thickveins

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of tkv at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

tkv expression is uniform in early embryos, but by stage 5, expression is more apparent on the dorsal side. By late stage 5,TKV is detected in seven stripes on the ventral side and in a discountinuous pattern on the dorsal side. By stage 9 [Images], expression is restricted to the mesoderm. By stage 16, TKV is restricted to the gastric caeca primordia, first and fourth midgut chambers, hindgut and the head region (Penton, 1994).

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), signaling was followed directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage, high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp (Dorfman, 2001).

Normally Sog may form a graded distribution in the dorsal region, which is essential for patterning. When the Sog/Scw complex is cleaved by Tld, Scw is released and can bind either Sog or Sax. The data suggest that in regions closer to the neuroectoderm, the levels of Sog are high and titrate the free ligand. In the dorsal-most region however, where Sog levels are low, the released Scw has a greater probability of binding and activating the Sax receptor, rather than being trapped again by Sog. Thus, the graded distribution of Sog is critical for generating the reciprocal distribution of Scw, and the ensuing activation profile (Dorfman, 2001).

Activation of Tkv by Dpp is essential for the appearance of the early pMad pattern, corresponding to the future amnioserosa cells. At this stage, distinct cell fates are also induced in the dorsolateral cells, as reflected by expression of pnr and repression of msh expression. It is assumed that low levels of activation that may be induced by Dpp alone, but not detected by pMad antibodies, are responsible for these fates. Elimination of Dpp or Tkv leads to complete absence of early, as well as late, pMad patterns. Thus, Scw is not sufficient for the early activation phase, and the presence of Dpp is crucial. Cooperativity between Scw and Dpp occurs at the level of receptor activation. One possibility is that the observed pMad levels reflect only an additive effect of Scw and Dpp signaling. Indeed, the number of dpp copies has a profound effect on signaling levels and the shape of the early pMad distribution. Alternatively, it is possible that there is a synergistic interaction between Scw and Dpp signaling. In this case, the requirement of both ligands for the production of the early pMad pattern may indicate that synergy occurs at the level of receptor activation. Phosphorylation of Mad may require the formation of heterotetrameric receptors, containing both Sax/Put and Tkv/Put pairs. Cross linking experiments of the vertebrate receptors support this model (Dorfman, 2001).

Scw is required for generating the pMad pattern only in the early phase. All subsequent patterns rely only on Dpp. This feature may be explained differently by each of the above two models. If Scw and Dpp are required additively in the early phase, higher levels of Dpp may suffice to induce the pMad pattern at later stages. The autoregulatory effects of Dpp on its transcription may account for the elevation in Dpp levels. Alternatively, if Scw and Dpp signaling is synergistic, why is such a synergism necessary only in the early phase? In the early embryo, a maternal transcript encoding an inhibitor of BMP signaling may be translated, to block signaling by Sax/Put or Tkv/Put dimers. Such inhibitor(s) may be displaced only in ligand-bound heterotetrameric receptor complexes. The maternal transcripts of the inhibitor(s) may diminish by stage 9, to allow pMad production by activation of Tkv/Put alone (Dorfman, 2001).

A presynaptic endosomal trafficking pathway controls synaptic growth signaling

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila. This study shows that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. An alternative signal termination pathway was identified in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)-mediated internalization of receptors into the endosomal lumen. These results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments (Rodal, 2011).

Endocytic membrane traffic regulates signaling by synaptic growth factor receptors and may be used as a point of control in tuning receptor traffic in response to neuronal activity. The neuronal F-BAR/SH3 protein Nwk is involved in endocytic membrane traffic that attenuates synaptic growth signaling, but the mechanism by which it acts has been unknown. This study has identified a physical interaction between Nwk and the early endosomal protein SNX16 that is critical for down-regulating the synaptic growth-promoting activity of SNX16. This interaction may bring together the actin-polymerizing activity of the first SH3 domain of Nwk with the potential lipid-binding/tubulating activities of the F-BAR domain of Nwk and the PX domain of SNX16, driving tubule-based membrane flux from early to recycling endosomes (Rodal, 2011).

