Table of contents

Regulation in dorsal-ventral axis

The initial distribution of dpp in the dorsal ectoderm of the developing fly is established by a gradient of the maternal Dorsal protein, asymmetrically distributed to the ventral portion of the fly. (Dorsal is so called because in its absence, the fly becomes ventralized). The Dorsal protein regulatory gradient initiates through dpp the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the early Drosophila embryo. There are two primary dorsal target genes: snail and dpp. These genes define the limits of the presumptive mesoderm and dorsal ectoderm, respectively. In addition, the dorsal regulatory gradient defines the limits of inductive interactions between germ layers after gastrulation. Thus dorsal controls between the subdivision of the mesoderm and dorsal ectoderm (Maggert, 1995).

The Dorsal nuclear gradient initiates the differentiation of the mesoderm, neuroectoderm, and dorsal ectoderm by activating and repressing gene expression in the early Drosophila embryo. This gradient is organized by a Toll signaling pathway that shares many common features with the mammalian IL-1 cytokine pathway. A second signaling pathway, controlled by the Torso receptor tyrosine kinase, also modulates DL activity. The Torso pathway selectively masks the ability of DL to repress gene expression but has only a slight effect on activation. Intracellular kinases that are thought to function downstream of Torso, such as D-raf and the Rolled MAP kinase, mediate this selective block in repression (Rusch, 1994).

The Dorsal morphogen is a transcription factor that activates some genes and represses others to establish multiple domains of gene expression along the dorsal/ventral axis of the early Drosophila embryo. Repression by Dorsal appears to require accessory proteins that bind to corepression elements in Dorsal-dependent regulatory modules called ventral repression regions (VRRs). A corepression element has been identified in decapentaplegic (dpp), a zygotically active gene that is repressed by the Dorsal morphogen. This dpp repression element (DRE) is located within a previously identified VRR and close to essential Dorsal-binding sites. A factor from Drosophila embryo extracts has been identified that binds to the DRE but not to mutant forms of the DRE that fail to support efficient repression. This protein also binds to an apparently essential region in a VRR associated with the zerknullt (zen) gene. One of the DREs in the dpp VRR overlaps the binding site for a potential activator protein suggesting that one mechanism of ventral repression may be the mutually exclusive binding of repressor and activator proteins. The DRE-binding protein is identical to NTF-1 (equivalent to Elf-1, the product of the grainyhead gene), a factor originally identified as an activator of the Ultrabithorax and Dopa decarboxylase promoters. NTF-1 mRNA is synthesized during oogenesis and deposited in the developing oocyte where it is available to contribute to ventral repression during early embryogenesis. Previous studies have shown that overexpression of NTF-1 in the postblastoderm embryo results in a phenotype that is consistent with a role for this factor in the repression of dpp later in embryogenesis (Huang, 1995).

The Dorsal morphogen acts as both an activator and a repressor of transcription in the Drosophila embryo in order to regulate the expression of dorsal/ventral patterning genes. Circumstantial evidence has suggested that Dorsal is an intrinsic activator and that additional factors (corepressors) convert it into a repressor. These corepressors, however, have previously eluded definitive identification. Via the analysis of embryos lacking the maternally encoded Groucho corepressor and via protein-binding assays it has been shown that recruitment of Groucho to the template by protein:protein interactions is required for the conversion of Dorsal from an activator to a repressor. Specifically, Groucho is required for the Dorsal, mediated repression of Zerknullt and Decapentaplegic. Groucho is not required for the spatially regulated expression of genes that are activated by Dorsal such as twist and snail. Groucho is therefore a critical component of the dorsal/ventral patterning system (Dubnicoff, 1997).

Embryonic target gene activation in the absence of brinker is independent of SMAD activity. Thus brk acts either parallel to or downstream of SMADs as a specific repressor of low and intermediate level Dpp target genes. brk is expressed like another dpp antagonist, short gastrulation (sog), in ventrolateral regions of the embryo abutting the dorsal dpp domain, and in brk mutants dpp expression expands to cover the entire ectoderm. In this situation sog is largely responsible for Dpp gradient formation, since brk;sog double mutant embryos have almost no polarity information in the ectoderm. The double mutants consist mainly of mesoderm and unstructured dorsal epidermis. Thus, brk and sog together specify the neuroectoderm of Drosophila embryos (Jazwinska, 1999b).

