Table of contents

Wingless and muscle progenitors

The patterning of the Drosophila mesoderm requires Wingless. Little is known about how Wg provides patterning information to the mesoderm, which is neither an epithelium nor contains the site of Wg production. Studying specification of muscle founder cells as marked by the lineage-specific transcription factor Slouch, it was asked how mesodermal cells interpret the steady flow of Wg. Through the manipulation of place, time and amount of Wg signaling, it has been observed that Slouch founder cell cluster II is more sensitive to Wg levels than the other Slouch-positive founder cell clusters. To specify Slouch cluster I, Wg signaling is required to maintain high levels of the myogenic transcriptional regulator Twist. However, to specify cluster II, Wg not only maintains high Twist levels, but also provides a second contribution to activate Slouch expression. This dual requirement for Wg provides a paradigm for understanding how one signaling pathway can act over time to create a diverse array of patterning outcomes (Cox, 2005).

In wg mutant embryos, the heart and approximately half the body wall muscles are lost. One subset of these Wg-dependent body wall muscles can be visualized using an antibody to the NK-homeodomain protein Slouch (S59). Slouch expression arises in a precise, stereotypic pattern during embryonic development. It is first expressed in a single progenitor cell during early stage 11 of embryonic development; this cell divides to give rise to two founder cells (Ia and Ib) which together form cluster I (cI). During late stage 11, two additional Slouch-positive progenitors appear at a different ventral location and divide sequentially to form four founder cells that make up cluster II. Still later, at stage 12, a single progenitor arises dorsally and divides to give rise to cluster III. These muscle founder cells contain all the information needed to create a particular subset of muscles and contribute to the stereotypic set of larval muscles in each abdominal segment. After stage 12, Slouch expression is maintained in a subset of these founder cells that give rise, in the final muscle pattern, to muscle VT1 (from cI), VA2 (from cII) and DT1 (from cluster III). Maintenance of Slouch expression in these founder cells is crucial to the development of these muscles; removal of slouch leads to complete (VT1) and partial muscle transformations (VA2; DT1). In this study, focus was placed on the role of Wg in patterning the Slouch muscle founder cells. For simplicity, focus was placed solely on two ventral Slouch clusters (I and II), which develop independently and arise in a similar position along the dorsoventral axis but have different anterior-posterior positions within each abdominal hemisegment (Cox, 2005).

Through the manipulation of the amount and time of exposure to Wg signaling in the Drosophila mesoderm, it is shown that Slouch founder cell cII requires more Wg signaling than its neighbor, cI. Because cII arises in the mesoderm beneath the source of Wg signal, it was initially thought that the sensitivity that was detected would be due to Wg acting as a classic morphogen. Specifically, during stage 11, Wg would directly elicit concentration-dependent responses, leading to Slouch cI specification at low levels and cII at higher levels. Instead, the data suggest an alternative mechanism underlying this sensitivity. For Slouch cI, Wg signaling through Twist is sufficient for fate specification. However, for Slouch cII, a second, Twist-independent Wg signal is also necessary (Cox, 2005).

It has been shown that wg mutants fail to maintain high levels of Twist. Overexpression of Twist leads to expanded somatic mesodermal fates at the expense of other mesodermal fates, such as heart and gut muscle. Conversely, decreasing Twist levels leads to a reduction in somatic mesodermal fate, while heart and gut muscle remain largely unaffected. These findings underscore the importance of high Twist levels for the proper implementation of somatic muscle fate. Because loss of high Twist levels leads to loss of muscle founder cells, including all Slouch-positive clusters of founder cells, it has always appeared that each Slouch cluster requires the same amount of Wg signal (relayed through Twist) to assume its particular fate. In this study, the requirement for Wg in maintaining high Twist levels was uncoupled from the later role of Wg in specifying cII fate. The fact that Twist specifically rescues Slouch cI in a wg mutant background suggests that Slouch cII requires an additional, Twist-independent contribution from Wg for proper patterning. Consistent with these results, wg hypomorphs were found that provided sufficient signaling to maintain high Twist levels during early mesoderm development and therefore pattern cI, but that do not pattern cII. Temperature-shift experiments using wg temperature-sensitive alleles have shown that Slouch cII specification and engrailed expression in the ectoderm require Wg expression at later stages of embryonic development. Thus, the absence of Slouch cII in the different wg alleles, in hh mutant embryos and in a Twist rescued wg mutant embryo, all suggest that proper patterning requires not only an earlier Wg-dependent regulation of Twist, but also an additional Wg contribution to specify its identity (Cox, 2005).