Sorting nexins form a large family of proteins that share a common phosphoinositide-binding PX domain and are involved in diverse aspects of membrane traffic (Cullen, 2008). The best-characterized members of this family are the sorting nexin-BAR family, including Snx1 (tied to endosome-to-Golgi traffic) and Snx9 (tied to the internalization step of endocytosis), which each contains both a lipid-tubulating BAR domain and a PX domain. Crystal structures and functional studies of Snx9 have shown that its BAR and PX domains form a single lipid-binding and -deforming module with combined specificities that neither domain exhibits alone. As such, SNX16-Nwk interactions may form an analogous F-BAR/PX module via an intermolecular rather than intramolecular interaction. It has not been possible to purify sufficient amounts of SNX16 to directly test its effects on lipid binding by Nwk; therefore, further analysis of their interaction will require an approach to isolate SNX16 in vitro. Interestingly, the SNX16-Nwk interaction depends on a region of Nwk surrounding its second SH3 domain, raising the possibility that this region of Nwk may exert intramolecular effects on the amino-terminal F-BAR domain, as has previously been shown for other F-BAR proteins (Rao, 2010). Furthermore, Nwk binds to the coiled-coil region of SNX16, which is involved in SNX16 dimerization, suggesting that Nwk may act on SNX16 by affecting its dimerization state (Rodal, 2011).

Mammalian SNX16 has been implicated in the trafficking of the EGF receptor from early to late endosomes, which may mediate down-regulation of receptor signaling. However, the mechanism by which SNX16 promotes this trafficking step is not understood and no loss-of-function studies on SNX16 have been reported. A mutant of mammalian SNX16 that lacks 60 aa corresponding to the Drosophila SNX16 Nwk-binding site blocks trafficking of SNX16 and EGF to late endosomes, leading to increased EGF signaling. Interestingly, the F-BAR protein FBP17 has been reported to interact with SNX2. These results raise the possibility that BAR family-sorting nexin interactions may be broadly used to control membrane traffic in cells (Rodal, 2011).

Drosophila SNX16 localizes to an early endosomal compartment at the NMJ defined by the small GTPases Rab4 and Rab5. This compartment accumulates signaling receptors, such as the BMP receptors Tkv and Wit, and signaling is amplified when receptors are stalled in this compartment, suggesting that the SNX16 compartment is an active site of signaling. By identifying specific Snx16 mutants that disrupt interactions with Nwk, it was possible to separate the requirements for receptor down-regulation at early endosomes and show that activated receptors specifically require SNX16-Nwk-mediated traffic to recycle to the plasma membrane (Rodal, 2011).

Previous studies have shown that Nwk colocalizes with the recycling endosome marker Rab11 in fixed tissue and cooperates with Cdc42, which is thought to function at the recycling endosome, to activate WASp/Arp2/3-mediated actin polymerization. Together with the result that rab11 mutants exhibit synaptic overgrowth similar to nwk mutants (Khodosh, 2006), it is concluded that Nwk functions at recycling endosomes, which are downstream of early endosomes. This study shows that Nwk tagged with fluorescent proteins localizes to novel punctate structures in heterologous cells and nerve terminals. These puncta had not been observed using anti-Nwk antibodies against either endogenous or overexpressed Nwk in fixed tissue, as they are poorly preserved upon fixation. In living synapses, Rab11 colocalizes with Nwk to mobile puncta, SNX16-containing early endosomes interact transiently with Nwk puncta, and inhibition of the GTPase dynamin blocks separation of these compartments. Dynamin inhibition has previously been shown to disrupt endosomal function in the fly NMJ, it cannot be certain that the collapse of SNX16-Nwk compartments under these conditions is caused by the specific interactions of these proteins with dynamin. However, because SNX16-Nwk interactions lead to the down-regulation of synaptic growth signals, the data are consistent with a model in which the exchange of receptors from the SNX16 endosome to the Nwk/Rab11 endosome leads to the attenuation of receptor signaling. Binding of Nwk to the cytoplasmic tail of the BMP receptor Tkv may mediate this event (O'Connor-Giles, 2008), but further experiments will be required to directly examine cargo transfer in the future. Because there are significant cytoplasmic pools of both Nwk and SNX16, the contribution of these soluble proteins to membrane traffic in the nerve terminal or the possibility that overexpression of Nwk and Snx16 to visualize live trafficking events does not faithfully recapitulate the behaviors of endogenous proteins cannot be excluded. However, the transient interaction of Nwk and SNX16 puncta correlates well with genetic results showing that nwk attenuates a synaptic growth-promoting activity of Snx16 at early endosomes (Rodal, 2011).