The cuticle of brk mutant embryos has an enlarged region carrying dorsal hairs and a smaller region carrying ventral denticles. The number of sna-expressing neuroblasts in the ventral neurogenic region is reduced. This indicates that brk mutations lead to an expansion of dorsolateral fates and a reduction of ventrolateral fates. However, despite these lateral fate shifts, the number of Kr-expressing amnioserosa cells is not different from wild type. Thus, brk specifically affects cell fates depending on low or intermediate levels of Dpp signaling, while those that require peak levels are not altered. To identify the underlying causes of the visible changes in cell fate, the effect of brk was examined on the expression of two groups of dorsal/ventral (DV) patterning genes. The first group consists of dpp, zerknullt (zen) and tolloid (tld), whose expression is initiated very early in syncytial blastoderm stages. Since they are ventrally repressed by Dorsal (Dl) protein, their expression domains are confined to the dorsal 40% of the egg's circumference. In brk mutant embryos dpp, zen and tld expression is initiated normally. However, in contrast to wild type their expression domains expand ventrally during mid-cellularization. These data demonstrate that brk is not required for the early ventrolateral repression of these genes, but is essential to prevent their lateral expansion during cellularization. The second group of DV patterning genes includes rhomboid (rho), u-shaped (ush) and pannier (pnr), which are not direct targets of repression by maternal Dl. The initiation of their expression during cellularization requires prior formation of the Dpp activity gradient. Therefore, they are candidates for being direct targets of Dpp signaling in the embryo. They are expressed in domains straddling the dorsal midline that are 12 (rho), 14 (ush) and 32 (pnr) cells wide at cellular blastoderm (cell counts at approx. 50% egg length). The two narrowly expressed genes rho and ush are not changed in brk mutant embryos. This is also true for late zen expression, which in brk mutant embryos, as in wild type, refines to a narrow 5- to 6- cell-wide stripe along the dorsal midline despite the prior expansion. However, pnr expression expands in brk mutant embryos and low ectopic pnr levels can be seen in a broad lateral domain that stops about five cells short of mesodermal sna expression. Thus, brk does not affect the Dpp target genes that are expressed in dorsalmost regions and supposedly depend on highest Dpp levels. However, a target gene that is expressed in a wider domain, and is therefore presumably activated by intermediate levels of Dpp, is expanded. In summary, brk mutations affect the Dpp activity gradient in the embryo by expanding the domains of expression of dpp and one of its activators (tld) into ventrolateral regions. Despite the uniform expression of dpp in the entire ectoderm, Dpp activity levels appear to be only mildly increased in the ventrolateral region since only low-level (zen) or intermediate-level (pnr) target genes are ectopically expressed, causing a reduction in the size of the nervous system and ventral epidermis accompanied by an expansion of dorsal epidermis. Peak levels of Dpp in dorsalmost positions appear to be normal, judging from both target gene expression and cell type differentiation (Jazwinska, 1999b).

In the Drosophila embryo, Dorsal, a maternally expressed Rel family transcription factor, regulates dorsoventral pattern formation by activating and repressing zygotically active fate-determining genes. Dorsal is distributed in a ventral-to-dorsal nuclear concentration gradient in the embryo, the formation of which depends upon the spatially regulated inhibition of Dorsal nuclear uptake by Cactus. Using maternally expressed Gal4/Dorsal fusion proteins, the mechanism of activation and repression by Dorsal has been explored. A fusion protein containing the Gal4 DNA-binding domain fused to full-length Dorsal is distributed in a nuclear concentration gradient that is similar to that of endogenous Dorsal, despite the presence of a constitutively active nuclear localization signal in the Gal4 domain. Whether this fusion protein activates or represses reporter genes depends upon the context of the Gal4-binding sites in the reporter. A Gal4/Dorsal fusion protein lacking the conserved Rel homology domain of Dorsal, but containing the non-conserved C-terminal domain also mediates both activation and repression, depending upon Gal4-binding site context. A region close to the C-terminal end of the C-terminal domain has homology to a repression motif in Engrailed -- the eh1 motif. Deletion analysis indicates that this region mediates transcriptional repression and binding to Groucho, a co-repressor known to be required for Dorsal-mediated repression. As has previously been shown for repression by Dorsal, activation by Dorsal, in particular by the C-terminal domain, is modulated by the maternal terminal pattern-forming system (Flores-Saaib, 2001).

