Interactive Fly, Drosophila



Promoter Structure

A TATA box is located 25 bp upstream from the transcription start site (Rushlow, 1987).

The zen promoter shows a two-tier organization: Distal sequences mediate its initial response to maternal factors, whereas proximal sequences are responsible for the refinement of the pattern in older embryos. The distal regulatory element has the property of a silencer (or anti-enhancer) element and can act over a distance to repress ventral expression of a heterologous promoter. Proximal promoter sequences interact with factors that may be modulated by a cell-cell communication pathway (Doyle, 1989).

The region upstream from the zerknüllt gene contains three sites that specifically bind the ZEN protein. Evidence for these binding sites was obtained by the filter binding technique and the DNase footprinting technique. Two of the ZEN protein binding sites were spaced only 30 base pairs apart. These sites could be separated without any loss in their specific binding properties. It is concluded that these two sites function independently in the binding of ZEN protein (Chen, 1989).

The Dorsal (DL) protein gradient determines patterns of gene expression along the dorsal-ventral axis of the Drosophila embryo. DL protein is at peak levels in ventral nuclei of the embryo where it activates some genes (twist and snail) and represses others (zerknüllt , decapentaplegic and tolloid). T-rich sequences close to the DL binding sites in the silencer region of the zen promoter are conserved between three Drosophila species. A minimal element (the ventral repression element, VRE) can mediate repression of a heterologous promoter by interacting with at least two factors present in embryonic extracts, one being DL protein. The other factor binds to the T-rich site. Point mutations in either site abolish ventral repression in vivo. In addition, mutations in the T-rich site cause ectopic expression in ventral regions indicating that the minimal silencer was converted into an enhancer (Kirov, 1993).

Dorsal acts to repress zerknüllt in conjunction with an HMG-like protein. One protein can activate some genes and repress others in the same cell. A Drosophila HMG1 protein, called DSP1 (dorsal switch protein), converts Dorsal and NF-kappa B from transcriptional activators to repressors. This effect requires a sequence termed the negative ventral regulatory element (VRE), found adjacent to Dorsal-binding sites in the zen promoter and adjacent to the NF-kappa B-binding site in the human interferon-beta enhancer. Previous studies have shown that another type of HMG protein, HMG I(Y), can stimulate NF-kappa B activity. Thus, the HMG-like proteins DSP1 and HMG I(Y) can determine whether a specific regulator functions as an activator or a repressor of transcription (Lehming, 1994).

The VRE sequence is located between -1.6 and -1.0. kb upstream of the zen start site. In order to examine the range of action of the VRE, a evenskipped minimal stripe 2 enhancer (MSE) was placed upstream of a reporter gene, and a VRE was placed downstream of a reporter gene. In these experiments, the closest Dorsal binding-sites in the VRE map nearly 5 kb from the MSE activators. Nonetheless, stripe 2 repression is repressed in ventral regions of early embryos. Repression takes place irrespective of orientation of the VRE. This repression appears to be distinct from that mediated by snail, Krüppel and knirps repressors that function in a local fashion to inhibit, or quench nearby activators within the enhancer to which it is bound (Cai, 1996).

To determine whether the gypsy insulator can block silencer-promoter interactions, it was placed between the VRE of zen and the eve stripe 2 enhancer. The VRE contains both silencer elements and general activation sequences. Gypsy is a retrotransposon (movable genetic element) that can function as a boundary between distal enhancer type sequences and promoters of structural genes. Binding of Suppressor of Hairy wing, a zinc finger transcription factor, potentiates the insulating character of gypsy. The gypsy insulator does not substantially impede silencing from the VR600 element, so that stripe 2 expression is largely excluded from ventral regions. The insulator does block VR600 activation function, indicating that the gypsy element selectively blocks activator-promoter interactions compared with silencer-promoter interactions (Cai, 1995).

