Transcriptional Regulation

Hedgehog (Hh) and Decapentaplegic (Dpp) direct anteroposterior patterning in the developing Drosophila wing by functioning as short- and long-range morphogens, respectively. The activity of Dpp is graded and is directly regulated by a novel Hh-dependent mechanism. Dpp activity is monitored by visualizing the activated form of Mothers against dpp (Mad), a cytoplasmic transducer of Dpp signaling. Activated Mad levels are highest near the source of Dpp but are unexpectedly low in the cells that express dpp. Hh induces dpp in these cells; it also attenuates their response to Dpp by downregulating expression of the Dpp receptor thick veins (tkv). It has been suggested that regulation of tkv by Hh is a key part of the mechanism that controls the level and distribution of Dpp (Tanimoto, 2000).

To determine whether the low levels of phosphorylated Mad (p-MAD) in dpp-expressing cells reflect an autoregulatory influence on Dpp signaling that functions only in dpp-expressing cells, or whether p-MAD levels are controlled by another signal such as Hh, p-MAD was monitored in wing discs carrying clones that ectopically express hh or dpp. If Dpp has the potential to attenuate its own signal transduction, the level of p-MAD would be expected to decrease cell autonomously in cells that express dpp. However, the level of p-MAD is elevated around the clones with no obvious reduction within the clones. This observation is not consistent with an autoregulatory mechanism. In contrast, clones expressing hh in the A compartment cause both a nonautonomous reduction of p-MAD level as well as ectopic elevation of p-MAD around the clones, which is due to ectopic induction of dpp by Hh. No changes in p-MAD levels are observed when Hh is expressed in the P compartment. These observations suggest that Hh attenuates Dpp signal transduction by regulating the type I receptor-dependent phosphorylation of MAD. Thus, the activity of Dpp depends on both the concentration of Dpp and Hh (Tanimoto, 2000).

It was next asked whether Hh directly controls p-MAD levels. First, clones that ectopically express HhCD2, a membrane-tethered form of Hh, were compared to clones that express a diffusible form of Hh. P-MAD levels are reduced both in cells that expressed HhCD2 and in cells that are immediately adjacent. In contrast, the levels of p-MAD are reduced more broadly when cells express diffusible Hh. The distribution of p-MAD was examined when clones of cells mutant for either Protein kinase A (Pka) alone or both Pka and dpp were induced. Pka functions to antagonize Hh signaling: Pka- clones in the A compartment fail to repress Hh signaling in a cell-autonomous manner. Pka- clones in the A compartment have reduced levels of p-MAD. Moreover, p-MAD accumulates to high levels in cells surrounding Pka- clones, due, presumably, to the ectopic induction of Dpp by constitutive Hh signaling in the mutant cells. In clones mutant for both Pka and dpp, the reduction was evident, but it was not accompanied by elevated levels in the surrounding cells. Outside the double-mutant clones, p-MAD appears to respect the endogenous gradient of Dpp. Autonomous reduction in the level of Spalt is also seen in both types of mutant clones, confirming both that p-MAD accurately manifests the active state of Dpp signaling and that the reduction of p-MAD leads to the decrease of target gene expression. It is concluded that Hh signaling directly attenuates Dpp signal transduction regardless of the level of endogenous Dpp (Tanimoto, 2000).

The contribution of Cubitus interruptus (Ci) to Hh-dependent attenuation of Dpp signaling was examined. ci is expressed in the A compartment only. In clones that ectopically express Ci in the P compartment where Hh is abundant, the levels of p-MAD were reduced in a cell-autonomous manner. Levels of Sal are similarly reduced in these cells. Clones that ectopically express ci in A compartment regions that receive little or no Hh have no effect on p-MAD levels. It is concluded that Hh both induces dpp expression and downregulates Dpp signal transduction in the same cells (Tanimoto, 2000).

Because Ci is involved in regulating p-MAD levels, it is possible that Hh controls the expression of a component that functions upstream of Mad phosphorylation. The possibiliy that Hh regulates the transcription of the Dpp receptors was therefore investigated. Dpp preferentially signals through the Tkv receptor and also negatively regulates tkv expression. Therefore, the correlation between tkv expression and the activity of Dpp was examined. The level of tkv expression is higher in cells located peripherally and is lower in the central region. In addition, a sharp reduction in expression at the A/P border was observed, a pattern very similar to that of p-MAD. The level of tkv expression in the area between the peripheral region and the A/P border is referred to as 'basal' and the reduced level at the A/P border as 'hyperrepression'. Interestingly, the basal level in the P compartment is higher than it is in the A compartment. This may account for the steeper gradient of p-MAD in the P compartment, since high levels of Tkv limit the movement of Dpp; since the spread of Dpp would be less in the P compartment, its gradient of activity would be expected to be steeper (Tanimoto, 2000).

In order to investigate the role of Dpp in generating the complex pattern of tkv expression, tkv and p-MAD were monitored in discs that ubiquitously express dpp. These discs exhibit significant overgrowth. tkv-lacZ at the A/P border remains hyperrepressed, but the level of peripheral tkv is significantly reduced, apparently to the basal level. This suggests that the level of tkv in the peripheral regions is directly regulated by Dpp but that the level at the A/P compartment border is not. The basal level in the P compartment remains higher than in the A compartment. Distribution of p-MAD is consistent with the expression pattern of tkv: overexpression of dpp caused the ubiquitous elevation of p-MAD, except at the A/P border. In contrast, phosphorylation of Mad at the A/P border is unchanged in the presence of excess Dpp. This suggests that the level of tkv along the A/P border is limiting. When constitutively active Tkv (tkv*) is ubiquitously expressed, peripheral tkv expression is reduced to the basal level, comparable to Dpp overexpression. Unlike the discs in which Dpp is overexpressed, no reduction in p-MAD is observed at the A/P border in these discs. These results suggest that in middle regions of the A and P compartments in normal discs, Dpp signaling reduces tkv expression from the levels at the periphery to the basal level. However, Dpp signaling is not responsible for the hyperrepression at the A/P border or for the difference between the basal levels in the A and P compartments. Given that hyperrepression of p-MAD along the A/P border is not observed in the presence of TKV*, it is more likely that in this region, Hh modulates p-MAD levels by regulating tkv (Tanimoto, 2000).

To ask whether Hh controls tkv expression directly at the A/P border, clones of cells mutant for patched (ptc), expressed in all A cells, were examined. The Hh signal transduction is constitutively active in the absence of the ptc activity. p-MAD and tkv-lacZ were monitored in ptc- clones. The clones in the A compartment ectopically activate Hh signaling in a cell-autonomous manner; they also caused cell-autonomous repression of tkv. Outside of the clone, the level of tkv is reduced to the basal level, but this level is higher than within the clone. This behavior is likely due to ectopic expression of dpp. P-MAD levels are also reduced in the clone and are higher around the clone. This establishes that Hh signaling directly represses tkv and, as a consequence, represses phosphorylation of MAD. This conclusion is confirmed by examining discs carrying clones of cells in the P compartment that ectopically express ci. tkv is autonomously repressed in these clones, indicating that Ci-mediated Hh signaling directly represses tkv (Tanimoto, 2000).

In order to understand the significance of Hh-dependent repression of tkv, the level of tkv expression was changed. In the wing disc, when overexpressing tkv in the dorsal compartment under the control of apterous (ap) enhancer, the levels of the dorsal p-MAD and Spalt expression at the A/P border are elevated, and their distribution is significantly narrower than in the ventral compartment. This is probably due to the sequestration of Dpp protein by the elevated Tkv. In addition, the adult wing misexpressing tkv was examined in dpp-expressing cells where tkv is hyperrepressed. The overexpression caused small wings with abnormal patterning in the central region of the wing. These results are consistent with the proposal that Hh, but not Dpp, patterns the central wing region. It should be noted, however, that the level of tkv expression in the experimental clones is probably at least several fold higher than normal, since these experiments utilized the Gal4/UAS system (Tanimoto, 2000).

The activities of Dpp were examined when tkv expression differed from wild type by only 2-fold. Clones mutant for tkv were generated in a heterozygous background such that both mutant (0 copies of the wild-type tkv) and fully wild-type sister clones (2 copies of wild-type tkv) were produced. However, since clones of cells with no Tkv activity do not survive in the wing pouch because they need Tkv activity to grow, only their sister clones survive. Homozygous (+/+) clones at the A/P border increase p-MAD and Spalt levels autonomously. The same analysis was performed using a null sax allele: differences in the levels of p-MAD and Spalt between clones carrying two wild-type sax and cells lacking one copy of sax were negligible. Taken together, it is proposed that the precise regulation of the Tkv receptor level by Hh signal is necessary for Dpp morphogen to shape the correct activity gradient (Tanimoto, 2000).