The results presented here show that just as Dorsal sites function in a context-dependent manner in the presence of endogenous Dorsal, so too do Gal4 sites function in a context-dependent manner in the presence of a Gal4/Dorsal fusion protein. When Gal4/Dorsal*/nt1 binds to multiple tandemly repeated Gal4 sites upstream of a core promoter, the result is activation. In contrast, when Gal4/Dorsal*/nt1 binds a modified dpp VRR in which two critical Dorsal-binding sites have been replaced by Gal4-binding sites, the result is repression. Thus, bringing Dorsal to its target sites is sufficient for both activation and repression -- the rel homology domain (RHD) itself need not be directly engaged with the DNA. Perhaps Dorsal, other DNA-bound repressors (the assistant repressors) and co-repressors such as Gro cooperatively assemble at the ventral silencer to form a 'silencesome'. As might be expected if silencer function required the assembly of such a complex, silencing by the zen VRR is crucially dependent upon the spacing between the sites for the DNA-binding proteins. Changing the spacing (by a non-integral multiple of the DNA helical repeat distance) severely abrogates silencing, presumably by rotating DNA-bound proteins onto opposite faces of the helix. Very similar spacing effects have been observed for enhancesomes (Flores-Saaib, 2001).

The co-repressor Gro, which is required for Dorsal-mediated repression, interacts with the Dorsal RHD. This finding is consistent with the observation that truncated forms of Dorsal consisting of little more than the RHD are able to mediate partial repression of target genes such as zen and dpp. However, the repression directed by the RHD alone is weak relative to that directed by full-length Dorsal and it is therefore not surprising to discover an additional Gro-interaction domain in Dorsal, this one in the CTD. Although the CTD is not conserved between Rel family proteins, the Dorsal-related immunity factor (Dif) can partially substitute for Dorsal during embryogenesis. In addition, patterning of the chick limb may involve the regulation by NF-kappaB of the vertebrate orthologs of Dorsal-target genes. Given these similarities in function, how is it possible to explain the apparent absence of the eh1-like repression domain from Dorsal-homologs such as Dif and NF-kappaB? One possibility is that Rel family protein-mediated transcriptional repression is of relatively minor importance to pattern formation. This is possible because other redundant mechanisms involving Short gastrulation (Sog)-family inhibitors exist to ensure that Dpp-orthologs will not be active at inappropriate positions along the dorsal/ventral axis of the metazoan embryo. The additional Gro-interacting repression domain in the Dorsal CTD may have arisen relatively recently, perhaps as an evolutionary adaptation to allow more complete or more reliable repression of dpp and other genes that interact with dpp to pattern the dorsal ectoderm (Flores-Saaib, 2001).

Regulation in segment polarity (part 1/2)

The principal function of Hedgehog is to activate transcription of dpp at the boundary between anterior and posterior compartments of the developing wing, thereby establishing a source of dpp activity that is the primary determinant of antero-posterior patterning. In a remarkably similar fashion, the function and expression of the sonic hedgehog (shh) gene is closely associated with the 'zone of polarizing activity' (ZPA) that controls antero-posterior patterning of the vertebrate limb. Both of these functions suggest a role for Hedgehog family proteins as morphogens. The effects on Drosophila wing patterning were explored by ectopically expressing varying levels of hh and shh, as well as of the hh target gene, dpp. Different levels of hh activity can induce graded changes in the patterning of the wing. Zebrafish shh acts in a similar though attenuated fashion. Varying levels of ectopic hh and shh activity can differentially activate transcription of the patched and dpp genes. Ectopic expression of dpp alone is sufficient to induce the pattern alterations caused by ectopic hh or shh activity (Ingram, 1995).

The identity of anterior cells in the wing imaginal disc requires cubitus interruptus function. Anterior cells lacking ci express hedgehog and adopt posterior properties without expressing engrailed. Most clones cause an up-regulation of CI protein levels in surrounding cells, in a manner that is similar to that of CI along the A/P compartment boundary. Increased levels of CI can induce the expression of the HH target gene decapentaplegic in a HH-independent manner, suggesting that dpp is a target gene of CI. This is the first identified component of the HH-signaling cascade that is able to activate dpp transcription. Also, there is reason to believe that CI can also repress dpp, suggesting that CI can act as both a repressor and as an activator of dpp transcription in a concentration dependent manner. CI also positively regulates patched. Thus, expression of CI in anterior cells controls limb development by restriction HH transcription to posterior cells and by conferring competence to respond to HH by mediating the transduction of this signal. The multiple role of CI in the anterior compartment suggests that anterior cell identity is not a default fate that imaginal cells adopt in the absence of engrailed (Domínguez, 1996).

