The regulatory region also contains binding sites for transcription factors expressed and required only in specific veins, such as Ara (L3 and L5) and Kni (L2). Most of these sequences are located in highly conserved regions of the dpp gene in different Drosophila species, indicating a general conservation of dpp regulation in the Drosophilids (Sotillos, 2006).

Regulation of the retinal determination gene dachshund in the embryonic head and developing eye

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow a better understanding of how spatial and temporal control of gene expression occurs during early eye development. Conserved non-coding sequences (CNCSs) between five Drosophilids were compared along the ~40 kb genomic locus of the RD gene dachshund (dac). This analysis uncovers two separate eye enhancers, in intron eight and the 3' non-coding regions of the dac locus, defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. These results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac (Anderson, 2006).

The smallest fragment in the 3' dac eye enhancer that can respond to dpp, eya and so is 3EE194 bp, which is centered around two CNCS blocks of ~40 bp and 20 bp. These two CNCS blocks are also common to all active fragments of the 3' eye enhancer. These two evolutionarily conserved stretches were scanned for known, genetically upstream transcription factor binding sites. The 40 bp conserved stretch contains two putative consensus So-binding sites, S1-5'-CGATAT and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T) described previously. Each of these putative So-binding sites in 3EE were mutated individually and in combination to test their requirement for normal enhancer activity in vivo. Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo. However, simultaneous mutation of both So binding sites completely abolishes enhancer activity in vivo. These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3' eye enhancer directly and nucleates a protein complex that includes Eya to regulate 3EE. However, despite much effort using a wide variety of binding conditions, it was not possible to demonstrate specific, direct binding of So protein to oligos that contain these So-binding sites. The 5' eye enhancer, which has four CNCS blocks, were scanned for potential upstream transcription factor binding sites and no strong candidate binding sites were found within the CNCS blocks (Anderson, 2006).

Loss- and gain-of-function analyses with the two eye enhancers suggest that each enhancer is regulated by a distinct set of protein complexes. The 5' eye enhancer is activated by a combination of ey, eya and so, but is not activated by Dpp signaling. 5EE is activated by ectopic ey expression even in eya and so mutants, suggesting that it is regulated exclusively by ey. However, somewhat paradoxically, expression of 5EE, the intron 8 enhancer, is lost in eya and so mutants even though ectopic expression of a combination of dpp, eya and so does not activate this enhancer. Furthermore, driving high levels of ey in so1 mutant eye discs restores 5EE-lacZ expression. Coupled together, these results suggest that 5EE is primarily regulated by ey but that the regulation of 5EE by ey also requires eya and so (Anderson, 2006).

By contrast, the 3' dac eye enhancer is regulated by a combination of eya, so and dpp signaling, but is not directly dependent on ey. 3EE-GFP expression is lost in eya2 and so1 mutant eye discs, and in posterior margin mad1-2 mutant clones. Furthermore, ey cannot bypass the requirement for eya and so to activate 3EE. Conversely, 3EE is strongly induced by co-expression of eya and so. Moreover, dpp signaling via the tkv receptor can synergize with eya and so to induce 3EE in ectopic expression assays. Furthermore, neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays. Thus, it is concluded that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3' dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5' and 3' dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EE and 3EE remain to be determined (Anderson, 2006).

Morphogenetic furrow (MF) initiation is completely blocked in posterior margin dac3-null mutant clones. However, dac3 clones that do not include any part of the posterior margin develop and do not prevent MF progression, but do cause defects in ommatidial cell number and organization. This dichotomy in dac function is reflected in the two eye enhancers characterized in this study. Analysis of dac7 homozygotes demonstrates that the 3' eye enhancer is dispensable for MF initiation and progression. It is proposed that in dac7 mutants, the intact 5EE enhancer is sufficiently activated by ey to drive high enough levels of dac expression to initiate and complete retinal morphogenesis. However, dac7 mutants have readily observable defects in ommatidial organization. Thus, it is further proposed that this lack of normal patterning in dac7 mutants is most likely due to the loss of 3EE, which normally acts in concert with 5EE after MF initiation, to integrate patterning inputs from extracellular signaling molecules such as Dpp with tissue-specific upstream regulators such as ey, eya and so. However, it is not known if the 3' eye enhancer is sufficient to initiate dac expression in the absence of the 5' eye enhancer (Anderson, 2006).

Based on the results, a two-step model is proposed for the regulation of dac expression in the eye. First, the initiation of dac expression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but a model is favored in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dac expression is maintained by an Eya and So complex bound to 3EE. In addition, 3EE can integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dac in the eye. This two part retinal enhancer ensures that dac expression is initiated only after ey activates eya and so expression. Thus, the dac eye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes (Anderson, 2006).

The redundancy in dac enhancer activity also explains the inability to isolate eye-specific alleles of dac, despite multiple genetic screens. The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac (Anderson, 2006).

Despite much investigation, very few direct targets of RD proteins, especially for Eya and So, have been identified. One study suggests that So can bind to and regulate an eye-specific enhancer of the lz gene. However, lz is not expressed early during eye development and is required only for differentiation of individual cell types. The results suggest that regulation of dac expression occurs via the interaction of two independent eye enhancers that are likely to be bound by Ey, Eya and So, and respond to dpp signaling. This analysis of the 3' eye enhancer suggests that two putative conserved So-binding sites are essential for 3EE activity in vivo. Mutation of individual So-binding sites dramatically reduces, but does not completely eliminate, reporter expression in the eye. Mutating both predicted So-binding sites completely blocks enhancer activity in vivo. Thus, it is concluded that So binds to 3EE via these conserved binding sites. However, it has not been possible to demonstrate a direct specific interaction of either So alone or a combination of Eya and So with oligos that contain these putative So-binding sites in vitro. It is possible that other unidentified proteins are required for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that responds to ectopic expression of dpp, eya, and so contains no conserved or predicted Mad-binding sites. This raises the intriguing possibility that dpp signaling activates other genes, which then directly act with eya and so to regulate the 3' eye enhancer. Alternatively, a large complex that includes Eya, So and the intracellular transducers of dpp signaling, such as Mad and Medea, may be responsible for activation of 3EE. Similarly, the results suggest that the 5' eye enhancer is regulated primarily by ey. However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey is fully functional only in the presence of Eya and So. Thus, Ey either independently recruits Eya and So into a 5' complex or is activated by virtue of its proximity to the So/Eya complex bound to the 3' enhancer or both (Anderson, 2006).

