Mothers against dpp


Protein Interactions

MAD is required downstream of DPP receptors in eye morphogenesis. dpp expression in the morphogenic furrow is dependent on Hedgehog which diffuses anteriorly and antagonizes the repression of dpp by Patched and PKA, the catalytic subunit of Protein kinase A. The DPP response mediated by MAD plays a central role in the initiation of the morphogenetic furrow but is largely dispensable for its subsequent anterior propagation (Wiersdorff, 1996).

The furrow is generally more curved in discs of mutant Mad alleles, possible due to a delay in initiation at the lateral edges of the disc. Normally, the furrow seems to continue to initiate along the posterior margin faster than it is propagated in the center of the disc, thus ensuring that it forms a straight line across the eye disc during later stages. In wild type discs, dpp is expressed at the lateral margin until the morphogenetic furrow has passed. This expression is absent at this stage in the central part of Mad mutant discs, suggesting that Mad function is required for the expression of dpp at the margin. Once a partial initiation of the furrow has occurred, furrow progression appears unaffected across the anterior eye field. Clones of Mad mutant cells that include the posterior margin of the eye disc completely abolish initiation of the furrow within the mutant tissue; furthermore, furrow progression in neighboring wild-type tissue is unable to spread laterally into the clone, suggesting that MAD plays a minor role in the propagation of the furrow (Wiersdorff, 1996).

In cells transfected with Medea, the protein is predominantly localized to the cytoplasm; this localization was not altered by coexpression of activated TKV type I receptor. Thus, activation of DPP type I receptor alone is not sufficient to cause accumulation of Medea in the nucleus. In contrast, Mad protein is detected throughout the cell in the absence of signal, and coexpression with activated TKV receptor results in accumulation of Mad in the nucleus. Mutation of the putative phosphorylation sites in Mad, Mad(3SA), prevents nuclear accumulation in the presence of activated TKV receptor, indicating that phosphorylation of Mad is essential for accumulation in the nucleus. Since Mad is expressed at extremely high levels in these cells, it is unlikely that stoichiometric levels of a Smad4-like protein are required for Mad to accumulate in the nucleus. To determine whether entrance of Medea into the nucleus might require physical association with Mad, the subcellular localization of both proteins was examined when coexpressed in the same cell. Although most cells show Medea localized to the cytoplasm when it is expressed alone, coexpression of this protein with Mad increases the number of cells with nuclear Medea staining. Coexpression with both Mad and activated TKV results in strong Medea nuclear staining. In contrast, when Medea is coexpressed with Mad(3SA), which does not accumulate in the nucleus and does not interact with Medea, no shift of Medea from the cytoplasm to the nucleus is detected. Thus, the entrance of Medea into the nucleus requires physical association with phosphorylated Mad (Wisotzkey, 1998).

Under conditions that result in Dpp receptor activation, Mad is able to translocate to the nucleus, while Medea remains cytoplasmic. In the presence of activated Mad, however, Medea translocates to the nucleus. These observations suggest that Mad, but not Medea, is a direct target of the receptor signal, and that the signal from the activated receptor complex to Medea is mediated by Mad. Thus it is likely that Medea, unlike Mad, does not interact with the type I receptor. The distinct responses of these two closely related proteins to stimulation in cell culture, provide a biochemical explanation for the genetic requirement for Mad and Medea in dpp signaling. The basis of this difference in response to receptor activation may lie in the major sites of phosphorylation for the Smads. The class I Smads have been shown to be phosphorylated in response to stimulus at C-terminal serines (consisting of the SSXS motif), an event that is important for signaling. This motif is absent in Medea and the other class II Smads, as well as in the class III Smads. From these observations, it is possible to draw a model whereby the activation of Mad occurs before the activation of Medea during Dpp signal transduction. The levels of Mad that become activated (Mad*) determine the potential of the next, equally important step, which is its hetero-oligomerization with Medea. Thus, the higher levels of signaling achieved by the Dpp/Punt/Tkv activation system in cell culture, yield higher levels of Mad*, and cause high levels of nuclear Medea, while the lower Tkv* stimulus yields low levels of Mad*, and hence undetectable levels of nuclear Medea. Since the formation of the Mad*-Medea complex is important for signaling, from this model it is also conceivable that a quantitative increase in the levels of Medea protein can compensate for a reduction of Mad, by increasing the likelihood of the hetero-oligomerization of Mad* with Medea, thereby explaining the ability of Ubi-Medea to rescue the maternal effect lethality of Mad 12 /+ flies with dpp hr27 (Das, 1998).

Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. A common mediator Smad, Medea, is described, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Medea upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp (Inoue, 1998).

Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. The effect of Dad on Mad phosphorylation by Tkv was studied. Various combinations of Mad, Dad, and constitutively active Tkv were introduced into COS cells. In the first experiment, cells were labeled with [32P]orthophosphate in vivo, and incorporation of radioactivity into Mad was detected. Dad inhibits phosphorylation of Mad by constitutively active Tkv. Next, anti-phosphoserine antibody was used. As in the orthophosphate labeling, phosphorylation of Mad diminishes in the presence of Dad. In vertebrates, inhibitory Smads such as Smad6 and Smad7 have been shown to stably associate with type I receptors. The interaction of Mad or Dad with Tkv was studied: cells were transfected with an appropriate combination of expression plasmids, affinity labeled with iodinated BMP-2, and subjected to immunoprecipitation with antibodies against Mad or Dad. Pathway-specific Smads are known to associate with type I receptors upon ligand stimulation, but this interaction is too brief to detect under natural conditions. The interaction can be observed when the type I kinases are rendered inactive or when the C-terminal phosphorylation sites of the Smads are modified to be resistant to phosphorylation. Mad interacts with the kinase-defective form of Tkv, whereas the interaction of Mad with wild type Tkv is also detectable. The interaction of Mad with Tkv might thus be more stable than that of mammalian Smads with receptors. Dad interacts with wild-type Tkv as efficiently as with the kinase-defective form of Tkv. Notably, almost the same amount of Tkv is immunoprecipitated with Mad and Dad, although the expression level of Mad is much higher than that of Dad. Thus the affinity of Dad with Tkv seems to be higher than that of Mad. It was found that the interaction of Mad with Tkv was hampered by expression of Dad. Dad thus inhibits the Tkv phosphorylation of Mad by competing with Mad in association with the receptor. Constitutively active Tkv causes hetero-oligomerization of Mad with Medea. The effect of Dad on the hetero-oligomerization was examined: could Dad inhibit the constitutively active Tkv-induced complex formation of Mad and human Smad4? The hetero-oligomerization of Mad with Smad4 was shown to be efficiently blocked. Dad thus blocked a critical step in the activation of Mad (Inoue, 1998).

The following model is suggested for Dpp signaling by Mad, Medea, and Dad: Dpp induces phosphorylation of Mad through Tkv and Punt. Mad then forms homo-oligomeric complexes and/or hetero-dimerizes with Medea. Oligomers of Mad and Medea translocate into the nucleus where they transactivate target genes, such as vestigial. Dad is one such target, and its expression is induced by Dpp. Dad stably binds to Tkv and interrupts phosphorylation of Mad by Tkv (Inoue, 1998).

In the visceral mesoderm, dpp is expressed in parasegment (ps) 7 under the control of the homeotic gene Ultrabithorax (Ubx). In this cell layer, dpp stimulates its own expression and the expression of Ubx. dpp also stimulates the expression of wingless (wg), an extracellular signaling molecule of the Wnt family, in the neighbouring ps8. wg in turn feeds back to stimulate Ubx and dpp expression in ps7. Thus, dpp is part of a parautocrine feedback loop by which Ubx maintains its own expression indirectly through controlling dpp and wg. Dpp also diffuses from its mesodermal source through the endodermal cell layer of the embyonic midgut, where it stimulates the expression of D-Fos and of the homeotic gene labial. These inductive steps ultimately specify the differentiation of distinct cell types in the larval midgut epithelium. In order to understand the mechanism by which dpp stimulates transcription, a short enhancer fragment of Ubx, called Ubx B, has been characterized that contains response sequences for dpp and wg signaling in the embryonic midgut. The dpp response sequence of this enhancer is bipartite, consisting of a tandem repeat of Mad binding sites and a cAMP response element (CRE). The presence of the latter raised the question whether the co-activator CBP (CREB-binding protein, binding to CREs) might participate in Dpp-induced transcriptional activation (Waltzer, 1999).

Drosophila CBP loss-of-function mutants show specific defects that mimic those seen in mutants that lack the extracellular signal Dpp or its effector Mad. CBP loss severely compromises the ability of Dpp target enhancers to respond to endogenous or exogenous Dpp. CBP binds to the C-terminal domain of Mad. These results provide evidence that CBP functions as a co-activator during Dpp signaling, and they suggest that Mad may recruit CBP to effect the transcriptional activation of Dpp-responsive genes during development (Waltzer, 1999).

The embryonic midgut of nejire (nej) mutants (whose CBP function is reduced) show phenotypes related to wg gain-of-function phenotypes: increased labial expression in the endoderm, and derepression of the Ubx B enhancer in the visceral mesoderm. These phenotypes do not resemble those seen in dpp or Mad mutants: in Mad mutants, labial expression is strongly reduced, and so is the beta-galactosidase (lacZ) staining mediated by the Ubx B enhancer in the middle midgut. However, the narrow band of lacZ staining normally visible in the visceral mesoderm of the gastric caeca (in ps3) is absent in nej mutant embryos. Indeed, closer inspection reveals that the gastric caeca frequently fail to elongate in these mutants. A similar phenotype is observed in Mad and in dpp mutants. Thus nej, like dpp, is required for the formation of the gastric caeca, and also for the activity of the Ubx B enhancer in the caecal primordia. The activity of this enhancer in these primordia coincides with Dpp expression and depends on dpp function. The formation of the first midgut constriction is often impeded. While this could reflect overactive Wg signaling, it also mimics loss of glass bottom boat (gbb) signaling: Gbb is a Dpp homolog expressed in the visceral mesoderm and whose function is required for the formation of the first midgut constriction (Waltzer, 1999).

The hypothesis that CBP is a co-activator of dpp-induced transcription was tested by examining the Dpp response of the Ubx enhancer in nej mutants. Because it was expected that the repressive effect of CBP on this enhancer would mask a possible activating effect of CBP in cells in which the enhancer is stimulated by Wg signaling, a mutant version of Ubx B, called B4, was used whose positive response to Wg is abolished. B4 activity in the midgut is reduced compared with the wild-type enhancer; however, B4 still contains a fully functional dpp-response sequence and can be efficiently stimulated by ectopic Dpp. B4 can thus be used to selectively monitor the stimulation of Ubx by Dpp in the visceral mesoderm. The activity of Ubx B4 is significantly reduced in nej mutants. LacZ staining is particularly weak in ps6/7 (near the Dpp source), but also in ps10, and is barely detectable in the gastric caeca. Furthermore, in nej mutant embryos derived from nej mutant germlines (nej), lacZ staining mediated by B4 is even weaker than in the zygotic nej mutants: although these nej GLC embryos are somewhat variable in terms of their phenotypes the most severely mutant embryos show lacZ staining in only a few cells in the ps8 region. Similarly, in Mad12 mutant embryos, lacZ staining is much reduced, with some staining remaining in ps6 and ps8. This implies that CBP, like Mad, is required for the Dpp response of the Ubx B4 enhancer (Waltzer, 1999).

The response of B4 to GAL4-mediated ectopic Dpp was examined in nej mutant embryos. If Dpp is expressed throughout the mesoderm, B4-mediated lacZ staining is increased and detectable throughout the midgut mesoderm. In nej mutants, this response of B4 to ectopic Dpp is strikingly disabled: there is barely any lacZ staining in the anterior midgut, and only a moderate increase of lacZ staining in the ps8/9 region, indicating a residual Dpp response in this region. These results strongly support the conclusion that CBP is required for the transcriptional response of the Ubx enhancer to Dpp signalling. They argue that CBP functions downstream of the Dpp signal (Waltzer, 1999).

In the early blastoderm embryo, dpp mediates the subdivision of the dorsal ectoderm into two embryonic tissues: the amnioserosa and the dorsal epidermis. High Dpp levels in the dorsal-most cells specify amnioserosa while lower Dpp levels in dorsolateral regions specify epidermis. Expression of the gene Race (related to angiotensin converting enzyme; the earliest known marker for the amnioserosa) in the dorsal blastoderm embryo depends on dpp signaling. Thus it was asked whether the activity of the Race enhancer depends on CBP function. This enhancer mediates lacZ staining in the presumptive amnioserosa and in the anterior midgut primodium: the former, but not the latter, staining requires dpp. In nej GLC embryos, there is no detectable lacZ staining in the presumptive amnioserosa, although staining remains, and is even slightly enhanced, in the head and in the anterior midgut primordium. This demonstrates that the Race enhancer depends on an activating function of CBP exclusively in a subset of the blastoderm cells, namely in the dorsal-most cells of the embryonic trunk. It suggests that CBP is required for the response of this enhancer to dpp (Waltzer, 1999).

To see whether CBP may be required in other developmental contexts in which dpp functions, the developing tracheae were examined in nej mutant embryos. The tracheal system develops from segmentally repeated clusters of ectodermal cells, the tracheal placodes. These cells undergo a complex process of migration and fusion to generate the final branched structure of the tracheal system. dpp signaling plays a crucial role in this process, and has been implicated in the dorsoventral migration of certain tracheal branches. For example, in punt or thick veins mutants, the branches that normally migrate dorsally or ventrally (the dorsal and ganglionic branches, respectively) fail to develop, whereas the branches that grow out anteriorly (the dorsal trunk and the visceral branches) are essentially not affected. The tracheae in nej mutant embryos were examined using an antibody that stains the lumina of the tracheal trees (2A12). The dorsal trunk and the visceral branches are essentially normal in these mutants. However, in most nej mutant embryos, branching defects are seen: usually, one or two dorsal branches fail to form at each side, and ganglionic branches fail to fuse. Essentially the same defects are also seen in in nej GLC embryos. These defects resemble those found in punt hypomorphs and in Mad12 mutant embryos, although the most apparent defects in the latter mutants are the fusion defects in their ganglionic branches. Once again, the similarity of the tracheal phenotypes of nej mutants when compared to dpp, punt and Mad mutants suggests that CBP may be required during Dpp signaling (Waltzer, 1999).

dpp promotes vein development during pupal stages, and a subclass of dpp mutant alleles cause loss of veins. In particular, in dppS1 homozygous flies, vein 4 fails to reach the margin. This weak dpp allele was exploited to see whether there would be a genetic interaction between dpp and nej. Indeed, while nej heterozygosity on its own shows no abnormality whatsoever in the wing, this condition clearly enhances the vein phenotype of dppS1 homozygotes: in many of the wings from flies of this genetic constitution, neither vein 4 nor vein 2 reaches the margin. This synergy in the wing between nej and dpp loss-of-function alleles is consistent with the notion that CBP functions during Dpp signaling. To clarify the position of CBP in this Dpp response in the wing, it was asked whether the mild dpp overactivation phenotype due to overexpression of a constitutively active form of Sax (Sax*), a Dpp type I receptor, depends on nej gene dosage. Expression of Sax* under the control of engrailed.GAL4 induces ectopic venation and overgrowth of the posterior part of the wing. Moreover, removal of one copy of genes required for Sax signaling, such as Mad or Medea suppresses this phenotype. Likewise, nej heterozygosity suppresses to a considerable extent the wing phenotypes caused by Sax*. This result is consistent with CBP being required for Sax signaling, and it indicates that CBP functions downstream of this Dpp receptor (Waltzer, 1999).

Since Mad mediates transcriptional activation by dpp and appears to be a transcription factor required for every aspect of dpp signaling, it was asked whether CBP might be recruited by Mad as a transcriptional co-activator. To test whether CBP might bind to Mad, the yeast two-hybrid system was used. When these binding studies were begun, Drosophila CBP had not yet been discovered. So fragments of mouse CBP were used to test whether these might bind to Mad, assuming that a putative interaction between the two proteins would be conserved. Indeed, there is a strong degree of homology between mouse and Drosophila CBP. A set of fragments of mouse CBP were used that cover the whole protein and these were fused to a transcriptional activation domain (the 'prey'). This series of prey was tested in two-hybrid assays in yeast with full-length Mad protein fused to the LexA DNA-binding domain (the 'bait'). The C-terminal domain of mouse CBP (CBP1678 to 2441) interacts specifically with Mad in this assay. This interaction was confirmed using a similar set of prey with fragments from the Drosophila CBP protein, which was tested against a series of baits containing different Mad domains. This reveals that a fragment of Drosophila CBP that spans amino acids 2240-2608 (which overlaps the above mentioned C-terminal domain of mouse CBP) interacts specifically with the MH2 domain of Mad (Mad219-455). These specific interactions in the yeast two-hybrid assay between CBP fragments and Mad almost certainly reflect direct binding since yeast does not encode any proteins homologous to either of these. Interactions between CBP fragments and Mad are significantly stronger if the N-terminal domain of Mad is removed, suggesting that MH1 inhibits the binding of CBP to MH2. Inhibitory interactions between MH1 and MH2 have been described previously (Waltzer, 1999).

