Protein Interactions

Thickveins binds DPP protein and vertebrate bone morphogenetic protein 2 with high affinity (Penton, 1994).

Axis formation in the Drosophila wing depends on the localized expression of the secreted signaling molecule Decapentaplegic (Dpp). Dpp acts directly at a distance to specify discrete spatial domains, suggesting that it functions as a morphogen. Expression levels of the Dpp receptor thick veins (tkv) are not uniform along the anterior-posterior axis of the wing imaginal disc. tkv is expressed at low levels in the center of the disc and at higher levels toward the edges of the disc. Although tkv levels are low in the center of the disc, clonal analysis has shown that tkv activity is stringently required in this region for growth and for target gene expression. Receptor levels are low where Dpp induces its targets Spalt and Omb in the wing pouch. Receptor levels increase in cells farther from the source of Dpp in the lateral regions of the disc (Lecuit, 1998).

Evidence is presented that Dpp signaling negatively regulates tkv expression and that the level of the receptor influences the effective range of the Dpp gradient. High levels of tkv sensitize cells to low levels of Dpp and also appear to limit the movement of Dpp outside the wing pouch. Thus receptor levels help to shape the Dpp gradient. To answer whether Dpp signaling regulates tkv expression an examination was made of the effects of clones of cells expressing Dpp at lateral positions in the disc, where the level of tkv is normally high. Dpp-expressing clones were marked indirectly by their ability to induce ectopic Spalt expression. tkv transcript levels are reduced where Spalt is misexpressed, suggesting that Dpp can act at a distance to repress tkv expression These results suggest that the reduced levels of tkv transcript in the center of the disc are due to downregulation by Dpp acting at a distance (Lecuit, 1998).

Are the reduced levels of the Tkv expression important for the formation of the Dpp activity gradient? To address this, an examination was made of the effects on the expression of the Dpp-target genes Spalt and Omb in clones of cells that overexpress wild-type Tkv. Tkv-expressing clones well inside the endogenous domains show little effect on either Spalt or Omb expression. Clones near the edge of the endogenous Spalt domain show increased Spalt expression and those near the edge of the Omb domain show elevated Omb expression. Tkv-expressing clones located outside but near the endogenous Spalt domain show ectopic induction of Spalt. Clones located farther from the Spalt domain do not show ectopic activation of Spalt. Together, these observations suggest that overexpressing Tkv can increase the sensitivity of cells to low levels of Dpp (Lecuit, 1998).

Tkv was overexpressed to assess the consequences of broadly elevating Tkv expression levels in the central region of the disc. Wings with elevated Tkv expression are reduced in size. The effect is stronger in the posterior compartment, with the region between veins 4 and 5 being more reduced than the region between veins 2 and 3. The region between veins 3 and 4 is relatively normal, possibly because the size of this intervein region is specified directly by Hedgehog, not by Dpp. These observations suggest that the long-range activity of Dpp in the vein 2-3 and 4-5 regions is compromised by overexpression of the Dpp receptor. Overexpressing Tkv strongly reduces the size of the Spalt domain in the Posterior (P) compartment. The effect on Spalt expression is much stronger in the P compartment than in the A compartment and the Spalt domains in both compartments appear to be less graded at their edges than in wild type. The effective range of the Dpp activity gradient appears to be limited to a few cells in the P compartment in mid- and late-third instar discs. This suggests that overexpression of receptor can limit the spread of Dpp in the P compartment. These observations suggest that high levels of the receptor might sequester ligand and limit its movement across the wing disc. The difference observe between A and P compartments when Tkv is overexpressed probably reflects the fact that cells originating in the Dpp expression domain can contribute to formation of a large part of the anterior compartment, but not to the posterior compartment. Thus cells originating in the Dpp domain could ‘carry’ Dpp protein away from the source as they and their progeny are displaced by addition of new cells (the displacement process can be directly visualized by lineage tracing cells originating in the dpp-expression domain). It is concluded that artificially high levels of Thick veins outside the wing pouch appear to limit the spread of Dpp and thereby modulate the shape of the ligand gradient. In addition, the level of Tkv expression modulates the sensitivity of cells to Dpp. Thus regulation of receptor levels by Dpp modulates the shape of the Dpp gradient (Lecuit, 1998).

Midway through embryogenesis, decapentaplegic is expressed in the visceral mesoderm, and enhances the expression of labial in the underlying midgut endoderm. Earlier in development, however, dpp expression is limited to the dorsal ectoderm. At this stage in development, thickveins is expressed in the mesoderm. This suggests that ectodermal DPP might not only be required for development of dorsal ectoderm, but could also act inductively to mediate pattern formation in the underlying mesoderm. By expressing dpp ectopically in the ectoderm and mesoderm and by examining dpp null mutant embryos, it has been shown that DPP regulates expression of mesodermal genes by way of Thickveins (Staehling-Hampton, 1994).

Drosophila punt gene encodes atr-II, a type II receptor that on its own is able to bind activin but not BMP2, a vertebrate ortholog of DPP. Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. Furthermore, Punt will bind BMP2 in concert with TKV or SAX, forming complexes with these receptors. It has been suggested that Punt functions as a type II receptor for DPP and has been proposed that BMP signaling in vertebrates may also involve sharing of type II receptors by diverse ligands (Letsou, 1995).

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).

The immunophilin FKBP12 binds to the cytoplasmic domain of TGFß type I receptors and is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with immunophilin FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGFß family ligands. Overexpression of FKBP12 specifically inhibits pathways activated by TGFß, and point mutations in FKBP12 abolish its inhibitory activity. FKBP12 functions as an immunosuppressive in vertebrates by binding macrolides FK506 and rapamycin and recruiting and thereby inactivating calcineurin and the serine kinase FRAP, respectively, resulting in the blockage of the signaling pathways mediated by calcineurin or FRAP. Since calcineurin is a serine/threonine phosphatase while type I receptors are serine/threonine kinases, and phosphorylation of the type I receptor as well as its downstream substrates is essential for signaling via the type I receptor, one plausible mechanism is proposed for FKBP12 action whereby calcineurin could inhibit type I signaling activity by dephosphorylating type I receptor or its bound substrates. A novel cDNA that is 66% identical to mouse FKBP12 was isolated as the predominant interactor for Drosophila type I receptor Thickveins. FKBP12 interacts with Saxophone as well (Wang, 1996).

Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. A method for identifying genes expressed in the follicle cells with potential roles in axis formation is described. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from pilot screens include (1) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers; (2) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (3) the identification of a Drosophila homolog of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers (Bryant, 1999).

FKBP-12 has been shown to bind to TGF-beta type I receptors and has been proposed, on the basis of cell culture studies, to act as a regulated inhibitor of TGF-beta type I signaling. The Drosophila TGF-beta homolog dpp is expressed in anterior follicle cells and is required for the formation of anterior chorion structures. Follicle cell patterning may provide an instructive system in which to study the interactions between TGF-beta signaling, FKBP-12-like proteins, and the Egfr pathway, which is required for the induction of the FKBP-12 homolog and other genes in the dorsal anterior follicle cells. Consistent with a role for the FKBP-12 homolog in modulating dpp signaling, preliminary overexpression studies show defects in anterior chorion structures (Bryant, 1999).

Members of the transforming growth factor-beta superfamily bind to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptors act as downstream components of type II receptors in the receptor complexes. Therefore, intracellular proteins that interact with the type I receptors are likely to play important roles in signaling. Drosophila inhibitor of apoptosis (Diap) 1 has been identified as an interacting protein of Thick veins (Tkv), a Dpp type I receptor. Diap1 associates with Tkv in vivo. The binding region in Diap1 is mapped to its C-terminal RING finger region. Diap2, another Drosophila member of the inhibitor of apoptosis protein family, also interacts with Tkv in vivo. These data suggest that Diap1 and Diap2 may be involved, possibly as negative regulators, in the Dpp signaling pathway, which leads to cell apoptosis. Tkv may induce apoptosis by suppressing the DIAP1 function (Oeda, 1998).

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGF beta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties that generate distinct cell fates along the A/P axis. Dpp expression and Dpp signaling have been monitored in endocytosis-mutant wing imaginal discs that develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion does not result in a concommittant expansion of the functional range of Dpp activity but rather, results in its reduction, as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells (Gonzalez-Gaitan, 1999).

Mutations in the Drosophila alpha-adaptin gene (DAda) disrupt clathrin-mediated endocytosis prior to vesicle formation at the cell membrane. Embryos that are homozygous for a lack-of-function allele, such as DAda3, develop into normal looking larvae that die while still in their eggshells. alpha-adaptin is also expressed at high levels at the plasma membrane of developing wing imaginal disc cells during larval stages. To address a possible role for alpha-adaptin during wing development, a hypomorphic allele, D-Ada4, was generated to overcome embryonic lethality. The strongest non-lethal allelic combination, D-Ada3/D-Ada4, causes a temperature-dependent wing phenotype. At 18 degrees C, the mutant wings are normal. At 25 degrees C, wings are reduced in size and show vein pattern defects along the A/P axis. At 29 degrees C, only wing remnants with strongly enhanced pattern defects along the A/P axis are observed. Such remnants develop diagnostic dorsoventral pattern elements, such as sensilla campaniformia on the hinge and the dorsal surface of the wing blade, the dorsal and ventral hairs of the wing margin triple row, and specific dorsoventral aspect of the veins. Thus, no discernible dorsoventral wing pattern defects were found. The mutant pattern formation along the A/P axis of the endocytosis-mutant wings is affected in a manner similar to hypomorphic decapentaplegic mutants (Gonzalez-Gaitan, 1999).

It was next asked whether wing pattern defects are also observed when clathrin-mediated endocytosis is impaired by double mutant combinations as has been shown for mutants where alpha-adaptin and dynamin activities are jointly reduced. In double heterozygous mutants for clathrin heavy-chain (D-Chc) and alpha-adaptin, wings develop a temperature-dependent phenotype. At 25 degrees C and 29 degrees C, the A/P pattern defects of D-Chc/1;DAda3/1 mutant wings resemble those observed with DAda mutant wings. Furthermore, such wings developed at 18 degrees C a thickened posterior cross-vein similar to mutants of the Dpp receptor thick veins (tkv). The dpp- and tkv-like phenotypes obtained with the endocytosis-mutant combinations are consistent with the proposal that clathrin-mediated endocytosis is necessary for proper Dpp action during wing development (Gonzalez-Gaitan, 1999).

The conclusions drawn from the mutant phenotype are consistent with the finding that despite the enlarged dpp expression domain found in endocytosis impaired mutants, the range of sal-activating Dpp activity is significantly reduced to 3±4 cell diameters from the source of the signal. Recent results suggest that gradient formation and long-range signaling by secreted signaling proteins such as Dpp, Hedgehog and Wingless are modulated by regulatory feedback loops involving the receptors of these genes. Here, Dpp acts like Wingless: it negatively regulates the expression of its receptor Tkv. Since endocytosis has been shown to be a prerequisite for receptor clearance at the cell membrane, and in view of the genetic interactions between clathrin and the Dpp receptors Tkv and Put shown here, it is possible, among other explanations, that impaired endocytosis interferes with Dpp receptor levels and/or the formation of the Dpp gradient, as well as with the need to recycle receptors in order to keep signaling working effectively. Increased Tkv is likely to sequester free Dpp and thereby hinders Dpp migration, resulting in an altered shape of the Dpp gradient. This genetic link between the Dpp receptors and clathrin suggests that a process involving receptor-mediated endocytosis might participate in mediating Dpp action over distance, extending its functional range beyond some 4 cell diameters. However, the results obtained with double mutant wing discs do not distinguish between a signaling defect, a transport defect or unrelated defects such as the need to recycle receptors to maintain effective signaling (Gonzalez-Gaitan, 1999).

Mosaic analysis carried out with endocytosis-deficient wing disc cells establishes that the reception of the Dpp signal is not dependent on endocytotic events. This is clearly shown by the fact that the endocytosis-deficient cells express sal normally, whereas cell clones of comparable size lacking the Dpp receptor Tkv, which disrupts signal reception, fail to express sal. Furthermore, the results establish that the intracellular processing of Dpp signal between the activated receptors and the nuclear factor(s) required to activate the target gene sal is not dependant on clathrin-mediated endocytosis, as has been reported for Egf signaling. This leaves the possibility that impaired endocytosis affects the secretion or the propagation of the Dpp signal over distance, for example by transcytosis, or that both processes are affected at the same time. Once Dpp antibodies or functional Dpp-GFP fusions are available to visualize the Dpp gradient and the subcellular distribution of Dpp directly, these question can be addressed in the mutant combinations described here (Gonzalez-Gaitan, 1999).

Hrs mediates downregulation of Thickveins in Drosophila

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).

Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homolog, Vps27p, also have a UIM and bind to ubiquitin (Bilodeau, 2002; Lloyd, 2002; Polo, 2002; Raiborg, 2002). The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Bilodeau, 2002; Raiborg, 2002; Jékely, 2003 and references therein).

To determine whether Hrs is generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the Drosophila ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation at the cell cortex could also be observed. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).

An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27 (Piper, 1995). Genetic studies in mice and Drosophila have also shown that cells mutant for hrs have enlarged endosomes (Komada, 1997; Lloyd, 2002), possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes (Gaullier, 1998; Christoforidis, 1999), and can also be used to specifically label endosomes (Wucherpfennig, 2003). The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells is compared, an enlargement of FYVE-positive structures is observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).

Hrs affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs that were analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures were also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).

To test whether the requirement for Hrs was limited to RTKs, other types of signalling receptors were analysed. The Hedgehog receptor Patched and the Hedgehog signal transducer Smoothened are multi- and seven-pass transmembrane proteins, respectively. Thickveins (Tkv) is a type-I serine-threonine kinase receptor for the TGF-ß family ligand Dpp. Notch is a single-pass transmembrane protein that undergoes specific proteolytic cleavage upon activation. Interestingly, hrs mutant follicle cells show a marked accumulation of each of these receptors. As for RTKs, most of the receptor molecules accumulate intracellularly and show significant colocalization with the ubiquitinated protein signal. Thus, Hrs has a general role in regulating the sorting and degradation of diverse classes of signalling receptors. The homotypic adhesion molecule Shotgun is not affected visibly in hrs mutant cells. The latter observation is in agreement with observations that nonsignalling transmembrane proteins are not upregulated in hrs mutant animals (Lloyd, 2002). Either the trafficking of these proteins is independent of Hrs function or they have a low turnover rate in the examined tissues (Jékely, 2003).