Formation of the trachea occurs by the migration and fusion of clusters of ectodermal cells specified in each side of ten embryonic segments. Morphogenesis of the tracheal tree requires the activity of many genes, among them breathless (btl) and ventral veinless (vvl), whose mutations abolish tracheal cell migration. Activation of the btl receptor by branchless (bnl), its putative ligand, exerts an instructive role in the process of guiding tracheal cell migration. decapentaplegic determines vvl expression along the embryonic dorsoventral axis; expansion of dpp expression results in an increased recruitment of cells to express vvl. These cells are allocated in the expanded tracheal placodes, indicating that expansion of dpp expression causes a concomitant enlargement of the traceal placodes and of vvl expression. vvl is also required for the maintenance of btl expression during tracheal migration (Llimargas, 1997).

vvl is independently required for the specific expression in the tracheal cells of thick veins (tkv) and rhomboid (rho), two genes whose mutations disrupt only particular branches of the tracheal system. Expression in the tracheal cells of an activated form of tkv, the Decapentaplegic receptor, induces shifts in the migration of these cells, asserting the role of the dpp pathway in establishing the branching pattern of the tracheal tree. In addition, by ubiquitous expression of the btl and tkv genes in vvl mutants it is shown that both genes contribute to vvl function. These results indicate that through activation of its target genes, vvl makes the tracheal cells competent to further signaling and suggest that the btl transduction pathway could collaborate with other transduction pathways also regulated by vvl to specify the tracheal branching pattern (Llimargas, 1997).

The Drosophila tracheal system is a network of epithelial tubes that arises from the tracheal placodes, lateral clusters of ectodermal cells in ten embryonic segments. The cells of each cluster invaginate and subsequent formation of the tracheal tree occurs by cell migration and fusion of tracheal branches, without cell division. The combined action of the Decapentaplegic (Dpp), Epidermal growth factor (EGF) and breathless/ branchless pathways are thought to be responsible for the pattern of tracheal branches. It is asked how these transduction pathways regulate cell migration and the consequences on cell behaviour of the Dpp and EGF pathways is examined. rhomboid (rho) mutant embryos display defects not only in tracheal cell migration but also in tracheal cell invagination unveiling a new role for EGF signaling in the formation of the tracheal system. These results indicate that the transduction pathways that control tracheal cell migration are active in different steps of tracheal formation, beginning at invagination (Llimargas, 1999).

These observations also illustrate the role of vvl in tracheal formation. Since btl expression is normally initiated in vvl mutants, early but not sustained activity of the Btl pathway could cause the tracheal phenotype in vvl mutant embryos. Since vvl is also required for the tracheal expression of tkv and rho, failure to activate the Dpp and EGF pathways could also be the source of the cell shape phenotypes in vvl mutant embryos. This latter possibility is substantiated by the observation that vvl and rho mutant embryos show abnormalities in tracheal invagination that are not present in btl mutant embryos. Finally, the tkv;rho double mutant tracheal phenotype is very similar to the vvl phenotype (Llimargas, 1999).

Multiple signaling pathways interact to determine the formation of the different tracheal branches. However, even though they all affect the directed migration of the tracheal cells, they are active in different steps in the morphogenesis of the tracheal tree. In particular, the results show that while the Bnl/Btl pathway is specifically required for migration, EGF signaling is active in tracheal cell invagination. These observations also indicate that the accurate invagination of the tracheal cells inside the embryo is an important factor in order to follow a particular direction of migration. In particular, different levels of invagination could predetermine whether cells would migrate in one or the other direction. In this regard, it is worth noting that while in rho mutant embryos some cells remain at the embryonic surface and do not invaginate, in tkv mutant embryos some cells remain in an intermediate position, indicating that they are able to invaginate but do not reach their final location. Altogether, these observations suggest that the precise topology of the invaginating cells controlled by EGF and Dpp signaling could be determining how the tracheal cells will respond to guiding cues, such as Bnl (Llimargas, 1999).

The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some downstream genes but not others. While tracheal expression of tkv depends on vvl, it appears to be independent of trh. The opposite appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight (hnt)], which code for two putative transcription factors. Both genes appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes seem to be common targets of vvl and trh but others seem to depend only on one of them (Boube, 2000).

In addition to essential myogenic functions, mutant Mef2 adult females are weakly fertile and produce defective eggs. Mef2 is expressed in nurse and follicle cells of the wild-type egg chamber. The Mef2 oogenic phenotype has been analyzed and it has been shown that the gene is required for the normal patterning and differentiation of the centripetally migrating follicle cells (CMFCs) that are crucial for development of the anterior chorionic structures. Mef2 alleles exhibit a genetic interaction with a dominant-negative allele of thick veins (tkv), which encodes a type I receptor of the Decapentaplegic-signaling pathway. TKV mRNA is overexpressed in Mef2 mutant egg chambers, and, conversely, forced expression of Mef2 represses tkv expression. These results indicate roles for Mef2 in the regulation of tkv gene expression and Decapentaplegic signal transduction that are essential for proper determination and/or differentiation of the anterior follicle cells. Mef2 is also expressed in both nurse and follicle cells. No defects have been observed in the germ line, either the number of germ cells or the location of the oocyte within the egg chamber. Therefore, a possible requirement for Mef2 in germ-line cells remains to be elucidated (Mantrova, 1999).

Mef2 appears to function in the somatic follicle cells, particularly in subpopulations of the oocyte-associated follicle cells (O-FCs), by negatively regulating TKV mRNA levels. It is not known whether Mef2 directly represses tkv gene transcription. Curiously, the expression patterns of Mef2 and TKV RNA are not mutually exclusive. Whereas Mef2 is expressed in all follicle cells, TKV is absent only in different populations of follicle cells at different times. Perhaps subtle changes in Mef2 levels can have different effects on tkv expression. For example, at stage 10A, Mef2 is more abundant in the leading CMFC than in the other O-FCs, whereas tkv is not expressed in CMFCs and expressed at a low level in the rest of the follicle cells. Alternatively, Mef2 may be a constitutive repressor of tkv, whereas other tissue-specific factors can counteract Mef2 and induce tkv expression (Mantrova, 1999).

In wild type, tkv expression is dynamic during oogenesis and appears to highlight a specific group of follicle cells, the leading front of the CMFCs. At stage 10A just before the commencement of centripetal migration, these cells form a ring marking the boundary between the oocyte and the nurse cell complex. After stage 10B, this ring of cells migrates inward until it reaches the border cells located at the center of the oocyte anterior. At stage 10A, tkv is expressed in O-FC but not in the leading CMFCs. This pattern is opposite that of the dpp expression pattern, which is highly expressed in the leading CMFCs but not in the rest of the O-FCs. It will be of interest to examine whether or not tissue-specific expression of dpp and tkv in the egg chamber is autoregulated by DPP signaling (Mantrova, 1999).

At stage 10B, tkv is expressed in the ventral half of the CMFC in addition to two short stripes in the dorsal region of the oocyte-associated follicular epithelium. This expression pattern appears to be complementary to that of the Egfr blocker argos, which forms a T-shaped pattern along the dorsal CMFCs and dorsal midline. argos expression is induced by the highest level of Egfr signaling; Egfr in turn, reduces the signaling strength by blocking the interaction between the receptor and its ligands. Thus, the initial graded distribution of Egfr signaling, extending laterally from the anterodorsal midline of the O-FCs, is transformed into two ridges of the Egfr-signaling level just lateral to the dorsal midline. These two ridges define the two lines of O-FCs that ultimately produce the two dorsal appendages. Interestingly, argos expression is diminished in the Mef2 mutant, consistent with the observed mutant egg chambers possessing broad and fused appendages. Although the notion is favored that argos expression is modulated by Mef2 through the action of Tkv, it cannot be ruled out that Mef2 may directly control the transcription of argos (Mantrova, 1999).

In addition to regulating the expression pattern of argos, Mef2 may play a more general role in modulating the Egfr-signaling level. This is suggested by the presence of Mef2 mutant egg chambers with reduced and fused dorsal appendages, a phenotype typical of hypomorphic Egfr-signaling pathway mutants. Indeed, reduced expression of Egfr-signaling components such as rhomboid has been observed in Mef2 mutants. More detailed and expansive studies are needed to elucidate the possible interaction between the Dpp- and Egfr-signaling pathways with Mef2 as a potential mediator (Mantrova, 1999).

Nevertheless, this report does demonstrate that the dpp-expressing CMFCs are poorly defined in D-mef2 mutant egg chambers. CMFCs are responsible for forming the operculum and, together with the border cells, specifying the construction of the micropile. Formation of these structures is also essential to closing the anterior end of the egg chamber. Because Dpp is critical for specifying anterior chorion production, the disrupted patterning of CMFCs in the Mef2 mutant may explain, at least in part, the chorion phenotypes observed (Mantrova, 1999).