Krü, snail, and knirps do not appear to require DNA binding cofactors to mediate transcriptional repression. The VRE contains binding sites for a number of proteins suggesting that the repressor may function as a complex. Some of these components may be redundant. One putative corepressor, DSP1 (dorsal switch protein 1), may be necessary for optimal silencing, but does not appear to be necessary for VRE activity, because minimal VRE sequences lacking DSP-1 binding sites can still mediate ventral repression. Another protein, NTF-1/Elf-1, was recently reported to bind to the ventral repression elements of the zen and dpp promoters. However, as with DSP1, it is still unclear whether the NTF-1/Elf-1 protein itself is involved in VRE activity. Interaction of DL and corepressor require fixed spacing between DL and corepressor binding sites, as separation of DL and corepressor sites with a 5-bp spacer, abolishes the silencer activity, while separation with a 10-bp spacer restores repression. In addition, zen VRE can silence multiple enhancers in a modular promoter, containing the VRE placed between eve stripe 2 and stripe 3 enhancers (Cai, 1996 and references).

Grainyhead protein binds to and regulates, in conjunction with the transcription factor Dorsal, an essential ventral repression region associated with the zerknüllt gene (Huang, 1995). zerknüllt is repressed by Dorsal in the ventral ectoderm of the developing fly. In contrast with the zerknüllt ventral repressor element, which contains a few high-affinity DL-binding sites, dpp contains multiple relatively low-affinity sites that function together to bring about complete ventral repression. Because dpp and zen have nearly coincident early expression domains, these results indicate that the same boundary of repression can be specified by DL-binding sites of different affinity (Huang, 1993).

Dorsal activates twist, and it also functions as a direct transcriptional repressor of zerknüllt. By exchanging DL binding sites between the promoters it can be shown that activator sites from twi can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo (Jiang, 1992). When multiple copies of a Dorsal binding site from the zerknüllt ventral repressor element are fused to a heterologous basal promoter, the resulting construct is activated by Dorsal to give a ventral specific expression pattern. Thus, the ability of a Dorsal binding site to mediate repression rather than activation is not an intrinsic property of the site, but depends upon its context (Pan, 1992).

Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknullt (zen) contains Dorsal binding sites as well as an essential element termed AT2. An AT2 DNA binding activity has been identified (called ZREB) and purified in embryos. It consists of cut (ct) and dead ringer (dri) gene products. dri was isolated as a novel gene encoding a sequence-specific DNA-binding protein. Dri is a founding member of a growing protein family whose members share a conserved DNA binding domain termed the A/T-rich interaction domain. dri is developmentally regulated, being expressed in a restricted set of cells including some neural cells and differentiating cells of the gut and salivary gland ducts. Dri is a member of the recently defined ARID family of DNA binding proteins, a family that includes the B-cell-specific factor Bright and the Drosophila factor Eyelid. Although Bright is thought to function as a transcriptional activator, genetic data suggest that Eyelid functions to repress transcription in response to activation of the wingless pathway (Valentine, 1998 and references).

Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, all of which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors (Valentine, 1998).