The high degree of overlap between the signal for each of the receptors and the signal for ubiquitinated proteins means that the receptors accumulate in roughly the same endosomal compartment. This, together with the increase in receptor levels in hrs mutant cells, suggests that these receptors are degraded through the same Hrs-dependent pathway. Ubiquitination of the inhibitory Smad7 by the E3 ubiquitin ligase Smurf2 has been shown to target the Smad7-TGF-ß receptor complex for lysosomal degradation. In follicle cells, a similar complex may be sorted for degradation in an Hrs-dependent manner. It has been argued that the turnover of Hedgehog receptors is strongly regulated and may be critical for signalling, but a role of ubiquitination in this event has not been reported. The observation that Patched and Smoothened accumulate in compartments highly enriched in ubiquitinated proteins in hrs mutant cells suggests that trafficking of Patched and Smoothened is also regulated by ubiquitination (Jékely, 2003).

When analysing hrs mutant clones, an increase of ubiquitinated proteins at the cell cortex was occasionally noticed in addition to the intracellular accumulation. Some cortical accumulation could also be observed directly for the signalling receptors, in particular for Tkv. This accumulation could be due to inefficient endocytosis from the plasma membrane or increased recycling of endocytosed proteins. Hrs does not appear to be required directly for endocytosis (Lloyd, 2002), but downstream defects may 'clog up' the endocytosis machinery. Hrs can also affect receptor recycling. Overexpression of Hrs in tissue culture cells increases the retention of ubiquitinated transferrin receptors (Raiborg, 2002). The strong intracellular accumulation of receptors in hrs mutant cells could therefore either be due to defective sorting towards lysosomal degradation or due to defective post-endocytic retention, a concomitant general increase in the steady-state levels of the receptors at the plasma membrane, and therefore in endosomes. The first explanation is favored because increased surface levels of receptors or ubiquitinated proteins were often not detected even when strong intracellular accumulation was evident. Receptors therefore seem to be retained intracellularly, rather than recycled, in hrs mutant cells. Hrs is most likely not the only factor responsible for the post-endocytic retention of receptors. Redundancy in sorting to the vacuole has been reported for the yeast alpha-factor receptor Ste3p. In this case, Vps27p and Hse1 have overlapping roles to sort Ste3p to the vacuolar lumen (Bilodeau, 2002; Jékely, 2003).

Hrs has been suggested to play a critical positive role in TGF-ß signal transduction in mice by stimulating the recruitment of Smad2 to the receptor (Miura, 2000). A test was performed to see whether Hrs is required for TGF-ß/Dpp signalling in Drosophila and thus might serve a conserved role in this pathway. In the egg chamber, Dpp is expressed in the anterior follicle cells and contributes to the patterning of the follicular epithelium. The receptor Tkv is expressed uniformly in the epithelium. Active signalling downstream of the receptor can be monitored by the presence of the phosphorylated form of MAD (P-MAD) in the nucleus (anti-P-MAD). In wild-type stage 10 egg chambers, P-MAD is detected in the Dpp-producing anterior follicle cells and 1-2 rows of follicle cells immediately adjacent to the source. In hrs mutant follicle cells close to the Dpp ligand source, MAD phosphorylation and nuclear translocation still occurs efficiently. Thus, Hrs is not required for Dpp signalling in this context (Jékely, 2003).

The P-MAD expression domain is expanded to 3-4 rows of follicle cells if the epithelium is mutant for hrs. The P-MAD signal is graded, indicating that signalling is still dependent on the Dpp ligand gradient. Apparently, the hrs mutant follicle cells have increased sensitivity to Dpp. Since a higher level of Tkv protein is known to sensitize cells to low levels of Dpp, the expansion of the P-MAD domain can most simply be explained by the increased amount of Tkv at the surface of hrs mutant cells (Jékely, 2003).

The effect of Hrs on Dpp target gene activation was determined. The wing disc was used for this analysis since Dpp signalling and target gene activation are well characterized. Dpp is expressed in the middle of the wing disc (at the anterior-posterior (A-P) boundary) and forms a morphogen gradient. Spalt, a target of Dpp signalling, is expressed in a characteristic band at both sides of the A-P boundary. hrs mutant patches within the endogenous Splat domain show a slight increase in Spalt levels. When hrs clones are located at the edge of the Spalt domain, a modest expansion of the expression is observed. Thus, Hrs is not required for Dpp target gene activation in the wing disc. Instead, Hrs has a slightly negative effect on the pathway. As for the P-MAD signal in follicle cells, the Spalt signal is still graded in hrs mutant clones and no ectopic Spalt expression was observed in hrs mutant cells far from the Dpp source. This indicates that Spalt activation is still dependent on endogenous Dpp. The effects of hrs appear to be cell autonomous and positive in all parts of the Spalt expression domain, suggesting that hrs mutant wing disc cells are simply more sensitive to Dpp (Jékely, 2003).

Hrs mutant mouse embryonic cells show dramatically decreased responses to TGF-ß stimulation (Miura, 2000). Hrs was not required for Dpp signalling in Drosophila wing disc cells and ovarian follicle cells. The difference between these results may reflect an acquired aspect of TGF-ß pathway regulation in mammals or a specific regulation in mouse embryonic stem cells. However, it is clear that Hrs does not play a conserved general role in this otherwise quite conserved signalling pathway (Jékely, 2003).

To analyse the importance of ligand stimulation for Hrs-dependent downregulation of receptors, Tkv accumulation in hrs mutant cells was compared close to, and far from, the endogenous Dpp source. Follicle cells that allow a clear detection of intracellular as well as cortical Tkv staining were examined. There could be other TGF-ß-related ligands in the ovary, but the P-MAD staining indicates that the only significant source of signal stimulating this pathway comes from the anterior. Interestingly, high levels of Tkv accumulation are observed even in those hrs mutant follicle cells that are farthest from the Dpp source (100-120 micrometers away), experiencing no or very little Dpp ligand and signalling (no MAD phosphorylation). Tkv accumulation is apparently uniform in all hrs mutant follicle cells, that is, cells closest and farthest from the Dpp source accumulate similar amounts of the receptor. These observations indicate that the bulk of Hrs-dependent downregulation of Tkv is constitutive in these cells, independent of ligand. This does not rule out the possibility that ligand-induced endocytosis also occurs. In the follicular epithelium, the spread of Dpp may be limited to a few cell diameters by high levels of receptor. It is possible that only a small fraction of the receptor molecules bind Dpp ligand. In this case, given the high rate of constitutive receptor turnover, stimulation would not affect visibly the bulk of receptor trafficking (Jékely, 2003).

The bulk of Hrs-dependent downregulation of other signalling receptors appears to be constitutive as well. Hedgehog acts very early in egg chamber development, and patched-lacZ, which reflects Hedgehog activity, has very restricted expression. Downregulation of Patched in the stage 10 egg chamber should therefore be ligand independent. Smoothened protein is, in turn, controlled by Patched. EGFR ligands are highly enriched and active at the dorsal side of the egg chamber, whereas a PVR ligand is present throughout the oocyte. However, for both receptors, the level of receptor accumulation in hrs mutant cells is similar throughout the follicular epithelium. Signal-induced endocytosis is well established for acute stimulation of signalling receptors, in particular RTKs. Yet signalling does not appear to control the bulk of receptor turnover in follicle cells. The physiological levels of stimulatory ligands may be relatively low compared to the levels used for acute stimulation experiments (Jékely, 2003).

Precise regulation of signalling strength is essential for interpreting morphogen gradients and thus for correct patterning during development. The control of signalling receptor levels at the cell membrane is an important aspect of this regulation. It is therefore of interest to know how receptor levels are regulated under physiological conditions. The results presented here indicate that diverse classes of signalling receptors undergo constitutive (ligand-independent) ubiquitination, endocytosis and Hrs-dependent degradation. The efficiency of this traffic affects the responsiveness of cells to patterning signals: blockage of trafficking in hrs mutants can sensitize cells to a low level of signalling molecules, whether RTK ligands (Lloyd, 2002) or Dpp (this study). However, it does not lead to ligand-independent signalling, supporting the conclusion that most endocytosed receptor molecules are not activated. Ligand-induced endocytosis may also occur, but affects only a minority of receptor molecules in this in vivo context. Constitutive turnover of receptors may serve as quality control by removing damaged receptors or receptors in partially formed signalling complexes. A constant flux of all receptor molecules may also facilitate the efficient clearance of activated receptors (Jékely, 2003).

Dpp receptor (Tkv) signaling is upregulated in vps25 mutant cells and is responsible for generating ectopic DV organizer activity and consequent ventral leg outgrowths

Clones mutant for vps25 in the leg disc mimic those expressing an activated form of the Dpp receptor Thickveins, Tkv). Activated Tkv signaling represses expression of Wingless, allowing Hedgehog to activate expression of the Dpp ligand in ventral cells and generate ventral leg outgrowths. vps25 mutant clones located on the ventral side of the leg imaginal disc similarly upregulate expression of the Dpp ligand, observed with a dpp-lacZ transgene. In contrast, the dpp-lacZ transgene is not activated in vps25 mutant clones in wing discs, where the antagonistic Dpp-Wg regulatory loop does not operate and dpp-lacZ is not induced by Dpp signaling. However, mutant cells in wing discs still mimic overexpressed Tkv, revealed by upregulation of the Dpp target gene, omb-lacZ. These results indicate that Dpp receptor (Tkv) signaling is upregulated in vps25 mutant cells and is responsible for generating ectopic DV organizer activity and consequent ventral leg outgrowths (Thompson, 2005).

The interaction between PP1 and Punt requires the type I receptor Thick-veins

Two proteins have been identified that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. Modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants (Bennett, 2002; Parker, 2002). These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFß superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, this study shows that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1 (Bennett, 2003).

I-2Dm and NIPP1Dm are potent inhibitors of PP1 in vitro. Ectopic I-2Dm and ectopic NIPP1Dm also inhibit PP1c in vivo, resulting in titration of PP1c from its other functions. First, PP1 activity is reduced in extracts from flies ectopically expressing I-2Dm or NIPP1Dm. NIPP1Dm has a larger effect on PP1 activity than I-2Dm does, consistent with the potent inhibition of PP1c by NIPP1Dm in vitro. NIPP1Dm degradation in tissue extracts releases NIPP1 from PP1c, thereby restoring PP1 activity. Consequently, the reduction in PP1c activity in ectopic NIPP1Dm flies is probably higher than that measured in extracts. This is consistent with the phenotypic effect of NIPP1Dm overexpression. Second, ectopic expression of I-2Dm or NIPP1Dm results in phenotypes resembling those of PP1c loss-of-function mutations. In conjunction with arm-GAL4, ectopic NIPP1Dm flies phenotypically resemble PP1alpha87B-/- and PP1ß9C-/- mutants. In conjunction with vg-GAL4, which expresses only weakly in the wing, ectopic NIPP1Dm resembles strong PP1alpha87B-/+ mutants in combination with type II TGFß superfamily receptor Punt. In contrast, overexpression of I-2Dm in the wing resembles the effect of weak PP1alpha87B-/+ mutants in combination with Punt, indicating that the effects of I-2Dm overexpression are similar to, but weaker than, the effects of ectopic NIPP1Dm. Lastly, PP1 activity is reduced in flies modestly overexpressing I-2Dm, but can be restored by reactivation of I-2Dm:PP1c with GSK3ß + MgATP. This implies that I-2Dm and NIPP1Dm sequester PP1c away from other functions and suggests that there is no compensation for titration of endogenous PP1c by production of additional active PP1c. This might be because PP1 is normally in excess, which would also explain why PP1c overexpression in a wild-type background has no phenotypic effect. However, the effects of ectopic I-2Dm and NIPP1Dm on their own, in combination with each other, or in a sensitized background, can be suppressed by coexpression of PP1c, indicating that exogenous PP1c can restore PP1c levels by titrating additional inhibitor (Bennett, 2003).

Co-overexpression of either I-2Dm or NIPP1Dm with PP1 has no phenotypic effect. This implies that neither inhibitor:PP1c complex (I-2Dm:PP1c or NIPP1Dm:PP1c) has any significant function when in excess, and may simply be inactive, as expected if the binding proteins are simply inhibitors of PP1. The ability of I-2 to convert recombinant PP1 to more native-like activity upon phosphorylation of I-2 has led to the suggestion that I-2 may be a molecular chaperone of PP1 as well as an inhibitor of PP1. This biochemical property is also conserved in I-2Dm (Bennett, 1999). However, the net effect of I-2Dm overexpression in flies is to reduce PP1 activity. This indicates that phosphorylation of I-2Dm and reactivation of PP1 is rate limiting, at least when I-2Dm is in excess. Alternatively, reactivation of PP1 in vivo might serve another purpose, namely to allow a pool of inactive (I-2Dm-bound) PP1 to be recruited by targeting subunits to specific subcellular loci. Indeed there is some precedent for cycling of PP1c from inhibitors to targeting subunits: for instance phosphorylation and inactivation of Inhibitor-1 (I-1) by PKA releases I-1-bound PP1 and might be coordinated with phosphorylation and activation of glycogen-binding subunit of PP1 to recruit PP1 to glycogen particles. The isolation of loss-of-function mutations in I-2Dm and NIPP1Dm will help to resolve the question of whether these proteins act solely as inhibitors of PP1c (Bennett, 2003).

To explore the utility of PP1c inhibitors in vivo the effect of I-2Dm and NIPP1Dm on TGFß signaling was examined. Ectopic expression of I-2Dm or NIPP1Dm in the cells expressing ectopic Punt had the same effect as PP1alpha87B mutants, which reduce PP1 levels across the whole wing disc. Since genetic analysis indicates that both Babo signaling and Tkv/Sax signaling have a role in regulating growth of the wing, attempts were made to identify the relevant type I receptor responsible for these effects. In a sensitized genetic background in which Punt is not limiting (vg-GAL4, UAS-punt), the wing phenotype is sensitive to the level of Tkv but not the other type I receptors. The overgrowth phenotype is enhanced by extra Tkv (UAS-tkv), and this closely resembles the phenotype of extra Punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1-/+). Furthermore, the wing phenotype of elevated punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1-/+) is suppressed by reducing the level of Tkv (tkv-/+) and enhanced by elevating the level of Tkv (UAS-tkv) but is not affected by similar manipulation of the levels of Sax or Babo. Therefore the overgrowth wing phenotypes are entirely regulated by Tkv (Bennett, 2003).

Displacement of PP1 from Sara has been shown to induced TGFß superfamily signaling (Bennett, 2002), implying that the role of Sara-bound PP1 may be to prevent inappropriate ligand-independent signaling by the receptors. This study shows that the interaction between PP1 and Punt is not sensitive to the levels of the ligand Dpp and that reduction of PP1 activity results in ectopic expression of Bi, a target of Tkv/Dpp signaling, beyond the region in which Dpp is known to elicit signaling (reported to be up to 25 cell diameters from its source). Since PP1 loss of function has the same effect on TGFß/Dpp signaling as a PP1c-nonbinding mutant of Sara, the possibility can be ruled out that free PP1 goes off and does something else, ultimately leading to the increased Dpp signaling by some other mechanism. Taken together, this suggests that reduction of PP1 activity can activate the downstream Dpp signaling pathway in cells that receive low levels or no Dpp and is consistent with a role for PP1 in preventing inappropriate activation of the signaling pathway where the ligand is low or absent (Bennett, 2003).