One of the pleiotropic functions of scribbler (sbb) is an effect on wing morphogenesis. This function has been addressed by Funakoshi (2001), who shows that sbb shapes the activity gradient of the Dpp morphogen through regulation of thickveins. Drosophila wings are patterned by Decapentaplegic, which is expressed along the anterior and posterior compartment boundary. The distribution and activity of Dpp signaling is controlled in part by the level of expression of its major type I receptor, thickveins. The level of tkv is dynamically regulated by Engrailed and Hedgehog. sbb, termed master of thickveins (mtv) by Funakoshi, downregulates expression of tkv in response to Hh and En. mtv expression is controlled by En and Hh, and is complementary to tkv expression. mtv integrates the activities of En and Hh that shape tkv expression pattern. Thus, mtv plays a key part of regulatory mechanism that makes the activity gradient of the Dpp morphogen (Funakoshi, 2001).

Dpp signaling activity can be visualized by using the antibody against phosphorylated Mothers against dpp (p-Mad): the distribution of the Dpp morphogen activity largely depends on the levels of the Tkv receptor. tkv expression, which is monitored by expression of beta-galactosidase in the tkv-lacZ enhancer trap line, is downregulated by Hh along the A/P border where dpp expression is induced by the same signal. The basal level of tkv is higher in the P compartment than it is in the A compartment. This complex pattern appears to shape the activity gradient of Dpp directly. The p-Mad level is low along the A/P border where tkv is downregulated. The gradient of the p-Mad distribution is steeper in the P compartment than it is in the A compartment, probably because high levels of Tkv limit the movement of Dpp; since the spread of Dpp would be less in the P compartment, its gradient of activity would be expected to be steeper (Funakoshi, 2001).

mtv was identified by characterizing the enhancer trap lines, 1E1 and l(2)k00702, that generate expression patterns largely complementary to that of tkv in wing discs except at the dorsoventral compartment border in the peripheral region, where both genes are expressed at high levels. Distribution of the transcript revealed by in situ hybridization with a probe prepared from the corresponding cDNA is consistent with the pattern of the enhancer trap lines. Only the longer form of bks/sbb/mtv mRNA is predominantly detected in imaginal discs (Funakoshi, 2001).

In order to know whether mtv has a role in regulating tkv, a deletion mutant allele, mtv6, was made by imprecise excision of the P-element and used for clonal analysis. It is believed that mtv6 is a strong hypomorphic allele, because its transcript can only encode a 19 amino acid polypeptide, which lacks most of putative functional domains of the Mtv protein. In mtv6 clones, tkv-lacZ levels are autonomously upregulated indicating that Mtv represses tkv. When a large mtv clone is induced in the area including the A/P border, tkv-lacZ levels become uniform within the clone, suggesting that mtv plays an important role in regulating a dynamic pattern of tkv expression throughout the wing pouch. p-Mad levels are also upregulated in a graded manner. This is consistent with the fact that tkv is derepressed within mtv mutant clones because ectopically induced Tkv upregulates p-Mad levels. No significant changes in dpp transcription levels were observed within mtv mutant clones, thus it is concluded that mtv shapes p-Mad spatial distribution through regulation of Tkv levels (Funakoshi, 2001).

The development of the Drosophila wing is governed by the action of morphogens encoded by decapentaplegic and wingless that promote cell proliferation and pattern the wing. Along the anterior/posterior (A/P) axis, the precise expression of dpp and its receptors is required for the transcriptional regulation of specific target genes. The function of the T-box gene optomotor-blind (omb), a dpp target gene, was analyzed. The wings of omb mutants have two apparently opposite phenotypes: the central wing is severely reduced and shows massive cell death, mainly in the distal-most wing, and the lateral wing shows extra cell proliferation. Genetic evidence is presented that omb is required to establish the graded expression of the Dpp type I receptor encoded by the gene thick veins (tkv) to repress the expression of the gene master of thick veins and also to activate the expression of spalt (sal) and vestigial (vg), two Dpp target genes. optomotor-blind plays a role in wing development downstream of dpp by controlling the expression of its receptor thick veins and by mediating the activation of target genes required for the correct development of the wing. The lack of omb produces massive cell death in its expression domain, which leads to the mis-activation of the Notch pathway and the overproliferation of lateral wing cells (del Alamo Rodriguez, 2004).

Hox control of organ size by regulation of morphogen production and mobility: Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins

Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. This study investigated how the Drosophila Hox gene Ultrabithorax (Ubx) limits the size of the haltere, which, by the end of larval development, has ~fivefold fewer cells than the wing. It was found that Ubx controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp). Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes (Crickmore, 2006).

Changes in body part sizes have been critical for diversification and specialization of animal species during evolution. The beaks of Darwin's finches provide a famous example for how adaptation can produce variations in size and shape that allowed these birds to take advantage of specialized ecological niches and food supplies. Sizes also vary between homologous structures within an individual. For example, vertebrate digits and ribs vary in size, likely due to the activities of selector genes such as the Hox genes. Although the control of organ growth by selector genes is likely to be common in animal development, little is known about the mechanisms underlying this control (Crickmore, 2006).

The two flight appendages of Drosophila, the wing and the haltere, provide a classic example of serially homologous structures of different sizes. Halteres, appendages used for balance during flight, are thought to have been modified from full-sized hindwings during the evolution of two-winged flies from their four-winged ancestors. All aspects of haltere development that distinguish it from a wing, including its reduced size, are under the control of the Hox gene Ultra-bithorax (Ubx), which is expressed in all haltere imaginal disc cells but not in wing imaginal disc cells. At all stages of development, haltere and wing primordia (imaginal discs) are different sizes. In the embryo, the wing primordium has about twice as many cells as the haltere primordium. By the end of larval development, the wing disc has ~five times more cells (~50,000) than the haltere disc (~10,000). The wing and haltere appendages will form from the pouch region of these mature discs. The final step that contributes to wing and haltere size differences occurs during metamorphosis, when wing, but not haltere, cells flatten, thus increasing the surface area of the final appendage (Crickmore, 2006).

To confirm that Ubx has a postembryonic role in limiting the size of the haltere disc, Ubx clones were generated midway through larval development. Haltere discs–bearing large Ubx clones generated at this time become much larger than wild-type discs. Ubx could limit haltere size cell-autonomously by, for example, slowing the cell cycle of haltere cells relative to wing cells. This was tested by comparing the sizes of isolated Ubx clones in the haltere with those of their simultaneously generated wild-type twin clones. Contrary to the prediction of a cell-autonomous function for Ubx in size control, Ubx mutant clones did not grow larger than their twins, a result that is consistent with earlier experiments suggesting that wing and haltere cells have similar mitotic rates during development. Hence, Ubx limits the size of the haltere during larval development by modifying pathways that control organ growth cell-nonautonomously (Crickmore, 2006).

In the fly wing, Decapentaplegic (Dpp) [a long-range morphogen of the bone morphogenetic protein (BMP) family] has been shown to promote growth. In both the wing and the haltere, Dpp is produced and secreted from a specialized stripe of cells called the AP organizer, which is induced by the juxtaposition of anterior (A) and posterior (P) compartments, two groups of cells that have separate cell lineages. The AP organizer is a stripe of A cells that are instructed to synthesize Dpp by the short-range morphogen Hedgehog (Hh) secreted from adjacent P compartment cells. Dpp has a positive role in appendage growth. When more Dpp is supplied to the wing disc, either ectopically or within the AP organizer, more cells are incorporated into the developing wing field. Conversely, mutations that reduce the amount of Dpp lead to smaller wings (Crickmore, 2006).

A comparison of the expression patterns of Dpp pathway components in the wing and the haltere demonstrates that Ubx is modifying this pathway. Compared with the wing, the stripe of dpp expression in the haltere was reduced in both its width and intensity, as reported by a lacZ insertion into the dpp locus (dpp-lacZ). There was also a difference in the profile of Dpp pathway activation, as visualized by an antibody that detects P-Mad, the activated form of the Dpp pathway transcription factor Mothers against Dpp (Mad). In the wing, P-Mad staining was low in the cells that transcribe dpp. Immediately anterior and posterior to this activity trough, P-Mad labeling peaked in intensity and then gradually decayed further from the Dpp source, revealing a bimodal activity gradient. In contrast, in the haltere intense P-Mad staining was detected only in a single stripe of cells that overlaps with Dpp-producing cells of the AP organizer (Crickmore, 2006).

Because of the coincidence between dpp transcription and peak P-Mad staining in the haltere, it was hypothesized that Dpp might be less able to move from haltere cells that secrete this ligand. This idea was tested by generating clones of cells in both wing and haltere discs in which the actin5c promoter drove the expression of a green fluorescent protein (GFP)–tagged version of Dpp (Dpp:GFP). By using an extracellular staining protocol to analyze simultaneously generated clones, Dpp:GFP and P-Mad were observed much further from producing cells in the wing than in the haltere. These observations strongly suggest that, compared with the wing, Dpp's mobility—and consequently the range of Dpp pathway activation—is reduced in the haltere (Crickmore, 2006).