To determine if cut and dir are required for the activity of the AT2 site in vivo, the effects of mutations in these genes were examined on the activity of the lacZ transgene under control of the minimal zen VRR. For both cut and dir, germ line clones were generated to test the effects of eliminating maternally contributed gene products, and, in addition, the effects of eliminating zygotically produced gene products were examined. A null mutation in ct (which is an X-linked gene) results in strong ventral derepression of the transgene. This ventral derepression is observed in about one-half the embryos derived from a cross between females containing ct germ line clones and hemizygous males. It was never observed in a cross between heterozygous females and hemizygous males, suggesting that derepression requires simultaneous elimination of both maternal and zygotic Ct. A strong hypomorphic mutation in dri (which is an autosomal gene) also results in strong derepression. In contrast to the results observed with ct, this effect is strictly zygotic. It is observed in a cross between heterozygous dri males and females but not in a cross between females carrying dri germ line clones and wild-type males. Most strikingly, in the absence of zygotic Dri, the zen VRR directs strong ventral expression in the blastoderm embryo, reminiscent of the results observed when the AT2 element is mutagenized. These results strongly suggest that, in the context of the minimal zen VRR, Dri plays an essential role in converting Dorsal from an activator into a repressor. The dri mutation results in a significant weakening of the transverse eve stripe (generated by the minimal even skipped (eve) stripe 2 enhancer (MSE) as well as a shift in the position of the stripe toward the anterior pole of the embryo, presumably due to a role for Dri in anteroposterior pattern formation. Despite the strong effects of the cut and dir mutations on the activity of the minimal zen VRR, both genes make only minor contributions to the ventral repression of the endogenous zen gene in the stage 4 embryo. In the absence of both zygotic and maternal Ct or in the absence of zygotic Dri, zen expression in the stage 4 embryo is still largely restricted to the dorsal 40 to 50% of the embryo, although weak ventral patches of zen expression are observed with high frequency. Such patches are never observed in wild-type embryos stained in parallel with these embryos. The contrast between the strong effect observed for the minimal VRR and the weak effect observed for the endogenous zen gene suggests redundancy in the zen locus. In other words, there may be additional unidentified ventral repression regions in the zen locus that function in a Ct- and Dri-independent manner. Although neither Ct nor Dri is essential for ventral repression of the endogenous zen gene in the stage 4 embryo, both factors appear to play essential roles in the refinement of the zen pattern that normally occurs in stage 5 embryos. Normally, zen expression refines during cellularization to a stripe approximately three to five cells in width. However, in the absence of both maternal and zygotic Ct or in the absence of zygotic Dri, a severe refinement defect is observed (Valentine, 1998).

Both Dorsal and Dri bind to the corepressor Gro in vitro, suggesting a possible mechanism for repression in which Dorsal and Dri recruit Gro to the template. This model is strengthened by results showing that Dorsal and Dri bound to DNA can cooperatively recruit Gro to the zen VRR in vitro. However, the magnitude of the cooperativity observed in vitro is small (twofold) and therefore does not completely account for the absolute requirement for the Dorsal and AT2 sites observed in germ line transformation assays. This suggests that factors in addition to Dorsal and Dri are required for the efficient recruitment of Gro in vivo. For example, it is possible that the addition of Ct would enhance cooperative recruitment, an idea that could not be tested due to difficulty obtaining sufficient amounts of recombinant Ct. It is also likely that elements in addition to Dorsal sites and AT2 are required for efficient Gro recruitment and therefore for efficient repression, since previous experiments indicate that, while these sites are required for repression, they are not sufficient for repression. Finally, it is possible that the cooperativity of Gro recruitment would be enhanced in the context of chromatin templates rather than naked DNA templates (Valentine, 1998).

Genome-wide analysis of clustered Dorsal binding sites was used to examine the distribution of Dorsal recognition sequences in the Drosophila genome. The homeobox gene zerknullt (zen) is repressed directly by Dorsal, and this repression is mediated by a 600-bp silencer, the ventral repression element (VRE), which contains four optimal Dorsal binding sites. The arrangement and sequence of the Dorsal recognition sequences in the VRE were used to develop a computational algorithm to search the Drosophila genome for clusters of optimal Dorsal binding sites. There are 15 regions in the genome that contain three or more optimal sites within a span of 400 bp or less. Three of these regions are associated with known Dorsal target genes: sog, zen, and Brinker. The Dorsal binding cluster in sog is shown to mediate lateral stripes of gene expression in response to low levels of the Dorsal gradient. Two of the remaining 12 clusters associated with genes that exhibit asymmetric patterns of expression across the dorsoventral axis. These results suggest that bioinformatics can be used to identify novel target genes and associated regulatory DNAs in a gene network (Markstein, 2002).

zen is an immediate target of the maternal Dl gradient. The gene is activated initially at nuclear cleavage cycle 11-12 within 1 h after the Dl gradient is formed. zen initially exhibits a broad pattern of expression in the presumptive dorsal ectoderm and at the termini. High and low levels of the Dl gradient keep zen off in ventral and lateral regions. sog exhibits a complementary pattern of expression because it is activated by Dl, whereas zen is repressed. As seen for zen, sog expression is detected shortly after the formation of the Dl gradient (Markstein, 2002).

