Using full length and truncated forms of Neurexin (Dnrx) and Neuroligins (Dnlg) together with cell biological analyses and genetic interactions, this study reports novel functions of dnrx and dnlg1 in clustering of pre- and postsynaptic proteins, coordination of synaptic growth and ultrastructural organization. dnrx and dnlg1 extracellular and intracellular regions are required for proper synaptic growth and localization of dnlg1 and Dnrx, respectively. dnrx and dnlg1 single and double mutants display altered subcellular distribution of Discs large (Dlg), which is the homolog of mammalian post-synaptic density protein, PSD95. dnrx and dnlg1 mutants also display ultrastructural defects ranging from abnormal active zones, misformed pre- and post-synaptic areas with underdeveloped subsynaptic reticulum. Interestingly, dnrx and dnlg1 mutants have reduced levels of the BMP receptor Wishful thinking (Wit), and dnrx and dnlg1 are required for proper localization and stability of Wit. In addition, the synaptic overgrowth phenotype resulting from the overexpression of dnrx fails to manifest in wit mutants. Phenotypic analyses of dnrx/wit and dnlg1/wit mutants indicate that Dnrx/Dnlg1/Wit coordinate synaptic growth and architecture at the NMJ. These findings also demonstrate that loss of dnrx and dnlg1 leads to decreased levels of the BMP co-receptor, Thickveins and the downstream effector phosphorylated Mad at the NMJ synapses indicating that Dnrx/Dnlg1 regulate components of the BMP signaling pathway. Together these findings reveal that Dnrx/Dnlg are at the core of a highly orchestrated process that combines adhesive and signaling mechanisms to ensure proper synaptic organization and growth during NMJ development (Banerjee, 2016).
Neurexins and Neuroligins have emerged as major players in the organization of excitatory and inhibitory synapses across species. Mutational analyses in Drosophila uncovered specific functions of dnrx and dnlg1 in NMJ synapse organization and growth, where dnrx is expressed pre-synaptically and dnlg1 post-synaptically. Deletion of the extracellular and intracellular regions of dnrx or dnlg1 revealed that both regions are necessary for dnrx and dnlg1 clustering and function at the synapse. Most importantly, the data presented here suggest that dnrx and dnlg1 genetically interact with wit as well as the downstream effector of BMP signaling, Mad to allow both the organization and growth of NMJ synapses. The surprising finding that loss of dnrx and dnlg1 leads to decreased wit stability and that dnrx and dnlg1 are required for proper wit localization raises the possibility that these proteins function to coordinate trans-synaptic adhesion and synaptic growth. This is further strengthened from the observations that, in the absence of Wit, the synaptogenic activity from overexpression of dnrx did not manifest into an increased synaptic growth as is seen in the presence of wit. Loss of dnrx and dnlg1 also led to a reduction in the levels of other components of the BMP pathway, namely the Tkv and pMad. Together these findings are the first to demonstrate a functional coordination between trans-synaptic adhesion proteins dnrx and dnlg1 with wit receptor and BMP pathway members to allow precise synaptic organization and growth at NMJ. It would be of immense interest to investigate whether similar mechanisms might be operating in vertebrate systems (Banerjee, 2016).
The trans-synaptic cell adhesion complex formed by heterophilic binding of pre-synaptic Neurexins (Nrxs) and post-synaptic Neuroligins (Nlgs), displayed synaptogenic function in cell culture experiments. In vertebrates and invertebrates alike, Nrxs and Nlgs belong to one of the most extensively studied synaptic adhesion molecules with a specific role in synapse organization and function. In Drosophila, dnrx and dnlg1 mutations cause reductions in bouton number, perturbation in active zone organization and severe reduction in synaptic transmission. These phenotypes are essentially phenocopied in both dnrx and dnlg1 mutants, and double mutants do not cause any significant enhancement in the single mutant phenotypes illustrating that they likely function in the same pathway. From immunohistochemical and biochemical analyses, it is evident that lack of dnrx or dnlg1 causes their diffuse localization and protein instability in each other's mutant backgrounds. These data suggest that trans-synaptic interaction between dnrx and dnlg1 is required for their proper localization and stability, and that these proteins have a broader function in the context of general synaptic machinery involving Nrx-Nlg across phyla. It also raises the possibility that the trans-synaptic molecular complex involving Nrx-Nlg may alter stability of other synaptic proteins and lead to impairments in synaptic function without completely abolishing synaptic structure and neurotransmission. It is therefore not surprising that Nrx and Nlg have been recently reported to be associated with many non-lethal cognitive and neurological disorders, such as schizophrenia, ASD, and learning disability (Banerjee, 2016).
The rescue studies of dnrx and dnlg1 localization using their respective N- and C-terminal domain truncations emphasize a requirement of the full-length protein for proper synaptic clustering. Given that dnrx and dnlg1 likely interact via their extracellular domains, it is somewhat expected that an N-terminal deletion as seen in genotypic combinations of elav-Gal4/UAS-DnrxΔ N;dnrx-/- and mef2-Gal4/UAS-dnlg1Δ N;dnlg1-/- would fail to rescue the synaptic clustering of Dnlg1 and Dnrx, respectively. However, the inability of the C-terminal deletions of these proteins as seen in elav-Gal4/UAS-DnrxΔ c;dnrx-/- and mef2-Gal4/UAS-dnlg1Δ Cdnlg1-/- to cluster Dnlg1 and Dnrx, respectively, to wild type localization and/or levels suggest that the lack of the cytoplasmic domains of these proteins may render the remainder portion of the protein unstable, thereby leading to its inability to be either recruited or held at the synaptic apparatus (Banerjee, 2016).
Finally, the subcellular localization of Dlg at the SSR and its levels in the dnrx and dnlg1 single and double mutants as well as in the rescue genotypes provides key insights into the stoichiometry of Dnrx-Dnlg1 interactions, and how Dnrx-Dnlg1 might be functioning with other synaptic proteins in their vicinity to organize the synaptic machinery. A significant reduction in Dlg levels in dnrx mutants raises the question of whether Dlg localization/levels are controlled presynaptically? Alternatively, it is possible that Dnrx-Dnlg1 is not mutually exclusive for all their synaptic functions and there might be other Neuroligins that might function with Dnrx. One attractive candidate could be Dnlg2, which is both pre- and postsynaptic. dnrx could also function through the recently identified Neuroligins 3 and 4. Although dnlg1 mutants did not show any significant difference in Dlg levels, a diffuse subcellular localization nevertheless raised the possibility of a structural disorganization of the postsynaptic terminal and defects in SSR morphology, which were confirmed by ultrastructural studies. The rescue analysis of the subcellular localization of Dlg demonstrates that Dlg localization and levels could be restored fully in a presynaptic rescue of dnrx mutants by expression of full length Dnrx, however the localization of Dlg could not be restored by expression of DnrxΔNT. These observations suggest that the extracellular domain of dnrx is essential for the localization and also the levels of Dlg. Whether the extracellular domain influences Dlg via dnlg1 or any of the other three Dnlgs (2, 3 and 4) at the NMJ remains to be elucidated (Banerjee, 2016).
Most studies on Nrx/Nlg across species offer clues as to how these proteins assemble synapses and how they might function in the brain to establish and modify neuronal network properties and cognition, however, very little is known on the signaling pathways that these proteins may potentially function in. It has been previously reported that Neurexin 1 is induced by BMP growth factors in vitro and in vivo and that could possibly allow regulation of synaptic growth and development. In Drosophila, both dnrx and dnlg1 mutants showed reduced synaptic growth similar to wit pathway mutants. Reduced wit levels both from immunolocalization studies in dnrx and dnlg1 mutant backgrounds and biochemical studies from immunoblot and sucrose density gradient sedimentation analyses present compelling evidence towards the requirement of dnrx and dnlg1 for wit stability. Reduction of synaptic dnrx levels in wit mutants argue for interdependence in the localization of these presynaptic proteins at the NMJ synaptic boutons. The findings that synaptic boutons of dnrx and dnlg1 mutants also show reduction in the levels of the co-receptor Tkv suggest that this effect may not be exclusively Wit-specific, and possibly due to the overall integrity of trans-synaptic adhesion complex that ensures Wit and Tkv stability at the NMJ (Banerjee, 2016).
Genetic interaction studies displayed a significant reduction in bouton numbers resulting from trans-heterozygous combinations of wit/+,dnrx/+ and wit/+,dnlg1/+ compared to the single heterozygotes strongly favoring the likelihood of these genes functioning together in the same pathway. Although double mutants of wit,dnrx and wit,dnlg1 are somewhat severe than dnrx and dnlg1 single mutants, they did not reveal any significant differences in their bouton counts compared to wit single mutants. Moreover, genetic interactions between Mad;dnrx and Mad;dnlg1 together with decreased levels of pMad in dnrx and dnlg1 mutants provided further evidence that dnrx and dnlg1 regulate components of the BMP pathway. Interestingly, reduced levels of pMad were observed in dnrx and dnlg1 mutants, in contrast to a recent study that reported higher level of synaptic pMad in dnrx and dnlg1 mutants. The differences in pMad levels encountered in these two studies could be attributed to differences in rearing conditions, nature of the food and genetic backgrounds, as these factors have been invoked to affect synaptic pMad levels. These findings strongly support that dnrx, dnlg1 and wit function cooperatively to coordinate synaptic growth and signaling at the NMJ (Banerjee, 2016).
Loss of either dnrx or dnlg1 does not completely abolish the apposition of pre- and post-synaptic membrane at the NMJ synapses but detachments that occur at multiple sites along the synaptic zone. These observations point to either unique clustering of the Dnlg1/Dnrx molecular complexes or preservation of trans-synaptic adhesion by other adhesion molecules at the NMJ synapses. Indeed, recent studies show nanoscale organization of synaptic adhesion molecules Neurexin 1Β, NLG1 and LRRTM2 to form trans-synaptic adhesive structures (Chamma, 2016). In addition to dnrx and dnlg1 mutants, synaptic ultrastructural analysis showed similarity in presynaptic membrane detachments with a characteristic ruffling morphology in wit mutants as well suggesting that these proteins are required for maintaining trans-synaptic adhesion. Interestingly, double mutants from any combinations of these three genes such as dnlg1,dnrx or wit,dnrx and wit,dnlg1 did not show any severity in the detachment/ruffling of the presynaptic membrane suggesting that there might be a phenotypic threshold that cannot be surpassed as part of an intrinsic synaptic machinery to preserve its very structure and function. It would be interesting to test this possibility if more than two genes are lost simultaneously as in a triple mutant combination. Alternatively, there could be presence of other distinct adhesive complexes that remain intact and function outside the realm of Dnrx, dnlg1 and wit proteins thus preventing a complete disintegration of the synaptic apparatus (Banerjee, 2016).
The same holds true for most of the ultrastructural pre- and postsynaptic differentiation defects observed in the single and double mutants of dnrx, dnlg1 and wit. Barring dnrx,dnlg1 double mutants in which the SSR width showed a significant reduction compared to the individual single mutants, all other phenotypes documented from the ultrastructural analysis showed no difference in severity between single and double mutants. Another common theme that emerged from the ultrastructural analysis was that loss of either pre- or postsynaptic proteins or any combinations thereof, showed a mixture of defects that spanned both sides of the synaptic terminal. For example, presynaptic phenotypes such as increased number of active zones or abnormally long active zones as well as postsynaptic phenotypes such as reduction in width and density of the SSR were observed when presynaptic proteins such as dnrx and wit and postsynaptic dnlg1 were lost individually or in combination. These studies suggest that pre- and postsynaptic differentiation is tightly regulated and not mutually exclusive where loss of presynaptic proteins would result only in presynaptic deficits and vice-versa (Banerjee, 2016).
The postsynaptic SSR phenotypes seen in the single and double mutants of dnrx, dnlg1 and wit might be due to their interaction/association with postsynaptic or perisynaptic protein complexes such as Dlg and Fasciclin II. Alternatively, the postsynaptic differentiation or maturation deficits in these mutants could also be due to a failure of postsynaptic GluRs to be localized properly or their levels maintained sufficiently. It has been shown previously that lack of GluR complexes interferes with the formation of SSR. Deficits in postsynaptic density assembly have been previously reported for dnlg1 mutants, including a misalignment of the postsynaptic GluR fields with the presynaptic transmitter release sites. GluR distribution also showed profound abnormalities in dnrx mutants. These observations suggest that trans-synaptic adhesion and synapse organization and growth is highly coordinated during development, and that multiple molecular complexes may engage in ensuring proper synaptic development. Some of these questions need to be addressed in future investigations (Banerjee, 2016).
In Drosophila, the postsynaptic muscle-derived BMP ligand, Glass bottom boat (Gbb), binds to type II receptor Wit, and type I receptors Tkv, and Saxophone (Sax) at the NMJ. Receptor activation by Gbb leads to the recruitment and phosphorylation of Mad at the NMJ terminals followed by nuclear translocation of pMad with the co-Smad, Medea, and transcriptional initiation of other downstream targets. It is interesting to note that previously published studies revealed that a postsynaptic signaling event occurs during larval development mediated by Type I receptor Tkv and Mad. Based on findings from this study, it is speculated that Dnrx and Dnlg1-mediated trans-synaptic adhesive complex allows recruitment and stabilization of wit and associated components to assemble a larger BMP signaling complex to ensure proper downstream signaling. Loss of dnrx and/or dnlg1 results in loss of adhesion and a decrease in the levels of Wit/Tkv receptors as well as decreased phosphorylation of Mad. Thus a combination of trans-synaptic adhesion and signaling mediated by Dnrx, Dnlg1 and components of the BMP pathway orchestrate the assembly of the NMJ and coordinate proper synaptic growth and architecture (Banerjee, 2016).
The data presented in this study address fundamental questions of how the interplay of pre- and postsynaptic proteins contributes towards the trans-synaptic adhesion, synapse differentiation and growth during organismal development. dnrx and dnlg1 establish trans-synaptic adhesion and functionally associate with the presynaptic signaling receptor wit to engage as a molecular machinery to coordinate synaptic growth, cytoarchitecture and signaling. dnrx and dnlg1 also function in regulating BMP receptor levels (Wit and Tkv) as well as the downstream effector, Mad, at the NMJ. It is thus conceivable that at the molecular level setting up of a Dnrx-Dnlg1 mediated trans-synaptic adhesion is a critical component for molecules such as Wit and Tkv to perform signaling function. It would be of immense interest to investigate how mammalian Neurexins and Neuroligins are engaged with signaling pathways that not only are involved in synapse formation but also their functional modulation, as the respective genetic loci show strong associations with cognitive and neurodevelopmental disorders (Banerjee, 2016).
Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. This study shows that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. During starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. These results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity (Kamimura, 2019).
Although Wit shows a strong overall similarity to the vertebrate BMP type II receptor, its signaling mechanism remains untested. To examine whether Wit actually mediates a BMP type signal, use was made of an antibody that specifically recognizes the phosphorylated form of Mad (P-Mad) (Tanimoto, 2000), the major transducer of BMP signals in Drosophila. In late embryos, this antibody reveals an intricate developmental pattern of BMP signaling, including P-Mad accumulation in the developing midgut and gastric cecae that closely parallels dpp expression and is thought to be indicative of cells receiving a Dpp signal. In addition to these sites of accumulation, a novel pattern of P-Mad accumulation is noted in a subset of neurons. Staining is first evident at late stage 15 and then becomes more elaborate and intense as the embryos continue to develop. Ultimately about 35-40 neurons per hemisegment show strong staining by stage 17. This expression continues into the first instar stage but is not detectable in late third instar larvae. The pattern of P-Mad accumulation in the CNS is completely abolished in wit mutant embryos whereas all other patterns of P-Mad accumulation appear normal. Conversely, with the exception of the CNS pattern, all other sites of P-Mad accumulation in late embryogenesis are absent in zygotic punt null mutants. Maternal null embryos cannot be examined since they do not develop an organized CNS. These observations are consistent with Wit function being required specifically in the CNS to transduce BMP signals whereas other sites of P-Mad accumulation result from signaling by Punt, the primary Dpp type II receptor (Marqués, 2002).
The BMP receptor Wishful thinking (Wit) is required for synapse stabilization. In the absence of BMP signaling, synapse disassembly and retraction ensue. Remarkably, downstream Smad-mediated signaling cannot fully account for the stabilizing activity of the BMP receptor. LIM Kinase1 (DLIMK1)-dependent signaling has been identified as a second, parallel pathway that confers the added synapse-stabilizing activity of the BMP receptor. DLIMK1 binds a region of the Wit receptor that is necessary for synaptic stability but is dispensable for Smad-mediated synaptic growth. A genetic analysis demonstrates that DLIMK1 is necessary, presynaptically, for synapse stabilization, but is not necessary for normal synaptic growth or function. Furthermore, presynaptic expression of DLIMK1 in a wit or mad mutant significantly rescues synaptic stability, growth, and function. DLIMK1 localizes near synaptic microtubules and functions independently of ADF/cofilin (Twinstar), highlighting a novel requirement for DLIMK1 during synapse stabilization rather than actin-dependent axon outgrowth (Eaton, 2005).
The canonical bone morphogenic protein (BMP) signaling system has been implicated in diverse cellular and developmental processes ranging from cell growth to tissue patterning. At the Drosophila NMJ, a BMP signaling system has been identified that controls synaptic growth via canonical Smad-mediated signaling to the cell body. It has been demonstrated that mutations in the BMP ligand glass bottom boat (gbb), the type I and type II BMP receptors thick veins (tkv) and wishful thinking (wit), and the Smad homologs mad and medea all significantly impair synaptic growth and function. These data define a retrograde trophic signaling system that functions through transcriptional mechanisms in the cell soma to control motoneuron synaptic growth. This study demonstrates that BMP signaling at the Drosophila NMJ is not only required for normal synaptic growth, but also for synaptic stabilization. In the absence of BMP signaling, significant increases in synapse retraction and disassembly are observed. Signaling downstream of the BMP receptors can be genetically separated into two pathways: Smad-dependent synaptic growth and LIM Kinase1-dependent synaptic stability (Eaton, 2005).
LIM Kinase1 (LIMK1) is a cytoplasmic serine/threonine kinase that was originally isolated in screens for novel kinases expressed in the nervous system. Findings in LIMK1 knockout mice reveal defects in dendritic spine morphology and activity-dependent plasticity, although neither synaptic growth nor synaptic stability has been specifically analyzed (Endo, 2003). In the Drosophila central nervous system, DLIMK1 has been implicated in the mechanisms of axonal outgrowth during metamorphosis, acting through ADF/cofilin to modulate the actin cytoskeleton, a mechanism also documented in other tissues (Arber, 1998; Ng, 2004; Ohashi, 2000; Eaton, 2005 and references therein).
Genetic analyses have defined a new function for DLIMK1 during synaptic stability in comparison with its function in axonal outgrowth. Synaptic DLIMK1 is closely associated with the synaptic microtubule cytoskeleton. In addition, genetic manipulation of ADF/cofilin activity does not affect synaptic stability at the NMJ. These data highlight differences in LIMK1 function during the rapid, dynamic process of axon outgrowth compared to the slower, more prolonged mechanisms that govern synapse stabilization at the NMJ. Together, these data define genetically separable signaling pathways downstream of the BMP receptor that could allow a single trophic signaling event to coordinately control synaptic growth and synaptic stabilization (Eaton, 2005).
An assay to quantify synaptic retraction at the Drosophila NMJ was developed and used to identify molecules involved in synaptic stability (Eaton, 2002). This assay is based on the demonstration that the formation of organized postsynaptic muscle membrane folds, termed the subsynaptic reticulum (SSR), requires the presence of the presynaptic nerve terminal. Therefore, the SSR and proteins that localize to this structure will be present only at sites where the nerve terminal resides, or where it has recently resided. Thus, observed sites of organized postsynaptic SSR that lack opposing presynaptic neuronal markers identify regions of the neuromuscular junction (NMJ) where the nerve terminal once resided and has since retracted. This interpretation has been confirmed in previous studies using light-level, ultrastructural, and electrophysiological analyses (Eaton, 2002; Pielage, 2005). Sites of synapse retraction are referred to as 'retraction events' or 'synaptic footprints' and represent a quantitative assay for synaptic stability. A wide array of pre- and post-synaptic markers have been used to clearly define synaptic retraction events. Synaptic retractions can be identified with equal efficiency using antibodies that recognize diverse presynaptic antigens, including cytoplasmic, membrane-associated, cytoskeleton, or vesicle-associated proteins (Eaton, 2002; Pielage, 2005). Several postsynaptic markers have also been used to quantify synapse retractions, including Discs-large, Shaker-GFP, and the clustered postsynaptic glutamate receptors (Eaton, 2002; Pielage, 2005; Eaton, 2005 and references therein).
The retraction assay was used to test whether BMP signaling in Drosophila motoneurons is required for synapse stabilization. First it was confirmed that mutations in the BMP type II receptor Wit and the BMP ligand Gbb cause a significant decrease in bouton number. These mutations also cause a significant increase in synaptic footprints, demonstrating that synaptic stability is significantly compromised in the absence of BMP signaling. Synaptic footprints were identified in equal numbers using multiple presynaptic markers, including anti-Synapsin and anti-nc82, which recognizes an antigen at the presynaptic active zone. Synaptic footprints can be rescued in the wit mutant background by neuronal expression of the full-length wit transgene, demonstrating that synapse destabilization is caused by the absence of the presynaptic Wit receptor. The number of synapse retractions is slightly, but statistically significantly, less in gbb compared with that in wit. This could be due to the fact that wit is a null mutation, whereas the gbb genotype that was used is not. Null mutations in gbb do not survive through larval development and, therefore, the genetic combination previously used in studies of its effects on synaptic growth was used (Eaton, 2005).
This analysis was extended to mutations that disrupt additional downstream components of the canonical BMP signaling cascade. Mutations in the BMP type I receptor thick-veins (tkv), the Smad homolog mad, and the co-Smad medea, were used. All three mutations decrease bouton numbers to levels that are statistically identical to those observed in the wit and gbb mutations. All three mutations also cause a statistically significant increase in synaptic footprints compared to wild-type, demonstrating that canonical BMP signaling is necessary for synaptic stability as well as for growth (Eaton, 2005).
The fact that synaptic footprints in wit can be rescued by neuronal expression of UAS-wit suggests that BMP signaling is necessary in the motoneuron for synaptic stability. To confirm that Smad-mediated signaling is also required within the motoneuron for synaptic stabilization, the inhibitory Smad dad was neuronally overexpressed (dad gain of function, DAD GOF). Genetic evidence suggests that this manipulation can block both Mad and DSmad2 signaling. Neuronal DAD GOF was found to decrease bouton numbers and increases synaptic footprints to levels that are near those observed in tkv, mad, and medea. Finally, in order to examine whether Smad signaling is required in the cell soma for synaptic stabilization, a genetic interaction was examined between the wit mutation and the overexpression of a dominant-negative Glued transgene (DN-Glued) that disrupts retrograde axonal transport. Previous work has demonstrated that impairment of dynactin function in motoneurons disrupts retrograde axonal transport and causes synapse retraction at the NMJ. The number of retractions in the DN-Glued; wit double mutant is not additive with respect to the wit mutation or DN-Glued overexpression. Although the overexpression of DN-Glued cannot be considered a null mutant condition, it is sufficient to block the accumulation of nuclear phospho-Mad and therefore prevents BMP signaling to the nucleus. Thus, it is concluded that Smad-mediated signaling to the motoneuron cell soma is necessary for both synaptic growth and synaptic stability. Together, these data establish an in vivo link between retrograde axonal transport, an essential trophic signaling system, and the mechanisms of synaptic stabilization (Eaton, 2005).
Additional experiments tested whether mutations that decrease the number of boutons always increase the number of footprints. In these experiments, mutations were analyzed that decrease bouton number but that have not been implicated in the BMP signaling system. Bouton numbers are significantly decreased in these three independent mutations: the cell adhesion molecule fasciclin II (fasII); the tyrosine phosphatase Dlar, and the microtubule binding protein futsch. In each of these three mutations, bouton numbers are significantly decreased whereas the number of synaptic footprints remains unchanged. It has also been shown that impaired synaptic growth in a presynaptic calcium channel mutant does not alter synaptic stability. From these data it can be concluded that impaired synaptic growth does not necessarily impair synaptic stabilization. By extension, it is concluded that BMP signaling is required for two separable processes, synaptic growth and synaptic stability (Eaton, 2005). While all of the mutations in canonical BMP signaling molecules decrease bouton numbers to the same extent, there are significantly fewer synaptic footprints in the tkv, mad, and medea mutations (as well as in DAD GOF) when compared to the wit mutation. Thus, while canonical BMP signaling is required for both synaptic growth and stability, the Wit receptor has an additional stabilizing influence on the NMJ that cannot be accounted for by the downstream Smad signaling system. Therefore experiments were pursued to investigate the mechanism of Wit-mediated synaptic stability that appears to be independent of Smad-mediated signaling (Eaton, 2005).
It was first determined whether the signaling associated with synaptic stabilization and synaptic growth might map to different regions of the cytoplasmic tail of the Wit receptor. To do so, a transgenic rescue approach was taken that involves neuronal expression of either the full-length wit transgene or a truncated wit transgene in the wit mutant background. The truncated Wit receptor lacks a C-terminal portion that is not required for Smad signaling in mammalian systems and has been shown to restore viability to the wit mutation. First, it was demonstrated that presynaptic expression of the full-length wit transgene in the wit mutant background rescues synaptic growth and synaptic stability. However, while presynaptic expression of the truncated wit transgene (wit-dCT) completely rescues synaptic growth, it is unable to fully restore synaptic stability to wild-type levels. It was confirmed that the Wit-dCT receptor is able to activate downstream Smad signaling, by showing that neuronal expression of the wit-dCT construct rescues the presence of nuclear phospho-Mad in the wit mutant background.
These data identify a region of the Wit receptor that is necessary for synapse stabilization, but is not required for nuclear Smad signaling, synaptic growth, or synaptic function. Experiments were performed to investigate how this region of the Wit receptor influences synaptic stability (Eaton, 2005).
The C-terminal tail of mammalian BMP type II receptors has been shown to interact with and regulate the activity of LIMK1 in vitro (Foletta, 2003; Lee-Hoeflich, 2004). This study demonstrated that Drosophila LIM Kinase1 (DLIMK1) binds the C-terminal tail of the Wit receptor that is deleted in the wit-dCT transgene. Peptides corresponding to the N-terminal region of DLIMK1 containing either the tandem LIM and PDZ domains together (LIM-PDZ) or containing the tandem LIM domains alone (LIM only) interact with the C-terminal region of the Wit receptor (C-Term), but not with the kinase domain (Kinase) in a yeast two-hybrid binding assay. No interactions were detected between the PDZ domain of DLIMK1 and the Wit receptor in this analysis. In addition, an interaction was detected between the C-terminal region of the Wit receptor (Wit-CT-FLAG) and full-length DLIMK1 (DLIMK1-HA) when these proteins were coexpressed in Drosophila S2 cells. These observations suggest that the LIM domains of DLIMK1 mediate specific binding to the C-terminal tail of the Wit receptor, consistent with findings (Foletta, 2003; Lee-Hoeflich, 2004) reported for LIMK1 binding to mammalian BMP receptors (Eaton, 2005).
Next, whether DLIMK1 is required for synaptic growth and/or stabilization was tested. A P element insertion (DLIMKP1) was acquired that resides in a 5′ untranslated exon of the DLIMK1 gene as well as two deficiency chromosomes that uncover the DLIMK1 locus. Northern analysis demonstrates that DLIMKP1/DLIMKP1 results in the near absence of detectable message, indicating that this is a strong-hypomorphic or null mutation. The DLIMK1 mutations, including DLIMKP1/DLIMKP1, DLIMKP1/Y, DLIMKP1/Df(1)JA26, and DLIMKP1/Df(1)HF368 are all viable. This is consistent with the observation that LIMK1 knockout mice are homozygous viable (Meng, 2002). This study demonstrates that DLIMKP1/DLIMKP1, DLIMKP1/Y, DLIMKP1/Df(1)JA26, and DLIMKP1/Df(1)HF368 all cause a significant increase in synaptic footprints (p <0.001) without decreasing synaptic bouton number. These genetic data indicate that DLIMK1 is necessary for synaptic stability, but is not required for normal synaptic growth. Since the number of synaptic footprints is comparable when DLIMKP1 is analyzed in trans to deficiency chromosomes that uncover the DLIMK1 locus, it is concluded that this P element insertion represents a strong loss-of-function mutation (Eaton, 2005).
