In imaginal discs brk is expressed complementary to regions of Dpp expression. The same principle applies for its expression during embryonic development. In late syncytial and in cellular blastoderm embryos brk is expressed in a ventrolateral stripe, which is initially 9-10 nuclei wide and expands to encompass 18 nuclei shortly before gastrulation. A narrow gap separates the initial stripe dorsally from the expression domain of dpp. On the ventral side, expression stops one cell row short of the snail domain. Thus, the brk domain largely overlaps short gastrulation (sog) expression, in agreement with the finding that brk and sog together are required for the establishment of the ventral neurogenic ectoderm. Furthermore, the domain also corresponds to the region of the embryo showing ectopic dpp, zerknullt (zen) and tolloid misexpression in the absence of brk. This expression pattern is thus consistent with the view that brk may directly repress the transcription of these genes. During germ band extension brk continues to be expressed in the ventral ectoderm where it appears to be restricted to neuroectodermal cells during neuroblast segregation. New sites of expression appear in the ventral mesoderm at stage 10, when Dpp-signaling from the ectoderm induces dorsal mesoderm. During stage 11, some ectodermal cells surrounding the tracheal pits express brk. The tracheal pits lie in between the dorsolateral and the ventral stripe of dpp expression, and patterning of the invaginating tracheal cells is known to be influenced by dpp. During germ band retraction the developing midgut broadly expresses brk. In stage 13, three localized domains of expression can be seen in the visceral mesoderm and endoderm of the developing gut: (1) a domain slightly anterior to the region of the developing gastric caeca; (2) a domain approximately at the location of parasegment 5, and (3) a posterior domain at the border between hindgut and midgut. These regions lie between domains of dpp expression in the visceral mesoderm where dpp mediates inductive processes between visceral mesoderm and endoderm. Finally, brk is expressed during dorsal closure in a broad stripe of ectodermal cells localized ventrally to the leading edge cells in which dpp expression controls morphogenesis. In summary, these expression patterns suggest a potential role of brk in all major aspects of Dpp signaling in the embryo (Jazwinska, 1999b).

Dorsoventral patterning of the Drosophila hindgut is determined by interaction of genes under the control of two independent gene regulatory systems, the dorsal and terminal systems

Dorsoventral (DV) patterning in the trunk region of Drosophila embryo is established through intricate molecular interactions that regulate Dpp/Scw signaling during the early blastoderm stages. The hindgut of Drosophila, which derives from posterior region of the cellular blastoderm, also shows dorsoventral patterning, being subdivided into distinct dorsal and ventral domains. engrailed (en) is expressed in the dorsal domain, which determines dorsal fate of the hindgut. This study shows that a repressor Brk restricts en expression to the dorsal domain of the hindgut. Expression domain of brk during early blastodermal stages is defined through antagonistic interaction with dpp, and expression domains of dpp and brk in the early blastoderm include prospective hindgut domain. After stage 9, dpp expression in the dorsal domain of the hindgut primordium disappears, but, the brk expression in the ventral domain continues. It was found that Dorsocross (Doc), which is a target gene of Dpp, is responsible for restricting brk expression to the ventral domain of the hindgut. On the other hand, activation of en is under the control of brachyenteron (byn) that is regulated independently of dpp, brk, and Doc. The cooperative interaction of common DV positional cues with byn during hindgut development represents another aspect of mechanisms of DV patterning in the Drosophila embryo (Hamaguchi, 2012).

A model is presented of the genetic pathway leading to the DV subdivision of the Drosophila hindgut. The dorsal fate of the hindgut is finally determined by a selector gene en that is expressed in the dorsal domain of the hindgut. The present study revealed that DV patterning of the hindgut is based on antagonistic interaction of Dpp and Brk in the early cellular blastoderm. Dpp expression disappears in the hindgut primordium after stage 9, but, Dpp target gene Doc takes over the repressive effect on Brk, and restricts Brk expression to the ventral domain. In other words, primary role of Dpp in the hindgut development is to activate Doc genes in the dorsal domain for repression of Brk. Eventually, Brk represses the selector gene en in the ventral domain, restricting it to the dorsal domain. This is the outline of gene regulatory pathway of DV patterning of the hindgut. It should be noted that Doc represses brk in the dorsal domain, while Brk does not regulate Doc expression in the hindgut in normal development, since brk mutation does not affect Doc expression in the hindgut. Dpp and Doc do not determine the dorsal fate directly, but, act indirectly by repressing brk in the dorsal domain. In fact, brk; DocA (null) double-mutant embryos, as well as brk; dpp double mutant embryos, expressed en in both dorsal and ventral domains of the hindgut. This regulatory interrelation between brk and Dpp/Doc is partially reminiscent of that in the wing discs, in which primary role of Dpp is repression of brk, and the latter is responsible for defining antero-posterior pattern of gene expression in the wing discs. Repression of brk by Dpp signals has been reported to depend on the zinc finger protein Schnurri (Shn) in some tissues. However, brk expression in the hindgut did not expand dorsally in the shn mutant embryo, and also, en expression was observed in the hindgut in shn mutant embryo. Thus, shn may not be essential for regulation of brk in the hindgut (Hamaguchi, 2012).

However, activation of en in the hindgut, is under the control of byn, the process of which is independent of DV patterning. The expression domain of byn is included in the region where intricate interaction of dpp, brk, and Doc proceeds. The dpp, brk, and Doc genes are all under the control of the dorsal system, while byn is activated under the control of the terminal system that provides AP positional cues in terminal regions of the early blastoderm. In other words, AP positional cues activate en, while DV positional cues repress en. Thus, cooperative interaction of the two independent gene regulatory systems establishes the expression pattern of en in the hindgut (Hamaguchi, 2012).

Larval and Pupal

The brinker gradient controls wing growth in Drosophila

The Dpp morphogen gradient controls growth and patterning in the Drosophila appendages. There is recent evidence indicating that the Dpp gradient is converted into an inverse gradient of activity of the gene brinker (brk), which encodes a transcriptional repressor and is negatively regulated by the Dpp pathway. How alterations in the Brk gradient affect the growth of the wing disc has been studied. There is a negative correlation between brk activity and growth of the disc: high levels of brk prevent or reduce growth, whereas loss of brk activity results in excessive growth. This effect is concentration dependent: different amounts of Brk produce distinct rates of growth. Furthermore, these results demonstrate that although brk is able to induce apoptosis where there is a sharp difference in Brk levels, its role as a growth repressor is not achieved by inducing apoptosis but by reducing cell proliferation. Brk appears to downregulate the activity of genes that control cell proliferation, such as bantam (Martín, 2004).

Alterations of brk expression may have two different consequences: (1) Activation of the JNK pathway and (2) Alterations of cell proliferation rate. Activation of the JNK pathway occurs when an alteration of brk expression generates a sharp border of brk activity. This phenomenon was observed both in experiments inducing ectopic brk activity and in others in which brk function is eliminated in clones of cells. The local induction of JNK results in apoptosis that can be visualised by the activation of caspase 3. This local apoptosis induced by Brk is probably the mechanism of cell elimination during cell competition and suggests that brk is involved in the elimination of slow dividing cells or of cells that are not able to read or interpret efficiently the Dpp pathway. This function may be aimed to keep the general fitness of the cell population. However, it does not appear to be involved in growth control, because apoptosis inhibition (by means of puc or p35 overexpression) does not eliminate the effect on size caused by Brk (Martín, 2004).

Previous work has shown that loss of brk activity results in increased growth: in mutant brk discs there is an enlargement of the lateral region, and cells mutant for brk produce outgrowths. The cause for the additional growth associated with the loss or reduction of brk activity is due to an increase in the cell proliferation rate: brk- clones incorporate BrdU more actively than surrounding cells. Conversely, the repression of growth caused by elevated levels of Brk is associated with reduced mitotic activity and BrdU incorporation (Martín, 2004).

Given the nature of the Brk protein, it would be expected that its role in growth be mediated by transcriptional repression of genes involved in cell division and proliferation. The results indicate that it acts as a repressor of bantam, although this control may not be direct. Given that Bantam protein is itself a post-transcriptional regulator of cell division genes, this observation suggests that Brk occupies a high position in the genetic hierarchy controlling cell proliferation. Its activity links Dpp signalling and cell proliferation (Martín, 2004).

The Dpp pathway is known to be involved in the control of growth of the wing (and of other appendages). The activity of the Dpp pathway has a positive effect on growth, and, furthermore, the growth response of the disc correlates with its levels of activity. This graded response is of interest, since it suggests that growth control mechanisms recognise different concentrations of inducing or repressing factors. This result has implications in the understanding of these mechanisms; classically, it has been argued that proliferation in the imaginal discs is a response to confrontation of cells with different positional values. The current results in the wing disc do not support this view, since they suggest that growth is a lineal response to Dpp/Brk activity (Martín, 2004).

The current results also indicate that the role of Dpp on growth is mediated by brk. The simplest view is that as the Dpp gradient is converted into an inverse Brk gradient, the concentration-dependent stimulus of Dpp on growth should be converted into a concentration-dependent repression by Brk. The demonstration that the effect of Brk on wing size depends on the amount of protein supports this view (Martín, 2004).

There are several arguments that implicate brk as a principal factor controlling growth. (1) Loss of brk activity leads to increased proliferation. This is consistent with previous observations showing that brk wing discs are bigger than wild-type discs. Furthermore, this excessive proliferation can occur in absence of Dpp activity. (2) Increased or ectopic brk levels block or reduce growth, even though brk does not alter dpp expression. (3) The stimulation caused by the Dpp pathway on growth requires repression of brk. This is demonstrated by the finding that the presence of Brk protein suppresses the excessive growth caused by Dpp hyperactivity (Martín, 2004).

Together, these observations indicate that growth does not require direct input from Dpp, but simply its repression of brk. However, the repression of brk by Dpp is an important developmental phenomenon because in the absence of such control brk would become constitutively active, thus repressing all or the majority of Dpp targets. Two control elements have been identified in the brk regulatory region: a Dpp-regulated silencer that contains binding sites for the Mad/Medea complex; and a constitutive enhancer. This enhancer is probably responsible of the generalised brk expression in the absence of Dpp activity (Martín, 2004).

What is the role of brk in normal development? The results demonstrate that Brk has the properties of a growth repressor and can perform this function all over the wing. However, in wild-type wing discs, brk is expressed only in the lateral region and therefore its repressing role is limited to this region. This is agreement with the observation that brk- clones overgrow only on the sides of the disc (Martín, 2004).

The restriction of the role of brk to the lateral region is intriguing, because if it were the only repressor it would be expected that the central region, where there is no brk activity, would grow more than the lateral one. The overall growth of the different wing regions is uniform; not only does clone size fail to change in the different wing regions but BrdU incorporation and PH3 staining are also uniform. This suggests that there another factor located in the center of the disc should exist that represses growth in the absence of brk. This hypothetical gene would fulfil in the center of the wing the role that brk performs in the lateral region (Martín, 2004).

In principle, a candidate could be daughters against dpp (dad), a Dpp target that is expressed at high levels in the center of the disc. dad overexpression reduces growth. However, this appears to be achieved by allowing high brk levels subsequent to slackening of Dpp activity, indicating that the effect of dad is mediated by brk. Thus, dad appears to be a Dpp modulator with no direct role in growth. The finding that brk- clones containing high levels of dad activity can overgrow also supports this view (Martín, 2004).

Dpp of posterior origin patterns the proximal region of the wing

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila. The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments. A new site of dpp expression has been found in the posterior wing compartment that appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity that is independent of that originating in the anterior compartment. This posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. It was further shown that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, it is suggested that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs (Foronda, 2009).

This study was triggered by a consistent observation of a small region in the P compartment of the wing disc that appeared to be active in dpp transcription. This P compartment expression of dpp has not been properly analysed in previous works about Dpp function in wing disc. dpp expression was carefully examined in third instar wing and haltere discs by in situ hybridization and also with a P-element insertion (P10638) at the disk region of the dpp gene. Transcriptional activity was identified close to and anterior to the A/P border. In addition, dpp transcripts were found in a proximal region of the posterior compartment. A homologous zone of dpp expression was also found in the posterior region of the haltere disc. This dpp posterior transcriptional domain appears during the third larval period; wing discs from early 3rd larval instar do not show it (Foronda, 2009).