To confirm these results, direct binding between Mad and CBP in vitro were tested with pull-down assays. In these assays, [35S]methionine-labelled Mad domains and various fragments from Drosophila CBP expressed as GST fusion proteins and immobilized on GST-Sepharose beads were used. Either full-length Mad or its MH2 domain binds to the same Drosophila CBP fragment that interacts with Mad in the yeast assay while Mad's MH1 domain does not bind CBP. Interestingly, deletion of the C-terminal of Mad's MH2 domain (amino acids 372-455) abolishes CBP interaction, demonstrating that the C-terminal 84 amino acids of Mad are required for binding to CBP. The linker domain (L) between MH1 and MH2 seems to be dispensable for Mad interaction with CBP. Weak binding of full-length Mad to CBP2 (CBP2240-2507), the highly conserved domain of Drosophila CBP that overlaps the Mad-binding fragment of CBP, was observed. However, the significance of this binding is uncertain, as this binding activity could not be detected in the reciprocal assay nor in yeast. Finally, to characterize more precisely the mutual binding domains within Mad and Drosophila CBP, the reciprocal experiment was performed using [35S]methionine-labelled C-terminal fragments of CBP and various GST-Mad fusion proteins. CBP binds to GST-MH2, but not to GST alone, nor to GST-MH1+L fusion proteins, which include extended MH1 fragments (Mad1-241), nor to GST-MH2C fusion protein in which the last 84 amino acids of Mad's MH2 domain are deleted. Deletion mapping of the C-terminal region of CBP reveals a minimal fragment of CBP (CBP2413-2608) that is sufficient for binding to Mad. This domain partially overlaps the highly conserved CBP2 domain but in most binding assays, CBP2 by itself is not sufficient for binding to Mad. Altogether, these experiments demonstrate that CBP and Mad bind to one another, and that the stretch between amino acids 2507 and 2640 within Drosophila CBP is critical for CBP's binding to the MH2 domain of Mad (Waltzer, 1999).

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. This approach was applied to assay the transactivation potential of proteins in transgenic Drosophila embryos. A chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaves as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) requires an unexpected degree of context to function as a transcriptional activator. Evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, these data suggest that context-dependence of transcription factors may be more prevalent than previously thought (Szuts, 2000).

Which factors provide the context for Dfos function? Several lines of evidence implicate JNK and Dpp signaling and their transcriptional target factors Djun and Mad as the essential context. (1) The only embryonic cells in which LexFos functions reliably and robustly to stimulate transcription are the dorsal leading edge cells which experience both of these signals. (2) Neither of the LexFos derivatives (LexFosN, LexFosC) function in these cells, strongly implicating the basic leucine zipper domain of LexFos (the only domain absent from both derivatives) in its function. As this domain mediates dimerization with Djun, the only known dimerization partner of Dfos in Drosophila, this indicates that the activity of LexFos depends on Djun. Recall that Djun is present and activated by JNK signaling in the leading edge cells. (3) JNK signaling as mimicked by overexpression of constitutively acitive Drac* or Dcdc42*, potently synergizes with LexFos to mediate widespread transactivation in the embryo. A very similar widespread synergy has also been seen between LexFos and Jun*, a mutant form of c-Jun that mimics signal-activation of this protein. The embryonic territories in which these synergies are observed appear to correspond to sites of Dpp stimulation. Consistent with this, a limited synergy between Dpp and LexFos has also been observed in some embryonic cells. These synergies strongly implicate JNK and Dpp as necessary context signals for LexFos function. (4) LexFos activity strictly depends on the context sequence in the MadL target reporter; under no conditions does it transactivate a reporter that contains four tandem LexA binding sites (albeit LexGAD very efficiently does so). The context sequence in MadL essentially consists of a binding site for the Dpp response factor Mad, which is thus a likely partner for the putative LexFos/Djun* dimer (Szuts, 2000).

These results indicate that JNK-activated Djun and Dpp-activated Mad may be critical and widespread context partners of Dfos. Consistent with this, Dfos function is required for dorsal closure of the embryo and, by implication, functions normally in cells that experience JNK and Dpp signaling. In the embryonic midgut, Dfos functions in cells that experience Dpp and Egfr signaling. Because the LexFos/JNK synergy in the mesoderm implies that JNK signaling is normally absent from this tissue, this suggests that the normal partner of Dfos in the midgut visceral mesoderm may be a factor, as yet unidentified, that is activated by Egfr signaling. Interestingly, synergy between the c-Jun/c-Fos dimer and TGF-beta activated Smad has also been observed in mammalian cells. Furthermore, Jun proteins have recently been shown to bind directly to Smad3/4. Thus, the partnership between signal-activated Jun/Fos dimers and Smads may be fairly widespread and fundamental (Szuts, 2000).

Schnurri and Mad can interact directly through discrete domains. Smads characteristically contain an amino-terminal Mad homology region 1 (MH1) and a carboxy-terminal Mad homology region 2 (MH2) separated by a poorly conserved linker region. Mad and Shn interactions were assayed using the yeast two-hybrid assay. Prey plasmids were generated that express full-length Mad (FL), Mad MH1, or the MH2 domain along with the linker (MH21L). In the two-hybrid assay, interaction of the bait and prey allows yeast expressing both proteins to grow on selective media and activate transcription of a beta-galactosidase reporter. Based on both these criteria, it was observed that Shn associates with Mad FL and Mad MH21L, but not with Mad MH1. The failure of Mad MH1 to interact with Shn cannot be ascribed to lower levels of expression of this domain, since antisera directed against an HA epitope present in each prey fusion detect comparable levels of all three Mad polypeptides on Western blots (Dai, 2000).

In order to delineate the domains in Shn that interact with Mad, GST pull-down assays were used. Mad MH21L domain was expressed as a glutathione S-transferase (GST) fusion protein. Sixteen overlapping subclones encompassing the entire Shn coding region were used to generate 35Smethionine-labeled protein fragments using coupled in vitro transcription and translation. The GST-Mad affinity matrix was tested for its ability to retain the 35S-labeled Shn peptides. Mad MH21L interacts most strongly with two overlapping regions, Shn 1069-1776 and Shn 1463-2318. Further deletions have allowed the interaction to be narrowed to Shn 1441-1635, a region of 194 residues preceding the second set of paired zinc fingers. Two additional nonoverlapping fragments, Shn 341-1069 and Shn 1776-2529, show a moderate interaction with Mad. Subdivision of these fragments significantly reduce their association with Mad. Thus, a total of one strong and two weaker Mad interaction domains (MIDs) has been detected in Shn. Shn 1441-1776 shows strong binding to itself with an affinity comparable to that displayed for Mad MH21L. In addition significant binding was detected to Shn 341-1069 and Shn 1776-2529, fragments that contain the other MIDs. The binding of Shn 1441-1776 is specific since it fails to bind either Shn 1-968 (a fragment that lacks a MID). Thus these results suggest that regions of the protein that are involved in association with Mad may also mediate homomeric Shn interactions (Dai, 2000).

Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad has been shown to bind to the Dpp-responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown. The biochemical functions of Shn have been characterized in this study. Shn forms homo-oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Shn interacts with Mad in a Dpp-dependent manner. The present results argue that Shn may act as a nuclear component of the Dpp signaling pathway through direct interaction with Mad (Udagawa, 2000).

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. mSin3A-histone deacetylase complexes (see Drosophila Sin3A) are altered in the presence of dCtBP and dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, it is proposed that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function (Phippen, 2000).

The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis; Shn is not an obligate co-factor for Mad

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. During embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk;shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are also expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo (Torres-Vazquez, 2001).

Genetic evidence implicates both Shn and Mad in dpp-dependent repression of brk. In the wing disc, cells that lack Mad or shn ectopically express brk and fail to activate the Dpp-responsive genes optomotor-blind, vestigial, spalt and Dad. Abolition of shn or Mad activity results in upregulation of brk in the embryo and in the absence of shn ectopic Dpp cannot suppress brk expression. Since Shn and Mad interact directly, an attractive hypothesis is that a Shn/Mad complex is involved in the Dpp-dependent repression of brk. It has recently been suggested that Dpp signaling bifurcates downstream of Mad/Med into a Shn-dependent pathway, leading to brk repression and a Shn-independent pathway that triggers gene activation. According to this model, Shn acts primarily as a dedicated repressor that switches Mad from a transcriptional activator to a transcriptional repressor on the brk promoter. However several lines of evidence from this study are incompatible with such an interpretation (Torres-Vazquez, 2001).

A strong argument that shn has additional roles beyond brk repression comes from the fact that simultaneous loss of brk and shn activity results in a phenotype that is distinct from that of brk-null animals. If the sole function of shn is to mediate brk repression, then shn activity should be redundant in a brk mutant background. However, both at the overt phenotypic level as well as in the regulation of individual target genes, brk;shn double mutants display defects consistent with lower levels of Dpp signaling, compared with embryos that lack brk alone. These results indicate that shn participates in gene activation through brk-independent mechanisms as well. The finding that Shn is not obligately required to suppress brk transcription prior to germband elongation, while Mad is essential in this process, also argues against an exclusive role for Shn as a Mad co-repressor. In dpp- and Mad-null embryos, brk is upregulated at stage 8, while in embryos lacking shn function, derepression occurs approximately 3 hours later than the transition of brk regulation from maternal to zygotic control. Thus, brk transcription is insensitive to the absence of shn function at a time when it is responsive to Dpp and Mad. This idea is reinforced by the fact that ectopic Dpp signaling (through a constitutively activated form of Tkv called TkvA) can repress brk transcription at stage 5/6 in both wild-type and shn- animals, but not in Mad-null embryos. Collectively these data provide compelling evidence that refutes a model in which all aspects of the shn mutant phenotype result from derepression of brk transcription (Torres-Vazquez, 2001).

The unexpected result that at high levels TkvA mediates activation of brk promoter, while at low levels it causes repression reveals a possible mechanism by which Shn contributes to Mad activity. One explanation for these concentration-dependent effects of TkvA could be that the default mode of Mad action is transcriptional activation, and interaction with a co-repressor (perhaps present in limiting amounts) is crucial to bring about repression. Cells that receive very high levels of signaling could experience 'squelching', owing to excess nuclear Mad that binds to the brk promoter without recruitment of the co-repressor, thus promoting activation rather than repression. Supporting this idea, injection of TkvA into embryos that lack Mad does not induce either brk activation or repression. The increased frequency of ectopic brk expression in shn- embryos could indicate that Shn stabilizes a Mad/co-repressor complex on the brk promoter. It is worth bearing in mind that even in shn- embryos, ectopic activation did not occur independent of brk repression in the peripheral cells. Thus, it appears that Shn does not determine whether Mad acts as an activator or as a repressor, but may promote its interaction with other factors that determine the polarity of Mad transcriptional activity (Torres-Vazquez, 2001).

Analysis of Dpp-responsive gene expression in brk; shn double mutants has allowed an assessment the brk-independent input from shn to gene activation at different developmental stages in a range of tissues. Although it has not been demonstrated that each of these markers is a direct target of Dpp signaling, three categories of responses can be distinguished based on these studies. In the first group (class A), exemplified by dpp in the leading edge of the dorsal ectoderm, expression in the double mutant is indistinguishable from that in brk- embryos. Thus, shn contributes to class A gene expression primarily by relief of brk repression. Promoters belonging to class B include Dad and pnr in the dorsal ectoderm during germband extension. Expression of class B genes is downregulated in the double mutant compared with brk- embryos, but is equivalent to wild-type levels. It is inferred from this result that in the absence of Brk and Shn, Mad-mediated activation may be sufficient for expression within the normal domain, but cannot sustain the lateral expansion encountered in brk mutants. A third category of responses (class C) includes dpp and Ubx in the midgut, and sna in the primordia of the wing/haltere imaginal discs. Genes in this class show significantly reduced levels of expression in the double mutant, not only relative to brk- but also compared with wild-type animals. Class C promoters incorporate a brk-independent positive input from shn that is necessary for wild-type levels of expression. The inability of ectopic Dpp to induce sna expression in shn mutants demonstrates the essential nature of the requirement for Shn in activation of class C genes (Torres-Vazquez, 2001).

It is evident that repression of brk is crucial for expression of all three classes of genes described, and as such accounts for a significant part of the positive input from shn to gene activation. In addition, the data suggest that Mad and Shn contribute equally to repression of brk and regulation of class A genes. However, the fact that brk activity is only partially epistatic with respect to class B and C promoters, indicates that the majority of genes examined in this study integrate positive inputs from shn, as well as negative inputs from brk. The near wild-type expression of class B genes in double mutant embryos suggests that the brk-independent input from shn may be crucial at the margins of the expression domains and may be less significant in regions of the embryo that receive moderate to high levels of Dpp signaling. In contrast, the positive input from shn to class C targets appears to be important throughout the domain of expression. The observation that genes such as dCreb-A and Scr, which are repressed by dpp signaling, and which are also sensitive to loss of brk, raises the possibility that Dpp regulates their expression indirectly. In this event, the dpp target genes that mediate repression of dCreb-A and Scr would belong to classes A and C, respectively (Torres-Vazquez, 2001).

The partial restoration of dpp target gene expression in the double mutants relative to shn- embryos provides a basis for interpreting the cuticle phenotype. Homozygous brk;shn animals as well as brk;tkv mutants have an intermediate phenotype in that they show rescue of the dorsal closure defect observed in shn and tkv mutants, but they also display a reduced dorsal epidermis compared with brk-null embryos. Both dpp and pnr have been implicated in dorsal closure, which results from movement of the epidermal cells over the amnioserosa and their suturing at the midline. In light of this, the recovery of their expression in the dorsalmost ectodermal cells in the double mutants correlates well with the restoration of dorsal closure. Likewise, the compromised expression of dorsal ectodermal markers such as Dad and pnr in brk;shn embryos relative to brk null animals, provides molecular correlates for the ventralization observed in the double mutants (Torres-Vazquez, 2001).

The data presented in this study indicate that Shn can mediate both gene activation and brk repression in response to Dpp signaling. An important question is whether Shn has a Mad-independent role in activation. Shn contains a potential activation domain, and the human ortholog of Shn (PRDII-BF1) can elicit a 10-fold increase in gene expression in transfection assays. However, a Shn-Gal4 fusion protein does not activate transcription in yeast, and Shn is only marginally effective in stimulating a Dpp-responsive reporter in the absence of Mad in cell culture assays. Taken together these results suggest that Shn acts by promoting Mad binding to DNA and/or its interactions with the transcriptional machinery. There is ample precedent for such a mechanism, since several vertebrate DNA-binding Smad partners such as FAST1, OAZ, Mixer and Milk, do not have an innate ability to stimulate transcription, but potentiate gene activation by Smads in a pathway specific manner. A prediction from this data is that promoters of class B and class C genes are likely to contain binding sites for Shn as well as Mad, and that Shn increases Mad specificity by recruiting it to a subset of promoters that contain binding sites for both proteins. Analysis of gene expression in brk;tkv mutants demonstrates that for class B and class C genes Mad provides a greater brk-independent input compared with shn, consistent with the idea that Mad plays a primary role in Dpp-dependent gene activation and that shn facilitates Mad activity. Further support comes from the observation that deletion of Mad sites in the Ubx midgut enhancer had a more profound effect than abolition of Shn binding (Torres-Vazquez, 2001).

It has been shown that Mad interacts with Nejire (Nej), the Drosophila homolog of the co-activator p300/CREB binding protein (CBP). Reduction in nej activity affects the expression of ush, pnr and Ubx, and disrupts events that are dpp and shn-dependent, like tracheal migration and imaginal disc patterning. It is interesting to speculate that Shn may interact directly with Nej and stabilize complex formation between Mad/Medea and Nej (Torres-Vazquez, 2001 and references therein).

The requirement for Shn and Mad in both aspects of Dpp signaling suggests that Shn does not confer the ability to activate or repress transcription. It appears more likely that the activity of the Mad/Shn complex is modulated in a promoter specific fashion analogous to the mechanisms that convert Dl from an activator to a repressor. Similarly, the presence of binding sites for factors that bring co-repressors into proximity with Mad/Shn could permit inhibition of transcription at the brk promoter while target genes that lack these sites could be activated in the same cells. It has been shown that Smad4 interacts with the co-repressor TGIF and the co-activator CBP in a mutually exclusive manner. Thus, the ability to recruit co-activators as opposed to Smad co-repressors (such as cSki and SnoN), or more general transcriptional repressors like Groucho or CtBP, would be crucial to determining whether Dpp stimulation resulted in activation or repression of the target gene (Torres-Vazquez, 2001).

It is conceivable that in addition to repressing brk transcription, Shn and Mad could prevent residual Brk protein in the nucleus from binding to target gene promoters through steric hindrance or direct competition for common binding sites. Related anti-repression mechanisms have been postulated for Smad1 and Smad2 that interact with the transcriptional repressors Hoxc-8 and SIP1, respectively, triggering their dissociation from the osteopontin and X-Bra promoters. Although such a mechanism could potentially enhance the efficiency with which Shn and Mad antagonize brk activity, it does not account for the brk-independent input from shn observed in brk;shn embryos, since there is no Brk protein in the double mutants.