The zen VRE contains four optimal Dl recognition sequences within a span of 400 bp. Three of the four Dl binding sites contained within the zen VRE conform to the following consensus sequence for high-affinity Dl binding sites: GGG(W)nCCM (where W = A or T, M = C or A, and n corresponds to either four or five W residues). The fourth recognition sequence (binding site 3 within the VRE) contains a G residue in the AT-rich central region and is represented by the optimal consensus sequence GGGWDWWWCCM (where D = A, T, or G). To determine whether a similar density of optimal Dl sites might account for the regulation of sog, the entire Drosophila genome was scanned for clusters of any of the 208 unique Dl sequences that conform (either directly or by reverse complement) to two degenerate sequences: GGG(W)4CCM and GGGWDWWWCCM (Markstein, 2002).

Transcriptional Regulation

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. 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. Normally, the Toll and Torso pathways are both active only at the embryonic poles, and consequently, target genes (zen and dpp) that are repressed in middle body regions are expressed at these sites. Constitutive activation of the Torso pathway causes severe embryonic defects, including disruptions in gastrulation and mesoderm differentiation, as a result of misregulation of dl target genes. These results suggest that RTK signaling pathways can control gene expression by antirepression, and that multiple pathways can fine-tune the activities of a single transcription factor (Rusch, 1994).

Both maternal and zygotic genes contribute to the establishment of the expression patterns of the following four zygotic patterning genes: decapentaplegic (dpp), zerknüllt (zen), twist (twi), and snail (sna). All of these genes are initially expressed either dorsally or ventrally in the segmented region of the embryo, and at the poles. The DL gradient appears to be interpreted on three levels: dorsal cells express dpp and zen, but not twi and sna; lateral cells lack expression of all four genes; ventral cells express twi and sna, but not dpp and zen. DL appears to activate the expression of twi and sna and repress the expression of dpp and zen. Polar expression of dpp and zen requires the terminal system to override the repression by DL, while that of twi and sna requires the terminal system to augment activation by DL (Ray, 1991).

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).

The Drosophila protein DSP1, an HMG-1/2-like protein, binds DNA in a highly cooperative manner with three members of the Rel family of transcriptional regulators (NF-kappaB, the p50 subunit of NF-kappaB, and the Rel domain of Dorsal). This cooperativity is apparent with DNA molecules bearing consensus Rel-protein-binding sites and is unaffected by the presence of a negative regulatory element, a sequence previously proposed to be important for mediating repression by these Rel proteins. The cooperativity observed in these DNA-binding assays is paralleled by interactions between protein pairs in the absence of DNA. In HeLa cells, as assayed by transient transfection, expression of DSP1 increases activation by Dorsal from the twist promoter and inhibits that activation from the zen promoter, consistent with the previously proposed idea that DSP1 can affect the action of Dorsal in a promoter-specific fashion (Brickman, 1999).

DSP1 has opposite effects on the activity of Dorsal assayed with regulatory sequences excised from the twist and zen promoters. These experiments were performed by transiently transfecting mammalian cells in culture. Thus, reporters containing either a 180-bp fragment from zen (a fragment sufficient to mediate repression in Drosophila) or the entire regulatory region of twist (from -1,438 to +38) were activated by cotransfection with DNA encoding Dorsal. Cotransfection with DNA encoding DSP1 has just the opposite effects on this Dorsal mediated activation of the two promoters: activation from the twist promoter is stimulated 4-fold, whereas that from the zen promoter is inhibited 3-fold. DSP1's stimulation of Dorsal-mediated activation from the twist promoter can be mapped to the defined enhancer elements or VARs. Thus, DSP1 also stimulates Dorsal-mediated activation if the template bears, instead of the intact twist promoter, a cassette that contains the two VARs that drive ventral-specific expression of the twist gene in the Drosophila embryo. The two VARs together constitute approximately 300 bp and contain multiple Rel-protein-binding sites (Brickman, 1999).