According to the fate map, the posterior region containing dpp expression gives rise to proximal adult wing structures, including the alula and the axillary cord. This domain in adult wings was delimited by X-gal staining freshly emerged flies carrying the dpp-lacZ insertion. The area with lacZ activity corresponds mostly to the axillary cord (Foronda, 2009).

To test whether the posterior dpp expression activates the Dpp transduction pathway, use was made of an antibody raised against the phosphorylated (active) form of Mad, an indicator of Dpp pathway activity. As expected from previous work there are high levels of pMad in the centre of the disc, but in addition a domain of pMad activity was observed in the posterior compartment, which includes the dpp expression domain. The zone expressing pMad is bigger than that expressing dpp, consistent with the formation of a diffusion gradient of Dpp activity (Foronda, 2009).

In addition to pMad levels, whether other elements of the Dpp pathway are expressed in the posterior dpp domain was also examined. The gene daughters against dpp (dad) is a target that requires moderate levels of Dpp signalling. dad is expressed in the posterior wing compartment in the same region where pMad is active. Other Dpp-target genes, like optomotor blind and spalt, are also expressed in this region (Foronda, 2009).

The brinker (brk) gene is a special case as it is negatively regulated by Dpp activity. Therefore, it is expressed in the lateral regions of the wing disc where the levels of Dpp activity are lower. The domain of Dpp activity in the proximal posterior compartment should therefore repress brk activity in that region. The comparison of pMad and brk activities in this region clearly shows they occupy mutually exclusive domains, supporting the idea that brk is repressed by the posterior pMad activity. The staining of adult wings of brk-lacZ genotype also argues in the same direction because the region of brk expression does not coincide but abut with that containing Dpp activity. Although the haltere disc was not analyzed with the same detail, there is also a posterior region of pMad activity in the zone where dpp is expressed (Foronda, 2009).

pMad and brk expression were examined in wing discs from second instar larvae. These exhibit the central domain of pMad activity but there is no detectable staining in the posterior compartment. This result is consistent with the late appearance of the posterior dpp transcription domain and indicates that this domain functions only during the second half of the proliferation phase of the disc (Foronda, 2009).

Having shown that the posterior dpp expression domain acts as a source of morphogen the question was addressed whether it has a functional role. Previous experiments analysing dpp mutant clones did not report significant alterations in the posterior wing. However, given the diffusible nature of the Dpp product it is possible that the lack of Dpp in the mutant clones could be rescued by Dpp emanating from neighbour wildtype cells (Foronda, 2009).

An experiment was designed in which all cells in the posterior compartment would be homozygous for the dppd12 mutation, which eliminates dpp activity in the wing disc without affecting embryonic or pupal expression. In discs of genotype dppd12 ck FRT40A/M (2)24F arm-Z FRT40A; hh-gal4/UAS-Flp the high levels of flipase generated by the hh-gal4 driver would induce FRT-mediated mitotic recombination in virtually all the cells in the posterior compartment. The dpp M+ clones produced will have proliferation advantage and are expected to fill the posterior compartment. They can be identified in the disc because they lose the arm-lacZ transgene, and in the adult wing because they are homozygous for the cuticular marker crinkled (ck) (Foronda, 2009).

These clones entirely fill the posterior compartment. Staining for pMad activity shows the normal pattern in the centre of the disc, but the posterior pMad domain is completely absent, in clear contrast with wildtype discs (Foronda, 2009).

Since the genotype used allows good viability of the flies containing dpp M+ clones, a large number of adult wings was examined. In nearly every case the entire posterior compartment is marked with ck, indicating that it lacks dpp expression. The pattern and size of these wings is normal except in the proximal posterior region: the alula does not form and the pattern that appears in its place resembles that of a more distal region, which is specified by Dpp of anterior origin. The axillary cord is much reduced in size and has lost all its characteristic long hairs; some sclerites are also missing. This loss of structures does not appear to be due to death of proximal cells; no indication of caspase activity was found in this region. Moreover, the addition of the apoptosis inhibitor P35 in the posterior compartment does not modify the phenotype of the dpp M+ compartments (Foronda, 2009).

The favored interpretation of the wing phenotype is that in absence of the organising activity of the posterior Dpp, the proximal region is only patterned by Dpp of anterior provenance. It is interesting to note that even though dpp is not expressed in the alula cells, this structure is affected. This result illustrates the role of the posterior Dpp as an organizer, since it affects patterning in a non-autonomous manner (Foronda, 2009).

One intriguing question was if this posterior Dpp could form these proximal structures without Dpp of anterior origin. A transgenic strain carrying UAS-shmiR-dpp2 construct was used which has been shown to degrade the mRNA of Dpp. The smhiR-dpp over-expression in the anterior Dpp domain (by using ptc-gal4 or dppblnk-gal4) is able to silence efficiently Dpp gene activity, as shown by absence of pMad staining and the extended brk expression domain. Neither the posterior dpp expression nor the formation of alula and axillary cord are affected in both size and morphology. It is concluded that Dpp of posterior origin is necessary and sufficient to pattern these structures (Foronda, 2009).

dpp transcription in the P compartment is an intriguing finding and suggests a novel mode of dpp activation. The normal activation of dpp in the A compartment requires Hh signalling, which is blocked in the P compartment. Alternatively, it was possible that a local diminution of en activity in the proximal region of the P compartment would allow Hh and dpp activation by the standard mechanism. This question was addressed by examining in this region the expression of Cubitus interruptus (ci), a transcription factor that is essential for Hh signalling, and of patched (ptc), a Hh target gene. It was found that neither ci nor ptc are expressed in the posterior. However, there was the possibility that these two genes were expressed at low, undetectable levels. To test this possibility in full, Dpp activity was examined in clones of cells mutant for the smoothened (smo) gene, which would be unable to transduce the Hh signal. The result demonstrates that the posterior expression of dpp does not require Hh signalling and must therefore be activated by a different mechanism (Foronda, 2009).

The approach taken to identify the factor/s behind this posterior Dpp expression was to look for candidate genes or signalling pathways which are expressed in the corresponding place in the posterior compartment. The first one was vein, a ligand of EGFR signalling pathway, which coexpresses with Dpp in late 3rd instar wing disc. vein is the only EGFR ligand required for a proper wing development, so it was a good candidate. The elimination of all posterior vein function has effect neither on posterior Dpp function nor on hinge morphology. Other genes were tested based on expression and/or mutant phenotype in the alula, i.e., homothorax, Zfh2 and empty spiracles, among others. None of them affected Dpp expression (Foronda, 2009).

Another likely candidate was mirror, a member of the Iroquois complex (iro-C), for which a role in alula and axillary cord formation has been described. mirr expression was examined using the mirr-lacZ line and it was found that mirr is expressed in the presumptive alula and axillary cord region (Foronda, 2009).

M+ mirr clones were made to generate posterior compartments that were wholly mirr. They show an adult phenotype more extreme than that of dpp compartments: the alula and the axillary cord are entirely missing. In the discs dpp expression (shown by pMad staining) in the posterior compartment is lost, and consequently brk expression is up-regulated in the presumptive alula region. This result indicates that mirr is necessary for posterior dpp expression. In contrast, Dpp is not required for mirr expression, since the lack of posterior Dpp does not have an effect on mirr-LacZ transcription (Foronda, 2009).

Since the preceding results might suggest a mirr-mediated dpp activation gain of function clones of mirr were generated and whether they gave rise to ectopic Dpp activity was checked. NI significant up-regulation of dpp associated with those clones was detected. These experiments demonstrate that mirr activity is necessary for posterior dpp expression, but it is not sufficient to induce it. Therefore there must be other factors involved in posterior dpp activation (Foronda, 2009).

These results report a novel organizer role of Dpp that occurs during the third instar and is necessary and sufficient to pattern the proximal posterior region of the wing. This is achieved by an hh-independent, mirr-dependent activation of dpp in the posterior compartment (Foronda, 2009).

These findings provide a more complete picture of the development of the wing disc. There is evidence that the three signalling Dpp, Wg and Hh pathways are necessary for normal wing pattern and originate at compartment borders. The results indicate an unforeseen complexity of the function of the Dpp pathway. dpp becomes active in two different body domains, which have independent temporal and spatial regulation. The anterior domain is required for distal wing growth and patterning, whereas the P compartment domain is responsible for the formation of the posterior-proximal structures of the wing. Moreover, the source of Dpp in the posterior domain does not appear to be a compartment border, since no lineage restriction has been reported in the region. A comparable situation has been described for leg development, in which the Dpp and Wg signals originate at the A/P border, but the source of the EFGR signal is not a lineage border (Foronda, 2009).

The fact that this new dpp expression domain is common to wings and halteres may have some evolutionary significance, since this may be an attribute of dorsal thoracic appendages. These results suggest that the posterior tier of dpp expression may have appeared before the mesothoracic and metathoracic appendages diverged. It is therefore, possible that the new model of dorsal appendage development proposed may occur in other species of insects (Foronda, 2009).

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. This study asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, it was found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. It was also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone (Pent: see Flybase Magu), an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth (Hararatoglu, 2011).

This study measured Dpp pathway activity using an antibody specific to the phosphorylated form of Mad, and compared the P-Mad levels in space and time with the activity levels of direct target genes, such as brk, which plays key roles in both growth and patterning. Transcription of brk is directly repressed via P-Mad binding at defined silencer elements (SEs), resulting in inversely graded brk expression. Brk is the only known regulator affecting the positioning of the expression boundary of omb, while sal and dad translate input from both P-Mad and Brk into their expression boundaries. The dynamics of all of these readouts were analysed using antibodies where possible, to avoid potential misinterpretations due to reporter stability (Hararatoglu, 2011).

P-Mad levels scale very well posterior to 0.4 Lp (Lp stands for the length of the posterior compartment) with the exception of TC5 profiles near the D/V boundary. Previous studies that examined P-Mad scaling reached contradictory conclusions: the P-Mad gradients in late stage discs were reported to correlate with tissue size in a previous study and to have no correlation in another. Similarly, examination of P-Mad gradients across discs of different sizes led to the conclusion that the gradient did not expand, but in another study the P-Mad gradient was shown to scale with tissue size. It is believed that most of the confusion can be attributed to different profile extraction protocols as well as to the use of various definitions of scaling, as discussed below (Hararatoglu, 2011).

Since P-Mad is an early signature of the activation of the Dpp signaling pathway, it was of interest to find out how its scaling properties translate to its immediate key target, the brk gene. In addition to brk being directly repressed by P-Mad, the Brk protein itself is necessary for graded brk transcription. The range of Brk expression was found to strictly follow P-Mad in both wild-type and pent mutant discs. Similar to what was observe for P-Mad, Brk also shows very good scaling for positions posterior to 0.4 Lp. By contrast, levels of Brk increase steadily as the discs grow and cannot be explained by P-Mad dynamics alone. This increase in Brk levels could be due to the build-up of the unknown activator of brk transcription or, alternatively, the SEs in brk could become desensitized to the repressive input of P-Mad. Regardless of the cause of the increase, cells at a given relative position experience increasing levels of the Brk repressor over time (Hararatoglu, 2011).

Traditionally, Dpp and P-Mad gradients have been described by a decaying exponential with characteristic decay length λ. This decay length is different for each profile and corresponds to the position at which the protein levels have decreased by a factor e. The correlation between the decay length and the tissue size has been used as a proxy for scaling, e.g. one study found no significant scaling for the Dpp and P-Mad profiles at the end of 3rd instar stage. Similarly, the width of the P-Mad profile has been used to characterize the spread of the gradient and it was concluded that the width of the P-Mad profile is constant during growth. In contrast, the current study detected that the P-Mad profile expands as the tissue grows. This discrepancy may be due to the fact the previous study lacked time classes TC3 and TC4 in their sample collection, the period where the P-Mad gradient expands before contracting again when measured close to the D/V boundary. Thus, possibly the measurements of the P-Mad profiles were done in the vicinity of the D/V boundary, where at TC5 P-Mad has a sharp profile reminiscent of 30 h younger discs. Hence, the choice of position can significantly alter the final interpretations of the data (Hararatoglu, 2011).