Despite the fact that shn transcripts are present from the precellular blastoderm stage onwards, loss of shn activity does not affect either brk repression or the expression of Dpp target genes until germband extension. Germline clonal analysis and ds-RNAi experiments indicate that the insensitivity of Dpp target gene expression to loss of shn during early embryogenesis is unlikely to result from perdurance of maternal message. Thus, the 'weakness' of the shn mutant phenotype may reflect a limited temporal requirement for shn in dpp signaling, rather than a lesser requirement for shn activity throughout development. The functional redundancy of shn during early patterning could be due to the presence of another protein that contributes a Shn-like activity to Dpp signal transduction. Alternatively, Mad activity alone could be sufficient for induction of early D/V patterning genes if they contain promoter elements that are more sensitive to Mad. It is also possible that the higher levels of nuclear Mad resulting from the synergy between Scw and Dpp in early embryogenesis renders the potentiation of Mad by Shn unnecessary. Finally, given the conserved nature of the BMP signal transduction pathway and the identification of Shn homologs in humans, frogs and worms, it is possible that Shn-like proteins in other systems potentiate Smad activity in an analogous manner (Torres-Vazquez, 2001).

The Smurf1 ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis

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).

To identify novel negative regulators of BMP signaling in Drosophila, a genetic selection was conducted for mutations that result in elevated Dpp activity during embryonic D-V pattern formation. From 65,000 mutagenized genomes, six extragenic mutations were recovered that suppressed the lethal, partially ventralized embryonic phenotype caused by the hypomorphic maternal-effect Medea mutation, Med15. Both the molecular and genetic characterization of two of these mutations, 11R and 15C, which disrupt the previously unrecognized DSmurf locus, are presented in this study. Both mutations act as largely recessive maternal-effect suppressors that restore viability and wild-type pattern to Med15 embryonic progeny, either as homozygotes or in trans-heterozygous combination. Because these mutations act in the same fashion to suppress the partially ventralized embryonic phenotype caused by dpp haploinsufficiency, they effect a general elevation of Dpp signaling activity during embryonic D-V pattern formation (Podos, 2001). 2001).

The activities of vertebrate Smurf1 and Smurf2 in opposition to BMP and TGF-ß signals are mediated in part by direct binding interactions with their R-Smad substrates. Whether Drosophila Smurf1 interacts physically with the R-Smad encoded by Mad was examined. In a yeast two-hybrid assay, it was found that Smurf1 binds Mad but not its co-Smad Medea. Similar to the vertebrate Smurf-Smad interactions, the interaction between Smurf1 and Mad was disrupted by deletion of the PY motif from Mad. Smurf1 therefore shares substrate binding properties with its vertebrate homologs, likely reflecting a common function in restricting Dpp/BMP signals by promoting the proteolysis of the BMP-specific R-Smad proteins (Podos, 2001).

While mutation of Smurf1 does not cause overt alterations in D-V cuticular pattern, the cuticle presents a snapshot of embryonic development that does not necessarily reflect initial D-V pattern and does not incorporate the dorsal-most tissue, the amnioserosa. To obtain a direct readout of the Dpp activity gradient that is sensitive to subtle changes in its strength and spatial parameters, wild-type and mutant embryos were examined for changes in the spatial extent of staining with the phosphorylated form of MAD (P-Mad) antibody and changes in the expression domains of direct Dpp target genes (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 (28%, P < 0.001) 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).

Despite the intensive study of Dpp-dependent developmental events, there are multiple reasons why the role of Smurf1 eluded prior notice: (1) both the maternal and zygotic components of Smurf1 must be mutated to uncover a lethal phenotype; (2) Smurf1 mutants are relatively dosage insensitive, as Smurf1 mutations do not exert significant dominant phenotypes even in sensitized backgrounds such as Med15, precluding their isolation in most genetic screens. Such dosage insensitivity might be a general feature of enzymes, as opposed to stoichiometric components of signaling pathways such as Mad and MED. (3) Smurf1 mutations do not cause overt defects in dorsal-ventral patterning. Possibly, the activity of Smurf1 is partially redundant with another ubiquitin-protein ligase. In support of this hypothesis, Dpp signals are eventually downregulated in Smurf1 mutants. Moreover, an analysis of human Smad2 turnover has implicated a ubiquitination activity that does not require the Smad2 linker domain and therefore is likely independent of the Smurf proteins (Podos, 2001).

The spatial modulation by Smurf1 of graded Dpp signaling is evident prior to the onset of gastrulation. The abrogation of Smurf1 activity causes a sensitization to Dpp signals at all positions in the D-V activity gradient, as indicated by expanded domains of P-Mad staining, target gene transcription, and tissue differentiation. Similar global expansions of Dpp-dependent territories have been observed in embryos with elevated dpp gene dosage. Since an increase in ligand concentration is likely to result in the phosphorylation of additional cytoplasmic Mad, this spatial control over Dpp target gene expression is likely to derive from the unregulated ubiquitin-mediated proteolysis of Mad throughout the embryo. Similar properties have been established for human Smurf1, from demonstrations that BMP receptor activation does not alter the rate of Smad1 ubiquitination and degradation mediated by Smurf1 (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 Med15 mutation is a missense lesion in the MH2 domain, within the L3 structural loop that has been implicated in the signal-dependent interaction between trimers of Mad and MED. Because an experimental elevation of wild-type Mad levels is sufficient to restore the specification of amnioserosa to embryos derived from Med15 mothers (Hudson, 1998), it is hypothesized that Smurf1 mutations suppress Med15 because the resulting elevation of Mad is sufficient to overcome its reduced affinity for the mutant MED protein (Podos, 2001).

Phenotypic analysis has identified a second requirement for Smurf1 in the temporal downregulation of Dpp signaling. With the exception of the amnioserosal cells, all of the descendants of cells with high levels of P-Mad at the onset of gastrulation downregulate P-Mad staining by stage 10. In contrast, Smurf1 embryos of the same stage retain P-Mad staining in all these cell types, leading to deleterious consequences in hindgut morphogenesis. A causal link has been established between the prolonged Dpp signaling in the dorsal hindgut primordium, ectopic zen transcription, and the subsequent breakdown in the epithelial integrity of the hindgut cells (Podos, 2001).

This second function of Smurf1 is distinct, and possibly mechanistically separable, from its ability to target Mad for destruction. Although the incremental spatial expansion of P-Mad staining along the dorsal-ventral axis at the blastoderm stage is consistent with an overall increase in the amount of Mad protein in Smurf1 mutants, the complete lack of temporal downregulation of P-Mad staining in stage 8-10 Smurf1 embryos is more consistent with a specific effect of Smurf1 on P-Mad. Because the level of P-Mad is a readout of both the amount of Mad protein within a cell and the intensity of Dpp signaling that the cell receives, one attractive hypothesis is that Smurf1 downregulates the level of P-Mad by antagonizing Dpp signaling, independent of Mad degradation. One possible mechanism is suggested by demonstrations that vertebrate Smurf1 and Smurf2 can use the I-Smad, Smad7, as an adaptor to promote the degradation of activated TGF-ß receptors. It is proposed that Smurf1 might similarly target activated Dpp receptors for degradation, using Mad or, more likely, the I-Smad protein DAD as an adaptor. Such an activity would serve as a feedback mechanism of attenuation, whereby receptors are targeted for degradation only upon activation by ligand. Feedback mechanisms are integral to many developmental signaling processes; the proposed feedback activity of Smurf1 on the Dpp receptors would be one of the few instances where temporal downregulation of a signaling system has been shown to be necessary for the proper differentiation of cells previously exposed to the signal (Podos, 2001).

Involvement of Smurf1 have been demonstrated in the control of multiple aspects of Dpp signaling. Further analysis will be required to determine whether Smurf1 acts bifunctionally to mediate the degradation of Mad and of activated Dpp receptors. Smurf1 might also have additional activities that have not been uncovered by these mutants. For example, although homozygous Smurf1 adults have normal cuticular morphology, an examination of Dpp target gene expression might reveal Smurf1 function in imaginal disc patterning. By analogy to the vertebrate Smurf proteins, Smurf1 also might modulate the activin/Smad2 signaling pathway, which has been implicated in the control of cell proliferation. Lastly, Smurf1 might promote the ubiquitin-mediated degradation of other substrates, independently of the Smads. More generally, further characterization of the relationship between Smurf1 and other modulators of Dpp signaling will yield insights into the precise regulation that underlies intercellular signaling systems during normal development (Podos, 2001).

The Drosophila BMP type II receptor Wishful thinking regulates neuromuscular synapse morphology and function

Although Wishful thinking shows a strong overall similarity to the vertebrate BMP type II receptor, its signaling mechanism remains untested. To examine whether Wit actually mediates a BMP type signal, use was made of an antibody that specifically recognizes the phosphorylated form of Mad (P-Mad) (Tanimoto, 2000), the major transducer of BMP signals in Drosophila. In late embryos, this antibody reveals an intricate developmental pattern of BMP signaling, including P-Mad accumulation in the developing midgut and gastric cecae that closely parallels dpp expression and is thought to be indicative of cells receiving a Dpp signal. In addition to these sites of accumulation, a novel pattern of P-Mad accumulation is noted in a subset of neurons. Staining is first evident at late stage 15 and then becomes more elaborate and intense as the embryos continue to develop. Ultimately about 35-40 neurons per hemisegment show strong staining by stage 17. This expression continues into the first instar stage but is not detectable in late third instar larvae. The pattern of P-Mad accumulation in the CNS is completely abolished in wit mutant embryos whereas all other patterns of P-Mad accumulation appear normal. Conversely, with the exception of the CNS pattern, all other sites of P-Mad accumulation in late embryogenesis are absent in zygotic punt null mutants. Maternal null embryos cannot be examined since they do not develop an organized CNS. These observations are consistent with Wit function being required specifically in the CNS to transduce BMP signals whereas other sites of P-Mad accumulation result from signaling by Punt, the primary Dpp type II receptor (Marqués, 2002).

The number and the positions of the phosphorylated form of Mad (P-Mad)-positive cells, per hemisegment at stage 17, indicative of cells undergoing Wit signaling, correlates well with the estimated number and positions of motoneurons at this stage. To better characterize the cells that accumulate P-Mad in the nervous system, wild-type embryos were double stained for P-Mad and several other markers, including Even-skipped and Lim-3, two transcription factors expressed in discrete subsets of embryonic motoneurons and interneurons. Staining was also performed for Fasciclin II (Fas II), a neural adhesion molecule expressed in a subset of interneurons as well as most motoneurons. There is colocalization of Eve and P-Mad in the RP2 and U/C motoneurons but not in the EL interneurons, while P-Mad and Lim-3 colocalize in a lateral cluster of cells that include motoneurons 28 and 14/30. In addition, Lim-3 and P-Mad colocalize in RPs 1, 3, 4, 5. The Fas II antibody highlights axon tracts and cell bodies of most motoneurons as well as some interneurons. Most, if not all, P-Mad cells also show colocalization of Fas II. These findings suggest that Wit is required to transduce BMP signals in motoneurons and is consistent with the ability of UAS-wit constructs to rescue wit mutants when expressed with a motoneuron-specific driver (Marqués, 2002).

Combinatorial signaling by an unconventional Wg pathway and the Dpp pathway requires Nejire (CBP/p300) to regulate dpp expression in posterior tracheal branches

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

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

The CBP coactivator, interacting with Mad, functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo

The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-ß molecules Dpp and Screw in the early embryo. The expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-ß signaling pathway to a decline in CBP concentration (Lilja, 2003).

These results suggest that several transcription factors that regulate expression of Dpp/Screw signaling components require the dCBP coactivator for their function in Drosophila embryos, and implicate dCBP in regulation of the Dpp/Screw pathway independently of an interaction with Smad proteins. An additional role of dCBP is to regulate Dpp-target genes, acting at a step downstream of Smad protein phosphorylation. It is likely that direct interactions between dCBP and Mad/Medea contribute to regulation of Dpp target genes). Such interactions have been observed in vitro, both in mammalian systems and using Drosophila proteins. However, a major cause of impaired Dpp/Screw signaling in dCBP mutant embryos is due to reduced tolloid expression. This prevents Dpp/Screw from initiating a signaling event in cells that would normally receive the Dpp/Screw signal, presumably by a failure to cleave the Dpp-Sog and/or Screw-Sog complexes. In fact, a majority of embryos that do not express the Dpp/Screw-target gene rhomboid in dorsal cells, also do not contain phosphorylated Smad proteins. Furthermore, the pattern of phosphorylated Smad proteins correlates closely with that of tolloid expression. For example, in many early, cellularizing dCBP mutant embryos, an anterior patch of both tolloid expression and phosphorylated Smad staining remains. At later stages, tolloid expression recovers in dCBP mutant embryos, as does Dpp/Screw signaling as revealed by Smad protein phosphorylation. This recovery of tolloid expression at later stages of development might explain the recovery of phosphorylated Smad proteins in dCBP mutant embryos, by allowing Dpp/Screw to signal. For these reasons, regulation of tolloid expression appears to be a major means of controlling Dpp/Screw signaling by dCBP (Lilja, 2003).

It is likely that reduced screw expression also contributes to the reduction of phosphorylated Smad proteins observed in dCBP mutant embryos. In both screw and tolloid mutants, phosphorylation of Mad is eliminated. Furthermore, progressive reduction in Screw activity leads to a corresponding progressive deletion of dorsal-most cell fate, the amnioserosa. Tsg is required together with Sog to generate peak Dpp activity in dorsal midline cells. Reduced tsg expression in dCBP mutants may therefore contribute to the lack of Dpp/Screw-target gene expression. However, it is not believe that this lack can explain the defects in dCBP mutants, because in tsg mutants, low levels of Dpp signaling persist in a broad dorsal domain, leading to expanded rhomboid expression in dorsal cells. By contrast, in dCBP mutant embryos, expression of genes in response to a low threshold of Dpp activity, such as U-shaped and the dorsal rhomboid pattern, is eliminated (Lilja, 2003).

These experiments do not address whether dCBP regulation of tolloid, screw, and tsg expression is direct or indirect. However, since expression of these genes begins at about the time when zygotic transcription initiates in the embryo, and the effect of dCBP is evident from the onset of expression of tolloid and screw, the notion is favored that dCBP is acting directly on the enhancers of these genes. It is not yet understood whether HATs such as CBP primarily act to acetylate large chromosomal domains, or are directed to specific genes. In the case of the tolloid gene, the results indicate that dCBP is being recruited to the enhancer by a DNA-binding protein, since the isolated enhancer removed from its normal chromosomal location requires dCBP for its activity (Lilja, 2003).

Given its central position in gene regulation and the great number of mammalian transcription factors shown to interact with CBP, relatively few genes are affected by the dCBP mutation. For example, activation and repression mediated by the Dorsal protein are unaffected in the dCBP mutant embryos, as demonstrated by the expression patterns of Dorsal target genes. Also, no defects in early segmentation gene expression could be observed in germline clone mutants. However, the nej1 mutation used in this study to create dCBP mutant germline clone embryos is a weak mutation that results in a very modest reduction in dCBP levels. Since other means of reducing the dCBP amount by approximately two-fold results in similar gene expression defects, Smad proteins and the unidentified activators of tolloid, tsg, and screw expression are particularly sensitive to a decline in dCBP concentration. It may not be a coincidence that screw, tsg, tolloid, and Dpp-target gene expression are all specifically affected by a small dCBP reduction. Perhaps components of the Dpp/Screw signal transduction pathway have evolved to be coordinately regulated by a common coactivator. Given the phylogenetic conservation of the CBP protein and the TGF-alpha signal transduction pathway, as well as the ability of CBP and Smad proteins to interact in vitro, CBP is likely to play an equally important role in TGF-ß signaling in other metazoans (Lilja, 2003).

Analysis of a brinker enhancer reveals that a simple molecular complex mediates widespread BMP-induced repression during Drosophila development

The spatial and temporal control of gene expression during the development of multicellular organisms is regulated to a large degree by cell-cell signaling. A simple mechanism has been uncovered through which Dpp, a TGFß/BMP superfamily member in Drosophila, represses many key developmental genes in different tissues. A short DNA sequence, a Dpp-dependent silencer element, is sufficient to confer repression of gene transcription upon Dpp receptor activation and nuclear translocation of Mad and Medea. Transcriptional repression does not require the cooperative action of cell type-specific transcription factors but relies solely on the capacity of the silencer element to interact with Mad and Medea and to subsequently recruit the zinc finger-containing repressor protein Schnurri. These findings demonstrate how the Dpp pathway can repress key targets in a simple and tissue-unrestricted manner in vivo and hence provide a paradigm for the inherent capacity of a signaling system to repress transcription upon pathway activation (Pyrowolakis, 2004).