The exact order and dynamics of protein complex assembly at 5EE and 3EE requires further investigation. However, the two dac eye enhancers are extremely useful tools with which to investigate fundamental issues about the mechanism of RD protein action. One significant issue concerns the mechanism of Eya function during eye development. Eya consists of two major conserved domains, an N-terminal domain that has phosphatase activity in vitro and a C-terminal domain that can function as a transactivator in cell culture assays. So contains a conserved Six domain and a DNA binding homeodomain. However, it is unclear if Eya provides phosphatase activity, transactivator function, or both, in this complex. Characterization of the components of the protein complexes that regulates dac expression may uncover the targets of Eya phosphatase activity during eye development. Thus, the isolation of two eye enhancers with distinct regulation provides very useful tools with which to study protein complex formation and function during Drosophila retinal specification and determination (Anderson, 2006).

Decapentaplegic-responsive silencers contain overlapping mad-binding sites

Smad proteins regulate transcription in response to transforming growth factor-β signaling pathways by binding to two distinct types of DNA sites. The sequence GTCT is recognized by all receptor-activated Smads and by Smad4. The subset of Smads that responds to bone morphogenetic protein signaling recognizes a distinct class of GC-rich sites in addition to GTCT. Recent work has shown that Drosophila Mad protein, the homologue of bone morphogenetic protein rSmads, binds to GRCGNC sites through the same MH1 domain β-hairpin interface used to contact GTCT sites. However, binding to GRCGNC requires base-specific contact by two Mad proteins, and this study provides evidence that this is achieved by contact of the two Mad subunits that overlap across the two central base pairs of the site. This topology is supported by results indicating that His-93, which is located at the tip of the Mad β-hairpin, is in close proximity to base pairs 2 and 5. Also consistent with the model is disruption of binding by mutation of Glu-39 and Glu-40, which are predicted to lie at the interface of the two overlapping Mad MH1 domains. As predicted from the overlapping model, binding is disrupted by insertion of 1 bp in the middle of the site, whereas insertion of 2 bp creates abutting sites that can be bound by the Mad-Medea heterotrimer without requiring Glu-39 and Glu-40. Overlapping Mad sites predominate in Decapentaplegic response elements, consistent with a high degree of specificity in response to signaling (Gao, 2006).

DNA contact by Smad proteins has been shown to play an important role in many instances of target regulation by TGFβ pathways. For the consensus Smad3/Smad4-binding site, GTCT can also be bound by Mad- and BMP-specific Smad1, but Smad3 does not bind to GC-rich Mad/Smad1-binding sites, leaving open the question of whether such sites are contacted by a different mechanism. Recent work had shown that Mad-binding sites within the brk and bam silencers are bound by two Mad subunits and that in each case two Mad MH1 domains simultaneously contact a single 6-bp site using the same three β-hairpin residues that are responsible for base-specific contact by Smad3. By using mutational analysis and directly measuring binding, this study provides evidence that two Mad MH1 domains bind to the 6-bp site by overlapping across the two central base pairs. Smad1-binding sites match this 6-bp motif, a likely indication that overlap is also a feature of BMP-response elements (Gao, 2006).

The overlapping structure of Mad sites explains the seeming discrepancy between the Mad consensus and that of Smad3/Smad4. Smad3 differs from Mad at two positions that influence binding to brkS. Arg-58 at the C terminus of helix 2 is absolutely essential for binding to brkS; in Smad3 this position is a threonine, whereas the adjacent Lys-59 of Mad is absent in Smad3. Glu-39 at the N terminus of helix 2 contributes substantially to binding affinity for brkS, and Smad3 has instead a glutamine at this position. In addition, the loop between helices 1 and 2 is three residues shorter in Mad than in Smad3, a difference that modeling suggests will affect the structure of the alpha-carbon backbone and side chains near the N terminus of helix 2. Each of these differences is conserved between Smad3 and Smad2 and between Mad and the vertebrate BMP-specific rSmads (Gao, 2006).

Mutational analysis indicated the optimal sequence for an overlapping Mad site is GGCGCC, meaning each Mad MH1 prefers GGCG in the context of overlap. However, even when the two Mad sites are spaced such that they do not overlap, GGCG is still bound by Mad with about the same affinity as GTCT. The structural basis for this compatibility with two distinct sites remains to be determined, but the differential effects of helix 2 alanine substitutions suggest distinct docking geometries. Individual GGCG motifs occur in Dpp and BMP-response elements, and the results indicate that these are likely sites for contact by Mad and Smad1 (Gao, 2006).

Although the natural brkS element was specific for the Mad-Medea heterotrimer, changing the Mad site to abutting SBEs allowed binding by Mad alone or by Medea alone. The ability of such a site to be bound by Medea oligomers (putatively homotrimers) without Dpp signaling seemingly would make it ill-suited to function as a Dpp-response element, although signaling-induced activation was observed by reporter analysis (perhaps an indication that Medea alone is a poor activator). However, the brkS derivative with abutting GGCG sites (i.e. GGCGCGCC) shows little or no Medea binding in the absence of active Mad, is able to recruit Shn, and causes repression in response to signaling. Similar sites in the Dpp-response element of Race (GACGCGAC), which does not respond to repression by Brk protein, and in a BMP-response element of Smad7 (GGCGCGCC) appear to be examples of functional nonoverlapping Mad/Smad1 sites. In Drosophila a potentially significant difference between overlapping and nonoverlapping Mad sites is that the overlapping motif allows for competitive binding by the Brinker protein and thus dual control of Dpp targets, whereas the nonoverlapping motif does not. This may account for the predominance of overlapping Mad sites in Drosophila. The predominance of overlapping sites in BMP-response elements may reflect specificity for Smad1 but not Smad3 (Gao, 2006).