Manipulations of Wg signaling also revealed two additional aspects of Wg signaling to the mesoderm. (1) It was found that the mesoderm, in general, has a different threshold for Wg signaling when compared with the ectoderm. Conditions that completely rescue the ventral ectoderm and epidermis (wgPE6 at the permissive temperature) fail to completely rescue the mesoderm. (2) It was found that different mesodermal targets respond differently to Wg signaling. For example, expression of the DeltaNTcf (dominant negative form of Pangolin) has mild effects on Twist but significant effects on Slouch cII. Although it is predicted that TCF binds slouch regulatory regions directly, it was found that Wg regulates Twist both directly through TCF and indirectly through the pair-rule gene sloppy-paired. Whether or not the difference in Wg regulation of twist and slouch is due to the structure of the regulatory regions, additional factors that integrate on these promoters in these contexts and the activity of the Arm/dTCF complex remains to be uncovered (Cox, 2005).

This study also underscores the contribution that other factors make to position the Slouch clusters: ectopic Wg expression in the mesoderm does not produce uniform Slouch expression. This aspect of Wg signaling is reflected in other tissues such as the epidermis. The size of Slouch cII could not be further enlarged beyond that seen when Wg signaling was initially increased. This suggests a prepatterning mechanism, perhaps involving the activity of the pair-rule genes that have been shown to be responsible for segmentation of the mesoderm, as well as the integration of other signal transduction pathways, such as EGF/FGF and Notch signaling. The data suggest that Wg signaling then works on this prepattern to regulate the domain of Slouch expression (Cox, 2005).

The effect of Wg on muscle patterning is similar to that described for even-skipped muscle progenitor specification; that is, Wg signaling (in collaboration with such signals as Decapentaplegic) is first required to set up a region of 'competence' through activation of mesoderm-specific factors such as Twist and Tinman. Wg then later cooperates with these intrinsic factors to induce the expression of even-skipped in dorsal muscle progenitors, much as would be suggested for Slouch cII. However, the observations suggest an important variation of Wg signaling in mesodermal patterning. In the case of Slouch patterning, Wg creates temporal as well as spatial diversity, while in patterning eve it only acts temporally. Wg signaling contributes to the expression of Slouch in its two discrete ventral patches by two distinctive mechanisms: through the regulation of an upstream transcription regulator (Twist), which is sufficient for one domain of expression; and through the cooperation of this factor with a second, temporally distinct Wg input for the second domain of expression. The expression of the same gene but at two different times and places, through two Wg-dependent means, gives insight into how an organism may generate diverse tissues in response to the same signal (Cox, 2005).

Work carried out in the wing imaginal disc suggests that Wg acts as a morphogen. In this tissue, Wg protein was visualized in a graded distribution and it appears to activate multiple target genes directly, in a concentration-dependent manner. Based on these criteria, Wg has been labeled as a classical morphogen. However, careful inspection of the molecular mechanisms underlying Wg activation of both short- and long-range targets in the wing have revealed that the pattern of Wg expression changes during wing imaginal disc development, and that Wg collaborates with other pathways to set up the expression of these genes. These studies have cast doubt on whether Wg is a true morphogen in this tissue (Cox, 2005).

Investigating the molecular mechanisms that govern patterning of the embryonic mesoderm, similarly suggests that Wg does not act on Slouch clusters I and II as a classical morphogen. Wg does not activate cI directly, but instead maintains high levels of Twist, which sets up a somatic mesodermal competency domain that is sufficient to create cI. Additional Wg is then required later to pattern cII. It can be argued that Wg acts as a morphogen to regulate Twist expression (at low levels), and then to control Slouch expression (at high levels) within cells of cII. However, the precise regulation and dependence of Slouch clusters I and II on Wg within both the dorsoventral and anteroposterior axes suggest that there must be additional patterning information available to properly place these two cell types. As more putative morphogens are held up to the lens of molecular biology, it will be interesting to see whether there are unexpected, new twists in the molecular underpinnings of morphogens (Cox, 2005).

The Wingless pathway and heart development

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

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

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

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

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

The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the Egfr- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).

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

Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).