To support the conclusion that DLIMK1 function is necessary for synapse stabilization and to determine whether DLIMK1 functions in the nerve or the muscle to control synaptic stability, transgenic rescue experiments and overexpression experiments were pursued using a dominant-negative DLIMK1 transgene. It was found that neuronal expression of a dominant-negative, kinase-inactive DLIMK1 (DN-DLIMK1) transgene significantly increases synaptic retractions while muscle-specific expression of DN-DLIMK1 has no effect. There was a slight decrease in bouton number observed when DN-DLIMK1 was expressed neuronally that was not observed in the DLIMK1 mutations. This may represent a dominant effect of this transgene. However, this decrease in bouton number is significantly less than that observed following disruption of BMP signaling. Finally, in agreement with experiments using a dominant-negative transgene, it was found that neuronal expression of a wild-type DLIMK1 transgene (UAS-DLIMK1) restores synapse stability to the hemizygotic DLIMKP1/Y loss-of-function mutation without altering other aspects of synapse development. Together, these data support the conclusion that DLIMK1 is specifically required in the presynaptic motoneuron for synaptic stabilization (Eaton, 2005).
The observation that DLIMK1 binds the Wit receptor and is required presynaptically for synapse stabilization suggests that DLIMK1 functions downstream of the Wit receptor and may confer the added synapse-stabilizing activity of the Wit receptor. To address this possibility, genetic interactions were examined between the wit and DLIMK1 mutations. Animals harboring one mutant copy of wit and one mutant copy of DLIMK1 (DLIMKP1/+; witA12/+) show a significant increase in synaptic footprints without a change in bouton number and without a change in synaptic function compared to wild-type. In comparison, heterozygous mutations in either gene alone do not show a significant increase in synaptic footprints compared to wild-type and have normal bouton numbers. The DN-DLIMK1 was neuronally overexpressed in the wit mutant background and no additive effect was found on the number of NMJs with retractions with respect to the wit mutation or the overexpression of DN-DLIMK1 alone. The strong transheterozygous interaction between the witA12 mutation and DLIMK1P1, as well as the lack of an additive effect in the number of NMJs with footprints when DN-DLIMK1 is overexpressed in the wit mutant background, supports the conclusion that DLIMK1 functions in the same genetic pathway as wit to control synaptic stability (Eaton, 2005).
Next whether overexpression of UAS-DLIMK1 could rescue synaptic stability in the wit mutant background was investigated. To do so, the wild-type DLIMK1 transgene (UAS-DLIMK1) was neuronally expressed in the wit mutant background. When UAS-DLIMK1 was expressed in the wit mutant background using either a pan-neuronal (elav-GAL4) or motoneuron-specific (OK6-GAL4) GAL4 driver, defects were rescued in both synaptic growth and synaptic stability. Importantly, strong presynaptic expression of UAS-DLIMK1 by elav-GAL4 raised at 29°C resulted in complete rescue of synaptic footprints to wild-type levels. In all rescue experiments, including overexpression of DLIMK1 in the wit background, the presence of the wit mutation was confirmed. These data, in combination with the demonstration that DLIMK1 binds the Wit receptor and the genetic interactions observed between wit and DLIMK1, support the conclusion that DLIMK1 functions downstream of wit to control synaptic stability (Eaton, 2005).
Remarkably, rescue of the wit mutation by UAS-DLIMK1 not only restores synaptic growth and stability, but also rescues animal viability. wit mutant animals normally die during midlarval life and are never observed to mature into adult flies. Neuronal expression of DLIMK1 (with elav-GAL4) in the wit mutant background restores adult viability. Adult flies emerge in large numbers and are able to climb normally the vertical sides of a plastic vial (Eaton, 2005).
A further analysis has demonstrated that DLIMK1 expression restores synaptic function to the wit receptor mutation. Electrophysiological analysis of the wit mutant synapse demonstrates a severe impairment of synaptic transmission including a dramatic decrease in the excitatory postsynaptic potential (EPSP) amplitude, a significant decrease in the average amplitude of the spontaneous miniature release events (mEPSP), and a dramatic decrease in presynaptic release as estimated by the average EPSP/average mEPSP. Expression of UAS-DLIMK1 restores all aspects of synaptic function to near wild-type levels, including EPSP amplitude, mEPSP amplitude, and quantal content. The degree of rescue with UAS-DLIMK1 is not statistically different from that observed when wit is rescued by the full-length wit transgene. A slight but significant decrease in quantal content was observed in the DLIMKP1/DLIMKP1 homozygotes compared to wild-type, although this decrease is much less than that observed in wit or mad mutants. It is therefore concluded that DLIMK1 is able to rescue the wit electrophysiological defects even though it is not normally involved in BMP-dependent regulation of synaptic function. Thus, DLIMK1 expression can restore all of the essential functions of the Wit receptor in the wit mutant background (Eaton, 2005).
Next to be tested was whether the rescue of synaptic growth and stability by DLIMK1 expression in the wit mutant background is caused by activation of downstream Mad signaling that is independent of the Wit receptor. It was first demonstrated that mad mutations have a significant increase in synaptic footprints and a significant reduction in bouton number. It was found that neuronal expression of DLIMK1 in the mad mutant background rescues both synaptic growth and stability to wild-type levels. However, unlike expression of DLIMK1 in the wit mutant background, expression of DLIMK1 does not restore viability to the mad mutant background. This difference is likely due to the expression of mad outside of the nervous system, whereas wit expression is largely restricted to the nervous system. Thus, DLIMK1 is sufficient to promote synaptic growth and stability in the absence of Mad-mediated signaling. These data support the conclusion that DLIMK1 functions in parallel to Mad-mediated signaling in the motoneuron to control synaptic stability and growth (Eaton, 2005).
The localization of DLIMK1 was investigated in the presence and absence of the Wit receptor. The UAS-DLIMK1 transgene used in the rescue experiments harbors an HA epitope tag that was used to visualize DLIMK1 at the NMJ. Staining for HA in animals with neuronal expression of UAS-DLIMK1 revealed a filamentous DLIMK1 localization throughout the presynaptic nerve terminal. This staining pattern is identical in wild-type or wit mutant animals that neuronally express UAS-DLIMK1. Surprisingly, DLIMK1-HA was found in very close association with the synaptic microtubules identified by anti-Futsch (Map1B-like protein). However, although DLIMK1-HA is closely associated with anti-Futsch, the two markers do not colocalize. Rather, the DLIMK1-HA staining appears as an independent continuous filament that extends throughout the NMJ. Two additional experiments were pursued to test whether the DLIMK1-HA staining pattern requires the integrity of the synaptic microtubule cytoskeleton. (1) DLIMK1-HA was overexpressed in the futsch mutant background, which severely disrupts the synaptic microtubule cytoskeleton. Filamentous DLIMK1-HA staining remains in the futsch mutation, indicating that DLIMK1 filaments are independent of stable synaptic microtubules. (2) Whether DLIMK1-HA is perturbed following treatment of the synapse with nocadozole was examined at a concentration that eliminates dynamic microtubule plus ends. It was found that nocadozole treatment slightly decreases the intensity of DLIMK1-HA staining, but clear filamentous staining remains. These data suggest that DLIMK1-HA filaments are closely associated with synaptic microtubules, but can be stable in the absence of a continuous synaptic microtubule cytoskeleton. This staining pattern does not resemble the synaptic localization of actin-GFP, which concentrates into randomly distributed patches throughout the NMJ. Thus, DLIMK1 concentrates into a unique compartment within the presynaptic nerve terminal that has not been previously described. In addition, instances were found in which this compartment approached the synaptic plasma membrane, where it could associate with membrane receptors such as the Wit receptor. Finally, the possibility that low levels of DLIMK1 are present throughout the cytoplasm and in association with synaptic actin cannot be ruled out, since steady-state protein distribution was examined in fixed tissue (Eaton, 2005).
The role of LIMK1 during axon outgrowth and growth cone motility is largely due to LIMK1 phosphorylation and inactivation of ADF/cofilin, which alters actin turnover (Endo, 2003; Ng, 2004). In these studies, the phosphorylation of ADF/cofilin by LIMK1 is antagonized by the Slingshot (Ssh) family of phosphatases (Niwa, 2002). Therefore, if ADF/cofilin is the target of LIMK1, then the overexpression of the Ssh phosphatase should mimic LIMK1 loss of function in this signaling cascade. It was found that overexpression of the ssh cDNA in the motoneuron (elav UAS-SSH wt) has no effect on synaptic growth, synaptic stability, or synaptic function. It was confirmed that Ssh protein traffics to the synapse in these experiments and that Ssh has a cytoplasmic localization that does not resemble what was observed for DLIMK1-HA. Finally, overexpression of a constitutively activated cofilin transgene (elav UAS-tsr-S3A) also has no effect on synaptic growth or stabilization. In combination with the observation that the distribution of synaptic DLIMK1 does not resemble the distribution of synaptic actin, these data suggest that LIMK1 is not acting through ADF/cofilin to control synaptic stability. These data highlight differences in LIMK1 function during the rapid, dynamic process of axon outgrowth versus the slower, more prolonged mechanisms that govern synapse stabilization at the NMJ (Eaton, 2005).
This study demonstrates that BMP-receptor signaling at the
Drosophila NMJ controls both synaptic growth and synaptic stabilization. The data support a model in which signaling from the BMP receptor can coordinately activate two genetically separable developmental programs: (1) cell-wide regulation of neuronal growth via nuclear Smad signaling and (2) LIMK1-dependent synaptic stabilization. This organization of synaptic signaling systems involved in synaptic growth versus synaptic stabilization could have important consequences for neural development. Since a single trophic receptor can increase both cell-wide growth and synaptic stabilization, the efficiency of trophic signaling could be increased due to the coupling of new synapse growth with synaptic stabilization and retention. Another scenario is also possible that could help to explain how synaptic growth and elimination could occur simultaneously at the terminals of a single motoneuron. If LIMK1 functions locally at the synapse, then cell-wide growth signaling could be uncoupled from the local control of synaptic stability. Thus, the loss of trophic signaling at a single muscle target could lead to target-specific synapse destabilization while the same trophic signaling system at other muscle targets could simultaneously promote synaptic growth throughout the motoneuron via cell-wide growth signaling. While there is no direct evidence that LIMK1 functions locally at the synapse, the synaptic activation of kinase signaling downstream of the BMP receptor could support such a model (Eaton, 2005).
The data support a model in which DLIMK1 functions downstream of the Wit receptor and in parallel to Smad-mediated nuclear signaling in order to achieve wild-type synaptic stability. Several lines of evidence indicate that DLIMK1 functions downstream of the Wit receptor. DLIMK1 was shown to bind to a region of the Wit receptor that is necessary for synaptic stabilization, but which is dispensable for Smad-mediated synaptic growth. DLIMK1 mutations were shown to specifically disrupt synaptic stabilization and it was found that both DLIMK1 and Wit are necessary within the presynaptic neuron for synapse stability. A strong transheterozygotic genetic interaction was demonstrated between mutations in DLIMK1 and wit, resulting in a specific loss of synaptic stability. It was further shown that neuronal overexpression of a dominant negative DLIMK1 in the wit mutant background does not result in an additive increase in the number of synapse retractions. From these data, it is concluded that DLIMK1 functions downstream of Wit to control synaptic stability. It was also shown that the stabilizing activity of DLIMK1 does not require the presence of the Smad signaling system, since expression of DLIMK1 in either the wit or the mad mutant background is able to restore synaptic stability to the NMJ. While these data are consistent with DLIMK1 functioning either downstream or in parallel to Smad-mediated signaling, the conclusion is favored that DLIMK1 functions in parallel to Smad because DLIMK1 binds a region of the Wit receptor that is dispensable for Smad-mediated synaptic growth (Eaton, 2005).
These data demonstrate that signaling via the Wit receptor stabilizes the synapse via both Smad-dependent and LIMK1-dependent signaling. Mutations in mad, medea, and expression of the inhibitory Smad dad (DAD-GOF) all cause an increase in synapse retractions as well as a decrease in bouton number. The data indicate that Smad-mediated signaling accounts for approximately 50% of the stabilizing activity of the Wit receptor, and LIM Kinase functions in parallel to mediate the other 50% of the Wit receptor's stabilizing activity. It is hypothesized that the stabilizing functions of Smad signaling and LIMK1 signaling are quite different. First, it is suspected that Smad-dependent synaptic stabilization is directly coupled to the growth-promoting function of the nuclear Smad signaling system. It is hypothesized that Smad-dependent growth regulation involves transcriptional programs that produce the necessary raw material for synaptic growth. In the absence of Smad signaling, these raw materials may become limiting, not only for new growth but also for the maintenance of the existing synapse, since synaptic proteins will need to be turned over and replaced at some rate. According to this logic, the synapse retractions caused by mutations in the Smad signaling system are related to synaptic atrophy. In contrast, DLIMK1 is necessary for synaptic stability, but is not required for normal synaptic growth. Thus, it is suspected that synapse retractions observed in DLIMK1 mutants are caused by modulation of the synaptic stabilization machinery resident at the synapse (Eaton, 2005).
Smad-independent signaling of TGF-β, including the BMP receptors, has been observed in a number of systems and includes the activation of other diverse signaling cascades such as MAPK, PI3K, Rho-like GTPases, and LIMK1. However, only LIMK1 has been associated biochemically with the region of the BMP type II receptor that was found to be specifically required for synaptic stabilization in the Wit receptor. In vitro kinase assays demonstrate that the interaction of LIMK1 with the BMPRII tail leads to changes in LIMK1 kinase activity (Foletta, 2003; Lee-Hoeflich, 2004). Although these studies reached different conclusions about the effects of receptor binding on LIMK1 activity, both studies support a model in which binding of BMP to the receptor complex leads to an increase in LIMK1 activity. It is proposed that ligand binding to the Wit receptor activates DLIMK1 to stabilize the NMJ (Eaton, 2005).
It is remarkable that the overexpression of DLIMK1 rescues all aspects of the wit receptor mutant phenotype, including synaptic growth, synaptic function, and animal viability. DLIMK1 expression is also sufficient to restore synaptic growth and stability to the mad mutation. These data contrast with the genetic analysis demonstrating that DLIMK1 is necessary for synaptic stability but is not required for normal synaptic growth and has only a minor role in functional synapse development. How can DLIMK1 expression restore synaptic growth, function, and viability in the absence of the BMP receptor? Even if DLIMK1 independently signals to the nucleus (Yang, 2004), it seems unlikely that DLIMK1 activity would be sufficient to restore the entire transcriptional program normally mediated by nuclear Smad signaling. Instead, it is proposed that the overexpression of DLIMK1 in the wit receptor mutant background hyperstabilizes the synapse, consistent with synaptic stabilization being the primary function of DLIMK1. If UAS-DLIMK1 hyperstabilizes the synapse, a second, parallel growth factor signaling system may thereby be allowed to assume the growth-promoting functions normally mediated by BMP signaling. This hypothesis invokes the existence of an unidentified second growth factor signaling cascade at the Drosophila NMJ. It seems likely that additional growth factor signaling exists since the synapse still grows to nearly 50% of its normal size in the wit mutant background. One candidate for a second synaptic growth signaling system is the activin signaling system. In the Drosophila central nervous system, ecdysone-regulated axonal remodeling during metamorphosis is regulated, in part, by signaling via the activin type I receptor Babo, the type II BMP receptor Put, and DSmad2. It should be noted, however, that the Put type II receptor lacks the C-terminal DLIMK1 binding region found in the Wit receptor. Therefore, the Put receptor probably does not normally signal via direct interactions with DLIMK1 during the remodeling of the CNS or during stabilization at the NMJ (Eaton, 2005).
In lower motor diseases, such as ALS, two events are thought to contribute significantly to disease progression: (1) the loss of nuclear trophic signaling due to impaired axonal transport, and (2) the loss of access to trophic signal due to synapse retraction. The ability of LIMK1 to stabilize the NMJ in the absence of trophic signaling might suggest a model in which activation of stability-promoting proteins, such as LIMK1, may counteract inappropriate synaptic disassembly during disease. It is interesting to note that LIMK1 has been shown to accumulate within the presynaptic nerve terminal during the late maturation of the mammalian NMJ, after the period of developmental plasticity observed during embryonic and early postnatal development (Wang, 2000). This observation supports an intriguing model in which the maturation of the mammalian NMJ from a highly plastic synapse early in development to a more stable adult synapse involves both the reduction of plasticity-associated proteins, such as CAP-23 and GAP-43, and the enrichment of stability-promoting proteins such as LIMK1. It remains to be determined whether synaptic loss in degenerative disease is directly related to the loss of stabilizing factors such as LIMK1 and, indeed, whether enhanced signaling from these factors could lead to restabilization of diseased synapses. However, the possibility that LIMK1 may function locally at the NMJ to promote synaptic stabilization, even in the absence of an essential source of trophic signaling, suggests a potent activity of LIMK1 in the nervous system that may have important therapeutic value as a future avenue for intervention in neuromuscular degenerative disease (Eaton, 2005).
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)
To understand the functions of NIPA1, mutated in the neurodegenerative disease hereditary spastic paraplegia, and of ichthyin, mutated in autosomal recessive congenital ichthyosis, their Drosophila melanogaster ortholog was studied. Spichthyin (Spict) is found on early endosomes. Loss of Spict leads to upregulation of bone morphogenetic protein (BMP) signaling and expansion of the neuromuscular junction. BMP signaling is also necessary for a normal microtubule cytoskeleton and axonal transport; analysis of loss- and gain-of-function phenotypes indicate that Spict may antagonize this function of BMP signaling. Spict interacts with BMP receptors and promotes their internalization from the plasma membrane, implying that it inhibits BMP signaling by regulating BMP receptor traffic. This is the first demonstration of a role for a hereditary spastic paraplegia protein or ichthyin family member in a specific signaling pathway, and implies disease mechanisms for hereditary spastic paraplegia that involve dependence of the microtubule cytoskeleton on BMP signaling (Wang, 2007).
Axonal abnormalities, including impairment of transport, are a hallmark of many neurological and neurodegenerative diseases. These include the hereditary spastic paraplegias (HSPs), a heterogeneous set of diseases characterized by degeneration of corticospinal tract axons and spasticity of the lower extremities. Different forms of the disease are termed either pure or complicated, depending on whether other mainly neurological symptoms are present. The mechanisms of degeneration in HSPs are unknown, but over twenty causative loci (SPG loci) have been mapped and thirteen cloned. Some SPG products are implicated in microtubule function or transport, including the microtubule motor protein kinesin, and the microtubule-severing protein spastin. Since microtubules are the route for fast axonal transport, the most distal portions of axons are likely to be most sensitive to impairments of microtubule function. A second class of SPG products are mitochondrial proteins, but it is not known how mutations in these cause axonal degeneration. A third class of SPG products are apparently associated with endosomes, judged by immunolocalization or the presence of domains such as MIT or FYVE. HSP is also caused by some mutations in the amyotrophic lateral sclerosis gene ALS2, which encodes alsin, a guanine-nucleotide-exchange-factor for the early endosomal GTPase Rab5. However, the mechanism by which impairment of endosomal membrane traffic might cause axonal degeneration is unknown. (Wang, 2007).
One membrane protein encoded by an SPG gene is SPG6, mutations in which cause a dominant pure form of HSP, and which is widely expressed, although enriched in brain tissue. SPG6 is a member of a protein family (Pfam: DUF803) predicted to have between seven and nine transmembrane (TM) domains. Three different amino-acid substitutions are known, one of which is found in ethnically disparate families and another caused by different nucleotide substitutions in the same codon, suggesting a dominant gain-of-function disease mechanism that can be mediated by only a few mutations in the protein. This protein family includes another human disease protein, ichthyin, mutations in which cause autosomal recessive congenital ichthyosis (ARCI), a skin disorder whose cellular basis is not understood. Ichthyin is widely expressed, although with high expression in keratinocytes, and little or no expression in brain, and at least six recessive alleles are known that cause substitutions of mainly conserved amino acids in different parts of the protein. In summary, little is known of the cellular roles of the SPG6 and ichthyin family (Wang, 2007).
To understand the normal role of the SPG6 and ichthyin protein family, and how changes in their function might lead to cellular defects, their Drosophila homolog, spichthyin (Spict) was have studied. Spict shows preferential localization on early endosomes. It regulates growth of the neuromuscular junction (NMJ) presynaptically, by inhibition of BMP (Bone Morphogenic Protein)/TGF-β (Transforming Growth Factor-β) signaling. BMP signaling regulates synaptic growth, function and stabilization at the NMJ. This study shows a novel role for BMP signaling in maintenance of microtubules and axonal transport, and that this function is also inhibited by Spict. These data suggest that Spict inhibits BMP signaling by regulating BMP receptor traffic. These findings provide a cellular role for the Spict family of proteins, and suggest potential mechanisms for the pathology of HSPs and ARCI that include dependence of microtubules on BMP signaling (Wang, 2007).
A BLASTP search using human SPG6 identified one Drosophila homolog, CG12292. A search using CG12292 identified four predicted human proteins that were 40-50% identical to it: SPG6 (NIPA1), NIPA2, ichthyin and NPAL1. Two more distantly related human proteins, NPAL2 and NPAL3 are more closely related to plant and fungal homologs than to CG12292, and probably represent a subfamily lost from the Drosophila lineage. Since Drosophila CG12292 appears orthologous to both SPG6 and ichthyin, it was designated spichthyin (spict).
To generate spict mutant flies, transposase-mediated imprecise excision was used of a P element, EP(2)2202, inserted in the spict 5' untranslated region. One imprecise excision, spictmut, had lost the entire coding region, and was therefore a null allele of spict. Several precise excision events were recovered; one of these was used as a wild-type control in most subsequent experiments, and is referred to as spict+. Homozygous spictmut flies were viable and fertile, and took about a day longer than spict+ flies to reach adulthood (Wang, 2007).
To determine where Spict might act, its expression pattern and subcellular localization was examined. spict mRNA was found ubiquitously during embryogenesis, with elevated expression in some tissues, including CNS and muscles. EGFP-Spict and Spict-EGFP fusions both showed punctate distributions in Drosophila S2 cells, that overlapped substantially with the early endosome compartment detected using anti-Rab5, but showed no striking overlap with the late endosomal/multivesicular body marker Hook, the recycling endosomal marker Rab11, or the late endosomal/lysosomal markers Spinster and LysoTracker. A Spict-mRFP fusion protein also showed a punctate cytoplasmic distribution in wild-type and spictmut third instar larvae, which also overlapped substantially with Rab5, but not with late endosomal/lysosomal markers, in muscles and NMJs. Trypsin digestion of N-terminally and C-terminally tagged Spict, redistibuted to the plasma membrane by blockage of endocytosis, suggested that the N-terminus of Spict is in the endosome lumen, and its C-terminus in the cytosol. This result is consistent with previous suggestions that Spict family members might either have nine transmembrane domains, or be divergent members of the 7-TM superfamily. Attempts to raise an antibody that recognized endogenous Spict in immunomicroscopy were unsuccessful. However, since Spict-EGFP and EGFP-Spict fusions had apparently identical localizations in S2 cells, the Spict-mRFP fusion could rescue a spictmut phenotype and cause the same overexpression phenotypes as wild type Spict, these fusions are likely to have the same localization as endogenous Spict (Wang, 2007).
Since tagged Spict proteins localized with Rab5, tests were performed to see whether Rab5 staining is normal when Spict is lacking. Rab5 staining was less intense in spictmut NMJ boutons compared to wild-type; these phenotypes were rescued by ubiquitous expression of UAS-spict. Rab5 staining was also reduced in muscles but not obviously affected in neuronal cell bodies and axons of spictmut larvae, or in S2 cells treated by spict RNAi. Therefore, Spict is essential for a normal Rab5 compartment at the NMJ, but not in all situations (Wang, 2007).
One of the signaling pathways with the largest effects on synaptic size at the Drosophila NMJ is the BMP pathway, which stimulates synaptic growth. The expanded NMJ phenotype of spictmut is similar to that of spinster (also known as benchwarmer), which also shows defects in endosomal-lysosomal trafficking and requires an active BMP/TGF-β signaling pathway for NMJ expansion. It is also similar to the increase in bouton number of highwire NMJs. Highwire encodes a putative E3 ubiquitin ligase that appears to affect multiple signaling pathways including JNK and BMP. To determine whether the synaptic overgrowth of spictmut larvae requires BMP signaling, key BMP signaling components were genetically removed from spictmut larvae. Mutations affecting the type I receptor subunits Tkv (Thickvein) and Sax (Saxophone), the type II receptor subunit Wit (Wishful Thinking), the type II receptor ligand Gbb (Glass Bottom Boat), or the co-Smad Med (Medea) all suppressed the NMJ overgrowth of spictmut larvae. In all cases, the synaptic undergrowth in larvae that were doubly homozygous for spictmut and BMP pathway mutations was indistinguishable from that of homozygous BMP pathway mutations alone. In addition, all heterozygous BMP pathway mutations tested partly suppressed the NMJ expansion of spictmut larvae, but had no effect on NMJ bouton number in a wild type background. Therefore, BMP signaling is essential for the excessive NMJ growth of spictmut larvae (Wang, 2007).
The contrasting phenotypes of spictmut and loss of BMP signaling, and the genetic interactions between spict and BMP signaling mutants, suggest that Spict antagonizes BMP signaling in the control of NMJ growth. Nevertheless, alternative models are possible: for example, highwire mutations interact with BMP signaling mutations, but Highwire affects synaptic size primarily through a MAPK signaling pathway. However, evidence strongly supports a direct effect of Spict on BMP signaling. During BMP signaling in neurons, the R-Smad protein Mad is phosphorylated by active BMP receptors, and phosphorylated Mad (PMad) is then translocated to the nucleus and acts as a transcription factor. At the NMJ, PMad overlaps mainly with the presynaptic marker cysteine string protein (CSP), but also with the largely postsynaptic marker Discs-large (Dlg). PMad is also found in cell body nuclei in the larval CNS. PMad levels were significantly higher in spictmut than in spict+ larvae, both at the NMJ and in CNS cell bodies, and this phenotype was fully rescued by neuronal expression of UAS-spict. Therefore, BMP signaling is upregulated at spictmut neurons, in contrast to highwire neurons. Next the possibility of upregulation of BMP receptors at spictmut NMJs was tested. HA-tagged Tkv was found mainly in a punctate distribution in the periphery of synaptic boutons, at or close to the plasma membrane, and at higher levels in spictmut than in spict+ boutons. Wit was barely detectable in spict+ boutons, but was present at higher levels in spictmut boutons, also in a punctate pattern mainly at or close to the plasma membrane. The effect of spictmut on Tkv-HA and Wit levels was rescued by neuronal expression of UAS-spict. No effect was found of spictmut on levels of other neuronal membrane proteins (Fasciclin II, Syntaxin), or on the neuronal surface antigen recognized by anti-Horseradish Peroxidase (HRP) at the NMJ. Therefore, Spict action specifically lowers the levels of BMP receptors at the presynaptic NMJ (Wang, 2007).