Antagonistic and cooperative actions of the EGFR and Dpp pathways on the iroquois genes regulate Drosophila mesothorax specification and patterning

In Drosophila, restricted expression of the Iroquois complex (Iro-C) genes in the proximal region of the wing imaginal disc contributes to its territorial subdivision, specifying first the development of the notum versus the wing hinge, and subsequently, that of the lateral versus medial notum. Iro-C expression is under the control of the EGFR and Dpp signalling pathways. To analyze how both pathways cooperate in the regulation of Iro-C, several wing disc-specific cis-regulatory elements of the complex were isolated. One of these (IroRE2) integrates competing inputs of the EGFR and Dpp pathways, mediated by the transcription factors Pointed (downstream of EGFR pathway) and Pannier/U-shaped and Mothers against Dpp (Mad), in the case of Dpp. By contrast, a second element (IroRE1) mediates activation by both the EGFR and Dpp pathways, thus promoting expression of Iro-C in a region of elevated levels of Dpp signalling, the prospective lateral notum near the anterior-posterior compartment boundary. These results help define the molecular mechanisms of the interplay between the EGFR and Dpp pathways in the specification and patterning of the notum (Letizia, 2007).

The Iro-C genes ara and caup show similar patterns of expression in the wing disc. In early second instar larvae, they are expressed in the whole prospective mesothorax region. Later, in the third instar, their expression is restricted to the lateral notum. In addition, at this developmental stage, novel domains of expression appear in the prospective regions of the L1, L3 and L5 veins, tegula, dorsal radius, dorsal and ventral pleura and alula. The expression of mirr is slightly different, being absent from the L3, L5 and tegula domains but present at the other domains. The Iro-C harbours two additional transcription units, lincoyan (linc), whose pattern of expression at the notum is identical to that of ara and/or caup and quilapan (quil), which is ubiquitously expressed. Previous genetic analysis suggested the existence of enhancer-like REs that would drive the coincident expression of ara and caup in the wing disc. Thus, In(3L)iroDFM2, associated with a breakpoint within the ara transcription unit, removes ara expression in the wing disc except in the L3 vein domain, in contrast to caup expression which is only lost from that domain. This suggests the existence of vein L3-specific RE(s) distal to the In(3L)iroDFM2 breakpoint and other RE(s), specific for the remaining domains of Iro-C expression, located proximal to such breakpoint. To identify notum-specific REs, the regulatory potential of 31 different genomic fragments, spanning approximately 110 kb of genomic Iro-C DNA was analyzed (Letizia, 2007).

Only five of those fragments drove lacZ expression at specific regions of the imaginal wing disc. One of them, 3.3 kb in length and named Iro regulatory element2. The IroRE2 was reduced to a 1.6 kb subfragment (sequence of the IroRE2-B fragment), which maintained enhancer activity in the notum and was activated by EGFR and repressed by Dpp signalling. Thus, IroRE2-lacZ was expressed in the proximal region of early third instar wing discs (the presumptive notum region) and at the presumptive lateral notum in third instar wing discs. Note, however, that the pattern of IroRE2-mediated lacZ expression does not exactly coincide with that of ara/caup. Thus, ß-gal was not detected in a triangular area, located near the notum/hinge border and centred around the AP compartment boundary, where expression of ara/caup is enhanced. This is precisely the region where expression of lacZ was driven by another Iro-C genomic fragment of 3.9 kb, IroRE1. Accordingly, an IroRE1-IroRE2 composite RE was found to drive lacZ expression in a pattern very similar, albeit not identical, to that of the endogenous ara/caup genes (Letizia, 2007).

Two other genomic fragments, IroRE3 and IroRE4 (3.4 and 3.7 kb), adjacent to each other, drove lacZ expression in a stripe of cells located at the proximal region of the presumptive lateral notum, which partially overlapped with the caup expression domain. Finally, IroRE5 (2.8 kb) drove expression mainly in the prospective alula and peripodial membrane (Letizia, 2007).

A common theme in development is the convergence of different signalling pathways to implement a given developmental program. For instance during embryonic development, the antagonistic activity of the EGFR and Dpp pathways sets the limits between the neuroectoderm and the dorsal ectoderm. A similar situation applies to the specification of prospective body regions within the wing imaginal disc. During the early second instar, EGFR and Dpp pathways act antagonistically on the regulation of the Iro-C restricting its expression to the prospective notum region where it specifies notum development rather than hinge. Later, at the early third instar, again the concomitant activity of EGFR and Dpp signals (the latter now also emanating form the most proximal region of the wing disc) partition the prospective notum into two different subdomains, the medial and the lateral notum, the latter being specified by ara/caup expression. Thus, to understand how regionalization of the adult fly body is achieved it is important to elucidate the mechanisms responsible for the joint interpretation of both signalling pathways (Letizia, 2007).

This study shows that the opposing effects of the EGFR and Dpp pathways on Iro-C expression result from the convergence of both pathways on at least two distinct Iro-C regulatory elements, IroRE1 and IroRE2. These two REs drive gene expression in two complementary domains of the prospective notum region of the wing disc, and appear to mediate most of the regulation of the Iro-C genes by the Dpp and EGFR pathways in this region of the wing disc. Furthermore, IroRE1 provides a regulatory mechanism for the coexistence at the prospective lateral notum of Iro-C expression and Dpp pathway activity, notwithstanding the negative regulation of Iro-C by such pathway (Letizia, 2007).