Consistent with these results, another study (Wartlick, 2011) recently showed that the decay lengths of Dpp-GFP, P-Mad, brk-GFP, and dad-RFP do correlate with the length and the area of the posterior compartment during tissue growth. Importantly, the Wartlick study also assessed scaling qualitatively in the whole field by looking at the collapse of the profiles in relative positions and normalized intensities. This method has two advantages: it does not require any fitting of the profiles, and it shows scaling at all positions and not just at the characteristic decay length position (i.e. x = λ) (Hararatoglu, 2011).

A recent mathematical model termed 'expansion-repression integral feedback control' suggests that scaling can emerge as a natural consequence of a feedback loop (BenZvi, 2010). The hypothetical 'expander' molecule facilitates the spread of the morphogen and in turn is repressed by it; scaling is achieved given that the expander is stable and diffusible. The known properties of Pent fit the requirements of this hypothetical agent: Pent is secreted, required for Dpp spreading, and pent transcription is directly inhibited by Dpp signaling. However, it is not known how stable Pent is, and pent transcription is never abolished in the entire field in which the gradient acts during larval stages. To test whether Pent could be a key player involved in scaling of Dpp activity during disc growth, the analyses were repeated at all time points in the absence of Pent. It was found that the P-Mad and Brk gradients indeed fail to scale with the tissue size in this mutant background. Scaling of dad-GFP and Omb are also strongly affected, while Sal still exhibits some degree of Pent-independent scaling in the anterior compartment. Importantly, while the function of Pent is essential for proper scaling of the Dpp activity gradient, it is noted that Pent alone cannot account for the observed selective scaling of Omb and Sal domain boundaries. Scaling of these target genes specifically in those regions in which they have a patterning function points to the involvement of additional players, which will be the subject of future research. Hence, the current findings strongly suggest that Pent is a very good candidate to be the expander in the 'expansion-repression integral feedback control' model and therefore provide the first mechanistic insights into the question of scaling in wing patterning. The exact biochemical functions of Pent have to be determined in order to get a more mechanical view of gradient scaling in the developing wing imaginal disc (Hararatoglu, 2011).

More than 40 years ago, Lewis Wolpert proposed the French flag model to explain pattern formation by morphogens. This study tested whether the activity gradients downstream of Dpp, namely P-Mad and Brk, are read out by their target genes at constant concentration thresholds. Thus, average P-Mad and Brk concentrations were measured at Dad, Omb, and Sal expression boundaries across development. It was found that the amount of P-Mad at these boundaries slightly increased (Dad), slightly decreased (Sal), or was constant throughout development (Omb). Among these three targets, the Omb domain is the widest and it corresponds to a region where the P-Mad gradients scale perfectly; as a result, P-Mad levels fluctuate very little at the Omb domain boundary. Interestingly, the domain boundary of Omb is thought to solely depend on Brk and hence constant P-Mad levels might be a mere coincidence. Remarkably, all the target genes that were considered respond to significantly increasing levels of Brk, suggesting that the target genes desensitize to Brk over time, so that more and more Brk can be tolerated at the domain boundary. Alternatively, if it is considered that the domain boundaries of dad-GFP and Sal do not respond to constant P-Mad levels either, another explanation could be that Brk and P-Mad signals are combined in a non-additive fashion in order to define the boundary position of the target genes. Following this assumption, a simple combination of these signals was sought that is constant at the target gene domain boundary for all TCs. It is proposed that Brk and the unknown activator of Omb could be similarly combined in order to determine the Omb domain boundary (Hararatoglu, 2011).

This paper's data was used to further test a model that was recently proposed to explain the uniform growth in the wing imaginal disc (Wartlick, 2011). The model poses that the temporal changes in Dpp signaling levels drive tissue growth; cells divide when they experience a relative increase of 50% in the levels of Dpp signaling. Since it is the relative differences and not the absolute amount of Dpp signal that regulate cell divisions, the model can account for the uniform growth of the wing disc. Since the relative increase in Dpp activity slows down, the cell cycles lengthen as the disc grows. Growth stops when the cell division time exceeds 30 h. The model of Wartlick is based on the finding that Dpp activity scales with tissue size and that cells at a given relative position experience increasing levels of Dpp signaling over time. In contrast, a general temporal increase was not observed in the level of Dpp signaling at a given relative position in this study. P-Mad is the most upstream and the most dynamic readout available for the activity of the Dpp pathway and it was found that the relative increase in P-Mad levels throughout development is not significantly different from zero at most relative positions. Why is the increase in Dpp-GFP levels not reflected in P-Mad levels? A potential explanation for this might be that the observed accumulation of Dpp-GFP was due to the stability and accumulation of Gal4 since Dpp-GFP was under UAS control. The Wartlick study showed that the half-life of the Dpp-GFP fusion protein is only 20 min, but the Gal4 stability was not considered. Alternatively, the system could get desensitized over time and more and more Dpp would be required to lead to similar P-Mad levels. Finally, increases in Dad levels could counteract the increase in Dpp levels, since Dad is an inhibitory Smad (Hararatoglu, 2011).

Wartlick (2011) monitored Dpp signaling levels using a dad-RFP reporter and found a 5-fold increase in the course of 36 h. In the current analysis, a similar tool, dad-GFP, was used and the Wartlick results could not be fully reproduced. Though it was also found that dad-GFP scales with tissue size, its levels increase merely 2-fold over 40 h, and this increase takes place only in the medial 25% region of the disc, while cells in the lateral part experience a decrease in dad-GFP levels. This disparity in the fold increases is likely due to the higher stability of RFP, since the enhancer used was identical in both studies. Additionally, it was found that the levels of Brk, another direct target of the pathway with a very well-established role in suppressing growth in lateral regions, increase in average 4-fold in the interval studied, an observation not reported by Wartlick. In the lateral areas, increase in Dpp activity (if present) is below detection levels and would be opposed by increasing Brk levels. Importantly, increasing Brk levels, if they were to depend solely on Dpp, would suggest decreasing Dpp activity in lateral areas as Brk expression is directly suppressed by Dpp activity. Hence, the data raise serious questions about the validity of this uniform growth model, especially in the lateral regions of the pouch. An alternative model is favored that does not rely on Dpp activity alone to explain uniform growth in the wing disc (Hararatoglu, 2011).

Effects of Mutation, Deletion or Mis-expression

Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker

In an enhancer trap screen designed to identify genes whose expression may be regulated by Dpp in the imaginal discs, one potential line, X47, was selected. This enhancer trap is located adjacent to the brk gene and its expression is identical to that of brk. In X47, ß-gal is expressed at the anterior and posterior extremes of the wing disc but not in the central region. Dpp is expressed along the compartment border within this central region, suggesting X47 expression may be negatively regulated by Dpp signaling. To test this, ubiquitous expression of Dpp was induced and a complete loss of X47 expression was observed. X47 expression was examined in dpp mutant discs and ubiquitous expression was observed. Mad clones were induced in the developing wing and autonomous activation of X47 expression within these clones was observed, indicating X47 may be a direct target of Dpp signaling. X47 expression is graded along the A-P axis within the wing pouch and overlaps with optomotor blind (omb) but not spalt (sal). Although the outgrowths generated by brkXH mutant clones are autonomous, they are reminiscent of outgrowths generated by misexpression of Dpp. Consequently, the expressions of dpp and Dpp target genes were examined in brk discs and clones. Initially, this was done in brkXA hemizygotes because these survive through larval life and allow the examination of gene expression in whole discs. brkXA mutant wing discs have a dramatic overgrowth phenotype showing expansion of the wing pouch along the A-P axis. The expression domains of sal, omb, and vestigial (vg) are expanded in the enlarged wing pouch. This phenotype is very similar to that produced by ubiquitous expression of Dpp (Campbell, 1999).

Wing discs from the pupal lethal hypomorph brkXA are greatly enlarged along the A-P axis, phenocopying the ubiquitous expression of Dpp. These discs show expanded domains of sal, omb, and vg expression in the expanded wing pouch. Null mutants, including brkXA, are embryonic lethal, but mutant clones can result in outgrowths in adult wings when the clone is located in the anterior or posterior extremes of the wing. These outgrowths are comprised entirely of mutant tissue but are similar to outgrowths produced by misexpression of Dpp. Examination of such clones in wing discs reveals autonomous activation of sal, omb, and vg outside of their normal expression domains. Thus, Brk functions in the developing wing to repress the expression of Dpp targets such as sal, omb, and vg (Campbell, 1999).

The observation that Dpp targets are expressed in brk clones must be reconciled with previous studies that suggest these targets are directly activated by Dpp signaling via Mad. There are at least two explanations for these observations: (1) the conclusions of the previous studies are correct, and brk functions simply to reduce sensitivity to Dpp so that in brk mutants lower levels of Dpp are required to activate target gene expression; this would predict that loss of Dpp signaling in brk clones would prevent target gene expression. (2) Target gene expression does not require a direct positive input from Dpp/Mad, and brk functions to directly repress target gene expression independent of Dpp signaling; this would predict that loss of Dpp signaling in brk clones would not prevent target gene expression. This was tested directly by analyzing sal or omb expression in cells mutant for both brk and dpp, thick veins (tkv), or Mad (Campbell, 1999).

Initially it was shown that expression of dpp in discs containing brkXH clones is normal, demonstrating that any phenotypes associated with these clones do not depend upon a secondary source of dpp. dpp disc-specific mutations, such as the dppd12/d14 combination, result in discs in which growth is markedly reduced and in which Dpp target gene expression is lost. In contrast, the brkXA; dppd12/d14 double mutant wing disc closely resembles the brkXA single mutant disc: there is some overgrowth when compared to wild-type discs, although not as much as the single mutant, and there is ectopic sal expression in the wing pouch. This is strong evidence that Dpp is not required for sal expression in the absence of brk activity. The tkv7 mutant has no signaling activity and results in the loss of expression of all Dpp targets. However, cells mutant for both brk and tkv7 still show omb expression. This demonstrates that omb expression does not require Dpp signaling activity in cells mutant for brk. Single mutant clones of Mad1-2 show loss of sal expression in the wing pouch, but cells mutant for both brk and Mad1-2 express sal in the wing pouch. Mad1-2 is not a null; however, expression of sal in brk single mutant cells is identical to that in brk;Mad1-2 double mutant cells, indicating Mad activity is not required for sal expression in brk clones. These epistasis experiments support the second possibility, that brk functions independently of Dpp signaling to repress Dpp targets and that in the absence of both brk and Dpp activity, Dpp targets are expressed (Campbell, 1999).

Why is the indirect method involving Brk used to activate expression of Dpp targets? In other words, if Dpp can directly activate these genes via Mad, then why is this not sufficient? It is speculated that it is directly related to Dpp acting as a morphogen. Activation of sal, omb, and vg is not simply all or none, but each is induced above a distinct threshold concentration of Dpp, with sal requiring the highest level and vg the lowest. The gradient of Dpp will be transduced into a gradient of activated Mad, but it is possible that cells cannot perceive small differences in activated Mad reliably enough to faithfully define the expression domains of Dpp targets and that the introduction of the Brk intermediary provides the necessary information. This type of dual control of gene expression may turn out to be a common feature of many morphogen systems. The possibility is raised that other TGFßs may also use indirect mechanisms to control expression of target genes, possibly even Brk-related proteins, especially if they also induce multiple targets in a concentration-dependent manner. One relevant observation in this regard is that brk is also expressed in the early embryo where its expression also appears to be regulated by Dpp; null mutant embryos are partially dorsalized, suggesting it has a similar function here as in the wing. Unlike the wing, control of D-V patterning by TGFßs is probably a conserved feature of almost all animal embryos and strengthens the possibility that brk homologs will be typical regulators of TGFß target genes (Campbell, 1999).