The opposing effects of Spict and BMP signaling on NMJ and neuronal microtubules suggest that Spict is a novel antagonist of BMP signaling. BMP signaling acts both presynaptically and postsynaptically at the NMJ; rescue experiments show that Spict acts presynaptically to regulate NMJ expansion. The data suggest a direct effect of Spict on the presynaptic BMP signaling machinery. First, elevated levels of PMad and BMP receptors are seen at spictmut NMJs. Second, Spict can be co-immunoprecipitated with Wit. Third, Spict shows partial colocalization with the BMP receptors Tkv-HA or Wit at NMJ boutons. Fourth, Spict promotes relocalization of Wit from the surface of S2 cells to the Rab5 early endosomal compartment. Therefore, these data suggest strongly that Spict antagonizes BMP signaling by regulating its receptor traffic. This is in contrast to Highwire - while synaptic overgrowth in highwire mutants can be suppressed by BMP signaling mutants, the highwire phenotype is more completely suppressed by loss of the Wallenda MAP kinase kinase kinase, and there is no apparent upregulation of PMad in highwire mutants (Wang, 2007).
The posterior crossveinless phenotype in some spictmut adult wings is also typical of reduced BMP signaling in pupal wing discs. At first sight a crossveinless phenotype is inconsistent with Spict being an antagonist of BMP signaling. However, lowered BMP signaling in the posterior crossvein primordium could be due not only to direct downregulation of signaling, but also to upregulation of receptors that reduces diffusion of BMP ligands. No changes were detected in the level of BMP signaling about the time when the posterior crossvein primordium develops, but this could be due to either the partial penetrance of the phenotype, or the robustness of the regulatory and feedback mechanisms that translate smooth gradients of BMP ligands into more sharply defined developmental features (Wang, 2007).
How might an endosomal protein regulate BMP signaling? Membrane trafficking from the plasma membrane to lysosomes regulates many signaling pathways including BMP/TGF-β. For example, mutations that impair endosome to lysosome traffic cause an increase in BMP signaling, in at least some cases accompanied by increased levels of Tkv. However, the predominant localization of Spict on early endosomes, and its ability to internalize Wit to this compartment suggest that Spict functions at some step of plasma membrane to endosome traffic. (1) Rab5 compartments fail to accumulate at spictmut NMJs, rather than enlarge as in Hrs mutants. (2) Spict overexpression in S2 cells redistributes Wit mainly to early endosomes, rather than to late endosomes or lysosomes. (3) There is no obvious degradation of Wit in Spict-overexpressing cells that internalize Wit, suggesting that Spict does not directly target Wit for degradation, at least in S2 cells. While levels of BMP receptors are elevated locally in NMJ boutons that lack Spict, this could be either to altered trafficking or degradation, and BMP signaling in S2 cells can be affected by Spict, without detectable changes in levels of BMP receptors. Therefore, Spict might inhibit BMP signaling by internalizing vacant receptors and thus preventing them from responding to ligand; since clathrin RNAi treatment redistributes Spict to the plasma membrane, Spict probably appears at least transiently at the plasma membrane. However, more complex models are possible. For example, Spict might sequester BMP receptors in a compartment from which they cannot signal; Notch receptors apparently have to reach a specific endosomal compartment before they can signal (Wang, 2007).
By studying Spict, this study has identified a role for BMP signaling in maintenance of axonal microtubules. Notably, local loss of presynaptic microtubules has also been seen in loss of BMP signaling at the NMJ, and apical microtubule arrays are eliminated in tkv mutant clones in wing imaginal discs. Since BMP signaling promotes synaptic growth and synaptic strength at the NMJ, it would be logical for it also to stimulate the additional transport of materials and organelles that a larger more active synapse requires (Wang, 2007).
If human SPG6 alleles are dominant gain-of-function, then the HSP that they cause would resemble the situation of Spict overexpression in Drosophila, and axonal degeneration in HSP could then be caused by inhibition of BMP signaling, loss of axonal microtubules, and impaired axonal transport. Given the effect of BMP signaling on axonal microtubules, other HSP gene products apart from SPG6 may affect BMP signaling and thus maintenance of axonal microtubules. (Wang, 2007).
In contrast to SPG6, ARCI appears to be caused by loss of ichthyin function (Lefevre, 204). Identification of a role for the ichthyin ortholog Spict in inhibiting BMP signaling suggests upregulation of BMP signaling as a possible disease mechanism in ARCI. Indeed, the BMP-like ligand TGF-β1 has complex roles in maintenance of skin, and its overexpression can cause psoriasis, a condition that bears some resemblance to ichthyosis. Inhibitors of BMP signaling may therefore be candidates for therapeutic purposes in ARCI or similar conditions. (Wang, 2007).
In conclusion, this study has established a cellular role for the SPG6 and ichthyin family of proteins, thus identifying a novel group of players in BMP signaling, and providing a framework for future understanding of diseases caused by mutations that affect these proteins (Wang, 2007).
miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, its single mir-124 locus was deleted, and it was found to be dispensable for gross aspects of neural specification and differentiation. In contrast, a variety of mutant phenotypes were detected that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124-expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology (Sun, 2012).
microRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs that function primarily as post-transcriptional repressors. In animals, miRNAs have propensity to target mRNAs via 6-7 nt motifs complementary to their 5' ends, termed 'seed' regions. This limited pairing requirement has allowed most miRNAs to capture large target networks. Analysis of multigenome alignments indicates that typical human miRNAs have hundreds of conserved targets, and that a majority of protein-coding genes are under miRNA control. The extraordinary breadth of animal miRNA:target networks has been extensively validated by transcriptome and proteome studies (Sun, 2012).
miR-124 is strictly conserved in both primary sequence and spatial expression pattern, being restricted to the nervous system of diverse metazoans, including flies, nematodes Aplysia, and all vertebrates studied. Such conservation implies substantial functions of miR-124 in controlling neural gene expression. miR-124 has been a popular model for genomewide investigations of miRNA targeting principles. For example, studies of miR-124 yielded the first demonstration of the downregulation of hundreds of direct targets detected by transcriptome analysis, and that this activity was driven by the miRNA seed region. In addition, miR-124 provided one of the first illustrations of spatially anticorrelated expression of a miRNA and its targets and direct identification of Ago-bound target sites (Sun, 2012).
Functional studies have connected vertebrate miR-124 to various aspects of neural specification or differentiation. Studies in chick ascribed miR-124 as a proneural factor that inhibits the anti-neural phosphatase SCP1. However, no substantial effect of miR-124 on chick neurogenesis was found in a parallel study, although miR-124 was observed to repress neural progenitor genes such as laminin gamma1 and integrin beta1. In the embryonic mammalian brain, miR-124 was reported to direct neural differentiation by targeting polypyrimidine tract binding protein 1 (PTBP1), a global repressor of alternative splicing in non-neural cells. In the adult mammalian brain, miR-124 promoted neural differentiation of the immediate progenitors, the transit-amplifying cells (TAs). Here, miR-124 was shown to directly target the transcription factor Sox9, which maintains TAs and is downregulated during neural differentiation. Other mammalian studies bolster the concept that miR-124 promotes neurogenesis or neural differentiation. One mechanism involves direct repression by miR-124 of Baf53a, a neural progenitor-specific chromatin regulator that must be exchanged for a neural-specific homolog to consolidate neural fate. However, complicating the picture is the recent report that Xenopus miR-124 represses neurogenesis by directly targeting the proneural bHLH factor NeuroD1 (Sun, 2012 and references therein).
All vertebrate miR-124 loss-of-function studies have relied on antisense strategies and have yet to be validated by bona fide mutant alleles. However, as the three vertebrate mir-124 loci are co-expressed in the nervous system, analysis of the null situation will require a triple knockout. So far, a mir-124 knockout has only been described in C. elegans, which harbors a single copy of this gene. Like most other miRNA mutants in this species, the loss of miR-124 did not cause obvious developmental, physiological or behavioral phenotypes. Nevertheless, comparison of gene expression in mir-124-expressing cells from wildtype and mir-124 mutant animals revealed strong enrichment in miR-124 target sites amongst upregulated transcripts, revealing the impact of miR-124 on neuronal gene expression (Clark, 2010). The broad, but phenotypically-tolerated, misregulation of miR-124 targets in this species is potentially consistent with the 'fine-tuning' model for miRNA regulation (Sun, 2012).
This study analyzed a knockout of the sole mir-124 gene in Drosophila. Although this mutant is viable and exhibits grossly normal patterning, numerous phenotypes were documented, including short lifespan, increased variation in the number of dendritic branches of sensory neurons, decreased locomotion and aberrant synaptic release at CNS motoneuron synapses. All of these phenotypes were rescued by a single copy of a 19 kilobase (kb) genomic transgene encompassing the mir-124 locus. A transcriptional reporter of mir-124 was generated that recapitulated the CNS expression of endogenous pri-mir-124, and this was used to purify mir-124-expressing cells from stage-matched wild-type and mir-124-mutant embryos. Transcriptome analysis revealed strong enrichment of direct miR-124 targets amongst genes upregulated in mir-124-mutant cells. The miR-124 target network included coordinate repression of multiple components in the retrograde BMP signaling pathway, whose activity controls synaptic release. Loss of miR-124 further correlated with increased activity of other neural miRNAs and the neural translational regulator Pumilio, and had the net effect of impairing transition from the neuroblast to neuronal gene expression signature. Altogether, it was demonstrated that endogenous miR-124 has substantial impact on CNS gene expression, which underlie its requirement for organismal behavior and physiology (Sun, 2012).
These studies of Drosophila mir-124 demonstrate that its loss is compatible with grossly normal neural development and differentiation, despite broad changes in gene expression and global upregulation of direct miR-124 targets. Nevertheless, many clear defects are detected in these mutants, including short lifespan of adult males, defective larval locomotion, and aberrant synaptic transmission. The latter phenotype is perhaps reminiscent of reports that inhibition of Aplysia miR-124 similarly results in an increase in evoked EPSP amplitude. These phenotypes were confirmed phenotypes to be due to miR-124 loss, as shown by their rescue by a mir-124 genomic transgene. Importantly, these phenotypes were obvious even under optimal culture conditions, demonstrating palpable requirements for this miRNA in the intact animal. It remains to be seen if synaptic overactivity in the mir-124 mutant can be directly linked to the behavioral defects observed at the organismal level. The electrophysiological defects in mir-124 mutants phenocopy activation of BMP signaling at the synapse, and miR-124 directly targets multiple components of this pathway. Still, it remains possible that the many other gene expression changes in mir-124 mutant neurons contribute to its loss of function phenotype. The detailed in vivo transcriptome-wide analysis of endogenous miR-124 targets sets the stage for future studies of how individual targets might affect different settings of miR-124 function (Sun, 2012).
Only a handful of other miRNA mutants are lethal or exhibit overt morphological defects, suggesting that many miRNAs serve as robustness factors. For example, a Drosophila mir-7 mutant exhibits minor cell specification defects, but these are enhanced by heat shock. In addition, the introduction of many C. elegans 'benign' miRNA mutants into genetically sensitized backgrounds uncovers a high frequency of phenotypes. Interestingly, miR-124 is not required for normal dendrite formation per se, but its absence caused a broader distribution of dendrite numbers on ddaD and ddaE neurons, i.e. a 'robustness' defect. It is speculated that environmental or genetic stress may reveal additional requirements for miR-124 in development and differentiation of the nervous system (Sun, 2012).
In light of the broad roles ascribed to endogenous miR-124 in neurogenesis, neural differentiation, and neural physiology (Gao, 2010), all from antisense strategies, the extensive negative data from the current Drosophila mir-124 knockout are equally compelling. While the relevant neural subpopulation may not have been examined, these studies indicate that miR-124 is not required for gross aspects of neurogenesis and differentiation in the embryonic and larval nervous system. Similarly, C. elegans deleted for mir-124, which is expressed mostly in ciliated sensory neurons, do not reveal obvious defects in neural development (Clark, 2010). Given that these invertebrate orthologs of miR-124 are identical in sequence to their vertebrate counterparts, and are highly and specifically expressed in their respective nervous systems, there is not strong reason a priori to suspect that miR-124 should not have comparable requirements amongst different animals. The analysis of vertebrate mir-124 knockouts is therefore highly anticipated (Sun, 2012).
The Drosophila system has been critical for elucidating fundamental features of miRNA target recognition in animals, and for studying specific miRNA-target interactions that mediate phenotype. However, it has been little-used to analyze the effects of miRNA-mediated gene regulation in the animal at the transcriptome-wide level. Perhaps the clearest example is the broad upregulation of maternal transcripts in early embryos lacking the mir-309 cluster. However, most miRNAs are tissue or cell-specific, and while it is much simpler to profile transcripts from whole flies, the inclusion of irrelevant cells can mask the action of the miRNA. For example, only 4/200 transcripts upregulated in mir-8 mutant pupae appeared to be direct conserved targets (Sun, 2012).
By purifying cognate miRNA-expressing cells from wild-type and miRNA-mutant backgrounds, this study succeeded in assessing transcriptome-wide effects of genetic removal of miR-124 with precision. The data provide a new perspective on the utilization of 'anti-targeting' in Drosophila. Previously, miR-124 was selected as a particularly compelling case in which its Drosophila targets were depleted for in situ terms related to nervous system development, and enriched for terms related to epidermal development. Since these tissues derive from a common developmental progenitor, the neuroectoderm, this led to a model in which miR-124 may solidify the neural fate by widespread suppression of epidermal genes that should be absent from neurons. This bioinformatic correlation has not been confirmed using an independently-derived set of miRNA targets (Sun, 2012).
Nevertheless, two observations suggest that the feature of mutual exclusion in the Drosophila miR-124 network is of subtle consequence. First, derepressed target genes were not enriched for epidermally-expressed genes. This is consistent with the view that on the transcriptome-wide level, the exclusion of epidermal genes from miR-124-expressing cells is primarily enforced by transcriptional mechanisms. Second, miR-124 targets were preferentially amongst the higher-expressed transcripts in miR-124+ cells, even in wild-type. Moreover, as well-conserved targets were expressed at overall higher absolute levels than poorly-conserved targets in miR-124+ cells, it is concluded that a dominant feature of the miR-124 target network has selected for substantial co-expression of the miRNA and its targets, perhaps to fine-tune their levels. This viewpoint is consistent with analyses of miR-124 targets in human, indicating a unifying theme for this particular miRNA across animals (Sun, 2012).
Early manifestations of the miRNA world emerged from pervasive control of the C. elegans heterochronic pathway and the D. melanogaster Notch pathway by miRNAs, and a few similar situations have been documented, i.e. direct targeting throughout the branched amino acid catabolism pathway by miR-277 or repression of multiple components of fatty acid metabolism by miR-33. Nevertheless, it is rare for such dedicated target networks to be seen amongst the miRNA oeuvre. Amongst the broad network of miR-124 targets, coordinate targeting of multiple components of the retrograde BMP signaling pathway is striking, including all three receptors (Sax/Tkv/Wit), the downstream transcription factor (Mad) and its cofactor (Medea). It was recently shown that misexpression of activated Sax and Tkv receptors in motoneurons increases evoked excitatory junctional potentials without affecting spontaneous activity, very similar to that of mir-124 mutants. This study extends this finding by analysis of activated Tkv alone. Therefore, deregulation of BMP signaling may contribute to the electrophysiological defects observed in mir-124 mutants (Sun, 2012).
Still, a 'one size fits all' description of miR-124 activity is not appropriate, since a number of functional miR-124 targets were observed whose predominant activities are in epidermal or other non-neural derivatives. Thus, the large miR-124 network accommodates a range of target properties. Derepression of a sufficient number of such non-neural transcripts may contribute collectively to the incomplete capacity of mir-124 mutant cells to transition from a neuroblast to neuronal gene expression signature (Sun, 2012).
One may speculate that dysfunction of miRNAs, which have large networks of targets, may trigger global changes in other modes of gene regulation. For example, overexpression of individual miRNAs or siRNAs can de-repress endogenous regulation via non-cognate miRNAs, possibly reflecting a titration mechanism. In addition to a global effect on neuroblast-to-neural transition, it was observed that genes downregulated upon in vivo loss of miR-124 were enriched for seeds of K box miRNAs and miR-10-5p. This is potentially consistent with a model in which absence of this abundant miRNA frees up AGO1 complexes to accept other neural miRNAs, yielding their overactivity. Another plausible mechanism might be that miR-124 represses a transcriptional repressor of these other miRNAs (Sun, 2012).
Pumilio binding sites were strongly associated with downregulated transcripts in mir-124 mutants. Pumilio is well-characterized as a neural RNA binding protein and translational regulator, and affects synaptic function and dendrite morphogenesis, which was also observed to be miR-124-regulated settings. Predictions of conserved miRNA binding sites (e.g., TargetScan or mirSVR) did not identify miR-124 target sites in the annotated pumilio 3' UTR or CDS; however modENCODE data revealed that pumilio transcription extends ~2 kb downstream of its annotated 3' end. The regulatory potential of such long pumilio 3' UTR isoforms remains to be studied. Other possibilities are that miR-124 regulates a transcriptional regulator of pumilio, or that Pumilio activity is altered in mir-124 mutants. Future studies should address the cross-talk of post-transcriptional regulation in neurons mediated by miR-124, neuronal miRNAs and Pumilio (Sun, 2012).
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).
wit expression pattern was studied using in situ hybridization. In embryos, wit transcription is first observed at stage 12 within a subset of cells in the developing central nervous system (CNS). There is no maternal deposition, consistent with the absence of a requirement for wit expression in the germline. wit remains restricted to the CNS, where it is expressed for the rest of embryonic development. A larger number of cells express wit in the CNS by stage 14, and the message can also be detected in the PNS and in leading edge cells during dorsal closure. After dorsal closure is complete, continued expression of wit is seen in the CNS and PNS as well as in portions of the gut. By stage 17, expression of wit is seen in the prothoracic component of the ring gland (Marqués, 2002).
The number and the positions of the phosphorylated form of Mad (P-Mad)-positive cells, per hemisegment at stage 17, indicative of cells undergoing Wit signaling, correlates well with the estimated number and positions of motoneurons at this stage. To better characterize the cells that accumulate P-Mad in the nervous system, wild-type embryos were double stained for P-Mad and several other markers, including Even-skipped and Lim-3, two transcription factors expressed in discrete subsets of embryonic motoneurons and interneurons. Staining was also performed for Fasciclin II (Fas II), a neural adhesion molecule expressed in a subset of interneurons as well as most motoneurons. There is colocalization of Eve and P-Mad in the RP2 and U/C motoneurons but not in the EL interneurons, while P-Mad and Lim-3 colocalize in a lateral cluster of cells that include motoneurons 28 and 14/30. In addition, Lim-3 and P-Mad colocalize in RPs 1, 3, 4, 5. The Fas II antibody highlights axon tracts and cell bodies of most motoneurons as well as some interneurons. Most, if not all, P-Mad cells also show colocalization of Fas II. These findings suggest that Wit is required to transduce BMP signals in motoneurons and is consistent with the ability of UAS-wit constructs to rescue wit mutants when expressed with a motoneuron-specific driver (Marqués, 2002).
Expression pattern of wit mRNA was examined by in situ hybridization. At early embryonic stages, no wit mRNA was detected, suggesting that wit is not maternally deposited. Zygotic wit expression was detected first at stage 11 in a reiterated subset of CNS neurons and in the labral sensory cluster of the peripheral nervous system. In stages 11 to 14, wit expression persists in these spatially restricted clusters. Co-staining with the monoclonal antibody BP102 that labels the axon scaffold reveals that wit is expressed in the motoneurons of the Ventral-Unpaired Median (VUM) cluster. Co-staining with anti-Even-Skipped antibodies reveals that Wit is expressed by several identifiable motoneurons including the RP2 motoneurons but not by the EL interneurons. From stage 15 to 17, the boundaries of the CNS clusters gradually disappear, as more CNS neurons appear to express wit. At stage 16, wit mRNA is also detectable in the furrows of the developing midgut (Aberle, 2002).
Wit protein is expressed in a restricted subset of CNS neurons as detected with a monoclonal antibody (23C7) generated against Wit. Wit protein is detected in a reiterated pattern in the CNS from stage 13 onward. Beginning at stage 15, Wit is also seen in the CNS axon scaffold in both the commissures and longitudinal tracts. Weak expression is also detected in the sensory neurons of the peripheral nervous system. At stage 16, Wit staining forms a pattern resembling the staining pattern of the motoneuron marker Late Bloomer (Lbl). Similar to Lbl, Wit protein is predominantly found in a stripe of cells in a region close to the posterior commissure in each segment where motoneurons are located (Aberle, 2002).
Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).
FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).
Rotund, a zinc finger protein of the C2H2 Krüppel-type belongs to a conserved subfamily of zinc finger proteins together with Drosophila CG5557, C. elegans Lin-29, and rat CIZ. Squeeze is most closely related to Rotund, with identity greater than 90% throughout the zinc finger region; Squeeze is 78% identical to LIN-29 in the conserved zinc finger region. Both rotund and CG5557 are expressed in subsets of cells in the developing CNS. CG5557 has a larval lethal phase. Mutants eclosed at a low frequency as immotile adults that died within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant and scored with 100% accuracy (sqzlacZ/sqzDf), CG5557 was renamed squeeze (sqz) (Allan, 2003).
What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).
Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).
Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).
Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). Mutations in wishful thinking (wit), a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. Gbb, a Bmp ligand expressed in the segmentally repeated neurohemal organ associated with the ventral cord, provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. It is proposed that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity (Marqués, 2003).
wishful thinking is primarily expressed in, and required for, proper nervous system function. Mutations in wit result in pharate lethality caused, in part, by defects in the growth and physiology of motoneuron synapses. Mutations in wit also affect the peptidergic phenotype of certain FMRFa-expressing cells found in the ventral cord. In particular, FMRFa expression is eliminated in the Tv neurons that contribute to the systemic supply of FMRFa peptides through release at the neurohemal organ. The regulation of FMRFa expression in Tv neurons is mediated by the Bmp ligand Gbb, since gbb null mutations also eliminate FMRFa expression in Tv neurons. Furthermore, supplying Gbb to the dorsal neurohemal cells restores FMRFa expression in Tv neurons. Since Tv neuron axons arborize onto the neurohemal cells, this strongly suggests that Gbb signals in a retrograde manner to specify the peptidergic phenotype of Tv neurons. Consistent with this view, overexpression in neuroendocrine cells of Dynamitin or a dominant-negative form of p150/Glued, both components of the Dynactin/Dynein motor complex was found to eliminate FMRFa expression in the Tv neurons. Finally, it is shown that providing FMRFa in neuroendocrine cells using the Gal4/UAS system partially rescues the lethal phenotype of wit mutants, even though they still exhibit structural and physiological synaptic defects. It is suggested that Bmp signaling provides a global cue that not only regulates the growth of the NMJ synapses locally but also controls their systemic modulation by the neuroendocrine system (Marqués, 2003).
The Drosophila genome contains seven TGFß type ligands. Three of these, Dpp, Screw and Gbb, have been shown to transduce Bmp-type signals (Mad) and to use the type I receptors Tkv and Sax. Two others, Activin and Activin-like protein, transduce signals through Smad2. The signaling pathways used by Maverick and Myoglianin remain untested. Among the three Bmp-type ligands, Gbb seemed a likely candidate for controlling expression of FMRFa, since it is broadly expressed, at least in embryos, and can signal through Wit to regulate P-Mad accumulation in motoneurons and tissue culture cells. gbb is strongly expressed in the larval brain lobes and much more weakly in the ventral ganglia. Interestingly, gbb shows enriched expression in the NHO relative to other ventral ganglia neurons. Thus, Gbb is expressed in the correct place to be a FMRFa regulating ligand (Marqués, 2003).
In Drosophila, FMRFamide peptides have been shown to enhance synaptic transmission and muscle twitch tension when perfused onto standard larval nerve-muscle preparations; however, their in vivo role(s) are not known as no mutations in the FMRFa gene have been identified. As with most neuropeptides, FMRFamide related peptides are thought to act as neuromodulators and neurohormones. The Tv-produced FMRFamide related peptides are released into the hemolymph through the neurohemal organ and hence are able to act on every tissue in the animal that is not blocked to hemalymph contact. It has been hypothesized that the lethality of wit mutants is due to the lack of proper synaptic transmission at the NMJ, resulting in the animals not being able to eclose from the pupal case. The lack of systemic FMRFamide described in this study would be expected to further decrease synaptic efficiency and the ability of wit mutants to eclose. The fact that loss of FMRFa does contribute to the lethal phenotype is supported by the partial rescue of wit mutants by overexpression of FMRFa. These results are consistent with the view that in vivo, FMRFa peptides probably enhance NMJ synaptic activity similar to their in vitro documented effects on standard larval electrophysiological preparations (Marqués, 2003).
It is important to note that although the lethal phenotype is partially reversed, the morphological and physiological synaptic defects reported for wit mutants are not rescued by overexpression of FMRFa. The simplest interpretation is that the excess of FMRFamide related peptides enhances the efficiency of wit mutant synapses in vivo without correcting the underlying developmental defects. Although one might expect a significant improvement of the electrophysiological phenotype, this is not detected, probably because the excess FMRFamide related peptides are either washed off the preparation during standard dissection prior to recording or act for only short periods (Marqués, 2003).
How Wit signaling regulates FMRFa expression is not clear. Since Smads are well known to act as transcriptional co-activators or co-repressors, the simplest explanation is that Mad directly regulates activation of FMRFa transcription, perhaps by forming a complex with Ap. However, other indirect mechanisms are also possible and this issue will only be resolved once the FMRFa promoter is fully characterized. It is also not clear whether Gbb is the only ligand that regulates FMRFa expression through Wit. In some developmental contexts, such as wing imaginal disc patterning, Gbb acts in combination with Dpp, another Bmp-type ligand. No expression of dpp has been detected in the NHO. However, it could be that one of the as yet uncharacterized ligands, Maverick or Myoglianin, could be a partner with Gbb in regulating FMRFa expression. Conversely, it seems clear that regulating the peptidergic phenotype of the six Tv neurons is not the only role of Gbb signaling. There are hundreds of neurons that receive Bmp signaling as indicated by P-Mad nuclear localization Most of them appear to be motoneurons, which require Wit/Gbb signaling to achieve proper synaptic growth but not to specify their neurotransmitter phenotype. Given that Smads act as co-transcriptional regulators, the fact that the same signal (nuclear translocation of P-Mad) results in different phenotypic outcomes in different neurons can probably be ascribed to the presence of a different set of transcription factors available in each cell type. The Tv neurons receiving the Bmp signal express apterous, a transcription factor required in those cells for FMRFa transcription, and maybe other factors that are required, in addition to the Wit signal, to activate FMRFa (Marqués, 2003).
Another important issue to resolve is whether Gbb is constitutively released from the NHO, or is synthesized and released as part of a feedback mechanism to modulate muscle contractions. It might be that efficient muscle contraction under normal conditions requires a constant level of FMRFamide related peptides that are produced in response to a constitutive Gbb signal. Alternatively, Gbb production or release might be regulated by a sensing mechanism that would activate the pathway in response to an increased demand for FMRFamide related peptides, owing to increased locomotor activity or other stimuli, such as compensating for a synaptic developmental defect. Muscle-derived Gbb acts through neuronal Wit to convey a retrograde signal essential for NMJ synapse growth and maturation. In that context, it appears that the role of Bmp signaling is to coordinate muscle growth with synapse maturation to ensure proper synaptic efficiency. Thus, the Wit/Gbb pathway acts as a two-step regulator of NMJ function. First, there is a developmental role in which Wit signaling is required for proper synaptic growth during larval development. Second, Wit signaling is required to achieve the neuromodulatory effect of circulating FMRFamide related peptides that are required for optimal synaptic transmission. Lack of either one of these inputs probably results in a substantial decrease of the EJCs. These two examples suggest that the Gbb/Wit pathway is of general importance in neural retrograde signaling and it is speculated that it may be used in the nervous system for other as yet uncharacterized developmental and physiological purposes (Marqués, 2003).