One of the primary events controlled by the Dpp morphogen gradient during growth and patterning of imaginal discs is the establishment of an inverse gradient of brk expression. brk expression is controlled by two opposing activities, a ubiquitous enhancer and a Dpp-dependent silencer. The minimal requirements for a functional silencer complex, both at the DNA and at the protein level, have been determined. Importantly, it has been demonstrated that the minimal element functions in vivo when assayed in the vicinity of a strong enhancer (the brk enhancer) or when present in a single copy in chimeric transgenes (brk enhancer-bamSE fusions) or from within an endogenous gene (gsb-enhancer lacZ fusions). The minimal functional silencer contains a distinct, single binding site for each of the two signal mediators, Mad and Med. Med binds to a GTCTG site, previously recognized as a high-affinity site for Smad binding. Mad binds to a different, GC-rich sequence. Upon binding of Mad and Med, the zinc finger protein Shn is recruited to the protein-DNA complex, bringing along a highly effective repression domain. Although ShnCT contains three essential zinc fingers, it does not bind the silencer element in the absence of Mad and Med. These data suggest that even in the triple protein complex, Shn might bind DNA with moderate sequence specificity, since only a single nucleotide position was identified that is essential for Shn recruitment. However, a number of other cis-regulatory elements that bind Mad and Med (derived from the vestigial, labial tinman, and ubx genes failed to recruit Shn, demonstrating the exquisite selectivity of the element defined in this study (Pyrowolakis, 2004).

Part of this selectivity is accounted for by the specific spacing and orientation of the Mad and Med binding sites in the silencer. Deletion and insertion of single base pairs between the two sites abolish Shn recruitment in vitro and Dpp-dependent repression in vivo, although such alterations still allow the efficient formation of a Mad/Med complex. These findings suggest that Shn recruitment requires a specific steric positioning of amino acid residues in the Smad signal mediators. Strikingly, GTCTG- and GC-rich elements were also found to be crucial for the activation of the Id gene by BMP signaling, but in this case the spacing between the GTCTG- and the GC-rich sites is much larger, and additional factors might be involved in the signal-dependent activation of the Id gene. A more recent study also links these two elements to transcriptional activation of the BMP4 synexpression group in Xenopus. It is tempting to speculate that simple sequence elements similar to the one identified here in several Drosophila genes might be involved in the repression of genes by BMP signaling. Interestingly, human Smad1/5 and Smad4 do form a complex with ShnCT on the Drosophila silencer element from brk; however, a mammalian protein sharing clear homology with Shn in the C-terminal three zinc fingers has not been identified (Pyrowolakis, 2004).

The Dpp-dependent SE allows cells in the developing organism to read out the state of the Dpp signaling pathway. This readout is relatively straightforward because the SE participates in a single switch decision, that is, either to repress (bind Mad/Med and recruit Shn along with its repression domain) or not to repress (not bind Mad/Med, thus failing to recruit Shn). This decision is critically dependent upon one major parameter: the amount of available nuclear Smad complex. For the SE to be functional in vivo, it only needs to interact with a Mad/Med heteromer in those regions of the genome that are actively transcribed; genes that are not active in a given tissue do not need to be repressed by Dpp signaling. This might be one of the main characteristics explaining why such a simple sequence element can have operator-like function in vivo; the element only needs to be recognized by the relevant trans-acting factors in open and active chromatin regions (Pyrowolakis, 2004).

A minimal Dpp-dependent silencer element derived from the brk gene has been identified and demonstrated to function in vivo in a single copy. Its interaction with relevant trans-acting factors have been identified. Based on the results of this analysis, it was possible to derive a consensus sequence, GRCGNCN(5)GTCTG, which allowed scanning of the entire Drosophila genome for potential additional elements. Approximately 350 sites were identified, that, when assayed using transgenic approaches in vivo or in cell culture, should function in a manner analogous to the SEs isolated from the brk regulatory region. Strikingly, and likely significantly, in silico search revealed that the brk gene contains a total of ten SEs, three of them in regions that have been shown to respond to Dpp-dependent repression. Since brk transcription responds to (or can respond to) Dpp signaling in all tissues examined so far, brk might require a SE in the vicinity of each of the different enhancers driving expression in distinct tissues. Alternatively, the readout of the Dpp morphogen gradient might require several SEs, each contributing to the graded repression by Dpp signaling (Pyrowolakis, 2004).

Interestingly, subsequent analysis of two genes containing such Dpp-dependent SEs has demonstrated that these elements function in these transcription units the same way as they do in the brk regulatory region. Therefore, the same molecular principle underlies morphogen readout (brk repression), germline stem cell maintenance (bam repression), and restriction of gene expression to the ventral side of the developing embryo (gsb repression). When the SEs from these three genes are aligned, all the parameters determined to be important for complex formation and for repression are conserved; at all other positions, different base pairs were found in different SEs. In addition, several genes harboring silencer elements are expressed in the wing imaginal disc in a pattern similar to brk or are known to be repressed by Dpp signaling. In contrast, SEs were not found in the vicinity of enhancers known to be activated by Dpp signaling (Pyrowolakis, 2004).

Clearly, these findings implicate that Dpp-induced, Shn-dependent repression via SE elements is a key aspect of development. The readout of the brk gradient contributes to growth and patterning of appendages, and the repression of bam in the germline is essential for the maintenance of germline stem cells. To what extent the repression of gsb contributes to proper cell fate determination along the dorsoventral axis will have to be determined by rescuing the gsb phenotype with a transgene lacking the gsbSE. However, it has been observed that wingless (wg) expression expands from ventral positions to the dorsal side in shn mutant embryos. Since gsb activates wg transcription, the expansion of gsb (in the absence of the gsbSE) possibly leads to the expansion of wg and subsequently to the alteration of dorsoventral cell fate assignments (Pyrowolakis, 2004).

It is important to note that genes repressed by a signaling pathway will not easily be identified in genetic screens because the loss-of-signaling phenotype does not correspond to the loss-of-function phenotype of a repressed gene; in the absence of the signal, such genes are ectopically expressed, leading to a locally restricted gain-of-function phenotype of the corresponding gene. Moreover, since these specific, local patterns of misexpression are likely to result in different phenotypes than widespread overexpression would, simple gain-of-function screens for candidate targets of signal-mediated repression are unlikely to offer straightforward results. Since the target sequence of Dpp/Shn-mediated repression have been identified, the genome can now be scanned and potential target genes can be identified by expression studies and enhancer dissection. It is likely that additional Dpp-repressed genes will be identified using this approach, and this will allow the painting of a much clearer picture of the gene network controlled by Dpp signaling (Pyrowolakis, 2004).

Only a few cases of signal-induced repression have been studied at the molecular level. In most of these cases, repression relies on cooperative action of cell type-specific transcription factors with nuclear signal mediators. The DNA elements that have been demonstrated to mediate repression of particular genes have not been demonstrated to be important for the regulation of other genes, and genome-wide identification of potential target genes using a bioinformatic approach might therefore be difficult, if not impossible (Pyrowolakis, 2004).

The Dpp-dependent repression system identified in this study relies on the organization of Smad binding motifs into Smad/Shn complex-recruiting SEs. The simplicity of these SEs and their capacity to repress transcription in different tissues argues that they function in the absence of tissue-restricted factors. The simple consensus sequence of the SE provides a signature for Dpp-dependent repression, allowing for a genome-wide analysis of potential target genes. Confirmed Dpp-repressed target genes can then be expressed ectopically under the control of the appropriate SE-mutated enhancers to assess the biological importance of repression in a given tissue (Pyrowolakis, 2004).

Identification of phosphatases for Smad in the BMP/DPP pathway

Phosphorylation of the SSXS motif of Smads is critical in activating the TGF-ß and bone morphogenetic protein (BMP) pathways. However, the phosphatase(s) involved in dephosphorylating and hence inactivating Smads has remained elusive. Through RNA interference (RNAi)-based screening of serine/ threonine phosphatases in Drosophila S2 cells, pyruvate dehydrogenase phosphatase (PDP) was identified as required for dephosphorylation of Mothers against Decapentaplegic (MAD), a Drosophila Smad. Biochemical and genetic evidence suggest that PDP directly dephosphorylates MAD and inhibits signal transduction of Decapentaplegic (DPP). The mammalian PDPs are important in dephosphorylation of BMP-activated Smad1 but not TGF-ß-activated Smad2 or Smad3. Thus, PDPs specifically inactivate Smads in the BMP/DPP pathway (Chen, 2006).

In Drosophila S2 cells, the level of DPP-induced phosphorylation of MAD remains in the continuous presence of DPP. However, upon removal of DPP, the amount of phospho-MAD decreases rapidly while the total protein level of MAD is unchanged. Thus, it was reasoned that phospho-MAD is dephosphorylated once the DPP signal subsides. To identify phosphatase(s) for MAD, a library of double-stranded RNAs (dsRNAs) against all 44 Ser/Thr phosphatases in the Drosophila genome was screened using RNA interference (RNAi). The dsRNA against CG12151 (CG12151-A, targeting the Drosophila PDP) consistently impeded dephosphorylation of MAD. A similar observation was made with a second dsRNA for PDP (CG12151-B) targeting a different region. It was noticed that eventually (i.e., 2 h after removal of DPP), the level of phospho-MAD decreased substantially even when PDP was knocked down. This could reflect activities from residual PDP or other phosphatases that can compensate partially. Another possibility is that if phospho-MAD constitutes a very small fraction of the total MAD, then degradation of phospho-MAD specifically may also result in reduction of phospho-MAD without grossly changing the level of total MAD (Chen, 2006).

In parallel to the decrease in phospho-MAD, the expression of Daughters against Decapentaplegic (DAD), a DPP-dependent target gene, dropped substantially from the peak DPP-induced level 2 h after DPP removal. dsRNA against PDP significantly reduced such decrease in DAD expression, consistent with its ability to delay dephosphorylation of MAD (Chen, 2006).

To further determine the importance of PDP in regulating the C-terminal phosphorylation state of MAD, Drosophila strains carrying genetic lesions that affect the PDP locus on the X chromosome were examined. The strain PBacRBCG12151e02351 (PBacCG12151) harbors a piggyback transposon 7 base pairs upstream of the first exon of the PDP gene. Another strain, Df(1)ct4b1, carries a chromosome deletion that includes the PDP gene. Whole embryos were stained with the PS1 antisera that specifically recognize phospho-MAD in Drosophila embryos. In wild-type blastoderm-stage embryos, phospho-MAD is distributed only in the dorsal-most cells and was predominantly nuclear. In the PBacCG12151 and Df(1)ct4b1 embryos, in addition to the dorsal nuclei, punctate staining was observed for phospho-MAD throughout the embryo. The ectopic PS1 staining was mostly outside the nucleus, and was detected in embryos at most, if not all, developmental stages. In cleavage-stage embryos, which undergo rapid mitosis, aggregated signals were detected for phospho-MAD in PBacCG12151 and Df(1)ct4b1 embryos. Double staining with DAPI showed that in these mutant embryos, phospho-MAD accumulated on both sides of the condensed chromosomes (Chen, 2006).

Genetic analysis showed that the ectopic phosphorylation of MAD is a maternal effect phenotype. All embryos from crosses between heterozygous mutant females and wild-type males displayed phenotypes. When heterozygous males were crossed with wild-type females, no embryos showed any ectopic PS1 staining. Therefore, haploinsufficiency of PDP has a pronounced maternal effect on the level and distribution of phospho-MAD in the early embryos, suggesting that PDP is a critical regulator of the C-terminal phosphorylation state of MAD in vivo (Chen, 2006).

Thickveins (TKV) and Punt are the receptor kinases upstream of MAD that are themselves activated by phosphorylation. Conceivably, phosphatases toward either TKV or Punt could also impact the level of phospho-MAD. It was therefore determined if PDP directly dephosphorylates MAD as its substrate. Recombinant GST-PDP effectively dephosphorylated phospho-MAD, at as low as 0.3 microM. Removal of the GST moiety by thrombin did not affect the efficiency of dephosphorylation. Asp93 in PDP is highly conserved and critical for metal ion chelating. When Asp93 was mutated to Ala, the phosphatase activity toward MAD was largely abolished. Therefore, C-terminally phosphorylated MAD is a bona fide substrate of PDP. In eukaryotes, Ser/Thr phosphatases are grouped into PPP and PPM families. PDPs belong to the PPM family whose catalytic domains are similar to that of PP2C, and pyruvate dehydrogenase was the only known substrate of PDPs. This study has thus identified a novel substrate and function for PDP (Chen, 2006).

The C-terminal phosphorylation is critical for MAD to regulate gene transcription. Therefore whether PDP could inhibit MAD-mediated transcriptional activation was tested. In the Drosophila S2R+ cells, 2xUbx-lacZ, a reporter controlled by a DPP response element from the Ultrabithorax promoter, was activated upon expression of MAD, Medea (MED), Punt, and TKV. The expression of this reporter was significantly repressed when PDP was overexpressed in a dose-dependent manner. Moreover, dsRNA against PDP, but not dsRNAs against PP1-like (CG8822) or PP4-like (CG11597) Ser/Thr phosphatases, enhanced the expression of 2xUbx-lacZ upon activation of MAD. Either overexpression or knockdown of PDP had little effect on the basal 2xUbxlacZ expression. Thus, the level of PDP is an important determinant of the strength of DPP signaling (Chen, 2006).

After DPP stimulation, endogenous phospho-MAD was readily coimmunoprecipitated with V5-tagged PDP. In S2 cells transfected with Flag-MAD and PDP-V5, anti-Flag immunoprecipitation brought down PDP-V5, demonstrating that the coimmunoprecipitation of MAD and PDP works in reciprocal order. Anti-V5 immunoblotting revealed two V5-containing proteins in cells transfected with PDP-V5, suggesting the presence of different forms of PDP. The contribution of C-terminal phosphorylation of MAD in its interaction with PDP was further investigated. Phospho-MAD was bound equally well by wild-type and the D93A mutant form of GST-PDP. Unphosphorylated MAD interacted with PDP. However, when compared with the input, ~3% of phospho-MAD was bound by GST-PDP; while <1% of unphosphorylated MAD was bound. Thus, while not being required for interaction, the C-terminal phosphorylation substantially enhanced MAD interaction with PDP (Chen, 2006).

In live S2 cells, PDP-GFP not only distributed throughout the cell, but also exhibited a punctate pattern that overlapped with mitochondria as revealed by Mito-Tracker. Such distribution of PDP was not changed upon activation of the DPP pathway. Overexpression of PDP-GFP did not apparently prevent nuclear accumulation of MAD, possibly because the receptor activation overrode PDP activity under the condition or because changes in the kinetics of MAD nuclear import/export require more sensitive and quantitative methods to measure. These observations suggest that PDP has broad subcellular localization and can therefore gain access to substrates in mitochondria, cytoplasm, and nucleus. Indeed, in both the cytosolic and nuclear fractions, coimmunoprecipitation of phospho- MAD and PDP could be detected. Less phospho-MAD was coimmunoprecipitated with PDP in the nuclear fraction, which could be due to the fact that less PDP was present in the nucleus and that the buffer used to extract nuclei contained a higher salt and detergent concentration, which is more stringent for protein-protein interaction (Chen, 2006).

Whether mammalian PDPs has similar functions to their Drosophila counterpart was investigated. Two orthologs of Drosophila PDP, PDP1 and PDP2, have been identified in the human genome, and both share ~40% identity with the Drosophila PDP in amino acid sequences. Coimmunoprecipitation between PDP2 and Smad1 was detected in 293T cells, suggesting that the functional interaction between PDP and Smad is conserved in mammalian cells. To further test this, two siRNA duplexes were designed for each of PDP1 and PDP2; these correspondingly reduced the mRNA levels of PDP1 and PDP2. The TGF-ß receptor kinases can be inhibited by the compound SB431542, while SB202190 was shown to block BMP receptor kinase activity toward Smad1. In HeLa cells, TGF-ß- and BMP2-induced phosphorylation of Smad2/3 and Smad1 decreased considerably after SB431542 or SB202190 treatment, respectively, reflecting dephosphorylation of these Smads. Therefore, these two kinase inhibitors were used to monitor phosphatase activities toward Smad1, Smad2, and Smad3 (Chen, 2006).

Compared with the control, siRNA against PDP1 or PDP2 resulted in reduced Smad1 dephosphorylation. However, the same siRNA treatment did not significantly change the dephosphorylation of Smad2 and Smad3. Even when siRNAs against PDP1 and PDP2 were combined, dephosphorylation of Smad2 and Smad3 was largely unaffected. Moreover, the expression of one BMP target gene, Smad6, was enhanced in cells transfected with siRNA against PDP1 and PDP2, both at the basal state and after BMP2 stimulation. The increase in Smad6 expression without added BMP2 could reflect enhancement of low-level autocrine BMP signaling. These observations suggest that PDP1 and PDP2 are important for dephosphorylation of Smad1. Therefore, the mechanism of Smad dephosphorylation in vertebrates is similar to that in Drosophila. BMP is more closely related to DPP, and Smad1 is more similar to MAD than are Smad2/3. Interestingly PDP1 and PDP2 did not appear to be rate-limiting in dephosphorylation of Smad2 and Smad3. This raised the possibility that different phosphatases are used to inactivate different R-Smads (Chen, 2006).

The activation state of various signal transduction pathways is often dictated by phosphorylation-dephosphorylation control of key signaling molecules. This study reports that PDPs are phosphatases for R-Smads in the DPP pathway in Drosophila and the BMP pathways in mammals. PDP can act to reduce the concentration of phospho-R-Smads in the nucleus and consequently weaken the transcriptional responses to BMP. Moreover, these findings also raised the possibility that MAD may be phosphorylated by kinases outside of the DPP pathway, and the role of PDP is to remove such aberrant phosphorylation and prevent ectopic DPP signaling. TGF-ß/BMP cytokines control the biology of many cell types in different physiological contexts. In addition to the receptors and Smads, other factors must participate to modify and fine-tune the signaling. The identification of phosphatases that inactivate R-Smads provides a new angle to study how TGF-ß/BMP signaling is modulated (Chen, 2006).