Given the importance of Ras/MAPK signaling in Eve progenitor determination, a determination was made of whether Pnt, an Ets domain transcriptional activator that functions downstream of MAPK, is also involved in this process. In pnt mutant embryos, there is a severe reduction in the number of both Eve progenitors, although this loss is more pronounced for the P15 lineage. Since mesoderm migration is normal in pnt embryos, Pnt must only be required for the progenitor specification function of Htl. Consistent with this conclusion, an activated form of Pnt induces extra Eve progenitors (Halfon, 2000).

In embryos mutant for yan, which encodes a MAPK-regulated Ets-domain transcriptional repressor, there is an increase in the number of Eve progenitors and their differentiated derivatives. Conversely, a constitutively activated form of Yan inhibits Eve progenitor formation. Thus, two MAPK-regulated transcription factors are involved in the development of Eve progenitors (Halfon, 2000).

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

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

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

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

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

The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).

Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).

An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).

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

The T-box genes midline and H15 are conserved regulators of heart development: The expression of midline and H15 is dependent on Wingless signaling and tinman and pannier

The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. The selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart (Miskolczi-McCallum, 2005).

In order to determine where mid and H15 fit in the genetic hierarchy controlling heart development, their expression was examined in several mutant backgrounds. The initiation of mid expression in the dorsal mesoderm in early stage 12 occurs after the expression of tin and pnr, as well as after the period of Wg signaling in the dorsal mesoderm, suggesting that mid and H15 are regulated downstream of the factors that confer cardiac fate. Indeed, the dorsal vessel expression of mid and H15 is completely lost in both wgcx4 and tinec40 mutant embryos, which fail to specify dorsal mesoderm. Embryos mutant for pnr have greatly decreased numbers of cardioblasts. Accordingly, mid and H15 expression is variably lost in pnrvx6 null mutant embryos, with most embryos completely lacking mid expression in the dorsal mesoderm. Ectopic expression of pnr throughout the mesoderm using the GAL4/UAS system is able to induce ectopic expression of mid and H15. These results indicate that the initiation of mid and H15 in the dorsal mesoderm is downstream of factors required for the specification of cardiac fate (Miskolczi-McCallum, 2005).

The Dorsocross T-box genes are key components of the regulatory network controlling early cardiogenesis in Drosophila

Cardiac induction in Drosophila relies on combinatorial Dpp and Wg signaling activities that are derived from the ectoderm. Although some of the actions of Dpp during this process have been clarified, the exact roles of Wg, particularly with respect to myocardial cell specification, have not been well defined. The present study identifies the Dorsocross T-box genes as key mediators of combined Dpp and Wg signals during this process. The Dorsocross genes are induced within the segmental areas of the dorsal mesoderm that receive intersecting Dpp and Wg inputs. Dorsocross activity is required for the formation of all myocardial and pericardial cell types, with the exception of the Eve-positive pericardial cells. In an early step, the Dorsocross genes act in parallel with tinman to activate the expression of pannier, a cardiogenic gene encoding a Gata factor. Loss- and gain-of-function studies, as well as the observed genetic interactions among Dorsocross, tinman and pannier, suggest that co-expression of these three genes in the cardiac mesoderm, which also involves cross-regulation, plays a major role in the specification of cardiac progenitors. After cardioblast specification, the Dorsocross genes are re-expressed in a segmental subset of cardioblasts, which in the heart region develop into inflow valves (ostia). The integration of this new information with previous findings has allowed drawing a more complete pathway of regulatory events during cardiac induction and differentiation in Drosophila (Reim, 2005b).

In vertebrate species, genetic studies with loss-of-function alleles have implicated Tbx1, Tbx2, Tbx5 and Tbx20 in the control of heart morphogenesis and the regulation of cardiac differentiation markers. In the case of Tbx5, a small number of cardiac differentiation genes have been identified as direct downstream targets. However, owing to the complexity of the system, the respective positions of these genes within a regulatory network during early cardiogenesis are still poorly understood (Reim, 2005b).

Drosophila offers a simpler system to study regulatory networks in cardiogenesis. The Tbx20-related T-box genes mid and H15 have been shown to play a role in cardiac development downstream of the early function of the NK homeobox gene tin and the Gata gene pannier (pnr). Whereas the role of these genes in the morphogenesis of the cardiac tube is minor, they are involved in processes of cardiac patterning and differentiation during the second half of cardiogenesis, which includes the activation of tin expression in myocardial cells (Reim, 2005a). The present report characterizes the roles of the Tbx6-related Dorsocross T-box genes (which may actually have arisen from a common ancestor of the vertebrate Tbx4, Tbx5 and Tbx6 genes), in Drosophila cardiogenesis. The Doc genes have a fundamental early role; they are required for the specification of all cardiac progenitors that generate pure myocardial and pericardial lineages. They are not required for generating dorsal somatic muscle progenitors and lineages with mixed pericardial/somatic muscle, even though their early expression domains also include cells giving rise to these lineages (Reim, 2005b).