It is not known what DNA sequences in the zen and twist promoters determine the opposite effects of DSP1 on dorsal-mediated activation. The finding that a negative regulatory element (NRE) has no effect on cooperative binding to DNA of DSP1 and various Rel proteins prompted a reexamination of the earlier claims that DSP1 converts Dorsal, the p50 homodimer, and the NF-kappaB heterodimer into repressors and that this effect requires the NRE. In each case, DSP1 inhibits Rel-protein-dependent activation both in the presence and absence of an NRE. In no case was NRE-dependent conversion of the Rel protein to a repressor by cotransfection with DSP1 observed. It is not understood why the current results differ from those reported previously (Brickman, 1999 and references therein).

Sites of the described protein-protein interactions are found in the conserved Rel domains and in the fragment of DSP1 that bears both HMG domains. The Rel domains of p65 and of Dif differ from those of Dorsal and of p50 in that they lack the HMG-domain-interaction site. The HMG domain of DSP1 also interacts with the TATA-binding protein. Similar interactions have been reported for HMG-1 and HMG-2 with the steroid hormone receptors, for HMG-1 with p53, for HMG-1 with HOXD9, and for HMG-2 with Oct2. Thus, the HMG domain may contain a common structural motif for cooperative DNA binding and interaction with other transcription factors. The interaction between TATA-binding protein and DSP1 also seems to be influenced by the glutamine-rich amino-terminal domain in that the full-length DSP1 interacts more avidly with TATA-binding protein than does the HMG-1 domain. These experiments suggest that the amino-terminal glutamine-rich domain may also potentiate the DSP1-Rel protein interaction as well, because all DSP1-Rel interactions seem stronger with full-length DSP1, particularly the weak interactions seen between DSP1 and p65 or Dif, which are observed only with GST-DSP1 and not with GST-DSP1 (178-393) (Brickman, 1999).

capicua (cic) is involved in gene repression in Drosophila terminal and dorsoventral patterning. Torso signaling at the embryonic poles regulates repressor processes that operate during dorsoventral patterning. Such patterning depends on Dorsal: Dorsal activates ventral-specific genes [for example, twist (twi)] and represses dorsal-specific genes, such aszerknullt. Repression by Dorsal requires its association with Gro and other postulated corepressors that bind next to Dorsal in the zen promoter. This repressor complex is under negative regulation by Tor signaling at the embryonic termini, allowing zen expression at each pole of the embryo (Jimenez, 2000 and references therein).

The mechanism of repression by Dorsal is not fully understood. Dead-Ringer (Dri) and Cut (Valentine, 1998) function as corepressors that assist Dorsal (and Gro) in Dorsal's function as a repressor. However, the effects of removing either of these two factors appears weaker than those caused by the loss of Dorsal or Gro function, suggesting that other factors may also contribute to Dorsal repression. Because cic is involved in a Gro-mediated process that is inactivated by Tor signaling, it was of interest to see if cic could also be involved in Dorsal repression. Consistent with this idea, zerknullt expression is expanded ventrally in cic1 mutant embryos. Although this expansion is not as strong as in dorsal or gro mutants, ectopic zen transcripts are clearly detected in lateral and ventral regions of the embryo, especially in its posterior half. In contrast, activation of twi by Dorsal is normal in cic1 embryos, suggesting that cic only participates in repression, not activation, by Dorsal (Jimenez, 2000).