Whether the decreased production of Dpp in the haltere contributes to the different pattern of pathway activation observed in this tissue compared with the wing was tested. This is unlikely because, even in haltere discs that overexpress Dpp in its normal expression domain, peak P-Mad staining was still observed close to Dpp-expressing cells. Despite increased dpp expression, no P-Mad activity trough was observed in these haltere discs. Further, although they become larger, these discs remained smaller than wild-type wing discs. It is concluded that the decreased Dpp production in the haltere contributes to its reduced growth, but there must be mechanisms that also limit the extent of Dpp pathway activation, even in the presence of increased Dpp production (Crickmore, 2006).

One way in which Dpp's activation profile can be modified is by varying the production of the type I Dpp receptor, Thick veins (Tkv). In the wing, tkv expression is low within and around the source of Dpp, resulting in low Dpp signal transduction in these cells and robust Dpp diffusion. Low tkv expression in the medial wing is due to repression by both Hh and Dpp. Accordingly, tkv expression is highest in lateral regions of the wing disc, where Hh and Dpp signaling are low. In contrast to the wing, tkv transcription and protein levels were high in all cells of the haltere. Thus, the more restricted Dpp mobility and P-Mad pattern in the haltere may result from a failure to repress tkv medially. To test this idea, all cells of the wing disc were supplied with uniform UAS-tkv+ expression, to mimic the haltere pattern. The resulting P-Mad pattern in these wing discs was very similar to that found in the wild-type haltere: The P-Mad trough was gone, and the activity gradient was compacted into a single stripe that coincides with Dpp-producing cells. Conversely, lowering the amount of Tkv in the haltere by expressing an RNA interference (RNAi) hairpin construct directed against tkv (UAS-tkvRNAi) in Dpp-producing cells induced a bimodal pattern of P-Mad staining similar to that of the wild-type wing disc. Thus, different amounts of Tkv result in qualitative differences in the P-Mad profiles of the wing and the haltere (Crickmore, 2006).

It was hypothesized that the more limited pathway activation in the haltere might contribute to its smaller size. If correct, increasing tkv expression in the wing should reduce its size. Adult wings from flies expressing uniform UAS-tkv+ were ~30% smaller than control wings; however, wing cell size remained the same. Similar results were seen in staged imaginal discs and when UAS-tkv+ expression was limited to the wing and the haltere. Conversely, reducing Tkv amounts by uniformly expressing UAS-tkvRNAi in wings and halteres increased haltere size by 30 to 60%. In a complementary experiment, tkv transcription was reduced in the haltere by expressing a known tkv repressor, master of thickveins (mtv). In this experiment, haltere discs were measured instead of the adult appendage; it was consistently found that the appendage-generating region of these discs increased in size by ~40%. Thus, different amounts of Tkv not only affect Dpp pathway activation but also affect organ size. The fact that manipulating only Tkv production does not fully transform the sizes of these appendages suggests that additional mechanisms, such as the reduced amounts of dpp transcription and the modulation of other morphogen pathways by Ubx, also contribute to size regulation. Consistently, when Dpp production is decreased in wing discs that uniformly express UAS-tkv+, wing size was reduced more than it was by either single manipulation (Crickmore, 2006).

Next, how Ubx up-regulates tkv in the haltere was addressed. tkvlacZ expression and amounts of Tkv protein were cell-autonomously reduced in medial Ubx clones, whereas lateral Ubx mutant tissue retained high amounts of Tkv. Because tkv is repressed by Dpp and Hh signaling in the wing, these results suggest that, in the haltere, these signals are not able to repress tkv. Consistently, activation of the Dpp pathway by expressing a constitutively active form of Tkv (TkvQD) resulted in cell-autonomous tkv-lacZ repression in the wing pouch, whereas repression is not observed in the corresponding region of the haltere disc (Crickmore, 2006).

In Ubx mosaic haltere discs, it was also found that medial Ubx+ tissue showed stronger P-Mad staining than Ubx tissue at the same distance from the Dpp source. This observation is interpreted as evidence that Ubx+ tissue is more effective at trapping and transducing Dpp than Ubx tissue because of higher Tkv production in Ubx+ cells (Crickmore, 2006).

To further understand the control of tkv by Ubx, the known tkv repressor, mtv, was examined. In medial wing disc cells, mtv expression is approximately complementary to tkv expression, and mtv clones in this region of the wing disc cell autonomously derepressed tkv. In the haltere, very low mtv-lacZ expression was detected in the cells that stained strongly for P-Mad, suggesting that mtv is repressed by Dpp in this appendage. Accordingly, strong repression of mtv-lacZ was seen in UAS-tkvQD-expressing haltere pouch clones, whereas weak or no repression was seen in analogous wing clones. It was also found that, as expected, Ubx clones in the medial haltere cell autonomously derepressed mtv-lacZ (Crickmore, 2006).

In the wing, Dpp and mtv are mandatory repressors of tkv: In the absence of either, tkv expression is high. In the haltere in the presence of Ubx, Dpp is a repressor of mtv. Consequently, high levels of these obligate tkv repressors (Dpp signaling and mtv) do not coexist in the haltere, resulting in tkv derepression. Consistent with this model, when mtv expression was forced in the medial haltere, where it coexists with Dpp signaling, it repressed tkv-lacZ. It is noted, however, that Ubx is likely to control tkv through additional means, because mtv mutant wing clones did not derepress tkv-lacZ expression to haltere levels, and ectopic mtv in the haltere did not repress tkv-lacZ expression to the extent seen in the medial wing (Crickmore, 2006).

Because of high Tkv production in the wild-type haltere disc, peak Dpp signal transduction occurs in the AP organizer, the same cells that transduce the Hh signal. Thus, in the haltere, the activity profiles for these two signal transduction pathways coincide with each other. In contrast, low tkv expression in the wing AP organizer results in two peaks of Dpp signaling that are on either side of Hh-transducing cells. This difference will have important consequences for the expression of genes that are targets of both pathways. For example, dpp is activated by Hh and repressed by Dpp signaling. In the haltere, these two conflicting inputs occur in the same cells, possibly contributing to reduced dpp expression compared with the wing. Ubx clones cell-autonomously up-regulated dpp-lacZ in the haltere. To test whether Ubx lowers dpp transcription in part by aligning Dpp and Hh signaling, uniform UAS-tkv+ was expressed in the dorsal half of the wing disc. As a result, in this region of the wing disc both signals peaked in the same cells, and dpp-lacZ expression was reduced compared with the ventral half of these wing discs. Conversely, expressing tkvRNAi in dorsal haltere cells increased dpplacZ expression. Thus, Ubx reduces dpp transcription in part by changing where peak Dpp signaling occurs in the disc. Ubx is likely to reduce dpp expression in additional ways, because increasing tkv expression does not lower dpplacZ expression to that observed in wild-type haltere. Nevertheless, varying the relative spatial relationships between signal transduction pathways is a potentially powerful mechanism for modifying the outputs from commonly used pathways. It is suggested that selector genes may work through molecules that control ligand distribution to vary the spatial relationships between these and other signal transduction pathways in diverse contexts during development (Crickmore, 2006).

The finding that increased tkv expression results in decreased dpp transcription reveals an unexpected link between Dpp mobility and Dpp production. Because of this link, the above experiments do not discriminate between growth effects due to differences in Dpp mobility per se as opposed to secondary consequences on Dpp production. To distinguish between these scenarios, use was made of a compartment-specific Ubx regulatory allele, posterior bithorax (pbx), that lacks detectable Ubx in the P compartment when paired with a Ubx null allele but still has normal Ubx expression in the A compartment. Consequently, in pbx/Ubx haltere discs, the P compartment increased in size such that the P:A size ratio was 1.45; the P:A ratio of +/Ubx haltere discs was ~0.35. It is suggested that Dpp more readily diffuses into and through the P compartments of pbx/Ubx discs because of the wing-like expression pattern of tkv and that this wing-like diffusion results in its robust growth (Crickmore, 2006).

To test whether differences in Tkv-regulated Dpp diffusion affect tissue growth independently of an effect on Dpp production, the consequences of expressing UAS-tkv+ uniformly in pbx/Ubx haltere discs were examined. If Tkv's effect on growth is mediated only by lowering Dpp production, both compartments should be reduced in size and thus maintain the same size ratio. However, if reducing Dpp mobility directly affects growth, the P compartment should be reduced in size more than the A compartment, which, in pbx/Ubx discs, already has high tkv expression. It was found that expressing uniform tkv+ in pbx/Ubx discs decreased the size of the P compartment more than the A compartment, resulting in a P:A ratio of 0.83. Because uniform tkv+ returned the P:A ratio back to the wild-type ratio by ~56%, these results suggest that this single variable is sufficient to provide ~50% rescue of the size of an otherwise Ubx mutant P compartment (Crickmore, 2006).

This study has investigated the mechanism underlying a classic yet poorly understood phenomenon in biology: how size variations are genetically programmed in animal development. Many experiments show that organ size is not governed by counting cell divisions but instead depends on disc-intrinsic yet cell-nonautonomous mechanisms, possibly relying on morphogen signaling. The results support this idea by showing that alterations in a morphogen gradient contribute to size differences between appendages. In the example investigated here, Ubx limits the size of the haltere by reducing both Dpp production and Dpp mobility. Moreover, both of these effects are due, in part, to higher tkv expression in the medial haltere. In many morphogen systems, the receptors themselves have been shown to control the distribution of the ligand and, consequently, pathway activation. This study shows that a selector gene exploits this phenomenon to modify organ size (Crickmore, 2006).