Drosophila Nemo antagonizes BMP signaling by phosphorylation of Mad and inhibition of its nuclear accumulation

Drosophila Nemo is the founding member of the Nemo-like kinase (Nlk) family of serine/threonine protein kinases that are involved in several Wnt signal transduction pathways. Nemo performs a novel function in the inhibition of bone morphogenetic protein (BMP) signaling. Genetic interaction studies demonstrate that nemo can antagonize BMP signaling and can inhibit the expression of BMP target genes during wing development. Nemo can bind to and phosphorylate the BMP effector Mad. In cell culture, phosphorylation by Nemo blocks the nuclear accumulation of Mad by promoting export of Mad from the nucleus in a kinase-dependent manner. This is the first example of the inhibition of Drosophila BMP signaling by a MAPK and represents a novel mechanism of Smad inhibition through the phosphorylation of a conserved serine residue within the MH1 domain of Mad (Zeng, 2007).

This study demonstrates a novel regulatory role for the Drosophila Nlk family member Nemo in a TGF-ß-superfamily signal transduction pathway. Evidence is provided that Nemo is an antagonist of BMP signaling in Drosophila by examining its role in wing development through genetic analysis and monitoring of BMP-dependent gene expression. The genetic interaction studies show that phenotypes caused by activation of the BMP pathway can be suppressed by ectopic nmo and enhanced by loss of nmo. The data suggest that Nemo participates in the BMP pathway by modulating Mad activity. This is seen in the inhibition by Nemo of Mad-dependent gene expression and in the elevated expression of Mad target genes observed in nmo mutant clones. Nemo can bind to and phosphorylate Mad and this phosphorylation has direct consequences on the nuclear localization of Mad in cell culture. The single Nemo target residue maps to serine 25 within the MH1 domain of Mad, a site distinct from those previously implicated in the regulation of Mad activity and nuclear localization (Zeng, 2007).

The vertebrate Mad ortholog Smad1 normally shuttles between the cytoplasm and nucleus in the absence of signal, but upon receptor activation becomes phosphorylated at its C-terminus, binds the Co-Smad and accumulates primarily in the nucleus. Such nucleocytoplasmic shuttling is observed with R-Smads participating in both BMP and TGF-ß signaling. The shuttling provides a tightly regulated mechanism for monitoring the activation status of the receptors. Receptor-phosphorylated Smads are dephosphorylated in the nucleus, most likely causing them to detach from Co-Smads and DNA and allowing them to shuttle back to the cytoplasm. Their nuclear retention is aided by the formation of the R-Smad-Co-Smad complex and DNA binding. Thus, receptor activation leads to elevated nuclear retention. The actual rates of nuclear import are not altered by receptor-mediated phosphorylation (Zeng, 2007).

From these findings it is concluded that under normal conditions, endogenous Nemo acts to modulate the level of active Mad that is retained in the nucleus. Since Nemo is expressed ubiquitously at low levels and is enriched in cells with elevated levels of pMad, it fulfils the requirements for such a molecule involved in fine-tuning the BMP response. The phosphorylation by Nemo might control a delicate balance between promoting cytoplasmic localization of Mad, while allowing certain levels of Mad signaling to proceed in a receptor-dependent manner (Zeng, 2007).

Nemo can inhibit BMP signaling by antagonizing the nuclear localization of Mad in a kinase-dependent manner. Such a mechanism has been attributed previously to crosstalk between Erk MAPK signaling and TGF-ß/BMP signaling. This research presents Nemo as the first MAPK-like protein to attenuate Drosophila BMP pathway activity through phosphorylation of Mad. It has also been found that murine Nlk can bind to Mad, raising the intriguing possibility that this mechanism is conserved across species (Zeng, 2007).

MAPK can repress TGF-ß-superfamily signaling by targeting several Smads. The BMP-specific Smad1 is a target of cross-regulation by EGF signaling through the Erk MAPK pathway. Erk phosphorylates Smad1 in the linker domain and inhibits both the nuclear accumulation and transcriptional activity of Smad1 in cell culture and, in consequence, the in vivo function of Smad1 in neural induction and tissue homeostasis. Ras-stimulated Erk also phosphorylates two R-Smads involved in TGF-ß/Activin signaling and prevents their nuclear accumulation. The phosphorylation sites within these Smads differ, thus providing a mechanism for preferentially selective inhibition of one subtype. Thus, the distinct Nemo phosphorylation site in the MH1 domain represents an additional level of regulation of these proteins (Zeng, 2007).

Interestingly, in these studies, the Drosophila Erk MAPK does not inhibit Mad during wing development. In fact, Erk and Mad appear to synergize in the wing blade, as would be predicted given that both Egfr and BMP signaling are required for vein specification (Zeng, 2007).

The phosphorylation of serine 25 in the MH1 domain of Mad represents a novel site of regulation of Smads. This protein domain is involved in nuclear localization, DNA binding and association with transcriptional regulators. Based on known protein structures of Smads, one can predict that the Mad MH1 domain is composed of several elements. The most N-terminal sequence predicts a flexible region, then a short alpha-helix followed by a linker region and a longer, second alpha-helix. The second alpha-helix contains the predicted nuclear localization sequence (NLS). Serine 25 is located just N-terminal to the first alpha-helix. The added negative charge following phosphorylation by Nemo could modify the interaction between the two alpha-helical regions by potentially neutralizing the positively charged NLS and thereby influencing nuclear localization of Mad. Such a model is also supported by the finding that mutation of serine to alanine renders Mad constitutively nuclear. Interestingly, a similar constitutively nuclear localization has been observed when the Erk phosphorylation sites is mutated in Smad1. This suggests that both Nemo and Erk MAPK are involved in the inhibition of BMP signaling and that their distinct sites of action function to block the nuclear accumulation of Smads. Thus, the cellular factors that induce either Nlk or Erk activity can oppose the functions of BMP signaling (Zeng, 2007).

In addition to the biochemical and cell culture evidence that Nemo targets the MH1 domain of Mad to promote its nuclear export, in vivo evidence is presented that clearly demonstrates that the expression of Nemo or absence of nmo has a measurable effect on the readout of the BMP pathway in terms of Mad target gene expression, wing size, wing vein spacing and vein patterning. Specifically, elevated Nemo can attenuate the expression of vgQ and salm, whereas nmo somatic clones and mutant discs show elevated or expanded target gene expression. Genetic interaction studies confirm such an antagonistic role, as elevated Nemo can suppress the mutant phenotypes induced by elevated BMP signaling, and reductions in nmo enhanced the penetrance of activated BMP phenotypes. Thus, the phenotypic analyses support and extend the biochemical model of the inhibition of Mad and BMP signaling by Nemo (Zeng, 2007).

Modulation of Nemo does not affect the levels of pMad found at the peaks of the BMP response gradients, suggesting that the effect of Nemo is at the level of the nuclear function of Mad. Studies with leptomycin B (LMB), which acts to inhibit Crm1-dependent nuclear export of Smads, demonstrate that Nemo can affect the nuclear localization of Mad. Thus, it is proposed that Nemo promotes the nuclear export of Mad and that this results in a fine-tuning of the levels of target genes in regions where nmo is expressed (Zeng, 2007).

It is proposed that one role for nmo is in refining the level of BMP signaling regulating proliferation. This early role for BMP signaling also relies on Mad and is therefore a candidate for Nemo-mediated inhibition. The effect on proliferation may affect the spacing, but not levels, of the pMad gradient. It is consistently observed that the genotypes in which wing width is affected do have a mild effect on the spacing of pMad stripes, and it is suggested this might be due to actual changes in cell number in the disc. Additionally, nmo mutations manifest in alterations in wing size, wing shape and cell density (Zeng, 2007).

nmo mutations also affect the later larval and pupal patterning and differentiation functions of BMP, and these can be correlated to changes in target gene expression and with vein patterning abnormalities. Thus, it appears that Nemo can modulate levels of BMP signaling at several developmental stages in wing growth and patterning (Zeng, 2007).

It has been demonstrated that Nemo can antagonize Drosophila Wg signaling during wing development. In this study it was demonstrated that Nemo also acts to attenuate BMP signaling by targeting the activity of Mad. In both of these signaling pathways the net outcome is the inhibition by Nemo of pathway-dependent target gene expression. These results demonstrate that Nemo (and by extension the Nemo-like kinases) play important roles in refining signaling pathways during development (Zeng, 2007).

An intriguing but still incomplete picture is emerging regarding the regulation of both Nlk expression and activity; this regulation represents a potential point of crosstalk between signaling pathways. nmo is transcriptionally regulated by Wg signaling. The kinase activity of Nlk is stimulated by Tak1 after Wnt induction and that Tak1 can be activated by BMP signaling. Activated Nlk can inhibit Tcf/Lef proteins and modulate Wnt-dependent gene expression. In this study, it was found that Drosophila Nlk is playing an important role in modulating BMP signaling and Mad-dependent gene expression, revealing an additional point of cross-regulation and refinement between signaling molecules (Zeng, 2007).

Postsynaptic mad signaling at the Drosophila neuromuscular junction

Cell-to-cell communication at the synapse involves synaptic transmission as well as signaling mediated by growth factors, which provide developmental and plasticity cues. There is evidence that a retrograde, presynaptic transforming growth factor-β (TGF-β) signaling event regulates synapse development and function in Drosophila. This study shows that a postsynaptic TGF-β signaling event occurs during larval development. The type I receptor Thick veins (Tkv) and the R-Smad transcription factor Mothers-against-dpp (Mad) are localized postsynaptically in the muscle. Furthermore, Mad phosphorylation occurs in regions facing the presynaptic active zones of neurotransmitter release within the postsynaptic subsynaptic reticulum (SSR). In order to monitor in real time the levels of TGF-β signaling in the synapse during synaptic transmission, a FRAP (fluorescence recovery after photobleaching) assay was established to measure Mad nuclear import/export in the muscle. Mad nuclear trafficking is shown to depend on stimulation of the muscle. These data suggest a mechanism linking synaptic transmission and postsynaptic TGF-β signaling that may coordinate nerve-muscle development and function (Dudu, 2006).

Molecular signals are often transmitted in the reverse direction across the synapse to regulate the structure and function of the presynaptic neuron. There is now good evidence that BMP-type TGF-β ligands are essential retrograde signaling ligands in the Drosophila nervous system and that they control synaptic growth, synaptic function, and in some cases the specificity of the particular neurotransmitter released at the synaptic terminal. These data show that there is an additional postsynaptic TGF-β signaling cascade at the NMJ that is consistent with anterograde and/or autocrine signaling. Four lines of evidence support the existence of a postsynaptic signaling event: (1) core transduction components of the BMP-type signaling pathway, such as the receptor Tkv and the transcription factor Mad, are present in the muscle where they accumulate in the subsynaptic reticulum (SSR), (2) phosphorylation of Mad takes place in the PSD, opposite to the presynaptic active zones where exocytosis of neurotransmitter takes place, (3) a FRAP-based assay that monitors signaling-dependent nuclear import/export and nuclear retention of Mad revealed that Mad-mediated signaling can take place in the muscle, and (4) upon stimulation of the muscle, Mad targeting to the terminal is enhanced and the levels of postsynaptic Mad signaling are increased, as monitored with the FRAP assay (Dudu, 2006).

Taken together, these data are consistent with the existence of an anterograde signaling event that is initiated in the presynaptic terminal through exocytosis of a TGF-β-type ligand. Binding of the ligand to the Tkv receptor at the PSD would then lead to activation of the receptor and thereby phosphorylation of Mad. Phosphorylation of Mad causes a decrease in the nuclear export of the transcription factor and thereby its accumulation in the muscle nuclei. Nuclear P-Mad in turn mediates transcriptional control of the target genes of the signaling pathway. Thus, while retrograde BMP signaling instructs the neurons about the physiological and developmental state of the muscle, such an anterograde signaling event may provide the muscle with information about the activity of the neuron in the medium/long term (Dudu, 2006).

However, these observations do not rule out an autocrine BMP signaling event: a ligand (perhaps, again, Gbb) released from the muscle at the synaptic site could activate signaling in the muscle itself. Clearly, further analysis needs to be carried out to discriminate between these two possibilities (Maitra, 2006).

A FRAP assay was established to determine in real time the state of activation of TGF-β pathway in the muscle. This assay is useful to study rapid events such as synaptic transmission, where it is desirable to capture fast changes in the state of signaling. The FRAP experiments allow determination the rates of nuclear import/export of Mad and the size of the nuclear Mad pool that does not exchange (or exchanges slowly) with the cytosol: the immobile fraction. Experiments in which the levels of signaling are affected reveal that both the effective import/export rates and the size of the immobile fraction correlate with the signaling state: higher levels of signaling can be associated with a larger Mad immobile pool in the nucleus and higher import-to-export ratio (Dudu, 2006).

What is the molecular meaning of the correlation between signaling and the immobile pool? It is first considered that the nuclear P-Mad binds/unbinds to the DNA with rates that are slower than other rates during signaling (phosphorylation/dephosphorylation, import/export) and does not significantly contribute to the recovery in the 1000 s time of the experiments. Bound to the DNA, P-Mad could not exchange with the cytosol, and the relatively slow rates of binding/unbinding would generate an immobile fraction as shown in the FRAP experiment. Further experiments, however, did not support this hypothesis. After bleaching GFP-Mad in a small region within the nucleus in a TkvQD-expressing muscle (where 67% of the nuclear Mad does not exchange with the cytosol), the fluorescence fully recovered within this bleached region, revealing that Mad is not immobilized within the nucleus. Is then the DNA moving within the nucleus? FRAP experiments with Histone2A-GFP discard the possibility that the DNA in the nucleus is moving rapidly and show that it remains immobile during the time scale of the FRAP experiments (Dudu, 2006).

What could then explain the correlation between signaling and immobile pool? It is speculated that P-Mad associates with other cofactors in the nucleus with slow binding and unbinding rates. Engaged in a macromolecular complex, P-Mad would be unable to exit the nucleus through the nuclear pore and thereby would generate the immobile pool revealed in the FRAP experiments (Dudu, 2006).

Retrograde signaling has been proposed to convey information to the neuron about the development of the muscle, so that the presynaptic terminal and the muscle grow synchronously and the increase of the SSR is coordinated with the appearance of new boutons and active zones. What could then be the role of anterograde Mad signaling in the muscle? It is speculated that upon synaptic transmission, a quantum of neurotransmitter is released together with a “quantum” of growth factor from the presynaptic vesicles. If this is true, postsynaptic signaling coupled to synaptic activity could endow the muscle with information about the activity of the neuron and thereby control the growth and development of the NMJ. It will be interesting, therefore, to study at the ultrastructural level whether the same vesicles that contain the neurotransmitter contain the TGF-β growth factor (Dudu, 2006).

These data show that postsynaptic TGF-β signaling occurs at the NMJ. Furthermore, the data suggest a coupling between muscle stimulation and postsynaptic TGF-β signaling. Such coupled processes may confer information to the muscle about the activity of the neuron during development (Dudu, 2006).

dSno facilitates baboon signaling in the Drosophila brain by switching the affinity of Medea away from Mad and toward dSmad2

A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno; termed snoN in FlyBase). The SnoN locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for SnoN function. This is an intriguing possibility from the perspective of developmental evolution. SnoN is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. SnoN mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and Smad2 mutants. This suggests that SnoN is a mediator of Baboon signaling. SnoN binds to Medea and Medea/SnoN complexes have enhanced affinity for Smad2. Alternatively, Medea/SnoN complexes have reduced affinity for Mad such that, in the presence of SnoN, Dpp signaling is antagonized. It is proposed that SnoN functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFß signaling and a novel function for Sno/Ski family proteins (Takaesu, 2006).

Studies in mammalian cells showed that, when overexpressed, Sno is an antagonist of TGFß/Activin signaling. Overexpression of dSno with A9.Gal4 (throughout the presumptive wing blade) resulted in small wings with multiple vein truncations at 100% penetrance. A9.Gal4:UAS.dSno pupal wing discs were examined for Drosophila serum response factor (dSRF) expression, an intervein marker repressed by Dpp signaling. In A9.Gal4:UAS.dSno pupal discs, dSRF expression is highly irregular with no obviously downregulated regions corresponding to vein primordia. These wing and disc phenotypes are strongly reminiscent of those expressing the dominant-negative allele Mad1 (DNA binding defective but competent to bind Medea) with a variety of drivers, including A9.Gal4 (100% penetrant) and 69B.Gal4. Mad1 dominant-negative effects are due to the titration of Medea into nonfunctional complexes. The similarity of dSno and Mad1 phenotypes suggests that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

This was further tested this by coexpressing dSno with Medea or Mad or dSmad2. Coexpression of dSno with Medea or Mad rescues the dSno phenotype to nearly wild type in size and vein pattern. In dSno and Medea coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 28% penetrance. In dSno and Mad coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 19% penetrance. Alternatively, coexpression of dSno with dSmad2 has little effect on the dSno phenotype. In dSno and dSmad2 coexpressed wings, reduced size and multiple vein defects remained 100% penetrant. Coexpression of Mad1 and dSno significantly enhanced the dSno phenotype. One hundred percent of Mad1 and dSno coexpressing the wings are more abnormal than those expressing either dSno or Mad1. The coexpressing wing is very small and veinless and resembles wings expressing UAS.Dad (Dpp antagonist) or dpp class II disc mutants (e.g., dppd5). The enhanced phenotype suggests that dSno and Mad1 antagonize BMP signaling in distinct ways that have additive effects (Takaesu, 2006).

