Table of contents

Targets of Dpp in tracheal cell migration and cell fate determination

Dpp signaling is required for directed tracheal cell migration during Drosophila embryogenesis. This requirement is twofold (Vincent, 1997): (1) Dpp first acts to induce Branchless, the ligand for Breathless, the FGF receptor. Branchless functions as a tropic factor to direct tracheal cell migration. (2) Dpp then controls localized gene expression in the tracheal system. Dpp is first expressed in a large band covering the entire dorsal ectoderm. Somewhat later, but still prior to the invagination of the tracheal primordia, DPP is expressed in a segmentally repeated pattern in the lateral ectoderm aligned with the ventral margin of the tracheal pits. Dorsal to the tracheal placode, DPP is expressed along the entire anteroposterior axis in the dorsal-most ectodermal cell. This dorsal expression domain is regulated by Jun related antigen and by Anterior open (also known as Yan), both proteins acting downstream of Rac1, hemipterous and basket (Riesgo-Escovar, 1997).

Dpp controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling. In contrast to sal the gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling. The requirement for Dpp for the correct expression of the ligand branchless (bnl) was revealed using tkv and punt mutants. The dorsal-most patches of bnl expression which prefigure the formation of the dorsal branches, are severely reduced in punt mutant embryos and absent in tkv mutants. Thus, Dpp plays a dual role during tracheal cell migration. It is required to control the dorsal expression of the Bnl ligand. In addition, the Dpp signal recruits groups of dorsal and ventral tracheal cells and programs them to migrate in dorsal and ventral directions (Vincent, 1997).

Cell migration during embryonic tracheal system development in Drosophila requires Dpp and Egf signaling to generate the archetypal branching pattern. Two genes encoding the transcription factors Knirps and Knirps related are shown to possess multiple and redundant functions during tracheal development. knirps/knirps related activity is necessary to mediate Dpp signaling that is required for tracheal cell migration and formation of the dorsal and ventral branches. Dpp signaling is required for the directed migration of dorsal and ventral tracheal cells. It activates kni expression and has been proposed to control target gene expression via Kni. Dpp signaling is required for dorsal and ventral tracheal branch formation and for kni expression. The tracheal mutant phenotypes of embryos lacking the Dpp receptors Tkv and Put are reminiscent of the kni tracheal phenotype, suggesting that kni is necessary to mediate functional aspects of Dpp signaling. To link kni activity and Dpp signaling more directly, kni was expressed in a tkv mutant background by using the tracheal-specific driver. Since tkv mutant embryos lack the dorsalmost patches of branchless expression that are necessary for dorsal branch outgrowth, the analysis was focused on ventral branch formation in the presence of kni expression. These embryos develop a rudimentary ventral tracheal system that is indistinguishable from the branching of tkv mutants. Thus, the activation of kni expression by Dpp is necessary but not sufficient for ventral branch formation. This result also suggests that Dpp signaling controls additional genes different from kni/knrl that are necessary for branch outgrowth. Ectopic Dpp expression in all tracheal cells leads to dorsoventral cell migration, which causes the lack of dorsal trunk and visceral branches that are normally formed by anteroposterior migration. It also leads to ectopic kni expression in all tracheal cells. The finding that ectopic kni expression also interferes with dorsal trunk formation suggests a role of kni activity in mediating ectopic Dpp signaling. Thus, the tracheal phenotypes generated by either ectopic dpp or ectopic kni expression were examined. Ubiquitous tracheal dpp expression causes the lack of anterioposterior branch formation and the dorsal migration of supernumerary cells. Ubiquitous tracheal expression of one copy of kni leads to a reduced dorsal trunk and an increased number of cells migrating dorsally, whereas ubiquitous expression of two copies of kni results in the absence of the dorsal trunk and the migration of supernumary cells towards dorsal positions. Thus, kni activity leads to a dorsal tracheal cell migration, as observed for Dpp. In summary, these results indicate that the role of Dpp in directing tracheal cells to adopt a dorsoventral migration behaviour is mediated by kni/knrl activity, but kni/knrl is not sufficient to mediate Dpp-dependent branch formation (C.-K. Chen, 1998).

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).