Tv neurosecretory cells form part of a cluster of four apterous-expressing neurons on each side of the three thoracic ganglia. The axons of the Tv neurons extend proximally and dorsally to join the contralateral axon, and form a median nerve that swells and arborizes onto a group of neurons and glial cells that constitute the neurohemal organ. In wit mutants, these structures develop normally, but the Tv neuron fail to activate FMRFa transcription. Using the Gal4/UAS system Wit's requirement for FMRFa expression was narrowed down to the Tv neurons. Since these neurons accumulate nuclear P-Mad, the results strongly suggest that Wit is required in the Tv neurons themselves, as opposed to forming part of an indirect signal relay mechanism. It appears likely that the source of Gbb in this signaling system is the NHO, since gbb is expressed in the NHO and replenishing Gbb in the NHO of gbb mutants rescues FMRFa expression in the Tv neurons. These experiments do not exclude the possibility that signaling might occur at the cell soma of the Tv neurons in vivo or that the source of the diffusible ligand could be a different tissue under physiological conditions. However, the dependence of nuclear P-Mad accumulation and FMRFa expression in Tv neurons on Dynein-mediated retrograde transport strongly suggests that signaling is taking place at the Tv axon terminal. This dependency on Dynein motors is not a general requirement for FMRFa expression in all neurons because subesophageal ganglion neurons are not affected by overexpression of dominant-negative Glued or Dynamitin. Nor is the consequence of disrupting this motor likely to exert its effect at the level of P-Mad translocation to the nucleus, since nuclear accumulation of P-Mad in epithelial and mesodermal cells is not effected by retrograde transport disruption. Only in the nervous system is P-Mad accumulation specifically affected, consistent with a role for a retrograde transport mechanism in moving some component of this signaling pathway from the synapse to the nucleus (Marqués, 2003).
In larvae, wit is expressed strongly in the brain lobes and more weakly in the ventral ganglia. All imaginal discs also express wit, however, with the exception of the wing disc, without a prominent spatial pattern. In the wing disc , abundant transcription is seen in the wing pouch and also a broad band of cells along the anterior-posterior margin, particularly in the presumptive notum (Marqués, 2002).
In first and second instar larvae, Wit is expressed throughout the brain. In third instar larvae, Wit expression in the ventral nerve chord ceases but is still prominent in the synaptic centers and the lamina of the developing brain lobes. Wit protein is also detected in all imaginal discs. Endogenous Wit could not be detected at the larval NMJ using the 23C7 antibody; however, when Wit was overexpressed in all neurons, it was readily detected at the NMJ with this antibody. This analysis of the expression pattern demonstrates that wit is expressed specifically in the nervous system from embryonic stages until third instar larvae, indicating that Wit signaling is continuously required during these developmental periods (Aberle, 2002).
Metamorphosis of the Drosophila brain involves pruning of many larval-specific dendrites and axons followed by outgrowth of adult-specific processes. From a genetic mosaic screen, two independent mutations were recovered that block neuronal remodeling in the mushroom bodies (MBs). These phenotypically indistinguishable mutations affect Baboon function, a Drosophila TGF-ß/activin type I receptor, and Smad on X (Smox, or dSmad2), its downstream transcriptional effector. Punt and Wit, two type II receptors, act redundantly in this process. In addition, knocking out Activin-beta (dActivin) around the mid-third instar stage interferes with remodeling. Binding of the insect steroid hormone ecdysone to distinct Ecdysone receptor isoforms induces different metamorphic responses in various larval tissues. Interestingly, expression of the Ecdysone receptor B1 isoform (EcR-B1) is reduced in activin pathway mutants, and restoring EcR-B1 expression significantly rescues remodeling defects. It is concluded that the Drosophila Activin signaling pathway mediates neuronal remodeling in part by regulating EcR-B1 expression (Zheng, 2003).
In Drosophila, recent data suggest that a BMP signaling pathway controls synaptic growth and function at the neuromuscular junction (NMJ). Whether this pathway also contributes to activity-dependent remodeling at the NMJ remains to be determined. It is interesting to note, however, that in this pathway Wit acts as a BMP receptor, and it can not be substituted for by Punt. In contrast, the activin pathway described here appears to be able to utilize either Punt or Wit for signaling. This may reflect selectivity in the binding of some ligands to one receptor, but not the other. Additional studies will be required to resolve this issue. Since many components of several different TGF-β signaling pathways show pronounced expression in different parts of the developing and postnatal rodent brain, the demonstration that TGF-β/Activin signaling cell-autonomously controls plasticity of MB neurons may provide novel insights into how neuroplasticity is dynamically regulated in higher organisms (Zheng, 2003).
Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).
Once growing axons reach the correct postsynaptic target, axon outgrowth terminates and synaptogenesis begins. These studies suggest that TGFβ signals play a role. When Babo is inactivated, MB axon growth does not terminate properly and overextends across the midline. Consistent with this, CA Babo expression results in precocious termination, forming axon truncations. How Babo is spatially and temporally regulated remains to be determined. Analogous to the Drosophila NMJ, MB axon growth might be terminated through retrograde signalling. Target-derived TGFβ ligands could signal to Babo (on MB axon growth cones) and stop axons growing further. In an alternative scenario, TGFβ ligands might act as a positional cue that prevents MB axons from crossing the midline. Recent data have shown that Babo acting through Smad2 restricts individual R7 photoreceptor axons to single termini. Loss of Babo, Smad2, or the nuclear import regulator Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons invading neighbouring R7 terminal zones. With the phenotype described in this study, Babo could similarly be restricting MB axons to appropriate termination zones, its loss resulting in inappropriate terminations on the contralateral side (Ng, 2008).
In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted in axon extension and targeting defects. This might reflect cell-intrinsic differences in the response in different neurons to a common Babo signalling program. This may be the case for MB axon pruning and DC axon extension, which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic properties, or from Babo signal transduction, they underline the importance of Smad-independent signals in many aspects of axonal development (Ng, 2008).
The results suggest that Smad-independent signals involve Rho GTPases. One caveat in genetic interaction experiments is that the loss of any given gene might not be dosage-sensitive with a particular assay. Nevertheless, all the manipulations together suggest that Babo-regulated axon growth requires Rho1, Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully determined. In addition to LIMK1, which binds to Wit, one possibility, as demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).
The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).
Do mutations in Rho1 and Rac components phenocopy babo phenotypes? β lobe overextensions are observed in Rok, Rho1 and Rac mutant neurons. In MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is activated. LIMK1 inactivation in MB neurons was reported previously. Axon overextensions were not observed as LIMK1 loss results in axon outgrowth and misguidance phenotypes. This suggests that LIMK1 mediates multiple axon guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).
Although their phenotypes are similar, several lines of evidence indicate that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First, whereas LIMK1 genetically interacts with most Rho family members and many Rho regulators, CA babo is dosage-sensitive only to Rho1 and Rac and specific Rho regulators, suggesting that Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).
Second, the LIMK1 misexpression phenotype is suppressed by expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).
CA Babo might not simply misregulate LIMK1 but also additional cofilin regulators. Recent data suggest that extracellular cues (including mammalian BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In different cell types, cofilin phosphorylation and phospholipid binding (which also inhibits cofilin activity) states vary and potently affect cell motility and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).
Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).
This study differs significantly from the canonical model of Smad signalling, in which type 1 receptors function downstream of the ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit and Put suppress the babo axon overextension phenotype, this implies that Smad-dependent and -independent signals have distinct type 1/type 2 receptor interactions. How these interactions propagate Smad-independent signals remains to be fully determined. Babo could act as a ligand-binding co-receptor with Wit and Put. In addition, Babo kinase activity could regulate type 2 receptor or Rho functions. The results suggest, however, that provided that Wit or Put signals are sufficiently high, Babo is not required. Whatever the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1 interaction to relay cofilin phosphoregulatory signals. How Put functions is unclear. Since the put135 allele (used in this study) carries a missense mutation within the kinase domain, this suggests that kinase activity is essential. put does not genetically interact with LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary for LIMK1 binding, this suggests that Put acts independently of LIMK1. One potential effector is Rac, which, in the context of Babo signalling, also appears to be Pak1- and thus LIMK1-independent (Ng, 2008).
In MB neurons, Wit and Put can function interchangeably. In other in vivo paradigms, type 2 receptors are not interchangeable. However, since the Wit C-terminal tail is required to substitute for Put, this suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).
This study shows that Babo mediates two distinct responses in related MB populations. How do MB neurons choose between axon pruning and axon growth? The babo rescue studies suggest that whereas Baboa or Babob elicits Smad-independent responses, only Baboa mediates Smad-dependent responses. Since Babo isoforms differ only in the extracellular domain, differences in ligand binding could determine Smad2 or Rho GTPase activation. However, it is worth noting that in DC neurons, either isoform mediates axon extension through Smad2 and Medea. In addition, although expressed in all MB neurons, CA babo misexpression (which confers ligand-independent signals) perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).
Many TGFβ ligands signal through Babo. For example, Dawdle, an Activin-related ligand, patterns Drosophila motor axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear. Taken together, the evidence suggests that Babo signalling is varied in vivo and is involved in many aspects of neuronal development (Ng, 2008).
TGFβ signals are responsible for many aspects of development and disease and, throughout different models, Smad pathways are closely involved. Although Smad-independent pathways are known, their mechanisms and roles in vivo are unclear. TGFβ signals often drive cell shape changes in vivo. During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial structure and adopt a fibroblast-like structure that is essential for cell migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin breakdown through Rho1 degradation. In other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which can be regulated by Smad activity (Ng, 2008).
Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).
At the Drosophila neuromuscular junction (NMJ), the loss of retrograde, trans-synaptic BMP signaling causes motoneuron terminals to have fewer synaptic boutons, whereas increased neuronal activity results in a larger synapse with more boutons. This study shows that an early and transient BMP signal is necessary and sufficient for NMJ growth as well as for activity-dependent synaptic plasticity. This early critical period was revealed by the temporally controlled suppression of Mad, the SMAD1 transcriptional regulator. Similar results were found by genetic rescue tests involving the BMP4/5/6 ligand Glass bottom boat (Gbb) in muscle, and alternatively the type II BMP receptor Wishful Thinking (Wit) in the motoneuron. These observations support a model where the muscle signals back to the innervating motoneuron's nucleus to activate presynaptic programs necessary for synaptic growth and activity-dependent plasticity. Molecular genetic gain- and loss-of-function studies show that genes involved in NMJ growth and plasticity, including the adenylyl cyclase Rutabaga, the Ig-CAM Fasciclin II, the transcription factor AP-1 (Fos/Jun), and the adhesion protein Neurexin, all depend critically on the canonical BMP pathway for their effects. By contrast, elevated expression of Lar, a receptor protein tyrosine phosphatase found to be necessary for activity-dependent plasticity, rescued the phenotypes associated with the loss of Mad signaling. Synaptic structure and function develop using genetically separable, BMP-dependent mechanisms. Although synaptic growth depended on Lar and the early, transient BMP signal, the maturation of neurotransmitter release was independent of Lar and required later, ongoing BMP signaling (Burke, 2013).
This study investigated how retrograde BMP signaling by Gbb, Wit, and Mad influences the development of the Drosophila NMJ. The experiments examined the timing of retrograde signaling and the relationship between BMP signaling and the activity-dependent modulation of NMJ development. The results indicate that an early and transient period of BMP signaling, acting through Mad, activates key developmental programs necessary for synapse maturation. Transcriptional regulation by Mad in the first larval instar (L1, 24 h) is necessary and sufficient for robust NMJ growth during the second (L2) and third (L3) instars (72 h). Mad signaling during L1 also allows activity to enhance the growth process. By contrast, Mad signaling in L1 through L3 is required for normal active zone morphology and the developmental increase in quantal content. The results therefore indicate that retrograde BMP signaling 'gates' NMJ development and plasticity by initiating two genetically separable programs for growth and physiology (Burke, 2013).
In the absence of retrograde BMP signaling, the NMJ shows only residual growth, forming weak connections that are insensitive to activity-dependent modulation. BMP signaling mutations do not disrupt axonal guidance, target selection, or the initiation of synaptogenesis. They instead have profound effects on later aspects of synaptic development, affecting NMJ expansion and bouton stabilization. Expression of Mad1, the protein encoded by the strong dominant-negative mad1 allele, phenocopies BMP signaling mutants, affecting both NMJ growth and physiology. By driving Mad1 expression at various times during development, it was found that Mad-dependent signaling during L1 is both necessary and sufficient for subsequent NMJ growth. The results are consistent with an L1 critical period for BMP signaling. Inducing Mad1 expression at all times except L1 produced a WT-sized NMJ, whereas induction only during L1 reduced NMJ size to that seen in mad mutants. The genetic rescue of gbb and wit mutants also revealed the importance of the retrograde BMP pathway signaling during embryogenesis and L1. The timing of retrograde BMP signaling, after synaptogenesis yet before growth commences, suggests a model where the muscle uses BMP signaling to inform the motoneuron nucleus of a successfully formed synapse, activating subsequent growth and plasticity programs. The exact timing of this critical period, however, requires knowing when the Mad1 transgene inhibits transcription of Mad's major effectors of NMJ growth, which are unknown. The data indicate that a 24 h exposure to RU-486 during L1 expresses enough Mad1 to suppress NMJ growth. It also suggests that the perdurance of the dominant negative plus the time needed for its activation must be less than 24 h, as expression during embryogenesis, L2, and L3 led to NMJs that were nearly WT in size. Therefore, although the critical period may be shifted later in development than the data indicate, it is likely to begin as early as 5 h after the onset of L1 (Burke, 2013).
The data provide an entrance into the mechanisms regulating the critical period. Based on its timing between the embryonic and L2 stages, it is possible that molting hormones influence Mad activity. Recent work also indicates that anterograde activin signaling induces Gbb expression in body wall muscles, whereas postsynaptic dCIP4 signaling (Drosophila Cdc42 Interacting Protein 4) and the activity of dRich, a conserved Cdc 42-selective guanosine triphosphatase-activating protein, inhibits Gbb secretion from these muscles (Nahm, 2010a; Nahm, 2010b). Furthermore, the secretion of a TGF-β ligand (Maverick) from peripheral glia strongly regulates Gbb signaling from the postsynaptic muscle, affecting both pMad levels within motoneurons and NMJ growth. It would therefore be interesting to perform phenocritical analyses on activin, dCIP4, dRich, and Maverick and to address whether postsynaptic depolarization influences the activity of dCIP4 or dRich. Genetic interaction experiments found no evidence to suggest that moderate increases in BMP signaling modulate the final size of the NMJ or synergize with presynaptic activity. The findings of an early critical period for NMJ growth differ slightly from a previous study, which overexpressed an inhibitory SMAD. In an effort to reconcile the current results with these earlier data, Dad was transiently expressed during L1 but no alteration was found in NMJ size, perhaps resulting from different mechanisms of BMP pathway inhibition (Burke, 2013).
An unexpected result from this study is that the early BMP signal is necessary and sufficient for subsequent growth and structural plasticity, but not for synaptic function. In the absence of later BMP signaling, the NMJ grows to its structurally normal size but has reduced neurotransmission. The later requirement for BMP signaling is consistent with the observation that pMad levels remain high in motoneuron nuclei throughout larval development. In the absence of continual Mad activity, the number of active zones is reduced and their size is aberrantly enlarged. The separable roles of BMP signaling for NMJ growth and function are also consistent with the distinct actions of the two known targets of retrograde BMP signaling in motoneurons, Trio and Target of wit (Twit) (Burke, 2013).
It is not known whether the active zone phenotype arising from the loss of BMP signaling late in development results from defects in the formation or the maintenance of these structures. The active zone protein Sunday driver (dSyd) and the Teneurin-a adhesion molecule are potentially involved in Mad's physiological program, as their loss causes physiological and ultrastructural phenotypes that overlap with those of BMP mutants. Future studies of how Mad simultaneously and separately regulates NMJ growth and physiology could lead to a deeper understanding of how dSyd and Teneurin-a might act dowstream of Mad. The separable downstream effects of Mad on growth versus physiology may depend on post-translational modifications that can occur at the linker between the MH1 and MH2 domains, changes that can affect the affinity of Mad's binding partners. Unfortunately, Mad's noncanonical binding partners in motoneurons are entirely uncharacterized (Burke, 2013).
Elevated action potential firing in motoneurons resulting from the loss of repolarizing K+ channels or by high-temperature rearing substantially enhances NMJ growth and synaptic transmission. Mutations affecting the BMP signaling pathway block this plasticity without suppressing presynaptic hyperactivity. The results indicate that retrograde BMP signaling allows motoneuron growth to be responsive to increased levels of activity. Indeed, the level of excitability during Mad's critical period for growth strongly affected synaptic size in mature larvae. Understanding how this early activity engages BMP-dependent programs could be very insightful given that cAMP, AP-1, and Fas-2 signaling failed to rescue the effects of Mad1 expression (Burke, 2013).
Instead, the receptor protein tyrosine phosphatase Lar rescued both normal and activity-dependent NMJ growth, and Lar was required for activity-dependent developmental plasticity. Lar family members are essential, well-conserved regulators of synaptogenesis from worms and flies to mammals. Despite the prior identification of extracellular ligands for Lar family RPTPs, there has been no significant insight into the temporal regulation of Lar signaling during synaptogenesis in any system. Current evidence suggests that the Lar pathway regulates cytoskeletal assembly and active zone formation while antagonizing the activity of the highly conserved Abelson (Abl) tyrosine kinase. At the larval stage, Abl negatively regulates NMJ size and abl mutations have phenotypes reciprocal to those of Lar. Lar may relieve the growth-inhibitory action of Abl, promoting synaptic expansion in response to elevated activity or postsynaptic growth. Lar acts via the actin-modulating protein Ena for NMJ growth, whereas Trio and Lar regulate growth and the presynaptic cytoskeleton by interacting with Diaphanous, a member of the formin family of proteins. As Trio levels are reduced in BMP mutants and the expression of Trio only partially rescues NMJ growth, it is possible that Ena or Diaphanous act independently of Mad signaling (Burke, 2013).
In the absence of BMP signaling, the small NMJs can be genetically rescued by increased expression of Lar. This suggests that reduced BMP signaling in some fashion reduces Lar activity or function at the NMJ. qPCR analyses show that Lar transcript levels remain normal despite reduced pMad activity, leaving open the possibility that post-transcriptional mechanisms reduce Lar expression, NMJ localization, or activity, either by changes to the receptor itself, to Lar's binding partners (for e.g., Liprins), or to the heparin sulfate proteoglycan ligands. The observations support a model where retrograde BMP signaling allows synaptic growth to be modulated by neural activity, with Lar acting as the downstream 'gain controller' to establish the specific level of synaptic efficacy. In this model, postsynaptic BMP release initiates competence of the presynaptic terminal to respond to the matrix via Lar. Lar's heparin sulfate proteoglycan ligands and its anchoring proteins (Liprins) might then provide spatial information or couple Lar function to synaptic activity. Heparin sulfate proteoglycans play important roles during critical periods, and they modulate the signaling of BMPs, Wnts, and fibroblast growth factors. It is therefore possible that the extracellular matrix provides a key integrator that coordinates multiple trans-synaptic signals in a developmental and activity-dependent manner (Burke, 2013).
The Drosophila neuromuscular junction (NMJ) is capable of rapidly budding new presynaptic varicosities over the course of minutes in response to elevated neuronal activity. Using live imaging of synaptic growth, this dynamic process was characterized, and it was demonstrated that rapid bouton budding requires retrograde bone morphogenic protein (BMP) signaling and local alteration in the presynaptic actin cytoskeleton. BMP acts during development to provide competence for rapid synaptic growth by regulating the levels of the Rho-type guanine nucleotide exchange factor Trio, a transcriptional output of BMP-Smad signaling. In a parallel pathway, it was found that the BMP type II receptor Wit signals through the effector protein LIM domain kinase 1 (Limk) to regulate bouton budding. Limk interfaces with structural plasticity by controlling the activity of the actin depolymerizing protein Cofilin. Expression of constitutively active or inactive Cofilin (Twinstar) in motor neurons demonstrates that increased Cofilin activity promotes rapid bouton formation in response to elevated synaptic activity. Correspondingly, the overexpression of Limk, which inhibits Cofilin, inhibits bouton budding. Live imaging of the presynaptic F-actin cytoskeleton reveals that activity-dependent bouton addition is accompanied by the formation of new F-actin puncta at sites of synaptic growth. Pharmacological disruption of actin turnover inhibits bouton budding, indicating that local changes in the actin cytoskeleton at pre-existing boutons precede new budding events. It is proposed that developmental BMP signaling potentiates NMJs for rapid activity-dependent structural plasticity that is achieved by muscle release of retrograde signals that regulate local presynaptic actin cytoskeletal dynamics (Piccioli, 2014).
Activity-dependent changes in synaptic structure play an important role in developmental wiring of the nervous system. The Drosophila larval neuromuscular junction (NMJ) has emerged as a model glutamatergic synapse that is well suited to study activity-dependent structural plasticity. The NMJ can be imaged in vivo during developmental periods of rapid synaptic growth when the axonal terminal expands ~5- to 10-fold in size over 5 d. Forward genetic screens to identify mutations that alter synaptic growth have revealed essential roles for retrograde bone morphogenic protein (BMP) signaling mediated by the secreted ligand Glass bottom boat (Gbb). Mutations that disrupt BMP signaling lead to synaptic undergrowth and neurotransmitter release defects. Multiple pathways downstream of retrograde BMP signaling through the type II receptor Wishful thinking (Wit) have been linked to synaptic growth, synapse stability, and homeostatic plasticity in Drosophila. BMP signaling via the Smad transcription factor Mothers against Dpp (Mad) regulates the expression of the Rho-type guanine nucleotide exchange factor (GEF) trio to control normal synaptic growth. Wit also interacts with LIM domain kinase 1 (Limk) to enhance synaptic stabilization in a pathway parallel to canonical Smad-dependent signaling. BMP signaling through Wit also potentiates synapses for homeostatic plasticity in a pathway that is independent of limk and synaptic growth regulation (Piccioli, 2014).
The NMJ displays acute structural plasticity in the form of rapid presynaptic bouton budding in response to elevated levels of neuronal activity. These rapidly generated presynaptic varicosities, referred to as ghost boutons, lack presynaptic and postsynaptic transmission machinery when initially formed. The budding of ghost boutons requires retrograde signaling mediated by the postsynaptic Ca2+-sensitive vesicle trafficking regulator synaptotagmin (Syt) 4 (Korkut, 2013). Syt4 also participates in developmental synaptic growth and controls retrograde signaling that mediates enhanced spontaneous release at the NMJ (Yoshihara, 2005; Barber, 2009). Beyond the role of Syt4 in ghost bouton budding, little is known about the signaling pathways that underlie this rapid form of structural synaptic plasticity. In particular, it is unclear whether pathways that regulate synaptic growth over the longer time scales of larval development also trigger acute structural plasticity. To address these issues, this study identified synaptic pathways that are required for rapid structural plasticity at Drosophila NMJs. Ghost bouton budding was found to be locally regulated at the synapse level, occurring in axons that have been severed from the neuronal cell body. In addition, activity-induced ghost bouton formation requires Syt1-mediated neurotransmitter release and postsynaptic glutamate receptor function. Like developmental growth, retrograde BMP signaling is required for ghost bouton budding. BMP signaling functions through a permissive role mediated by developmental Smad and Trio signaling, as well as through a local Wit-dependent modulation of Limk and Cofilin (Twinstar) activity that alters presynaptic actin dynamics (Piccioli, 2014).
Experimental analysis of ghost bouton budding at the Drosophila NMJ indicates that rapid activity-dependent synaptic growth requires retrograde BMP signaling at this synapse. The current data support a model in which BMP signaling through the type II receptor Wit is required developmentally to potentiate synapses for budding in response to elevated synaptic activity. This pathway requires Smad-dependent expression of the Rho-type GEF trio, and parallels a requirement for BMP signaling and Trio in developmental synaptic growth that occurs during the larval stages. In a parallel pathway, Wit interaction with Limk inhibits bouton budding through regulation of Cofilin activity. Both pathways regulate the synaptic actin cytoskeleton and may converge on similar actin regulatory molecules such as Limk and Cofilin via Rac1 or RhoA. Manipulating Cofilin activity levels by the overexpression of Limk or the expression of constitutively active/inactive Cofilin demonstrates that high Cofilin activity favors bouton budding, while low Cofilin activity inhibits budding. Local changes in the actin cytoskeleton that accompany activity-dependent bouton budding were also observed at sites of new synaptic growth. In addition, pharmacological disruption of normal actin turnover inhibits budding, suggesting that increased actin turnover mediated by Cofilin potentiates rapid activity-dependent synaptic plasticity (Piccioli, 2014).
Multiple genetic perturbations of BMP signaling were identified that altered the frequency of activity-dependent bouton budding at the NMJ. Although several of these mechanisms are shared with those previously characterized to control BMP-mediated developmental synaptic growth, several manipulations separated rapid activity-dependent BMP-mediated bouton budding from the slower forms of developmental growth. In the case of wit mutants or motor neuron overexpression of dad, a reduction in baseline bouton number was observed that showed varying degrees of severity. Wit mutants displayed strongly undergrown synapses, while dad overexpression animals had only modest synaptic undergrowth. In contrast, both these manipulations strongly suppressed ghost bouton budding. Additionally, synaptic undergrowth with partial knockdown of Gbb using postsynaptic RNAi was not observed, while this manipulation caused a strong reduction in ghost bouton budding. These observations indicate that rapid ghost bouton budding is more sensitive to modest perturbations in BMP signaling compared with developmental synaptic growth. One explanation for this differential sensitivity is that BMP signaling potentiates NMJs for activity-dependent bouton budding via transcriptional regulation of molecular components that are not required for normal synaptic growth. Alternatively, similar molecular pathways are required, but at different levels of output. In particular, trio mutants display a less severe synaptic undergrowth phenotype than wit mutants, but show similarly severe defects in ghost bouton budding. Because trio expression is strongly dependent on BMP signaling (Ball, 2010), a modest reduction in BMP output could reduce Trio levels such that ghost bouton budding is significantly reduced, while normal synaptic growth is less affected. It will be interesting to determine in future studies whether the developmental role for BMP signaling for acute structural plasticity shares a critical period as has recently been found for BMP function during developmental synaptic growth (Piccioli, 2014).
Given the requirement of the postsynaptic Ca2+ sensor Syt4 for normal levels of ghost bouton budding, an attractive model is that BMP is released acutely in response to elevated activity through the fusion of Syt4-positive postsynaptic vesicles. However, the current analysis indicates that retrograde BMP signaling through trio transcriptional upregulation is unlikely to be an instructive cue for bouton budding, as the severing of axons and the inhibition of retrograde trafficking of P-Mad before stimulation does not reduce budding in response to elevated activity. It is possible that synaptic P-Mad may play an instructional role in ghost bouton budding, as a local decrease in budding frequency was observed when Gbb expression was specifically reduced in muscle 6. Neuronal overexpression of dad also reduced synaptic P-Mad. Therefore, dad overexpression could inhibit ghost bouton budding by decreasing synaptic P-Mad signaling, in addition to decreasing nuclear Smad signaling. However, no dosage-dependent genetic interactions were observed between syt4 and wit, suggesting that Syt4 may participate in a separate pathway to regulate ghost bouton budding. Activity-dependent fusion of Syt4 postsynaptic vesicles (Yoshihara, 2005) could release a separate unidentified retrograde signal that provides an instructive cue for budding that would function in parallel to a developmental requirement for retrograde BMP signaling (Piccioli, 2014).
In addition to instructive cues from the postsynaptic compartment that trigger ghost bouton budding, the presynaptic nerve terminal must have molecular machines in place to read out these signals and execute the budding event. The regulation of Rho GTPases via Rho GEFs and GAPs downstream of extracellular cues is an attractive mechanism, as these proteins play critical roles in the regulation of neuronal morphology and axonal guidance. Several studies have shown that retrograde synaptic signaling regulates Rho GTPase activity to alter synaptic function and growth in Drosophila (Tolias, 2011). Ghost bouton budding mediated by developmental BMP signaling also shares some similarities with mechanisms underlying homeostatic plasticity at Drosophila NMJs. The Eph receptor is required for synaptic homeostasis at the NMJ, and it interfaces with developmental BMP signaling via Wit. While Eph receptor-mediated homeostatic plasticity predominantly requires the downstream RhoA-type GEF Ephexin, the Eph receptor may also signal through Rac1. Drosophila VAP-33A may also act as a ligand for synaptic Eph receptors, as it has been shown to regulate NMJ morphology and growth, while preferentially localizing to sites of bouton budding. The current analysis indicates that the levels of Trio, which functions as a Rho-type GEF, are bidirectionally correlated with ghost bouton budding activity and that overexpressed Trio is localized to ghost boutons after budding. As such, acute Trio regulation represents another attractive pathway for rapidly modifying bouton budding activity (Piccioli, 2014).