The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation

dpp is required for both cell proliferation and A-P patterning in wing imaginal discs. Ectopic expression of dpp leads to outgrowths and pattern duplications in both the anterior and posterior compartments of the wing. These outgrowths and duplications are highly nonautonomous, that is, small patches of dpp-expressing cells have long-range organizing effects on the surrounding tissue, reflecting the fact that Dpp spreads from the local source of its production into neighboring regions. brk mutant clones also cause outgrowths and pattern duplications in anterior and posterior regions of the wing, indicating that removal of brk leads to ectopic activation of the Dpp pathway. However, the outgrowths are entirely cell autonomous, meaning that all cells belonging to the outgrowth are mutant for brk. This suggests that brk is not a negative regulator of dpp expression. Accordingly, dpp expression is not changed in third instar larval discs containing brk clones irrespective of their position within the disc (Jazwinska, 1999a).

The effects of brk mutant clones are similar to those of an activated version of the Dpp receptor Thick veins (Tkv*). Both are cell autonomous, and in both cases outgrowths are often accompanied by notches. This similarity indicates that loss of brk results in locally restricted pathway activation. However, a detailed comparison of the cuticular markers present in outgrowths induced by brk, as compared to those induced by Tkv*, suggests that loss of brk leads to a lower level of pathway activation than Tkv*. Some structural elements of the adult wing, including wing veins and the bristle types along the margin, can be correlated with certain levels of Dpp activity. In the anterior compartment, vein L2 and the triple row bristles of the margin depend on low levels of Dpp, while higher levels are required for double row margin bristles. brk mutant clones close to the A-P compartment boundary in regions of peak levels of Dpp (posterior to vein L2 and anterior to the L4/L5 intervein region) have no phenotypic effects. Outgrowths, venation defects, and notches are observed if brk clones are located in regions more distant from the A-P boundary, corresponding to low Dpp levels. Anterior outgrowths frequently harbor a vein L2 recognized by corrugation at the ventral side of the wing, and their margins carry triple row bristles. Thus, anterior outgrowths are composed of structures dependent on low levels of Dpp. In contrast, outgrowths induced by ectopic Tkv* do not carry a vein L2, and their margins are occupied by an irregular array of double row bristles characteristic of high levels of Dpp activity. This comparison indicates that removal of brk leads to the formation of structures corresponding to low or intermediate Dpp levels (Jazwinska, 1999a).

To investigate the developmental origin of the pattern rearrangements seen in brk mutants, the expression of the Dpp target genes omb and sal were examined in third instar larval imaginal discs that contained brk mutant clones. In wild-type discs, omb and sal are expressed in nested domains centered around the stripe of dpp expression; omb being activated by low levels of Dpp has the broader expression domain, and sal requiring higher levels has the narrower domain. brk clones located in the endogenous omb and sal regions have no visible effect on omb and sal expression, respectively. However, all clones outside the respective domains but within the wing pouch primordium show strong ectopic omb and weak sal expression, both in a strictly cell-autonomous manner. If brk mutant clones start at the endogenous sal domain and extend laterally, sal expression within the clone is often observed to decline continuously with increasing distance from the A-P compartment boundary. In contrast, patches of cells expressing Tkv* always showed high levels of sal expression. This again indicates that loss of brk does not result in maximal activation of the Dpp pathway. In summary, loss of brk in the wing disc leads to an activation of target genes dependent on low or intermediate levels of Dpp signaling and to an expansion of corresponding fates. However, fates depending on highest Dpp levels are not affected. Thus, brk function is most important in regions where the Dpp gradient has diminishing levels or where a further spreading of Dpp signaling has to be prevented (Jazwinska, 1999a).

The cell autonomy of brk clones suggests that brk acts as an intracellular negative regulator of the Dpp pathway. As such, brk could be a negative modulator of signaling strength. In that case, the brk phenotype would result from amplification of residual levels of pathway activity. Alternatively, brk might act completely independently of pathway activation. In this case, the phenotype would be expressed even in the absence of pathway components. Furthermore, brk could act as an antagonist either at the receptor level, at the level of the SMAD proteins, or directly at the level of target gene promoters. To genetically address these questions, double mutants of brk with mutations in Dpp pathway components were constructed and their effects on the expression of the Dpp targets omb and sal were examined (Jazwinska, 1999a).

To test whether ectopic expression of omb and sal in brk clones requires Dpp ligands, a trans-heterozygous combination of two hypomorphic dpp alleles was used that leads to rudimentary wings and elimination of omb expression in the wing blade primordium. Induction of brk clones in this background leads to omb and sal expression and to small outgrowths composed of wing blade material, demonstrating that target gene activation in brk mutant cells occurs in the absence of normal Dpp levels (Jazwinska, 1999a).

tkv and Mad have been demonstrated to be required in a cell-autonomous manner for the expression of omb and sal. To test whether this requirement is maintained in a brk mutant background, double mutant clones were induced. Both brk;tkv and brk;Mad double mutant clones express high levels of omb and low levels of sal, similar to expression observed in brk single mutant clones. In the case of sal, the expression level in double mutant clones is low even if the clone is located in the endogenous sal domain. Thus, removal of brk leads only to a certain level of sal expression, and the higher levels normally seen in the sal domain must reflect some additional signaling input from the Dpp pathway. These data indicate that in the absence of brk, neither tkv nor Mad are required for omb and low-level sal expression. Since for both tkv and Mad null alleles (tkva12 were used, it is concluded that removal of brk leads to Dpp target gene activation by a mechanism independent of Dpp pathway activity. The fact that brk acts downstream or in parallel to Mad suggests that brk itself may act at the transcriptional level. Target gene activation by Dpp would then be accomplished by inhibiting brk's repressor function (Jazwinska, 1999a).

brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes

To investigate the role of brk, mutant clones were analyzed in wings. brk mutant clones cause outgrowth of wing tissue, a phenotype that is similar but not identical to the phenotype caused by ectopic dpp expression. Both brk and dpp cause outgrowths, but the consequences of ectopic dpp expression is non-autonomous, whereas the effect of brk is autonomous in the mutant cells. This was confirmed by examining the expression of the Dpp-target genes omb, sal and dad in brk-mutant clones. All three Dpp target genes are expressed autonously in mutant clones located within the normal domain of brk expression. The normal domain of sal expression is narrower than that of omb or dad and is nested in the domain where brk expression is very low; brk-mutant cells adjacent to the normal domains of sal expression ectopically upregulate sal expression, indicating that the brk expression gradient is functional -- it represses omb expression at high levels and sal expression at low levels. Clones within the domain of high brk expression are larger than those elsewhere, probably because of the overexpression of Dpp targets in these cells. The patterns of brk-lacZ are normal in mutant clones, not detectable in clones near the A/P compartment border, and high in peripheral clones, suggesting that brk expression is not regulated by Brk itself. Expression of brk in Xenopus embryos indicates that brk can also repress the targets of BMP-4, the vertebrate homolog of Dpp. The evolutionalry conservation of Brk function underscores the importance of its negative role in proportioning Dpp activity (Minami, 1999).

Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification

Salivary glands are simple-structured organs that can serve as a model system in the study of organogenesis. Following a large EMS mutagenesis a number of genes required for normal salivary gland development have been identified. Mutations in the locus small salivary glands-1 (ssg-1) lead to a drastic reduction in the size of the salivary glands. The gene ssg-1 was cloned and subsequent sequence and genetic analysis shows identity to brinker. The salivary gland placode in brinker mutants appears reduced along both the anterior-posterior and dorso-ventral axis. Analysis of the brinker cuticle phenotype reveals a similar loss of anterior-posterior as well as lateral cell fates. The abdominal ventral denticle belts show a reduced number of setae in the first denticle row. Furthermore, a preferential loss of lateral neuroblasts has been observed in the anterior parasegment. Together, these phenotypes suggest that brinker not only plays a role in dorso-ventral but also in anterior-posterior axis patterning (Lammel, 2000).

The function of Dpp is required to restrict the dorsal extent of the developing salivary gland placode, and in dpp mutants enlarged salivary gland placodes are found. Similarly, the Dpp receptor encoded by punt is required to restrict the dorsal boundary of the salivary gland placode. Mutations in brinker lead to a phenotype opposite that of dpp or punt. The salivary gland placodes of brinker mutant embryos are dorsally and anteriorly restricted and as a result smaller salivary glands are formed. The placode reduction in the D-V axis could be expected, since brk is known to repress Dpp activity. The smaller extension of the placodes along the A-P axis was unexpected and is either a consequence of unrepressed Dpp activity in brk mutant embryos, or may be explained by a function of brk in specification of the A-P axis. In sog mutant embryos with four copies of dpp, the salivary gland placodes are reduced in their dorsal expansion but their size in the A-P direction is unaffected. The dpp expression expands ventrally to a similar degree in sog mutant embryos with four copies of dpp as in brk mutants. Thus, the difference in the placode phenotype is unlikely to be a result only of altered Dpp signaling (Lammel, 2000).

brinker expression is initially found in the salivary gland placode but during the invagination, this brinker expression declines in the presumptive salivary gland cells. Thus, brinker function might be required to repress dpp target gene expression only in the placode stage. In line with this notion, an expansion of the salivary gland placode was observed following ubiquitous expression of brinker using the GAL4 system. In this experiment dpp function is reduced in the entire ectoderm, which subsequently leads to a dorsal expansion of the salivary gland placode. Analysis of the cuticle phenotype shows that the function of dpp as a dorsally located morphogen is generally affected in these embryos. The finding that ubiquitous expression of brk does not result in expansion of the placodes along the A-P axis is due to the restricted expression domain of the gene Sex combs reduced (Scr). Scr is not expressed anterior to parasegment 2 and the gland fate is suppressed in the more posterior parasegments by teashirt (tsh) and Abdominal-B (Abd-B) genes. Thus, brk has no instructive function in gland development but is necessary for positioning the anlage along the D-V axis (Lammel, 2000).

The A-P patterning defect in salivary gland placodes of brk mutant embryos suggests that an analysis of the larval cuticle for a similar phenotype would be of interest. In wild-type embryos, the anterior rows of setae have a smaller dorso-lateral extent than the posterior rows of setae, resulting in the characteristic trapezoid shape of the abdominal denticle belts in segments A2-A7. In brk mutant larvae, the trapezoid form of the abdominal denticle belts is lost and denticle belts appear slightly narrowed in their D-V extent when compared to wild-type. The anterior row of setae is preferentially missing and a few single scattered setae are formed instead. The loss of setae in the first abdominal denticle rows is more pronounced in the anterior abdominal segments than in the more posterior segments. To exclude the possibility that the loss of setae of the anterior denticle rows in abdominal segments is a general feature of mutations in genes functioning as antagonists of DPP signaling in the ventrolateral body region, an examination was made as to whether comparable defects appear in sog mutant larvae. The anterior rows of abdominal denticle belts are not found in sog mutant larvae. Thus, the reduction of the anterior denticle rows in the abdominal segments is a feature typical of brk. In addition to the lateralization observed in brk mutants, an expansion of naked cuticle is found, suggesting that brk may affect more than one signaling cascade (Lammel, 2000).

Further evidence that brk mutants are additionally affected along the A-P axis comes from analysis of the CNS phenotype. In the CNS of brk mutant embryos the connectives are reduced and are often interrupted. This phenotype led to the identification of an EMS-induced allele of brk in a screen for mutants with CNS defects. In wild-type embryos, neuroblasts (NBs) delaminate from the neuroectoderm in a characteristic spatial and temporal pattern and express a specific combination of molecular markers. Delamination of neuroblasts occurs in five waves (S1-S5) between late stage 8 and late stage 11 of embryonic development to form a stereotypical array of seven antero-posterior rows and three columns along the D-V axis: medial, intermediate and lateral. brk null mutants have a severely reduced number of neuroblasts in the outer two rows, corresponding to the lateral and intermediate rows of neuroblasts. The phenotype has been analyzed in greater detail using markers that enable the identification of specific neuroblasts. With respect to the D-V axis, as expected, many of the late delaminating lateral and intermediate NBs, but not the medial NBs, are reduced in number in this allele. For example, NB2-4, a neuroblast that delaminates in the intermediate row at stage 11, is the only neuroblast that expresses both Eagle and Pox-neuro. Double labelling with anti-Poxn and anti-Eagle antibodies shows that NB2-4 is often missing in brk. Evenskipped (Eve) is expressed in the progeny of several neuroblasts in wild-type, namely EL neurons, RP2 (NB4-2 progeny), CQ neurons (NB7-1 progeny) and aCC/pCC neurons (NB1-1 progeny). Of these, NB1-1 and NB7-1 delaminate medially in S1; NB4-2 and NB3-3 delaminate later and more laterally. Labelling of brk mutant embryos with anti-Eve antibody reveals that the progeny of these intermediate and lateral neuroblasts are missing in some hemisegments, whereas progeny from the medial NBs (NB1-1 and NB7-1) are unaffected. Using markers for Eagle and Inv expression shows that only NB6-4 and NB7-3 coexpress both genes in wild-type. Staining of brk embryos for Eagle and Inv expression at the same stage reveals some reduction in NB6-4 and a strong loss of NB7-3. However, in addition to this loss of specific neuroblasts along the D-V axis, brk mutants also show a differential loss of NBs along the A-P axis: when brk mutant embryos are analyzed at SI for expression of the general neuroblast marker, Hunchback, there is drastic loss of the most posterior neuroblast in the lateral row (NB7-4). In contrast, the other lateral NBs are relatively unaffected. Their presence has been confirmed with more specific markers (e.g. NB3-5, Empty spiracles marker; NB5-6, Ladybird early marker). Similar results were seen with brk null mutants. Interestingly, NB7-4 originates in the Invected stripe and it is these cells that give rise to the anterior setae in the larval cuticle. Therefore, as with the salivary gland placodes and the larval cuticle, loss of brk also affects patterning along the D-V and A-P axes in the neuroectoderm (Lammel, 2000).