The new information on the regulation and function of Doc fills a major gap in the understanding of early Drosophila cardiogenesis. Previous data have shown that the combinatorial activities of Wg and Dpp are required for the formation of both myocardial and pericardial cells. In addition, the homeobox gene even-skipped (eve) is a direct target of the combined Wg and Dpp signaling inputs in specific pericardial cell/dorsal somatic muscle progenitors. Current data identify the Doc genes as downstream mediators and potential direct targets of combined Wg and Dpp signals during the induction of myocardial and Eve-negative pericardial cell progenitors. The induction of Doc expression by Wg and Dpp occurs concurrently with the induction of tin by Dpp alone, at a time when the mesoderm still consists of a single layer of cells. As a result, tin and Doc are co-expressed in a segmental subset of dorsal mesodermal cells that include the presumptive cardiogenic mesoderm. Conversely, in the intervening subset of dorsal mesodermal cells (the presumptive visceral mesoderm precursors) tin is co-expressed with bagpipe (bap) and biniou (bin), which are both negatively regulated by Wg via the Wg target sloppy paired (slp). Ultimately, these shared responses to Dpp, differential responses to Wg and the specific genetic activities of Doc versus bap and bin lead to the reciprocal arrangement of cardiac versus visceral mesoderm precursors in the dorsal mesoderm (Reim, 2005b and references therein).

Although the Dpp signaling pathway (and likewise, the Wg pathway) is activated in both ectodermal and mesodermal germ layers, tin and bap respond to it only in the mesoderm. The germ layer-specific response of these genes to Dpp relies on two probably interconnected mechanisms. The first of these involves the additional requirement for Tin protein as a mesodermal competence factor for Dpp signals, which is initially produced in the mesoderm downstream of twist. The second involves the specific repression of the responses of tin and bap to Dpp in the ectoderm by yet unidentified factors that bind to the Dpp-responsive enhancers of these two genes. By contrast, the Doc genes are induced by Dpp and Wg with the same spatial and temporal expression patterns in both germ layers. This implies that the (yet unknown) Dpp and Wg-responsive enhancer(s) of the Doc genes are not subject to the ectodermal repressor activities acting on the tin and bap enhancers, and fits with the observation that induction of Doc in the mesoderm does not require Tin as a mesodermal competence factor. However, because of the distinct roles of Doc in the ectoderm and mesoderm, this situation also implies that Doc must act in combination with germ layer-specific co-factors to exert its respective functions. These data suggest that, in the early mesoderm, Doc acts in combination with tin (Reim, 2005b).

A key gene requiring combinatorial Doc and Tin activities for its activation in the cardiac mesoderm is the Gata factor-encoding gene pannier (pnr). pnr expression is activated in the cardiac mesoderm shortly after the induction of Doc and tin, at a time when Doc expression has narrowed to the mesodermal precursors giving rise to pure cardiac lineages. The mechanisms restricting Doc expression to the cardiac mesoderm are currently not known, but as a consequence, pnr expression is also limited to the cardiac mesoderm. It is conceivable that Doc receives continued inputs during this period from the ectoderm through Dpp, whose expression domain narrows towards the dorsal leading edge by then. Together with the observed feedback regulation of pnr on tin and Doc, this situation leads to a prolonged co-expression of Tin, Doc and Pnr in the cardiac mesoderm of stage 11 to stage 12 embryos. Based upon the onset of the expression of early markers such as mid and svp, this is precisely the period when cardiac progenitors become specified (Reim, 2005b).

It is anticipated that the activation of some downstream targets in presumptive cardiac progenitors requires the combination of two, or perhaps all three, of these cardiogenic factors. Potential target genes include mid, svp and hand. However, none of these candidates is essential for generating cardiac progenitors, although mid and svp are known to be required for the normal diversification of cardioblasts within each segment (Reim, 2005b).