To test further the role of cic in ventral repression of zen, an examination was carried out of a lacZ transgene carrying an even-skipped (eve) stripe 2 enhancer coupled to a silencer from the zen promoter: the zen Ventral Repression Element (VRE), which includes binding sites for Dorsal and adjacent regulatory sites. In wild-type embryos, lacZ expression directed by the eve stripe 2 enhancer is repressed ventrally by the VRE. This repression is clearly attenuated in cic1 mutant embryos, permitting stripe 2 activation in the ventral-most side of the embryo. In addition, significant ectopic lacZ expression is observed in ventral and lateral regions of the embryo, as expected if repression by Dorsal bound to the VRE is switched in favor of activation. These results suggest that cic encodes one of the cofactors required for VRE activity and the conversion of Dorsal from an activator to a repressor of transcription. Because Dri and Cut also function as Dorsal corepressors, it appears that this role is shared by several factors with overlapping activities (Jimenez, 2000).

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic and Screw. short gastrulation, which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Sog has a second function, which is to promote BMP signaling on the dorsal side of the embryo. A weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and this lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of Sog is directed against Scw, data are presented that suggest that the positive activity of Sog is directed towards Dpp. Chordin shares the same apparent ligand specificity as does Sog, preferentially inhibiting Scw but not Dpp activity. However, in Drosophila assays Chordin, does not have the same capacity to elevate BMP signaling as does Sog, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates (Decotto, 2001).

Morphogen gradients, once a purely theoretical concept, are now viewed as central players in the establishment of cell identity in a broad range of developmental processes. However, the exact biological mechanisms used to establish and maintain a morphogen gradient vary, depending on the biological context. In the Drosophila embryo, while Dpp can act in a dose-dependent fashion to specify different cell fates along the DV axis, in vivo its activity is modulated spatially by other components of the patterning system. In particular, Sog, a diffusible BMP-binding protein, has been shown to inhibit BMP signaling ventrally by preventing ligand access to the BMP receptors. A novel aspect of Sog’s function has been characterized in this study. Specifically, Sog functions cell non-autonomously to elevate BMP signaling on the dorsal side of the embryo. Thus, the interpretation of any experiment to elucidate the role of Sog in the control of dorsoventral patterning must take into account the two apparently opposing functions of the protein (Decotto, 2001).

Loss-of-function mutations in Mad or Medea have been identified as dominant enhancers of a weak homozygous-viable sog mutation, and the enhanced embryos have been shown to have defects in amnioserosa specification. Furthermore, synthetic lethality between weak homozygous-viable alleles of sog and zen has been demonstrated, indicating that both are required for maximal production of amnioserosa. Lastly, there was a dramatic decrease in the level of zen transcription in sogP129D embryos that were derived from Mad/+ females, compared to the level of zen transcription in either genotype alone. Taken together, these results unambiguously demonstrate that the positive action of Sog is exerted before gastrulation to attain the maximal expression of a direct BMP target gene (Decotto, 2001).

Drosophila Smurf1 is a negative regulator of signaling by the BMP2/4 ortholog Decapentaplegic during embryonic dorsal-ventral patterning. Smurf1 encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog in Drosophila Mothers against dpp (Mad) and likely promotes its proteolysis. The essential function of Drosophila Smurf1 is restricted to its action on the Dpp pathway. Smurf1 has two distinct, possibly mechanistically separate, functions in controlling Dpp signaling. Prior to gastrulation, Smurf1 mutations cause a spatial increase in the Dpp gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, Smurf1 mutations cause a temporal delay in downregulation of earlier Dpp signals, resulting in a lethal defect in hindgut organogenesis. The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, and may have additional functions in regulating the levels of Dpp receptors (Podos, 2001).

In wild-type embryos at the onset of gastrulation, a stripe of P-Mad staining is visible in a dorsal subset of dpp-expressing cells and in the cells at either pole of the embryo. In Smurf115C mutant embryos, there is a small but statistically significant increase in the width of the dorsal P-Mad stripe as well as a nonquantitated increase in the intensity of staining. In wild-type embryos at this stage, the Dpp target genes zen and Race are activated by high levels of Dpp signaling in the presumptive amnioserosa, while the intermediate threshold target gene u-shaped (ush) is activated in a broader domain by lower levels of Dpp activity. All three transcriptional domains showed significant lateral expansion in Smurf115C mutant embryos; a lesser but significant expansion of zen was also observed in Smurf111R mutant embryos. Later, Smurf115C mutant embryos differentiate a nearly 2-fold excess of amnioserosa cells compared to wild-type. A 2-fold increase in dpp gene dosage effects a similar expansion of zen transcription and a comparable increase in amnioserosa cell number. These observations indicate that disruption of Smurf1 gene activity elicits an expansion of multiple Dpp signaling thresholds in the early embryonic ectoderm comparable to the phenotype caused by a doubling of dpp gene dosage (Podos, 2001).