In summary, a method has been developed of inhibiting PP1 activity in a cell- and tissue-specific manner. I-2Dm and NIPP1Dm do not discriminate between different PP1c isoforms; therefore the role of PP1 in a given pathway can be easily tested without having to test the separate isoforms individually. The role of PP1c has been examined in wing development using ectopic inhibitors of PP1c; that reduction of PP1c activity enhances the effect of the TGFß superfamily type II receptor Punt, giving rise to overgrowth of the wing. The basis of this interaction was examined using I-2Dm; this interaction is shown to require the type I receptor Tkv and is accompanied by induction of a downstream target Bi, suggesting that PP1c negatively regulates Dpp/Tkv signaling during wing morphogenesis. It is anticipated that ectopic inhibitors of PP1c can be used in a wide variety of contexts to test for the effect of reducing PP1 activity on specific developmental processes. While these inhibitors are very useful for identifying a role for PP1 in a particular developmental process, they are not able to dissect the role of a specific PP1c species. The demonstration of the usefulness of ectopic expression of PP1c inhibitors in Drosophila, together with the highly conserved nature of both PP1 and the inhibitors of PP1, suggests that this approach is also applicable to other, less genetically tractable systems (Bennett, 2003).

Drosophila p24 homologues eclair and baiser are necessary for the activity of the maternally expressed Tkv receptor during early embryogenesis

p24 proteins are assumed to play an important role in the transport of secreted and transmembrane proteins into membranes. However, only few cargo proteins are known that partially, but in no case completely require p24 proteins for membrane transport. Here, it is shown that two p24 proteins are essential for dorsoventral patterning of Drosophila melanogaster embryo. Mutations in the genes, eclair (eca) and baiser (bai), encoding two p24 proteins reduce signalling by the TGF- homologue, Dpp, in early embryos. This effect is strictly maternal and specific to early embryogenesis, as Dpp signalling in other contexts is not notably affected. Genetic evidence is provided that in the absence of eca or bai function in the oocyte, the maternally expressed type I TGF- receptor Tkv is not active. It is proposed that during early embryogenesis eca and bai are specifically required for the activity of the maternal Tkv, while the zygotic Tkv is not affected in the mutant embryos. Mutations in either eca or bai are sufficient for the depletion of Tkv activity and no enhancement of the phenotypes is observed in embryos derived from oocytes mutant for both genes. The dependence of maternal Tkv protein on the products of p24 genes may serve as an in vivo model for studying p24 proteins (Bartoszewski, 2004).

p24 proteins are very abundant in the endoplasmic reticulum (ER) and Golgi apparatus. They bind both COP I and COP II coat proteins and thus are thought to promote formation of both COP I and COP II vesicles, which transport cargo between the Golgi apparatus and ER, and between different compartments of Golgi. p24 proteins have been suggested to function as receptors for cargo proteins. However, until now only the transport of some proteins, like Gas1p and Invertase, has been shown to be reduced but not abolished in yeast lacking either one or all eight p24 genes. This defect does not have severe consequences, since all eight p24 genes can be removed in yeast without affecting the growth rate. Also, p24 mutants in C. elegans are viable and show no obvious defects on their own. In contrast, however, knocking out a p24 gene in the mouse results in an early embryonic lethality (Bartoszewski, 2004 and references therein).

Another proposed role for p24 proteins has been proposed (Wen, 1999) based on observations that p24 proteins prevent membrane localisation of the C. elegans Notch homologues, Lin12 and Glp1 proteins, which have mutations in their extracellular domains, but do not affect the wild type Lin12 and Glp1 proteins. It has been proposed that the lumenal domains of p24 proteins could interact with the misfolded extracellular domains of Lin12 and Glp1 proteins and prevent them from entering the COP II vesicle. This model could explain how highly conserved p24 proteins could be redundant if their only function would be to remove misfolded forms of secreted or membrane proteins. These misfolded proteins might normally only slightly reduce a cell's fitness but from an evolutionary point of view their removal could have a crucial meaning (Bartoszewski, 2004).

Mutations in eca and bai specifically reduce Dpp signalling during early embryogenesis. Genetic evidence is provided that this reduction is caused by affecting specifically the activity of maternally expressed Tkv protein, while the otherwise normal development of the embryo suggests that other membrane proteins are not affected. Both p24 genes are not redundant; removing of either eca or bai completely abolishes the signalling by maternally expressed tkv gene and no enhancement of the phenotype is observed when both genes are mutated (Bartoszewski, 2004).

The major question is how eca and bai affect the activity of the tkv gene. Three possible models are proposed. The simplest model is that both p24 proteins participate in the formation of the COP II bud in ER and recruit Tkv to the bud. However, no difference was seen in Tkv localisation in eca and bai mutant compared to wild type embryos; this would argue against this model. Since this was a negative result, potential subtle differences in Tkv localisation, which were not detectable, cannot be excluded. One could also imagine that the correctly transported Tkv is seen in the endocytic pathway while in eca or bai embryos Tkv accumulates in the secretory pathway and both stainings might look very similar (Bartoszewski, 2004).

In the second model, a fraction of Tkv would be misfolded under physiological conditions but p24 proteins would prevent the misfolded Tkv from being transported into membranes, which is in agreement with the model proposed by Wen, (1999). Normally, therefore, only active Tkv receptor would be present in the plasma membrane, but in eca or bai embryos the misfolded Tkv would reach the membrane and by a dominant negative interaction it would block Tkv activity. In this case no clear difference in Tkv localisation would be expected, which is consistent with the results. The weakness of this model is that rather a high proportion of Tkv should be expected to be misfolded (Bartoszewski, 2004).

For the third model, Eca/Bai would play an essential role in folding or posttranslational modification of Tkv. This contradicts the implications that p24 proteins act in transport of vesicles between ER and Golgi compartments. But, since there is no strong evidence that p24 proteins are real cargo receptors in vivo, this model should be considered in the future research. Also, p24 proteins might act here indirectly, for example, they might be necessary for localisation of an unknown factor, which modifies Tkv protein and this modification is necessary for the receptor's activity. Although this model is quite speculative, it would fit well the results -- Tkv protein would be expressed and localised correctly in the eca/bai embryos, but would not be active (Bartoszewski, 2004).

Although eca and bai mutants show reduced viability, the flies homozygous or hemizygous for either mutation can survive until adulthood and apart from slight defects in wing development they do not differ from wild type flies. The most characteristic and penetrant phenotype of eca and bai mutations is that they lead to the lack of the maternal tkv activity during early embryogenesis. It is striking that tkv activity is so severely disrupted at this stage, while it is not notably affected in many other developmental contexts in which the gene is required. No phenotypes characteristic of hypomorphic tkv mutations were observed: short legs, split thorax, and lack of wing veins in the homozygous or hemizygous mutants. Since there are two Tkv isoforms and one of them is predominantly maternal, it seems likely that only the maternal isoform might be affected in eca and bai cells. Since the zygotic Tkv lacks the leader peptide it would be folded in the cytoplasm, using different chaperones, and subsequently translocated to ER by a different mechanism. Also, the two isoforms have different N-termini. These molecular differences could be the reason why only the maternal form is affected in eca and bai embryos (Bartoszewski, 2004).

A second possibility is that tkv is expressed maternally at the levels only slightly exceeding the requirement and thus its activity would be affected if the overall rate of membrane transport were decreased in eca and bai embryos. But then, the zygotic Tkv would have to be transported and act very rapidly, since it induces pnr expression only slightly later than under normal conditions. However, it could be still possible, since the zygotic Tkv protein lacks the leader peptide and it would be transported to the ER through a different mechanism and possibly also with different kinetics (Bartoszewski, 2004).

A third possibility is that tkv mRNA would be translated only for a limited time, while during the development of the oocyte and early embryogenesis there would be three phases of the protein transport. The first phase would be independent of eca and bai and majority of the maternal secretory and transmembrane proteins would be translated at this stage. The second phase would be dependent on eca and bai, and maternally deposited tkv mRNA would be translated only at this stage. The third phase would be independent of eca and bai and coincide with the beginning of the zygotic transcription after the maternal tkv mRNA would have been degraded (Bartoszewski, 2004).

p24 genes exist in multiple copies in different eucaryotic organisms, including seven genes in Drosophila. p24 proteins are highly conserved among different organisms, for example, eca is 73% identical and 87% similar to its vertebrate and worm homologues, and bai is 60% identical and 73% similar to its vertebrate homologues. The high similarity of p24 proteins suggests that they play a conserved role. However, their importance is not clear, since all yeast paralogs could be deleted without affecting the fitness of the cells. Also mutations in p24 genes in C. elegans are viable and fertile (Wen, 1999). In contrast, knocking out a p24 gene in the mouse (Denzel, 2000) results in an early embryonic lethality and probably is cell lethal (Bartoszewski, 2004).

In the fly, both p24 genes identified in this study are required maternally for embryonic patterning, yet not essential for viability, although it cannot be rule out that the maternally expressed eca and bai genes are sufficient until adulthood. In two cases they are, however, essential: in follicular epithelium, and for the activity of maternally expressed tkv. If the maternal activity of tkv, and maybe also of a limited number of other proteins, were independent of eca and bai, both p24 genes would also not be required in germline. This explains the apparent difference between the yeast and mouse mutants: if activity of one essential protein depends on p24 proteins, the p24 knock out will be lethal, which could be the case in the mouse. Regardless of the role of p24 proteins in either recruiting cargo into COP vesicles or preventing misfolded proteins from being transported, p24 proteins seem to be functionally redundant with other factors acting in a parallel pathway. However, some proteins might require specifically the p24 pathway for their proper secretion or activity, like the maternal tkv (Bartoszewski, 2004).

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFβfamily signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-β (TGFβ) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, a biochemical approach was undertaken to establish the ligand binding repertoire and downstream signaling pathway. It was observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor Thickveins and Saxophone to activate a BMP-like signaling pathway. Further it was demonstrated that both Myoglianin and its most closely related mammalian ligand, Myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFβ/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFβ-like signaling pathways. Given that Myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate Myoglianin-dependent signals in the nervous system (Lee-Hoeflich, 2005).

To provide insight into the molecular mechanisms of Wit that contribute to the biological functions of Wit, this study has characterized the Wit interacting ligands, their compatible type I receptor partners, and their downstream signaling pathways. The binding of BMP7, the mammalian ligand most related to Gbb, to Wit is in agreement with and gives biochemical evidence for results obtained from genetic analysis indicating that Wit mediates Gbb-activated BMP signaling in collaboration with the type I receptors, Tkv and Sax. The demonstration of the binding of BMP4, a functional ortholog of Dpp, to the receptor complex also suggests the possibility of Wit mediating Dpp signals. Dpp is not expressed in muscle or motoneurons but Wit is widely expressed in the central nervous system from embryonic stages suggesting that this putative Dpp signaling might regulate early developmental processes other than NMJ formation. The data showing that a receptor complex comprised of Wit and Tkv can activate MAD phosphorylation is also in agreement with the observation of impaired phosphorylation of MAD in Wit deficient flies and provides further support for a role of Wit in mediating BMP signaling (Lee-Hoeflich, 2005).

While the expression of dActivin in the developing nervous system and its proposed function in neuronal remodeling downstream of Wit or Punt have led to a suggestion that dActivin might induce Wit-mediated activin signaling, this study observed that mammalian activin, which is most closely related to dActivin, does not bind to Wit. One possible explanation for this discrepancy is that Wit might bind ligands other than dActivin and that this indirectly compensates for the lack of Punt-dActivin interaction. An attempt to produce dActivin in mammalian cells using a heterologous system was unsuccessful, thus the possibility cannot be eliminated that mammalian and Drosophila activins have different binding specificities. Generation of dActivin null flies or cell clones and testing for functional equivalence in rescue experiments should help resolve this issue (Lee-Hoeflich, 2005).

Alternatively, it is speculated that Wit might mediate activin signaling via Myoglianin since myostatin, the mammalian ligand most closely related to Myoglianin, activates a TGFβ/activin-like pathway. Accordingly, it was found that both myoglianin and myostatin bind to the Wit and Babo receptor complex. Furthermore, it was observed that coexpression of Wit and Babo induces dSmad2 phosphorylation and mediates myostatin-induced transcriptional activation of a TGFβ/activin-responsive reporter. In agreement to these observations, ectopic expression of Wit induces dSmad2 phosphorylation in insect S2 cells. Retrograde signaling between target-derived factors and the presynaptic terminal is crucial for NMJ development. Since Drosophila Myoglianin is abundantly expressed in muscle at late developmental stages and since Wit-mediated retrograde signaling had been identified previously, it is postulated that Myoglianin might activate a novel retrograde Wit signaling pathway. Interestingly, myostatin inhibits the BMP7 signaling response by competitive binding to type II receptor, ActRIIB, thus the binding of Myoglianin to Wit might also affect Gbb-mediated signaling and thus contribute to NMJ formation. Generation of flies harboring myoglianin loss-of-function mutations will shed light on these issues. These observations underscore the diverse mechanisms controlling Wit signaling and add impetus to further experiments in the context of the Wit receptor (Lee-Hoeflich, 2005).

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. This study indicates that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients (Bangi, 2006a).

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. This study shows that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. It is proposed that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, this model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs (Bangi, 2006b).

These data clarify the respective roles of Sax and Tkv in mediating Bmp signaling during wing patterning. This analysis shows that Tkv is responsible for mediating both Dpp and Gbb signals, and that Sax has a much more complex role in wing patterning than previously appreciated; Sax not only promotes signaling but also antagonizes signaling by limiting the availability of primarily the Gbb ligand. Both the antagonistic and signal promoting functions of Sax were revealed not only by gain-of-function studies but importantly, also by loss-of-function analyses. Loss of the antagonistic function of endogenous sax is evident: (1) as a broadening the pMad profile when the wing disc completely lacks sax function; and (2) as a non-autonomous increase in pMad levels in wild-type cells abutting the boundary of sax null clones. Loss of Sax-mediated signaling itself is evident: (1) in sax mutant discs as a reduction in the peak pMad levels along the AP boundary; and (2) in sax clones as a cell-autonomous reduction in pMad accumulation. Gain-of-function or overexpression studies indicate that the balance of Sax and Tkv levels in wing disc cells is crucial for proper signaling and, thus, wing patterning. Altogether, these results indicate that Sax is important in modulating Bmp signaling across the wing disc by both mediating and blocking Bmp signals, and, thus, shaping the Bmp activity gradient. How can the novel function of Sax as an antagonist be reconciled at the molecular level with the ability of Sax to promote signaling (Bangi, 2006b)?