Although the mechanism by which Dpp controls proliferation is not fully understood, recent results argue that, in the medial wing disc, cells may compare the amount of Dpp transduction with their neighbors, whereas lateral cells proliferate in response to absolute Dpp levels. The results suggest several ways in which the altered Dpp gradient in the haltere could limit its growth. First, proliferation of lateral haltere cells may be limited because they perceive less Dpp. Second, the narrower Dpp gradient results in fewer cells exposed to the gradient in the medial haltere. Another notable difference is that, because there are two peaks of Dpp signaling in the wing but only one in the haltere, the wing has four distinct slopes whereas the haltere has only two. The less complex Dpp activity landscape of the haltere may also contribute to its reduced growth (Crickmore, 2006).

On the basis of these results, it is suggested that altering the shape and intensity of morphogen gradients may be a general mechanism by which selector genes affect tissue sizes in animal development. Consistent with this view, wingless (wg), another long-range morphogen in the wing, is partially repressed in the haltere. Intriguingly, some of the size and shape differences in the beaks of Darwin's finches are controlled by alterations in the production of the Dpp ortholog BMP4. The results suggest that differences in the diffusion of this ligand may also contribute to the range of beak morphologies that have evolved in these species (Crickmore, 2006).

Drosophila eggshell is patterned by sequential action of feedforward and feedback loops

During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad (Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. The resulting changes in the Br pattern affect the expression of Dpp receptor thickveins (tkv), which is essential for Dpp signaling. By controlling tkv, Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative-feedback loop. These observations have been synthesized into a model, whereby the dynamics of Br expression are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br expression, while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene expression can emerge from simple inputs, through the interaction of regulatory network motifs (Yakoby, 2008).

These results provide new insights into the dynamics and function of the Dpp pathway in oogenesis. First, it was demonstrated that, contrary to the current model of Drosophila eggshell patterning, the pattern of Dpp signaling in oogenesis is not static, and undergoes a clear transition from purely AP to DV pattern in late stages of eggshell patterning. This transition reflects the change in the expression of the type I Dpp receptor and is conserved in fly species separated by more than 40 million years of evolution. Second, it was shown that the early and late patterns of Dpp signaling control the dynamic pattern of br, a transcription factor expressed in the roof of future dorsal appendages. While the AP phase of Dpp signaling represses br in the anterior region of the egg chamber, the DV phase of Dpp signaling limits the duration of br expression in the roof cells. Third, it was established that, in addition to being regulated by Dpp, Br actively controls Dpp signaling, thus regulating its own repression via a negative-feedback loop (Yakoby, 2008).

The results lead to a new model for the dynamics of Br expression in the roof cells. Within the framework of this model, the rising phase of Br expression is due to an incoherent feedforward loop, a network in which the input activates both the target and its repressor. In this case, the feedforward loop, formed by EGFR, Pointed, and Br, determines the spatial pattern of Br. This pattern is then modulated in time by a negative-feedback, which depends on the Br-mediated increase of tkv expression and Dpp signaling. The feedforward part of the model is supported by the previously published gain- and loss-of-function experiments with Pointed and EGFR signaling, and by the current analysis of Br expression in ras- mosaics. The negative-feedback loop is supported by the correlation of patterns of Br, Tkv, and P-Mad, by published experiments with manipulation of the levels of Dpp, by analysis of Br protein and br transcript in the Dpp pathway loss-of-function experiments, and by the effects of br- clones and Br overexpression on tkv and Dpp signaling (Yakoby, 2008).

Four phases in the dynamics of the Br pattern are distinguished. (1) Low levels of Br before stage 9 of oogenesis are independent of EGFR signaling and insensitive to repression by Dpp. (2) Following the formation of the DV gradient of EGFR activation, Br is repressed in the midline and in the dorsoanterior cells. The midline repression is due to Pointed, a transcription factor induced by high levels of EGFR activation in the dorsal midline. The dorsoanterior repression is due to the early phase of Dpp signaling, which reflects the anterior secretion of Dpp and uniform expression of Tkv. (3) Levels of Br begin to rise in the roof cells. Changes in the Br pattern have two effects on the spatial pattern of Dpp signaling: higher levels of Br lead to higher levels of tkv in the roof cells. Second, the dorsoanterior and midline repression of Br generates a corresponding repression of tkv. (4) As a result, the anteriorly produced Dpp can diffuse over the 'Tkv-free' area to the roof cells. A combination of the arrival of the anteriorly produced ligand and a higher level of receptor expression leads to a higher level of Dpp signaling in the roof cells and subsequent repression of br. Another layer of regulation is provided by Brk, a transcriptional repressor of Dpp signaling, which is induced by Gurken and repressed by Dpp signaling in the dorsal follicle cells. Brk antagonizes the repressive effect of Dpp in the roof cells until the level of Dpp signaling in the roof cells becomes high enough to repress Brk expression (Yakoby, 2008).

The network characterized in this study can interact with a number of previously discovered feedback loops. For instance, Argos, which provides negative-feedback control of EGFR signaling in the dorsal midline, is a potential target of Dpp signaling. Future work is required to explore the extent to which this feedback loop, which had been proposed to affect dorsal midline patterning, interacts with the mechanism established in this paper (Yakoby, 2008).

Haemocytes control stem cell activity in the Drosophila intestine

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

Targets of Activity

In the embryonic midgut, mutations affecting thickveins block the expression of two decapentaplegic-responsive genes, dpp and labial (Penton, 1994).

While maternal tkv product allows largely normal dorsoventral patterning of the embryo, zygotic tkv activity is indispensable for the dorsal closure of the embryo after germ band retraction. Furthermore, tkv activity is crucial for patterning the visceral mesoderm; in the absence of functional tkv gene product, visceral mesoderm parasegment 7 cells fail to express Ultrabithorax, but instead accumulate Antennapedia protein. The TKV receptor is therefore involved in delimiting the expression domains of homeotic genes in the visceral mesoderm.

Interestingly, tkv mutants fail to establish a proper tracheal network. Tracheal braches formed by cells migrating in dorsal or ventral directions are absent in tkv mutants. The requirements for tkv in dorsal closure, visceral mesoderm and trachea development assign novel functions to DPP or a closely related member of the TGF beta superfamily (Affolter, 1994).

Decapentaplegic, through its receptors Thickveins and Punt targets optimotor blind and spalt transcription in the wing imaginal disc. The range of DPP action is wide, affecting spalt and omb expression on both sides of the anterior-posterior compartment boundary. The finding of an extended range of action for DPP is unexpected, yet DPP diffusion away from its site of expression may be limited by its tendency to be sequestered by components of the extracellular matrix. spalt and omb respond differently to the DPP concentration gradient, with omb showing a wider range of response due to its greater sensitivity to low DPP concentrations (Nellen, 1996)

brinker expression in the imaginal discs is not uniform but shows complementarity to regions of Dpp signaling. In wing discs, brk is highly expressed in lateral regions that are distant from the Dpp source in the center of the disc. In leg discs, brk expression is lowest in the dorsal compartment, which is specified by high levels of Dpp signaling. Double stainings for brk-lacZ and Omb protein demonstrate the complementarity between high levels of brk transcription and the expression of a low-threshold target gene of Dpp in wing and leg imaginal discs. They also reveal a narrow zone of overlap between low brk levels and omb expression in the wing pouch, suggesting that brk expression extends into regions of low-level Dpp signaling. In this region of overlap between Omb and brk, brk levels are declining in a graded fashion and become undetectable at positions where Sal expression starts. The complementarity between brk expression and regions of Dpp signaling may reflect a negative regulation of brk by Dpp. Consistent with this view, clones of mutant cells missing the Dpp receptor Tkv express high levels of brk, irrespective of their location within the wing pouch. Thus, brk expression would occur evenly throughout the wing pouch in the absence of a Dpp gradient emanating from the center of the disc. An important function of Dpp signaling in the wing disc might be to generate the asymmetric distribution of a repressor (such as brk) of Dpp's target genes (Jazwinska, 1999).