The transcriptional regulation of the Iro-C genes is modular. Thus, the non-coding Iro-C genomic DNA contains a series of five separate enhancers that control the expression of a reporter gene in sub-domains within the realm of Iro-C expression in the prospective notum region of the wing disc. None of the identified fragments reproduces on its own the entire pattern of expression of Iro-C in the prospective notum. However, IroRE1 and IroRE2 promote expression in complementary domains that entirely cover the territory of the presumptive lateral notum. Furthermore, IroRE2-mediated transcription recapitulates expression of Iro-C at the whole prospective notum at the second larval instar. It is hypothesized that the combined activity of both REs would be responsible for a great part of the regulation of Iro-C expression in the notum territory. Moreover, although IroRE3, IroRE4 and IroRE5 mediate lacZ expression in patterns only partly related to that of the Iro-C genes, these REs probably contribute to the complex regulation of the Iro-C. In addition, the possibility cannot be excluded of other RE(s) located outside the tested region that would help to establish the final pattern of Iro-C expression. Indeed, IroDFM3, a deficiency obtained by imprecise excision of the irorF209 P-lacZ element that extends up to the mirr promoter, maintains some lacZ expression in part of the central notum (Letizia, 2007).

The identified REs might act simultaneously on ara and caup expression to give rise to their almost coincident patterns of expression. Such coincidence cannot be attributed to cross-regulation between ara and caup since in irorF209 mutant discs (irorF209 is an ara null allele expression of caup is unmodified. Regulation of ara/caup would be, accordingly, similar to that of the achaete-scute genes of the AS-C, which show identical patterns of expression due to the use of shared enhancers. Expression of the vertebrate Iroquois (Irx) genes appears to be similarly regulated. Thus, the analysis of the regulatory potential of highly and ultra conserved non-coding regions present in the intergenic regions of the Irx clusters suggests these genes to be regulated by partially redundant enhancers shared by the components of each cluster (Letizia, 2007).

Expression of mirr in the notum region of the wing disc largely coincides with that of ara/caup and most likely is under the control of the same REs. Thus, activity of the IroRE2 may account for the unmodified expression of mirr in iro1 imaginal discs (associated with an inversion breakpoint located within the caup transcription unit). In addition, differences in the expression of ara/caup and mirr might be due to the presence of repressor RE(s) or insulator sequences that would prevent the action of the RE(s) controlling ara/caup on the mirr promoter. This is consistent with the previous observation of ectopic expression of mirr in Mob1 mutants, a regulatory mutation mapped within the Iro-C (Letizia, 2007).

The identification of REs present in the Iro-C has allowed unveiling of some of the molecular mechanisms of its transcriptional regulation at the level of DNA-protein interaction and analysis of the interplay of positive and negative inputs from convergent signalling pathways (Letizia, 2007).

EGFR activation in the proximal region of the wing disc leads to expression of Iro-C. This study demonstrates that both IroRE1 and IroRE2 mediate positive regulation by the EGFR pathway. It is shown that Pnt mediates activation of IroRE2-lacZ by the EGFR pathway. Furthermore, EGFR-dependent activation is cell context dependent. This suggests the existence, in the cells receiving EGFR signalling, of presently unknown factors that would contribute to ara/caup activation and/or the presence of counteracting repressing mechanisms, which should prevent their activation. Clearly, the Dpp pathway is so far the best candidate, since it has been shown that it can repress Iro-C and the IroRE2-lacZ transgene (Letizia, 2007).

The molecular mechanism of Dpp-dependent regulation of Iro-C expression appears to be more complex. The Dpp pathway can repress or activate Iro-C through different REs and different effector proteins. IroRE2 appears to mediate Dpp-dependent repression at the medial notum (most probably through direct binding of the heterodimer Pnr/Ush and Mad) and at the hinge and lateral notum (independently of Pnr, Ush and the GATA factor Grn in these domains). Dpp-dependent repression of Iro-C may be mediated, in addition, through a different RE, namely, through a brk silencer element (brkSE), shown to mediate Dpp-dependent repression of brk by binding of a Medea/Mad/Schnurri repressor complex, which is present at the Iro-C within IroRE5 (Letizia, 2007).

Despite the Dpp-mediated repression through IroRE2, a high level of Iro-C proteins accumulates in the lateral region of the notum, near the strong source of Dpp at the AP border. Furthermore, in this region of the wing disc Iro-C expression is refractory to Dpp-dependent repression. It is noteworthy that, IroRE1 mediates lacZ expression exclusively in that region of the wing disc and it appears to provide a regulatory mechanism for the co-existence of Iro-C expression and Dpp pathway activity, since the Dpp pathway does not repress but, on the contrary, activates IroRE1-mediated lacZ expression. Activation is restricted to the lateral notum, most likely because of the presence, in the hinge and medial notum territories, of repressors [Muscle segment homeobox, Msh; also known as Drop and Pnr/Ush, respectively] that would counteract activation. Putative binding sites for both Msh (consensus sequence G/C TTAATTG) and GATA proteins are indeed present in IroRE1. Thus, IroRE1 and IroRE2 represent two different REs in the same gene that respond in opposite ways to the same positional information, i.e. Dpp signalling. In addition a Dpp-independent mechanism based in the mutual repression between Iro-C and the homeoprotein Msh helps to maintain the distal border of Iro-C expression. This repression could be mediated by direct binding of Msh to one putative Msh binding site present in the Iro-RE2-B sequence (Letizia, 2007).

A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila

Analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF/Pangolin and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung (Takaesu, 2008).

These data show that within the dppMX region there is a combinatorial enhancer that contains binding sites recognized by TCF and Mad that are essential for activating dpp expression in the spiracular chambers, in the spiracular branches and in the dorsal trunk branches. There is also a binding site recognized by Brinker that is likely employed to repress dpp expression late in spiracle development (Takaesu, 2008).