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. Dpp is expressed in dorsolateral epidermal clusters located near the tips of the outgrowing dorsal branches. The Dpp receptor, Thick veins, is expressed in all tracheal cells during embryogenesis and is required in dorsal branch outgrowth ectopic activation of fusion markers in cells of the dorsal branch. Dpp signaling is required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Delta is among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling. In conditional Notch loss of function mutants, additional tracheal cells adopt the fusion cell fate. Ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch ts1 mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches (Steneberg, 1999).

Several alleles of Delta were identified using an enhancer trap screen for genes that are selectively expressed in the tracheal fusion cells. In these strains, the Delta-lacZ marker is initially expressed homogeneously in all tracheal cells, but becomes up-regulated in the fusion cell from embryonic stage 13, approximately at the same time that the first fusion marker genes initiate their expression in the fusion cell. Inactivation of Notch signaling by a conditional allele of the receptor results in more cells acquiring the fusion cell fate. Delta-lacZ is also up-regulated in the extra fusion cells, indicating that Notch signaling in the trachea also regulates the expression of Delta, as has been suggested for other Notch signaling events. In Notchts1 mutants the total number of cells in the dorsal branch remains the same; the number of cells expressing the terminal cell marker is not decreased. Thus, Notch is required for the suppression of the fusion cell program in the three cells that will remain in the stalk of the branch. Accordingly, expression of an activated form of Notch in all tracheal cells, or in the fusion cells only, inhibits branch fusion and suppresses the expression of fusion marker genes. Cell counts in these affected dorsal branches show that the number of cells is unchanged; this suggests that the presumptive fusion cell does not undergo apoptosis but instead remains in the stalk of the branch. However, in embryos expressing the activated form of Notch in all trachea, a few additional cells express the terminal marker genes and sprout off the dorsal branches, but this is likely due to the inability of the presumptive fusion cell to produce the Hdc inhibitor of tracheal cell branching (Steneberg, 1999).

The localized expression of Dpp in the dorsal epidermis is required for the induction of the fusion cell fate, whereas Notch signaling has the opposite effect in the expression of fusion marker genes. Since the number of cells that acquire the fusion cell fate in response to Dpp becomes higher when the amount of functional Notch is reduced, it has been proposed that Notch signaling in the trachea is required for the selection of a single fusion cell among the group of cells that receive the Dpp signal at the tip of the dorsal branch. Furthermore, Dpp signaling can activate expression of Delta suggesting that Delta expression in the fusion cell may be directly activated by downstream effectors of Dpp signaling. Such transcription factors include Mad, Knirps and Knirps-like, all of which act downstream of Dpp signaling during primary branching. Alternatively, the activation of Delta in the cells in the stalk of the branch may be suppressed by negative regulators of Dpp signaling (such as Daughters against dpp (Dad) or Brinker, both of which are regulated by Dpp signaling). The requirement for Dpp signaling in branch fusion does not seem to be a general theme for all tracheal branches. Dpp is not expressed in the proximity of the dorsal trunk fusion cells and dorsal trunk fusion is not affected in embryos mutant for Mad, tkv or in embryos expressing the dominant negative forms of the receptors in all tracheal cells. These results argue that the induction of fusion cell fate in different branches employs different signaling mechanisms. Such a signaling event that may be responsible for the activation of fusion genes in the dorsal trunk may be provided by the Spi/EGFR pathway. Mutations in members of this pathway are defective in dorsal trunk formation, and in the developing Drosophila wing; rhomboid, a component of EGF signaling, has been shown to regulate the expression of Delta. In contrast to the local requirement of Dpp in the dorsal branch, Notch signaling is a prerequisite for the selection of the fusion cell in all primary branches that will extend a fusion sprout. Thus, even though the inductive signals required for the expression of the fusion cell-specific marker genes may be different, they all appear to result in the up-regulation of Delta; they signa through Notch to select a single cell at tip of the branch that will execute the complex cellular dynamics of branch fusion (Steneberg, 1999).

Targets of DPP in salivary gland morphogenesis

Expression of Creb-A in salivary glands depends on Sex combs reduced, the master regulator of salivary gland fate, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by dpp (Andrew, 1997).