The role of TGFß signaling in the formation of the dorsal nervous system is conserved between Drosophila and chordates

Transforming growth factor ß signaling mediated by Decapentaplegic and Screw is known to be involved in defining the border of the ventral neurogenic region in the fruitfly. A second phase of Decapentaplegic signaling occurs in a broad dorsal ectodermal region. The dorsolateral peripheral nervous system forms within the region where this second phase of signaling occurs. Decapentaplegic activity is required for development of many of the dorsal and lateral peripheral nervous system neurons. Double mutant analysis of the Decapentaplegic signaling mediator Schnurri and the inhibitor Brinker indicates that formation of these neurons requires Decapentaplegic signaling, and their absence in the mutant is mediated by a counteracting repression by Brinker. Interestingly, the ventral peripheral neurons that form outside the Decapentaplegic signaling domain depend on Brinker to develop. The role of Decapentaplegic signaling on dorsal and lateral peripheral neurons is strikingly similar to the known role of Transforming growth factor ß signaling in specifying dorsal cell fates of the lateral (later dorsal) nervous system in chordates (Halocythia, zebrafish, Xenopus, chicken and mouse). It points to an evolutionarily conserved mechanism specifying dorsal cell fates in the nervous system of both protostomes and deuterostomes (Rusten, 2002).

The embryonic abdominal (A) PNS of Drosophila consists of three bilateral clusters of neurons (ventral, lateral and dorsal) per segment, which can be most especially appreciated in the serially homologous segments A1-A7. In order to investigate whether the second phase of Dpp signaling is necessary for patterning the PNS, mutant alleles for a gene involved in the Dpp signaling pathway, schnurri (shn), were examined. This gene encodes a zinc-finger transcription factor that is necessary for the transcription of some Dpp target genes and binds directly to the main Dpp mediator Mothers against Dpp (Mad). Unlike the zygotic mutants of dpp, scw, tolloid (tld) or mad, shn mutants have no effect on the initial dpp/scw governed dorsoventral patterning of the blastoderm. They express normally the early Dpp target genes, such as pannier (pnr, stage 7), dpp itself in the dorsal ectoderm (stage 9) and Krüppel (Kr) (which is a marker for the amnioserosa), showing that the dorsal ectoderm is correctly specified. By contrast, several Dpp target genes that are expressed following the second phase of Dpp signaling are affected in shn zygotic mutants: at stage 11, the expression of genes responsive to Dpp signaling, such as dad, pnr, spalt or dpp itself is reduced or lost. Thus, any failures in PNS formation, which are observed in shn mutant embryos, must originate from the second rather than the first phase of Dpp signaling and are likely to be mediated by Shn. PNS malformations were sought in strong shn zygotic mutant embryos using the ubiquitous PNS neuronal marker 22C10. Homozygous shn1 and shnk00401 fail to undergo dorsal closure and show severe defects of PNS development. A strong reduction in number of neurons is observed, especially in the dorsal and lateral PNS clusters, although it is difficult to determine exactly which neurons are affected because of the dorsal closure failure. Therefore, another allele, shnk04412, which does undergo dorsal closure, was also examined. In these embryos, position and identity of PNS neurons could be more clearly assigned. In homozygosity, as well as in transheterozygosity over shn1, this mutant shows a reduction in the number of dorsal and lateral neurons, similar to the other mutants analyzed. These results are consistent with a role for Shn-mediated Dpp signaling in the formation of the dorsal and lateral PNS (Rusten, 2002).

A different way to interfere with the second phase of Dpp signaling is to express specific inhibitors once the initial dorsoventral patterning is accomplished. Brk is a nuclear protein that negatively regulates Dpp-induced genes and is expressed ventrally in a complementary pattern to Dpp in the embryo. Sog is a secreted protein that can bind to Dpp and inhibit it from signaling, and Supersog (Ssog) is a hyperactive inhibitory fragment of Sog. In order to avoid interference with the first wave of Dpp signaling (stage 5 to 7, 2.10-3.10 hours AEL), brk and ssog were misexpressed from stage 8 (3.10 hours AEL) to interfere with the second phase (stage 9 to 10/11, 3.40-5.20 hours AEL). UAS-brk expression in segments T2-A3, which is driven by the Kr-Gal4 driver, and ubiquitous expression of ssog in the entire embryo, produced using a HS-ssog construct, leads to reduced number of neurons in the dorsal and lateral PNS. The effects are less severe for Ssog misexpression than for UAS-brk misexpression and notable for shn mutations. Approximately 20% of the embryos expressing ubiquitous ssog do not undergo dorsal closure, similar to the phenotype observed when strong alleles of shn are analyzed. The HS-ssog produces a manifest decrease in phosphorylated-Mad (p-Mad) in the dorsal region. This indicates a reduction in Dpp signaling responsible for the phenotype. The residual p-Mad staining observed in some embryos might be the reason why Ssog misexpression leads to less severe effects than UAS-brk misexpression or shn mutations (Rusten, 2002).

In all these mutant backgrounds the dorsal and lateral PNS clusters show a severe reduction in the number of neurons. No major differences are found depending on the neuronal type: the percentage of external sensory organ neurons lost is similar to the loss of neurons in the chordotonal organs. The penetrance of this effect, as measured in the differentiated PNS clusters, varies among abdominal segments. The average reduction in neuronal number ranges from 25% (HS-ssog) to 41% (shnk00401) in the dorsal cluster and 8% (HS-ssog) to 52% (shnk00401) in the lateral cluster. By contrast, the ventral cluster is less affected because it shows 2% (HS-ssog) to 18% reduction (shnk00401). The lateral pentascolopodial organ shows migration defects in these embryos, but the other sensory organs are located in their expected relative positions (Rusten, 2002).

The reduced number of neurons observed in the dorsal and lateral PNS when Dpp signaling is impeded could result from lack of proneural gene expression, which is known to be necessary for PNC and SOP formation. The expression of ato and ac was analyzed to examine the specification of progenitor cell subclasses in mutant backgrounds defective for Dpp signaling. The development of the serially homologous abdominal segments A1 to A7 is similar and very synchronous. Thus, in the wild type, whenever a specific number of PNCs and SOPs appear in one abdominal segment, a similar pattern is observed in the other abdominal segments as well. This is not true for shnk04412 mutants and for embryos expressing ubiquitous ssog, where the numbers of Ac and Ato positive SOPs and PNCs vary among the abdominal segments. This is consistent with the variably penetrant phenotypes observed in differentiated PNS among abdominal segments. In embryos expressing Kr-Gal4;UAS-brk, loss of Ato- and Ac-positive PNCs and SOPs was observed specifically in the abdominal segments A1-A3 where brk was misexpressed, when compared with abdominal segments A4-A7 that served as an internal reference. The reduced numbers of Ato- and Ac-positive neuronal progenitors appear to result from failure of PNC formation rather than an increase in cell death ratio: apoptosis does not appear to increase in segments expressing brk compared with the other abdominal segments. Taken together, these results suggest that reduction in the number of neurons is produced by failure in proneural gene expression (Rusten, 2002).

brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).

The ab gene, which encodes a zinc finger protein containing a BTB/POZ domain, is required for L5 development as revealed by viable alleles such as ab1, which bypass the early embryonic requirement for this gene in motor neuron axon guidance and result in distal truncation of the L5 vein (Hu, 1995). Four additional viable ab alleles have been recovered in a genome-wide screen for new wing vein mutants, one of which results in a somewhat stronger phenotype in which the L5 vein is consistently truncated proximal to the posterior cross-vein. Expression of ab in the wing disc was examined; it is expressed as a single stripe in the posterior compartment. The viable ab1 allele is likely to be a regulatory mutation, since ab expression is greatly reduced in ab1 mutant wing discs. ab expression is similarly reduced or undetectable in the other four independently isolated viable ab alleles. Double-label experiments with the vein marker Delta (Dl), which is expressed in L1 and L3-L5, reveal that ab is co-expressed with Dl in the L5 primordium (Cook, 2004).

Extension of a previous analysis of ab in initiating L5 development (Biehs, 1998; Sturtevant and Bier, 1995) has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in ab1 mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).

Whether restricted expression of ab in small clones is sufficient to induce vein development was also investigated. The flip-out misexpression system was used to generate clones of cells ectopically expressing ab in the wing disc; these cells (identified by Ab or ß-Gal expression) ectopically express the vein marker Dl and downregulate expression of the intervein marker Bs in a cell-autonomous fashion when located anywhere within the wing pouch. Adult wings containing small ab-expressing clones marked with forked also produce ectopic vein material cell autonomously. These results demonstrate that ab is necessary to control known gene expression in the L5 primordium, and is sufficient to induce vein development when expressed in a restricted number of cells. These data are consistent with ab acting in a vein-organizing capacity to direct L5 development (Cook, 2004).

The L2 primordium forms along the anterior boundary of the sal expression domain, in cells expressing low levels of sal and facing those expressing high levels of sal. The symmetrical disposition of the L2 and L5 veins, and the positioning of both of these veins by Dpp rather than Hh signaling, suggests that the L5 vein might form along the posterior border of the sal expression domain in much the same way that L2 is induced along its anterior border. However, two lines of evidence indicate that sal is not likely to be directly involved in determining the position of L5. (1) The posterior border of the sal expression domain is located several cells anterior to the L5 primordium (Sturtevant, 1997). (2) Although salm- clones do occasionally result in the formation of ectopic posterior veins, they do so non-autonomously at a distance of several cell diameters from the clone border (Sturtevant, 1997). This phenotype is entirely different from the ectopic L2 veins that form at high penetrance immediately within the borders of anterior sal- clones, located between the L2 and L3 veins (Sturtevant, 1997). Clones of a deficiency removing both salm and the related salr gene also result in the production of an ectopic vein, but this vein forms within the interior of such clones between L4 and L5, in a position corresponding to a cryptic vein, or paravein, which has a latent tendency to form along the posterior border of the sal domain (Cook, 2004).

Since the L5 primordium forms approximately four to six cell diameters posterior to the sal expression domain (Sturtevant, 1997), the expression was examined of other BMP target genes, omb and brk, relative to the L5 primordium. The borders of these gene expression domains are known to form posterior to that of the sal domain. Previous studies revealed that the domains of cells expressing high levels of omb and brk are largely reciprocal, although these genes are co-expressed at lower levels in cells along the border. Therefore the relative positions of the border of high level omb/brk expression was determined with respect to vein primordia marked by Dl (L1, and L3-L5) and Kni (L2). These experiments revealed that the L5 stripe of Dl expression forms inside and along the posterior border of the domain expressing high levels of omb, whereas the anterior border of the omb domain extends well beyond the L2 primordium. A complementary pattern was observed in wing discs of brk-lacZ flies double stained for ß-Gal and Dl, in which the L5 Dl stripe runs outside and along the border of the high level brk expression domain. Similar results were obtained using ab as a marker for the L5 primordium, in which the stripe of ab-expressing cells was found to lie within the omb domain, adjacent to high level brk-expressing cells. These expression studies reveal that omb and brk are expressed in the right location to play a role in positioning the L5 primordium (Cook, 2004).