Given that Tkv is required for all Bmp signaling in the wing disc, the simplest explanation for the fact that Sax signaling appears to depend on the presence of Tkv is that Sax can only promote signaling in a receptor complex also containing Tkv. Three different forms of Bmp receptor complexes can potentially form in wing disc cells, those composed of two type II receptor molecules and either two Tkv, two Sax or one molecule of each: Tkv-Tkv, Sax-Sax and Tkv-Sax. Overexpressing Tkv or Sax in wing disc cells enabled shifting of the balance between the relative levels of these two molecules, artificially enriching for the formation of receptor complexes homomeric for type I molecules Tkv-Tkv or Sax-Sax. Disrupting the balance of endogenous Tkv to Sax levels by overexpressing Sax immediately reveals the antagonistic function of Sax, consistent with the idea that excess Sax could be sequestering ligand in Sax-Sax receptor complexes which signal either very poorly or not at all. However, overexpression of Tkv, enriching for Tkv-Tkv complexes with high affinity for Dpp and lower affinity for Gbb, leads to increased signaling given sufficient ligand. The third receptor complex, Tkv-Sax, probably accounts for the contribution of Sax to the promotion of Bmp signaling and probably signals in vivo more efficiently than Tkv-Tkv, based on the fact that pMad levels are lower inside clones devoid of Sax than the pMad levels seen in cells at an equivalent position along the AP axis elsewhere on the disc. Loss of Tkv, by definition, eliminates signaling by both Tkv-Tkv and Tkv-Sax, leaving only Sax-Sax containing receptor complexes, which are clearly unable to elicit a pMad-mediated signal on their own. Thus, the model predicts that removing Sax function results in two opposing consequences: (1) a reduction in total Bmp signaling caused by loss of Tkv-Sax complexes, and (2) an increased availability of Bmp ligand and potential signaling caused by loss of Sax-Sax complexes. Several biochemical studies support the putative existence of functional Sax-Tkv receptor complexes. Heteromeric complexes involving different vertebrate type I receptors have been shown to contribute to a single signaling receptor complex and in Drosophila S2 cells both Sax and Tkv appear to be necessary to produce a synergistic signal (Bangi, 2006b).

It is important to note that increasing wild-type Tkv levels in the presence versus absence of excess ligand results in very different phenotypic outcomes. In contrast to Sax, increasing Tkv in the presence of excess ligand leads to a larger increase in Bmp signaling. However, at endogenous ligand levels, as Tkv levels are experimentally increased, a loss of Bmp signaling is seen that is indicative of the preference of Tkv for binding Dpp over Gbb. Clearly, both Gbb and Dpp become limiting in the presence of excess Tkv, with low level Tkv overexpression preferentially limiting Dpp-dependent signaling, while higher levels of overexpression limit both. Clearly, although overexpression of ligand and receptor together reveals a significant difference in the signaling ability of Tkv and Sax, overexpression of receptor alone in the absence of increased ligand appears to reflect only receptor ligand-binding preference (Bangi, 2006b).

Such experimental manipulations of Tkv levels can lead to the loss of Bmp signaling by limiting the range of Bmp signaling, but unlike sax, loss of endogenous tkv function never leads to an increase in Bmp signaling. Furthermore, there is no indication that Tkv is required for or involved in the antagonistic function of Sax. At endogenous levels, Sax-Sax complexes, unlike Tkv-Tkv or Tkv-Sax complexes, appear to modulate the range of Bmp signaling by sequestering ligand without any associated signaling, and, thus, Sax identifies a new previously unrecognized Bmp modulator whose signaling ability appears to depend on which receptor it partners (Bangi, 2006b).

The fact that both Dpp and Gbb are dependent on Tkv for signaling has significant implications regarding the Bmp activity gradient, given that removal of Tkv at any point along the gradient results in the loss of both Gbb and Dpp signaling, not just Dpp signaling. When both ligands are present at similar levels, the higher affinity of Dpp for Tkv means the contribution of Dpp to total Bmp signaling will be more significant than that of Gbb, and movement of Dpp across the wing disc will be affected more strongly by Tkv than that of Gbb. Thus, Gbb should and does contribute more significantly to the low points of the Bmp activity gradient, especially since competition with Dpp for binding to Tkv will also be lower in these regions (Bangi, 2006b).

These findings from receptor and ligand overexpresion experiments suggest that both the antagonistic and signal promoting functions of Sax impact Gbb signaling most significantly because of their preferential interaction. For example, although localized loss of Sax from the peripheral cells of the wing pouch leads to ectopic induction of brk, loss in more central cells does not, suggesting that the relative contribution of Sax to overall Bmp signaling is less in the central cells where Tkv must contribute more significantly given the higher level of Dpp near the AP boundary. The greater contribution of Sax to total signaling in the more peripheral cells of the wing pouch is consistent with its higher affinity for Gbb and the long-range nature of Gbb versus Dpp (Bangi, 2006b). Similarly, removal of Sax from just anterior compartment cells results in brk repression in both the anterior and posterior compartments suggesting that in the absence of Sax, anteriorly expressed Gbb can signal to the posterior-most cells of the wing pouch to effectively repress brk expression beyond its normal domain. This result indicates that endogenous Sax normally functions to not only restrict the level of Gbb signaling but also the range of Gbb. The role that Sax plays in promoting Gbb function, in particular, is detected only when sax function is completely eliminated and gbb function is also significantly compromised (Bangi, 2006b).

Given that Tkv is also required for mediating Gbb signals, of the two proposed receptor complexes that could mediate Gbb signaling (Tkv-Tkv and Tkv-Sax), which is preferentially used by Gbb in wild-type cells? It is clear that Tkv-Sax complexes are not obligatory for Gbb signaling since Gbb signaling is not abolished in sax mutants. The fact that removing Sax does not cause a gbb loss-of-function phenotype indicates that enough Gbb is made available by the loss of Sax antagonism and can signal to compensate for losing that region of total signaling that Sax normally promotes. The fact that pMad levels within a sax clone are lower then endogenous levels indicates that signaling in the clone cells containing only Tkv-Tkv is less efficient than the neighboring cells that have wild-type levels of both Sax and Tkv (Bangi, 2006b).

A synergy has been observed between co-expressed constitutively active (CA) Tkv and Sax in the early embryo and between Tkv and Sax in S2 cells in response to Dpp-Scw heterodimers, since only Dpp homodimers are able to signal efficiently in the absence of Sax. A likely, albeit minimal, contribution of Dpp-Gbb heterodimers to long-range wing patterning has been detected (Bangi, 2006a) making it is possible that Tkv-Sax complexes could respond to Dpp-Gbb heterodimers and such complexes could be particularly efficient at signaling. Given the dual function of Sax, the relative levels of Sax to Tkv are likely to be crucial for establishing a synergistic interaction. The ability of Tkv-Sax containing complexes to mediate ligand homodimers has not yet been determined in vivo and it is also not yet completely clear if the antagonism by Sax can affect heterodimers as well as homodimers. The current data indicate that the ability of Sax to promote signaling must reside with Tkv-Sax-containing complexes and the strong contribution of Gbb to the low points of the gradient with a minimal contribution by Dpp leaves open the possibility that Dpp-Gbb can signal, in addition to Gbb-Gbb, to cells far from the AP boundary (Bangi, 2006b).

Overexpression studies in the follicle cells of the Drosophila ovary produce the same results as those in the wing, indicating that the ability of Sax to block Gbb signaling is not limited to the developing wing. However, in contrast to studies in the wing disc, loss of sax from the follicle cells, as well as the embryonic midgut and neuromuscular synapse produces mutant phenotypes indicative of a loss of ligand function. It is possible that the contribution of Sax to signal promotion in these tissues may be stronger than its antagonistic function. The phenotypic outcome of sax loss of function in a particular process probably depends on the relative numbers of Sax-Sax and Sax-Tkv complexes on the cell surface and the relative binding affinity of a given Bmp ligand for these two complexes. What regulates the composition of type I receptors in a signaling complex is not yet known (Bangi, 2006b).

The ability of the Sax to block Bmp signaling may reflect its requirement to have input from another molecule to activate its kinase domain. When activated by in vitro mutagenesis, Sax and its vertebrate orthologs Alk1/Alk2 (Acvrl1 and Acvr1 - Mouse Genome Informatics) are able to phosphorylate Bmp specific R-Smads, but ligand-induced activation of Sax or Alk1/2 kinase has not been reported. Interestingly, a ligand-induced Bmp receptor complex containing Alk2 and ActRII is unable to phosphorylate Smad1. Furthermore, Alk1 has been shown to require a different type I receptor (Alk5) to activate its kinase domain. Although it has been suggest that the Alk2/ActRII complex might be unstable in vitro, it is also possible that activation of Alk2 (and of its Drosophila ortholog Sax) may depend on its partner type I receptor and/or which ligand is bound, or some other protein. Although Gbb fails to activate Sax-Sax, perhaps another Bmp ligand (i.e. Scw) can. Similarly, endoglin, related to the co-receptor betaglycan, could be important in modulating Alk1-dependent signaling given that mutations in either gene give rise to hereditary hemorrhagic telangiectasia. Sax may require a different type I receptor partner, i.e. Tkv, to activate its kinase or transduce a signal, and such a requirement may be a universal feature of the Alk1/Alk2/Sax subgroup of Bmp type I receptors (Bangi, 2006b).

The robustness of morphogen gradients may depend on negative-feedback mechanisms to buffer against environmental and genetic fluctuations. Clearly, Sax plays a crucial role in modulating the range of the Bmp activity gradient from analysis at both the level of Bmp-dependent target gene expression and the final pattern of the adult wing. The identification of the antagonistic nature of a Bmp type I receptor to modulate signaling activity by sequestering ligand without transducing a signal provides a new mechanism that contributes to the robustness of the Bmp activity gradient. It is proposed that the dual function of Sax is crucial for buffering the wing disc Bmp activity gradient against local fluctuations in ligand levels (environmental, genetic or experimentally induced). Whether this mechanism of signal modulation is evolutionarily conserved remains to be determined, but the fact that the vertebrate Sax orthologs Alk1 and Alk2 have been shown biochemically to exhibit antagonistic behaviors in vitro is interesting. Detailed analysis of these orthologs in developmental contexts will be crucial to determine whether the robustness of vertebrate Bmp activity gradients also depends on the modulation of ligand availability by specific receptors (Bangi, 2006b).

The BMP-binding protein Crossveinless 2 is binds Tkv and is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila

In Drosophila, the secreted BMP-binding protein Short gastrulation (Sog) inhibits signaling by sequestering BMPs from receptors, but enhances signaling by transporting BMPs through tissues. Crossveinless 2 (Cv-2) is also a secreted BMP-binding protein that enhances or inhibits BMP signaling. Unlike Sog, however, Cv-2 does not promote signaling by transporting BMPs. Rather, Cv-2 binds cell surfaces and heparan sulfate proteoglygans and acts over a short range. Cv-2 binds the type I BMP receptor Thickveins (Tkv), and this study shows that the exchange of BMPs between Cv-2 and receptor can produce the observed biphasic response to Cv-2 concentration, where low levels promote and high levels inhibit signaling. Importantly, the concentration or type of BMP present can determine whether Cv-2 promotes or inhibits signaling. Cv-2 expression is controlled by BMP signaling, and these combined properties enable Cv-2 to exquisitely tune BMP signaling (Serpe, 2008).

Cv-2 modulates BMP signaling in the Drosophila wing by a mechanism distinct from that of Sog. BMP signaling in the early stages of PCV development depends, in large part, on BMPs being produced in the adjacent longitudinal veins, and endogenous Sog acts over a long range to promote signaling in this context, likely by transporting BMPs from the longitudinal veins into the PCV region. Both Sog and Cv-2 are biphasic, as low levels promote and high levels inhibit BMP signaling. However, Cv-2 acts over a short range within the PCV, precluding a direct role in the long-range transport of ligands from the longitudinal veins. The short-range action of Cv-2 is likely to involve binding to cell surface proteins such as Dally, and strongly suggests that Cv-2 acts on cells receiving the BMP signal. Moreover, Cv-2 can stimulate signaling in vitro, where the transport or stability of BMPs in the medium is unlikely to be an issue (Serpe, 2008).

Consistent with a role in reception, it was found that Cv-2 binds not only BMPs, but also the type I BMP receptor Tkv and vertebrate BMPR-IA and -IB. It is therefore proposed that the binding between Cv-2 and receptor facilitates transfer and signaling of BMPs via formation of a transient, nonsignaling complex containing Cv-2, type I receptor, and BMPs. At moderate levels, Cv-2 moves ligand from the extracellular space onto receptors via this complex, while at higher levels Cv-2 antagonizes signaling by sequestering ligand in the complex. The inability of this complex to signal is consistent with studies suggesting that Cv-2 binds to the BMP “knuckle” epitope used to bind type II BMP receptors (Serpe, 2008).

Computational analyses also predict that the relative affinities of different BMPs for Cv-2 or receptors will influence the effect of Cv-2 upon signaling. Although the vertebrate counterparts of BMP ligands appear to have similar affinities for Cv-2, they have different affinities for their receptors, and the model predicts that this alone can alter the activity of Cv-2. Indeed, in cell culture assays Cv-2 only antagonizes Dpp signaling, but has biphasic effects on Gbb signaling. This could explain why a vertebrate member of the Cv-2/Kielin-like family, mouse KCP, stimulates BMP-2 signaling but inhibits TGF-β and Activin signaling in vitro. Likewise, in the early Drosophila embryo, where a different set of BMP ligands act, it was found that loss of endogenous cv-2 actually expands BMP signaling, opposite to the effects of Cv-2 loss in the PCV. Thus, Cv-2 activity is highly context dependent (Serpe, 2008).

Fundamental to the proposed model is the formation of a transient complex containing Cv-2, BMP, and the receptor. Tripartite complexes have been demonstrated to form between follistatin, type I receptor, and BMP ligands, and this study found that Cv-2 and the extracellular portion of BMPR-IB simultaneously coimmunoprecipitate with Dpp. Similarly, the vertebrate type I receptor can coprecipitate both BMP and mouse KCP. Although the tripartite intermediate was not directly demonstrated, this might reflect the transient nature of this complex due to very rapid on-off kinetics. In fact, modeling predicts the intermediate is a low-affinity, transient complex (Serpe, 2008).

It is important to recognize that Cv-2 does not act as an obligate coreceptor in the described model. Rather, Cv-2 is modulatory, consistent with the fact that Cv-2 does not participate in BMP signaling in many contexts. In fact, the model requires that the tripartite complex does not signal, and it is only after Cv-2 is displaced that the type I receptor is free to signal. This is in contrast to the activity of coreceptors like Cripto, which is required for binding of the TGF-β family member Nodal to type I receptors and formation of signaling complexes with type II receptors. While Cripto can antagonize signaling, this involves non-Nodal ligands. In contrast, Cv-2 can promote or antagonize the signaling mediated by a single type of ligand such as Gbb (Serpe, 2008).