The identification of mutations in Tgfbeta-60A as dominant enhancers of tkv 6 in the imaginal discs raises the possibility that Tgfbeta-60A is required for optimal signaling by the dpp pathway. To determine if there is a general requirement for Tgfbeta-60A in dpp signaling, the effects of Tgfbeta-60A mutations were examined on dpp signaling in the visceral mesoderm where both dpp and Tgfbeta-60A are expressed. dpp is expressed in two discrete domains in the visceral mesoderm. The anterior domain of dpp coincides with the gastric caecae primordia, which are immediately anterior to the expression domain of Sex combs reduced (Scr) in parasegment (ps) 4. The failure to initiate dpp expression in ps3 in dpp shv mutants results in anterior expansion of Scr expression and arrested outgrowth of the gastric caecae, indicating a role for dpp in repressing Scr in ps3. tkv 6 homozygotes are homozygous viable, so it is not surprising that all the midgut gene expression patterns examined are essentially normal. Scr expression in tkv 6 and Tgfbeta-60A mutants is normal. However, in tkv 6 and Tgfbeta-60A double mutants, the Scr expression extends anteriorly into ps3 as it does in dpp shv mutants, suggesting that Tgfbeta-60A activity is required in ps3 for optimal dpp signaling (Chen, 1998).

During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted Tgf-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative Tgf-beta-like receptors Thickveins and Punt activates a Tgf-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the Tgf-beta-like signaling pathway can disrupt dorsal closure. Although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the Tgf-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK (Ricos, 1999).

Thick veins is likely to target p38b, a MAP kinase implicated in Dpp signal transduction. Two Drosophila homologs of p38, Mpk2 (also known as p38a or simply p38) and p38b, have been identified on the basis of their homology to mammalian p38 and to one another. p38b is maternally expressed and is present ubiquitously during embryonic development (Han, 1998). The chromosomal region around the p38b locus has been well characterized genetically. However, a p38b transgene was unable to rescue any of the known mutations mapping to this region. Likewise, attempts to isolate a mutant of p38b were unsuccessful. These failures are possibly due to the functional redundancy of the two p38 homologs. Various alternative methods were therefore use to interfere with endogenous p38(s) in order to investigate its function. A dominant-negative allele of p38b, designated D-p38bDN, was generated by replacing the Thr-183 of the MAPKK target site with Ala, analogous to the change in ERK2 that produces a dominant-negative allele (Adachi-Yamada, 1999).

Two lines were prepared which express D-p38bDN at different levels: D-p38bDN-S (Strong), which expresses high levels, and D-p38bDN-W (Weak), which expresses low levels. When two copies of the D-p38bDN-S transgene are expressed in the wing, a certain fraction of adult flies that escape death exhibit ectopic vein fragments around the end of the longitudinal vein L2 and a reduction in the distance between L4 and L5. Both of these features have also been observed with some mutant alleles of decapentaplegic and thick veins. This wing phenotype is rescued by coexpression of the wild type p38b+ transgene. When two copies of the D-p38bDN-S transgene are weakly expressed in the wing of a dpp mutant, the vein phenotype of dpp is strongly enhanced. These phenotypes suggest the involvement of Drosophila p38(s) in Dpp function in the early and late stages of wing pattern development. Dpp is known to play a dual role during wing development, acting as a morphogen and mitogen at early stages, while activating vein differentiation at later stages (Adachi-Yamada, 1999).

To examine whether p38(s) functions in the Dpp signaling pathway, the genetic interaction was examined between p38(s) and a constitutively active mutant of Tkv (TkvCA). Two classes of tkvCA insertions, tkvCA-S (Strong) and tkvCA-W (Weak), were used. When tkvCA-S is expressed, normal wing venation is severely distorted and extensive production of fragments of vein material is observed. The abdominal-cuticle pattern also appears irregular. This wing phenotype suggests that TkvCA may influence Dpp action during vein formation. Ectopic coexpression of dpp+ and tkv+ causes similar phenotypes, indicating that these TkvCA-induced aberrations are indeed the result of an increase in Dpp signaling. It was thus expected that reducing the levels of downstream components would suppress tkvCA. In fact, reducing by one-half the gene dosage of Mothers against dpp (Mad), a well-documented Dpp-signaling factor, significantly suppresses the tkvCA wing phenotype (Adachi-Yamada, 1999).

The effect of the imidazole compound SB203580, a p38 inhibitor, was tested on the tkvCA wing phenotype. SB203580 has been reported to inhibit both p38a and p38b (Z. Han, 1998), and penetration of various imidazole compounds through the insect epidermis is well known. Exposure of growing larvae to SB203580 indeed results in suppression of the phenotype. Tests were performed to see whether endogenous p38 genes are involved in the tkvCA wing phenotype by reducing endogenous gene dosage using chromosomal hemizygosity. Interestingly, reduction of p38b suppresses the tkvCA wing phenotype, while reduction of p38a is not effective. Suppression by reduction of the p38b gene dosage is abrogated by the introduction of a transgene for p38b+. Thus, the gene within the deficiency that suppresses tkvCA is indeed p38b. These results suggest that p38b plays a major role in this morphogenetic process, and attention was focussed on this gene in further analyses (Adachi-Yamada, 1999).

Antisense RNA can often be used to mimic the effects of mutation. When antisense p38d RNA is coexpressed with TkvCA-S, the tkvCA-S phenotype is markedly suppressed. Four of five independently established p38bantisense lines showed significant suppression. Suppression affects the various pleiotropic phenotypes associated with the tkvCA allele, including wing blade morphology, abdominal-cuticle morphology, and wing posture. Similar suppression of the tkvCA phenotype is also achieved by coexpression of p38bDN-W in a dose-dependent manner. This suppression is greater when the strong dominant negative p38b (p38bDN-S) is coexpressed instead of p38bDN-W. Furthermore, this suppression is abrogated by simultaneous coexpression of wild type p38d, demonstrating that p38bDN and wild type p38b competitively sequester endogenous factors essential to signaling. These results suggest either that p38b functions downstream of Tkv or that inhibition of p38b causes a reduction in endogenous dpp activity. Since the expression pattern of dpp in the developing wing of the D-p38bDN-S producer has been found to be indistinguishable from that of the wild type, and reduction in the gene dosage of dpp is not effective in suppressing the tkvCA phenotype, it is concluded that p38b does not affect Dpp production per se but rather acts as a downstream component of the Dpp-Tkv signaling pathway, operating late in wing development. The fact that the weak phenotype of tkvCA-W is significantly enhanced by wild type p38b is also consistent with this conclusion (Adachi-Yamada, 1999).

The effect of p38b on optomotor blind transcription was examined in order to study the involvement of p38b in the Dpp signaling pathway. The omb gene encodes a T-box family transcription factor, and its expression in the wing imaginal disc is dependent on early Dpp-Tkv signaling. In the wing discs of flies ectopically expressing tkvCA-5, the omb expression domain is greatly expanded and overgrowth of the disc is evident. Expression of p38bDN or p38bantisense markedly suppresses both omb expression and disc overgrowth. Induction of omb in the tkvCA-expressing clones in regions outside those where dpp is expressed is also inhibited by coexpression of p38bDN, consistent with the possibility that p38b functions downstream of Tkv. Furthermore, while p38bDN slightly affects omb expression in a tkv+ genetic background, the wing phenotype of a hypomorphic omb allele is clearly enhanced by expression of p38bDN or p38bantisense, as observed in the wing phenotype of severe omb alleles. These results suggest that p38b is also involved in early Dpp-Tkv signaling in wing development to activate omb transcription. Evidence is presented that p38b, or possibly both p38s, are phosphorylated in vivo downstream of ectopically expressed constitutively active Tkv (Adachi-Yamada, 1999).

To investigate whether p38b is activated by Tkv signaling, a preliminary biochemical characterization of p38b was carried out. Immediately after heat treatment of flies, the amount of p38b immunoprecipitated by anti-p-Tyr antibody was found to increase considerably, demonstrating that p38b is tyrosine phosphorylated following heat shock, like mammalian p38. The site of tyrosine phosphorylation is expected to be in the 'activation loop' region recognized by MAPKK, as is the case in mammalian p38. Thus, a test was performed to see whether an anti-phospho-p38 (anti-p-p38) antibody raised against a phosphorylated peptide from the activation loop of mammalian p38 could cross-react with p38b. This anti-p-p38 antibody detects a protein with a calculated size of 42 kDa whose amount increases immediately after heat shock. This protein is also more abundant in the flies overproducing p38b regardless of heat treatment. Therefore, it has been concluded that anti-phospho-p38 can cross-react with the phosphorylated from of p38b and can be used to assay recombinant p38b phosphorylation in vitro. Treatment of p38b with recombinant human MKK6, a MAPKK that activates p38, causes a marked increase in the level of p38b, as detected with anti-phospho-Tyr and anti-p-p38 antibodies, and a drastic increase in the level of Drosophila p38-dependent phosphorylation of recombinant human activating transcription factor 2 (ATF2), a physiological substrate for mammalian p38. The correlation between the phosphorylation state and kinase activity of p38b indicates that the anti-p-p38 antibody recognizes the active form of p38b. This allowed activation of p38b by TkvCA to be examined in vivo. The amount of active p38b was found to be slightly but significantly higher in larvae carrying ectopically expressed tkvCA relative to that in wild-type Canton-S larvae. However, it has been reported that p38a protein expressed in yeast, which was presumed to have the same molecular mass as p38b, is also recognized by anti-p-p38 antibody. It is therefore possible that p38b, or both D-p38's, may be activated by Tkv signaling in vivo (Adachi-Yamada, 1999).