As a first step in determining whether omb or brk play a role in L5 development, genetic interactions between these genes and ab were tested. Several viable or lethal ab alleles were crossed to stocks carrying the brkm68 allele or a deficiency of brk, and trans-heterozygous brk-/+;ab-/+ F1 flies were examined for L5 phenotypes. None of the combinations of brk and ab alleles tested resulted in any dominant vein-loss phenotype in trans-heterozygotes. In addition, no enhancement of the homozygous ab1/ab1 L5 truncation phenotype was observed in brk-/+; ab1/ab1 flies. By contrast, when trans-heterozygous interactions between ab and omb alleles were tested, consistent genetic interactions were observed. For example, omb1/+; ab1/+ flies exhibit truncations in the distal portion of L5 (with 3% penetrance, whereas neither ab1/+ nor omb1/+ heterozygotes ever show any L5 phenotype. Moreover, the omb1 allele, which causes notching of the wing margin when homozygous but has no associated L5 phenotype, strongly enhances the ab1/ab1 L5 truncation phenotype. This interaction is evident in omb1/+; ab1/ab1 females, and is very pronounced in omb1/omb1;ab1/ab1 double homozygous females or hemizygous omb1/Y; ab1/ab1 males. These results suggest that omb and ab function in concert to promote L5 formation (Cook, 2004).

Additional detailed experiments have shown that (1) misexpression of omb and brk shifts or eliminates the L5 and L2 veins; (2) omb is required cell autonomously for L5 development; (3) brk is required for the production of an L5 inductive signal, and (4) ab acts downstream of brk in L5 development (Cook, 2004).

Thus, this study examined the role of two Dpp target genes, brk, which is expressed in a domain abutting the L5 primordium, and omb, which is expressed in a domain just including the L5 primordium, in establishing the position of this vein. The results suggest a model for how the BMP activity gradient induces formation of the L5 primordium in the posterior compartment of the wing. According to this model, L5 development is initiated within the posterior region of the wing where brk and omb are expressed in adjacent domains with a sharp border between them. Since brk- clones induce vein development within the clone along the border with brk+-neighboring cells, it is suggested that brk-expressing cells produce a short-range vein-inductive signal, Y, to which they cannot respond. This signal acts on neighboring omb-expressing cells to initiate vein development. The additional cell-autonomous requirement for Omb activity to respond to this Brk-derived signal suggests that the intracellular effector of the vein inductive signal Y must act in combination with Omb to induce vein formation. Because Brk is a repressor of omb expression, the combined requirement for the short-range Brk-derived vein-inductive signal and Omb activity within responding cells constrains L5 initiation to omb-expressing cells adjacent to brk-expressing cells. In this scheme, Brk plays at least two distinct roles in L5 induction. First, as a repressor of omb, Brk defines the border between the brk and omb expression domains, and, second, brk-expressing cells are the source of a vein-inductive signal required to initiate L5 development within adjacent omb-expressing cells (Cook, 2004).

A key mediator of L5 induction is the Ab transcription factor, which is expressed in a narrow stripe along the brk/omb border, just within the omb expression domain. ab is required for expression of all known vein genes and for downregulation of intervein genes in the L5 primordium (Biehs, 1998). Similarly, the ability of brk- clones to induce an ectopic posterior vein depends on ab function. In addition, localized misexpression of ab in small flip-out clones leads to induction of vein markers in wing imaginal discs and to the formation of ectopic patches of vein material. The vein-organizing activity of ab depends on its being expressed in a localized pattern, since ubiquitous expression of ab suppresses vein development throughout the wing disc. This effect of ubiquitous ab misexpression is similar to that observed previously for ubiquitous expression of kni or knrl, in which all distinctions between vein and intervein regions are lost although expression of other genes in the wing disc are not perturbed. One explanation for this vein-erasing phenotype is that kni/knrl and ab control the expression of a lateral inhibitory signal. Consistent with this possibility, small ab flip-out clones autonomously express the lateral inhibitory signal Dl. According to the model, establishment of the L5 primordium requires input from both omb (cell autonomous) and brk (cell non-autonomous), which collaborate to initiate ab expression in a narrow stripe along their borders (Cook, 2004).

A curious phenotype associated with some brk- clones generated in an ab1/ab1 background is the formation of diffuse wandering veins within the interior of the clone. A similar disorganized ectopic vein phenotype is also observed in a fraction of omb- brk- double mutant clones. This phenotype may reflect the lack of a lateral inhibitory factor (e.g. Dl) produced by ab-expressing cells to suppress vein formation in neighboring cells. The observation that ubiquitous expression of ab suppresses vein formation throughout the wing disc is consistent with this possibility. It is also possible that omb plays a role in promoting intervein development as well as in activating ab expression. Additional analysis will be needed to address this question (Cook, 2004).

Previous analysis of L2 initiation lead to a model in which sal-expressing cells produce a short-range vein-inductive signal (X) to which they cannot respond (Sturtevant, 1997). In response to signal X, neighboring cells outside of the sal domain express the L2 vein-organizing genes kni and knrl. In addition, analysis of an L2-specific cis-regulatory element of the kni/knrl locus provided indirect evidence for negative regulation by a repressor, possibly Brk, expressed in peripheral/lateral regions of the wing disc (Cook, 2004).

An interesting question regarding veins forming within more anteriorly located brk- clones is whether they have an L2- or an L5-like identity. In one case, these veins express kni, but not Dl, suggesting that they have an L2-like identity. In the other, the ectopic veins induced anteriorly by brk- clones require omb function, as do L5-like veins generated in the posterior compartment of the wing. This latter observation suggests that the brk- border in anterior regions acts as it does in posterior regions of the wing disc, but that its effect may be mediated by the L2 organizing kni/knrl locus rather than the L5 organizing gene ab. This hypothesis might provide an explanation for why ectopic veins that form in various mutant backgrounds tend to form along a line running between the L2 vein and the margin (which is referred to as the P2 paravein) (Sturtevant, 1997). This sub-threshold vein promoting position may be defined by the anterior border of brk and omb expression. Further analysis of the identity of these ectopic veins will be required to resolve this question (Cook, 2004).

Since the L2 and L5 veins form at similar lateral positions within the anterior and posterior compartments of the wing, respectively, it is informative to compare the mechanisms by which positional information is converted into vein initiation programs in these two cases. The positions of these two veins are determined by precise dosage-sensitive responses to BMP signaling emanating from the center of the wing; these responses are mediated by the borders of the broadly expressed, Dpp signaling target genes sal and omb. Brk also plays a role in initiating both L2 and L5 development. In the posterior compartment, Brk leads to the production of a hypothetical vein-promoting signal Y, which has a function and range similar to the putative L2 vein-inducing signal X, produced by sal-expressing cells. It is not clear whether the signals X and Y are the same or different; however, an important difference between L2 and L5 initiation is that only L5 has an additional requirement for omb function. This dual requirement for omb function within the L5 vein primordium and a short-range inductive signal in neighboring brk-expressing cells provides a stringent constraint on where the L5 primordium forms. Brk may also directly repress expression of the vein-organizer gene ab in cells posterior to the L5 primordium, analogous to the proposed role as a repressor of kni/knrl anterior to L2. One possible rationale for induction of the L5 vein depending on inputs from both omb and brk is that these genes are expressed in partially overlapping patterns and neither pattern may carry sufficiently detailed information to specify the position of the L5 primordium alone. Although the omb and brk expression levels fall off relatively steeply (i.e. over a distance of six to eight cells), these borders are not as sharp as the anterior sal border (two to three cells wide), which alone is sufficient to induce the L2 primordium (Cook, 2004).

A final similarity between the initiation of L2 and L5 formation is that induction of both veins is mediated by a vein-organizing gene that regulates vein and intervein gene expression in the vein primordium. Although kni and ab are members of different subfamilies of Zn-finger transcription factors, they are both expressed in a narrow stripe of cells along their respective inductive borders, and ubiquitous misexpression of either gene results in elimination of vein pattern in the wing disc. Thus, the L2 and L5 veins are induced by remarkably similar mechanisms and principles of organization. Further comparison of the mechanisms of these developmental programs should provide insights into the degree to which general and specific vein processes define the L2 versus the L5 vein identity (Cook, 2004).

Induction of Drosophila wing veins at borders between adjacent gene expression domains provides a simple model system for studying how information provided by morphogen gradients is converted into the stereotyped pattern of wing vein morphogenesis. Each of the four major longitudinal veins (L2-L5) is induced by a for-export-only mechanism in which cells in one region of the wing produce a diffusible signal to which they cannot respond. In the case of L3 and L4, an EGF-related signal (Vein) is produced between these veins in the central organizer where expression of the EGF receptor is locally downregulated. With respect to L2, response to the vein-inductive signal X is repressed in Sal-expressing cells that produce the hypothetical signal X. Finally, the L5 vein-inductive signal produced by brk-expressing cells depends on omb, the expression of which is repressed by Brk (Cook, 2004).

For-export-only mechanisms also underlie the induction of boundary cell fates in many other developmental settings. In the well-studied Drosophila wing, the earliest and most rigorously defined boundaries are the AP and DV borders, which are determined by Hh and Notch signaling, respectively. These compartmental borders define domains of non-intermixing groups of cells, and function as organizing centers by activating expression of the long-range morphogens Dpp and Wingless (Wg), respectively. In both cases, cells in one compartment produce a signal to which they cannot respond. This signal is constrained to act only on neighboring cells in the adjacent compartment. Other well-studied examples of for-export-only signaling include: induction of the mesectoderm in blastoderm stage Drosophila embryos by a likely cell-tethered Notch ligand expressed in the mesoderm; induction of parasegmental expression of stripe via Wg, Hh and Spi signaling in gastrulating Drosophila embryos; induction of mesoderm in Xenopus embryos by factors produced in the endoderm under the control of VegT, and formation of the DV border of leaves in plants controlled by the PHANTASTICA gene. The similar but distinct mechanisms for inducing the L2 and L5 vein primordia offers a well-defined system for examining these relatively simple cases in depth. These inductive events take place at the same developmental stage but within separate compartments of a single imaginal disc, and should provide general insights into the great variety of mechanisms that can be co-opted to accomplish for-export-only signaling (Cook, 2004).

The role of Dpp and its inhibitors during eggshell patterning in Drosophila

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor ß [TGF-ß; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages (DAs). Evidence is presented that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells (Shravage, 2007).

The results show that Dpp has Grk-independent and Grk-dependent functions in the follicular epithelium. Even in the absence of Grk, Dpp is required to specify a group of anterior follicle cells that surround the micropyle. All dorsal follicle cells that contribute to a morphologically visible polarization of the eggshell require the combined action of Grk and Dpp. Within the region giving rise to dorsal follicle cells, Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types (Shravage, 2007).

In the absence of Dpp, Grk can still activate kekkon and repress pipe. Thus, Dpp is not required for Grk signaling per se. It is suggested that Dpp signaling rather activates transcription factors or causes chromatin modifications that allow Grk to induce dorsal target genes involved in follicle cell specification (Shravage, 2007).

Mirror might be such a transcription factor that is activated by Dpp and confers the ability to adopt dorsal fates to a ring of anterior follicle cells. mirror acts downstream of Grk and probably also downstream of Dpp in specifying dorsal follicle cells. However, mirror expression alone leads only to the formation of DA material. Thus, it is likely that mirror only provides the general potential to produce dorsal follicle cells. Additional inputs from Dpp and EGF signaling are needed to produce the full set of dorsal follicle cell fates. This scenario suggests two phases of Dpp signaling. An early phase demarcates the region in which Grk induces dorsal follicle cell fates. This might require only one (low level) threshold of Dpp signaling and is likely to be mediated through activation of mirror. A later phase establishes distinct dorsal follicle cell fates. Here, Dpp acts as a morphogen in combination with EGF signaling (Shravage, 2007).