The functional, structural, and regulatory aspects of Drosophila Cv-2 show remarkable conservation with its vertebrate homologs in terms of HSPG binding, cleavage, and feedback by BMP signaling. Despite these similarities, a different mechanism was recently proposed to explain the ability of zebrafish Cv-2 to either promote or inhibit signaling; the cleavage of Cv-2 was proposed to convert Cv-2 from an antagonist to an agonist (Rentzsch, 2006a). In support of this model was the observation that an uncleavable form of Cv-2 was more potent at dorsalizing zebrafish embryos (indicating a loss of BMP signaling) than was the full-length cleavable form, and that an N-terminal fragment lacking the vWFD domain ventralized embryos (indicating a gain in BMP signaling). Processing did not dramatically alter the KD of zebrafish Cv-2 for BMP binding, but apparently blocked its ability to bind HSPGs. Thus, the authors proposed that uncleaved Cv-2 binds HSPGs to sequester BMPs, while cleaved Cv-2 promoted signaling in a tissue-specific manner by an unknown mechanism (Serpe, 2008).

Little support was found for this model in Drosophila. Blocking cleavage did not create a strictly inhibitory molecule, since both wild-type and uncleavable Drosophila Cv-2 acted in a biphasic fashion. Moreover, both cleaved and uncleaved forms of Drosophila Cv-2 bound Dally and cell surfaces. Also no evidence was found of differential cleavage among cell types or developmental stages. Evidence from other secreted proteins suggests that GD-PH cleavages like that in Cv-2 occur via an autocatalytic process triggered by the low pH found within the late secretory compartments. Indeed, evidence was found of constitutive, pH-dependent Cv-2 cleavage in vitro, suggestive of an unpatterned, autocatalytic process in vivo (Serpe, 2008).

Nonetheless, conservation of the cleavage site among species suggests that cleavage plays an important role, and it was found that cleavage of Drosophila Cv-2 lowers its affinity for BMPs in vitro. However, similar manipulations of zebrafish Cv-2 did not greatly affect its KD for BMP. These may represent true species-specific differences, or they may result from differences in the binding assays used: the immobilization of proteins in the Biacore analyses of zebrafish Cv-2, or the presence of additional factors in the conditioned S2 cell medium present in coimmunoprecipitation assays. Since Drosophila Cv-2 can rescue the knockdown of zebrafish Cv-2, any species-specific differences are likely quantitative, rather than qualitative (Serpe, 2008).

In zebrafish, Chordin largely antagonizes BMP signaling, and thus Cv-2 and Chordin have essentially opposite effects on BMP signaling. However, loss of Cv-2 ameliorates only a subset of the gain-of-signaling phenotypes caused by loss of Chordin. Thus, Cv-2 has been proposed to promote signaling by two distinct mechanisms, one that depends on Chordin and one that is independent of Chordin. The current model can explain the Chordin-independent effect of Cv-2 and suggests that the Chordin-dependent effect may result from competition between Chordin and Cv-2 for BMPs. Since Cv-2 can block binding between BMPs and Chordin, the presence of Cv-2 will impact the amount of Chordin-bound BMP. In the absence of Chordin, the amount of free BMPs is likely to be higher, and the effect of Cv-2 in promoting signaling would not be as prominent (Serpe, 2008).

The situation is different in the Drosophila wing, where both Sog and Cv-2 promote signaling in the developing PCV. A model has emerged in which Sog and Cv (Tsg2) facilitate transport of BMPs into the PCV competent zone, where processing by Tlr leads to release of BMPs, and capture by Cv-2 for presentation to receptors. Thus, Sog and Cv-2 act coordinately, through independent mechanisms, to promote BMP signaling during PCV specification. Intriguingly, binding between Cv-2 and Sog have been detected in vitro, and this may provide a direct connection between the two systems by facilitating the exchange of BMPs from Sog to Cv-2 and thus onto the receptor (Serpe, 2008).

The data presented in this study indicate that Cv-2 can have remarkably versatile effects on signaling depending on the particular context in which it acts, providing an explanation for the contradictory effects observed for members of Cv-2/Kielin family in different developmental contexts. In addition, it was demonstrated that coupling the extracellular effects with positive feedback on the production of Cv-2 itself can lead to bistable signaling wherein a very sharp transition can be generated between cells that receive high versus low levels of signal. This positive feedback thus provides a mechanism for positionally refining signaling. However, the ability of Cv-2 to promote signaling apparently does not rely solely on spatial patterns of Cv-2, Sog, and Cv expression: Cv-2 promotes signaling in cell culture, and the PCV is formed in wings in which Cv-2, Sog, and Cv are overexpressed throughout the posterior compartment. The current model of Cv-2 function shows how a cell surface ligand-binding molecule can act locally to either promote or inhibit signaling. It is noted that this model may be applicable to other molecules such as the HSPGs that have been proposed to both activate and inhibit signaling (Serpe, 2008).

Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth

Regulation of synaptic growth is fundamental to the formation and plasticity of neural circuits. This study demonstrates that Nervous wreck (Nwk), a negative regulator of synaptic growth at Drosophila NMJs, interacts functionally and physically with components of the endocytic machinery, including dynamin and Dap160/intersectin, and negatively regulates retrograde BMP growth signaling through a direct interaction with the BMP receptor, thickveins. Synaptic overgrowth in nwk is sensitive to BMP signaling levels, and loss of Nwk facilitates BMP-induced overgrowth. Conversely, Nwk overexpression suppresses BMP-induced synaptic overgrowth. Analogous genetic interactions were observed between dap160 and the BMP pathway, confirming that endocytosis regulates BMP signaling at NMJs. Finally, a correlation existes between synaptic growth and pMAD levels and Nwk regulates these levels. It is proposed that Nwk functions at the interface of endocytosis and BMP signaling to ensure proper synaptic growth by negatively regulating Tkv to set limits on this positive growth signal (O'Connor-Giles, 2008).

Nwk interacts functionally and physically with a number of known endocytic proteins, notably dynamin and Dap160. Uptake experiments showed that Nwk does not function in an internalization step of synaptic vesicle endocytosis, suggesting that Nwk affects a later step in endocytic trafficking. In agreement, colocalization was observed between Nwk and the recycling endosome-associated Rab GTPase Rab11, but not between Nwk and Rab proteins associated with either early or late endosomes. Consistent with this observation, it has been found that hypomorphic mutations in Drosophila rab11 result in a synaptic overgrowth phenotype that very closely resembles nwk (O'Connor-Giles, 2008).

Importantly, Nwk, Dap160, and dynamin are all linked to regulation of actin assembly. Recent experiments demonstrate that dynamin-mediated vesicle fission requires actin polymerization. For example, inhibiting actin polymerization blocks fission. Nwk likely facilitates a critical interaction between the endocytic machinery and actin polymerization at NMJs because it directly binds both dynamin and Wasp. Nwk also contains an F-BAR domain, which promotes membrane invagination. These domains are found almost exclusively in adaptor proteins that associate both with actin regulators and the endocytic machinery, highlighting the important links between these cellular processes (O'Connor-Giles, 2008).

The critical role of endocytic accessory proteins in linking the core endocytic machinery to cell signaling molecules is becoming increasingly clear. In addition to attenuating signaling by targeting receptors for degradation, endocytic adaptor proteins play key roles in spatial and temporal regulation of signal transduction from ligand-activated receptors. Recent work in other systems also suggests a critical role for endocytic trafficking during TGF-β/BMP signal transduction. For example, Smad phosphorylation and nuclear translocation in vertebrates depend on localization of the endosomal protein Smad anchor for receptor activation (SARA) and activated type-I and -II receptors to EEA1-positive endosomal compartments. Similarly, in the Drosophila wing, targeting of Sara, Tkv, and the ligand decapentaplegic (Dpp) to early endosomes is required for productive signaling. Nwk is ideally situated to bridge endocytosis and growth signal regulation at presynaptic terminals because of its links to actin assembly and endocytosis as well as its capacity for binding a number of proteins (including Tkv) through its multiple protein-protein interaction domains (O'Connor-Giles, 2008).

Loss of endocytic proteins results in the specific morphological phenotype of excessive satellite bouton formation, as do mutations in actin-associated proteins, including Nwk, Wasp, and components of the Scar complex. These observations suggest that satellite bouton formation may result from impairment of an actin-dependent step in endocytosis. Misregulation of a signaling pathway responsible for bouton growth and morphology in endocytic mutants may occur and, because satellite bouton formation had not been linked with any known pathway, the existence of either an unidentified positive growth signal downregulated by endocytosis or an endocytosis-dependent negative growth signal has been postulated (O'Connor-Giles, 2008).

This study show that BMP signaling regulates satellite bouton formation. Increasing levels of BMP signaling, either by expressing UAStkvACT or by reducing endogenous negative regulation of the pathway, generates a significant increase in satellite bouton formation. Further, overexpression of Nwk or Dap160 suppresses BMP-induced overgrowth, including satellite bouton formation. Finally, a direct correlation was observed between pMAD levels and satellite bouton formation in each of the genetic backgrounds analyzed. Together, these results indicate that impaired endocytic regulation of retrograde BMP signaling results in generation of satellite boutons at NMJs. In the case of endocytic and Dad mutants, downregulation of endogenous BMP signaling is impaired, while in TkvACT-expressing larvae, ectopic BMP signaling apparently overwhelms the usual mechanisms of negative regulation (O'Connor-Giles, 2008).

Although BMP signaling is required for NMJ growth, it has remained unclear whether the signal acts merely as a switch to initiate or permit growth or instead plays a more instructive role in regulating and coordinating synaptic growth. This study demonstrates a direct relationship between levels of BMP signaling and extent of synaptic growth. Neuronal expression of a single copy of TkvACT results in a modest increase in pMAD and no significant increase in synaptic growth, whereas expression of two copies induces a dramatic increase in pMAD levels and extensive synaptic overgrowth, both of which are suppressed by overexpression of Nwk. Further, it was found that mutations in the endogenous negative regulator Dad also cause increased synaptic growth. These data indicate that the level of BMP signaling has an instructive role in governing synaptic size and complexity and reveal the importance of interactions between positive and negative regulators that modulate the growth signal in response to internal and external cues (O'Connor-Giles, 2008).

The results demonstrate that endocytosis is an important regulatory mechanism for attenuating BMP signaling at synapses. Previous work suggested that Hiw, an E3 ubiquitin ligase and negative regulator of synaptic growth, also acted to limit BMP signaling. However, subsequent work demonstrated that pMAD levels are not increased in hiw, and no effects of Hiw overexpression on BMP signaling have been described. In addition, hiw synapses are extremely expansive, elaborately branched, and contain numerous small boutons, but no satellite boutons. This phenotype is distinct from that associated with TkvACT overexpression or other genotypes believed to elevate BMP signaling, including Dad, nwk, and known endocytic genes -- all of which exhibit satellite boutons. Together with the recent finding that Hiw regulates MAPKKK-dependent Fos activity, these observations suggest that Hiw and BMP signaling may regulate different aspects of synaptic growth (O'Connor-Giles, 2008).

In their recent study of Spict, Wang (2007) demonstrated the localization of Spict to early endosomes along with data suggesting that Spict plays a role in BMP receptor trafficking (Wang, 2007). For example, Spict overexpression in S2 cells caused Wit to relocalize to early endosomes, suggesting negative regulation of BMP signaling by sequestering Wit receptor and/or Wit-Gbb signaling complexes. However, studies of Wit trafficking at spict NMJs, which are limited by the small size of boutons and the lack of reliable antibodies, did not uncover this trafficking defect but instead revealed increased Wit levels consistent with a role for Spict in the degradation of Wit. Nonetheless, the results from the analysis of Spict support the idea that endocytic regulation of BMP signaling is required for proper synaptic growth. It will be interesting to determine whether Spict interacts with Nwk or plays a distinct role in the regulation of BMP signaling at synapses (O'Connor-Giles, 2008).

Overall, the current findings support a model in which Nwk constrains synaptic growth by regulating endocytic trafficking of Tkv to attenuate positive retrograde growth signaling. While the simplest model is that Nwk targets Tkv for degradation, gross differences were not observed in levels of ectopically expressed Tkv-GFP in otherwise wild-type, nwk, and C155GAL4; UAS-nwk larvae (unpublished data). A caveat is that, without the necessary antibody reagents, it was not possible to look at endogenous Tkv. Thus, these observations might not accurately reflect normal receptor trafficking but rather the fact that high levels of Tkv can override endogenous regulation. Nonetheless, together with the observation that Nwk colocalizes with Rab11, this finding is consistent with a role for Nwk in BMP receptor recycling. For example, Nwk might attenuate BMP signaling levels by regulating the rate at which vacant Tkv receptors are recycled back to the plasma membrane following activation and internalization. Nwk might also regulate the trafficking of unbound receptors from the plasma membrane. Previous work in vertebrates demonstrates that TGF-β receptors are recycled through a Rab11-dependent mechanism independent of ligand binding, possibly as a means of rapidly and dynamically regulating surface receptor number and, thus, sensitivity to TGF-β. Interestingly, the relocalization of Nwk from a more uniform to a more punctate expression pattern was observed upon overexpression of Tkv, consistent with recruitment of Nwk to regulate trafficking of ectopic Tkv. Nwk might also have localized effects within boutons, for example, by restricting sites of BMP signaling through spatial regulation of receptor recycling. It is intriguing to speculate that disruption of spatial constraints on BMP signaling results in ectopic bouton division and, thus, satellite bouton formation. Such a mechanism could provide a critical means for effecting localized changes to existing synapses that underlie neural plasticity. A conceptually similar Rab11-dependent process for the asymmetric activation of Notch signaling in the developing nervous system has recently been described. A future challenge will be to further dissect the regulatory mechanisms that control the levels, timing, and localization of BMP signaling at synapses. These studies will advance understanding of the dynamic regulation of synaptic growth and plasticity and likely provide additional general insights into the intricate role of endocytosis in signal transduction (O'Connor-Giles, 2008).

Formation of the long range Dpp morphogen gradient

Dpp acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion, and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. This study analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. Novel genetic tools were then used, experimentally monitoring the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Importantly, results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Together these results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution (Schwank, 2011).

One outcome of the modeling was the prediction that RMT and RED mechanisms could be discriminated by analyzing Dpp levels behind receptor mutant clones. While in the transcytosis model these levels should be significantly decreased, they would be almost unaltered in the diffusion model. This difference stems from the uptake of Dpp by its receptors, which is an essential feature for morphogen transport by RMT, but not by RED. The experimental results revealed that neither GFP:Dpp levels nor Dpp signaling activity is reduced behind receptor mutant clones, excluding a significant role for receptor-mediated transcytosis in Dpp gradient formation. Important support for this conclusion was provided by situations where 'islands' of wild-type cells received Dpp signal despite being surrounded by mutant tissue, ruling out the possibility that Dpp reaches the distal side of receptor mutant clones by being transported around the clones. When analyzing the GFP:Dpp distribution in mosaic tissues, it was also found that the Dpp levels are not significantly reduced within receptor mutant clones. While this outcome further argues against the RMT model, it is consistent with the 'external-unbound limit case scenario,' representing RED with the majority of Dpp not being bound to Tkv. Indeed, in the GOF experiments the ratio of unbound Dpp could be narrowed down to approximately 60%-80% (Schwank, 2011).