Experiments with a constitutively activated form of the Dpp type I receptor Thickveins (CA-Tkv) are also consistent with this hypothesis. One hundred percent of A9.Gal4:UAS.CA-Tkv wings are overgrown and have numerous ectopic veins as well as vein truncations. This phenotype is suppressed in 98% of the individuals when UAS.dSno is coexpressed with UAS.CA-Tkv. In fact, the coexpression phenotype is not much different from A9.Gal4:UAS.dSno alone, indicating that dSno antagonism of Dpp signaling is fully epistatic to activated Tkv. Finally, ubiquitous overexpression of dSno in the embryonic ectoderm with 32B.Gal4 resulted in discless larvae—a phenotype seen in Mad and Medea null genotypes and in dpp class V disc mutants ( e.g., dppd12. It is concluded that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

In Drosophila, as in vertebrates, two TGFß subfamilies are present. The bone morphogenetic protein (BMP) subfamily member Dpp signals through its type I receptor Thickveins to its dedicated transducer Mad (Smad1 homolog) and the Co-Smad Medea (Smad4 homolog). The TGFß/Activin subfamily member activin signals through its type I receptor Baboon to its dedicated transducer dSmad2 and Medea. This study shows that dSno binds Medea and then functions as a mediator of Activin signaling by enhancing the affinity of Medea for dSmad2. Antagonism for BMP signaling likely arises as a secondary consequence of dSno overexpression. Examination of dSno loss-of-function mutants shows that dSno is required in cells of the optic lobe of the brain to maintain proper rates of cell proliferation. Given that Dpp signaling is essential for neuronal differentiation in the optic lobe, these data suggest that dSno functions as a switch that shunts Medea from the Dpp pathway to the Activin pathway to ensure a proper balance between differentiation and proliferation in the brain (Takaesu, 2006).

To resolve the apparent contradiction between the effect on signaling of dSno overexpression (antagonism) and the role identified in dSno mutants (mediation), dSno was examined biochemically. Expression constructs encoding Flag or T7 epitope-tagged Medea, Mad, and dSmad2 were used and various combinations were co-expressed in COS1 cells. It was possible to clearly detect interaction of Medea with both dSmad2 and Mad by co-immunoprecipitation. A T7-tagged dSno expression construct was generated and coexpressed with Flag-dSmad2, Mad, or Medea or with a control vector. Complexes were isolated on Flag agarose and analyzed for the presence of coprecipitating dSno by T7 Western blot. T7-dSno was readily detectable in complexes isolated from cells expressing Medea, but not Mad or dSmad2 (Takaesu, 2006).

Since Medea is a shared partner for both Mad and dSmad2, whether co-complexes containing Medea and dSno together with either Mad or dSmad2 was tested. COS1 cells were transfected with T7-dSno and Flag-Mad or dSmad2 with or without T7-Medea. T7-dSno was present in a complex with Flag-dSmad2 only when T7-Medea was also present. Interestingly, approximately equal amounts of Medea and dSno appeared to coprecipitate with dSmad2, suggesting that much of the Medea that interacts with dSmad2 in this assay is also bound to dSno. In contrast, dSno was not detected in complex with Mad, even when Medea was present, even though Medea clearly interacted with Mad in this assay. These results suggest that dSno interacts specifically with Medea and that the dSno-Medea complex can interact with dSmad2 but not with Mad (Takaesu, 2006).

To test whether incorporation of dSno affected formation of the Medea-dSmad2 complex, Flag-Medea and T7-dSmad2 were coexpressed with or without dSno. The amount of dSmad2 that coprecipitated with Flag-Medea was clearly increased in the presence of dSno. In the reverse of this experiment, it was also observed that an increase in T7-tagged Medea present in Flag-dSmad2 precipitates when dSno was coexpressed. These results suggest that dSno may promote the formation of Medea-dSmad2 complexes (Takaesu, 2006).

To test whether dSno has any effect on the formation of Mad-Medea complexes, similar experiments were performed in which Flag-Medea and Western blotted was isolated for coprecipitating T7-Mad or dSmad2 in the presence or absence of coexpressed dSno. The interaction of Medea with Mad was more readily detectable than with dSmad2. However, inclusion of dSno again increased the interaction between Medea and dSmad2. In contrast, no increase was seen in the Medea–Mad interaction when dSno was coexpressed and it appeared that increasing dSno expression decreased the amount of Mad that coprecipitated with Flag-Medea. Thus it appears that dSno not only may promote interaction of Medea with dSmad2, but also may compete with Mad for Medea interaction, suggesting that dSno may play a role in determining the pathway specificity of Medea (Takaesu, 2006).

Msk is required for nuclear import of TGF-β/BMP-activated Smads

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor β (TGF-β) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Mad as the readout, a whole-genome RNAi screening was carried out in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-β. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. Thus, evolutionarily conserved proteins have been identified that are important in the signal transduction of TGF-ß and BMP into the nucleus (Xu, 2007).

Genome-wide RNAi screening in this study offers a genetic approach to uncover new elements in TGF-ß signal transduction. Msk and its mammalian orthologues Imp7 and 8 are critical components in transporting TGF-ß-activated Smads into the nucleus. Biochemical evidence further suggests that Msk/Imp7/8 directly import phospho-Smads as cargoes (Xu, 2007).

Although there appears to be some discrepancy between these new findings and previous reports that importins are dispensable for the nuclear import of Smads, these observations can be reconciled. The present and previous studies, based on different approaches, may have revealed different nuclear import mechanisms used by basal and activated Smads to enter the nucleus. There are important differences comparing Smads import with or without TGF-ß stimulation. Unphosphorylated Smads are monomers, but phosphorylated Smads are assembled into complexes with Smad4 and are thus much larger in size. Moreover, as phospho-Smads accumulate in the nucleus they have to move across the nuclear pore against an ascending concentration gradient of Smads already in the nucleus, whereas unphosphorylated Smads never reach a higher concentration in the nucleus than in the cytoplasm. Thus, importing phospho-Smad complexes and unphosphorylated Smad monomers may entail different mechanisms, with or without the participation of importins. Indeed, RNAi data in both Drosophila and mammalian cells suggest that nuclear import of the two forms of Smads is very different regarding the requirement of Msk/Imp7/8. This type of differential requirement for import factors is not unique to Smads. In fact, STATs (signal transducers and activators of transcription) in the interferon pathway are another example in which the latent STATs are imported by an importin-independent mechanism, whereas the phosphorylated STATs depend on importins to accumulate in the nucleus. It is also interesting to note that phospho-Smads were still detected in the nucleus upon RNAi-mediated knockdown of Msk/Imp7/8. Although the trivial explanation that this may be due to incomplete depletion of the targeted proteins cannot be ruled out, this observation may also suggest additional import mechanisms for activated Smads. It is recognized that the previous finding of importin-independent nuclear import of Smads was largely based on an in vitro reconstituted nuclear import assay. Although this in vitro system is widely accepted, it may not fully recapitulate nuclear import of activated Smads in cells. Based on RNAi data, regarding the requirement of importins, the conclusion drawn from the in vitro import assay may not apply to phospho-Smads in intact cells. However, the current study does not necessarily contradict the previous suggestions that direct Smad-nucleoporin interaction is critical for nuclear import of Smads (Xu, 2007).

The data showed that Msk/Imp7/8 interacted with Smads regardless of their phosphorylation status; thus, additional factors must be involved to explain why only TGF-ß/BMP-activated Smads can accumulate in the nucleus. Because basal-state Smads are actively exported out of the nucleus, it is possible that retaining only phospho-Smads in the nucleus requires blocking Smads nuclear export, a scenario that has been demonstrated for Smad4. This hypothesis would be consistent with findings in live cells, in which TGF-ß signaling led to reduced mobility of Smad2 in the nucleus (Xu, 2007).

Because Msk, Imp7, and Imp8 are shown to be critical for targeting phospho-Smads into the nucleus, it is conceivable that regulatory inputs to this nuclear import factor would impact TGF-ß signaling. Although no changes were observed in subcellular localization of Msk or Imp7/8 in response to TGF-ß in cultured cells, during Drosophila embryonic development, Msk distribution changed between cytoplasm and nucleus in a dynamic fashion. Moreover, Msk is phosphorylated on tyrosine residues with yet-unknown functional consequences. If and how Msk localization is regulated and by what signals are completely open questions at present (Xu, 2007).

A number of mitogen-induced phosphorylation events in the linker region of Smad have been suggested to inhibit TGF-ß-induced nuclear translocation of Smads in Xenopus and mammalian cells. Because part of the Imp7/8 binding was mapped to the linker region of Smad3, it will be interesting to determine if linker phosphorylation would affect the interaction between Smads and Imp7/8 and hence the rate of nuclear import. It is also worth noting that Msk has been genetically implicated in the nuclear import of activated ERK in Drosophila. Such convergence on the same molecule for nuclear import raises the possibility of cross-talk between MAP kinase and TGF-ß pathways at the level of nuclear translocation of key signal transducers (Xu, 2007).

Specific nucleoporin requirement for Smad nuclear translocation

Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor beta (TGF-beta) signal transduction. This study identified a subset of nucleoporins that, in conjunction with Moleskin (Msk, Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. The roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, novel pathway-specific functions of Sec13 and Nup93 were uncovered; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, this study has delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism (Chen, 2010).

This study identified a distinct nucleoporin cohort, including both non-FG nucleoporins and FG-nucleoporins, that represents a unique trans-NPC mechanism for signal-activated MAD. Such specificity in nucleoporin utilization may reflect different demands of constitutive and signal-induced nuclear import events. Most unexpectedly, several non-FG nucleoporins, including Sec13, Nup93, Nup75, and Nup205, appear to act in concert with Msk to selectively transport MAD, but not the cNLS-cargo or basal-state Medea, into the nucleus. This is the first indication that beyond their involvement in the general assembly of the NPC, non-FG nucleoporins could play discrete roles in specific nuclear transport pathways. This study further identified the distinct functions served by two non-FG scaffold nucleoporins, Sec13 and Nup93, that are critical and specific for the nuclear import of MAD. These findings suggest a novel functional interplay between the MAD nuclear import machinery and the NPC (Chen, 2010).

Sec13 is part of the Nup107-160 complex, and Nup93 is part of the Nup53-93 complex; both are scaffolds of the NPC. It is emphasized that these findings are not in conflict with the established roles of Sec13 and Nup93 in general NPC assembly but broaden the functions of these non-FG nucleoporins to specific nuclear import pathways. It was somewhat surprising that depletion of Nup75 and Sec13 had little impact on MAb414 staining and nuclear envelope permeability, in contrast to the more severe phenotypes exhibited by the knockdown of other components in the Nup107-160 complex (i.e., nup145, nup107, and nup160). It was hardly possible to detect Sec13 after RNAi, so the lack of impact on MAb414 staining could not be attributed to incomplete depletion of Sec13. Therefore, these observations suggest that knocking down individual components of the Nup107-160 complex could lead to different phenotypes regarding MAD nuclear import, the MAb414 staining pattern, and the permeability of the NPC, arguing that each nucleoporin in the Nup107-160 complex serves distinct functions (Chen, 2010).

The challenging question ahead is how these non-FG nucleoporins mediate the nuclear import of MAD. Interestingly, Sec13 has been shown to dynamically transit between the cytoplasm and the nucleus, and endogenous Sec13 is partitioned among NPC, the intranuclear space, and the endoplasmic reticulum (ER). With theobservation that Sec13 preferentially interacts with phosphorylated/activated MAD, it is possible that Sec13 could act as an active trafficker rather than as a stationary component of the NPC to mediate the nuclear import of MAD. Whether phosphorylated MAD reaches the NPC via random diffusion or is guided by particular factors remains an open question, and it will be interesting to investigate whether Sec13 might be involved (Chen, 2010).

Msk has a characteristic nuclear rim localization pattern that is shown in this study to be important for its ability to transport MAD into the nucleus. Two of the nucleoporins that are required for MAD nuclear import, Nup93 and Nup358, appear to be responsible for targeting Msk to the nuclear periphery. Deletion of the C-terminal region of Msk disrupted its nuclear rim distribution and also significantly weakened the Msk-Nup93 interaction. It is unclear whether the same C-terminal deletion of Msk would affect the Msk-Nup358 interaction as well. Thus, the question remains, between Nup93 and Nup358, which one is more directly responsible for recruiting Msk to the nuclear periphery. Interestingly, Impβ is also concentrated to the nuclear periphery, like Msk, but such localization has been shown to depend on Nup153 instead of Nup93 and Nup358. Therefore, different importins are apparently recruited to the NPC through distinct nucleoporins, another direct indication that various nuclear import pathways operate through different modes of interaction with the NPC (Chen, 2010).

As exemplified by Sec13, Nup93, and Nup358, the nucleoporins implicated in MAD nuclear import serve distinct functions at different stages of the import process. One appealing model is that Msk is positioned by Nup93 and Nup358 to the vicinity of NPC, and perhaps Sec13 engages phosphorylated MAD and, through its own trafficking ability, delivers MAD to Msk, which completes the translocation across the NPC. While the data clearly suggest that Sec13 and Nup93 play roles distinct from those of the other components of the Nup107-160 or Nup53-93 complex, it is not suggested that they function in isolation from the other nucleoporins. Nor is it possible at this point to rule out a possible requirement for other nucleoporins in the nuclear import of MAD. Nevertheless, the direct physical interaction between Sec13/Nup93 and MAD or Msk, as well as the very selective impact of Sec13 and Nup93 RNAi on the nuclear import of MAD, but not other cargoes, is consistent with the interpretation that Sec13 and Nup93 are directly involved in the nuclear import of MAD (Chen, 2010).

Thisr genetic dissection of the Smad nuclear import pathway has important implications for the model of NPC structure and function. The findings in this study depart from the current dogma that puts only FG-nucleoporins at the center of the NPC-importin interplay. The diversity in trans-NPC routes and the pathway-specific involvement of non-FG nucleoporins need to be incorporated into models of NPC function in nuclear transport. It is increasingly clear that there are multiple distinct routes through the NPC that are taken by different importin/cargo complexes. The question, then, is how the NPC can accommodate these different passages. X-ray crystal structure analysis and electron microscopy have suggested that the Nup107-160 complex assumes a Y-shaped topography, raising speculations that such a porous assembly may leave room for additional trans-NPC passages besides the central tunnel, which is densely populated by FG-nucleoporins. One could also speculate that maybe the NPC can assume different configurations upon receiving different importin/cargo complexes to enable the translocation process (Chen, 2010).

Mad is required for wingless signaling in wing development and segment patterning in Drosophila

A key question in developmental biology is how growth factor signals are integrated to generate pattern. This study investigated the integration of the Drosophila BMP and Wingless/GSK3 signaling pathways via phosphorylations of the transcription factor Mad. Wingless was found to regulate the phosphorylation of Mad by GSK3 in vivo. In epistatic experiments, the effects of Wingless on wing disc molecular markers (senseless, distalless and vestigial) were suppressed by depletion of Mad with RNAi. Wingless overexpression phenotypes, such as formation of ectopic wing margins, were induced by Mad GSK3 phosphorylation-resistant mutant protein. Unexpectedly, Mad phosphorylation by GSK3 and MAPK was found to occur in segmental patterns. Mad depletion or overexpression produced Wingless-like embryonic segmentation phenotypes. In Xenopus embryos, segmental border formation was disrupted by Smad8 depletion. The results show that Mad is required for Wingless signaling and for the integration of gradients of positional information (Eivers, 2009; full text of article).

Transgenic flies expressing forms of Mad resistant to GSK3 phosphorylation displayed high BMP and Wg signaling phenotypes. Mad contains 74 serines/threonines, yet phosphorylation-resistant mutations of a single MAPK or of two GSK3 sites generated hyperactive transcription factors. Previous work in Drosophila had identified that phosphorylation by the Nemo/NLK kinase in the MH1 domain of Mad inhibits its activity, and that a neomorphic human Smad4 mutation can produce Wg-like phenotypes when overexpressed in the wing. During Drosophila early embryogenesis, Mad linker phosphorylations tracked the priming activity of MAPK/EGFR, particularly in the ventral side, suggesting that Mad may be regulated independently of dorsal Dpp signals. Drosophila EGFR activates MAPK in a broad ventral region which corresponds to the neurogenic ectoderm. Although this study focused on the role of Wg/GSK3 on Mad regulation, the priming phosphorylation for GSK3 is provided by EGFR signaling and is critical for Mad to be polyubquitinated and degraded in the centrosome (Eivers, 2009).