What makes this enhancer different from other enhancers in Drosophila also capable of integrating three inputs in the same cells. These enhancers integrate only positive signals. In all cases, PointedP2 binding displaces the Yan repressor that is constitutively bound to the enhancer in the absence of PointedP2. The difference is that the dppMX enhancer is actively repressed by Brk binding after being stimulated by positive input from the Dpp and Wg pathways. What makes this enhancer different from other enhancers in Drosophila that integrate positive and negative signals such as the enhancer of Ultrabithorax where positive input from TCF is associated with a competition between Mad (positive) and Brk (negative) inputs. The difference is that in the same cells the dppMX enhancer responds sequentially to positive combinatorial input from TCF and Mad and then to negative input from Brinker. The Ultrabithorax enhancer responds simultaneously to positive input from TCF and Mad in parasegment seven and to negative input from Brinker in the adjacent cells of parasegment 8 (Takaesu, 2008).

If combinatorial signaling by the Dpp and Wg pathways, via TCF and Mad, turn on the dppMX enhancer in posterior spiracle primordia of the dorsal ectoderm at stage 13, then where do the Dpp signals originate? One possibility is that Dpp signals derive from the adjacent region of the dorsal ectoderm -- leading edge cells located just anterior to the posterior spiracle primordia. In leading edge cells of the dorsal ectoderm, dpp expression is activated at stage 8. dpp leading edge expression is activated by enhancers distinct from the dppMX enhancer, and the leading edge enhancers are themselves stimulated, in part, by dpp blastoderm expression that sets up the embryonic dorsal/ventral axis. In this scenario, the activation of the dppMX enhancer in posterior spiracles by Dpp leading edge signaling represents the last step in a cascade, covering nearly all of embryogenesis, of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

The most likely the source of the Wg signal is a small group of cells in the spiracular chamber. wg expression in the spiracular chamber becomes visible at stage 11 and is present through the remainder of embryogenesis. This group of Wg expressing cells is required for the maintenance of Cut and Spalt expressions, genes shown in this study to be independent of Dpp signaling. The involvement of Wg in spiracle cell fate determination and dpp activation results in more severe spiracle defects in wg mutants than in brkF124 embryos or dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu, 2008).

The source of the signal that activates brk in the posterior spiracles is less easy to identify. However, one possibility is suggested by the mutant phenotype generated by ubiquitous expression of unpaired (a ligand of the Jak/Stat pathway with a role in posterior spiracle formation. These embryos display a U-shaped Filzkorper similar to brkF124 embryos and dpp null embryos with two copies of the dpp-ΔKX rescue construct (Takaesu,2008)

The data advance understanding of posterior spiracle development and the role that Dpp signaling plays in this process in three areas: (1) that dpp activity in dorsal/ventral patterning is genetically separable, in part, from its activity in posterior spiracle development; (2) that dpp signaling does not appear to influence posterior spiracle cell fate or external morphology but instead regulates spiracle internal morphology; and (3) that a functioning posterior spiracle is necessary for viability prior to hatching (Takaesu, 2008).

Regarding the separability of dpp dorsal-ventral patterning and posterior spiracle functions, this view contrasts with the prevailing wisdom that all dpp posterior spiracle defects are downstream consequences of dorsal-ventral patterning defects. Instead, the results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is necessary for setting up the posterior spiracle field in association with dorsal-ventral patterning at the blastoderm stage. The second begins during germ band retraction and appears to regulate the internal morphology of the spiracles. One possible explanation for why these distinct aspects of dpp function have been connected in the conventional wisdom is that the dppMX enhancer is located in an intron alongside dorsal/ventral patterning enhancers and is deleted in several widely studied dppHin alleles (Takaesu, 2008).

This two-round model for dpp signaling in posterior spiracle development fits well with analysis of Dpp signaling in heart development. Here, there is a second round of Dpp dorsal ectoderm to mesoderm signaling late in development that maintains the boundary between pericardial cells and the adjacent dorsal muscle cells. The second round of Dpp signaling in heart development is autoactivated by Dpp signals that also likely derive from dpp leading edge expression. Thus, in heart development, there is also evidence of a multi-step cascade of increasingly spatially restricted rounds of Dpp dorsal ectoderm signaling (Takaesu, 2008).

Regarding the function of the second round of Dpp signaling in posterior spiracle development, the data show that the expression of three transcription factors essential for cell fate determination in the spiracles is independent of Dpp signaling. In addition, pMad data show that the lumen of the spiracular chamber forms normally suggesting that spiracle external morphology and invagination, under the control of Rho signaling, is also independent of Dpp (Takaesu, 2008).

Cuticle data indicate that the primary defect in dpp posterior spiracle mutants is fully differentiated but U-shaped Filzkorper that do not appear to connect to the dorsal trunk branches. This phenotype plus the fact that dpp mRNA and pMad expressions normally span the spiracular chamber, spiracular branches and dorsal trunk branches suggests the hypothesis that Dpp regulates the internal morphology of the spiracles. Given the mutant phenotype and gene expression patterns, it is tempting to speculate that Dpp signaling via pMad directs the anterior outgrowth of the spiracles, the posterior outgrowth of the dorsal trunk branches and their eventual fusion into a coherent tracheal system (Takaesu, 2008).

Regarding posterior spiracle function in embryos, the fact that dpp posterior spiracle mutants do not hatch suggests that gas exchange through the posterior spiracles and the spiracular branches begins and is required to sustain the individual prior to hatching. This is an advance in the understanding of Drosophila embryonic and larval respiration (Takaesu, 2008).

Molecular integration of Wingless, Decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development

The development of the Drosophila leg requires both Decapentaplegic (Dpp) and Wingless (Wg), two signals that establish the proximo-distal (PD) axis by activating target genes such as Distalless (Dll). Dll expression in the leg depends on a Dpp- and Wg-dependent phase and a maintenance phase that is independent of these signals. This study shows that accurate Dll expression in the leg results from the synergistic interaction between two cis-regulatory elements. The Leg Trigger (LT) element directly integrates Wg and Dpp inputs and is active only in cells receiving high levels of both signals. The Maintenance (M) element is able to maintain Wg- and Dpp-independent expression, but only when in cis to LT. M, which includes the native Dll promoter, functions as an autoregulatory element by directly binding Dll. The 'trigger-maintenance' model describes a mechanism by which secreted morphogens act combinatorially to induce the stable expression of target genes (Estella, 2008).

