InteractiveFly: GeneBrief

Activin-β: Biological Overview | References


Gene name - Activin-β

Synonyms - dActivin

Cytological map position - 102D4-102D4

Function - ligand

Keywords - activin signaling, retrograde signaling, brain, axon tiling

Symbol - Act-β

FlyBase ID: FBgn0024913

Genetic map position - 4: 1,097,946..1,105,418 [-]

Classification - Transforming growth factor beta like domain

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Song, W., Cheng, D., Hong, S., Sappe, B., Hu, Y., Wei, N., Zhu, C., O'Connor, M. B., Pissios, P. and Perrimon, N. (2017). Midgut-derived Activin regulates glucagon-like action in the fat body and glycemic control. Cell Metab 25(2): 386-399. PubMed ID: 28178568
Summary:
While high-caloric diet impairs insulin response to cause hyperglycemia, whether and how counter-regulatory hormones are modulated by high-caloric diet is largely unknown. This study found that enhanced response of Drosophila adipokinetic hormone (AKH, the glucagon homolog) in the fat body is essential for hyperglycemia associated with a chronic high-sugar diet. The activin type I receptor Baboon (Babo) autonomously increases AKH signaling without affecting insulin signaling in the fat body via, at least, increase of Akh receptor (AkhR) expression. Further, it was demonstrated that Activin-β (Acβ), an activin ligand predominantly produced in the enteroendocrine cells (EEs) of the midgut, is upregulated by chronic high-sugar diet and signals through Babo to promote AKH action in the fat body, leading to hyperglycemia. Importantly, activin signaling in mouse primary hepatocytes also increases glucagon response and glucagon-induced glucose production, indicating a conserved role for activin in enhancing AKH/glucagon signaling and glycemic control.
Song, W., Owusu-Ansah, E., Hu, Y., Cheng, D., Ni, X., Zirin, J. and Perrimon, N. (2017). Activin signaling mediates muscle-to-adipose communication in a mitochondria dysfunction-associated obesity model. Proc Natl Acad Sci U S A. PubMed ID: 28739899
Summary:
Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. This study analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila. Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-beta signaling pathway were induced in the fat body. Strikingly, expression of the TGF-beta family ligand, Activin-&beta& (Act&beta&), was dramatically increased in the muscles by NF-kappaB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actβ expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling.
BIOLOGICAL OVERVIEW

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. The restriction of terminals to single columns is refered as tiling. In a genetic screen based on an R7-dependent behavior, the Activin receptor Baboon and the nuclear import adaptor Importin-α3 were identified as being required to prevent R7 axon terminals from overlapping with the terminals of other R7 projections in neighboring columns. This tiling function requires the Baboon ligand, dActivin (Activin-β), the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus (Ting, 2007).

Activin-β, the first invertebrate activin gene to be described (Kutty, 1998), is located 102 F region of the Drosophila chromosome 4, and has a multibasic protease site that would generate a mature C-terminal peptide containing 113 amino acids showing >60% similarity to the vertebrate activin βB(inhibin βB) sequences. A TGF-β family signature as well as all 9 cysteine residues conserved in the vertebrate activins are also present in this mature peptide sequence. Northern blot and RT-PCR analyses indicated that the activin β gene is expressed in embryo, larva and adult stages of Drosophila (Kutty, 1998). It is proposed that dActivin is an autocrine signal that restricts R7 growth cone motility, and it was demonstrated that dActivin acts in parallel with a paracrine signal that mediates repulsion between R7 terminals (Ting, 2007).

Previous anatomical studies have highlighted two prominent features of neuropil organization in the fly visual system: (1) the axons of most neuron classes arborize in characteristic layers of the brain and (2) they then remain restricted either to one column or a small number of adjacent columns. To gain insight into the developmental mechanisms that regulate these aspects of axon targeting, focus was placed on the R7 photoreceptor neuron. Previous studies have characterized mechanisms controlling the precise layer termination of R7 growth cones. This paper analyzed the mechanisms that restrict R7 terminals to the correct columns. The latter process is regulated by two partially redundant pathways: a paracrine signal that mediates repulsion between adjacent R7 axons and an autocrine Activin signal that is transduced by retrograde transport and import of the transcription factor Smad2 into the nucleus by a component of the classical nuclear import pathway, Importin-α3 (Ting, 2007).

A prominent organizing feature of the medulla is the restriction of axons and their terminals, including those of R7, R8, and the lamina monopolar neurons (except L4), to single columns. This phenomenon is similar to the tiling of processes observed in both the peripheral and central nervous systems. Ablation experiments in both fly and zebrafish support the view that repulsive interactions between processes of different cells of the same class prevent overlap of dendritic and axonal receptive fields. Consistent with this model, wild-type R7 terminals only invade adjacent columns from which R7s have been removed. However, an R7 axon does not invade even empty columns unless it is under 'competitive pressure' from additional R7 axons within its own column, suggesting that a second, intrinsic mechanism also restricts R7 terminals (Ting, 2007).