Rho GTPase signaling can produce distinct effects in differing systems and cell types depending on the presence or absence of downstream effectors, although most of these pathways ultimately impinge on regulation of the actin cytoskeletal. Indeed, this study has found a key role for Limk regulation of Cofilin activity in the control of ghost bouton budding. The current findings indicate that Limk activity normally functions to inhibit the formation of ghost boutons, as neuronal overexpression of Limk strongly suppressed activity-dependent bouton budding. Consistent with an inhibitory role for Limk, Cofilin activity promotes budding, while the overexpression of an inactive Cofilin inhibited budding. The expression of mutant Cofilin transgenes resulted in visible changes to the presynaptic actin cytoskeleton at NMJs, indicating that these manipulations likely alter rapid budding events by changing local actin dynamics at sites of potential growth. Using live imaging of F-actin dynamics before and after bouton budding, the formation of new F-actin puncta was observed at sites of bouton budding. Elevated Cofilin activity is sufficient to increase ghost bouton budding frequency, and is predicted to increase actin turnover and the formation of F-actin structures. Pharmacological disruption of actin polymerization dynamics also disrupts rapid bouton addition in response to elevated activity (Piccioli, 2014).
These findings support a model whereby Wit has opposing signaling roles with respect to bouton budding. Providing a permissive role via Smad signaling and an inhibitory role via Limk activation may provide for a system in which increased potential for rapid synaptic expansion is directly coupled to enhanced synaptic stability. This coupling could set a threshold for ghost bouton budding downstream of synaptic activity. In the background of moderate or low synaptic activity, Limk prevents ghost bouton budding. When synaptic activity is elevated, additional signaling events promote new synaptic growth by either reducing or outcompeting Limk activity, with a concurrent activation of Cofilin. Decreased Limk activity downstream of extracellular cues has been shown to regulate cell morphology in other systems as well, providing an attractive mechanism for rapid activity-dependent regulation of synaptic structure at Drosophila NMJs (Piccioli, 2014).
At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway-which modulates transcription of BMP target genes, and a noncanonical pathway-which connects local BMP/BMP receptor complexes with the cytoskeleton. This study describes a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches, it was demonstrated that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP's role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development (Sulkowski, 2016).
BMPs fulfill multiple functions during NMJ development via canonical and noncanonical pathways. In motor neurons, signaling by Gbb triggers a canonical BMP signaling that regulates transcription of BMP target genes and a noncanonical BMP pathway that connects Wit with LIMK1 and the cytoskeleton. This study describes a novel non-canonical BMP pathway, which induces selective accumulation of pMad at presynaptic sites. This pathway does not require Gbb, but depends on presynaptic BMP receptors Wit and Sax and postsynaptic GluRIIA. This novel pathway does not contribute to the NMJ growth and instead appears to set up a positive feedback loop that modulates the postsynaptic distribution of type-A and type-B receptors as a function of synapse activity (Sulkowski, 2016).
At the Drosophila NMJ, BMP signaling controls NMJ growth and promotes synapse homeostasis. BMP fulfills all these functions via canonical and noncanonical pathways. Canonical BMP signaling activates presynaptic transcriptional programs with distinct roles in the structural and functional development of the NMJ. For example, the BMP pathway effector Trio can rescue NMJ growth in BMP pathway mutants, but does not influence synapse physiology, whereas Target of Wit (Twit) can partially restore the mini frequency but has no effect on NMJ growth. It has been shown that both muscle and neuron derived Gbb are required for the structural and functional integrity of NMJ, and multiple mechanisms that regulate Gbb expression, secretion and extracellular availability have been described. Binding of Gbb to its receptors also triggers a noncanonical, Mad-independent pathway that requires the C-terminal domain of Wit. This domain is conserved among Drosophila Wit and vertebrate BMPRII and functions to recruit and activate cytoskeletal regulators such as LIMK1. In flies, Wit-mediated activation of LIMK1 mediates synapse stability and enables rapid, activity-dependent synaptic growth (Eaton, 2005; Piccioli, 2014; Sulkowski, 2016 and references therein).
This study uncovered a novel, noncanonical BMP pathway that triggers accumulation of presynaptic pMad in response to postsynaptic GluRIIA receptors. This pathway requires Wit and Sax, suggesting that various BMP pathways compete for shared components. Super resolution imaging mapped the pMad domains at active zones, in close proximity to the presynaptic membrane. These domains concentrate the pMad immunoreactivities into thin discs that reside mostly within individual synapse boundaries. The size and shape of pMad domains suggest that pMad could associate with membrane-anchored complexes at the active zone. Since BMP signals are generally short lived, these pMad domains likely represent pMad that, upon phosphorylation, remains associated with the BMP/BMPR kinase complexes at synaptic sites. Alternatively, pMad may accumulate in synaptic aggregates that protect it from dephosphorylation. While the second possibility cannot be excluded, several lines of evidence support the first scenario. First, excess Mad cannot increase the levels of synaptic pMad. Second, neuronal expression of activated Tkv/Sax but not Mad can restore the synaptic pMad at Importin impβ11 mutant NMJs. Finally, during neural tube closure, junctional pSmad1/5/8 and its association with PAR complexes depend on BMPs. Previous studies indicate a reduction of synaptic pMad signals in response to muscle-specific Mad RNAi. This study too has observed such a reduction. In addition, this study found a significant decrease of postsynaptic IIA/IIB ratio in Mad-depleted muscles: GluRIIA and GluRIIB synaptic levels were reduced to 49% and respectively 78% of control. Since GluRIIA is key to the synaptic pMad accumulation it is suspected that the muscle Mad RNAi phenotype is due to perturbation in synaptic GluRIIA levels, perhaps by interference with the Activin signaling pathway (Sulkowski, 2016).
How are the BMP/BMPR complexes stabilized at synaptic sites? Studies on single receptors demonstrate that the confined mobility of BMPRI on the plasma membrane is key to stabilize BMP/BMPR complexes and differentially stimulate canonical versus noncanonical signaling. Direct interactions between phosphorylated Smad5 and the Par3-Par6-aPKC polarity complex occur at the apical junctions. Similarly, synaptic pMad, which remains associated with BMP/BMPR complexes, may engage in interactions that restrict the mobility of BMP/BMPR complexes on the presynaptic membrane. Nemo-mediated phosphorylation of Mad-S25 could disrupt the pMad/BMPR association and expose the BMP/BMPR complexes, so they could dissociate and/or be internalized. The heteromeric BMPR complexes are transient; ligand binding greatly increases their lifespan and stability. Albeit Gbb is not essential for synaptic pMad, it may act redundantly with other ligands to stabilize BMP/BMPR local complexes. Several ligands secreted in the synaptic cleft have been shown to bind and signal via BMPRII; they include glia secreted Maverik, Myoglianin, which could be secreted from muscle and/or glia, and Activins. However, these ligands also appear to signal via a canonical Activin pathway, which regulates the postsynaptic GluRIIA/GluRIIB abundance at the Drosophila NMJ. Alterations in the Activin signaling pathway drastically alter the synaptic recruitment of both iGluR subtypes, in particular the GluRIIA, which controls synaptic pMad, making it difficult to identify the nature and the directionality of the signaling molecule(s) involved in the synaptic pMad accumulation. Interestingly, all of these ligands are substrates for BMP-1/Tolloid-type enzymes, which control their activity and distribution. Treatments that induce long-term stimulation up-regulate a BMP-1/Tolloid homolog in Aplysia neurons (Sulkowski, 2016).
An intriguing aspect of this novel BMP pathway is the dependence on active postsynaptic GluRIIA, which is both required and sufficient for pMad accumulation at active zones. Since pMad and BMP/BMPR complexes cluster at synaptic sites, it is speculated that trans-synaptic complexes may couple postsynaptic type-A glutamate receptors with presynaptic BMP/BMPRs. The synaptic cleft is 200 Å; the iGluR tetramer expands 135 Å in the synaptic cleft, and the BMP/BMPR complexes ~55 Å. The iGluRs auxiliary subunit Neto has extracellular CUB and LDLa domains predicted to expand 120-130 Å in the synaptic cleft, based on related structures. CUB domains are BMP binding motifs that may localize BMP activities and/or facilitate ligand binding to BMPRs. In this model, Neto provides the link between postsynaptic GluRIIA and presynaptic BMP/BMPR complexes. During receptors gating cycle, the iGluRs undergo corkscrew motions that shorten the channels as revealed by cryo-electron microscopy. Such movements may influence the stability of trans-synaptic complexes and allow synaptic pMad to function as a sensor for GluRIIA activity (Sulkowski, 2016).
While more components of this novel pathway remain to be determined, it is clear that this pathway does not contribute to NMJ growth and instead has a critical role in synapse maturation. Unlike canonical BMP signaling, loss of local pMad induces minor reductions in bouton number and does not rescue the NMJ overgrowth of endocytosis mutants. Local pMad accumulates independently of Wit-mediated LIMK1 activation and does not appear to influence synapse stabilization; in fact, nrx mutants have synapse adhesion defects but show increased synaptic pMad levels. The striking correlation between synaptic pMad levels and GluRIIA activity, together with previous findings that GluRIIA activity and gating behavior directly impacts receptor mobility and synaptic stabilization suggest a positive feedback mechanism in which active GluRIIA receptors induce stabilization of BMP/BMPR complexes at synaptic sites which, in turn, promote stabilization of type-A receptors at PSDs. In this scenario, presynaptic pMad marks active BMP/BMPR complexes and acts to maintain the local BMP/BMPR complexes in large clusters that evade endocytosis. Selective disruption of local pMad via a neuronal dominant-negative MadS25D presumably destabilizes the large presynaptic BMP/BMPR clusters and causes a significant reduction in the IIA/IIB ratio and quantal size (Sulkowski, 2016).
This positive feedback couples synaptic activity with synapse development and is controlled by (1) active GluRIIA receptors, (2) presynaptic BMP receptors, Wit, Sax, and likely Tkv, (3) mechanisms regulating BMPR heteromers assembly, endocytosis and turnover, and (4) the ability of pMad to remain associated with its own kinase upon phosphorylation. Perturbations of any of these components trigger variations in local pMad levels accompanied by changes in the IIA/IIB ratio and/or quantal size. For example, nemo mutants have increased synaptic pMad levels and increased mEJCs, while imp mutants have decreased synaptic pMad levels and decreased mEJPs. The assembly and function of these putative trans-synaptic complexes, in particular ligand availability, should be influenced by the composition and organization of the synaptic cleft. Indeed, local pMad and quantal size are increased in mutants lacking heparan sulfate 6-O-endosulfatase (sulf1), or 6-O-sulfotransferase (hs6st). Since this Mad-dependent, noncanonical pathway shares components with the other BMP signaling pathways, the balance among different BMP pathways may coordinate the NMJ development and function (Sulkowski, 2016).
The complexity of BMP signaling at the Drosophila NMJ is reminiscent of the neurotrophin-regulated signaling in vertebrate systems. Neurotrophins were first identified as neuronal survival factors. Like BMPs, they are secreted as pro-proteins that must be processed to form mature ligands. The active dimers bind to transmembrane kinase receptors and induce their activation through trans-phosphorylation. Neurotrophin/receptor complexes are internalized and transported along axons to the cell soma; signaling in the cell soma controls gene expression and promotes neuronal differentiation and growth. In addition, local neurotrophin signaling regulates growth cone motility, enhances the presynaptic release of neurotransmitter and mediates activity-dependent synapse formation and maturation. At the Drosophila NMJ, several neurotrophins have been implicated in neuron survival, axon guidance and synapse growth. It will be interesting to test for the crosstalk between neurotrophin and BMP signaling at these synapses (Sulkowski, 2016).
The novel noncanonical BMP pathway reported in this study is the first example of a BMP pathway triggered by selective neurotransmitter receptors and influencing receptor distribution at PSDs. It is expected that some of these functions will apply to mammalian glutamatergic synapses: First, as indicated in the Allen Brain Atlas, glutamate receptors and Neto proteins are widely expressed in mammalian brain structures where BMPs, BMPRs and Smads are expressed. Second, BMPs have been shown to rapidly potentiate glutamate-mediated currents in human retina neurons, presumably via a noncanonical pathway. Finally, mice lacking Chordin, a BMP antagonist, have enhanced paired-pulse facilitation and LTP and show improved learning in a water maze test. Such changes could not be explained by Smad-dependent transcriptional responses and were not accompanied by structural alterations in synapse morphology. Instead, presynaptic noncanonical BMP pathway may influence the activity of postsynaptic glutamate receptors by modulating their synaptic distribution and stability (Sulkowski, 2016).
Using the Drosophila larval neuromuscular junction, this study shows a PI3K-dependent pathway for synaptogenesis (a pro-syaptogenesis pathway) which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, the functional hierarchy within the pathway was determined. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, an antagonistic pathway (an anti-synaptogenesis pathway) was identified that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways (Jordan-Alvarez, 2017).
The epistasis assays have determined the in vivo functional links between PI3K and other previously known pro-synaptogenesis factors. Epistasis assays are based on the combined expression of two or more UAS constructs. Several double combinations in this study have produced a phenotype in spite of the apparent ineffectiveness of the single constructs. This type of results underscores the necessity to use epistasis assays in order to reveal functional interactions in vivo, hence, biologically relevant. In addition to the pro-synaptogenesis signaling, the study has revealed an anti-synaptogenesis pathway that composes a signaling equilibrium to determine the actual number of synapses. The magnitude of the synapse number changes elicited by the factors tested here are mostly within the range of 20%-50%. Are these values significant to cause behavioral changes? Reductions in the order of 30% of excitatory or inhibitory synapses in adult Drosophila local olfactory interneurons transform perception of certain odorants from attraction to repulsion and vice versa. In schizophrenia patients, a 16% loss of inhibitory synapses in the brain cortex has been reported. In Rhesus monkeys, the pyramidal neurons in layer III of area 46 in dorsolateral prefrontal cortex show a 33% spine loss, and a significant reduction in learning task performance during normal aging. Thus, it seems that behavior is rather sensitive to small changes in synapse number irrespective of the total brain mass (Jordan-Alvarez, 2017).
The signaling interactions analyzed here were chosen because they were reported in other cellular systems and species previously. Some of these interactions have been confirmed (e.g., Gbb/Wit), while others have proven ineffective in the context of synaptogenesis (e.g., Ras85D/PI3K binding). Likely, the two signaling pathways, pro- and anti-synaptogenesis, are not the only ones relevant for synapse formation. For example, in spite of the null condition of the gbb and wit mutant alleles used here, the resulting synaptic phenotypes are far less extreme than expected if these two factors would be the only source of signaling for synaptogenesis. Although it could be argued that the incomplete absence of synapses in the mutant phenotypes could result from maternal perdurance, Wit is not part of the oocyte endowment while Gbb is. Three alternative possibilities may be considered, additional ligands for Wit, additional receptors for Gbb, and a combination of the previous two. Beyond the identity of these putative additional ligands and receptors, the stoichiometry between ligands and receptors may certainly be relevant. Actually, Gbb levels are titrated by Crimpy. An equivalent quantitative regulation could operate on Wit. The reported data on Wit illustrate already the diversity of the functional repertoire of this receptor. Wit can form heteromeric complexes with Thick veins (Tkv) or Saxophone (Sax) receptors to receive Dpp/BMP4 or Gbb/BMP7 as ligands. However, the same study also showed that Wit could dimerize with another receptor, Baboon, upon binding of Myoglianin to activate a different and antagonistic signaling pathway, TGFβ/activin-like (Jordan-Alvarez, 2017).
The Gbb/Wit/PI3K signaling analyzed in this study is likely not the only pro-synaptogenesis pathway in flies and vertebrates. The ligand Wingless (Wg), member of the Wnt family, and the receptors Frizzled have been widely documented as relevant in neuromuscular junction development, albeit data on synapse number are scant. Interestingly, however, the downstream intermediaries can be as diverse as those mentioned above for Wit. Although generally depicted as linear pathways, a more realistic image would be a network of cross-interacting signaling events whose in situ regulation and cellular compartmentalization remains fully unexplored (Jordan-Alvarez, 2017).
The quantitative regulation of receptors is most relevant to understand their biological effects. In that context, is worth noting that Tkv levels are distinctly regulated from those of Wit and Sax through ubiquitination in the context of neurite growth. On the other hand, although the receptor Wit is considered a RSTK type, the functional link with PI3K is a feature usually associated to the RTK type instead. The link of Wit with a kinase has a precedent with LIMK1 that binds to, and is functionally downstream from, Wit in the context of synapse stabilization. Thus, Wit should be considered a wide spectrum receptor in terms of its ligands, co-receptor partners and, consequently, signaling pathways elicited. Actually, the Wit amino acid sequence shows both, Tyr and Ser/Thr motifs justifying its initial classification as a 'dual' type of receptor. In this report this study did not determine if Wit heterodimerizes with other receptors, as canonical RSTKs do, or if it forms homodimers, as canonical RTKs do. However, the lack of synaptogenesis effects by the putative co-receptors, Tkv and Sax, and the phenotypic similarity with the manipulation of the standard RTK signaling effector Cic, leaves open the possibility that Wit could play RTK-like functions, at least in the context of synaptogenesis (Jordan-Alvarez, 2017).
Consistent with the proposal of a dual mechanism for Wit, its activation seems to be a requirement to elicit two independent signaling steps, PI3K and Ras85D, that could reflect RTK and RSTK mechanisms, respectively. Both steps are independent because the mutated form of PI3K unable to bind Ras85D, PI3KΔRBD, is as effective as the normal PI3K to elicit synaptogenesis. PI3K and Ras85D signaling, however, seem to converge on Bsk revealing a novel feature of this crossroad point. The activity level of Bsk is known to be critical in many signaling processes. The peculiarities of Bsk/JNK activity include its coordinated regulation by p38a and Slpr in the context of stress heat response without interference on the developmental context. Another modulator, Puc, was described as a negative feed-back loop in the context of oxidative stress. The Puc mediated loop is operative also for synaptogenesis, while that of p38a/Slpr is relevant for p38a only, as shown here. Further, Ras85D represents an additional regulator in the neural scenario. The triple regulation of Bsk/JNK by Ras85D, Puc and the MAPKs seems to stablish a narrow range of activity thresholds within which normal number of synapses is determined (Jordan-Alvarez, 2017).
The concept of signaling thresholds is also unveiled in this study by the identification of another signaling pathway that opposes synapse formation. The pro- and anti-synaptogenesis pathways have similar constituents, including small GTPases, MAPKs and transcriptional effectors, Mad/Smad, which are canonical for RSTK receptors. The RSTK type II receptor Put, which can mediate diverse signaling pathways according to the co-receptor bound can be discarded in either the pro- or the anti-synaptogenesis pathways. Thus, the main receptor for the anti-synaptogenesis pathway remains to be identified (Jordan-Alvarez, 2017).
Concerning small GTPases, the pro-synaptogenesis pathway uses Ras85D while its counterpart uses the poorly studied Ras64B. The anti-synaptogenesis pathway includes an additional member of this family of enzymes, Rala. This small GTPase plays a role in the exocyst-mediated growth of the muscle membrane specialization that surrounds the synaptic bouton as a consequence of synapse activity. That is, Rala can influence synapse physiology acting from the postsynaptic side. The experimental expression of a constitutively active form of Rala in the neuron does not seem to affect the overall synaptic terminal branching. However, the null ral mutant shows reduced synapse branching and its vertebrate homolog is expressed in the central nervous system. This study found that Rala under-expression in neurons yields an elevated number of synapses. Thus, it is likely that this small GTPase acts as a break to synaptogenesis, hence its inclusion in the antagonistic pathway (Jordan-Alvarez, 2017).
Synaptogenesis and neuritogenesis are distinct processes since each one can be differentially affected by the same mutant (e.g.: Hiw). Both features, however, share some signals (e.g., Wnd, Hep). This signaling overlap is akin to the case of axon specification versus spine formation for constituents of the apico/basal polarity complex Par3-6/aPKC [127]. These and other examples illustrated in this study underscore the need to discriminate between synapses and boutons. This study is focused on the cell autonomous signaling that takes place in the neuron. Non-cell autonomous signals (e.g., originated in the glia or hemolymph circulating) have not been considered. The active role of glia in axon pruning and bouton number has been the subject of other studies. Considering the reported role of Hiw through the midline glia in the remodeling of the giant fiber interneuron it is not unlikely that the glia-to-neuron signaling may share components with the neuron autonomous signaling addressed here (Jordan-Alvarez, 2017).
The summary scheme (see Summary diagram of antagonistic signaling pathways for synaptogenesis and their interactions) describes the scenario where two signaling pathways mutually regulate each other. Epistasis assays are the only experimental approach for in vivo studies of more than one signaling component, albeit this type of assay is only feasible in Drosophila Thus, it is plausible that vertebrate synaptogenesis will be regulated by a similar antagonistic signaling (Jordan-Alvarez, 2017).
The regulatory equilibrium as a mechanism to determine a biological parameter is the most relevant feature in this scenario for several reasons. First, because this type of mechanism can respond very fast to changes in the physiological status of the cell, and, second because it provides remarkable precision to the trait to be regulated, synapse number in this case. Although bi-stable regulatory mechanisms are known in other contexts, the case of synapse number may seem unexpected because the highly dynamic nature of synapse number has been recognized only recently. Consequently, a molecular signaling mechanism endowed with proper precision and time resolution must sustain this dynamic process. The balanced equilibrium uncovered in this study, although most likely still incomplete in terms of its components, offers such a mechanism (Jordan-Alvarez, 2017).
To screen for structural alterations of the neuromuscular junction (NMJ), a transmembrane GFP-fusion protein (CD8-GFP-Sh) was used that specifically localizes to the NMJs of Drosophila larvae. All somatic muscles and their neuromuscular connections can be observed through the translucent cuticle of an intact animal from first to late third instar larvae using confocal microscopy. In larvae, the somatic musculature of each hemisegment is innervated by a major nerve bundle, which defasciculates into five major nerve branches. Each branch forms synapses on a subset of muscles: for example, the ISN branch innervates dorsal and dorso-lateral muscles, while the SNa branch innervates exclusively lateral muscles. The dorsal and lateral muscles are most easily visualized in CD8-GFP-Sh larvae and have therefore been the focus of this screen (Aberle, 2002).
On the third chromosome, third instar larvae of 4973 homozygous Drosophila lines carrying EMS-induced mutations for morphological alterations of the NMJ were screened. 66 lines were saved that fell into 17 complementation groups comprising 10 phenotypical classes. Among the most prominent phenotypes, mutations were found that lead to smaller and more expanded synapses. Mutations were also recovered that affect muscle innervation, muscle morphology, and localization of the CD8-GFP-Sh transgene (Aberle, 2002).
Five mutant alleles of one complementation group, named wishful thinking (wit) (Marqués, 2002), exhibit particularly small NMJs, while muscle size remains close to normal. All neuromuscular synapses are smaller in wit mutants with nearly 100% penetrance. The size of individual synaptic boutons appears normal, but boutons are often spaced further apart from one another. The specificity of the neuromuscular connections is not affected, and no other gross morphological defects were observed in wit mutant larvae. In addition, the size of sensory neurons appears normal and the dendritic tree of multidendritic neurons is not visibly reduced in size in the mutants. wit larvae pupate at the same time as their heterozygous siblings and die as pharate adults shortly before eclosion. Thus, mutations in wit seem to primarily affect the structure of the NMJ (Aberle, 2002).
Since the synapses are drastically reduced in size in wit mutants, it was of interest to determine if there were any changes in the subcellular distribution of synaptic proteins. Discs Large (Dlg), a synaptic clustering protein, and myc-tagged glutamate receptor IIB (GluRIIB), a subunit of postsynaptic GluRs, appear to localize correctly. In contrast, alterations were found in the synaptic level of the cell adhesion molecule Fasciclin II (Fas II) and in the distribution pattern of the synaptic vesicle-associated protein Synaptotagmin (Syt). In wild-type larvae, Fas II is detected on all motor axons and at higher levels on both the pre- and post-synaptic side of synaptic boutons. wit mutant synapses generally exhibit a greatly reduced synaptic staining for Fas II. Interestingly, Fas II appears to be selectively absent from the synaptic terminals but remains at normal levels on the motor axon bundles. In wild-type, the synaptic vesicle marker Syt localizes exclusively to the presynaptic terminal and is not detected normally in motor axons. In wit mutants, however, Syt staining is typically found in a punctate pattern in the nerve bundles (Aberle, 2002).
The structural growth defects in wit mutants were quantified by counting the synaptic boutons. The bouton numbers on dorsal muscle 1 are approximately 55% reduced in wit mutants. The muscle surface area is on average 12% smaller. Similar results were obtained for muscles 2, 9, and 10. Neuromuscular synapses were examined in wit mutant embryos and at each larval stage. While neuromuscular synapses in wit mutant stage 16 embryos appear similar to those of wild-type, those of first instar wit larva have visibly smaller synapses compared to wild-type, as do those of second instar wit mutants (Aberle, 2002).
The wit gene was sequenced of flies bearing five different mutant alleles. Point mutations were found in the coding region of four alleles, all of which were located in the kinase domain. Stop codons were identified in three wit alleles (K482Stop in witHA4, W491Stop in witHA3, and R524Stop in witHA5). These three alleles and witHA2 have similar phenotypes either transheterozygous over each other or hemizygous over deficiencies, suggesting they represent strong loss-of-function alleles. The witHA1 allele appears to be a hypomorph since the synaptic phenotype is slightly less severe. Interestingly, a missense mutation was found in witHA1 that changes a glutamate residue to lysine within the coiled-coil/leucine zipper domain at position 413 (E413K). These results show that synaptic undergrowth in wit mutants is caused by single point mutations in the Drosophila BMPRII homolog Wit (Aberle, 2002).
Additional evidence for a presynaptic function for Wit was provided by the examination of the effect of a dominant negative wit construct lacking the cytoplasmic domain of the receptor protein (witDeltaC). Overexpression of WitDeltaC in motoneurons using OK6-Gal4 reduces synaptic size in a wild-type background, whereas overexpression of the same protein in muscles using G14-Gal4 has no effect. Finally, whether overexpression of Wit is sufficient to promote synaptic growth in a wild-type background was tested. When Wit is overexpressed in all neurons or in all somatic muscles, no synaptic overgrowth is seen at the NMJ, even when the copy number is increased 2-fold. In summary, these results suggest that wit function is required presynaptically for viability and normal synaptic development at the NMJ (Aberle, 2002).
Since wit is required for normal synaptic development at the NMJ, it was of interest to see whether wit also plays a role in synaptic transmission. Both the spontaneous and the evoked release were examined using intracellular recordings from muscle 6 at segment A3 in wild-type and wit mutant third instar larvae. Evoked excitatory junctional potentials (EJP) are greatly reduced in wit mutants reaching only 10% of those of the wild-type. The amplitude of spontaneous miniature excitatory junctional potentials (mEJPs) is also slightly reduced in wit mutants. To further examine the effect of wit loss of function on neurotransmitter release, the frequency of spontaneous release was measured in the absence of stimulation. This measure of release is also significantly reduced in the mutants. Quantal content was measured and it was found that wit mutant synapses release 7-fold less neurotransmitter than wild-type synapses. Several allelic combinations of wit mutants gave similar results. These results suggest that Wit is required for normal synaptic function. All defects of neurotransmission were rescued when Wit was overexpressed either pan-neuronally or in motoneurons with elav-Gal4 or OK6-Gal4, respectively. This indicates that wit loss of function disrupts a presynaptic process, consistent with the genetic results for rescuing lethality and structural defects (Aberle, 2002).