This study provides evidence that Dll expression during Drosophila leg development is controlled by separate, synergistically interacting cis-regulatory elements. The first element, LT, activates transcription only in response to high levels of Wg and Dpp signaling. The second element, M, includes the Dll promoter and has the ability to activate transcription in a Wg- and Dpp-independent manner, but only when in cis to LT. Together, these results fit well with previous genetic experiments showing that the Wg and Dpp inputs into Dll are only required transiently, prior to ~60 hr AEL. Based on the data, it is hypothesized that LT, and perhaps other elements with similar properties, is responsible for activating the Wg- and Dpp-dependent phase of Dll expression. Further, the data suggest that the combination of LT+M executes the Wg- and Dpp-independent phase of Dll expression. The existence of a two-component cis-regulatory system for Dll expression has several interesting implications and provides a mechanistic understanding of how Wg, Dpp, and Dll inputs are integrated into Dll expression (Estella, 2008).

The requirement for multiple inputs for gene activation is a common theme in transcriptional regulation. Enhancer elements can be thought of as 'logic integrators' that are only active in the presence of the correct activators and in the absence of repressors. The LT element defined here behaves as such a logic integrator. To be active, at least three conditions must be met. (1) LT must be bound to a transcriptionally active form of Tcf, a condition which indicates high levels of Wg signaling. (2) LT must be bound to a transcriptionally active form of Mad, and, (3) LT must not be bound to Brk. The second and third of these three conditions both indicate high levels of Dpp signaling. This combination of inputs ensures that LT is triggered only only where Wg and Dpp signaling are both active. In addition, it is hypothesized that there must be another input that restricts LT's activity to the ventral discs (e.g., it is not active in other tissues where Wg and Dpp signaling intersect such as the wing disc). Such a ventral-specific input could be Dll itself, which is expressed before LT is active via the Dll304 enhancer, and/or another ventral-specific factor such as buttonhead (btd), which is also required for Dll expression. Consistent with this idea, LT-lacZ is lost in Dll clones and in Dll hypomorphic discs, suggesting that Dll input, in addition to Wg and Dpp, is required for its activity (data not shown) (Estella, 2008).

As noted above, Dpp signaling uses two mechanisms (Mad binding and absence of Brk) to control LT's activity. Because Brk, a transcriptional repressor, binds directly to LT, it restricts the domain in which Wg signaling can activate this element. This conclusion is best supported by the expression pattern of the LT reporter gene in which the Brk-binding sites were mutated. Specifically, the expression of this reporter (LTBrk–-lacZ) was expanded ventrally, indicating its potential to be activated more broadly by Wg signaling in the absence of this repressor. Thus, it is suggested that the primary role of Brk is to provide spatial information to LT activation. The absence of Brk, however, is apparently not sufficient for LT activation; Mad input into LT appears also to be essential. Several experiments support this conclusion. Most informatively, LT-lacZ was not expressed in Mad; brk clones, and LT-lacZ reporter genes with either Mad site mutated were not expressed in brk clones. Thus, even in the absence of Brk, LT requires Mad input. It is suggested that in contrast to providing spatial information, the Mad input into LT is important for boosting the level of its activation, together with Tcf, by providing an additional potent transcriptional activator. Further, LT is unlikely to be the only Dll cis-regulatory element that integrates Wg plus Dpp signaling during leg development. Although LT was the only fragment within the 14 kb of 5' DNA that drove strong expression in the leg disc in a standard reporter gene assay, thus allowing the dissection of Wg and Dpp signal integration, a second fragment was identified that was able to synergize with M to produce a Dll-like expression pattern. In summary, these data suggest that during the Wg- and Dpp-dependent stage, Dll expression is regulated by the direct binding of Tcf, Mad, and Brk to LT and, perhaps, additional regulatory elements (Estella, 2008).

As is the case for Dll, there are examples of other genes that have separable initiation and maintenance phases of expression. For many of these examples, expression is maintained by the trxG and PcG of epigenetic regulators. There are also examples of genes that require enhancer-promoter communication for maintenance. For example, a regulatory element from the Hoxb4 gene requires sequences from its own promoter for stable expression in the mouse hindbrain. In this case, a key input into the promoter-proximal sequences is the PcG protein, YY1. Dll expression is unaffected in trx mutant clones but is lost in a subset of Pc and Scm clones, raising the possibility that PcG functions play a role in maintenance. However, PcG functions are more typically associated with maintaining genes in a repressed state, not an expressed state. Moreover, because of PcG's widespread role in gene silencing, many genes are likely to be derepressed in these clones. In fact, the Hox gene Abd-B is derepressed in these clones, and Abd-B has the ability to repress Dll. Thus, on balance, it seems more likely that the loss of Dll expression observed in some Pc clones is an indirect effect. In contrast, the results strongly argue that positive autoregulation, by direct binding of Dll to the M element, plays an important role in Dll maintenance (Estella, 2008).

One conclusion drawn from these observation that both LT and M are required for maintenance is that LT requires the Dll promoter to be fully active. Such promoter-specific enhancer activation has been observed previously and is generally thought to be important for remote enhancers to stimulate transcription from the correct promoter in gene-dense regions of the genome. The LT+M synergy described in this study is distinct from these other examples. In this case, although enhancer-promoter compatibility may be part of the reason that LT works better with M (and over large distances), the results show that the combination of the two has properties that are not exhibited by either element on its own. Specifically, while M-lacZ is very weakly expressed in leg discs, and LT-lacZ requires continuous Wg and Dpp inputs, the combination of LT+M allows Dll autoregulation to occur in a Wg- and Dpp-independent manner. Moreover, LT+M is not simply a Dll autoregulatory element: even though Dll is expressed in the wing disc, transcriptional activation by LT+M remains restricted to the ventral imaginal discs. This observation implies that the Dll input into LT+M can only occur in cells where LT was activated, which itself only happens in ventral discs. Thus, LT+M is not only a two-component Dll autoregulatory element, but is an autoregulatory element that requires the prior Wg- and Dpp activation of LT (Estella, 2008).