Because Nwk- and SNX16-labeled compartments transiently interact in nerve terminals, temporal control of the interaction between their lipid-binding domains may provide a mechanism to acutely drive membrane tubulation in a regulated fashion. Furthermore, the association of SNX16 with endosomes depends on phosphoinositides, as the phosphatidylinositol 3-kinase inhibitor wortmannin disrupts localization of SNX16 in cultured cells, indicating that regulation of phospholipid composition may also contribute to the membrane-deforming activities of SNX16 and Nwk (Rodal, 2011).

This study found that Snx16 loss-of-function mutants suppress synaptic overgrowth resulting from loss of Nwk-mediated traffic as well as from activation of Wg and BMP signaling pathways. These data suggest that SNX16 plays an active role in promoting synaptic growth at the endosome, aside from its function in signal attenuation through Nwk. Snx16 was found to be required to restrict synaptic growth when MVB formation is hampered in hrs mutants, suggesting that receptor entry into the endosomal lumen is an alternative signal attenuation pathway. Furthermore, it was found that overexpression of a mutant Snx16 that cannot bind Nwk promotes the accumulation of endosomal structures at the NMJ and drives excess synaptic growth, suggesting that Snx16 may play a role in MVB maturation and acts at the branch point between endosomal sorting pathways. Defining the mechanism by which SNX16 promotes synaptic growth will require further structure-function analysis of the active domains of the protein aside from its Nwk-binding coiled coil (Rodal, 2011).

Traffic through endocytic compartments has proven to be a critical point of regulation in the nervous system. Synaptic vesicle endocytosis is controlled by synaptic activity through calcium-dependent dephosphorylation of endocytic proteins, and postsynaptic trafficking of AMPA receptors through the recycling endosome is increased in response to activity via the calcium-dependent motor myosin V. Rab5-positive compartments at the Drosophila NMJ have been previously characterized for their role in the synaptic vesicle cycle, and it will be interesting to determine how receptor-mediated endocytosis and synaptic vesicle endocytosis are coordinately or separately regulated in response to activity. Synaptic growth at the Drosophila NMJ is positively regulated by calcium influx through voltage-gated calcium channels, and endosome number increases in response to activity. It is tempting to speculate that calcium influx acts through conserved mechanisms for modulating membrane dynamics to delay the attenuation of receptor signaling by SNX16-Nwk-mediated traffic, leading to increased synapse growth in response to activity. A key future goal will be to determine specific points of activity-dependent regulation of membrane traffic in presynaptic endosomes (Rodal, 2011).

Larval

During vein differentiation dpp is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Both dpp and thick veins are differentially expressed in vein territories during pupal development. dpp and tkv regulate one another by a feedback mechanism in which Tkv activity represses dpp expression. Dpp, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Ectopic dpp expression or Tkv activation in the wing disc result in the differentiation of ectopic veins. Outside of vein territories, the repression of dpp by the widely expressed Tkv could participate in restricting dpp expression to the veins. It is possible that the observed down-regulation of tkv expression in vein cells participates in generating the levels of Tkv activation necessary to activate vein differentiation, but insufficient to repress dpp expression. The expression of dpp and tkv in vein territories depends (either directly or indirectly) on EGF-receptor activity, because the transcription of these genes is not activated when Egf-R is reduced (as in veinlet and vein mutant wings). Once Dpp is established in the veins, local activation of Tkv in these cells is required both for the maintenance of veinlet and Delta expression and for the veins to differentiate. In dpp mutants, the vein thickening observed in Notch mutants is elimated. Conversely, Notch gain-of-function alleles that lead to the truncation of veins results in very pronounced vein loss in combination with both dpp and tkv mutants. In dpp mutants, Delta and E(spl)mß, which normally takes place in vein territories, is lost. In summary, genetic combinations between mutations that increase or reduce Notch, veinlet and dpp activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways (de Celis, 1997).