Mad MGM (Mad GSK mutant; see Phosphorylation-Resistant Mad Proteins are Hyperactive), which mimics Mad receiving a maximal Wg signal, phenocopied known Wg overexpression phenotypes. In the wing, MGM caused the formation of ectopic rows of Senseless-expressing cells, sensory bristles, and entire ectopic wing margins. In larval cuticles, MGM caused the reduction of ventral denticle belts, which were replaced by naked cuticle regions, an indicator of Wg signaling. The mad gene product was demonstrated to be involved in Wg signaling in multiple in vivo assays. In the wing disc, Wg overexpression strongly increased senseless, distalless, optomotor blind, and vestigial transcripts, and co-expression of Mad RNAi inhibited this effect, without affecting Wg expression levels. The induction of ectopic neurogenic ectoderm tissue positive for SoxNeuro by RNAi was epistatic to the inhibition of SoxNeuro expression caused by Wg overexpression. Taken together, these results suggest that Mad is a required component for several Wg signaling events in Drosophila (Eivers, 2009).

Segmentation phenotypes were observed when Mad RNAi was expressed maternally using the pUASp vector. Segment fusions were generated in which larval naked cuticle was replaced by large denticles of the same type (row 5) as those seen in Wg nulls. In gain-of-function experiments, overexpression of GSK3-resistant Mad caused denticle belts to be replaced by naked cuticle, mimicking Wg signaling. Thus, depletion or overexpression of Mad generated Wg-like phenotypes, indicating that Mad functions in the Wg signaling pathway during segmental patterning (Eivers, 2009).

The MAPK pathway, which during Drosophila embryonic segmentation is regulated by EGFR activity, would decrease the duration of the Mad signal by promoting Mad polyubiquitination and degradation. The EGFR-activating genes rhomboid and spitz are activated in the anterior of each segment and Wg in the posterior border of the anterior compartment. Wg/Wnt signals would increase the duration of the signal by inhibiting GSK3 phosphorylations, generating a double gradient of GSK3 and MAPK activities that would regulate Mad stability and signaling within each segment. This may occur in a Dpp-independent fashion, but it is also possible that BMP signals might be active during larval segmentation, since the expression of the BMP receptor thickveins has a segmental pattern of expression. In addition, Dpp is expressed in the ectoderm during segmentation stages, and its promoter contains segmentation modulation elements (Eivers, 2009).

Finding a role for Mad in segmentation was remarkable, because this process has been extensively studied in Drosophila genetic screens and Mad had not been previously implicated as part of the segmentation machinery. This new role for Mad can be explained by the fact that Mad appears to also function independently of Dpp and that the Mad10 and Mad12 null alleles are nulls only for the BMP pathway. This persistence of a Mad linker regulation by phosphorylation could explain results in the literature showing that Mad10 mutant clones can result in Wg-like effects in the Drosophila wing. Overexpression of Mad12 synthetic mRNAs mutated in the GSK3 phosphorylation sites have strong posteriorizing activity in Xenopus embryos. This indicates that Mad mutants previously thought to be nulls retain BMP-independent functions (Eivers, 2009).

There were previous indications of a role for Dpp during segmentation in the Drosophila literature. It has been noted that in hypomorphic mutations of the BMP antagonist short gastrulation (sog, the homolog of Chordin), four copies of Dpp caused loss of some denticles and an increase in naked cuticle. In addition, it has been reported that in Dpp null mutants the posterior spiracles are replaced by an ectopic denticle belt. As noted in this study, Dpp nulls can present denticle belt fusions, a phenotype that has been observed previously in embryos injected with noggin mRNA. Dpp null phenotypes and those of Mad10 and Mad12 mutants (which lose C- terminal, but not linker phosphorylations) are not identical to those of Mad RNAi. It is suggested that this is because Mad also has Dpp-independent functions. Dissecting which effects of Mad are Dpp-dependent and which ones are independent will be an interesting area of future investigation. This study presents evidence for both taking place (Eivers, 2009).

Future work will have to address the level at which Mad regulation by MAPK and GSK3 interacts with other intracellular components of the Wg transduction pathway that result in similar phenotypes. The phenotypes observed for Mad loss-of function and mad phosphorylation-resistant linker mutants overexpression were very similar to those found for the armadillo/β-catenin, pangolin/lef1, legless/bcl9 and pygopus genes. The present study does not resolve the issue of whether the stabilized forms of Mad interact directly at the protein-protein binding level, thus modifying the core Wg pathway, or at level of DNA enhancers. Wnt responsive enhancers frequently contain Smad binding sites near TCF/Pangolin binding sites. In the vertebrates, direct binding between Lef1/Tcf and Smads 1 to 4 at the level of enhancer binding sites has been known for some time. What this study now shows is that Mad is also directly regulated at the level of its phosphorylation at GSK3 sites by Wg signaling. The possibility that Wg-stabilized Mad may bind to Armadillo/β-catenin, Pangolin/lef1, Legless/bcl9 and Pygopus independently of nearby Mad binding sites cannot be excluded at present. Mechanistic studies will have to explain the remarkable similarities between the stabilized Mad phenotypes and those of canonical Wg phenotypes in wing discs and bristles, segments and in neurogenic ectoderm in Drosophila, which suggest a widespread requirement for Mad in Wg signaling. Another aspect that will need to be addressed is why in Xenopus Wnt signaling through Smad1 has a complete requirement for β-Catenin, and in Drosophila the MGM can induce senseless only in regions in which β-catenin is also stabilized (Eivers, 2009).

Many developmental mechanisms have been conserved during evolution, but segmentation is one in which commonalities between Drosophila and the vertebrates have not been found. Segmentation in vertebrates relies on the cyclic oscillation of Notch pathway transcripts in the posterior paraxial mesoderm. In theory, Smad1/5/8 could provide an attractive regulator of the segmentation clock, because BMP signals have a duration of 1-2 hours in cultured cells, which can be extended by inhibiting GSK3. Wnt pathway genes cycle rhythmically in vertebrates, offering an interesting possibility for regulating Smad5/8 activity. Notch is required for segmentation in spiders, but not in Drosophila (Damen, 2007). Recently, it has been found that in the cockroach, an insect in which the segments are formed sequentially in a posterior growth zone (and not simultaneously as in Drosophila), stripes of Delta and Hairy mRNA (two genes of the Notch pathway) cycle rhythmically as in the vertebrates (Damen, 2007). This study has now found that Smad5/8 is required for the formation of segmental boundaries in Xenopus somites and that Mad is required for Drosophila segment patterning. However, the results do not establish whether similar molecular steps are affected in both organisms. The conservation of this unexpected conserved role for Mad/Smad is important from an Evo-Devo perspective because it suggests that the last common ancestor shared between Drosophila and vertebrates, Urbilateria, might have been segmented (Eivers, 2009).

These studies on Drosophila Mad have uncovered an unexpected role for Mad in the Wg signaling pathway. Mad/Smads are transcription factors that have low binding affinity for DNA and require other DNA binding proteins as co-factors in order to recognize the promoters and enhancers of hundreds of target genes. Future work will have to address how Mad or its partner Medea/Smad4 interact with proteins such as Armadillo/β-Catenin and Pangolin/Lef1 on Wnt-responsive promoters in Drosophila. The present study shows that Mad is required for Wg to signal, through its GSK3 phosphorylation sites, in a number of different in vivo assays. These include wing margin formation, sensory bristle induction in the wing, induction of the Wg induced gene senseless, the repression of neurogenic ectoderm, and segmental patterning. It is proposed that Mad serves as an integrator of patterning signals, which determine embryonic positional information. The finding that three major signaling pathways - MAPK, Wnt/GSK3 and BMP - are integrated at the level of Mad/Smad1/5/8 both in Drosophila and in the vertebrates has interesting implications for the evolution of animal forms through variations on an ancestral gene tool-kit (Eivers, 2009).

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

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. The Fused (Fu)/Smurf complex has been shown to degrade the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 2012).

In the feedback loop model to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 2012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre-CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits the proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 2012).

Preferential genome targeting of the CBP co-activator by Rel and Smad proteins in early Drosophila melanogaster embryos

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. Therefore Drosophila CBP (nejire) ChIP-seq peaks were compared with regions bound by 40 different transcription factors in early embryos, and high co-occupancy was found with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-β/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. It is concluded that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation (Holmqvist, 2012).

By comparison of CBP-bound regions in 2-4 hour old Drosophila embryos to previously mapped transcription factors, an extensive overlap of CBP peaks was found with the key activator of dorsal-ventral patterning, the Rel-family transcription factor Dorsal. The genome-wide distribution of CBP was determined in embryos where Dorsal cannot enter the nucleus (gd7 mutants), and it was found that CBP peaks that overlap regions where Dorsal, but few other factors bind in wild-type are selectively reduced in gd7 mutant embryos. Instead, strong CBP-bound regions in gd7 mutants overlap best with regions bound by the Smad protein Medea, a mediator of Dpp-signaling. Signaling by the TGF-β molecule Dpp is exceptionally sensitive to a small decline in the level of CBP in Drosophila embryos. The current results are consistent with a function for CBP in the genomic response to Dpp-signaling (Holmqvist, 2012).

Less overlap of the CBP peaks is found with mapped activators of anterior-posterior patterning such as Stat92E, Fushi-tarazu (Ftz), Paired, Caudal, and Bicoid. Previous work has indicated that CBP may function as a Bicoid co-activator. When Bicoid and CBP are expressed in S2 cells, they can interact, and Bicoid-mediated activation of reporter genes in these cells is influenced by CBP levels. 43% of the 300 strongest Bicoid-binding regions overlap a CBP peak in wild-type embryos, indicating that CBP may participate in Bicoid-mediated activation in vivo. However, many of the Bicoid peaks are found in HOT regions that bind several transcription factors. Therefore, it may not be Bicoid that targets CBP to these sites. Furthermore, although the shape of the Bicoid gradient is slightly changed in embryos from the CBP hypomorph nej1, activation of Bicoid-target genes is not compromised by the decrease in CBP levels in nej1 embryos. Consistent with a non-essential function for CBP in Bicoid-mediated activation, there is no co-occupancy of CBP and Bicoid at the known target genes hb, otd, kni, and eve. Thus, although CBP may contribute to Bicoid-mediated activation of some target genes, it seems to make a more widespread contribution to Dorsal-mediated activation. In conclusion, both genetic and genomic evidence points to a particularly important function for CBP in controlling the two key events in dorsal-ventral patterning of Drosophila embryos, the Dorsal gene regulatory network and Dpp-signaling. Perhaps CBP serves to coordinate the Dorsal and Dpp pathways in dorsal-ventral patterning (Holmqvist, 2012).

In embryos where Dorsal cannot enter the nucleus, places were found where CBP occupancy is increased, unchanged, decreased or lost. Regions that are unchanged bind several transcription factors, evident in their high HOTness, indicating that in the absence of Dorsal, other factors maintain CBP binding at these sites. Surprisingly, regions where CBP binding is increased are even HOTer, and therefore associated with even more factors in wild-type embryos. Although many CBP peaks in the genome are found where also GAF binds, the regions where CBP occupancy increases in gd7 embryos are lacking strong GAF binding, despite their high HOTness. Perhaps binding of GAF to these sites is not compatible with proper regulation of the corresponding genes. Instead, many of these regions bind Medea and Dichaete, especially the places where CBP binding is strong already in wild-type. In gd7 embryos, Dpp/Medea-regulated genes are expressed in more cells, resulting in increased CBP signal. These data indicate that also Dichaete-regulated genes are more highly expressed in gd7 mutants, and that CBP-binding therefore increases at these regions (Holmqvist, 2012).

Unexpectedly, median gene expression level of genes associated with gd7 Up regions is high in wild-type embryos. Most genes associated with these regions increase in expression even further in the absence of Dorsal, in most cases probably due to an expansion in the number of cells expressing the gene. It is therefore expected that these CBP-binding sites would be situated in promoter regions, and the increase in CBP binding a consequence of increased gene activity. However, it was found that these sites are mainly found in intronic and intergenic regions associated with H3K4me1, a mark of transcriptional enhancers. This indicates that CBP becomes recruited to these enhancers to mediate gene activation, rather than passively associating with active gene regions (Holmqvist, 2012).

CBP occupancy in gd7 embryos is reduced at regions where only few factors bind. The bigger the reduction in CBP occupancy compared to wild-type, the fewer the factors that are associated with such a region in wild-type, i.e. the lower the HOTness of the region. CBP peaks that are reduced in gd7 embryos are much more common at regions where Dorsal binds in wild-type compared to other factors, consistent with a requirement for Dorsal in targeting CBP to chromatin. Although not all of the gd7 Down CBP peaks overlap the top 300 Dorsal-binding regions, 92% overlap Dorsal when all Dorsal-binding regions are considered. Peaks where CBP is reduced in gd7 embryos are found in several known Dorsal target genes, such as twi, brk, htl, and Mef2. Furthermore, 10 of the 20 strongest Dorsal peaks overlap a region where CBP binding is reduced in gd7 embryos. Together, these data show that in early embryos, chromatin binding of CBP to many sites in the genome is dependent on Dorsal (Holmqvist, 2012).

A number of genomic regions were found where CBP occupancy in gd7 embryos is reduced to a level approaching background, the gd7 Lost regions. These regions are mostly devoid of histone modifications and occupied by very few or none of the 40 transcription factors. The factors found at these regions bind at very low levels, indicating that they may not contribute to regulation of the corresponding genes at this stage of development. Further, most genes associated with the gd7 Lost regions are expressed at very low levels or completely silent. These CBP-binding regions may therefore represent regulatory sequences that are poised for subsequent activation. Consistent with this interpretation, mean expression of the corresponding genes increases at later stages of development. Why is CBP occupancy lost from these regions in gd7 embryos? Perhaps these genes are not, and will not be expressed in the dorsal ectoderm, and are therefore not associated with CBP in gd7 mutants that convert the entire embryo into dorsal ectoderm. Alternatively, CBP binding to these regions is dependent on Dorsal. Although binding is weak, Dorsal occupies many of these regions in wild-type. It is possible that even small amounts of Dorsal is sufficient and necessary for CBP recruitment to these sites, and that CBP binding is consequently lost in the absence of Dorsal (Holmqvist, 2012).

Although CBP occupancy is reduced predominantly at Dorsal-binding regions in gd7 mutant embryos, expression of Dorsal target genes is also altered. The decrease in CBP occupancy in mutant embryos may therefore be a consequence of transcriptional inactivity, rather than a lack of recruitment by Dorsal. Indeed, CBP occupancy is on average reduced at down-regulated genes and increased at up-regulated genes. Therefore, although Dorsal and CBP occupancy often coincide, Dorsal may not directly recruit CBP to regulatory DNA sequences. However, there are also places where CBP occupancy is reduced without a corresponding change in gene expression. One such example is at the promoter of the caudal (cad) gene, which is co-occupied by Dorsal and CBP but where CBP binding is reduced more than two-fold in gd7 embryos, although the gene continues to be expressed. Furthermore, Dorsal and CBP associate in vivo. It is believed, therefore, that Dorsal may directly recruit CBP to many sites in the genome (Holmqvist, 2012).

There are also genomic sites where CBP occupancy is not dependent on either Dorsal or gene expression. Several known Dorsal target genes, including sna, neur, ind and ths, continue to associate with CBP in gd7 embryos. Although in general, HOTness is major determinant of CBP occupancy, there is no big difference in HOTness of the Dorsal target gene regions where CBP-binding is reduced (e.g., twi, htl, brk) compared to Dorsal target gene regions where CBP binding is not changed (e.g., sna, ind, ths). What maintains CBP binding on these genes in the absence of Dorsal is not clear. Presumably, other factors recruit CBP to these sites in the absence of Dorsal, but no common factor was found for the regions where CBP binding is unchanged. It is noted, however, that GAGA-factor (GAF) associates with many of the CBP-binding regions in wild-type embryos, but much less with CBP-binding regions in gd7 embryos. It is possible that GAF contributes to the recruitment of CBP to chromatin (Holmqvist, 2012).

Dorsal is converted to a repressor when it binds in proximity to AT-rich sequences, and thereby prevents expression of dorsal ectoderm target genes in the neuroectoderm and mesoderm. Consequently, these target genes, e.g. dpp, zen, and tld, are activated in all cells of gd7 mutant embryos. As expected, CBP occupancy increases at these target genes in gd7 embryos, since more cells express the genes. The Zelda protein is a maternally contributed activator of these genes. It has been previously shown that in nej1 embryos containing reduced amounts of CBP, tld expression is diminished, whereas dpp and zen expression remains unaffected. It is possible, therefore, that more activators than Zelda contribute to activation of tld, zen, and dpp in the dorsal ectoderm. Until these factors are identified, it may not be possible to explain why tld expression is particularly sensitive to a reduction in CBP amount in early embryos (Holmqvist, 2012).

When Dorsal functions as a repressor, it recruits the Groucho co-repressor. The yeast Tup1 protein, which is related to Groucho, was recently shown to block recruitment of co-activators to target genes. By contrast, it was found that CBP continues to associate with the tld and zen genes in the neuroectoderm although they are being repressed by Dorsal/Groucho. Groucho binds the histone deacetylase Rpd3 (HDAC1), which may be important for repression. Indeed, it was found that when tld and zen are repressed by Dorsal in the neuroectoderm and mesoderm, the genes are hypoacetylated despite the presence of CBP (Holmqvist, 2012).