These observations lead to the suggestion of two classes of models by which maintenance may occur. In one, an activated LT element changes the chromatin structure of M, for example, by changing the position of a repressive nucleosome so that it can function as an autoregulatory element. According to this model, the continued presence of LT is required to maintain this chromatin structure. A second model that would also accommodate these data is that the combination of LT plus M is required to increase the efficacy of transcriptional activation by, for example, providing additional Dll (or other activator) binding. According to this scenario, LT activation by Wg and Dpp triggers the initial interaction between the LT and M elements, which would then be stabilized in a Wg- and Dpp-independent manner. These models are not mutually exclusive and both can be tested by analyzing the chromatin status at the M and LT elements (Estella, 2008).

The results also raise the question of what purpose this two-step trigger-maintenance mechanism may serve. One possibility is that, by having only a transient requirement for Wg and Dpp, these morphogens are available for carrying out completely different tasks, without affecting Dll expression. In support of this idea, in addition to working together to create the PD axis, Wg and Dpp function independently to instruct ventral and dorsal leg fates, respectively. Some of these late Wg and Dpp patterning functions may also require Dll input. The trigger-maintenance logic described in this study in principle allows Wg and Dpp to execute functions in collaboration with their own downstream target, Dll (Estella, 2008).

It is also noteworthy that the transient nature of the Wg and Dpp inputs into Dll is not the typical way these morphogens regulate their target genes in other tissues. In the Drosophila wing, for example, Dpp and Wg are required to continuously activate their targets, such as vestigial, optormotor blind, and spalt. One signficant difference between the regulation of wing and leg target genes by these morphogens is that in the wing Wg and Dpp generally act independently, whereas in the leg they act combinatorially to activate PD genes. Specifically, although they are expressed in ventral and dorsal sectors, respectively, Wg and Dpp activate Dll and dac in circular or nearly circular domains whose centers are located where the Dpp and Wg expression domains touch, in the middle of the leg disc. The trigger-maintenance mechanism defined in this study avoids the need for target genes such as Dll to continuously integrate Wg and Dpp inputs as the disc grows in size, and provides a mechanism to generate circular domains of gene expression using dorsal and ventral morphogen inputs (Estella, 2008).

A conserved activation element in BMP signaling during Drosophila development

The TGF-β family member Decapentaplegic (Dpp) is a key regulator of patterning and growth in Drosophila development. Previous studies have identified a short DNA motif called the silencer element (SE), which recruits a trimeric Smad complex and the repressor Schnurri to downregulate target enhancers upon Dpp signaling. The minimal enhancer of the dad gene was isolated, and a short motif was discovered that was termed the activating element (AE). The AE is similar to the SE and recruits the Smad proteins via a conserved mechanism. However, the AE and SE differ at important nucleotide positions. As a consequence, the AE does not recruit Schnurri but rather integrates repressive input by the default repressor Brinker and activating input by the Smad signal transducers Mothers against Dpp (Mad) and Medea via competitive DNA binding. The AE allows the identification of hitherto unknown direct Dpp targets and is functionally conserved in vertebrates (Weiss, 2010).

A 520-bp fragment was discovered within the second intron of the dad gene. This fragment induces an expression pattern very similar to that of the endogenous dad gene in embryonic, larval and adult tissues and contains evolutionarily conserved and largely overlapping binding sites for Smad and Brk proteins within a short sequence element that was called activating element (AE). The Smad and Brk proteins bind in a competitive manner to the AE, a mechanism similarly proposed for zen and Ubx enhancer elements. By precise targeted mutations, Brk binding was selectively abolished, and it was possible to unlink Smad and Brk input. Notably, the AE assembles a high-affinity trimeric complex of full-length Mad and Medea proteins. In Drosophila, such complexes have so far only been demonstrated for a so-called silencer element (SE) (Pyrowolakis, 2004). Therefore, this study presents the first example of such complex formation on a short-sequence element in the context of a gene activated by Dpp (Weiss, 2010).

The AE very closely resembles the SE, but despite their analogy, AE and SE differ in several key aspects. Because of the arrangement of the Smad binding sites, they are both able to recruit a complex of Mad and Medea. However, only the SE includes the second thymidine, which is essential for the recruitment of the repressor Shn (Pyrowolakis, 2004). Furthermore, the AE identified in the dad enhancer is able to interact with the Brk repressor. Brk competes with Mad for binding to the AE, which fulfills the consensus sequence derived from analysis of the SE with regard to Mad binding (GRCGNC) as well as the sequence for Brk binding (TGGCGYY). In contrast, Brk does not bind to the SEs described (Pyrowolakis, 2004). Thus, the AE and SE use a very similar sequence to exert opposite effects. These results provide a striking molecular scenario for Dpp signaling readout, based on the assembly of a trimeric Smad complex and its recruitment of a corepressor (Shn) or its competition with a dedicated repressor of the pathway (Brk) (Weiss, 2010).

A transcription factor collective defines cardiac cell fate and reflects lineage history

Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. This study demonstrates that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this 'dormant' TF binding signature may represent a molecular footprint of these cells' developmental lineage (Junion, 2012).

Dissecting transcriptional networks in the context of embryonic development is inherently difficult due to the multicellularity of the system and the fact that most essential developmental regulators have pleiotropic effects, acting in separate and sometimes interconnected networks. This study presents a comprehensive systematic dissection of the cis-regulatory properties leading to cardiac specification within the context of a developing embryo. The resulting compendium of TF binding signatures, in addition to extensive in vivo and in vitro analysis of enhancer activity, revealed a number of insights into the regulatory complexity of developmental programs (Junion, 2012).