Three genetic criteria indicate that defects in Dpp signaling directly cause the hindgut phenotype in Smurf1 mutant embryos: (1) the hindgut defects are not observed in Smurf1 mutant embryos that lack one copy of dpp; (2) a complete loss of zen function substantially suppresses the Smurf115C phenotype, restoring the embryonic hindgut to a tubular morphology and an interior location. It is noted that the hindgut of Smurf115C; zen double mutant embryos often fails to adopt the normal hook-shaped trajectory, suggesting that the deregulation of other target genes also contributes to the Smurf1 phenotype. (3) The hindgut defect was also suppressed in sog; Smurf115C double mutant embryos. While Sog antagonizes BMP signaling in the ventrolateral ectoderm, a positive activity of Sog is also required at the onset of gastrulation to promote the Dpp-dependent specification of amnioserosa at the dorsal midline. It is proposed that the blastoderm-specific Dpp signaling in the dorsal-most region of sog;Smurf1 embryos is reduced to a level that, even in the absence of temporal downregulation of P-Mad, does not elicit the observed Smurf1 hindgut defect (Podos, 2001).

The results suggest that Smurf1 provides an important mechanism to maintain the available pool of Mad at limiting concentrations, the necessity of which has been supported by previous genetic observations. Although not normally haploinsufficient, the Mad gene is rendered so when the activities of other components of the Dpp pathway, including dpp, zen, and sog, are reduced. More generally, limiting amounts of Smad protein might be an essential feature of all graded TGF-ß superfamily signaling systems. Cytoplasmic Smad pools are similarly limiting in Xenopus embryos, according to quantitative studies of activin signaling. Experimental elevations in Smad2 concentration cause proportionate increases in Smad activation, as represented by both nuclear Smad2 import and transcriptional readout. Therefore, it is predicted that Smurf enzymes will prove to be essential to maintain Smad proteins at limiting concentrations to ensure appropriate responses to all graded BMP and activin/TGF-ß signals (Podos, 2001).

The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain (Chang, 2001).

Dorsal epidermal and visual system fates, in particular those of the posterior optic lobe and larval eye, are not expressed in dpp loss of function. It is likely that these abnormalities are the result of changes in early head gene expression. This was followed in detail by assaying the expression of several regulatory genes known to be required for the normal development of the visual primordium, including otd, tll, so and eya in dpp-null mutants:

  1. otd is normally expressed in a wide domain that spans the dorsal midline and encompasses the entire dorsal head ectoderm. In normal development, its expression is turned off in the head midline (the head epidermis precursors) and in the part of the visual primordium forming the posterior optic lobe and larval eye. In dpp mutants, expression persists in the entire dorsal head ectoderm until stage 11. Expression then becomes patchy as many cells undergo apoptotic cell death (Chang, 2001).
  2. tll appears in the protocerebral ectoderm, including the head midline ectoderm. Only later does expression spread to cover part of the visual primordium. In embryos that lack Dpp, expression is expanded from the beginning to include the entire dorsal head. As for otd, expression also persists in the head midline ectoderm (Chang, 2001).
  3. so is expressed in a transverse stripe spanning the dorsal midline. This unpaired domain defines the eye field. Around gastrulation, so expression ceases in the dorsal midline and becomes restricted to the bilateral visual primordia. In addition to the visual system, so appears in the anlage of the stomatogastric nervous system (SNS) and head mesoderm. In a dpp-null fly, so is never expressed in the anlage of the visual system, although expression in the SNS and head mesoderm is unchanged (Chang, 2001).
  4. eya is normally expressed in a complex pattern that essentially consists of three domains located in the anlage of the SNS, the anterior protocerebrum and the anlage of the visual system. In dpp-null embryos, eya expression in the primordia of the visual system and SNS is absent from the beginning. The anterior protocerebral expression is narrowed (Chang, 2001).