The results presented in this study suggest that high levels of EGF and Dpp signaling correspond to regions II and III of the operculum, whereas lower levels of both pathways correspond to the DAs. With regard to region III of the operculum that separates the two DAs, the assumption appears to contradict a model based on results that showed that Grk signaling induces the expression of rhomboid (rho), which in turn activates Spitz, a second TGF-α-like molecule. This leads to an amplification of EGF signaling. Highest signaling levels centered at the dorsal midline lead to the induction of the inhibitor argos (aos), which antagonizes Spitz. This in turn lowers the levels of EGF signaling along the dorsal mildline. According to this model, high levels of EGF signaling promote DA, lower levels operculum region III formation. However, the expression patterns of kek, which result from Grk or Dpp overexpression, appear to contradict this model. Indeed, it is believed that the regulatory loop of rho and aos is not required to establish the operculum or DA fates per se. The pattern of BR-C expression is not significantly altered in rho or aos mutant follicle cell clones. However, rho and aos might contribute to patterning processes that are required for the morphogenesis and, as a result of this, for splitting of the DAs. DA extension (tube formation) has been shown to require the collaboration of rho-expressing floor cells and BR-C-expressing roof cells. The rho-expressing floor cells are part of the Fas3 expression domain that separates the BR-C domains. These rho-expressing cells have to form a separate stripe on each side of the dorsal midline to allow the splitting of the DAs. It is suggested that the rho/aos regulatory loop is required to generate two distinct stripes of late-rho expression within the dorsal Fas3 domain. The result is a splitting of the DAs accompanied by the establishment of a region of Fas3 cells that do not express rho, and thus give rise to region III of the operculum (Shravage, 2007).

The establishment of the region giving rise to dorsal follicle cells and its subdivision into operculum and DA-producing cells is an intriguing problem of two-dimensional patterning. The pattern of cell fates depends on the concentration-dependent read-out of two orthogonal signaling gradients (EGF and Dpp). This read-out is complex because the signaling pathways themselves appear to influence each other. (1) There is evidence for a direct influence of Dpp on EGF signaling; (2) the Dpp inhibitors brk and dSno are targets of both pathways, and (3) rho is also a target of both pathways (Shravage, 2007).

Evidence for a direct crosstalk between both pathways is provided by the analysis of kek expression. kek appears to be a primary target gene of EGF signaling, since basal levels of its expression are independent of Dpp. However, an enhancement of kek expression was observed upon dpp overexpression in stage 10A prior to the activation of rho and aos. Moreover, the stage 10B expression patterns of rho and aos do not correlate with the observed changes in kek expression. Thus, these changes cannot be caused by the secondary modulation of the EGF signaling profile. Therefore, a direct crosstalk between both pathways is suggested. This could be because of a Dpp receptor-dependent activation of the ras/MAPK cascade. A TGF-ß receptor-dependent activation of the MAPK cascade has been observed in several vertebrate cell types. One could imagine that the triangular-shaped domain of Fas3 expression, which defines the anterior and dorsal borders of the BR-C domain, is specified by high levels of EGF signaling brought about by a Dpp-dependent enhancement of MAPK signaling. A confirmation of this model would necessitate direct monitoring of MAPK activity upon altered Dpp signaling (Shravage, 2007).

The border between operculum and DAs is also crucially dependent on brk. In brk mutant follicle cell clones, Fas3 expression expands at the expense of the BR-C domains. However, brk expression is upregulated within a broad domain at the dorsal side that also includes the Fas3-expressing region separating the BR-C domains. Although brk represses Fas3 expression in lateral regions allowing BR-C expression, brk is unable to repress Fas3 at the dorsal midline. This suggests that Fas3 expression, which is predominantly dependent on high levels of EGF signaling, cannot be repressed by brk, whereas Fas3 expression in more lateral regions predominantly dependent on Dpp signaling is repressed by brk (Shravage, 2007).

The hemi-circular boundary of the total region giving rise to dorsal chorion fates appears to be defined by a constant value reflecting the sum or the product of EGF and Dpp signaling. The cis-regulatory elements of dSno represent a sensitive sensor for this dual input. At the dorsal midline, lower amounts of Dpp signaling are required to activate dSno than in lateral regions, and the opposite holds true for EGF signaling. During brain development in flies and in several contexts in vertebrates Sno is involved in the control of cell proliferation that has been shown to be crucially dependent on the relative levels of TGF-ß and EGF signaling. It is conceivable that for spatial patterning of the follicular epithelium dSno uses regulatory elements that are derived from a more basic function in the control of cell proliferation in other tissues. The follicle cell expression of dSno might provide a convenient experimental setting to dissect such regulatory elements (Shravage, 2007).

The fact that loss of dSno causes only mild defects is because of redundancy. A combination of three Dpp inhibitors appears to be involved in establishing the border between dorsal follicle cells and the remainder of the mainbody follicular epithelium. brk clones alone have no effect on the position of this border because they cause only a replacement of the DAs by operculum. dad mutant clones seem to lack patterning defects altogether. However, already removing one copy of these inhibitors in a homozygous dSno mutant background leads to an enlargement of operculum and a posterior shift of the DAs. Weak phenotypic effects of dSno have recently been reported for wing vein formation. Wing vein formation, too, represents a developmental context in which several Dpp inhibitors collaborate (Shravage, 2007).

The dSno mutation that was generated deletes a highly conserved protein domain that is responsible for the interaction with Smad proteins in vertebrates and with Medea in flies. The knockout mutations in mice are based on the deletion of this domain. Thus, this dSno mutation should represent a null allele. However, an unusual complexity of the dSno locus has been reported and a deletion is described that suggests that dSno is lethal, in variance to other findings. However, a truncation allele has been described lacking an important part of the conserved Smad interaction domain that, like the currently described allele, is viable. Because the possibility exists that the previously described deletion affects other genes in the chromosomal region of dSno, the question of lethality of dSno requires further analysis (Shravage, 2007).

Loss of dSno in the follicular epithelium does not result in changes in dpp expression or pMAD distribution. Whereas a feedback on dpp expression was not expected, possible changes in pMAD distribution might be below the level of detection of staining protocol. However, there are two other possible explanations. First, in brain development dSno has been shown to be a mediator of Baboon (Activin), rather than Dpp signaling. To investigate whether this also holds true for the follicular epithelium large baboon (Activin type I receptor) mutant follicle cell clones were generated. These clones did not show patterning defects, suggesting that dSno does not act via Baboon signaling with regard to follicle cell patterning. Second, the failure to detect changes in pMAD distribution might follow from the molecular mechanism of Sno action. A core feature of the inhibitory function of Sno proteins results from their ability to bind to the common Smad (Smad4). This binding prevents (or modulates) the interaction with phosphorylated R-Smads required for the transcriptional control of target genes. If this mechanism applies to DSno, the loss of dSno would not change the phosphorylation state of MAD and, if the interaction between DSno and Medea occurred predominantly in the nucleus, there would also be no significant change in the nuclear accumulation of pMAD (Shravage, 2007).

Growth regulation by Dpp: an essential role for Brinker and a non-essential role for graded signaling levels.

Morphogens can control organ development by regulating patterning as well as growth. This study used the model system of the Drosophila wing imaginal disc to address how the patterning signal Decapentaplegic (Dpp) regulates cell proliferation. Contrary to previous models, which implicated the slope of the Dpp gradient as an essential driver of cell proliferation, it was found that the juxtaposition of cells with differential pathway activity is not required for proliferation. Additionally, the results demonstrate that, as is the case for patterning, Dpp controls wing growth entirely via repression of the target gene brinker (brk). The Dpp-Brk system converts an inherently uneven growth program, with excessive cell proliferation in lateral regions and low proliferation in medial regions, into a spatially homogeneous profile of cell divisions throughout the disc (Schwank, 2008).

Morphogen gradients play essential roles in pattern formation during animal development. They direct the transcriptional on and off states of genes in a concentration-dependent manner in various embryonic organ systems. The tight link between organ patterning and organ growth raised the notion that morphogens also determine cell proliferation rates and final tissue size. This latter aspect of the morphogen concept, however, is not well understood. Indeed, it is not clear whether the nuclear response to morphogen signals that directs the transcription of patterning genes also regulates growth. And what property of a morphogen signaling system explains how uniform growth rates can ensue in response to a graded input? This study addresses these questions in the experimental system of the Dpp gradient, a key determinant in pattern formation and growth of the Drosophila wing (Schwank, 2008).

Studies from the past decade have shown that the Dpp gradient in the wing disc does not define the expression boundaries of subordinate patterning genes directly via its nuclear mediators, but does so indirectly by setting up an inverse gradient of the transcriptional repressor Brk. This study investigated the potential role of this indirect mechanism in growth regulation; it was found to be equally important, and essential, for the ability of Dpp to promote growth. Clones of cells with a constitutively active Dpp signaling pathway exhibited qualitatively and quantitatively the same growth behavior as brk- clones, overgrowing when located in the lateral area. Moreover, the phenotype of discs in which Brk levels can no longer be regulated by Dpp (because brk is either lacking genetically, or controlled by a heterologous promoter) are insensitive to experimentally varying Dpp signaling levels. Thus, these experiments demonstrate that the growth output of the Dpp pathway is entirely funneled through the regulation of the brk gene (Schwank, 2008).

The paradigm of Dpp directing pattern formation via brk repression thus also explains how Dpp controls growth. This observation serves to validate the connection between morphogen-mediated patterning and the control of organ size. The results indicate that for the Dpp system, any mechanistic bifurcation of the two outputs occurs downstream of the first tier of transcriptional regulation (Schwank, 2008).

Discs lacking both dpp and brk functions grow to a larger size than wild-type discs. Importantly, in this state, in contrast to the normally uniform profile, cell proliferation also occurs unevenly across the disc, with higher rates in the lateral areas and lower rates in the medial area. Based on this difference, it is concluded that the Dpp-Brk system is not a growth promoter but is rather a growth-modulatory system, ironing out inherent regional differences in proliferation rates (Schwank, 2008).

The origin of the regional proliferative differences in discs devoid of the Dpp-Brk system is unknown. Since such discs lack Dpp, as the only agent known to impose mirror-symmetric differences along the AP axis, no pre-patterning mechanism that depends on it can be postulated. The smooth transitions to higher proliferation rates between medial and lateral areas would be consistent with a diffusible factor that acts in a concentration-dependent manner. This hypothetical factor could originate, for example, at the border between the disc proper and the adjacent peripodial membrane and promote growth laterally. Alternatively, the factor could be a growth inhibitor with high activity in the center of the disc and low activity peripherally. Expression of the factor could be controlled by Hedgehog in a Dpp-independent manner. But this is pure speculation because to date there is no evidence for the existence of such a factor(s) in the developing wing discs (Schwank, 2008).

An entirely different explanation for the experimental observations could be a growth-regulatory mechanism that depends on mechanical forces. It has been proposed that during growth, mechanical compression of cells increases in the center, while cells in the peripheral regions become stretched. Assuming a growth-stimulatory role for stretching and a growth-inhibitory role for compression, growth would be facilitated in the peripheral regions during normal development, and Brk would counter this advantage and thus ensure uniform growth. In the absence of the Dpp-Brk system, the amount and distribution of mechanical stresses are likely to differ significantly, which in turn could feed back on growth and lead to the observed differences between the lateral and medial regions of the disc (Schwank, 2008).