Finally, the electrophysiological defects in wit mutants were examined by measuring the Ca2+ dependence of neurotransmitter release. Loss of function of wit does not appear to affect the Ca2+ cooperativity at the neuromuscular junction. The electrophysiological examination implies that upon depolarization, wit mutant motor terminals release significantly less neurotransmitter (Aberle, 2002).
Given the dramatic light microscopic and electrophysiological defects, it was of interest to see whether the ultrastructure of wit neuromuscular synapses was also affected. Serial cross-sections of both wild-type and wit mutant boutons at muscles 6 and 7 of third instar larvae were examined by electron microscopy. It was found that wit mutant synapses have active zones with T-bars, synaptic vesicles both free within the bouton and clustered around the T-bars, and other features that appear wild-type. In addition to these features, also observed were a smaller population of vesicles larger than and distinct from the synaptic vesicles, an unusual ruffling of the presynaptic membrane in active zones, and a novel electron dense structure associated with a tight cluster of vesicles. The postsynaptic specialization of the muscle membrane, the subsynaptic reticulum (SSR), was indistinguishable from wild-type (Aberle, 2002).
A number of bouton characteristics was quantified from serial sections of 17 wit boutons and they were compared with similar data from 18 wild-type boutons. Although wit and wild-type boutons are approximately the same size, the bouton surface area per active zone is 2-fold greater in wit mutants. In addition, the number of T-bars per active zone was 2-fold greater in wit mutants. The bouton surface area per T-bar (an indicator of an active vesicle release site) was not statistically different between wit mutants and wild-type (Aberle, 2002).
The ultrastructural observations reveal two significant defects in wit mutant boutons. In wit mutants, the postsynaptic membrane and the electron dense material in the cleft appear normal, but the presynaptic membrane within the active zone displays regions of ruffling or detachment from the postsynaptic membrane/cleft material. These membrane detachments do not include the entire active zone since some regions maintain close apposition of the pre- and post-synaptic membranes. This adhesion defect is observed in nearly all active zones of wit mutants and is similar in severity in sections with, and without, T-bars. This defect was quantified by comparing the ratio of the length of presynaptic membrane in close apposition to the postsynaptic membrane to the overall length of postsynaptic membrane in active zones of wild-type and wit mutants. In wild-type boutons, this ratio is 0.97 ± 0.01. By contrast, this ratio in wit mutants is 0.56 ± 0.03, indicating that 44% of the presynaptic membrane is detached. In no instance, however, was a T-bar observed associated with a detached section of presynaptic membrane, even when the immediately adjacent presynaptic membrane was detached (Aberle, 2002).
The second defect in wit mutant boutons is the presence of a novel electron dense structure and its associated cluster of vesicles within the cytoplasm of the bouton. An average of 2.4 ± 0.7 such structures per bouton has been observed in wit mutants, whereas none were observed in wild-type boutons. These structures were named T-bodies, due to their shared ability to cluster synaptic vesicles in a similar fashion to T-bars. Unlike T-bars, T-bodies appear to float freely within the bouton with no attachment to the presynaptic membrane. T-bodies often spanned 2-3 adjacent 80 nm serial sections, had a 'C' or closed ring shape in 3D reconstruction, and did not resemble conventional T-bars seen in either cross-section or tangential section. Their association with tight clusters of synaptic vesicles suggests, however, that they contain vesicle-docking components similar, if not identical, to those in T-bars. The wit loss of function therefore appears to disrupt the proper adhesion of the presynaptic membrane to the postsynaptic membrane/cleft material within active zones, which could underlie the defects observed in synaptic function (Aberle, 2002).
The 64A region had been screened previously by EMS mutagenesis for the presence of lethal complementation groups. Transgenic lines that contain the entire wit transcription unit, as well as 1.8 kbp or more of upstream sequence, rescue the l(3)64Aa complementation group, while P lines that do not completely cover wit do not rescue this group of mutants. To confirm that l(3)64Aa does indeed contain lesions within wit, genomic DNA from five l(3)64Aa alleles was sequenced. In each case, a molecular lesion was identified in the wit coding sequence. Since all alleles of l(3)64Aa contain mutations in the wit transcription unit, they will henceforth be referred to as wit alleles. Among the various lesions isolated, witA12 likely represents a simple null allele since the C684T change introduces an early stop codon prior to the transmembrane segment. The proximal breakpoint of the small deficiency Df (3L) C175 was localized to the 3' portion of the wit transcription unit. Interestingly, no mutations were found in the carboxy-terminal extension of Wit, despite the fact that this region represents over 40% of the coding sequence and that mutations in the tail of human BMPRII account for some cases of primary pulmonary hypertension (Marqués, 2002).
To determine the wit lethal phase, the number of wit mutant embryos that survived to the first instar, third instar, and pharate stage was quantitated. All transheterozygous combinations of wit alleles are 100% lethal, with no adult escapees. Approximately 50% of the mutant animals survive to the pharate stage. The remaining animals die during larval and early pupal stages. The animals that do survive to the pharate stage exhibit the head expansion and abdominal peristaltic movements characteristic of normal pre-eclosion behavior, but neither rupture nor escape from the pupal case. Removing the operculum and underlying pupal cuticle is not sufficient to allow the animals to eclose. If the animals are dissected free of the pupal case and cuticle, they remain alive for several days but, for the most part, exhibit only sporadic leg movements. None of these animals inflate their wings. However, when the wings are artificially inflated by immersion in water, they show no patterning defects. Many of the dissected animals showed a bend in the femur or tibia, the phenotype being more pronounced and frequent in the metathoracic leg. A small fraction of the animals also exhibited truncations of the tarsal segments. These defects are unlikely to be the cause of the inability to eclose since the penetrance of such defects is incomplete, and animals rescued to viability by expression of Wit in the nervous system still showed leg defects yet were able to escape the pupal case (Marqués, 2002).
The general morphology of the wit pharates was analyzed by silver staining of 10 µm paraffin sections. General organization of neural centers and muscle groups is essentially normal, and neither misrouted axonal tracts nor fasciculation defects were found (Marqués, 2002).
Germline clones were generated in an attempt to uncover earlier phenotypes. Maternally depleted animals derived from null alleles showed the same lethal phase and range of defects as those found in the zygotic mutants, indicating that wit is not required in the germline or in the early embryo (Marqués, 2002).
The inability of wit mutants to eclose, together with the locomotion defects exhibited by dissected animals and the strong neural expression of wit, indicates that Wit's function is likely to be required in neurons. To determine if the nervous system is the primary tissue requiring wit activity, wit cDNA was expressed using a number of different promoters as well as with the conditional Gal4-UAS expression system. Several independent lines of both Hsp70-wit and ubiquitin-wit were able to rescue different wit allelic combinations indicating that low level, ubiquitous expression of wit is sufficient to ensure its function. Using the Gal4 system, it was found that drivers such as nervana and elav that are expressed in most or all differentiated neurons completely rescue wit mutant animals to full viability. Other drivers such as twist (pan mesoderm) or Mhc (myosin heavy chain promoter, muscles) showed no rescuing ability (Marqués, 2002).
Since wit mutants exhibit defects in locomotion, whether more limited expression of wit in motor neurons could rescue wit lethality was examined. Viability is restored to wit mutants when two different Gal4 drivers, BG380 and OK6 are used: their expression in larvae is primarily restricted to motor neurons. These results demonstrate that Wit activity is primarily required in differentiated motoneurons (Marqués, 2002).
Since mutations in the tail of the human BMPR type II receptor have been isolated that affect its function (Deng, 2000; Lane, 2000; Thomson, 2000), whether the carboxy-terminal extension of Wit is also required for viability was examined. To address this issue, a genomic rescue construct was generated in which the tail was truncated from 351 to 88 aa. This construct was able to rescue wit mutant animals to full viability. Using the Gal4-UAS system, a truncated form of Wit that had only 24 aa of the carboxy-terminal tail was expressed in the nervous system and similar results were observed. It is concluded that the carboxy-terminal extension is not required for viability, possibly accounting for the absence of lethal mutations mapping to this part of the protein (Marqués, 2002).
Since in many tissues Punt appears to be the primary type II receptor for mediating BMP signals, whether overexpression of Punt in neurons would rescue wit mutants was examined. Expression of UAS-punt using elav-Gal4 did not rescue wit mutant animals nor did ubiquitous expression of wit rescue punt mutations, suggesting that these two receptors are not functionally interchangeable for mediating TGF-ß type signals during development (Marqués, 2002).
Since mutations in the C. elegans unc-129 gene, which encodes a BMP type ligand, have been shown to exhibit axon guidance defects (Colavita, 1998), the pattern of axon tracts was examined in wit mutant embryos using the 22C10, BP102, and Fas II antibodies. No evidence was found that wit mutant embryos have defects in axon guidance, and the synapses of all larval motoneurons appear to be established with their proper targets (Marqués, 2002).
The lack of any evidence for cell fate or axon guidance problems within the nervous system of wit mutants prompted an examination of these mutants for alterations in their electrophysiological properties. Since specific expression of wit in motoneurons is able to rescue wit mutant animals and lack of wit function results in specific loss of P-Mad accumulation in embryonic motoneurons, attention was focused on analysis of glutamatergic motoneuron synapses in third instar larva NMJs. wit mutants show a dramatic decrease in transmitter release without any change in the average quantal size. The reduction in evoked junctional synaptic currents (EJCs) in wit mutants is significantly rescued by expression of Wit in neurons using elav-Gal4. This observation is consistent with the finding that Wit is required in nerve cells and not in muscle. A proportional decrease in quantal content was observed at multiple calcium concentrations, indicating that there is not a substantial alteration in the Ca2+ cooperativity of transmitter release in the mutant. In addition, failure analysis demonstrates that most nerve stimuli fail to evoke transmitter release at low extracellular [Ca2+] in wit mutants compared to wild-type and rescued wit larvae. Consistent with a high rate of failures, the average frequency of spontaneous vesicle release was dramatically reduced in mutants by nearly 4-fold compared to control animals. The amplitude of spontaneous miniature excitatory junction potentials (mEJPs), however, was not significantly altered in wit mutants. Taken together, these results indicate that wit mutants are defective for vesicle release from the presynaptic terminal (Marqués, 2002).
To determine if the reduced transmitter release observed in wit mutants is caused by changes in synapse morphology and/or ultrastructure, overall synapse size, bouton number, and active zone morphology were examined at several different muscles of segment A3 in third instar larvae. The number of synaptic boutons in ventral longitudinal muscles 6 and 7 was 57 ± 18 for wild-type and 31 ± 9 for wit mutants (p < 0.0001). Since it was noticed that wit mutant larvae are slightly smaller than wild-type animals, the decrease in synapse size must be corrected to account for a 25% decrease in ventral muscle volume. After correcting for decreased muscle size, the number of synaptic boutons in wit mutant animals is about 70% of wild-type. The synapse length in dorsal longitudinal muscle 9 was 88 ± 28 µm (all data expressed as mean ± standard deviation) for wild-type and 44 ± 23 µm for wit mutants (Marqués, 2002).
Although synapse size and bouton number are reduced in wit mutants, the overall percentage reduction was not proportionately as severe as the reduction in EJC magnitude. Therefore, the ultrastructure of the synapses was examined by serial section electron microscopy (EM). In wild-type animals, sites of presumptive transmitter release (active zones) are characterized by tight apposition of muscle and membrane at a number of discrete sites in each bouton and the presence of a specialized structure referred to as T-bar. Bouton size and the overall synaptic structure, including the subsynaptic reticulum, number of synaptic vesicles, and vesicle docking at T-bars, appears normal in wit mutants. However, it was noted that there is a dramatic alteration in the morphology of the active zone. In wit mutants, the pre- and post-synaptic membranes in certain areas are no longer closely aligned. The presynaptic membrane detaches completely from the postsynaptic membrane, forming large curvatures bending toward the cytosol of nerve terminals. Such membrane detachments also occur in areas near the active zones. Natural membrane curvatures are also observed in wild-type controls. However, presynaptic membranes do not separate from postsynaptic membranes. This ultrastructural defect in the active zone morphology has not been previously described and is consistent with the altered transmitter release properties exhibited by wit mutants, and suggests that BMP signaling plays an important role in synapse maturation or maintenance during development (Marqués, 2002).
spinster (spin), which encodes a multipass transmembrane protein, has been identified in a genetic screen for genes that control synapse development. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. Spin has been localized to a late endosomal compartment and evidence is presented for altered endosomal/lysosomal function in spin mutants. Evidence is presented that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-ß signaling. TGF-ß receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. A model is presented for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease (Sweeney, 2002).
To determine whether synaptic overgrowth in spin is caused by enhanced TGF-ß signaling, it was asked whether TGF-ß signaling is necessary for synaptic overgrowth in spin. The type II receptor mutation wishful thinking causes a severe decrease in bouton number at the NMJ. Type I TGF-ß receptors are known to function in concert with type II receptors, and the type I receptors tkv and sax participate in synaptic growth regulation in this system. Third instar larva mutant for sax or tkv have smaller neuromuscular synapses. This study confirms that there is a significant decrease in bouton number in wit, and that there is a similar decrease in bouton number in both tkv and sax. These receptors are shown to function in the larval motoneurons by demonstrating that pMAD staining in the cell bodies of larval motoneurons requires wit or sax. In this experiment, the larval CNS was costained with pMAD and anti-evenskipped, which labels a subset of motoneurons (Sweeney, 2002).
A genetic analysis of the TGF-ß receptor mutations wit, tkv, and sax in combination with spin demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Heterozygous mutations in tkv, sax, and wit do not alter synaptic bouton numbers at the NMJ. Heterozygous mutations in tkv, sax, and wit suppress synaptic overgrowth when placed in the spin mutant background. Bouton numbers are significantly reduced in each case where a single copy of a receptor is mutated in combination with spin. Bouton numbers were quantified in each of the double mutant combinations of tkv, sax, or wit with spin. In each case, when both copies of a receptor were removed, synaptic overgrowth was suppressed in the spin mutant background further than when only a single copy of a receptor was mutated. These data demonstrate that TGF-ß receptor mutations suppress synaptic overgrowth in spin in a dose-dependent manner. Furthermore, since bouton numbers return to wild-type, or below wild-type levels, it demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Taken together with the increase in bouton numbers seen in dad, these data support the conclusion that enhanced or misregulated TGF-ß signaling is a major determinant of synaptic overgrowth in spin. It is hypothesized that altered endosomal function due to loss of Spin causes enhanced TGF-ß signaling and subsequent synaptic overgrowth. Future experiments will be necessary to determine whether enhanced signaling is due to increased receptor number at the plasma membrane, or an inability to stop signaling within the late endosomal system (Sweeney, 2002).
The BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those observed in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth (McCabe, 2003).
Of particular relevance to the involvement of endosomes in mediating BMP signaling at synapses is the recent observation that mutations in the spinster (spin) gene greatly enhance synaptic growth. Spin is a putative multipass transmembrane protein localized to the late endosomes/lysosomes. Mutations in spin disrupt the morphology of late endosomes and lead to a 2-fold overgrowth in bouton number at the NMJ. This overgrowth can be suppressed by mutations in wit, suggesting that elimination of Spin leads to enhanced BMP signaling. Although the molecular mechanism responsible for the increased signal is unclear, the combined data support the idea that BMP signaling is likely modulated by intracellular trafficking of various signaling components at the level of the endosome. If these endosomes are capable of binding to dynein motors, then, like the Trk containing endosomes, they may also be transported along microtubules back to the cell body. Other models, such as wave activation of receptors, may also be possible, although if this is the case, then it is not clear why overexpression of ΔGl should interfere with BMP signaling as described above (McCabe, 2003).
Although loss-of-function and rescue experiments clearly demonstrate that Gbb is required for proper synaptic development at the NMJ, it is not certain that it is the only TGF-β-type ligand or indeed the primary ligand that regulates this process. The electrophysiological and ultrastructure defects observed in gbb mutant synapses are not as severe as those found in wit null mutants. This could simply reflect an inability to produce true null animals that survive to the third instar stage, or it may indicate that another ligand also provides a signal. In support of this view is the observation that P-Mad accumulation is not totally eliminated in gbb1/gbb2 null mutant embryos as it is in wit mutants. In addition, it is noted that while overexpression of Gbb in the CNS only weakly rescued P-Mad accumulation in the CNS, the pattern of accumulation does not appear to change. That is, P-Mad still seems to be found primarily in motoneurons. Thus, other neurons do not appear to be competent to respond to BMP-type ligands, perhaps because a specific cosignal is absent or because they do not express the right combination of receptors. It is interesting to note that in several other developmental contexts in Drosophila, it appears that at least two BMP ligands provide regulatory inputs into a common process (McCabe, 2003).
Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, it is shown that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, presynaptic BMP receptor Wishful thinking is required for the retrograde signal to function. These results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).
It has been demonstrated that a BMP type II receptor, wishful thinking (wit), is required for both growth and function of the NMJ in Drosophila. To further explore the mechanism by which motor neurons respond to the retrograde signal, whether the retrograde enhancement of quantal content can occur in wit mutants was examined. The results indicate that the retrograde signal cannot increase neurotransmitter release in the absence of Wit. Activation of the retrograde signal by either postsynaptic expression of GluRIIAM/R or postsynaptic inhibition of CaMKII did not lead to any increase in quantal content. These results indicate a requirement for wit presynaptically for the functioning of the retrograde mechanism that controls the homeostasis of neurotransmitter release at the NMJ of Drosophila and that postsynaptic inhibition of CaMKII requires the function of presynaptic BMP signaling to enhance quantal release (Haghighi, 2003).
Glass bottom boat (Gbb), a BMP ortholog, functions as a retrograde ligand for Wit at the Drosophila NMJ. Mutations in gbb lead to NMJ defects similar to those observed in wit mutants, and postsynaptic transgenic expression of Gbb can rescue many of these defects. In light of these findings, it is possible that there is a link between postsynaptic activity of CaMKII and the level and function of Gbb at the NMJ of Drosophila (Haghighi, 2003).
Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further an understanding of this aspect of the function of dystrophin, Drosophila mutants that lack the large dystrophin isoforms were generated and their role in synapse function at the neuromuscular junction was analyzed. In expression and rescue studies, lack of the large dystrophin isoforms in the postsynaptic muscle cell were shown to lead to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. These results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis (van der Plas, 2006).
This study finds that the presynaptically localized type II BMP receptor wit is required for the increased QC observed in the dystrophin mutant, as also shown for increases in QC induced by the postsynaptic inhibition of CaMKII or glutamate receptor function (Haghighi, 2003). Dystrophin and CaMKII are unlikely, however, to signal through the PMad-dependent BMP signaling pathway, because the expression levels and domains of PMad are unchanged when Dystrophin or CaMKII levels are either decreased or increased. The results address the question as to whether the retrograde BMP signal directly participates in homeostatic signaling ('instructive') or is required for the overall development of the synapse ('permissive'). The findings indicate that it is likely permissive, at least for the homeostatic mechanisms induced by perturbation of Dystrophin or CaMKII levels. BMP signaling may simply be required for the development of the presynaptic apparatus to a point at which it can respond to muscle-derived cues (van der Plas, 2006).
The BMP pathway plays an important role in retrograde signaling at the larval NMJ and in the Drosophila CNS. To evaluate the role of BMP signaling in the increase in neurotransmitter release induced by the absence of dystrophin, electrophysiological recordings were performed at NMJs lacking both dystrophin and wit. The transgenic RNA interference approach was used to reduce postsynaptic dystrophin levels (van der Plas, 2006).
Homozygous wit mutant NMJs postsynaptically expressing dsRNA directed against the Dystrophin large isoforms (UAS-RNAi-dysNH2/+; G14-Gal4/+; wit) displayed mEJP amplitude, EJP amplitude, and QC values similar to the wit mutant alone. The homozygous wit NMJ has very low EJP amplitudes but maintains wild-type level mEJP amplitudes. Thus, the absence of dystrophin failed to elicit an increase in QC in the wit background. Wit function and, by extension, BMP signaling is therefore required at the NMJ for the increase in neurotransmitter release elicited by the absence of dystrophin (van der Plas, 2006).
To evaluate whether the absence of postsynaptic dystrophin affects a known BMP target, immunofluorescence analysis was performed of mutant and wild-type embryonic ventral nerve cords, using an anti-phospho-Mad (PMad) antibody that recognizes the activated form of the Mad downstream effector of wit signaling. No differences were observed in PMad expression levels or domains between the dystrophin mutants or individuals postsynaptically overexpressing DLP2 and controls, whereas homozygous wit mutants showed significantly decreased levels of the protein (van der Plas, 2006).
The dystrophin mutants show similar electrophysiological and morphological phenotypes as larvae postsynaptically expressing CaMKII inhibitors. Both display increased QC and an increase in the ratio of active zones with T-bars versus the total number of active zones, without additional significant changes in synaptic morphology. Furthermore, the increase in QC when CaMKII is reduced postsynaptically is dependent on wit function. Therefore, embryonic ventral nerve cords that have reduced or elevated levels of CaMKII, UAS-Ala, and UAS-T287D, respectively, were examined for PMad staining. No changes in the expression of PMad was observed. These results suggest that, although wit is required for increased QC at NMJs postsynaptically deficient for either Dystrophin or CaMKII, alteration of either Dystrophin or CaMKII levels does not result in changes in embryonic PMad expression. Thus, the interaction of dystrophin or CaMKII and wit in NMJ homeostasis is unlikely to involve regulation of PMad expression (van der Plas, 2006).
Inhibition of postsynaptic glutamate receptors at the Drosophila NMJ initiates a compensatory increase in presynaptic release termed synaptic homeostasis. BMP signaling is necessary for normal synaptic growth and stability. It remains unknown whether BMPs have a specific role during synaptic homeostasis and, if so, whether BMP signaling functions as an instructive retrograde signal that directly modulates presynaptic transmitter release. This study demonstrates that the BMP receptor (Wit) and ligand (Gbb) are necessary for the rapid induction of synaptic homeostasis. Evidence is provided that both Wit and Gbb have functions during synaptic homeostasis that are separable from NMJ growth. However, further genetic experiments demonstrate that Gbb does not function as an instructive retrograde signal during synaptic homeostasis. Rather, the data indicate that Wit and Gbb function via the downstream transcription factor Mad and that Mad-mediated signaling is continuously required during development to confer competence of motoneurons to express synaptic homeostasis (Goold, 2007).
These data advance understanding of BMP signaling at the Drosophila NMJ in several important ways. First, it was demonstrated that BMP signaling is essential for the rapid, protein-synthesis-independent, induction of synaptic homeostasis identified at this NMJ. Because expression of UAS-wit in motoneurons restores synaptic homeostasis in the wit mutant and because suppression of Mad-mediated signaling in neurons blocks synaptic homeostasis, it is concluded that BMP signaling acts upon the motoneuron to enable the rapid induction of synaptic homeostasis. Next, it was shown that the requirement for BMP signaling during synaptic homeostasis is separable from BMP-dependent support of synaptic growth and baseline neurotransmission. Finally, the temporal and spatial requirements for BMP signaling was dissected. The data support the conclusion that Mad-mediated signaling is required constitutively, downstream of the Wit receptor, in order to maintain the competence of motoneurons to express homeostatic plasticity. Further, the data argue that Gbb is not the retrograde signal that directly acts upon the presynaptic motoneuron terminal to homeostatically modulate presynaptic release (Goold, 2007).
It has been hypothesized that Gbb could function as a homeostatic retrograde signal at the Drosophila NMJ. According to this model, Gbb would be released in proportion to the perturbation of postsynaptic muscle excitation in a glutamate receptor mutant and, thereby, instruct the degree of homeostatic compensation expressed by the presynaptic motoneuron terminal. In favor of this model, homeostatic compensation observed in a glutamate receptor mutant is blocked by the wit mutation. This study present two lines of evidence that are consistent with the necessity of BMP signaling for homeostatic compensation. First, it was confirmed that the rapid induction of homeostatic compensation following application of a use-dependent glutamate receptor antagonist, Philanthotoxin (PhTx) is blocked by null mutations in both wit and gbb. Furthermore, it was shown that muscle-specific rescue of the gbb null mutation is sufficient to restore the rapid induction of homeostatic compensation (Goold, 2007).
Despite these compelling genetic data, several experiments now argue against the possibility that Gbb functions as an instructive, retrograde signal that directly modulates presynaptic release during synaptic homeostasis. First, it was found that although muscle-specific rescue of the gbb null mutation is sufficient to restore synaptic homeostasis, so is neuron-specific rescue of the gbb null mutation. Thus, homeostatic compensation can occur even in the absence of muscle-derived Gbb. These data argue against a model in which Gbb functions as the instructive retrograde signal that directly modulates presynaptic release during synaptic homeostasis (Goold, 2007).
Next, it was demonstrated that homeostatic signaling is blocked by expression of DN-Glued in neurons, which disrupts retrograde axonal transport. In this experiment, Gbb signaling at the NMJ should, in theory, persist. Furthermore, it was established that an intact motor axon is not required for the rapid induction of synaptic homeostasis. Thus, it can be concluded that trans-synaptic Gbb signaling from muscle to nerve is not sufficient for the rapid induction of synaptic homeostasis (Goold, 2007).
Given that Wit and Gbb are necessary for synaptic homeostasis, how do they participate in the process if Gbb is not the instructive retrograde signal? This study demonstrates that Mad is necessary for synaptic homeostasis, and evidence is providied that Mad-mediated signaling is required in the motoneuron. In addition, neuronal expression of UAS-Gbb restores homeostatic compensation in the presence of the DN-Glued transgene. These results suggest that the reason DN-Glued disrupts synaptic homeostasis is because it interferes with the retrograde axonal transport of P-Mad downstream of the Wit receptor. This is consistent with the prior demonstration that neuronal expression of Gbb can restore nuclear P-Mad in the presence of UAS-DN-Glued. Because the induction of synaptic homeostasis does not require the motoneuron soma, it is concluded that Gbb does not function as an acute, retrograde signal. Rather, Gbb may be a muscle-derived signal that acts developmentally to confer the competence of motoneurons to express synaptic homeostasis. Thus, the identity of the homeostatic retrograde signal at the NMJ remains unknown. It remains possible that other TGF-β superfamily signaling molecules could function at the NMJ in this capacity, including myoglianin and maverick, though it has been shown that synaptic homeostasis is intact in the baboon receptor mutant (Goold, 2007).
There are several possible ways in which BMP signaling could confer competence for motoneurons to express homeostatic plasticity. One possibility is that the BMPs control a transcriptional program that is necessary for synaptic homeostasis. For example, BMPs are potent regulators of cell fate during embryonic development. Perhaps the ability of motoneurons to express synaptic homeostasis is related to the maintenance of their cellular or electrical identity. An alternate possibility is that BMPs control the expression of essential presynaptic proteins that are required for synaptic homeostasis. For example, it has been shown in other systems that target-dependent TGF-β signaling can modulate neuronal ion channel expression. It has been demonstrated that CaV2.1 calcium channels are required for synaptic homeostasis at the Drosophila NMJ. However, it is considered unlikely that BMPs control synaptic homeostasis through the regulation of CaV2.1 channel expression because there is not a strong correlation between altered baseline synaptic transmission and the expression of synaptic homeostasis. Furthermore, overexpression of a GFP-tagged CaV2.1 calcium channel (cacophony-GFP) is unable to restore synaptic homeostasis when coexpressed with UAS-dad. Finally, BMP signaling could influence the expression of synaptic homeostasis by targeting the rate of spontaneous miniature release. Spontaneous release events that persist in the absence of evoked neurotransmission are sufficient to induce homeostatic compensation at the Drosophila NMJ. However, no strong correlation is found between baseline mEPSP frequency and whether or not a mutant NMJ is able to express synaptic homeostasis. Although the wit mutants show a severe decrease in mEPSP rate compared to wild-type, the expression of UAS-dad or UAS-DN-Glued both block synaptic homeostasis without severely impairing baseline mEPSP rate. Ultimately, continued forward genetic investigation of homeostatic signaling may be required to identify the BMP-dependent mechanisms that control the expression of synaptic homeostasis (Goold, 2007).