The observation that forced expression of Pnr in the absence of any Doc partially rescues cardiogenesis could indicate that the early, combinatorial functions of tin and Doc are primarily mediated by pnr. Alternatively, or in addition, this observation and the fact that a few cardioblasts can be generated without Doc could point to the existence of some degree of functional redundancy among these three factors. In the context of the latter possibility, it is tempting to speculate that the functional redundancy among T-box, Nkx and Gata factors during early cardiogenesis has further increased during the evolution of the vertebrate lineages. This would explain the less dramatic effects of the functional ablation of Tbx5, Nkx2-5 and Gata4/5/6 on vertebrate heart development as compared to the severe effects of Doc, tin or pnr mutations on dorsal vessel formation in Drosophila. Like the related Drosophila genes, these vertebrate genes are co-expressed in the cardiogenic region and developing heart of vertebrate embryos, which at least for Nkx2.5 and Gata6 also involves cross-regulatory interactions that reinforce their mutual expression (Reim, 2005b).

The observed co-expression of Doc, Tin and Pnr allows for the possibility that, in addition to combinatorial binding to target enhancers, protein interactions among these factors play a role in providing synergistic activities during cardiac specification. Physical interactions of Tbx5 with Gata4 and Nkx2-5, as well between Nkx2-5 and Gata4 in vitro as well as synergistic activities cell culture assays have been demonstrated in mammalian systems and may be relevant to human heart disease. In Drosophila, the genetic interactions between Doc, tin and pnr observed both in loss- and gain-of-function experiments reveal similar synergistic activities of the encoded factors during early cardiogenesis. Altogether, these observations make it likely that these Drosophila factors also act through combinatorial DNA binding and mutual protein interactions to turn on target genes required for the specification of cardiac progenitors (Reim, 2005b).

Whereas pnr is expressed only transiently during early cardiogenesis, tin and Doc continue to be expressed in developing myocardial cells, suggesting that they act both in specification and differentiation events. Recently it was shown that the T-box gene mid is required for re-activating tin in cardioblasts (Reim, 2005a). Of note, owing to the action of svp, Doc and tin are expressed in complementary subsets of cardioblasts within each segment. This mutually exclusive expression of tin and Doc implies that they are not acting combinatorially but, instead, act differentially during later stages of myocardial development. Hence, their activities could result in the differential activation of some differentiation genes such as Sulfonylurea receptor (Sur), which is specifically expressed in the four Tin-positive cardioblasts in each hemisegment (Nasonkin, 1999; Lo, 2001), and wingless (wg), which is only turned on in the two Doc-positive cells in each hemisegment of the heart that generate the ostia. Surprisingly, even the activation of some genes that are expressed uniformly in all cardioblasts has turned out to result from differential regulation within the Tin-positive versus Doc-positive cardioblasts. For example, regulatory sequences from the Mef2 gene for the two types of cardioblasts are separable and those active within the four Tin-positive cells are directly targeted by Tin. Likewise, regulatory sequences from a cardioblast-specific enhancer of Toll have been shown to receive differential inputs from Doc and Tin, respectively, in the two types of cardioblasts. In parallel with this differential regulation, it is anticipated that yet unknown differentiation genes are activated uniformly in all cardioblasts downstream of mid/H15 and hand. The integration of the new information on the roles of Doc in cardiogenesis has now provided a basic framework of signaling and gene interactions through all stages of embryonic heart development, which in the future can be further refined upon the identification of new components and additional molecular interactions (Reim, 2005b).

The Wingless pathway and tracheal development

The tubular epithelium of the Drosophila tracheal system forms a network with a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching induced by the FGF homolog Branchless; it differentiates cells with specialized functions such as fusion cells, which perform target recognition and adhesion during branch fusion, and extends branches toward specific targets. Specification of a unique identity for each primary branch is essential for directed migration, because a defect in either the Egfr or the Dpp pathway leads to a loss of branch identity and the misguidance of tracheal cell migration. The role of Wingless signaling in the specification of cell and branch identity in the tracheal system has been investigated. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, including specifying the dorsal trunk through activation of Spalt expression and inducing differentiation of fusion cells in all fusion branches. Moreover, Wingless signaling regulates Notch signaling by stimulating Delta expression at the tip of primary branches. These activities of Wingless signaling together specify the shape of the dorsal trunk and other fusion branches (Chihara, 2000).