This study has confirmed and extended previous findings that in wing discs with uniform Dpp signaling, lateral cells proliferate faster, and medial cells slower, than cells of wild-type discs. The inhibition of cell proliferation in the medial region is an important pillar for the model which proposes that it is the slope of the Dpp morphogen gradient that serves as the driving force behind medial wing cell proliferation during normal development. Contradicting the proposed requirement for disparate Dpp signaling activities among adjacent cells, it was found that when uniform pathway activity is established in, and limited to, the medial area, no deficit in cell proliferation rates occurs. Indeed, the medial domain of discs with such even Dpp signaling levels expands, and proliferation is uniform. This finding is consistent with results from the twin-spot analysis, which showed that the growth rates of medial tkvQ235D and brkM68 clones are identical to those of wild-type clones. Thus, the transient effect of additional proliferation at clonal boundaries observed by Rogulja (2005) seems to be more important for situations such as wound healing, in which cells of different Dpp signaling levels become juxtaposed, than for the normal growth of a wild-type wing disc. A reduction in proliferation rates in the medial area was found to occur only when Dpp activity is driven in the lateral area, independent of the presence or absence of a Dpp signaling gradient. Ectopic Dpp pathway activation in lateral cells is not only necessary, but also sufficient, to impede proliferation of medial cells. Thus, overproliferating lateral cells appear to exert a proliferation-retardant effect on other cells. Whether this effect underlies a mechanism also used to control proliferation rates during wild-type development, or whether it is 'only' a back-up mechanism used if something goes wrong during development (e.g. wound healing and regeneration), is not known. Moreover, as noted earlier, the mechanistic nature of the communication between lateral and medial cell populations remains speculative. It is possible that high Dpp signaling in lateral cells not only provides them with a growth advantage, but also causes the expression of a factor that spreads within the entire disc to reduce proliferation of cells without an additional growth advantage. Other possible explanations include the competition among wing cells for a limiting proliferation factor (whereby ectopic Dpp-transducing cells prevail), or the negative impact that overproliferating cells might exert on remaining cells via metabolic side-products or increased mechanical compression. These models would also be consistent with the observation that proliferation is reduced in all cells of the wing disc except those with an additional growth advantage (Schwank, 2008).

Dpp-mediated growth control in the wing disc can be summarized as follows. The disc consists of at least two different cell populations, medial and lateral, which have distinct abilities to proliferate. The Dpp signal is required to even out these growth differences and establish a uniform pattern of cell proliferation within the wing primordium. Medial cells must sense high levels of Dpp to shut down brk expression, which consequently promotes medial proliferation. Lateral cells have a growth advantage and must receive little or no Dpp input to allow brk expression. The action of Brk curbs lateral proliferation. It is not knowm how intermediate Brk levels affect the proliferative behavior of cells situated between lateral and medial cells. However, it can be concluded from the present results that differential pathway activity between neighboring cells is not necessary to direct proliferation, since constitutively high Dpp levels in the medial area and nil or low levels in the lateral areas are sufficient for uniform and normal cell proliferation rates throughout the disc (Schwank, 2008).


Ashe, H. L., Mannervik, M. and Levine, M. (2000). Dpp signaling thresholds in the dorsal ectoderm of the Drosophila embryo. Development 127: 3305-3312. PubMed Citation: 10887086

Ben-Zvi, D. and Barkai, N. (2010). Scaling of morphogen gradients by an expansion-repression integral feedback control. Proc. Natl. Acad. Sci. 107: 6924-6929. PubMed Citation: 20356830

Biehs, B., Sturtevant, M. A. and Bier, E. (1998). Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs. Development 125: 4245-4257. 9753679

Bray, S. (1999). DPP on the brinker. Trends Genet. 15(4):140. PubMed Citation: 10203822

Campbell, G. and Tomlinson, A. (1999). Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96(4): 553-62. PubMed Citation: 10052457

Cande, J., Goltsev, Y. and Levine, M. S. (2009). Conservation of enhancer location in divergent insects. Proc. Natl. Acad. Sci. 106(34): 14414-9. PubMed Citation: 19666595

Cook, O., Biehs, B. and Bier, E. (2004). brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 131: 2113-2124. 15073155

Cordier, F., et al. (2006). DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding. J. Mol. Biol. 361(4): 659-72. Medline abstract: 16876822

Dunipace, L., Saunders, A., Ashe, H. L., Stathopoulos, A. (2013) Autoregulatory Feedback Controls Sequential Action of cis-Regulatory Modules at the brinker Locus. Dev Cell 26: 536-543. PubMed ID: 24044892

Estella, C., McKay, D. J. and Mann, R. S. (2008). Molecular integration of Wingless, Decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development. Dev. Cell 14: 86-96. PubMed Citation: 18194655

Foo, S. M., Sun, Y., Lim, B., Ziukaite, R., O'Brien, K., Nien, C. Y., Kirov, N., Shvartsman, S. Y. and Rushlow, C. A. (2014). Zelda potentiates morphogen activity by increasing chromatin accessibility. Curr Biol 24: 1341-1346. PubMed ID: 24909324

Foronda, D., Pérez-Garijo, A. and Martín, F. A. (2009). Dpp of posterior origin patterns the proximal region of the wing. Mech. Dev. 126(3-4): 99-106. PubMed Citation: 19118625

Gafner, L., Dalessi, S., Escher, E., Pyrowolakis, G., Bergmann, S. and Basler, K. (2013). Manipulating the sensitivity of signal-induced repression: quantification and consequences of altered Brinker gradients. PLoS One 8: e71224. PubMed ID: 23951114

Gao, S. and Laughon, A. (2006). Decapentaplegic-responsive silencers contain overlapping mad-binding sites. J. Biol. Chem. 281(35): 25781-90. 16829514

Hamaguchi, T., et al. (2012). Dorsoventral patterning of the Drosophila hindgut is determined by interaction of genes under the control of two independent gene regulatory systems, the dorsal and terminal systems. Mech. Dev. 129(9-12): 236-43. PubMed Citation: 22898294

Hamaratoglu, F., et al. (2011). Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc. PLoS Biol. 9(10): e1001182. PubMed Citation: 22039350

Hasson, P., et al. (2001). Brinker requires two corepressors for maximal and versatile repression in Dpp signalling. EMBO J. 20: 5725-5736. 11598015

Hu, S., Fambrough, D., Atashi, J. R., Goodman, C. S. and Crews, S. T. (1995). The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections. Genes Dev. 9: 2936-2948. 7498790

Jazwinska, A., et al. (1999a). The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96(4): 563-73. PubMed Citation: 10052458

Jazwinska, A., Rushlow, C. and Roth, S. (1999b). The role of brinker in mediating the graded response to Dpp in early Drosophila embryos. Development 126(15): 3323-3334. PubMed Citation: 10393112

Kirkpatrick, H., Johnson, K. and Laughon, A. (2001). Repression of Dpp targets by binding of Brinker to Mad sites. J. Biol. Chem. 276: 18216-18222. 11262410

Kwon, C., et al. (2004). Opposing inputs by Hedgehog and Brinker define a stripe of hairy expression in the Drosophila leg imaginal disc. Development 131: 2681-2692. 15128656

Lammel, U., Meadows, L. and Saumweber, H. (2000). Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification. Mech. Dev. 92: 179-191. PubMed Citation: 10727857

Markstein, M., et al. (2002). Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo Proc. Natl. Acad. Sci. 99: 763-768. 11752406

Markstein, M., et al. (2004). A regulatory code for neurogenic gene expression in the Drosophila embryo. Development 131: 2387-2394. 15128669

Martín, F. A., Pérez-Garijo, Moreno, A. E. and Morata, G. (2004). The brinker gradient controls wing growth in Drosophila. Development 131: 4921-4930. 15371310

Marty, T., et al. (2000). Schnurri mediates Dpp-dependent repression of brinker transcription. Nat. Cell Biol. 2(10): 745-9. 11025666

Minami, M., et al. (1999). brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes. Nature 398(6724): 242-6. PubMed Citation: 10094047

Müller, B., et al. (2003). Conversion of an extracellular Dpp/BMP morphogen gradient into an inverse transcriptional gradient. Cell 113: 221-233. 12705870

Nibu, Y., Zhang, H., and Levine, M. (1998). Interaction of short-range repressors with Drosophila CtBP in the embryo. Science 280: 101-104. PubMed Citation: 9525852

Patterson, G. I., Koweek, A., Wong, A., Liu, Y. and Ruvkun, G. (1997). The DAF-3 Smad protein antagonizes TGF-beta-related receptor signaling in the Caenorhabditis elegans dauer pathway. Genes Dev. 11: 2679-2690. PubMed Citation: 9334330

Pyrowolakis, G., et al. (2004). A simple molecular complex mediates widespread BMP-induced repression during Drosophila development. Dev. Cell 7: 229-240. 15296719

Rebeiz, M., et al. (2012). Ancestral and conserved cis-regulatory architectures in developmental control genes. Dev. Biol. 362: 282-294. PubMed Citation: 22185795

Rebeiz, M., Stone, T. and Posakony, J. W. (2005). An ancient transcriptional regulatory linkage. Dev. Biol. 281: 299-308. PubMed Citation: 1589398

Rogulja, D. and Irvine, K. D. (2005). Regulation of cell proliferation by a morphogen gradient. Cell 123: 449-461. PubMed Citation: 16269336

Rushlow, C., et al. (2001). Transcriptional regulation of the Drosophila gene zen by competing Smad and Brinker inputs. Genes Dev. 15: 340-351. 11159914

Rusten, T. E., et al. (2002). The role of TGFß signaling in the formation of the dorsal nervous system is conserved between Drosophila and chordates. Development 129: 3575-3584. 12117808

Saller, E. and Bienz, M. (2001). Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription. EMBO Reports 2: 298-305. 11306550

Saller, E., Kelley, A. and Bienz, M. (2002). The transcriptional repressor Brinker antagonizes Wingless signaling. Genes Dev. 16: 1828-1838. 12130542

Schwank, G., Restrepo, S. and Basler, K. (2008). Growth regulation by Dpp: an essential role for Brinker and a non-essential role for graded signaling levels. Development 135(24): 4003-13. PubMed Citation: 19029041

Sivasankaran, R., et al. (2000). Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19: 6162-6172. PubMed Citation: 11080162

Shravage, B. V., Altmann, G., Technau, M. and Roth, S. (2007). The role of Dpp and its inhibitors during eggshell patterning in Drosophila. Development 134(12): 2261-71. Medline abstract: 17507396

Sturtevant, M. A. and Bier, E. (1995). Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121: 785-801. 7720583

Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E. (1997). The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 124: 21-32. 9006064

Takaesu, N. T., Bulanin, D. S., Johnson, A. N., Orenic, T. V. and Newfeld, S. J. (2008). A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila. Dev. Biol. 313(2): 829-43. PubMed Citation: 18068697

Thatcher, J. D., Haun, C. and Okkema, P. G. (1999). The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx. Development 126: 97-107. PubMed Citation: 9834189

Torres-Vazquez, J., Warrior, R. and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Bio. 227: 388-402. PubMed Citation: 11071762

Torres-Vazquez, J., et al. (2001). The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis. Development 128: 1657-1670. 11290303

Upadhyai, P. and Campbell, G. (2013). Brinker possesses multiple mechanisms for repression because its primary co-repressor, Groucho, may be unavailable in some cell types. Development 140: 4256-4265. PubMed ID: 24086079

Wartlick, O., et al. (2011). Dynamics of Dpp signaling and proliferation control. Science 331: 1154-1159. PubMed Citation: 21385708

Weiss, A., et al. (2010) A conserved activation element in BMP signaling during Drosophila development. Nat. Struct. Mol. Biol. 17(1): 69-76. PubMed Citation: 20010841

Winter, S. E. and Campbell, G. (2004). Repression of Dpp targets in the Drosophila wing by Brinker. Development 131: 6071-6081. 15537684

Yang, L., Meng, F., Ma, D., Xie, W. and Fang, M. (2013). Bridging Decapentaplegic and Wingless signaling in Drosophila wings through repression of naked cuticle by Brinker. Development 140: 413-422. PubMed ID: 23250215

Yao, L.-C., (2008). Multiple modular promoter elements drive graded brinker expression in response to the Dpp morphogen gradient. Development 135: 2183-2192. PubMed Citation: 18506030

Zhang, H., Levine, M. and Ashe, H. L. (2001). Brinker is a sequence-specific transcriptional repressor in the Drosophila embryo. Genes Dev. Vol. 15: 261-266. 11159907

Ziv, O., et al. (2009). The co-regulator dNAB interacts with Brinker to eliminate cells with reduced Dpp signaling. Development 136(7): 1137-45. PubMed Citation: 19270172

Ziv, O., Finkelstein, R., Suissa, Y., Dinur, T., Deshpande, G. and Gerlitz, O. (2012). Inverse regulation of target genes at the brink of the BMP morphogen activity gradient. J. Cell Sci. [Epub ahead of print]. PubMed Citation: 22956540

brinker: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 April 2015

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.