Nkx (Tinman in Drosophila), GATA (Pannier in Drosophila), and T box factors (Doc in Drosophila) regulate each other’s expression in both flies and mice, where they form a recursively wired transcriptional circuit that acts cooperatively at a genetic level to regulate heart development across a broad range of organisms. The data demonstrate that this cooperative regulation extends beyond the ability of these TFs to regulate each other’s expression. All five cardiogenic TFs (including dTCF and pMad) converge as a collective unit on a very extensive set of mesodermal enhancer elements in vivo (Tin-bound regions) and also in vitro (in DmD8 cells). Importantly, this TF co-occupancy occurs in cis, rather than being mediated via crosslinking of DNA-looping interactions bringing together distant sites. Examining enhancer activity out of context, for example, in transgenic experiments and luciferase assays, revealed that the TF collective activity is preserved in situations in which these regions are removed from their native genomic 'looping' context (Junion, 2012).

In keeping with the conserved essential role of these factors for heart development, the integration of their activity at shared enhancer elements may also be conserved. Recent analyses of the mouse homologs of these TFs (with the exception of the inductive signals from Wg and Dpp signaling) in a cardiomyocyte cell line support this, revealing a signifcant overlap in their binding signatures (He, 2011; Schlesinger, 2011), although interestingly not in the collective 'all-or-none' fashion observed in Drosophila embryos. This difference may result from the partial overlap of the TFs examined, interspecies differences, or the inherent differences between the in vivo versus in vitro models. Examining enhancer output for a large number of regions indicates that this collective TF occupancy signature is generally predictive of enhancer activity in cardiac mesoderm or its neighboring cell population, the visceral mesoderm—expression patterns that cannot be obtained from any one of these TFs alone (Junion, 2012).

There are currently two prevailing models of how enhancers function. The enhanceosome model suggests that TFs bind to enhancers in a cooperative manner directed by a specific arrangement of motifs, often having a very rigid motif grammar. An alternative, the billboard model, suggests that each TF (or submodule) is recruited independently via its own sequence motif, and therefore the motif spacing and relative orientation have little importance. The results of this study indicate that cardiogenic TFs are corecruited and activate enhancers in a cooperative manner, but this cooperativity occurs with little or no apparent motif grammar to such an extent that the motifs for some factors do not always need to be present. This is at odds with either the enhanceosome (cooperative binding; rigid grammar) or billboard (independent binding; little grammar) models and represents an alternative mode of enhancer activity, which was termed a 'TF collective' (cooperative binding; no grammar), and likely constitutes a common principle in other systems (Junion, 2012).

The data suggest that the TF collective operates via the cooperative recruitment of a large number of TFs (in this case, at least five), which is mediated by the presence of high-affinity TF motifs for a subset of factors initiating the recruitment of all TFs. The occupancy of any remaining factor(s) is most likely facilitated via protein-protein interactions or cooperativity at a higher level such as, for example, via the chromatin activators CBP/ p300, which interact with mammalian GATA and Mad homologs. This model allows for extensive motif turnover without any obvious effect on enhancer activity, consistent with what has been observed in vivo for the Drosophila spa enhancer and mouse heart enhancers (Junion, 2012).

Integrating the TF occupancy data for all seven major TFs involved in dorsal mesoderm specification (the five cardiogenic factors together with Biniou and Slp) revealed a very striking observation: the developmental history of cardiac cells is reflected in their TF occupancy patterns. Visceral mesoderm (VM) and cardiac mesoderm (CM) are both derived from precursor cells within the dorsal mesoderm. Once specified, these cell types express divergent sets of TFs: Slp, activated dTCF, Doc, and Pnr function in cardiac cells, whereas Biniou and Bagpipe are active in the VM. Despite these mutually exclusive expression patterns, the cardiogenic TFs are recruited to the same enhancers as VM TFs in the juxtaposed cardiac mesoderm. Moreover, dependent on the removal of a transcriptional repressor, these combined binding signatures have the capacity to drive expression in either cell type. This finding provides the exciting possibility that dormant TF occupancy could be used to trace the developmental origins of a cell lineage. It also explains why active repression in cis is required for correct lineage specification, which is a frequent observation from genetic studies. At the molecular level, it remains an open question why the VM-specific enhancers are occupied by the cardiac TF collective. It is hypothesized that this may occur through chromatin remodeling in the precursor cell population. An 'open' (accessible) chromatin state at these loci in dorsal mesoderm cells, which is most likely mediated or maintained by Tin binding prior to specification, could facilitate the occupancy of cell type-specific TFs in both CM and VM cells. Such early 'chromatin priming' of regulatory regions active at later stages has been observed during ES cell differentiation. The current data provide evidence that this also holds true for TF occupancy and not just chromatin marks. On a more speculative level, this developmental footprint of TF occupancy may reflect the evolutionary ancestry of these two organs. Visceral and cardiogenic tissues are derived from the splanchnic mesoderm in both flies and vertebrates. These complex VM-heart enhancers may represent evolutionary relics containing functional binding sites that reflect enhancer activity in an ancestral cell type (Junion, 2012).

Taken together, the collective TF occupancy on enhancers during dorsal mesoderm specification illustrates how the regulatory input of cooperative TFs is integrated in cis, in the absence of any strict motif grammar. This more flexible mode of cooperative cis regulation is expected to be present in many other complex developmental systems (Junion, 2012).

Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryo

Pattern 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).

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-beta superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. This study examined the expression pattern and transcriptional regulation of Dtg (CG6234), a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. The expression of Dtg is controlled by Dpp and a 524-bp enhancer was characterized that mediates expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica (Hodar, 2014).

The full-length transcripts and promoter analysis of intergenic microRNAs in Drosophila melanogaster

MicroRNA (miRNA) transcription is poorly understood until now. To increase miRNA abundance, miRNA transcription was stimulated with CuSO(4) and Drosha enzyme was knocked down using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. A series of miRNA promoter analyses was conducted to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoters. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect (Qian, 2011).

Mothers against dpp: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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