The Drosophila wing is divided into two compartments along its anteroposterior (A/P) axis. The compartment boundary between these regions serves as the source of an organizing activity that patterns both anterior and posterior compartments. This activity is mediated, at least in part, by the long-range action of Dpp, which is expressed by cells along the A/P compartment boundary. Dpp is thought to act as a morphogen to inform target cells of their position along the A/P axis, but as yet, little is known about how cells interpret the distribution of Dpp protein. An enhancer trap screen was conducted to identify genes whose transcription is controlled by Dpp. Two enhancer trap lines in the same locus (89E/F), P1883 and 1(3)1E4, were identified whose expression patterns are similar to those of Dpp during embryonic and imaginal development. The gene whose expression is reflected in these enhancer traps has been named Daughters against dpp (Dad). In these enhancer trap lines, beta-galactosidase is expressed in a wide stripe that straddles the A/P compartment boundary of the imaginal discs, in contrast to Dpp, whose expression is confined to the anterior side. This pattern of expression suggests that Dad expression is positively regulated by the secreted Dpp molecule. To test whether Dad responds to Dpp signaling, its expression has been examined in P1883 wing discs in which a UAS-dpp transgene was transcribed in a ring around a wing pouch under the control of a Gal4 driver. Ectopic Dpp expression results in abnormally large discs and in ectopic expression of Dad in a broad ring around a wing pouch. Identical results were obtained when another transgene was used -- UAS-tkv Q253D -- which encodes a constitutively active form of the major type-I Dpp receptor, Thick veins. In addition, expression of Dad is not detected in cells that lack a functional Tkv Dpp receptor. These results indicate that Dpp signaling is necessary and sufficient for Dad expression in the developing wing (Tsuneizumi, 1997).

Drosophila LSD1-CoREST demethylase complex regulates DPP/TGFβ signaling during wing development

The choice and timing of specific developmental pathways in organogenesis are determined by tissue-specific temporal and spatial cues that are acted upon to impart unique cellular and compartmental identities. A consequence of cellular signaling is the rapid transcriptional reprogramming of a wide variety of target genes. To overcome intrinsic epigenetic chromatin barriers to transcription modulation, histone modifying and remodeling complexes are employed. The deposition or erasure of specific covalent histone modifications, including acetylation, methylation, and ubiquitination are essential features of gene activation and repression. This study has found that the activity of a specific class of histone demethylation enzymes is required for the specification of vein cell fates during Drosophila wing development. Genetic tests revealed that the Drosophila LSD1-CoREST complex is required for proper cell specification through regulation of the DPP/TGFβ pathway. An important finding from this analysis is that LSD1-CoREST functions through control of rhomboid expression in an EGFR-independent pathway (Curtis, 2012).

The Su(var)3-3 gene (CG17149) encodes the Drosophila LSD1 homolog. Mutations in Su(var)3-3 result in aberrant histone methylation and heterochromatin formation, with increased global levels of H3K4me2 and impaired heterochromatic gene silencing. A physical association between LSD1 and CoREST has been described in Drosophila, revealing that the critical relationship between these proteins is conserved. LSD1 has an important role in organogenesis and germ line maintenance, such as during mouse anterior pituitary development and Drosophila ovary and wing development. LSD1 also regulates neural stem cell proliferation by modulating signaling via the orphan nuclear receptor TLX and LSD1 appears to have distinct functions in mammalian neuronal morphogenesis as well as stem cell self-renewal and differentiation. In humans, loss of LSD1 has been strongly correlated with several types of cancer and high-risk tumors, including prostate cancer, breast cancer and neuroblastomas). In contrast, overexpression of LSD1 has also been linked to some cancers. As a consequence of the emerging links between histone demethylase functions and disease, an understanding how LSD1 contributes to specific cell-cycle regulation and developmental processes is crucial (Curtis, 2012).

The Drosophila wing provides an outstanding in vivo model system to identify factors that regulate cell-fate determination as alterations in cell-fate can often be observed at the single cell level. Multiple conserved signaling pathways contribute to wing patterning and development and are regulated, in part, by the coordinated activities of chromatin remodeling complexes and epigenetic modifying enzymes. Previously work has identified histone lysine demethylase enzymes as coregulators of Brm complex remodeling activities in a genetic screen for factors that influenced a wing patterning phenotype associated with a conditional loss-of-function mutation in the snr1 gene that encodes a core regulatory subunit of the Brm complex. Genetic interaction tests indicated that lsd1 (Su[var]3-3) most likely interacted with the PBAP subtype of the Brm complex (Curtis, 2011). This report further addresses how LSD1 contributes to the cell-type and developmental time-point specific regulation of conserved signaling pathways by understanding its contribution to wing patterning and development (Curtis, 2012).

Recently, it was suggested that LSD1 regulates notch signaling during Drosophila wing development (Mulligan, 2011). This study presents evidence from genetic interaction analyses and tissue or cell-type specific targeted depletion experiments that suggest LSD1 and CoREST/CG42687 (synonymous with CG33525) may also regulate the DPP/TGFβ signaling pathway in a noncanonical manner, by regulating expression of rhomboid, a key player in canonical EGFR signal transduction. This is the first demonstration of LSD1-CoREST regulated DPP/TGFβ signaling and the results further define important roles of the LSD1-CoREST complex in tissue patterning (Curtis, 2012).

The appropriate elaboration of wing vein and intervein cell fates depends on the interplay of factors that promote and those that repress or block vein cell differentiation. In this study, we provide genetic evidence suggesting an important role for lsd1 and CoRest in repressing vein-promoting genes in intervein cells. Ectopic vein development can result from either the loss of a factor required for repressing vein cell differentiation or the gain of a factor that promotes vein cell fate in intervein cells. The experimental results suggest that lsd1 and CoRest utilize the first mechanism, since the aos hypomorphic mutation (aosw11), a factor known to repress vein fate, is enhanced by CoRestEY14216 and lsd1ΔN and targeted depletion by shRNAi of lsd1 and CoRest throughout the entire developing wing imaginal disc resulted in ectopic veins rather than loss of vein phenotypes. It was reasoned that if the LSD1-CoREST complex normally functions as a positive factor to promote vein development as proposed by the second mechanism, then mutations in lsd1 and CoRest or shRNAi depletion in the wing imaginal disc should produce a loss of vein phenotype. Based on the evidence presented in this manuscript, and on the recent finding that LSD1 is important for the regulation of NOTCH signaling in the wing (Di Stefano, 2011), it is proposed that the requirements of LSD1-CoREST are temporal and cell-type specific, and possibly dependent on the physical associations between LSD1 and several multiprotein complexes (Curtis, 2012).

An elaborate signaling network regulates wing patterning, where considerable cross-talk and functional redundancy connects five developmental pathways. For example, during pupal development, the main role of EGFR and DPP activation is to coordinately promote and maintain differentiation into vein cells while NOTCH activation establishes the provein-intervein boundary. However, DPP and NOTCH pathways are codependent, since expression of the NOTCH ligand, DELTA (DL) and its downstream target, ENHANCER OF SPLIT, (E(spl)mβ), require DPP signaling. LSD1 has been shown to interact directly with the histone deacetylase SIRT1 to repress NOTCH targets, suggesting important epigenetic functions for these co-repressors in metazoan development. However, recently it was shown that CoREST could function as a positive regulator of NOTCH in Drosophila follicle cells and wings (Domanitskaya, 2012). Therefore, there is growing precedent for the LSD1-CoREST complex to have both positive and negative roles in regulating gene expression depending on developmental context (Curtis, 2012).

LSD1 and CoREST depletion in the developing wing causes bifurcated or duplicated crossveins, a phenotype previously observed with Hairless (H) loss of function mutations. Because H both antagonizes NOTCH and promotes EGFR signaling, it is difficult to decipher the individual pathway regulated by LSD1-CoREST. Furthermore, the broadened vein delta phenotype observed at the wing margin in wing-specific LSD1-CoREST depleted and lsd1ΔN null flies (Di Stefano, 2011) is similar to Notch and DPP receptor (tkv) loss of function phenotypes (Curtis, 2012).