If transcytosis is modeled in a receptor-independent manner, the effects on Dpp distribution by receptor mutant clones do not differ significantly from those in the restricted extracellular diffusion scenario. Thus, receptor-independent transcytosis, for example via fluid phase uptake, remains a possible mechanism for Dpp gradient formation. Several other studies, however, support the restricted extracellular diffusion model. Based on theoretical grounds, it has been proposed that diffusive mechanisms for Dpp gradient formation are more likely than non-diffusive ones. Moreover, experimental studies on heparan sulfate proteoglycans (HSPGs), in particular glypicans, demonstrated the necessity of an intact ECM for morphogen movement. In the Drosophila wing disc, clones mutant for the glypicans Dally and Dally-like (Dlp) disrupted the formation of the Dpp gradient. Dally was also shown to bind Dpp, to stabilize it on the cell surface, and to influence its mobility (Schwank, 2011).

However, although the evidence that glypicans assist extracellular diffusion of Dpp seems compelling, alternative or additional functions of glypicans in Dpp distribution cannot be excluded. For example, a recent study suggests that apically localized Dlp binds to the Wingless (Wg) morphogen in the Wg producing region, undergoes internalization, and thereby redistributes Wg to the basolateral compartment where Wg spreads to form a long-range gradient. It is possible that recycling of glypicans is also involved in Dpp relocalization and that this process is important for Dpp movement. Consistent with such a notion, another study reported that dynamin-dependent endocytosis is necessary for Dpp movement. Blocking such a ubiquitous cellular machinery, however, not only inhibits the recycling of receptors and glypicans, but may also change the composition and distribution of glypicans in the ECM, which in turn might impede extracellular diffusion. Given that the phenotypes of receptor clones fully conform to the simplest model of Dpp movement along the ECM (restricted extracellular diffusion), the view is favored that the main function of glypicans for Dpp gradient formation is to facilitate Dpp diffusion along the ECM (Schwank, 2011).

The observation that receptor mutant clones do not have a major effect on the Dpp gradient contradicts previous observations in which ablation of tkv in small lateral clones leads to an accumulation of Dpp at the side of the clone facing the source, arguing for a block of Dpp movement within such clones. The different results could be explained by the presence of brk in the previous study. The ectopic up-regulation of brk in tkv mutant clones, which in most cases leads to clone elimination, most likely also causes drastic changes in the transcriptional program in 'escaper' cells. Thus the sharp increase in GFP:Dpp levels at the proximal edge inside tkv mutant clones (facing the Dpp source) could be accounted for by increased levels of Dpp binding proteins, a theory which is supported by the fact that Dpp accumulation was strictly clone-autonomous and not in cells ahead of the clones. In the current experimental setup, such secondary effects were avoided by simultaneously removing tkv together with brk. As the negative control (Mad brk clones) shows, the signaling state of these cells (Dpp signaling off, no Brk) does not significantly alter the Dpp profile (Schwank, 2011).

Transport along cytonemes is another proposed model for the dispersal of Dpp. In its simplest implementation, this model assumes that imaginal disc cells form filopodial extensions towards the Dpp producing region and that Dpp is shuttled along these extensions by binding to Tkv. In this scenario, Tkv GOF clones would not only lead to an increase of receptors inside the clones, but also along the cytonemes, and thus affect the Dpp profile also ahead of the clones. This, however, was not observed in the current experiments, and the restricted extracellular diffusion model is favored over the cytoneme model for Dpp gradient formation (Schwank, 2011).

During development morphogens function as short-range or long-range signals in order to specify cell fates within a tissue. For example, during wing disc development the range of Hh signaling is relatively short compared to that of Dpp, with a functional range of approximately 10 cells versus 40 cells, respectively. It is likely that properties of the transport system are important determinants of the range of a morphogen. In the restricted diffusion model, morphogen spreading is impeded by ECM proteins and cell surface receptors, which efficiently trap their ligand at the cell surface and direct it to degradation. Thus one mechanism to control the range of a morphogen gradient is regulating the receptor levels. Indeed, the Hh as well as the Dpp system appear to make use of this strategy to regulate their range. The Hh signal limits its range by upregulating the expression of its binding receptor Patched (Ptc), while the Dpp signal broadens its range by downregulating the expression of its receptor Tkv. The effects of Tkv LOF and GOF clones on the Dpp profile suggest that the majority of Dpp is not bound to the receptor Tkv. It is tempting to speculate that the Dpp-Tkv binding properties represent an additional property of the Dpp signaling system that facilitates the formation of a long-range gradient, by assuring that the majority of Dpp remains in a free and unbound state. Just like lower receptor levels, a lower binding constant would contribute to the spread of Dpp, due to reduced immobilization and degradation of Dpp. It remains to be seen if the ratio of bound to unbound ligand differs for long- versus short-range morphogens and if this ratio represents a general means to regulate the range of morphogen gradients (Schwank, 2011).

Spinster controls Dpp signaling during glial migration in the Drosophila eye; Spinster appears to antagonize Dpp signaling by facilitating the routing of Dpp receptors toward the lysosome

The development of multicellular organisms requires the well balanced and coordinated migration of many cell types. This is of particular importance within the developing nervous system, where glial cells often move long distances to reach their targets. The majority of glial cells in the peripheral nervous system of the Drosophila embryo is derived from the CNS and migrates along motor axons toward their targets. In the developing Drosophila eye, CNS-derived glial cells move outward toward the nascent photoreceptor cells, but the molecular mechanisms coupling the migration of glial cells with the growth of the eye imaginal disc are mostly unknown. This study used an enhancer trap approach to identify the gene spinster, which encodes a multipass transmembrane protein involved in endosome-lysosome trafficking, as being expressed in many glial cells. spinster mutants are characterized by glial overmigration. Genetic experiments demonstrate that Spinster modulates the activity of several signaling cascades. Within the migrating perineurial glial cells, Spinster is required to downregulate Dpp (Decapentaplegic) signaling activity, which ceases migratory abilities. In addition, Spinster affects the growth of the carpet cell, which indirectly modulates glial migration (Yuva-Aydemir, 2011).

During development of the nervous system, glia cell migration is tightly regulated in time and space. This study has demonstrated that, within the Drosophila visual system, glial migration requires the late endosomal/lysosomal protein Spinster to restrict a migratory stimulus provided by Dpp signaling. Spinster is crucially involved in the coordination of eye disc growth and glial migration (see Schematic view on spinster function). In spinster mutant eye discs, late endosomes accumulate and a glial overmigration phenotype develops. This migration phenotype can be modified by reducing the early to late endosomal transfer or late endosome-to-lysosome transfer through decreasing the levels of hrs. Moreover, the Dpp receptor Thickveins accumulates in spinster mutant cells. Thus, Spinster appears to antagonize Dpp signaling by facilitating the routing of Dpp receptors toward the lysosome (Yuva-Aydemir, 2011).

Retinal glial cells are born in the CNS and migrate onto the eye disc through the optic stalk. A reverse migratory direction is taken by the photoreceptor cell axons, which are born in the periphery and navigate toward the brain through the optic stalk. Peripheral glial cells generally follow axonal growth cones during their migration phase (Klämbt, 2009), and thus within the developing Drosophila visual system, glial cells have to be guided to the nascent photoreceptor axons by other means. In Drosophila, this is accomplished by the carpet cells, very large subperineurial cells that cover the entire eye field (Silies, 2007). The carpet cell shields the navigating axons within the stalk from the proliferating and migrating glial population. At the beginning of eye imaginal disc development, the carpet cell prevents precocious migration of glial cells onto the eye disc in a process requiring early eye patterning genes and Hedgehog signaling. Ablation of the carpet glia as well as disruption of, for example, early Hedgehog signaling result in an overshooting glial migration (Silies, 2007). As soon as the morphogenetic furrow is initiated, the carpet glial cell starts to grow and extends anteriorly. However, the carpet cell never reaches the morphogenetic furrow but stops to grow at a few cell rows behind where nascent photoreceptor cell axons are formed. Piggybacking on the carpet cell are the perineurial glial cells that migrate toward the anterior edge of the carpet cell. As soon as they come in contact with photoreceptor axons, they drop from the carpet cell and now follow the sensory axon toward the brain. Thus, two distinct phases of glial cell migration can be defined. Initially, the carpet cell prevents precocious migration, whereas in later phases of eye imaginal disc development the carpet cells provide a permissive substrate for the migrating perineurial glia (Yuva-Aydemir, 2011).

Spinster is involved in the regulation of both of these migratory phases and helps to coordinate the growth of the eye disc with the migration of glial cells. The most dramatic consequences of spinster mutants as seen in eye disc duplications are presumably attributable to effects on Wingless and Dpp within the eye disc. The broad glial overshooting phenotypes correlate with a failure of the carpet cell to prevent early-onset, precocious glial migration. The later glial overmigration phenotypes correlate with late, carpet cell-independent, defects in the perineurial glia. Thus, it is not surprising that, in contrast to panglial expression of Spinster, glial cell type-specific expression is not able to rescue the spinster overmigration phenotype (Yuva-Aydemir, 2011).

The size of the carpet glial cell is reduced in spinster mutants, and MARCM analysis as well as cell type-specific RNAi experiments indicate that spinster controls carpet cell growth cell autonomously. Directed expression of Hid or RicinA specifically in carpet cells resulted in a reduced cell size and a concomitant induction of ectopic glial cell migration (Silies, 2007). In contrast, single spinster mutant carpet cells did not lead to a glial migration defects, suggesting that the reduced carpet cell only contributes to the glial overmigration phenotype. How spinster controls the size of the carpet cells is currently unknown. Possibly, within the carpet cell, spinster acts via hedgehog signaling. Hedgehog bound to the Patched receptor is normally internalized and degraded in lysosomes. Moreover, Patched is expressed in glial cells and Hh signaling was suggested to prevent precocious glial cell migration through a yet-unknown pathway. However, mutations in different hedgehog signaling components did not suppress the spinster phenotype and inhibition of hedgehog signaling in glial cells did neither alter the morphology of the carpet glia nor did they affect glial migration. Likewise, activation of Hedgehog signaling through expression of PKAdsRNA did not influence glial migration (Yuva-Aydemir, 2011).

Within the carpet cells, Spinster most likely does also not act through altered Dpp signaling, since no phospho-Mad expression was detected in the carpet cells. In contrast, enhanced Dpp signaling (Mad phosphorylation) was noted in the migrating perineurial glial cells. The analysis of additional mutations affecting different aspects of intracellular vesicle dynamics revealed the importance of endocytic processes in TGF-β signaling. The block of Rab5 function causes a reduction in the range of Dpp target gene activation, and Rab5 overexpression causes increased Dpp signaling range. Hrs, a component of ESCRT complex, has been shown to interact with Smad2 and participates in the recruitment of Smad2 to the activated receptor. Hrs seems to be involved in the constitutive ligand-independent receptor turnover since Tkv accumulates at the cell membrane even in the absence of Dpp. Hrs downregulation suppresses the spinster glial overmigration, possibly by inhibiting Tkv internalization and accumulation in the endosomes. Within the cell, endosome-to-lysosome transfer and subsequent lysosomal degradation also affects Dpp signaling. Enhanced degradation caused by expression of activated Rab7 reduces the Dpp signaling range, whereas the inhibition of lysosome function with chloroquine leads to an endosomal accumulation of ligands. In addition, pan-glial expression of a dominant-negative Tkv receptor, tkv?GS or tkvdsRNA, resulted in reduced glial migration and fewer glial cells. Similar results were obtained when put or mad expression were silenced using RNA interference, demonstrating that the regulation of Dpp signaling via controlling vesicle dynamics is required in glial cells to ensure normal proliferation and migration (Yuva-Aydemir, 2011).

Endocytosis and vesicle recycling is generally required in migrating cells to dynamically remodel their adhesive contacts and locate active signaling receptors to the front of the cell in response to extracellular signals. Integrins from the cell rear can be relocalized to the leading edge, and during border cell migration in the Drosophila ovary, spatial restriction of the receptor tyrosine kinase signaling by endocytosis ensures the localized intracellular response to guidance cues. Likewise, glial migration in the embryonic Drosophila PNS is regulated by the fine-tuning of Notch signaling by Numb-mediated endocytosis. Thus, endocytotic trafficking may affect cell migration through several pathways (Yuva-Aydemir, 2011).

In spin mutants, enhanced Dpp signaling is also observed in the eye imaginal disc and the most extreme eye disc phenotype appears as to be an induction of an ectopic morphogenetic furrow perpendicular to the normal furrow. Ectopic expression of the Dpp in the eye disc or expression of activated Tkv in the glial cells increases glial cell proliferation. In spin mutants, both increased Dpp expression and accumulation of Tkv are observed. Since spin controls glial cell proliferation cell autonomously, this is likely attributable to the accumulation of Tkv in glial cells. In addition, enhanced Mad phosphorylation is observed in spin mutants, especially in the optic stalk glia. In agreement with this, the increase in glial cell number is mostly noted in the optic stalk. Interestingly, the function of spinster in controlling glial differentiation is also required in the wrapping glia. Loss of spinster results in a reduced wrapping of axonal membranes, which can be rescued by reexpression of spinster in the wrapping glia. Thus, spinster function appears to be needed to extend cellular processes during migration and differentiation (Yuva-Aydemir, 2011).

In conclusion, Spinster affects glial migration on two different levels. Spinster regulates carpet glia differentiation, which indirectly affects early migration, and subsequently, Spinster acts in the perineurial glia to confer a general cell motility signal (Yuva-Aydemir, 2011).

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).

Relay of retrograde synaptogenic signals through axonal transport of BMP receptors

Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. This study shows that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. It was further demonstrated that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast with these results, no evidence was found for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations (Smith, 2012).

Axonal transport is essential for neuronal function and survival. In Drosophila motoneurons the BMP pathway is activated at the synaptic terminal but ultimately results in regulation of transcription in the nucleus, thus this signaling pathway is dependent on long-range signal propagation. The current results indicate that signal relay is mediated by a signaling endosome containing an activated receptor complex formed by Wit and Tkv (Smith, 2012).