BMP signaling is required for NMJ growth, baseline neurotransmission, and NMJ stability in addition to being required for synaptic homeostasis. It is a challenge, therefore, to determine whether BMP signaling has a specific function during synaptic homeostasis versus a more general role during synapse development. This study presents several lines of evidence that BMP signaling may have a separable function during synaptic growth versus synaptic homeostasis. First, it was demonstrated that synaptic homeostasis can occur at BMP mutant synapses that show severely impaired synaptic growth. For example, the gbb hypomorphic mutant has a decrease in bouton number that is just as severe as the gbb null mutant, but the gbb hypomorphic mutant shows normal homeostatic compensation. As another example, animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in bouton number but normal homeostatic compensation. Thus, it is concluded that normal BMP-dependent synaptic growth is not required for the expression of synaptic homeostasis (Goold, 2007).
It was also possible to dissociate BMP-dependent baseline transmission from both synaptic growth and synaptic homeostasis. (1) Muscle-specific rescue of the gbb null mutation significantly restores synaptic growth and rescues synaptic homeostasis, but baseline transmission remains at levels observed in the null mutant. (2) Motoneuron-specific rescue of the wit mutation (OK371-GAL4) similarly rescues bouton number and synaptic homeostasis, although baseline transmission remains severely impaired. (3) Animals in which UAS-gbb and UAS-DN-Glued are coexpressed have a severe decrease in baseline transmission but normal homeostatic compensation. (4) Results were obtained that show the converse effect. When UAS-dad is expressed for 1.5 days at the end of larval development, both synaptic homeostasis and baseline transmission are significantly impaired, but synaptic bouton numbers remain wild-type. From these data it is concluded that impaired synaptic homeostasis is not a secondary consequence of BMP-dependent functional NMJ development. It also appears that there may be distinct effects of BMP signaling on the anatomical versus functional development of the NMJ. One possibility, consistent with BMPs being a classical morphogen, is that different levels of the ligand could initiate specific transcriptional programs with distinct effects on bouton number, baseline transmission, and homeostatic plasticity. It is also possible that the site of action of BMP signaling will play an important role in specifying signaling outcome (Goold, 2007).
It has been speculated that synaptic homeostasis might function, over the course of development, to ensure that the muscle cell is normally depolarized by the NMJ. How can one explain the observation that csp and syx/+ mutations have decreased baseline neurotransmitter release but normal acute synaptic homeostasis in response to PhTx application, or other genotypes explored in this study that show impaired baseline transmission and normal acute synaptic homeostasis? It has been demonstrated that the acute induction of synaptic homeostasis is independent of evoked neurotransmission. Thus, synaptic homeostasis may not function to modulate the absolute amplitude of evoked neurotransmitter release. Rather, synaptic homeostasis might be a rapid system to offset acute perturbations of postsynaptic receptor function. In this case, developmental programs that specify NMJ anatomy and active zone addition would achieve the reproducible development of the NMJ. Alternatively, the mechanisms of acute homeostatic compensation following PhTx application may be separable, either temporally or molecularly, from the other potential mechanisms that monitor and homeostatically control evoked EPSP amplitudes (Goold, 2007).
The data also suggest a possible link between the expression of homeostatic plasticity and the mechanisms of neuromuscular degenerative disease. Genetic mutations that impair retrograde axonal transport have been shown to cause familial amyotrophic lateral sclerosis. It has also been shown that, in Drosophila and mice, mutations that disrupt dynein-dynactin complex function lead to neuromuscular synapse degeneration. It is hypothesized that impaired retrograde axonal transport deprives motoneurons of muscle-derived trophic support leading to motoneuron degeneration. This study has demonstrated that impaired retrograde axonal transport blocks the expression of homeostatic plasticity at the NMJ. This deficit can be restored by expression of BMPs in the central nervous system, bypassing retrograde axonal transport as the source of BMPs to the motoneuron cell body. It is tempting to speculate that impaired synaptic homeostasis at the NMJ may play a role in the progression of motoneuron disease associated with impaired retrograde axonal transport (Goold, 2007).
Finally, the data could have relevance to the sustained expression of homeostatic plasticity in regions of the adult nervous system. BMPs and downstream signaling proteins such as the Smads continue to be expressed in the adult nervous system. In particular, BMPs are secreted into the cerebral spinal fluid at concentrations that are relevant for neuronal signaling. It is, therefore, interesting to speculate that circulating levels of BMPs might sustain the competence of neurons to express homeostatic plasticity without driving morphological plasticity in the adult nervous system (Goold, 2007).
Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance were examined in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development (Knox, 2007).
TSC1 and TSC2 encoded proteins form a complex that regulates a small GTP-binding protein, Ras homolog enriched in brain (Rheb), promoting its endogenous GTPase activity and thereby limiting Rheb signaling. Rheb in turn controls the activity of Target of Rapamycin (TOR), a serine-threonine kinase. The TSC-Rheb-TOR pathway is a critical determinant of growth during development, regulating a number of cellular functions including translation, mRNA turnover, protein stability, and actin organization (Inoki, 2005). It is responsive to growth factors, such as insulin and insulin-like growth factors (ILGFs), and also serves as a nutrient sensor, thus integrating numerous signals related to cell and tissue growth. TOR plays a pivotal role in this signaling pathway, receiving regulatory inputs from Rheb and affecting downstream targets via two distinct molecular complexes. Tor complex 1 (TORC1) includes Raptor and mLST8, and regulates translation via phosphorylation of S6 kinase (S6K) and 4E-binding protein (4EBP). Tor complex 2 (TORC2) includes Rictor in addition to Tor and mLST8; in both yeast and mammalian cells TORC2 influences the actin cytoskeleton. Tor complex 1, but not Tor complex 2, is inhibited by the anti-proliferative and immunosuppressant compound rapamycin, emphasizing that TORC1 and 2 are pharmacologically separable entities. The distinct molecular outputs of TORC1 and 2 have also suggested that TORC2 may be the primary regulator of cell polarity and morphology. It is not known which functions of TSC-Rheb-TOR in the nervous system are mediated by either or both of the two Tor kinase-containing complexes, and if pharmacological intervention in tuberous sclerosis complex patients should best be directed at TORC1, with agents such as rapamycin, or if TORC2-specific agents will also be important (Knox, 2007).
While TSC mutations produce hamartomatous growths in the brain, recent evidence has suggested that these benign tumors may not be solely responsible for the nervous system dysfunction that is a hallmark of tuberous sclerosis complex. Loss of TSC2 in hippocampal neurons produces changes in neuronal morphology and synaptic transmission (Tavazoie, 2005). Heterozygosity for TSC2 in the rat compromises several measures of hippocampal long term potentiation (von der Brelie, 2006). Loss of Pten, an important upstream regulator of Tsc-Rheb-Tor signaling, in a limited set of neurons also affects neuronal morphology and socialization behavior (Kwon, 2006). These findings collectively provide evidence that Tsc-Rheb-Tor signaling is critical for the morphological and functional development of the nervous system. It is not clear, however, if the entire Tsc-Rheb-Tor signaling network is critical for nervous system development, or if neural function is strictly a consequence of altered growth regulation. It is also not known if loss of signaling is as detrimental to neuronal development as inappropriately elevated signaling, such as occurs with loss of TSC function. This study has taken advantage of the genetic and molecular tools available in the fruit fly Drosophila to address these questions. The findings demonstrate that appropriate levels of Tsc-Rheb-Tor signaling are critical for both NMJ development and for axon guidance in the visual system. In both these contexts, effects are independent of growth, implicating TORC2 rather than TORC1 as the complex mediating Tsc-Rheb-Tor signaling influences in the nervous system (Knox, 2007).
Given the importance of Tsc-Rheb-Tor signaling in regulating cellular and tissue growth, it was important to determine if disruption of this pathway affects neural development via its effects on growth or through signaling components independent of those that govern cellular size and growth. To address this issue pharmacological and genetic methods were used to block the increased growth produced by pathway activation. The immunosuppressant rapamycin is a TORC1-specific inhibitor that prevents activation of S6k and blocks growth mediated by loss of Tsc1. Rapamycin treatment retarded growth in larvae with pan-neuronal expression of Rheb, but failed to reduce the synapse expansion characteristic of these animals. Similarly, while rapamycin effectively reduced the retinal overgrowth of Tsc1 mosaic animals, it failed to suppress the photoreceptor axon guidance defects seen in the visual system. Loss of S6k function also failed to ameliorate axon guidance defects in Tsc1 mosaic animals. This contrasts with effects of Tor partial loss-of-function mutations, which effectively rescued axon guidance defects of Tsc1 mutants. Collectively, these findings demonstrate that the role of Tsc-Rheb-Tor signaling in synapse assembly and axon guidance is largely independent of TORC1, S6k, and their effects on growth. Indeed, while animals bearing null alleles of S6k have some axon pathfinding defects, the effects are relatively modest compared to Tsc1 mosaics, indicating that S6k does not provide the critical outputs affecting axon guidance (Knox, 2007).
These findings parallel recent work in the mouse, where neuronal hypertrophy produced by loss of Pten in granule neurons of the cerebellum and dentate gyrus was not rescued by loss of S6k1 (Chalhoubm 2006). It is also of note that some but not all Tsc1/2-mediated changes in dendritic morphology of hippocampal neurons in organotypic cultures were suppressed by rapamycin treatment (Tavazoie, 2005). These findings suggest that inhibition of growth regulatory components in tuberous sclerosis patients, such as achieved with rapamycin and related agents, may not affect all processes that are deranged in the nervous system (Knox, 2007).
Recent studies of Pi3 kinase, Akt and InR in Drosophila have shown that activation of signaling upstream of Tsc1/2 also produces increases in synapse size, both at the NMJ as well as central synapses. Expression of these components in adult neurons demonstrated that Pi3 kinase-mediated synaptogenesis is age-independent, and therefore not a developmentally restricted phenomenon. In agreement with the current studies, the expanded NMJs produced by activation of Pi3 kinase are functional, with increased stimulus-induced EJPs. Overexpression of the Drosophila ortholog of the epidermal growth factor receptor (EgfR) in central neurons increases neuronal cell size, without an increase in synapse number. These results are consistent with those reported here where it was possible to directly suppress growth mediated by Tsc-Rheb-Tor pathway activation without altering effects on synapse formation or axon guidance (Knox, 2007).
Recent studies have also demonstrated a link between Tsc1/Tsc2 and highwire, a gene known to effect synapse size and functionality in Drosophila. The highwire ortholog Pam was shown to bind Tsc2 in pull-down assays, and it has been suggested that Pam may function as an E3 ubiquitin ligase to regulate the intracellular levels of the Tsc1/Tsc2 complex. This concept of Highwire as a negative regulator of Tsc levels is consistent with the current findings, since highwire mutants have been shown to possess enlarged NMJs similar to was was seen for Rheb overexpression. Despite this, the enlarged synapses of highwire mutants display compromised synaptic function which is contrary to what was found when overexpressing Rheb, so Highwire is likely to have multiple functions at the synapse besides simply the regulation of Tsc (Knox, 2007).
Tor has a number of molecular outputs that influence many cellular processes; notable among these are cellular growth and cellular morphology. TORC1, which contains Raptor and is sensitive to the anti-proliferative agent rapamycin, is a major contributor to the regulation of cellular growth, in large measure due to its effects on protein synthesis. TORC2, which includes Rictor, is implicated in the control of cell morphology mediated by regulation of the actin cytoskeleton (Wullschleger, 2006). Both pharmacological and genetic studies presented in this study argue in favor of Tor complex 2 providing an essential regulatory component of both synapse growth and axon guidance in Drosophila. The results support recent work showing that changes in dendritic morphology of hippocampal neurons produced by loss of Tsc1 required regulation of the actin-depolymerizing factor Cofilin (Tavazoie, 2005), implicating TORC2-mediated processes. There is a considerable body of work demonstrating that control of the actin cytoskeleton is critical for NMJ growth and function and TORC2 may provide an important component of that control. Regulation of actin is also essential for axon guidance in the visual system, and disruption of Tor-mediated control of actin may be the underlying molecular deficit in Tsc1 mosaics (Knox, 2007).
A number of studies have suggested that TOR activation produced by loss of TSC1/2 affects neuronal morphology and synaptic function. The current findings support these observations; elevated Rheb activity produces synaptic enlargement and enhanced physiological function at the Drosophila NMJ. However, it was not evident from earlier studies whether loss of signaling through Rheb and Tor is also important for neural development. This study has provided evidence that this is the case. Partial loss-of-function mutations in Rheb compromise NMJ growth and function, as well as photoreceptor axon targeting in the visual system. Overexpression of Tsc1 and Tsc2 in the motoneuron also limit synaptic growth, supporting the conclusion that depressed levels of Rheb activity compromise synapse development (Knox, 2007).
The capacity of Tsc-Rheb-Tor signaling to affect neuronal morphology and synapse function begs the question of whether these effects are dependent on signaling systems known to be critical for synapse development. At the Drosophila NMJ, BMP signaling is critical for normal growth and function. Mutations in wit, a gene encoding a type II BMP receptor, produce a small and poorly functioning NMJ. These deficits can be rescued by motoneuron expression of wit+, demonstrating that BMP signaling in the motoneuron is critical for synaptic expansion during larval growth. To determine if Rheb-mediated synaptic growth requires BMP signaling, elav-Gal4 and UAS-Rheb transgenes were placed into a wit mutant background. While overexpression of Rheb and the accompanying activation of the Tor pathway partially rescued the defect in synapse growth produced by loss of wit function, it was unable to restore a normal EJP response or rescue quantal content. These findings establish that Tsc-Rheb-Tor mediated effects on synapse morphology are partially dependent on BMP signaling, and are fully dependent on BMP activity for a physiologically competent synapse. These findings also establish that the functional deficits in wit mutants are not simply the result of reduced synapse size, since restoration of synapse size by expression of UAS-Rheb does not restore physiological function. Intersection of BMP, and Akt/PTEN/TOR signaling has been noted for other systems, and the current results indicate the relationship between these pathways is important for synapse growth and plasticity as well (Knox, 2007).
Previous analysis of gigas/Tsc2 mutants has demonstrated that loss of this gene in mechanoreceptors affects axon targeting, producing projections to novel areas in the CNS in addition to innervation of normal targets (Canal, 1998). This study has used genetic mosaics to evaluate the function of Tsc-Rheb-Tor signaling in photoreceptor axon guidance. Animals homozygous for Tsc1 in the retina showed grossly aberrant photoreceptor projections to both the lamina and medulla. R7 and R8 projections to the medulla in 40h pupae fail to terminate correctly and projected beyond normal targets to inappropriate regions within the brain. Somatic mosaics bearing retinal neurons mutant for Pten also show photoreceptor axon guidance defects, but to a notably lesser degree. Since both Tsc1 and Pten alleles used for this analysis were nulls and show comparable effects on cellular growth and differentiation, it follows that Pten is not as critical for axon guidance as Tsc1. The distinctions between axon guidance phenotypes of Pten and Tsc1 null mutants indicate that altered timing of differentiation is not critical for axon guidance and that control of this pathway at the level of Pten or Tsc1 is not functionally equivalent. These findings that rapamycin arrests retinal overgrowth produced by loss of Tsc1 but not Pten in the retina supports earlier work demonstrating that retinal overgrowth mediated by loss of Tsc1, but not Pten, can be suppressed by reductions in S6k activity. Those results were interpreted as demonstrating that Pten is largely a regulator of Akt activity, whereas Tsc1/2 serves as a tumor suppressor and inhibitor affecting principally S6k. The results support these relationships and emphasize that in the nervous system regulation of Tsc1/2 targets other than S6k are critical (Knox, 2007).
This study has used two different genetic methods for activating the Tsc-Rheb-Tor pathway in the visual system; generating retinal mosaics with a loss of function allele of Tsc1, and pan-neuronal expression of Rheb using elav-Gal4 and UAS-Rheb. The comparison of these methods revealed that overexpression of Rheb produced milder axon guidance phenotypes in the visual system than complete loss of Tsc1 function. Of interest is that the degree of activation achieved with elav-Gal4>UAS-Rheb, a level that did not produce lethality, did result in discernable axon targeting defects in the visual system. This suggests that axon guidance controlled by Tsc-Rheb-Tor is sensitive to incremental changes in signaling. The range of neurological and behavioral phenotypes associated with loss of one copy of TSC1 or TSC2 is consistent with this model, where other environmental or genetic factors may affect signaling levels, producing a range of deficits. These findings indicate that Drosophila can serve as a useful model for identifying how graded changes in signaling can produce a spectrum of defects in neural development (Knox, 2007).
The BMP pathway is essential for scaling of the presynaptic motoneuron arbor to the postsynaptic muscle cell at the Drosophila neuromuscular junction (NMJ). Genetic analyses indicate that the muscle is the BMP-sending cell and the motoneuron is the BMP-receiving cell. Nevertheless, it is unclear how this directionality is established as Glass bottom boat (Gbb), the known BMP ligand, is active in motoneurons. This study demonstrates that crimpy (cmpy) limits neuronal Gbb activity to permit appropriate regulation of NMJ growth. cmpy was identified in a screen for motoneuron-expressed genes and encodes a single-pass transmembrane protein with sequence homology to vertebrate Cysteine-rich transmembrane BMP regulator 1 (Crim1). Cysteine-rich repeat (CRR)-containing single-pass transmembrane protein are present in a large number of BMP-interacting proteins in vertebrates and invertebrates. This structurally related family includes extracellular antagonists, such as Drosophila Short gastrulation (Sog) and vertebrate Chordin (Chrd), which are believed to interfere with receptor-ligand interactions. It also includes proteins such as gremlin and sclerostin that can interact with BMPs intracellularly and are thought to interfere with BMP activity, at least in part, by altering ligand activation or secretion. A targeted deletion of the cmpy locus was generated; loss-of-function mutants exhibit excessive NMJ growth. In accordance with its expression profile, tissue-specific rescue experiments indicate that cmpy functions neuronally. The overgrowth in cmpy mutants depends on the activity of the BMP type II receptor Wishful thinking, arguing that Cmpy acts in the BMP pathway upstream of receptor activation and raising the possibility that it inhibits Gbb activity in motoneurons. Indeed, the cmpy mutant phenotype is strongly suppressed by RNAi-mediated knockdown of Gbb in motoneurons. Furthermore, Cmpy physically interacts with the Gbb precursor protein, arguing that Cmpy binds Gbb prior to the secretion of mature ligand. These studies demonstrate that Cmpy restrains Gbb activity in motoneurons. A model is presented whereby this inhibition permits the muscle-derived Gbb pool to predominate at the NMJ, thus establishing the retrograde directionality of the pro-growth BMP pathway (James, 2011).
Gbb has been proposed to cue presynaptic motoneurons to the size of their postsynaptic muscle partners. However, muscles have not been established as the primary source of Gbb at the NMJ. In fact, motoneuron-derived Gbb has a crucial retrograde activity at the motoneuron-interneuron synapse, demonstrating that motoneuronal Gbb is active. The present work demonstrates that motoneurons express Cmpy, a Gbb antagonist. It is proposed that Cmpy restrains motoneuronal activity of Gbb at the NMJ, thus establishing the muscle as the predominant source of the pro-growth BMP signal. Potential mechanisms for Cmpy function at the NMJ and the relationship of Cmpy with intracellular and extracellular BMP antagonists are discussed (James, 2011).
Interest in CG13253/Crimpy was sparked by its restricted expression in the VNC and was reinforced by the presence of a predicted transmembrane domain and CRR. The presence of these two sequence elements renders Cmpy similar to vertebrate Crim1. In mice, Crim1 hypomorphs have been described and display pleiotropic defects in multiple organ systems (Pennisi, 2007). Notably, Crim1 is expressed in developing motoneuron and interneuron populations in the developing mouse and chick spinal cord, although LOF studies have not addressed a neuronal function. A Crim1 homolog has also been described in zebrafish, where it is linked to vascular and somitic development, and in C. elegans, where RNAi-mediated knockdown of crm-1 (cysteine-rich motor neuron protein 1) suggests a pro-BMP function in the control of body size (Fung, 2007). Cell culture studies provide evidence that Crim1 binds Bmp4/7 and antagonizes the production and processing of the preprotein in the Golgi (Wilkinson, 2003). Interestingly, Crim1 interacts with Bmp4/7 at the cell surface and inhibits BMP secretion into the medium (Wilkinson, 2003), raising the possibility that Crim1 antagonizes BMP signaling by multiple cellular mechanisms (James, 2011).
CRR-containing proteins are established modulators of BMP signaling in vertebrates and invertebrates. In Drosophila, posterior wing crossvein specification requires local activation of the BMP pathway, and loss of BMP signaling yields a crossveinless phenotype. BMP ligands are produced in neighboring longitudinal wing veins and are transported to the posterior crossvein. Ligand activity is differentially regulated by the secreted CRR-containing proteins Sog and Crossveinless 2 (Cv-2). Sog and Cv-2 both have pro- and anti-BMP activity, although their mode and range of action differ. Sog is proposed to act at long range, and its anti-BMP activity is thought to derive from sequestering BMPs from their receptors, whereas its pro-BMP activity is likely to arise from transporting BMP ligands through tissues. By contrast, Cv-2 is proposed to act at short range and binds heparan sulfate proteoglycans and the type I receptor Tkv (James, 2011).
The biphasic activities of Sog and Cv-2 serve to emphasize the complex modes of extracellular regulation of BMPs by CRR-containing proteins, as well as to draw attention to possible differences between BMP regulation in the wing and Cmpy-dependent BMP regulation at the NMJ. Although overexpression of Cmpy suppresses Gbb overexpression phenotypes in the wing, cmpy LOF mutants do not display wing vein phenotypes. Cmpy does not function during early embryogenesis, when the BMP homolog Decapentaplegic acts as a classical morphogen in dorsoventral patterning. In both the early embryo and the wing, BMP activity is shaped over many cell diameters by extracellular CRR-containing proteins. Sog and Cv-2 play essential extracellular roles in establishing the magnitude and directionality of BMP signaling. By contrast, Gbb is proposed to act locally at the NMJ to couple pre- and postsynaptic growth (James, 2011).
The close apposition of the cells that send and receive BMP at the NMJ might relieve a requirement for long-range extracellular regulation of the ligand. Instead, it is proposed that a primary challenge at the NMJ is to establish the cellular source of the BMP signal, as Gbb is present both in motoneurons and muscle. In this case, cell-autonomous regulation of the ligand could provide a mechanism for the motoneuron to discriminate between motoneuron- and muscle-derived pools. Consistent with this model, evidence is presented that Cmpy binds Gbb prior to processing and inhibits its growth-promoting activity in motoneurons. In this manner, the Cmpy-Gbb interaction might provide motoneurons with an effective mechanism for distinguishing autocrine and paracrine Gbb signals within the NMJ microenvironment (James, 2011).
CRR-containing BMP antagonists were initially identified from their extracellular roles in the establishment of BMP morphogenetic gradients. It will be interesting to determine whether additional CRR-containing proteins function intracellularly as more short-range BMP-dependent signaling interactions are thoroughly described. Consistent with this idea, several mammalian CRR-containing proteins bind precursor forms of BMP and inhibit BMP activity or secretion in a cell-autonomous manner. Gremlin is a BMP antagonist that is expressed in differentiated cells, including neurons. When co-expressed with Bmp4, gremlin binds to the precursor form of Bmp4 and inhibits secretion. sclerostin, another BMP antagonist, inhibits Bmp7 secretion when the proteins are co-expressed in osteocytes. These studies argue that intracellular modulation of ligand production contributes to BMP signaling directionality in vertebrates (James, 2011).
The work presented in this study suggests that Cmpy antagonizes Gbb activity in motoneurons prior to ligand secretion. To further delineate the Cmpy-Gbb relationship, it will be important to map their localization patterns in motoneurons using compartment-specific markers. Although attempts to generate anti-Cmpy antibodies have been unsuccessful, generation of transgenic flies carrying epitope-tagged Cmpy might enable an analysis of Cmpy subcellular localization. Cmpy-mediated inhibition of Gbb at the NMJ might rely upon restricted localization of Cmpy to this subcellular locale; however, the possibility that Cmpy regulates Gbb activity at the central synapse remains open. Investigation of the localization pattern of Cmpy in motoneurons will begin to address the issue of Cmpy function at these distinct synapses (James, 2011).
An analysis of Gbb distribution, trafficking and secretion in motoneurons in cmpy mutants will indicate the stage of Gbb processing at which Cmpy is likely to act. Studies on mammalian sclerostin provide precedent for an intracellular mechanism for BMP inhibition, as sclerostin sequesters Bmp7 preprotein, leading to its intracellular retention and proteasomal degradation. Interestingly, Cmpy contains only a single, low-threshold CRR. These motifs modulate interactions with mature secreted ligand, suggesting that sequences outside of the CRR mediate interactions with the precursor form of Gbb. Indeed, interaction of Cmpy with Gbb is dependent on C-terminal sequences, including an arginine/lysine-rich domain at the extreme C-terminus. Likewise, the intracellular interaction of gremlin with the precursor form of Bmp4 is not modulated by its cysteine-rich region, but rather by an arginine/lysine-rich domain. The sequence similarities between the BMP interaction domains in gremlin and Crimpy raise the possibility that these proteins antagonize BMP activity by a conserved mechanism (James, 2011).
This study has focused on Cmpy regulation of Gbb in the anatomical development of the NMJ. In addition, Gbb regulates baseline neurotransmission and synaptic homeostasis at the NMJ. Motoneurons precisely compensate for impaired postsynaptic neurotransmitter receptor sensitivity by increasing presynaptic neurotransmitter release. This homeostatic response requires Gbb, which is not itself the acute retrograde homeostatic signal but rather establishes the competence of motoneurons to receive the homeostatic signal. A number of genetic manipulations indicate that the roles of Gbb in regulating synaptic homeostasis, basal neurotransmission and NMJ morphology are separable. Perhaps surprisingly, neuron-specific Gbb rescues both synaptic homeostasis and baseline neurotransmitter release in gbb null animals. By contrast, whereas muscle-derived Gbb rescues synaptic homeostasis in gbb null animals, it does not significantly rescue baseline synaptic function, arguing that neuronal- and muscle-derived pools of Gbb serve distinct functions. Although the data indicate that Cmpy antagonizes autocrine Gbb signaling in motoneurons to restrain morphological expansion at the NMJ, it is likely that motoneuronal Gbb has an independent role in regulating functional development of the NMJ. If so, the Cmpy-Gbb complex might be active and could elicit a signaling outcome distinct from that of the muscle-derived pool of Gbb. Physiological analyses of cmpy mutants, as well as an investigation of Gbb trafficking and secretion at the NMJ in cmpy mutants, should provide crucial insight into this important question (James, 2011).
More broadly, this study is of relevance to the regulation of signal release in neurons. By definition, neurotransmitter is released from the presynaptic compartment and received by neurotransmitter receptors on the postsynaptic side. However, signaling pathway activity is not circumscribed in this way and may occur at short or long range at multiple subcellular positions. Hence, neurons are likely to possess fine-regulatory mechanisms controlling the release of, and response to, extracellular cues. The present work provides insight into the regulation of signaling molecules in neurons and suggests that the mechanisms that control signaling specificity in the developing nervous system are only beginning to be uncovered (James, 2011).
Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).
It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).
The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).
This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).
In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).
Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).
In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).
During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).
ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).
The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).
Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the fragile X mental retardation 1 (FMR1) gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. Previous studies found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and Drosophila models of FXS and that these molecular and cellular markers were correlated in patients with FXS. This study reports that larval locomotion is augmented in a Drosophila FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. This study demonstrates that (1) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (2) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics (Kashima, 2017).