It is proposed that during the initial stages of wing vein development and differentiation, LSD1 negatively regulates NOTCH signaling. This is based on the observation that loss of lsd1 function suppresses the notched wing phenotype associated with mutations in suppressor of hairless (Su[HT4]) (Mulligan, 2011). However, later in development during vein refinement and maintenance, LSD1 appears to undergo a regulatory switch to positively regulate NOTCH signaling, since lsd1ΔN suppresses the short vein phenotype associated with the gain-of-function NAx-16 mutation. Additionally, the increased expression of downstream E(spl) targets in NAx-16 mutants is reversed by lsd1ΔN (Di Stefano, 2011). It was also recently shown that a transheterozygous mutant allele of CoRest (CoRestGF60) could enhance the wing phenotypes of flies carrying alleles of Dl and N (Domanitskaya, 2012), suggesting positive functions in regulating NOTCH signaling. Concurrently, LSD1 and CoREST repress vein cell differentiation by regulating components of the DPP signaling pathway at multiple points. For example, lsd1ΔN and CoRestEY14216 genetically interact with both dpp and genes encoding its receptors (e.g., dpp, tkv, sax), consistent with upstream functions. Strong genetic interactions were observed with downstream DPP signaling components (e.g., mad, med, ara, caup, shn), which suggests that the LSD1-CoREST complex has important regulatory functions in controlling the expression of DPP pathway targets. This conclusion is further supported by ectopic expression of the DPP-specific downstream signaling component, p-MAD, was observed in LSD1-CoREST-depleted animals. Activated DPP signaling is confined to proveins largely by the overexpression of TKV, a member of the TGFβ receptor family, in intervein boundary cells. TKV binds and sequesters the DPP morphogen. When TKV is downregulated, DPP spreads into regions of the wing destined to become intervein cells, resulting in ectopic veins. It is predicted that TKV is the most likely target of LSD1-CoREST complex regulation, since genetic interactions were observed between lsd1ΔN and CoRestEY14216 and almost all loss of function mutations in DPP signaling components, and tissue-specific LSD1-CoREST depletion lead to the development of ectopic veins, similar to phenotypes observed with loss of function alleles of tkv. Because activation of NOTCH and repression of DPP signaling are both required to repress vein promoting genes in differentiating intervein cells, LSD1 appears to have cell-type and context-specific activities to differentially regulate these pathways (Curtis, 2012).

Coimmunoprecipitation experiments suggested a complex forms between the HDAC1/2 class protein RPD3, LSD1, CoREST, and two TTK splice variants TTK88 or TTK69. Complexes containing CoREST/TTK69 or CoREST/TTK88 independently localize on polytene salivary glands, suggesting differential gene targeting. TTK and REST are likely functional homologs. Orthologs of tramtrack only exist in invertebrates, whereas REST orthologs are vertebrate-specific. TTK69 is a transcription factor that can recognize and bind to a specific DNA RE-1 consensus sequence (CCAGGACG), resulting in gene transcription. Unpublished observations suggest that TTK69, but not TTK88, function to negatively regulate vein cell development, since an incomplete vein phenotype is observed when TTK69 is overexpressed, whereas overexpression of TTK88 results in the development of ectopic veins. Therefore, it is predicted that LSD1-CoREST-TTK69 form a complex in developing wing tissue to negatively regulate DPP signaling in intervein cells. Furthermore, in mammals, the Brg1 complex chromatin remodeling capacity and recruitment specificity depends on formation of a LSD1-CoREST-REST-BRG1 comple. Because LSD1 can physically associate with the Brm chromatin remodeling complex in Drosophila, it is predicted that the Brm complex-LSD1-CoREST-TTK69 super-complex regulates genes essential for wing patterning, possibly through co-localization or recruitment to RE-1 consensus binding sites. Intriguingly, RE-1 consensus sites are present in both the rho and tkv gene loci, making these exciting targets for future investigation (Curtis, 2012).

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. This study shows that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. Similar roles for Fu were observed during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism (Xia, 2010).

Previous studies have demonstrated that BMP/Dpp signals from the niche play primary roles in the self-renewal of GSCs by silencing bam transcription. However, the mechanism by which the differentiating CBs avoid the control of BMP/Dpp and activate bam remains poorly understood. This study has provided direct evidence that the differentiating daughter cells of GSCs, known as CBs, become resistant to BMP signaling through degradation of Tkv in CBs. Fu functions as an antagonistic factor in BMP/Dpp signaling by regulating Tkv degradation during the differentiation of CBs. Moreover, both genetic and biochemical evidence is provided that Fu acts in concert with Smurf, a HECT domain-containing ubiquitin E3 ligase, to regulate the ubiquitination of Tkv in the CB, thereby generating a steep gradient of response to BMP signaling between GSCs and CBs for their fate determination. Finally, a conserved role is shown for fu in antagonizing BMP/ TGFβ signals in zebrafish embryonic development as well as in human cell cultures. These findings not only reveal a conserved function of fu in controlling BMP/TGFβ signal-mediated developmental processes, but also provide a comprehensive view of mechanisms that produce both self-renewal and asymmetry in the division of stem cells (Xia, 2010).

Observations of the existence of a BMP resistance mechanism that controls the proper division of GSCs through the regulation of Tkv prompted an exploration of how Tkv was regulated. Using immunoprecipitation followed by mass spectrometry analysis, it was identified that Fu associates with the Tkv protein. Given that previous studies demonstrated that a loss of fu leads to early germ cell proliferation and a tumorous germarium phenotype and that biochemical evidence showed that Fu forms a complex with Tkv and affects its stability, it was subsequently identified that Fu as a component negatively regulates BMP/Dpp signaling by interacting with the BMP/Dpp type I receptor, Tkv (Xia, 2010).

BMP/TGFβ signals play pivotal roles in controlling diverse normal developmental and cellular processes. In the canonical BMP/TGFβ pathway, the receptors and Smad proteins are the essential components for BMP/TGFβ signal transduction. However, this pathway is known to be modulated by additional factors to reach physiological levels in a cellular context-dependent manner. Smurfs and HECT domain-containing proteins have been shown to antagonize BMP/TGFβ signals through the regulation of the stability of either receptors or Smads in vertebrates. In Drosophila, Smurf has previously been implicated in regulating proteolysis of phosphorylated Smad proteins in somatic cells. In the ovary, Smurf was also proposed to downregulate the level of BMP to promote CB differentiation. The mechanism underlying the action of Smurf in Drosophila early germline cells remains elusive. This study has shown that Fu, Smurf, and Tkv could form a trimeric complex in S2 cells. Importantly, both Fu and Smurf are required for ubiquitination of Tkv in S2 cells and for turnover of Tkv in germ cells. Combined with genetic evidence, it is proposed that Fu and Smurf likely function in a common biochemical process by controlling Tkv degradation. The present study reveals a mechanism by which Fu serves as an essential component in the Smurf-mediated degradation of the BMP/TGFβ receptor, thereby terminating BMP/TGFβ signaling and negatively regulating the downstream target genes of BMP/TGFβ (Xia, 2010).

Because Fu is a putative serine/threonine protein kinase, the question becomes how Fu acts on Tkv regulation in concert with Smurf. Given that knockdown of fu does not significantly change the pattern of autoubiquitination of Smurf itself, it is therefore likely that Tkv is a strong candidate substrate for Fu kinase. Although there is no assay system for analyzing the kinase activity of Fu presently, in this study, mutagenesis assays were perfomred and it was identified that the S238 in Tkv is important for Tkvca to respond to Fu and is critical for Tkvca ubiquitination and degradation. Of note, it was found that the ubiquitin- resistant form of Tkvca [TkvcaS238A] blocks CB differentiation. A previous study has shown that the S189 site in TGF-β type-I receptor, the corresponding site of S238 in Tkv, was phosphorylated in the cell culture system. The current results suggest that Fu likely acts on Tkv through targeting and phosphorylating the S238 site and subsequently leads to Tkv ubiquitination and degradation by Smurf. Nevertheless, it would be advantageous to develop a kinase assay system for Fu to determine whether the S238 site in Tkv is an authentic phosphorylation site for Fu kinase in the future (Xia, 2010).

Previous genetic analyses revealed that Fu plays an evolutionarily conserved role in the proper activation of the Hh pathway and functions downstream of the Hh receptor. Increasing evidence has shown that the kinase Fu regulates the Hh-signaling complex by targeting Cos2. However, the function of Fu as a component in the Hh pathway is not consistent with its spatiotemporal expression pattern during development. For example, Hh signaling only plays a role in zebrafish embryonic development at late stages, but Fu is expressed ubiquitously at both the early and the late stages of zebrafish embryonic development. These findings suggest that Fu may have Hh-independent functions in different physiological conditions. In this study, by using several different systems, including Drosophila germline, zebrafish embryo, and human tissue cultures, it was demonstrated that Fu is indeed required for balancing proper BMP/TGFβ signals in different developmental processes. Given that both Fu and Smurf are evolutionarily conserved proteins, it would be interesting to determine whether the Fu/Smurf complex also plays roles in other signaling pathways (Xia, 2010).

The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. It was recently revealed that the Fused (Fu)/Smurf complex degrades the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 1012).

The present data strongly imply that GSC/CB fate finely controlled by Fu protein regulation is important for generating BMP activity gradient between GSCs and CBs. However, the remaining important question is how to understand the mechanism by which the dynamic reciprocal antagonism between Tkv and Fu controls the GSC-to-CB fate switch during GSC division. To clearly answer this question, a mathematical network model was developed based on the experimental evidences with bistable behavior to elucidate how the feedback loop regulation determines the fate specification of GSCs (Xia, 1012).

On the basis of these data, a feedback loop model is proposed to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 1012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre- CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 1012).

thickveins: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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