Salivary gland formation in the Drosophila embryo is linked to the expression of the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere, salivary glands form in new places. However, not every cell that expresses Scr is recruited to a salivary gland fate. Along the anterior-posterior axis, the posteriorly expressed proteins encoded by the teashirt (tsh) and Abdominal-B (Abd-B) genes block Scr activation of salivary gland genes. Along the dorsal-ventral axis, the secreted signaling molecule encoded by decapentaplegic prevents activation of salivary gland genes by Scr in dorsal regions of parasegment 2. Five downstream components in the Dpp signaling cascade required to block salivary gland gene activation have been identified: two known receptors (the type I receptor encoded by the thick veins gene and the type II receptor encoded by the punt gene); two of the four known Drosophila members of the Smad family of proteins which transduce signals from the receptors to the nucleus [Mothers against dpp (Mad) and Medea (Med)], and a large zinc-finger transcription factor encoded by the schnurri (shn) gene. The expression patterns of d-CrebA and Trachealess were examined in embryos missing zygotic function of schnurri. In embryos homozygous for shn, a dorsal expansion of salivary gland protein expression is observed. The presence of amnioserosa, an extreme dorsal cell type, suggests that embryos lacking zygotic shn function are not ventralized, as are embryos missing maternal and zygotic function of tkv, pt, Mad, or Med or missing zygotic function of dpp. These results reveal how anterior-posterior and dorsal-ventral patterning information is integrated at the level of organ-specific gene expression (Henderson, 1999).

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies PS2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

Targets of DPP in abdomen development

In Drosophila, the Hox gene Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologs of Abdominal-B in other species are also needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila is shown to transform male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. It is proposed that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).

In the genital disc, the transcription of Dll depends, as in the leg disc, on dpp and wg signals. Abd-B represses Dll expression. Moreover, increasing Abd-B levels in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in many late Abd-B minus clones, derepression of either dpp or wg was not observed. It is concluded that there is an antagonism between the activation of Dll by dpp/wg signaling and its repression by Abd-B. This is not mediated by changes in the expression of either dpp, wg or Abd-B (Estrada, 2001).

To characterize this antagonism further, Abd-B minus clones that were made were also unable to transduce the dpp signal. This signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).

Dpp and heart development

Inductive signaling is of pivotal importance for developmental patterns to form. In Drosophila, the transfer of TGFß (Dpp) and Wnt (Wg) signaling information from the ectoderm to the underlying mesoderm induces cardiac-specific differentiation in the presence of Tinman, a mesoderm-specific homeobox transcription factor. Evidence that the Gata transcription factor, Pannier, and its binding partner U-shaped, also a zinc-finger protein, cooperate in the process of heart development. Loss-of-function and germ layer-specific rescue experiments suggest that pannier provides an essential function in the mesoderm for initiation of cardiac-specific expression of tinman and for specification of the heart primordium. u-shaped also promotes heart development, but unlike pannier, only by maintaining tinman expression in the cardiogenic region. By contrast, pan-mesodermal overexpression of pannier ectopically expands tinman expression, whereas overexpression of u-shaped inhibits cardiogenesis. Both factors are also required for maintaining dpp expression after germ band retraction in the dorsal ectoderm. Thus, it is proposed that Pannier mediates as well as maintains the cardiogenic Dpp signal. In support, it is found that manipulation of pannier activity in either germ layer affects cardiac specification, suggesting that its function is required in both the mesoderm and the ectoderm (Klinedinst, 2003).

Both tin and pnr have been shown to be targets of Dpp signaling at stage 9/10. It is proposed that dpp is necessary again at stage 11 to activate and maintain pnr and tin expression in the cardiogenic region of the mesoderm. First, pnr is activated with the help of early stage 11 tin, which is expressed broadly throughout the dorsal mesoderm, and dpp, which is expressed in a narrow dorsal ectodermal stripe. Then, at mid-stage 11, tin is restricted to the cardiogenic region with the help of mesodermal pnr as well as continuous ectodermal Dpp signaling. Once both are activated in the cardiogenic mesoderm, they are likely to contribute to the maintenance of each other's expression, probably aided again, but only moderately, by ectodermal Dpp signaling. This interpretation is consistent with mesodermal versus ectodermal expression of dominant-negative pnrEnR and the dpp target repressor encoded by brk. They are both equally effective in reducing cardiac-specific tin when expressed in the mesoderm, but ectodermal repression is more effective when dorsal-stripe dpp at stage 11 is also affected (as in the case of ZKr-Gal4>UAS-brk, but not with ZKr-Gal4>UAS-pnrEnR) (Klinedinst, 2003).

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).

scylla and charybde are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction

Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).