Hedgehog (Hh) proteins play diverse organizing roles in development by regulating gene expression in responding cells. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Hh target genes. A repressor form of Ci arises in the absence of Hh signaling by proteolytic cleavage of intact Ci. This cleavage is essential for limb patterning and is regulated by Hh in vivo. Evidence is provided for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling (Methot, 1999).

The function of Hh in controlling growth and pattern of the wing primordium is mediated to a large extent by the local expression of Dpp, which is secreted from a subset of anterior cells in response to Hh signaling. Dpp acts directly, at long range, and in a concentration-dependent manner to convey positional information to wing cells along the anteroposterior axis. Thus, the precise domain in which dpp is expressed and the absolute levels of Dpp secreted are consequential for the morphogenesis of the wing. It may not be coincidence, therefore, that it is precisely the dpp gene that is subject to both modes of Ci control. In the simultaneous absence of ci and en function, dpp is expressed at a constitutive basal level in all wing cells. From this is surmised the existence of a ubiquitous enhancer (B, for basal) that stimulates dpp transcription by default. The results presented in this paper indicate further that both regulatory inputs, Ci[act] and Ci[rep], act on dpp, and it is proposed that their superimposition serves to 'sharpen' the Dpp morphogen source. Two consequences can be invoked from the combination of the two regulatory mechanisms: (1) a narrowing of the dpp stripe, and (2) an increase in dpp expression levels. Finally, it is noted that the dual control of dpp expression by Ci necessitates a mechanism to prevent dpp transcription in P cells that contain neither form of Ci and would thus express dpp by default. This complication appears to be solved by subjecting dpp expression to repression by En. The result of all these regulatory measures is an exquisitely controlled system in which Dpp is secreted at high levels by a narrow strip of cells located along the A/P compartment boundary in the center of the wing primordium (Methot, 1999).

Different thresholds of Wg activity in the wing imaginal disc elicit different outcomes, which are mediated by regulation of decapentaplegic expression and result in alterations in the expression of homeotic genes. A high level of Wg activity leads to loss of all dorsal pattern elements and the formation of a complete complement of ventral pattern elements on the dorsal side of legs, and is correlated with repression of dpp expression. wg expression in dorsal cells of each disc also leads to dose-dependent transdetermination in those cells in homologous discs such as the labial, antennal and leg, but not in cells of dorsally located discs. When dpp expression is repressed by high levels of Wg, transdetermination does not occur, confirming that dpp participates with wg to induce transdetermination. These and other experiments suggest that dorsal expression of wg alters disc patterning and disc cell determination by modulating the expression of dpp. The dose-dependent effects of wg on dpp expression, ventralization of dorsal cells and transdetermination support a model in which wg functions as a morphogen in imaginal discs (Johnston, 1996).

The finding that Wingless and Decapentaplegic suppress each others transcription provides a mechanism for creating developmental territories in fields of cells. What is the mechanism for that antagonism? The dishevelled and shaggy genes encode intracellular proteins generally thought of as downstream of WG signaling. The effects of changing either DSH or SGG activity were investigated on both cell fate and wg and dpp expression. At the level of cell fate in discs, DSH antagonizes SGG activity. At the level of gene expression, SGG positively regulates dpp expression and negatively regulates wg expression while DSH activity suppresses dpp expression and promotes wg expression. Sharp borders of gene expression correlating precisely with clone boundaries suggest that the effects of DSH and SGG on transcription of wg and dpp are not mediated by secreted factors but rather act through intracellular effectors. The interactions described here suggest a model for the antagonism between WG and DPP that is mediated via SGG. The model incorporates autoactivation and lateral inhibition, which are properties required for the production of stable patterns. In the Dorsal part of the leg disc, DPP signalling predominates; DPP together with SGG inhibit wg expression and the consequencent lack of inhibition of SGG promotes further dpp expression. In the ventral part of the disc, WG signaling predominates and WG acts through DSH to inhibit SGG activity thus removing the activator of dpp (SGG) and promotes its own expression by removing the combinatorial inhibition of SGG and DPP. The regulatory interactions described exhibit extensive ability to organize new pattern in response to manipulation or injury (Heslip, 1997).