Formation of the longitudinal veins (LVs) of the Drosophila wing involves the interplay among Dpp, Egf and Notch pathways. Formation of crossveins (CVs: see Derivatives of the wing disc) present a paradoxical problem. As shown both morphologically and using molecular markers, the definitive CVs are not formed until long after the initial specification of the LVs. The CVs therefore must form within territory that has already been specified as intervein. The CVs must also interconnect with existing LVs at a time when the Delta expressed by the LVs is thought to inhibit vein formation in adjacent cells. Mechanisms must exist that override both intervein specification and the lateral inhibition of veins, allowing the formation of continuous, interconnected vein tissue. BMP-like signaling plays a special role in the formation of the CVs from within intervein territory. BMP-like signals also help maintain the connections between the LVs and the margin of the wing. crossveinless 2 (cv-2) is a critical factor in these processes, as it is expressed more highly in the CVs and the ends of the LVs and is required for the high levels of BMP-like signaling observed in these regions (Conley, 2000). The cv-2 mutation was first identified by Benedetto Nicoletti in 1962 (FlyBase: Cv-2 site). The structure of the Cv-2 protein strongly suggests that these effects are direct, and that Cv-2 is a novel player in the BMP-like signaling pathway (Conley, 2000).

Both Dpp and Gbb vein signals are mediated largely by the type I receptor Thickveins, rather than the alternate type I receptor Saxophone. Cells lacking Tkv do not form veins, but removal of Sax does not reliably remove veins. However, not all veins are equally sensitive to reductions in Dpp and Gbb signaling. The hypomorphic gbb⁴ mutation shows complete loss of the cross veins (CVs), but only slight loss of the ends of the LVs. Sog encodes a Chordin-like molecule that inhibits BMP-like signaling; both Sog and Chordin are thought to bind to and sequester ligands, preventing the activation of receptors. Overexpressing Sog in the wing specifically blocks formation of the CVs and the ends of the LVs. The secreted Tolloid proteases, similar to vertebrate BMP1s, can increase BMP signaling by cleaving and inactivating Chordin or Sog. Loss of tolkin (also known as tolloid-related) blocks formation of the CVs and the tips of the LVs. Overexpressing a dominant negative form of Sax again induces a similar phenotype (Conley, 2000 and references therein).

Such phenotypes are very reminiscent of the crossveinless class of mutations in Drosophila (reviewed in Garcia-Bellido, 1992). Strong reductions in crossveinless 2 (cv-2) function have been shown to remove the posterior CV (PCV), the anterior CV (ACV), and the ends of the LVs. However, despite the possibility that the crossveinless genes encode novel players in BMP-like signaling, none have been characterized and the sensitivity of CVs to BMP-like signaling has not been explained. Evidence is presented that cv-2 encodes a novel member of the BMP-like signaling pathway, expressed in and required for high levels of BMP-like signaling in the developing cross veins. The Cv-2 protein contains five cysteine-rich domains similar to those known to bind BMP-like ligands, strongly suggesting that Cv-2 directly modulates Dpp or Gbb activity (Conley, 2000 and references therein).

dpp and gbb mutations both disrupt CV formation. Weak cv-2 alleles are strengthened by dpp and gbb loss-of-function mutations. cv-2^225-3/cv-2³⁵¹¹ flies never lack the entire PCV, but 50% of gbb 4 cv-2^225-3/cv-2 3511 flies lack the entire PCV. Similarly, cv-2³⁵¹¹/Df(2R)Pu-D17 only rarely disrupt the ACV, but dpp^d6 cv-2³⁵¹¹/Df(2R)Pu-D17 commonly does. However, cv-2 cannot dominantly enhance earlier dpp-dependent patterning in the wings: dpp^d5 Df(2R)Pu-D17 /dpp^hr4 wings look no worse than dpp^d5/dpp^hr4 wings. To provide a more direct link between cv-2 and Dpp and Gbb signaling, Mad activation was examined in mutant pupal wings. In cv-2¹ adults, the PCV is more reliably disrupted than the ACV; the anti-p-Mad staining normally found near the PCV in 19, 22, 26 and 36 hours after pupariation wings is lost or disrupted in cv-2¹ homozygotes, as is the reduction of anti-DSRF in the PCV. In adults of the stronger allelic combination cv-2¹/Df(2R)Pu-D17, the ACV is also often lost along with the ends of some of the LVs. Interestingly, no disruption of the ACV or LV anti-p-Mad staining cv-2¹/Df(2R)Pu-D17 pupal wings is detected at 21 or 25 hours after pupariation; only at 36 hours after pupariation is staining lost from the ACV. This indicates that cv-2 is required not only to initiate Mad activity in the PCV, but also to maintain that activity in the ACV (Conley, 2000).

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