Contrary to the general trend, some genes recruit CBP even though they are silent. Why are these genes not activated? In the cases examined, histone acetylation is low despite the presence of CBP when the genes are not expressed. Since lysine methylation and acetylation are mutually exclusive, histone methylation was measured at CBP-bound regions and it was found that Polycomb-repressed H3K27me3 chromatin is present at Dorsal-target genes in some tissues where these genes are not expressed. Although H3K27me3-decorated chromatin restricts DNA accessibility, it was found that H3K27me3-chromatin does not preclude CBP binding, but restrains histone acetylation at these CBP-bound genomic sites. Interestingly, all histone acetylations that were measured are blocked by H3K27me3-chromatin, not only the mutually exclusive H3K27ac. This indicates that despite the ability of CBP to bind to genes enclosed in H3K27me3-chromatin, the histones are not accessible for acetylation by CBP and other HATs. The data are consistent with a model for Polycomb silencing that allows access of proteins and pol II to DNA, but that restrains pol II elongation. Perhaps high levels of histone acetylation are necessary for release of pol II from the promoter, for example by recruiting the bromodomain protein Brd4 that brings in the P-TEFb kinase to phosphorylate pol II (Holmqvist, 2012).

In cells depleted of CBP and p300, global levels of H3K18ac and H3K27ac are greatly diminished whereas other histone acetylations remain unaffected, suggesting that these are in vivo targets of CBP acetylation. CBP can also acetylate H3K56, which occurs in response to DNA damage. It was found that H3K18ac and H3K27ac levels do not always correlate with changes in CBP occupancy at Dorsal target genes, although H3K18ac levels are most similar to CBP abundance. In part, this can be explained by the presence of H3K27me3-chromatin, that precludes histone acetylation. However, in the neuroectoderm (Tollrm9/rm10 embryos), the twi promoter contains less histone acetylation than in the dorsal ectoderm (gd7 embryos) although H3K27me3 levels are reduced and CBP binding not decreased compared to dorsal ectoderm. Together, these results show that CBP's HAT activity is regulated by substrate availability, but that it may also be regulated by genomic context or signaling (Holmqvist, 2012).

Genome occupancy of CBP/p300 and H3K4me1 can be used to predict cis-regulatory DNA sequences. However, what fraction of regulatory sequences that can be identified in this way is not known. It was found that CBP binding to many known enhancer sequences that are active in early embryos is below the cut-off for high-confidence peaks, although average CBP occupancy was determined to be 1.73 times the genomic background at 97 previously described early embryonic enhancers. The results also show that CBP binding differs greatly between wild-type and mutant embryos, and that some gene regulatory networks rely on CBP to a much larger extent than others. Together, these results suggest that although CBP/p300 binding can be used to successfully identify transcriptional regulatory sequences, many enhancer sequences will be missed because they are not bound by CBP/p300 or bound at levels below criteria for high-confidence peaks. Even though mapping CBP/p300 binding in different cell-types will increase the number of putative regulatory sequences, it is anticipated that a substantial number of enhancers will require alternative strategies for their identification, e.g. genome occupancy of other HATs (Holmqvist, 2012).

In conclusion, this study shows that association of CBP with the genome is dependent on the number and types of transcription factors that bind the DNA sequence, that CBP preferentially associates with some gene regulatory networks, that CBP binding correlates with gene activity, but that CBP also binds silent genes without causing histone hyperacetylation (Holmqvist, 2012).

R-Smad competition controls activin receptor output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, 'BMP-specific' Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, this study found that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, it was found that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. It is proposed that the dual activity of TGF-β/Activin receptors is an ancient feature, and several ways this activity can modulate TGF-β signaling output are discussed (Peterson, 2012).

This report presents experimental results showing that the Baboon receptor can directly phosphorylate Mad in cell culture and in vivo, and that this cross-talk activity is tightly controlled by the availability of dSmad2. These findings extend the initial report describing canonical signaling between Baboon and dSmad2 where dSmad2, but not Mad, was shown to be a substrate of Baboon in mammalian cells. There are several possible reasons why Baboon-Mad activity is observed in Drosophila cells but was missed in the initial report. First, it is possible that Mad binding to Baboon may be too weak or transient to be detected by immunoprecipitation. Alternatively, endogenous Smad2/3 may have blocked the interaction in heterologous cell systems, or species-specific co-factors may facilitate Mad-Baboon binding (Peterson, 2012).

The strong functional conservation of TGF-β superfamily proteins prompts a comparison of the current results in Drosophila with studies describing cross-pathway signaling in mammalian systems. The limited number of pathway members and the efficacy of RNAi in Drosophila enabled distinguishing between competing models presented for mammalian epithelial cells. With regard to the key mechanistic question of whether TGF-β Type I receptors can directly phosphorylate the BMP R-Smads, the observation of Mad phosphorylation in the absence of BMP Type I receptors is inconsistent with the model of heteromeric TGF-β/BMP Type I receptor complexes proposed in a previous study, but is consistent with the model of direct phosphorylation of BMP R-Smads by TGF-β/Activin receptors. It is possible that both types of mechanisms exist, but are differentially utilized depending on the ligands and receptors present in the particular tissue being examined. Even in Drosophila, it is noteed that direct action of Baboon on Mad does not preclude the possibility that mixed receptors form active signaling complexes in some situations. Mixed receptor complexes have been detected in Drosophila cell culture under over-expression conditions, but their functionality is unknown. A similar point can be made regarding mixed Smad oligomers detected in mammalian epithelial cells. In adult wing assay, this study found that the Babo* phenotype depended primarily on Mad and that dSmad2 was not required. If the wing development defect was caused by the activity of a complex containing both P-Mad and P-dSmad2, then removal of either one should have blocked the Babo* phenotype. Again, this observation does not argue against the formation or activity of mixed R-Smads complexes in some contexts, but shows that productive signaling by cross-pathway phosphorylation can take place independently of such complexes (Peterson, 2012).

One key observation in this report concerning the mechanism of cross-pathway signal regulation is that the degree to which it occurs, both in cell culture and in vivo, appears to be regulated by competition between the R-Smads, likely for receptor binding. Further work is required to determine how general this mechanism might be. Epithelial cell culture models showed that cross-talk is important for the TGF-β-induced migratory switch. The results in the larval wing disc and gut represent the first examples of cross-talk in vivo, and it is expected that additional examples will be found in various animals and tissues. With regard to developmental studies, other systems should be evaluated to see if loss-of-function mutations of Smad2/3 orthologs lead to increased signaling through BMP R-Smads. Additionally, several human diseases have been attributed to mutations in TGF-β components. A cautionary implication of this work is that mutations in the TGF-β branch may have unanticipated loss- or gain-of-function influences on the BMP branch (Peterson, 2012).

Curiously, TGF-β/Activin Type I receptors appear to have gained or retained cross-phosphorylation activity throughout evolution, but BMP Type I receptors do not appear to have reciprocal activity. Phosphorylation of dSmad2 by activated Drosophila Type I BMP receptors, or in response to various ligands, has never been seen. Likewise, no phosphorylation of Smad2/3 by BMP receptors has been reported in vertebrate cells. Why is there this distinction? The growing number of complete genome sequences has allowed phylogenetic reconstruction of the evolution of TGF-β signaling. Apparently all metazoans, even the simple placazoan, have a complex TGF-β network containing both TGF-β/Activin and BMP subfamilies. It is tempting to speculate that a single receptor with dual Smad targets could have played a transitional role in the expansion of the signaling network. The all-or-none nature of the TGF-β-superfamily network (both BMPs and TGF-β/Activins present, or none) in extant organisms, however, does not provide support for this idea. What it does support is the possibility that relationships between core pathway proteins stabilized hundreds of millions of years ago. If the dual-kinase activity of Baboon orthologs has been available to all metazoans during the radiation of animal forms, this activity would be expected to be deployed by different animals and different cell types in diverse ways. Several phenotypes are currently being investigated that differ between baboon and dSmad2 mutants to determine which depend on Babo-Mad cross-talk and which might depend on other forms of Smad-independent signaling (Peterson, 2012).

The different responses of adult wings and larval imaginal tissue upon Baboon activation illustrate that TGF-β pathway wiring and output can vary with developmental context. Given the relationship between Babo and dSmad2, the cross-talk activity can be viewed two ways. From one perspective, the response to loss of dSmad2 depends on the level of Baboon signaling: only cells with Baboon activity can produce P-Mad by cross-talk. From the other perspective, the response of wildtype cells to Baboon stimulation depends on dSmad2 levels: efficient cross-talk will only occur in the absence of dSmad2 (Peterson, 2012).

Given the dominant control that dSmad2 exerts on the ability of Baboon to phosphorylate Mad, the simplest model is that output of TGF-β ligand stimulus in a given cell depends on the expression level of dSmad2. Although dSmad2 is widely expressed, as are Smad2/3 proteins in other animals, different subsets of cells might express different ratios of R-Smads that could influence the signaling output (Peterson, 2012).

The observation that Baboon activity can lower the overall level of dSmad2 protein offers an additional regulatory possibility, where the TGF-β/Activin signal itself can influence its response. In the substrate-switch model, a cell exposed to a prolonged Activin signal would eventually degrade enough of its dSmad2 pool to allow Baboon signaling through Mad. It is not known which tissues, if any, require Baboon-to-Mad signaling in normal developmental contexts. In some cases, dSmad2 over-expression leads to mad loss-of-function phenotypes, which could represent disruption of a sensor incorporating dSmad2 concentration as an input and Mad activity as an output. Proteosomal degradation of activated Smad proteins is a well documented mechanism of signal attenuation, but the current observation of bulk degradation adds a new dimension because it can redirect receptor signaling. The mechanism of signal-dependent bulk dSmad2 degradation is unknown, and preliminary experiments do not support a simple ubiquitylation-proteosomal pathway. Regardless of the molecular details, the observed reduction of total dSmad2 available for receptor competition is the key parameter in the competition model. Since activated R-Smad proteins can also be dephosphorylated to rejoin the pool of would-be substrates, the relative rates of recycling and bulk degradation would be predicted to influence the substrate switch to Mad (Peterson, 2012).

What could be gained by the substrate switch? A general answer is that multiple interactions permit diverse regulatory schemes. For example, cross-talk would permit Mad signaling in a cell that is not exposed to BMP ligands or is otherwise not competent to transduce such signals. Other modes of signal integration are intriguing, such as the conditional formation of mixed R-Smad complexes. Another type of regulatory link that could contribute to the evolutionary maintenance of an integrated dSmad2-Babo-Mad triad considered. If dSmad2 transcription were a target for P-Mad, this would affect the substrate switch. In once case, Baboon signaling to Mad would be triggered by dSmad2 depletion and serve to up-regulate Smad2 to return balance to the system. In contrast, if dSmad2 were down-regulated by P-Mad, this would stabilize the switch to the Baboon-Mad interaction. Additional studies are required to explore these intriguing possibilities (Peterson, 2012).

Drosophila Ortholog of Acyl-CoA Synthetase Long-Chain Family Member 3 and 4, Inhibits Synapse Growth by Attenuating Bone Morphogenetic Protein Signaling via Endocytic Recycling

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. This study reports that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated Mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, these results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation (Liu, 2014).

Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues

The BMP ligand Dpp, operates as a long range morphogen to control many important functions during Drosophila development from tissue patterning to growth. The BMP signal is transduced intracellularly via C-terminal phosphorylation of the BMP transcription factor Mad, which forms an activity gradient in developing embryonic tissues. This study shows that Cyclin dependent kinase 8 and Shaggy phosphorylate three Mad linker serines. Linker phosphorylations control the peak intensity and range of the BMP signal across rapidly developing embryonic tissues. Shaggy knockdown broadened the range of the BMP-activity gradient and increased high threshold target gene expression in the early embryo, while expression of a Mad linker mutant in the wing disc resulted in enhanced levels of C-terminally phosphorylated Mad, a 30% increase in wing tissue, and elevated BMP target genes. In conclusion, these results describe how Mad linker phosphorylations work to control the peak intensity and range of the BMP signal in rapidly developing Drosophila tissues (Aleman, 2014: PubMed).

Ter94/VCP is a novel component involved in BMP signaling

Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-beta family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating Decapentaplegic (Dpp)/BMP signals. Taken together, these data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling (Zeng, 2014: PubMed).

CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster

The transforming growth factor beta (TGF-beta) and bone morphogenic protein (BMP) pathways transduce extracellular signals into tissue-specific transcriptional responses. During this process, signaling effector Smad proteins translocate into the nucleus to direct changes in transcription, but how and where they localize to DNA remain important questions. This study has mapped Drosophila TGF-beta signaling factors Mad, dSmad2, Medea and Schnurri genome-wide in Kc cells and find that numerous sites for these factors overlap with the architectural protein CTCF Depletion of CTCF by RNAi results in the disappearance of a subset of Smad sites, suggesting Smad proteins localize to CTCF binding sites in a CTCF-dependent manner. Sensitive Smad binding sites are enriched at low occupancy CTCF peaks within topological domains, rather than at the physical domain boundaries where CTCF may function as an insulator. In response to Decapentaplegic, CTCF binding is not significantly altered, whereas Mad, Medea, and Schnurri are redirected from CTCF to non-CTCF binding sites. These results suggest that CTCF participates in the recruitment of Smad proteins to a subset of genomic sites and in the redistribution of these proteins in response to BMP signaling (Van Bortle, 2015).

TGF-β effector proteins have been shown to co-localize with mammalian CTCF in a CTCF-dependent manner at just 2 individual loci. This observation has been extended to Drosophila using a genome-wide approach, providing evidence that architectural protein CTCF and canonical Smad signaling proteins, both highly conserved from fly to humans, co-localize on a global scale. Context-specific features were uncovered in which Smad localization is dependent or independent of CTCF binding. Interestingly, genome-wide analysis identifies Mad, dSmad2, Medea, and Schnurri binding to previously characterized response elements even in the absence of DPP ligand, in which levels of phosphorylated Mad are undetectable. This signal-independent clustering of signaling proteins suggests that the genomic TGF-β signaling response is not as simple as regulating binary 'off vs. on' states, dependent on phosphorylated Mad. However, attempts to map the genomic landscape of phosphorylated-Mad before and after DPP stimulation were unsuccessful, likely due to issues with currently available p-Mad antibodies. Though it was not possible to determine the role of phosphorylation as a determinant in Mad localization, it is conceivable that phosphorylation of Mad might play a role in regulating the resident time of DNA-binding, the recruitment of additional regulatory partners, or the ability to establish functional long-range interactions (Van Bortle, 2015).

Smad co-binding at dCTCF sites is sensitive to dCTCF depletion at low occupancy dCTCF target sequences for which Smad consensus sequences are depleted, whereas high occupancy dCTCF binding sites co-bound by additional architectural proteins remain unaffected. The dCTCF-independent recruitment of Smads to high occupancy APBSs suggests that additional architectural proteins may redundantly recruit Smads, or simply provide an accessible chromatin landscape to which Mad, Medea, and dSmad2 can associate. Nevertheless, dCTCF-dependent localization of Smad proteins to specific low occupancy elements is consistent with the CTCF-dependent nature of Smad binding at both the APP and H19 promoters in humans. It is speculated that dCTCF-dependent Smad localization to low occupancy APBSs within topological domains may represent regulatory elements involved in enhancer-promoter interactions, whereas dCTCF-independent high occupancy APBSs are involved in establishing higher-order chromosome organization. What role Smads might play in establishing or maintaining such long-range interactions relevant to chromosome architecture, or whether Smads and other transcription factors simply localize to high occupancy APBSs due to chromatin accessibility, remains difficult to address. However, it has been recently shown that high occupancy APBSs are distinct from analogous transcription factor hotspots, suggesting some level of specificity, most likely governed by protein-protein interactions, decides which factors can associate and where. Alternatively, the enrichment of ChIP-seq signal at high occupancy APBSs may, to some degree, reflect indirect association via long-range interactions with regulatory elements directly bound by Smad proteins. This possibility raises a potential explanation for why Smad ChIP signal is independent of dCTCF binding at high occupancy APBSs (Van Bortle, 2015).

Surprisingly, DPP-activated phosphorylation of Mad does not lead to significant changes in dCTCF binding, whereas Mad, Medea, and Schnurri levels increase at regulatory elements away from dCTCF. These results suggest that TGF-β signaling in Kc167 cells redirects Smad binding to genomic loci independent of architectural proteins, and that architectural proteins may facilitate binding of nuclear Smad proteins in the absence of signaling. The complete loss of Smad ChIP signal at numerous dCTCF binding sites enriched for the core dCTCF consensus sequence nevertheless provides compelling evidence that recruitment of Smad proteins is directly governed by Drosophila CTCF at a subset of binding sites. These results establish CTCF as an important determinant of Smad localization and, depending on the cell-type specific binding patterns of CTCF, suggest that CTCF might also influence the tissue-specific localization of Smad proteins analogous to master regulatory transcription factors in multi-potent stem cells (Van Bortle, 2015).

Mothers against dpp: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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