The observed downregulation of head gap genes and early eye genes in the dorsal midline is an indirect effect of Dpp mediated by the Dpp target zerknüllt (zen). Previous studies have demonstrated that high levels of Dpp in the dorsal midline upregulate and focus the expression of zen in the amnioserosa and, further anteriorly, in the dorsomedial head epidermis. An RNA in situ probe revealed the expression of zen in the early eye field of a stage 5-7 embryo. Assaying the expression of head gap and early eye genes in a zen-null mutant background demonstrates that Zen acts as a repressor of these genes. Whereas in wild type, after an initial unpaired expression straddling the dorsal midline, tll, so and eya are turned off in the dorsal midline, they continue to be expressed in this domain in a zen mutant. At later stages, lack of zen results in a cyclops phenotype (Chang, 2001).

In the head region, highest levels of Dpp are required to promote mid dorsal fates (head epidermis, analogous to amnioserosa in the trunk). The activation of screw is involved in this process, similar to its role in the dorsomedial trunk. Intermediate Dpp levels promote dorsolateral fates (visual primordium). Low levels of Dpp are reached in the protocerebral neurectoderm and are permissive for the formation of protocerebral neuroblasts. Several of the regulatory genes expressed in the anterior brain and eye field may be direct targets of Dpp signaling. The findings show that so, eya and omb are activated by Dpp in the visual primordium. These regulatory genes initiate the fate of visual structures, in particular larval eye and outer optic lobe. It has recently been shown that eya and so are also targets of Dpp signaling in the eye imaginal disc (Chang, 2001).

The secondary restriction of so (and other genes with bilateral expression domains developing from unpaired domains, including tll and otd) is effected by the Dpp target zen in the dorsal midline. This homeobox gene is expressed as a response to peak levels of Dpp in the dorsal midline, including amnioserosa and, in the head of the embryo, in the dorsomedial head epidermis primordium. Loss of zen, similar to reduction of Dpp, results in the absence of amnioserosa and head epidermis, and a cyclops phenotype (Chang, 2001).

In view of these results, it is speculated that the interaction between Dpp and Hh is indirect and requires the function of so, eya and possibly other 'early eye genes' -- according to this model, Dpp activates so and eya in the eye field. Slightly later, expression of so and eya is lost dorsomedially, due to repression by Zen at this level. In a second step, the expression of Hh (which comes on later than Dpp) triggers larval eye fate in cells close to the Hh source. The response of a cell to Hh, that is, its expression of ato, depends on its previously expressing so and eya. Finally, Ptc inhibits the range of Hh action, similar to its alleged function in the trunk and imaginal discs (Chang, 2001).

A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:

  1. If the level of Dpp is reduced (in dpp null heterozygote, or dpp hypomorph), so and eya are stably expressed in the dorsal midline, since zen, which normally inhibits the early eye genes, is not expressed. As a result Hh can induce larval eye dorsomedially (Chang, 2001).
  2. In the cyclops phenotype that results from reduction of Dpp, the visual primordium develops as a double crested placode that spans the dorsal midline. In this placode, the posterior crest is formed by larval eye cells, in line with the tenet that Hh induces larval eye fate in the cells next to the Hh source (posteriorly). The anterior crest, which is further away from the Hh source, constitutes posterior optic lobe (Chang, 2001).
  3. In the cyclops phenotype induced by loss of Ptc or overexpression of Hh, larval eye cells are increased in number, compared with the Dpp reduction induced cyclops. At the same time, posterior optic lobe cells are reduced in number (Chang, 2001).

zerknüllt: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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