A target-derived factor that is critical to ensure the survival and growth of selected neurons, NGF, utilizes a receptor signaling endosome to propagate pathway activity. NGF binds its receptors at the synaptic terminal and the complex is endocytosed and retrogradely transported along the axon in a Dynein dependent manner to activate downstream effector molecules. This study has found evidence that the BMP receptors colocalize with endosomal markers and are likely endocytosed at the synaptic terminal. This is supported by the exclusive colocalization of the receptors with Rab4 at the synaptic terminal, indicating that the receptors are subject to rapid membrane recycling in this subcellular compartment. This result agrees with the source of the BMP ligand Gbb being the muscle and signaling locally to the synaptic terminal. The BMP receptors colocalize with each other and are actively transported along the axon. In addition, this study found that both motoneuron BMP signaling and BMP receptor traffic are dependent on retrograde motor activity, similar to NGF signaling. Several published reports support these findings. First, TGF-β was found to be transported along mammalian motoneuron axons. The dependence on Gbb for endosome motility strongly suggests that the ligand is part of the signaling endosome complex. Second, several studies have shown that the endocytic pathway regulates BMP signaling in Drosophila motoneurons. Spichthyin (Spict) and Nervous wreck (Nwk) down-regulate the BMP signal at the synaptic terminal and, when mutated, cause BMP dependent synaptic terminal overgrowth. The late endosomal/lysosomal protein Spinster, when mutated, causes enhanced/ misregulated BMP pathway signaling resulting in synaptic terminal expansion. Loss of Vps35, an endocytic sorting protein, leads to BMP dependent upregulation of synaptic size, and a similar phenotype is observed in mutants of the novel endosomal protein Ema, with increased levels of Tkv and pMad at the NMJ (Kim, 2010). Finally, a recent report has proposed that sorting nexin SNX16 interacts with Nwk to regulate BMP signaling-dependent synaptic growth through endocytic routing of activated Tkv (Rodal, 2011). Taken together, these reports show that endocytic proteins regulate BMP signaling and that a signaling endosome is a plausible mechanism for signal sorting and attenuation in this pathway. This again parallels the situation of neurotrophin receptors and the endocytic pathway they share with neurotoxins (Smith, 2012).

An important question is the mechanism to preferentially select and transport active signaling endosomes as opposed to other vesicles that contain BMP receptors. The receptors, Wit and Tkv, could be transported individually, they could be in the same endosome without being in a complex, or the receptors could be in an active complex. All of these endosomes can either be degraded or destined for long-range trafficking, and in the case of those containing active receptor complexes ultimately lead to the phosphorylation of Mad in the soma. The skewed directionality of receptor traffic (2:1 ratio for retrograde to anterograde) is difficult to interpret with the current knowledge, and a mechanism by which the transport machinery selects the endosome that is positive for an active receptor complex and carries it to its final destination in the cell body has yet to be determined. Somehow this mechanism must be linked to BMP signaling itself, considering the diminished receptor traffic in the absence of either Wit or Gbb. Adaptor proteins that function to attach cargo to motor proteins may provide a specialized mechanism to preferentially select and transport active signaling endosomes, as has been shown in other cases of axonal transport. In this regard it is worth noting that the regulatory light chain of the Dynein complex Tctex interacts physically with the mammalian orthologue of Wit, BMPR-II). Dynein light and intermediate chains also interact with the NGF receptors TrkA and TrkB, further supporting the parallelism between NGF and BMP signaling endosomes (Smith, 2012).

The existence of a BMP signaling endosome in Drosophila larval motoneurons also raises questions as to how general this mechanism is for sorting and regulating the TGF-β signal. Smad Anchor for Receptor Activation (SARA) is an endocytic protein that regulates the subcellular distribution of Smad proteins and presents these R-Smads for phosphorylation to the activated TGF-β receptor complex. In mammalian cells SARA regulates TGF-β signaling by sorting the receptor complex to degradation or signaling pathways. In Drosophila wing imaginal discs, BMP signaling depends on SARA to properly distribute a gradient of the morphogen Decapentaplegic (Dpp). SARA, the ligand Dpp, and the type I receptor Tkv were found in a population of endosomes that associated with the spindle machinery. This association allows equal distribution of the Dpp morphogen into the two daughter cells during mitosis and proper activation of Mad. It seems then that BMP signaling spatial regulation through endocytic traffic is a general phenomenon, and not a specialization of extremely long cells such as motoneurons (Smith, 2012).

The dual localization of pMad at the synaptic terminal and nucleus has been well characterized. This suggests that pMad is the molecular carrier of the signaling event, purportedly transported from synaptic terminal to cell nucleus. While this model has been proposed, no experimental evidence supports it, and the current results show that axonal transport of pMad is an unlikely mechanism for pathway relay. Similar to the situation with the BMP receptors, Mad was found in the cell body, nucleus, axon and synaptic terminal. However, no evidence was found of the active form pMad along the axon when ample signal is observed at the synaptic terminal and nucleus. Furthermore, while disrupting Dynein retrograde motors abrogates BMP signaling and substantially interrupts the axonal traffic of BMP receptors, axonal movement of Mad is not affected. It is possible that a small amount - undetectable by standard imaging techniques - of pMad is present along the axon, and it is also possible that a small fraction of the total YFP-Mad in the FRAP experiments is subject to active retrograde traffic towards the nucleus. In neurotrophin signaling the transcription factor CREB is transported along the axon with the receptor complex to act as a second messenger. However, efforts to detect axonal colocalization of YFPMad and Tkv-CFP containing endosomes have been unsuccessful (Smith, 2012).

The presence of Mad along the axons without detectable phosphorylation by the activated receptor complex brings up the issue of substrate accessibility. It is possible that factors that help Mad phosphorylation, such as SARA are not present or active in the axons. It is also possible that Mad cannot access the active receptor complex due to hindrance by the linkers between receptor and molecular motors. It is worth noting β the two pMad populations show different immunofluorescence patterns, punctate at the NMJ and diffuse in the nucleus. The molecular nature of these different pMad isoforms is unclear, but at least part of the difference may stem from single versus double phosphorylation of the SVS Mad sequence. This is conceptually similar to ERK isoforms that are differentially phosphorylated by neurotrophin receptors at the synaptic terminal and the cell soma. An alternative explanation is that identically phosphorylated forms of Mad bind different partners at NMJ and nucleus, and that the complex formed at the NMJ cannot be recognized by the monoclonal antibodies due to steric hindrance. It is proposed that synaptic pMad is a different population with a distinct local role, perhaps signaling through cross talk with other pathways, while nuclear pMad is the result of phosphorylation of Mad in the soma by activated receptors retrogradely transported from the synaptic terminal. Consistent with this model of different pMad isoforms with different roles in larval motoneurons, a recent study found that synaptic pMad is regulated differently than nuclear pMad. Synaptic pMad was found to be absent in importin-beta11 mutants, but nuclear pMad was unaffected (Smith, 2012).

Identifying the mechanism by which the BMP signal is relayed along the axon is a first step towards understanding the regulation of synaptic plasticity by this critical pathway. Intriguingly, a recent study has linked Spinal Muscular Atrophy (SMA) and the BMP pathway. Spinal Muscle Atrophy is a recessive hereditary neurodegenerative disease in humans that results in early onset lethality, motor neuron loss, and skeletal muscle atrophy. Using a Drosophila ortholog to model this disease, the Wit receptor and Mad transcription factor were identified to modify the disease phenotype. It was proposed that increasing BMP signaling could be a possible therapeutic approach for SMA patients. The current results describing the mechanism of BMP signaling in Drosophila motoneurons helps understand the pathological consequences of pathway disruption and will open new avenues to understand human neurodegenerative disorders that involve TGF-β signaling. Additionally, the current results suggest that signaling endosome traffic is a general mechanism in TGF-β signaling (Smith, 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).

Scribbled optimizes BMP signaling through its receptor internalization to the Rab5 endosome and promote robust epithelial morphogenesis

Epithelial cells are characterized by apical-basal polarity. Intrinsic factors underlying apical-basal polarity are crucial for tissue homeostasis and have often been identified to be tumor suppressors. Patterning and differentiation of epithelia are key processes of epithelial morphogenesis and are frequently regulated by highly conserved extrinsic factors. However, due to the complexity of morphogenesis, the mechanisms of precise interpretation of signal transduction as well as spatiotemporal control of extrinsic cues during dynamic morphogenesis remain poorly understood. Wing posterior crossvein (PCV) formation in Drosophila serves as a unique model to address how epithelial morphogenesis is regulated by secreted growth factors. Decapentaplegic (Dpp), a conserved bone morphogenetic protein (BMP)-type ligand, is directionally trafficked from longitudinal veins (LVs) into the PCV region for patterning and differentiation. These data reveal that the basolateral determinant Scribbled (Scrib) is required for PCV formation through optimizing BMP signaling. Scrib regulates BMP-type I receptor Thickveins (Tkv) localization at the basolateral region of PCV cells and subsequently facilitates Tkv internalization to Rab5 endosomes, where Tkv is active. BMP signaling also up-regulates scrib transcription in the pupal wing to form a positive feedback loop. These data reveal a unique mechanism in which intrinsic polarity genes and extrinsic cues are coupled to promote robust morphogenesis.

This study shows that the Scrib complex, a basolateral determinant, is a novel feedback component that optimizes BMP signaling in the PCV region of the Drosophila pupal wing (Gui, 2016).

During PCV development, limited amounts of Dpp ligands are provided by the Dpp trafficking mechanism. Furthermore, amounts of receptors appear to be limited since tkv transcription is down-regulated in the cells in which the BMP signal is positive, a mechanism that serves to facilitate ligand diffusion and sustain long-range signaling in the larval wing imaginal disc. To provide robust signal under conditions in which both ligands and receptors are limiting, additional molecular mechanisms are needed. Previous studies suggest that two molecules play such roles. Crossveinless-2 (Cv-2), which is highly expressed in the PCV region, serves to promote BMP signaling through facilitating receptor-ligand binding. Additionally, the RhoGAP protein Crossveinless-c (Cv-c) provides an optimal extracellular environment to maintain ligand trafficking from LVs into PCV through down-regulating integrin distribution at the basal side of epithelia. Importantly, both cv-2 and cv-c are transcriptionally regulated by BMP signaling to form a feedback or feed-forward loop for PCV formation (Gui, 2016).

Scrib, a third component, sustains BMP signaling in the PCV region in a different manner. First, to utilize Tkv efficiently, Scrib regulates Tkv localization at the basolateral region in the PCV cells, where ligand trafficking takes place. Regulation of receptor localization could be a means of spatiotemporal regulation of signaling molecules during the dynamic process of morphogenesis. Second, to optimize the signal transduction after receptor-ligand binding, Scrib facilitates Tkv localization in the Rab5 endosomes. Localization of internalized Tkv is abundant at Rab5 endosomes in the PCV region of wild-type, but not scrib RNAi cells. While the physical interaction between Scrib, Tkv and Rab5 in the pupal wing remains to be addressed, the data in S2 cells suggest that physical interactions between these proteins are key for preferential localization of Tkv at the Rab5 endosomes. Recently, Scrib has been implicated in regulating NMDA receptor localization through an internalization-recycling pathway to sustain neural activity. Therefore, Scrib may be involved in receptor trafficking in a context-specific manner, the molecular mechanisms of which, however, remain to be characterized. Third, BMP/Dpp signaling up-regulates scrib transcription in the pupal wing. Moreover, knockdown of scrib leads to loss of BMP signaling in PCV region but not loss of apical-basal polarity. These facts suggest that upregulation of Scrib is key for optimizing BMP signaling by forming a positive feedback loop (Gui, 2016).

Previous studies indicate that cell competition takes place between scrib clones and the surrounding wild-type tissues in the larval wing imaginal disc. Moreover, cell competition has been documented between loss-of-Dpp signal and the surrounding wild-type tissues. It is presumed that the mechanisms proposed in this study are independent of cell competition for the following reasons. First, scrib RNAi and AP-2μ RNAi data reveal that loss of BMP signal in the PCV region is produced without affecting cell polarity. Thus, cell competition is unlikely to occur in this context. Second, BMP signal is intact in scrib mutant clones of the wing imaginal disc, suggesting that cell competition caused by scrib clones is not a direct cause of loss of BMP signaling in scrib mutant cells (Gui, 2016).

Previous studies established that receptor trafficking plays crucial roles in signal transduction of conserved growth factors, including BMP signaling. Several co-factors have been identified as regulators of BMP receptor trafficking. Some of them down-regulate BMP signaling while others help maintain it. It is proposed that the Scrib-Rab5 system is a flexible module for receptor trafficking and can be utilized for optimizing a signal. During larval wing imaginal disc development, BMP ligands are trafficked through extracellular spaces to form a morphogen gradient. Although previous studies indicate that regulation of receptor trafficking impacts BMP signaling in wing imaginal discs, BMP signaling persists in scrib or dlg1 mutant cells in wing discs. Wing disc cells interpret signaling intensities of a morphogen gradient. In this developmental context, an optimizing mechanism might not be beneficial to the system. In contrast, cells in the PCV region use the system to ensure robust BMP signaling (Gui, 2016).

Taken together, these data reveal that a feedback loop through BMP and Scrib promotes Rab5 endosome-based BMP/Dpp signaling during PCV morphogenesis. Since the components (BMP signaling, the Scrib complex, and Rab5 endosomes) discussed in this work are highly conserved, similar mechanisms may be widely utilized throughout Animalia (Gui, 2016).

Neuroligin 4 regulates synaptic growth via the Bone morphogenetic protein (BMP) signaling pathway at the Drosophila neuromuscular junction

The neuroligin (Nlg) family of neural cell adhesion molecules is thought to be required for synapse formation and development, and has been linked to the development of autism spectrum disorders in humans. In Drosophila melanogaster, mutations in the neuroligin 1-3 genes have been reported to induce synapse developmental defects at neuromuscular junctions (NMJs), but the role of neuroligin 4 (dnlg4) in synapse development has not been determined. This study reports that the Drosophila Neuroligin 4 (DNlg4) is different from DNlg1-3 in that it presynaptically regulates NMJ synapse development. Loss of dnlg4 results in reduced growth of NMJs with fewer synaptic boutons. The morphological defects caused by dnlg4 mutant are associated with a corresponding decrease in synaptic transmission efficacy. All of these defects could only be rescued when DNlg4 was expressed in the presynapse of NMJs. To understand the basis of DNlg4 function, genetic interactions were sought, and connections were found with the components of the bone morphogenetic protein (BMP) signaling pathway. Immunostaining and western blot analyses demonstrated that the regulation of NMJ growth by DNlg4 was due to the positive modulation of BMP signaling by DNlg4. Specifically, BMP type I receptor Tkv abundance was reduced in dnlg4 mutants, and immunoprecipitation assays showed that DNlg4 and Tkv physically interacted in vivo. This study demonstrates that DNlg4 presynaptically regulates neuromuscular synaptic growth via the BMP signaling pathway by modulating Tkv (Zhang, 2017).

The formation, development, and plasticity of synapses are critical for the construction of neural circuits, and the Drosophila larval neuromuscular junction (NMJ) is an ideal model system to dissect these processes. In the past few decades, several subcellular events and signaling pathways have been reported to be involved in regulating synaptic growth at Drosophila NMJs, such as local actin assembly, endocytosis, ubiquitin-mediated protein degradation, the Wingless pathway, and the bone morphogenetic protein (BMP) pathway. Among these, BMP signaling is thought to be a major retrograde pathway that promotes the synaptic growth of NMJs (Zhang, 2017).