Egfr signaling at stage 10 induces the central region of the tracheal placode to give rise to dorsal trunk (DT) and the visceral branches (VB). DT then migrates beneath the ectoderm and forms the main anterior-posterior connecting tube. DT, expressing Sal, carries fusion cells at the tip, but lacks terminal cells. VB, however, migrates to the interior of the embryo, possessing multiple terminal cells instead of fusion cells. DT migrates in close proximity to the source of Wg and Wg signaling is necessary for DT formation. Hyperactivation of Wg signaling in all tracheal cells forces prospective VB cells to express Sal and Esg and to participate in DT, without affecting other branches under the influence of Dpp. From these results, it is proposed that tracheal cells in the Egfr-induced cells close to a Wg source are instructed to adopt the DT fate. Egfr-induced cells distal to Wg take the fate of VB as a default. Since ubiquitous activation of Wg signaling in tracheal cells during the migration phase has no effect on the direction of migration, it is concluded that Wg signaling does not play a chemoattractive role during primary branching (Chihara, 2000).

Fusion cells are located at the tips of primary branches, and express a transcriptional factor, Esg, after stage 13. The mechanism of Esg induction is not known, although Dpp signaling has been implicated. Ectopic activation of Dpp in all tracheal cells can induce the expression of Esg in all branches. In thick vein (Receptor for Dpp) null embryos, Esg expressing cells disappear from DB and LT but remain in DT. These results suggest that Dpp signaling is required for the expression of Esg in DB and LT but not DT. Wg signaling is required for the expression of Esg in all fusion cells. It is thus proposed that Wg signaling is the primary inducer of fusion cell fate and that Dpp signaling provides an additional stimulus that is required to maintain fusion cell fate in a subset of branches (Chihara, 2000).

During primary branching, Delta protein accumulates at the tips of primary branches, restricting the differentiation of excess fusion cells by stimulating Notch signaling. Wg signaling is required for localized Delta expression. Ectopic expression of an activated form of Arm in all tracheal cells can activate Delta as well as Delta-lacZ expression, suggesting that Wg signaling stimulates Delta expression at the transcriptional level. A similar conclusion has been drawn from studies of Wg function in dorsoventral patterning of Wing imaginal discs. Another mechanism whereby Wg signaling interacts with Notch has been proposed. Dishevelled (Dsh), which is a transducer of Wg signaling acting upstream of Arm, inhibits Notch activity in Drosophila wing discs and interacts with the intracellular domain of Notch in yeast cells. This mechanism is distinct from the proposed mechanism of Notch inhibition by Wg signaling, since activated Arm acting downstream of Dsh causes a phenotype of Notch inhibition. These mechanisms are not mutually exclusive, however, and may reflect a different aspect of complex self- and cross-regulatory interactions of the two signaling pathways (Chihara, 2000).

How does the localized Delta induced by Wg signaling act in tracheal cells? As revealed by the study on Drosophila wing disc development, Notch ligands have a cell-autonomous dominant-negative effect on Notch activity in addition to the well-established role of lateral inhibition of cell differentiation. Clones of cells lacking both Delta and Serrate show a sign of Notch hyperactivation and clones of cells expressing high level of Delta autonomously inhibit Notch target genes. The same relationship between Notch and Delta appears to exist in the trachea, since overexpression of Delta shows phenotypes similar to loss of Notch function. Since Esg is expressed in cells with the highest Delta expression in primary branches and is normally inhibited by Notch signaling, it was proposed that Delta-dependent inhibition of Notch provides permissive conditions for fusion cell differentiation. Thus regulation of Delta by Wg signaling is an important mechanism of fusion cell-fate determination (Chihara, 2000).

Delta expression in tracheal cells is also under the influence of Bnl/Btl signaling. Loss of Btl causes a reduction of Delta and overexpression of Bnl leads to excess Delta expression. These observations suggest that the localized expression of Delta in the developing trachea requires both Wg and Bnl signaling, implying that the two signals synergistically stimulate Delta expression. It is proposed that the two diffusible ligands Bnl and Wg, expressed in distinct special domains, separately exert an inductive influence on the tracheal primordium. Delta integrates the two inductive signals and elevates its expression in sharply defined regions at the tip of the primary branches, and initiates the cell-fate restriction program. This mechanism is likely to be useful for sharpening the response of cells to multiple diffusible ligands (Chihara, 2000).