Behavioral manifestations in the Drosophila FXS model have been reported and include abnormal crawling and locomotion of third-instar larvae. This study developed quantitative behavioral assays that showed that reduction of Wit gene dosage in dFMR1 mutant larvae reverts the locomotion phenotype and that oral administration of LIMK antagonists and a protein synthesis inhibitor restores normal crawling velocity and reduces NMJ bouton numbers. It was also confirmed that administration of a LIMK antagonist in the mouse FXS model rescues the rodent behavioral abnormalities. Thus, this study demonstrates that (1) the locomotion phenotype in dFMR1 mutant larvae serves as a readout of NMJ bouton phenotype; (2) the larval crawling assay system that was developed can be used for the genetic or chemical screening of therapeutic molecules for FXS as well as other synapse formation abnormalities; and (3) targeting the LIMK1 pathway, which is conserved from Drosophila to human, is a potential therapeutic strategy for FXS (Kashima, 2017).
In Drosophila, glass bottom boat (Gbb), which is produced by the postsynaptic muscle, binds to the presynaptic receptor Wit and plays a critical role in modulating neuromuscular synaptic growth, stability, and function. Upon Gbb binding, Wit forms a heteromeric receptor complex with Thickveins (Tkv) and Saxophone (Sax), which then phosphorylate Mothers against decapentaplegic (MAD), a Drosophila homolog of Smad1/5/8. It has been reported that loss of Spartin, a Drosophila homolog of SPG20 that promotes endocytotic degradation of Wit and represses the BMP-Wit signaling pathway, results in an increment of neuromuscular synapses (Nahm, 2013). The result with the dFMR1Δ113/+ mutant is consistent with the Spartin study and confirms the effect of increased Gbb-Wit signal on abnormal synapse development. Loss-of-expression mutants of Spartin develop age-dependent and progressive neuronal defects resembling hereditary spastic paraplegia (HSP). Because frameshift mutations in the SPG20 gene cause a form of HSP known as Troyer syndrome (Online Mendelian Inheritance in Man no. 275900), these results underscore the significance of a presynaptic BMP signal finely tuned by multiple regulatory molecules, including SPG20 and FMRP, for proper motor neuron development and function. Beyond the domain of the NMJ, multiple studies reinforce the notion that the correct intensity and spatiotemporal dynamics of the BMP signaling pathway are critical for axon regeneration upon neuronal and glial injury responses after CNS injury. Furthermore, BMP signaling specifies large and fast-transmitting synapses in the auditory system in a process that largely shares homologies with retrograde BMP signaling in Drosophila neuromuscular synapses. In line with these findings, the current results propose an essential role for the FMRP-BMPR2 axis in the development of the neuropathology of patients with FXS (Kashima, 2017).
The major obstacle against the development of drugs for neurodevelopmental and neurodegenerative diseases is the lack of proper animal models that recapitulate the range of intellectual disability and/or cognitive dysfunction found in human patients. The existing, inadequate models also lack quantitative and reproducible assays to examine cognitive phenotypes. The mouse model of FXS exhibits cognitive and behavioral phenotypes that are both consistent and inconsistent with the symptoms of patients with FXS. For example, a frequent FXS trait is a high-anxiety behavior, whereas the FMR1-KO mice exhibit lower anxiety-like behaviors in the 'light-dark compartment' test. Furthermore, the result of the OFT is confusing because the FMR1-KO mice tend to spend a longer period in the center. The number of crossings and their velocity are both augmented compared to control mice because of their hyperactivity, but this behavior is interpreted as lower anxiety-like. The results of the 'elevated plus maze (EPM)' test, which is frequently used to investigate anxiety-like behaviors in FXS mice, exhibit both a decrease and an increase in anxiety. Furthermore, the tests show great variability and, sometimes, opposing outcomes in behavior depending on the genetic background of the mice, for example, FVB versus C57BL/6J. Considering the variability and lack of reproducibility of behavioral test results in FMR1-KO mice, as well as the concerns of animal welfare and the cost of husbandry, there is a strong need for an animal model and phenotypic readout to screen for FXS drugs (Kashima, 2017).
There are multiple advantages of the Drosophila FXS model over the rodent models. Flies are invertebrates, which are inexpensive and easily cared for. They have a shorter life span and produce numerous externally laid embryos than rodent models. Their genome is small, minimally redundant, and easy to genetically manipulate in a tissue-specific manner. It is easy to orally administer drugs to larvae by adding compounds to the Drosophila medium Formula 4-24 (Carolina Biological Supply Company). Previously, small molecules had to be delivered through conventional fly food that requires boiling followed by the addition of propionic acid, which disables the effect of heat- or acid-sensitive molecules. The use of Formula 4-24, which can be dissolved in water at room temperature and does not require exposure to high temperature nor addition of acid, expands the range of molecules that can be delivered to larvae without loss of activity (Kashima, 2017).
Drosophila has contributed extensively to the discovery and validation of drug targets, as well as to the mechanistic understanding of their genetic cause. In the context of FXS studies, it has been reported that dFMR1 adult mutant flies exhibit defects in learning/memory assays, such as Pavlovian olfactory association and courtship conditioning. These behavioral abnormalities can be restored by various compounds known to target different FMRP targets, including protein synthesis inhibitors, such as puromycin and cycloheximide, the metabotropic glutamate receptor 5 antagonist MPEP, γ-aminobutyric acid agonists, phosphodiesterase-4 inhibitor, and glycogen synthase kinase 3 inhibitor. The current study demonstrates that several dFMR1 mutant larvae exhibit an abnormally high number of NMJ synaptic boutons that correlate with their locomotion abnormality. Both are reversed by LIMK-i treatment, similarly to the effect of this drug in the FMR1-KO mouse. Thus, the crawling assay in dFMR1 mutant Drosophila larvae is proposed as a rapid, quantitative, and reproducible preclinical screening strategy for potential FXS therapies that is alternative to behavioral tests using dFMR1 adult mutant flies or vertebrate FXS models. To facilitate the transition to a high-throughput screen of FXS drugs, the current assay will benefit from an improvement in the number of larvae that can be simultaneously assessed and in the robustness of the phenotype of dFMR1Δ113/+ mutants (Kashima, 2017).
Larval locomotion abnormalities are described in Drosophila models of CNS diseases, such as Alzheimer's and Huntington's. It has been reported that a different strain of dFMR1 mutant Drosophila larvae (dFMR14) exhibits frequent turnings compared to wild-type larvae. This study observed that various dFMR1 mutants, including dFMR1Δ113/+, dFMR1Δ50/+, dFMR13/+, and dFMR1Δ113/3, as well as the dFMR1-RNAi line, crawled from the center to the periphery in a linear manner with an enhanced velocity compared to wild-type larvae. It is speculated that this discrepancy might be due to the different nature of the mutations. For example, dFMR1Δ113 harbors a deletion of the first three exons of the dFMR1 gene, including exon 3 that contains the translation initiation methionine. Consequently, the dFMR1Δ113 allele results in a loss of dFMRP. On the contrary, the dFMR14 allele has a replacement of amino acid 289 with a stop codon; hence, it expresses a partial dFMRP missing the C terminus. Furthermore, in the process of creating the dFMR14 mutant, a Gal4-binding site was inserted into the first intron between exons 1 and 2 of the dFMR1 gene to overexpress a truncated dFMRP upon coexpression of Gal4 transcription factor. These differences might explain the distinct larval crawling behavior of the dFMR1Δ113 and dFMR14 mutants. The homozygous dFMR1Δ50 and homozygous dFMR13 mutants are viable and develop into adulthood similarly to the homozygous dFMR14 mutant. The homozygous dFMR1Δ50 and homozygous dFMR13 mutants exhibited frequent turns in the locomotion assay similar to a previous study of the homozygous dFMR14 mutant. The velocity of dFMR13 and dFMR1Δ50 homozygous mutants was slower than wild-type, presumably because of the frequent changes of direction, unlike the transheterozygous dFMR1Δ113/3 mutant, which crawled in a linear fashion with an augmented velocity. It is speculated that the turning phenotype observed in the homozygous mutants is due to complete loss of dFMRP, affecting the CNS neurons. FMRP activity has already been shown to be important for CNS neuron development and function in Drosophila (Kashima, 2017).
Performing the locomotion assay with larvae instead of adults is beneficial as they present an accessible, anatomically simple, and well-described peripheral nervous system (for example, NMJ boutons), which allows the molecular and biochemical assessment of the mechanism underlying the locomotion dysfunction and the therapeutic effects of drugs. For chemical screens of known pathways or targets, the NMJ synapses of larvae that exhibit an altered crawling phenotype should be subjected to synaptic bouton phenotype analysis as well as biochemical investigation to rapidly validate the 'on-target' and eliminate the 'off-target' effects of the candidate molecules. A Drosophila larvae locomotion assay has been proposed as a way to screen drugs for neurodegenerative diseases, such as Alzheimer's disease. They subjected Drosophila larvae expressing the human three-repeat tau gene in motor neurons to crawling assays, such as a five-lane assay and a four-plate open-field assay, video-recorded the locomotion with an Ikegami digital video camera and a 5-mm digital video camera lens, and analyzed locomotion using EthoVision 3.0 software. In comparison, the advantage of the current strategy is that the assay does not require specialized equipment, but a common video recording device, such as an iPhone camera, and an algorithm that is accessible and free to the scientific community. Furthermore, the system can simultaneously track and assess the crawling activities of multiple larvae through the open-access algorithm LarvaTrack, which was developed to trace and measure larval crawling activity. Up to 15 larvae have been simultaneously assessed using a 15-cm agarose plate. The method can be easily adapted to a larger number of larvae by using a larger agarose plate to avoid larvae to cross paths during crawling. Thus, the semiautomated assay of locomotion behavior described in this study can allow the higher-throughput assay that is essential for the screen of candidate molecules. In conclusion, activation of the FMRP-BMPR2 axis plays a role in synaptic abnormalities in both mouse and Drosophila models of FXS. The larval crawling assay is an easy, fast, and well-suited medium-throughput screen for genetic or chemical modulators of locomotion dysfunction in the Drosophila FXS model, which can be further evaluated in cognitive and behavioral tests using mammalian FXS models (Kashima, 2017).
Aberle, H., et al. (2002). wishful thinking encodes a BMP type II receptor that regulates synaptic growth in Drosophila. Neuron 33: 545-558. 11856529
Allan, D., St. Pierre, S. E., Miguel-Aliaga, I. and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113: 73-86. 12679036
Arber, S., et al. (1998). Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393: 805-809. 9655397
Awasaki, T. and Ito, K. (2004). Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr Biol 14: 668-677. Pubmed: 15084281
Awasaki, T., Huang, Y., O'Connor, M. B. and Lee, T. (2011). Glia instruct developmental neuronal remodeling through TGF-beta signaling. Nat Neurosci 14: 821-823. Pubmed: 21685919
Baarends, W. M., et al. (1994). A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct. Development 120: 189-197. 8119126
Ball, R. W., Warren-Paquin, M., Tsurudome, K., Liao, E. H., Elazzouzi, F., Cavanagh, C., An, B. S., Wang, T. T., White, J. H., Haghighi, A. P. (2010). Retrograde BMP signaling controls synaptic growth at the NMJ by regulating trio expression in motor neurons. Neuron 66: 536-549. PubMed ID: 20510858
Banerjee, S., Venkatesan, A. and Bhat, M. A. (2016). Neurexin, neuroligin and wishful thinking coordinate synaptic cytoarchitecture and growth at neuromuscular junctions. Mol Cell Neurosci 78:9-24. PubMed ID: 27838296
Banerjee, S. and Riordan, M. (2018). Coordinated regulation of axonal microtubule organization and transport by Drosophila Neurexin and BMP pathway. Sci Rep 8(1): 17337. PubMed ID: 30478335
Barber, C. F., Jorquera, R. A., Melom, J. E., Littleton, J. T. (2009). Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. J Cell Biol 187: 295-310. PubMed ID: 19822673
Beppu, H., Kawabata, M., Hamamoto, T., Chytil, A., Minowa, O., Noda ,T. and Miyazono, K. (2000). BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev. Biol. 221: 249-258. 10772805
Beppu, H., Lei. H., Bloch, K. D. and Li, E. (2005). Generation of a floxed allele of the mouse BMP type II receptor gene. Genesis 41(3): 133-7. 15736264
Berke, B., Wittnam, J., McNeill, E., Van Vactor, D. L. and Keshishian, H. (2013). Retrograde BMP signaling at the synapse: a permissive signal for synapse maturation and activity-dependent plasticity. J Neurosci 33: 17937-17950. PubMed ID: 24198381
Boulanger, A., Farge, M., Ramanoudjame, C., Wharton, K. and Dura, J. M. (2012). Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-beta/BMP signaling and orphan nuclear receptors. PLoS One 7: e40255. PubMed Citation: 22792255
Cameron, J. S., Lhuillier, L., Subramony, P. and Dryer, S. E. (1998). Developmental regulation of neuronal K+ channels by target-derived TGFß in vivo and in vitro. Neuron 21: 1045-1053. 9856460
Cameron, J. S., Dryer, L. and Dryer, S. E. (1999). Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFß. Development 126: 4157-4164. 10457024
Canal, I., Acebes, A. and Ferrus, A. (1998). Single neuron mosaics of the Drosophila gigas mutant project beyond normal targets and modify behavior. J Neurosci 18: 999-1008. PubMed Citation: 9437021
Chalhoub, N., Kozma, S. C. and Baker, S. J. (2006). S6k1 is not required for Pten-deficient neuronal hypertrophy. Brain Res, 1100: 32-41. PubMed Citation: 16777079
Chamma, I., Letellier, M., Butler, C., Tessier, B., Lim, K. H., Gauthereau, I., Choquet, D., Sibarita, J. B., Park, S., Sainlos, M. and Thoumine, O. (2016). Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nat Commun 7: 10773. PubMed ID: 26979420
Charytoniuk, D. A., Traiffort, E., Pinard, E., Issertial, O., Seylaz, J. and Ruat, M. (2000). Distribution of bone morphogenetic protein and bone morphogenetic protein receptor transcripts in the rodent nervous system and up-regulation of bone morphogenetic protein receptor type II in hippocampal dentate gyrus in a rat model of global cerebral ischemia. Neuroscience 100: 33-43. 10996456
Chin, J., Angers, A., Cleary, L. J., Eskin, A. and Byrne, J. H. (1999). TGF-1 in Aplysia: role in long-term changes in the excitability of sensory neurons and distribution of TR-II-like immunoreactivity. Learn. Mem. 6: 317-330. 10492013
Colavita, A., Krishna, S., Zheng, H., Padgett, R. W. and Culotti, J. G. (1998). Pioneer axon guidance by UNC-129, a C. elegans TGF-ß. Science 281: 706-709. 9685266
Délot, E. C., et al. (2003). BMP signaling is required for septation of the outflow tract of the mammalian heart. Development 130: 209-220. 12441304
Deng, Z., et al. (2000). Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am. J. Hum. Genet. 67: 737-744. 10903931
Eaton, B. A., Fetter, R. D. and Davis, G. W. (2002). Dynactin is necessary for synapse stabilization. Neuron 34: 729-74. 12062020
Eaton, B. A. and Davis, G. W. (2005). LIM kinase1 controls synaptic stability downstream of the type II BMP receptor. Neuron 47: 695-708. 16129399
Endo, M., et al. (2003). Control of growth cone motility and morphology by LIM kinase and Slingshot via phosphorylation and dephosphorylation of cofilin. J. Neurosci. 23: 2527-2537. 12684437
Foletta, V. C. et al., (2003). Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J. Cell Biol. 162: 1089-1098. 12963706
Frisch, A. and Wright, C. V. (1998). XBMPRII, a novel Xenopus type II receptor mediating BMP signaling in embryonic tissues. Development 125: 431-442. 9425138
Goda, Y. and Davis, G. W. (2003). Mechanisms of synapse assembly and disassembly. Neuron 40: 243-264. 14556707
Goold, C. P. and Davis, G. W. (2007). The BMP Ligand Gbb gates the expression of synaptic homeostasis independent of synaptic growth control. Neuron 56: 109-123. Medline abstract: 17920019
Guo, X., Rueger, D. and Higgins, D. (1998). Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons. Neurosci. Lett. 245: 131-134. 9605473
Haghighi, A. P., et al. (2003). Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction. Neuron 39: 255-267. 12873383
Hassel, S., et al. (2006). Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor. J. Cell Physiol. 206(2): 457-67. 16155937
Inoki, K., Corradetti, M. N. and Guan, K. L. (2005). Dysregulation of the TSC-mTOR pathway in human disease. Nat. Genet. 37: 19-24. PubMed Citation: 15624019
James, R. E. and Broihier, H. T. (2011). Crimpy inhibits the BMP homolog Gbb in motoneurons to enable proper growth control at the Drosophila neuromuscular junction. Development 138(15): 3273-86. PubMed Citation: 21750037
Jiang, Y., McLennan, I. S., Koishi, K. and Hendry, I. A. (2000). Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury. Neuroscience 97: 735-742. 11107165
Jordan-Alvarez, S., Santana, E., Casas-Tinto, S., Acebes, A. and Ferrus, A. (2017). The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila. PLoS One (9): e0184238. PubMed ID: 28892511
Kamimura, K., Odajima, A., Ikegawa, Y., Maru, C. and Maeda, N. (2019). The HSPG glypican regulates experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway. Cell Rep 28(12): 3144-3156. PubMed ID: 31533037
Kashima, R., Redmond, P. L., Ghatpande, P., Roy, S., Kornberg, T. B., Hanke, T., Knapp, S., Lagna, G. and Hata, A. (2017). Hyperactive locomotion in a Drosophila model is a functional readout for the synaptic abnormalities underlying fragile X syndrome. Sci Signal 10(477). PubMed ID: 28465421
Kawabata, M., Chytil, A. and Moses, H. L. (1995). Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-ß receptor. J. Biol. Chem. 270: 5625-5630. 7890683
Kim, S., Wairkar, Y. P., Daniels, R. W. and DiAntonio, A. (2010), The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation. J. Cell Biol. 188(5): 717-34. PubMed Citation: 20194640
Kirsch, T., Nickel, J. and Sebald, W. (2000). BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II. EMBO J. 19: 3314-3324
Knox, S., et al. (2007). Mechanisms of TSC-mediated control of synapse assembly and axon guidance. PLoS One. 2(4): e375. PubMed Citation: 17440611
Korkut, C., Li, Y., Koles, K., Brewer, C., Ashley, J., Yoshihara, M., Budnik, V. (2013). Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 77: 1039-1046. PubMed ID: 23522040
Kwon, C. H., et al. (2006). Pten regulates neuronal arborization and social interaction in mice. Neuron 50: 377-388. PubMed Citation: 16675393
Lane, K. B., et al. (2000). Heterozygous germline mutations in BMPR2, encoding a TGF- receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat. Genet. 26: 81-84. PubMed Citation: 10973254
Lee-Hoeflich, S. T., et al. (2004). Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO J. 23: 4792-4801. 15538389
Lee-Hoeflich, S. T., Zhao, X., Mehra, A., Attisano, L. T. (2005). The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways FEBS Lett. 579: 4615-21. PubMed Citation: 16098524
Lein, P., Johnson, M., Guo, X., Rueger, D. and Higgins, D. (1995). Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons. Neuron 15: 597-605. 7546739
Liu, F., et al. (1995). Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Mol. Cell. Biol. 15(7): 3479-3486. PubMed Citation: 7791754
Liu, Q. R., et al. (1997). A developmental gene (Tolloid/BMP-1) is regulated in Aplysia neurons by treatments that induce long-term sensitization. J. Neurosci. 17: 755-764. 8987797
Lo, P. C. and Frasch, M. (1999). Sequence and expression of myoglianin, a novel Drosophila gene of the TGF-ß superfamily. Mech. Dev. 86: 171-175. 10446278
Lorentzon, M., Hoffer, B., Ebendal, T., Olson, L. and Tomac, A. (1996). Habrec1, a novel serine/threonine kinase TGF-ß type I-like receptor, has a specific cellular expression suggesting function in the developing organism and adult brain. Exp. Neurol. 142: 351-360. 8934566
Machado, R. D., et al. (2003). Functional interaction between BMPR-II and Tctex-1, a light chain of Dynein, is isoform-specific and disrupted by mutations underlying primary pulmonary hypertension. Hum. Mol. Genet. 12(24): 3277-86. 14583445
Marmion, R. A., Jevtic, M., Springhorn, A., Pyrowolakis, G. and Yakoby, N. (2013). The Drosophila BMPRII, wishful thinking, is required for eggshell patterning. Dev Biol 375: 45-53. PubMed ID: 23274688
Marqués, G., et al. (2002). The Drosophila BMP type II receptor Wishful thinking regulates neuromuscular synapse morphology and function. Neuron 33: 529-543. 11856528
Marqués, G., et al. (2003). Retrograde Gbb signaling through the Bmp type 2 receptor Wishful thinking regulates systemic FMRFa expression in Drosophila. Development 130: 5457-5470. 14507784
McCabe, B. D., et al. (2003). The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron 39: 241-254. 12873382
McLennan, I. S. and Koishi, K. (1994). Transforming growth factor-ß-2 (TGF-ß2) is associated with mature rat neuromuscular junctions. Neurosci. Lett., 177: 151-154. 7824170
McPherron, A. C., Lawler, A. M. and Lee, S. J. (1997). Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387: 83-90. 9139826
Meng, Y., et al. (2002). Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 35: 121-133. 12123613
Nahm, M., et al. (2010a), The Cdc42-selective GAP rich regulates postsynaptic development and retrograde BMP transsynaptic signaling. J Cell Biol 191: 661–675. PubMed ID: 21041451
Nahm M, et al. (2010b). dCIP4 (Drosophila Cdc42-interacting protein 4) restrains synaptic growth by inhibiting the secretion of the retrograde Glass bottom boat signal. J Neurosci 30: 8138–8150. PubMed ID: 20554864
Nahm, M., Lee, M.-J., Parkinson, W., Lee, M., Kim, H., Kim, Y.-J., Kim, S., Cho, Y. S., Min, B.-M., Bae, Y. C., Broadie,K., Lee, S. (2013). Spartin regulates synaptic growth and neuronal survival by inhibiting BMP-mediated microtubule stabilization. Neuron 77: 680–695. PubMed ID: 23439121
Ng, J. and Luo, L. (2004). Rho GTPases regulate axon growth through convergent and divergent signaling pathways. Neuron 44: 779-793. 15572110
Ng, J. (2008). TGF-β signals regulate axonal development through distinct Smad-independent mechanisms. Development 135(24): 4025-35. PubMed Citation: 19004854
Nishihara, A., Watabe, T., Imamura, T. and Miyazono, K. (2002). Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension. Mol. Biol. Cell 13(9): 3055-63. 12221115
Nishitoh, H., et al. (1996). Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. J. Biol. Chem. 271(35): 21345-21352. PubMed Citation: 8702914
Niwa, K., et al. (2002). Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108: 233-246. 11832213
Ohashi, K., et al. (2000). Drosophila homolog of LIM-kinase phosphorylates cofilin and induces actin cytoskeletal reorganization. Biochem. Biophys. Res. Commun. 276: 1178-1185. 11027607
Piccioli, Z. D., Littleton, J. T. (2014). Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin. J Neurosci 34: 4371-4381. PubMed ID: 24647957
Pielage, J., Fetter, R. D. and Davis, G. W. (2005). Presynaptic spectrin is essential for synapse stabilization. Curr. Biol. 15: 918-928. 15916948
Rodal, A. A., et al. (2011). A presynaptic endosomal trafficking pathway controls synaptic growth signaling. J. Cell Biol. 193(1): 201-17. PubMed Citation: 21464232
Rosenzweig, B. L., et al. (1995). Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc. Natl. Acad. Sci. 92(17): 7632-7636. PubMed Citation: 7644468
Smith, R. B., Machamer, J. B., Kim, N. C., Hays, T. S. and Marqués, G.. (2012). Relay of retrograde synaptogenic signals through axonal transport of BMP receptors. J. Cell Sci. [Epub ahead of print]. PubMed Citation: 22573823
Soderstrom, S., Bengtsson, H. and Ebendal, T. (1996). Expression of serine/threonine kinase receptors including the bone morphogenetic factor type II receptor in the developing and adult rat brain. Cell Tissue Res. 286: 269-279. 8854897
Sulkowski, M.J., Han, T.H., Ott, C., Wang, Q., Verheyen, E.M., Lippincott-Schwartz, J. and Serpe, M. (2016). A novel, noncanonical BMP pathway modulates synapse maturation at the Drosophila neuromuscular junction. PLoS Genet 12: e1005810. PubMed ID: 26815659
Sun, K., Westholm, J. O., Tsurudome, K., Hagen, J. W., Lu, Y., Kohwi, M., Betel, D., Gao, F. B., Haghighi, A. P., Doe, C. Q. and Lai, E. C. (2012). Neurophysiological defects and neuronal gene deregulation in Drosophila mir-124 mutants. PLoS Genet 8: e1002515. Pubmed: 22347817
Sweeney, S. T. and Davis, G. W. (2002). Unrestricted synaptic growth in spinster -- a late endosomal protein implicated in TGF-ß-mediated synaptic growth regulation. Neuron 36: 403-416. 12408844
Tanimoto, H., Itoh, S., ten Dijke, P. and Tabata, T. (2000). Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. Mol. Cell 5: 59-71. 10678169
Tavazoie, S. F., et al. (2005). Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat. Neurosci. 8: 1727-1734. PubMed Citation: 16286931
Thomson, J. R., et al. (2000). Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-ß family. J. Med. Genet. 37: 741-745. 11015450
Tolias, K. F., Duman, J. G., Um, K. (2011). Control of synapse development and plasticity by Rho GTPase regulatory proteins. Prog Neurobiol 94: 133-148. PubMed ID: 21530608
van der Plas, M. C., Pilgram, G. S., Plomp, J. J., de Jong, A., Fradkin, L. G. and Noordermeer, J. N. (2006). Dystrophin is required for appropriate retrograde control of neurotransmitter release at the Drosophila neuromuscular junction. J. Neurosci. 26(1): 333-44. 16399704
Veverytsa, L. and Allan, D. W. (2011). Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery. Development 138(15): 3147-57. PubMed Citation: 21750027
von der Brelie, C., et al. (2006). Impaired synaptic plasticity in a rat model of tuberous sclerosis. Eur. J. Neurosci. 23: 686-692. PubMed Citation: 16487150
Wang, X., Shaw, W. R., Tsang, H. T., Reid, E. and O'Kane, C. J. (2007). Drosophila spichthyin inhibits BMP signaling and regulates synaptic growth and axonal microtubules. Nat. Neurosci. 10(2): 177-85. PubMed Citation: 17220882
Withers, G. S., Higgins, D., Charette, M. and Banker, G. (2000) Bone morphogenetic protein-7 enhances dendritic growth and receptivity to innervation in cultured hippocampal neurons. Eur. J. Neurosci. 12: 106-116. 10651865
Wong, W. K., Morse, J. H. and Knowles, J. A. (2006). Evolutionary conservation and mutational spectrum of BMPR2 gene. Gene 368: 84-93. 16361068
Wullschleger, S., Loewith, R. and Hall, M. N. (2006). TOR signaling in growth and metabolism. Cell 124: 471-484. PubMed Citation: 16469695
Yang, E. J., et al. (2004). LIM kinase 1 activates cAMP-responsive element-binding protein during the neuronal differentiation of immortalized hippocampal progenitor cells. J. Biol. Chem. 279: 8903-8910. 14684741
Yoshihara, M., Adolfsen, B., Galle, K. T., Littleton, J. T. (2005). Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310: 858-863. PubMed ID: 16272123
Zhang, F., Endo, S., Cleary, L. J., Eskin, A. and Byrne, J. H. (1997). Role of transforming growth factor-ß in long-term synaptic facilitation in Aplysia. Science 275: 1318-1320. 9036859
Zheng, X., et al. (2003). TGF-ß signaling activates steroid hormone receptor expression during neuronal remodeling in the Drosophila brain. Cell 112: 303-315. 12581521
Zhicheng, J., et al. (2004). Bone morphogenetic protein receptor-II mutation Arg491Trp causes malignant phenotype of familial primary pulmonary hypertension. Biochem. Biophys. Res. Commun. 315(4): 1033-8. 14985116
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