The effects of ectopic Cubitus interruptus on decapentaplegic and patched transcription was assayed using dpp and ptc reporter plasmids. In the third larval instar wing disc, expression of the dpp reporter is activated ectopically in all cells expressing high levels of CI protein in the anterior compartment, but is not activated in the posterior compartment. Expression of the ptc reporter is activate ectopically throughout both anteior and posterior compartments. Thus in the embryo, high levels of CI protein are sufficient to activate transcription of patched, even on the presence of Engrailed; however, ectopic CI activity apparently cannot overcome the repression of dpp transcription by EN (Alexandre, 1996).

The secreted protein Hedgehog (Hh) transmits a signal from posterior to anterior cells that is essential for limb development in insects and vertebrates. In Drosophila, Hh has been thought to act primarily to induce localized expression of Decapentaplegic and Wingless, which in turn relay patterning cues at long range. Hh plays an additional role in patterning the wing. Engrailed is expressed in the posterior compartment and in anterior cells close to the AP boundary. Anterior En levels decrease rapidly with distance from the posterior comparment. Expression of En in anterior cells is thought to be regulated by Hh. Consistent with this, anterior clones of smoothened fail to express En, whereas posterior smo clones express En normally. Likewise, anterior expression of Ptc is regulated by Hh. As Hh acts directly to induce Dpp, to upregulate Ptc in a broad domain of 8-10 cell diameters, and to induce En in a narrow band of cells close to the AP boundary, it is possible that the broad spatial domains of Ptc and Dpp and the narrow domain of En reflect requirements for different levels of Hh activity. Use of a temperature sensitive allele of hh shows that dpp can be activated by levels of Hh activity that are below the minimal levels required to activate En (Strigini, 1997).

By replacing endogenous Hh activity with that of a membrane-tethered form of Hh, it has been shown that Hh acts directly to pattern the central region of the wing, in addition to its role as an inducer of Dpp. Comparing the biological activities of secreted and membrane-tethered Hh provides evidence that Hh forms a local concentration gradient and functions as a concentration-dependent morphogen in the fly wing. Such tethered Hh can only induce En in immediately adjacent cells. Tethered Hh expressing cells also act on adjacent cells to induce dpp. It is noted that dpp expression is reduced in cells that express En at high levels, suggesting that En represses dpp. Membrane-tethered Hh flies develop to pharate adults that completely lack wings. Restoring Hh during development allows flies to form wings that show substantial rescue of anterior and posterior structures while missing structures from the cental region. These results suggest that Hh plays an important role in directly patterning the central region of the wing, while Dpp is primarily responsible for patterning at long range (Strigini, 1997).

Cyclic AMP (cAMP)-dependent Protein kinase A (PKA) is essential during limb development to prevent inappropriate decapentaplegic and wingless expression. A constitutively active form of PKA can prevent inappropriate dpp and wg expression, but does not interfere with their normal induction by hh. It seems that the basal activity of PKA imposes a block on the transcription of dpp and wg and that hh exerts its organizing influence by alleviating this block (Jiang, 1995).

patched inhibits decapentapletic expression in the anterior compartment of imaginal discs. This results in a restricted expression of dpp near the anterior-posterior compartment boundary. This is essential to maintain the wild-type morphology of the wing disc. Viable mutations in the segment polarity genes patched and costal-2 cause specific alterations in dpp expression within the anterior compartment of the wing imaginal disc. The interaction between ptc and dpp is particularly interesting; both genes are expressed with similar patterns at the anterior-posterior compartment boundary of the disc, and mis-expressed in a similar way in segment polarity mutants ptc and cos2. This mis-expression of dpp may be correlated with some of the features of the adult mutant phenotypes (Capdevila, 1994).

engrailed activity programs wing cells to express hh whereas the absence of en activity programs them to respond to hh by expressing dpp. As a consequence, posterior cells secrete hh and induce a stripe of neighboring anterior cells across the compartment boundary to secrete DPP. DPP can exert a long-range organizing influence on surrounding wing tissue, specifying anterior or posterior pattern depending on the compartmental provenance, and hence the state of en activity, of the responding cells. Thus, DPP secreted by anterior cells along the compartment boundary has the capacity to organize the development of both compartments. DPP may exert its organizing influence by acting as a gradient morphogen in contrast to HH which appears to act principally as a short range inducer of dpp (Zecca, 1995).

Table of contents

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

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