Wg signaling controls the formation of DT by regulating at least three target genes (sal, esg and Delta) in distinct ways. Sal is expressed in all DT cells and is required for directed migration along the anterior and posterior directions. Most of the cells in the Egfr domain can respond to Wg signaling by expressing Sal, and the expression of Sal is not affected by excess Delta. It is proposed that Sal expression is regulated by Wg signaling but not by Notch signaling, and that it serves as a major mediator of Wg signaling in determining DT identity. Regulation of Esg is more complex. Although Esg expression is stimulated by Wg signaling, it is normally limited to a single cell on each branch due to repression by Notch. Wg signaling activates Esg expression independently of Delta. It is proposed that Wg signaling bifurcates after activation of Arm, activating Esg on the one hand, and Delta on the other. Elevated Delta activates Notch in nearby cells, leading to repression of Esg in the stalk of tracheal branches. These combinatorial effects limit Esg expression to the tip of fusion branches. Stimulation of both positive and negative regulation of Esg by a single inductive signal comprises a self-limiting process of cell-fate determination and accounts for the assignment of single fusion cells that mark the end of the tracheal tubule. In combination with the specification of thick tubules through regulation of Sal, Wg signaling determines the shape of the tracheal tubule (Chihara, 2000).

dpp expression has been examined in two groups of dorsal ectoderm cells at the posterior end of the embryo, in abdominal segment 8 and the telson. These dpp-expressing cells become tracheal cells in the posterior-most branches of the tracheal system (Dorsal Branch10, Spiracular Branch10, and the Posterior Spiracle). These branches are not identified by reagents typically used in analyses of tracheal development, suggesting that dpp expression confers a distinct identity upon posterior tracheal cells. dpp posterior ectoderm expression begins during germ band extension and continues throughout development. The sequences responsible for these aspects of dpp expression have been isolated in a reporter gene. An unconventional form of Wingless (Wg) signaling, Dpp signaling, and the transcriptional coactivator Nejire (CBP/p300) are required for the initiation and maintenance of dpp expression in the posterior-most branches of the tracheal system. These data suggest a model for the integration of Wg and Dpp signals that may be applicable to branching morphogenesis in other developmental systems (Takaesu, 2002).

During early stages of embryogenesis, wg and dpp are expressed in undifferentiated dorsal ectoderm. wg mRNA expression, in 15 stripes along the entire dorsal-ventral axis of the embryo (including the dorsal ectoderm), begins at stage 8. wg expression persists in this striped pattern through stage 17. dpp mRNA is expressed on the dorsal side of the embryo along the entire anterior-posterior axis, beginning at stage 4. dpp mRNA expression persists in a large portion of the dorsal ectoderm through stage 8 and resolves into leading edge cell-specific expression in stage 12 embryos. At this time, the embryonic expression pattern of nej has not been reported. However, some information can be obtained from nej mutant phenotypes. nej zygotic mutant embryos show visible defects in the tracheal system at stage 12. The tracheal system is derived from the dorsal ectoderm, suggesting that nej is expressed in this tissue prior to stage 12 (Takaesu, 2002).

dpp expression in posterior tracheal branch anlagen appears to be initiated by prior episodes of wg and dpp expression in the undifferentiated dorsal ectoderm. The maintenance of dpp expression in posterior tracheal branches appears to require continuous input from wg and from a dpp feedback loop. The initiation and maintenance of dpp expression in posterior tracheal branches also requires continuous nej activity. Overall, the data are consistent with the following combinatorial signaling model. The transcriptional activator Medea (Med, signaling for the Dpp pathway) interacts with the transcriptional activator Arm (signaling for the Wg pathway) via the transcriptional coactivator Nej. This multimeric complex initiates and, with continuous signaling, maintains dpp expression in posterior tracheal branches with the help of Zw3. These data extend previous studies of dpp expression and Dpp signaling in several ways. nej has been reported to participate in Dpp signaling. Expression from Dpp responsive enhancers is reduced in nej zygotic mutant embryos. While they show that nej3 enhances dpp wing phenotypes, this study shows that Mad1 enhances nej3 embryonic phenotypes. The Dorsal Trunk Branch forms normally in Mad12 zygotic mutant embryos, and the Dorsal Trunk Branch appears normal in Med1 mutants. nej is involved in mediating combinatorial signaling by the Wg and Dpp pathways and the involvement of nej in morphogenesis of Dorsal Branch, Spiracular Branch, and the Posterior Spiracle is demonstrated. A region of the histone acetyltransferase domain of Nej binds to Mad. Further study is needed to reveal the mechanisms used by Nej to interact with Wg and Dpp signaling. Several questions remain about the regulation of dpp expression by Wg, Dpp, and Nej. Two questions arise about the mechanism of signal integration: how is zw3 involved and how is Nej recruited to bridge the two pathways? It is tempting to speculate that, in response to a Wg or a Dpp signal, Zw3 (a serine-threonine kinase) is involved in Nej recruitment. Numerous studies have shown that p300/CBP transcriptional coactivation functions are stimulated by its phosphorylation, but the site of phosphorylation has never been mapped. Other questions concern the molecular nature of the enhancers that direct dpp expression in the posterior tracheal branches. A 54-nucleotide region has been identified that contains two sets of conserved, overlapping consensus binding sites for dTCF and Mad/Med. Analyses of DNA-protein interactions predicted by the data involving this candidate combinatorial enhancer have begun (Takaesu, 2002).