The foundation for the current study was a survey of RNA expression patterns by automated whole-mount RNA hybridization in situ. This screening protocol led to the identification of scyl as a dorsally restricted transcript in blastoderm stage embryos. Based on its spatial and temporal expression properties, which represent a subset of the dpp expression pattern, it was postulated that scyl is a transcriptionally regulated target of the Dpp signaling cascade that specifies early embryonic dorsal fates. To test this hypothesis, scyl transcript distributions were compared in wild-type and dorsoventral patterning mutant embryos. Fate determining genes functioning downstream of the ventral morphogen Dorsal (Dl) and/or the dorsal morphogen Dpp are expected to exhibit altered expression patterns in Dl-deficient/Dpp-constitutive and Dl-constitutive/Dpp-deficient mutant embryos. Indeed, this was found to be the case for the scyl transcript. Transcription of scyl in wild-type and mutant embryos was similar to that of zen, the best characterized target of Dpp, indicating that Dpp is both necessary and sufficient for scyl transcription. In blastoderm stage embryos, zen is expressed at the posterior pole and in the dorsal-most 40% of the developing embryo. In like fashion, scyl is expressed at the posterior pole and is dorsally restricted. scyl transcripts, however, are confined to the subset of zen-expressing cells that correspond to the dorsal-most 10% of the developing embryo. Both scyl and zen are ubiquitously expressed in dorsalized embryos derived from dl mutant females and in which the dorsal morphogen Dpp is ubiquitously expressed. Neither scyl nor zen is expressed in the abdominal regions of ventralized embryos, which are derived from cactus (cact) mutant females and which lack zygotic Dpp (Scuderi, 2006).

Two observations that led to an examination of the regulation of scyl and chrb by downstream components of the Dpp signaling cascade are: (1) scyl, like zen, is a transcriptionally regulated target of Dpp-mediated signaling and (2) scyl, chrb and zen are expressed in overlapping dorsal fields. The gene encoding the divergent homeobox transcription factor Zen is itself activated by Dpp-mediated signaling in dorsal domains of the blastoderm stage embryo. In zen mutant embryos, dorsally restricted scyl and chrb transcripts are lost, placing both genes downstream of the Zen transcriptional effector of Dpp-mediated signal transduction (Scuderi, 2006).

In addition to the dorsal field of scyl and chrb expression in early embryos, segmental expression along the anteroposterior axis suggests that scyl and chrb may also be sensitive to regulation by homeobox genes other than zen. Both scyl and chrb transcripts are localized in anteroposteriorly segmented patterns in ventral regions of the blastoderm and in the three thoracic segments of stage 13 embryos. In stage 13 embryos, genes of the bithorax complex (BX-C) repress expression of target genes in abdominal segments, restricting their expression to the thorax. The expression of scyl and chrb was examined in BX-C mutant embryos: expansion was observed of thoracic expression into abdominal segments of mutant embryos, thereby placing scyl and chrb downstream of the BX-C homeobox transcription factors, Ubx, Abd-A and/or Abd-B. Consistent with this observation is the finding that Ubx binds to regulatory regions of both scyl and chrb (Scuderi, 2006).

Finally, as an extension of the observation that scyl and chrb are downstream targets of homeobox genes acting in distinct signaling pathways, bioinformatic tools were used to identify conserved elements of the scyl and chrb promoters in D. melanogaster and D. pseudoobscura. In a computational cross-genome comparison utilizing algorithms based on both Gibbs sampling and Artificial Neural Networks, one 24-nucleotide motif and three 16-nucleotide motifs were identified that are conserved in the promoter regions of both genes in both species. Motif 1 was found much more frequently than expected for a random sequence, suggestive of its role as a generic transcription factor binding site or regulatory element. Motifs 2-4 were observed much less frequently, and this statistic is interpreted to be indicative of more specific roles for motifs 2-4 in the co-regulation of scyl and chrb. With respect to the identification of defined binding sites, neither motif 1 nor motif 2 corresponds to any canonical transcription factor binding sites listed in the TRANSFAC transcription factor database. Motif 3, however, is GC-rich and contains canonical binding sites for two widely used transcriptional activators: SP1 (GCCCGCCCCCC) and AP2 (GCCCGCGGC). More notable, however, is the characterization of motif 4, which was found only twice in each of the Drosophila genomes (in the promoter regions of scyl and chrb). In scans of the TRANSFAC database, it was found that motif 4 harbors a 10-bp canonical binding site for Zen (ATTTAAATGA) (Scuderi, 2006).

Table of contents

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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