At the Drosophila NMJ, the BMP homolog Glass bottom boat (Gbb) is released by muscle cells and binds to the presynaptic type II BMP receptor Wishful thinking (Wit). Wit is a constitutively active serine/threonine kinase and, upon binding to Gbb, forms a complex with the type I BMP receptor Thickvein (Tkv) or saxophone (Sax), which results in their activation by phosphorylation. The activated type I receptor subsequently phosphorylates the downstream R-Smad protein Mothers against decapentaplegic (Mad). Phosphorylated Mad (pMad) then binds to the co-Smad Medea (Med). This complex translocates to the nucleus of motoneurons to activate or repress the transcription of target genes required for NMJ growth. Mutation of any component in the BMP signaling pathway results in a striking deficiency of NMJ growth. In addition, many molecules are reported to affect NMJ growth by negatively regulating BMP signaling at different points in the pathway. This study report that Drosophila Neuroligin 4 (DNlg4), a trans-synaptic adhesion protein, acts as a positive regulator of BMP signaling to regulate NMJ growth (Zhang, 2017).

Neuroligins (Nlgs) were initially reported to be the postsynaptic ligands of the presynaptic adhesion proteins neurexins (Nrxs), and loss of function of Nlgs in humans is thought to be associated with several mental disorders, including autism and schizophrenia. Nlgs are an evolutionarily conserved family of proteins encoded by four independent genes in rodents and five independent genes in humans. Nlgs have been reported to induce synapse assembly by co-cultured neurons when expressed in nonneuronal cells, and overexpression of Nlgs in neurons increases synapse density. These in vitro cell culture studies suggest a role of Nlgs in inducing the formation of synaptic contacts. However, an in vivo study showed, despite severe defects in synaptic transmission, that there was no alteration of synapse number in neurons from nlg1-3 triple knock-out mice. Similarly, loss of Nlg1 specifically in the hippocampus or amygdala did not alter the synapse number, suggesting that the role of Nlgs is not to trigger the initial synapse formation. Rather, it is more likely that upon binding to Nrx, Nlg functions in maturation of nascent synapses, including differentiation and stabilization by recruiting scaffolding proteins, postsynaptic receptors, and signaling proteins (Zhang, 2017).

In Drosophila, four Nlgs have been identified. Mutations in dnlg1-3 result in defective synapse differentiation that is primarily characterized by abnormal protein levels or the ectopic postsynaptic localization of glutamate receptors in larval NMJs. In addition, loss of dnlg1-3 separately leads to impairment in NMJ synapse development, as indicated by abnormal synaptic bouton number, but the precise underlying mechanism is poorly understood. A recent study showed that flies with a dnlg4 mutation exhibit an autism-related phenotype of behavioral inflexibility, as indicated by impaired reversal learning. DNlg4 also regulates sleep by recruiting the GABA receptor to clock neurons and thus modulating GABA transmission, which suggests a role of DNlg4 in synapse differentiation. However, potential molecular mechanisms underlying the behavioral defects caused by dnlg4 mutation and the role of DNlg4 in synapse development have not been reported (Zhang, 2017).

This paper reports the generation of an independent null allele of dnlg4 and characterized the role of DNlg4 in neuromuscular synaptic growth. Loss of DNlg4 led to impaired NMJ synapse growth, as indicated by decreased synaptic bouton numbers and increased bouton size. Presynaptic knockdown of DNlg4 mimicked these phenotypes. These morphological abnormalities in dnlg4 mutants led to corresponding impairment in synaptic transmission efficacy. Unexpectedly, all of these defects were only rescued when DNlg4 was expressed in presynaptic, instead of postsynaptic, areas of NMJs.DNlg4 genetically interacted with components of the BMP pathway and that the presynaptic BMP signaling at NMJs was decreased in dnlg4 mutants. A reduction of the BMP type I receptor Tkv was observed in dnlg4 mutants and DNlg4 physically interacted with Tkv in vivo. Altogether, this study revealed that DNlg4 regulated neuromuscular synaptic growth by positively modulating BMP signaling though Tkv (Zhang, 2017).

Sequence analyses showed that there are four nlg genes in the Drosophila genome, and all four DNlgs share significant amino acid sequence homology and protein structures with vertebrate Nlgs. DNlg1 and DNlg2 have a positive effect on synaptic growth of NMJs, as indicated by an obvious reduction in synaptic boutons in the dnlg1 and dnlg2 mutants. Conversely, loss of DNlg3 led to increased numbers of synaptic boutons at NMJs. In the present study, a dnlg4 null mutant was generated by gene targeting, and this mutant exhibited significant defects in NMJ morphology, including fewer synaptic boutons and increased bouton size. However, neuronal overexpression of two copies of the dnlg4 transgene induced a pronounced increase in bouton number. These results demonstrated a positive role of DNlg4 in regulating synapse development. Interestingly, neuronal overexpression of one copy of dnlg4 in the WT background did not induce an increase in bouton number, but it did when expressed in a dnlg4 mutant background. These results suggested a homeostatic adjustment during synapse development in Drosophila, which could somewhat counteract the effect caused by increased DNlg4. As a result, a moderate increase of DNlg4 in WT flies did not lead to increased synaptic growth of NMJs (Zhang, 2017).

In Drosophila, loss of DNlgs in the dnlg1-3 mutants also induced synaptic differentiation defects that were characterized by decreased protein levels or impaired distribution of glutamate receptors and other postsynaptic proteins at NMJs. In contrast to other dnlg mutants, statistical alteration in the distribution or protein level of glutamate receptors at NMJs in was not observed dnlg4 mutants. In addition, the distribution and protein level of some presynaptic proteins, such as BRP, CSP, and SYT, were normal in the dnlg4 mutants. However, the ultrastructural analyses of the NMJs showed that there were still some defects in synaptic ultrastructure in the dnlg4 mutants, including the increased bouton area per active zone, longer single PSD, and reduced postsynaptic SSR regions. One striking ultrastructural defect in the dnlg4 mutants was the partial detachment of presynaptic membranes from postsynaptic membranes within the active zone, which was rarely observed in WT flies, suggesting the adhesion function of DNlg4 during synaptogenesis. It was interesting that this defect also appeared in dnlg1 mutants and dnrx mutants, suggesting that the DNlg4 might affect the synaptic architecture by a mechanism similar to that of DNlg1 and DNrx (Zhang, 2017).

Functionally, dnlg4 mutants showed a mild impairment in transmitter release at NMJs, as characterized by slightly reduced evoked EJP amplitude and quantal contents. This phenotype was consistent with the morphological impairments and the ultrastructural defects of NMJs, suggesting a probable reduction in the number of total synapses or functional synapses at NMJs. The amplitude of mEJP was not changed in the dnlg4 mutants, which was consistent with the normal protein levels of postsynaptic glutamate receptors. Interestingly, the frequency of mEJPs in the dnlg4 mutants was dramatically increased, indicating that DNlg4 affects spontaneous transmitter release. The detailed mechanism underlying this should be addressed in future work (Zhang, 2017).

In mammals, Nlgs are generally considered to function as postsynaptic adhesion molecules and to help form trans-synaptic complexes with presynaptic Nrxs. In Drosophila, DNlg1 and DNlg3 are reported to be located in postsynaptic membranes. However, there are some exceptions to the postsynaptic localization of Nlgs. For example, an Nlg in Caenorhabditis elegans is reported to be present in both presynaptic and postsynaptic regions. DNlg2 is also required both presynaptically and postsynaptically for regulating neuromuscular synaptic growth. These studies support a more complex mechanism of Nlgs in synapse modulation and function. These data add to this complexity by suggesting a presynaptic role of DNlg4 in larval neuromuscular synaptic growth and synaptic functions (Zhang, 2017).

First, in the VNC of third-instar larvae, DNlg4 was concentrated in the neuropil region where the synapses aggregated. In NMJs, although endogenous DNlg4 was not detected by anti-DNlg4 antibodies, the exogenous DNlg4 that was expressed in motoneurons was located in type I synaptic boutons of NMJs, suggesting a reasonable presynaptic location of DNlg4 at NMJs. In another assay, DNlg4 was expressed using a DNlg4-Gal4 driver to mimic the endogenous expression pattern of the dnlg4 gene. The DNlg4 promoted by DNlg4-Gal4 was distributed in type I boutons of NMJs and was located in presynaptic areas of boutons, which also supported the presynaptic location of endogenous DNlg4 at NMJs, although a simultaneous postsynaptic localization of DNlg4 at NMJs could not be excluded. Second, knockdown of DNlg4 presynaptically led to morphological defects in NMJs similar to those observed in the dnlg4 mutants, indicated by decreased bouton numbers and increased bouton size. However, knockdown of DNlg4 postsynaptically did not cause the same phenotypes. These morphological defects of NMJs in the dnlg4 mutants could be completely rescued when DNlg4 was expressed in presynaptic neurons, but not when it was expressed in postsynaptic muscles. Third, dnlg4 mutants had a significant increase in spontaneous transmitter release frequency, which was usually interpreted as a presynaptic defect. In addition, all of the functional defects in the dnlg4 mutants, including decreased amplitude of EJPs, reduced quantal contents, and increased mEJP frequency, could be rescued by presynaptic expression of DNlg4. These results indicated that presynaptic DNlg4 was essential for proper proliferation and function of synapses at NMJs. Finally, the number of synaptic boutons at NMJs was significantly increased when two copies of UAS-dnlg4 were overexpressed in presynaptic neurons, whereas this phenomenon was not observed when two copies of UAS-dnlg4 were overexpressed in muscles, suggesting that presynaptic DNlg4 alone was sufficient to promote synaptic growth. Altogether, these data provided convincing evidence that DNlg4 functions as a presynaptic molecule in regulating synaptic growth and transmitter release at NMJs (Zhang, 2017).

The BMPs are major retrograde trans-synaptic signals that affect presynaptic growth and neurotransmission both in the CNS and at NMJs. This study presents immunohistochemical and genetic data showing that DNlg4 regulates NMJ growth via the BMP signaling pathway (Zhang, 2017).

First, the dnlg4 mutants shared similar phenotypes with the components of the BMP signaling pathway in synapse development, synapse architecture, and synapse functions of NMJs, including reduced synaptic bouton number, increased presynaptic membrane ruffles, and decreased synaptic transmission efficacy. Second, several genetic crosses followed by neuromuscular bouton number analyses showed a definite dosage-sensitive genetic interaction between dnlg4 and the components of the BMP signaling pathway, such as tkv, wit, mad, and dad, suggesting that DNlg4 is required for BMP signaling in regulating NMJ growth. Third, both immunohistochemical and Western blot analyses showed that the pMad, which serves as an indicator of BMP signaling, is decreased in both the synaptic boutons of NMJs and motoneuronal nuclei in the dnlg4 mutants, whereas it is increased in dnlg4-overexpressing flies. Finally, the expression of the BMP signaling target gene trio, is significantly reduced in the dnlg4 mutants, but it was increased in dnlg4-overexpressing flies. Together, these results supported the hypothesis that DNlg4 promotes synaptic growth by positively regulating BMP signaling (Zhang, 2017).

The critical question to be addressed, therefore, is the mechanism underlying this regulation. Through Western blot analyses of larval brain homogenates, it is found that the protein level of Tkv (as assessed by ectopically expressed Tkv-GFP) in the dnlg4 mutants is significantly decreased, whereas Wit and Mad, the other two components of the BMP pathway, were not altered. The immunohistochemical assay also showed that the Tkv protein in the dnlg4 mutants is reduced in both the VNC and the synaptic boutons of NMJs. These data indicated the specific positive regulation of Tkv by DNlg4. Previous studies reported that tkv mutants had decreased synaptic bouton number, increased presynaptic membrane ruffles, reduced amplitude of EJP, and unchanged mEJP amplitude. In addition, neuronal overexpression of one copy of transgenic tkv did not induce NMJ overgrowth, but it induced such overgrowth when two copies of transgenic tkv were expressed in neurons. These phenotypes were similar to what was observed in the dnlg4 mutants and dnlg4-overexpressing flies. All of these results strongly demonstrated that DNlg4 regulates BMP signaling by modulating Tkv protein levels (Zhang, 2017).

Because the dnlg4 mutants had a level of tkv mRNA comparable with that of the WT controls, DNlg4 did not affect the transcription of tkv. The possibility of Tkv trafficking defects from the cell body to the axon terminal could also be excluded because no retention of Tkv in the soma of motoneurons was observed. Thus, it seems reasonable to speculate that DNlg4 affects the recruitment or stability of Tkvs at the presynapse. In support of this hypothesis, co-localization of Tkv and DNlg4 at presynaptic regions of NMJs was observed by immunostaining. As further confirmation, it was shown that Tkv could be co-immunoprecipitated with an antibody against DNlg4, and a reverse co-immunoprecipitation assay using anti-GFP antibodies also resulted in the precipitation of DNlg4 by Tkv-GFP, suggesting a physical interaction between DNlg4 and Tkv in vivo. Furthermore, using pull-down assay, the acetylcholinesterase-like domain of the N terminus was shown to be essential for DNlg4 interacting with Tkv (Zhang, 2017).

If the recruitment of Tkv to the presynaptic membrane entirely depends on DNlg4, the existence of Tkv in the presynaptic membrane would not be detected, and accumulation of Tkv proteins in the presynaptic areas of NMJs would probably be observed in the dnlg4 mutants. However, minor Tkv protein level was detected at the presynaptic membrane, and no accumulation of Tkv was observed at NMJs in the dnlg4 mutants. Thus, a more reasonable hypothesis is that DNlg4 affected the stability of Tkv at the presynaptic membranes. The protein level of Tkv in the presynapses of NMJs was reported to be regulated by several pathways, including direct proteasomal degradation, ubiquitin-mediated degradation, and endocytosis. The simplest model is that the DNlg4 might stabilize Tkv via inhibiting its degradation. Several protein kinases, such as the ribosomal protein S6 kinase-like protein (S6KL) and the serine/threonine kinase Fused, have been reported to interact physically with Tkv in vitro and to facilitate its proteasomal degradation. In particular, S6KL has been demonstrated to degrade Tkv at NMJs. DNlg4 might stabilize Tkv by inhibiting these protein kinases, but the detailed mechanism of such activity still needs to be addressed (Zhang, 2017).

In summary, this study demonstrated that DNlg4 positively regulated neuromuscular synaptic growth by modulating BMP signaling through the maintenance of Tkv protein levels in the presynapse of NMJs. To accomplish this function, DNlg4 acted as a presynaptic molecule instead of a postsynaptic molecule. This study further suggested a relationship between Nlgs and BMP signaling and provided a new understanding of the exact role of Nlgs during synapse formation and development (Zhang, 2017).

thickveins: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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