From a broader perspective, mammalian homologs of Dpp and orthologs of Wg are important in branching morphogenesis in a variety of developing tissues. For example, BMP2 is involved in renal branching and Wnt4 plays a role in mammary gland branching. The widespread use of TGF-ß and Wnt signals in branching suggests that a greater understanding of the regulation of dpp tracheal expression and dpps role in specifying the unique identities of posterior tracheal branches will have wide relevance (Takaesu, 2002).

A fundamental question in developmental biology is how tissue growth and patterning are coordinately regulated to generate complex organs with characteristic shapes and sizes. This study shows that in the developing primordium that produces the Drosophila adult trachea, the homeobox transcription factor Cut regulates both growth and patterning, and its effects depend on its abundance. Quantification of the abundance of Cut in the developing airway progenitors during late larval stage 3 revealed that the cells of the developing trachea had different amounts of Cut, with the most proliferative region having an intermediate amount of Cut and the region lacking Cut exhibiting differentiation. By manipulating Cut abundance, it was shown that Cut functioned in different regions to regulate proliferation or patterning. Transcriptional profiling of progenitor populations with different amounts of Cut revealed the Wingless (known as Wnt in vertebrates) and Notch signaling pathways as positive and negative regulators of cut expression, respectively. Furthermore, the gene encoding the receptor Breathless (Btl, known as fibroblast growth factor receptor in vertebrates) was identified as a transcriptional target of Cut. Cut inhibited btl expression and tracheal differentiation to maintain the developing airway cells in a progenitor state. Thus, Cut functions in the integration of patterning and growth in a developing epithelial tissue (Pitsouli, 2013).

Wingless and salivary gland development

In the early Drosophila embryo, a system of coordinates is laid down by segmentation genes and dorsoventral patterning genes. Subsequently, these coordinates must be interpreted to define particular tissues and organs. To begin understanding this process for a single organ, a study has been carried out of how one of the first salivary gland genes, fork head (fkh), is turned on in the primordium of this organ, the salivary placode. A placode-specific fkh enhancer was identified 10 kb from the coding sequence. Dissection of this enhancer shows that the apparently homogeneous placode is actually composed of at least four overlapping domains. These domains appear to be developmentally important because they predict the order of salivary invagination, are evolutionarily conserved, and are regulated by patterning genes that are important for salivary development. Three dorsoventral domains are defined by Egf receptor (Egfr) signaling, while stripes located at the anterior and posterior edges of the placode depend on wingless signaling. Further analysis has identified sites in the enhancer that respond either positively to the primary activator of salivary gland genes, Sex combs reduced (Scr), or negatively to Egfr signaling. These results show that fkh integrates spatial pattern directly, without reference to other early salivary gland genes. In addition, a binding site for Fkh protein was identified that appears to act in fkh autoregulation, keeping the gene active after Scr has disappeared from the placode. This autoregulation may explain how the salivary gland maintains its identity after the organ is established. Although the fkh enhancer integrates information needed to define the salivary placode, and although fkh mutants have the most extreme effects on salivary gland development thus far described, it is argued that fkh is not a selector gene for salivary gland development and that there is no master, salivary gland selector gene. Instead, several genes independently sense spatial information and cooperate to define the salivary placode (Zhou, 2001).

In the anteroposterior dimension, establishment of parasegmental borders depends on interaction between wingless expressing cells at the posterior edge of one parasegment and hedgehog or engrailed expressing cells at the anterior edge of the next, more posterior parasegment. Since 507-1008:lacZ is expressed in anterior and posterior stripes adjacent to the borders of parasegment 2, its reliance on wg was tested. At the placode stage in wg-mutant embryos, the posterior stripe is not seen, and the anterior stripe may also be missing. At slightly earlier times, when only the stripes are seen in wild-type embryos, no expression of 507-1008:lacZ is seen in wg-mutant embryos. These results suggest that fkh responds to wg signaling by expression in both posterior and anterior stripes (Zhou, 2001).

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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