Activin signaling is required for tiling of R7 terminals: loss of babo, dSmad2, or dActivin causes R7 axons to invade adjacent occupied targets. Because these mutant axons invade even when they are not under competitive pressure, it is hypothesized that Activin affects an intrinsic property of R7 terminals such as their motility or ability to initiate synaptogenesis. In support of this model, it was found that virtually all R7 axons lacking babo, dSmad2, or dActivin initially extend filopodia beyond the R7-temporary layer at 17 hr APF, although these retract by 40 hr APF, and expression of a constitutively active Baboon in R7s affects growth cone morphology. Although models in which Activin signaling also mediates repulsion among R7 terminals cannot be ruled out, R7s unable to respond to Activin are still partly repelled by their neighbors, indicating the existence of repulsive mechanisms that are redundant with Activin. Recent studies have demonstrated that Dscam2 mediates repulsion between L1 growth cones in a layer immediately distal to the R7 terminals (Millard, 2007). Because Dscam2 is not expressed in R7, other cell-surface proteins must mediate the repulsive interactions between adjacent R7 terminals. The identification of Activin's involvement in R7 tiling paves the way to identifying such molecules by allowing the removal of a pathway that is functionally redundant with them (Ting, 2007).

Members of the TGF-β superfamily have been widely implicated in regulating axon guidance and synaptogenesis by both transcription-dependent and -independent mechanisms. This study found that loss of dSmad2 from R7s resembles loss of Activin, suggesting that the tiling of R7 terminals requires changes in transcription. In support of this model, it was found that restriction of R7 terminals also requires imp-α3, which is required for the accumulation of dSmad2 in R7 nuclei. Whereas some previous vertebrate studies have suggested that individual Smads are imported by Importin-α, others argue instead that active Smad complexes can enter the nucleus by an importin-independent mechanism. The results provide the first genetic evidence that Smad function can require Importin-α-mediated nuclear import and may help reconcile previous results by demonstrating that different cell types import dSmad2 by different mechanisms (although R7s require imp-α3, pigment cells do not). It is noted that imp-α3 mutant R7s have more frequent defects in tiling than babo or dSmad2 mutant R7s, suggesting that imp-α3 may transport additional nuclear proteins that, redundantly with the Activin pathway, restrict R7 terminals (Ting, 2007).

In addition to their classical nuclear import function, Importins have been implicated in mediating retrograde transport of signals from growth cones to the nucleus. Both Imp-α3 and dSmad2 are found throughout the length of R7 axons, and like loss of Activin signaling, disrupting retrograde axonal transport affects the intrinsic mechanism that restricts R7 terminals. These results are consistent with a model in which the Activin signal is received by R7 growth cones, and dSmad2 bound to Imp-α3 is transported through the axon and ultimately into the nucleus (Ting, 2007).

Surprisingly, Activin appears to be required in the R neurons and likely in R7s themselves: disrupting Activin in all R neurons causes R7 terminals to invade adjacent targets, and among the photoreceptors, only R7 and R8 express Activin. An attempt to test whether Activin is specifically required in R7s met with only partial success: sevenless-Gal4 (sev-Gal4) was used to express UAS-ActivinDN in R7s but not R8s and it was found that the resulting R7s temporarily overshoot their initial target layer, a phenotype also caused by loss of Babo or dSmad2. Thus, Activin can function as an autocrine effector. Unfortunately, the sev-Gal4 driver is no longer expressed by 40-50 hr APF, the time at which Activin prevents R7s from invading adjacent columns, and sev-Gal4/ActivinDN R7s appear normal at this time point (Ting, 2007).

Nonetheless, the finding that R7s and/or R8s are the source of Activin raises two mechanistic questions. First, if the R7 or R8 neurons themselves are providing the signal and the signal is simply transduced into the R7 nucleus, why might Activin, as has been argued, be secreted in the target region and received by the R7 growth cone (i.e., rather than being secreted and received by the cell body)? One possibility is that R7s use Activin to coordinate their developmental program with that of other cells within the medulla. For example, one could imagine that both R7 growth cones and their postsynaptic targets would encounter the Activin signal in the medulla at the same time, allowing them to coordinate their preparations for mutual synaptogenesis. A second question is, therefore, how might Activin signaling be coordinated with the R7 growth cones' arrival in the medulla? It was found that the Activin-processing enzyme Tolloid-related (Tlr) is located both at the R7-temporary target layer (at 17 hr APF) and at the final R7 target layer (at 50 hr APF) and that Tlr mutants exhibit severe R7 retinotopic map defects. One possibility is therefore that the medulla localization of Tlr might confer spatial and temporal specificity on Activin expressed by R7 and/or R8 (Ting, 2007).

Although Activin is also expressed in R8s, no evidence was found that Activin affects R8 axons, as shown by the fact that neither babo mutant R8s nor R8s expressing GMR-Gal4/UAS-ActivinDN exhibited connectivity defects. However, the possibility cannot be ruled out that redundancy obscures such a role (for example, there is no straightforward method of removing adjacent R8s) (Ting, 2007).

In the mushroom body, dActivin signaling results in upregulation of the ecdysone receptor gene, EcR-B1. Although EcR-B1 is expressed in essentially all photoreceptor neurons, three lines of evidence indicate that EcR-B1 is not the target gene of Activin signaling in R7s: the expression level of EcR-B1, as judged by anti-EcR-B1 staining, was not altered in babo mutant R7 clones; forced expression of EcR-B1 did not rescue babo mutant R7 defects; and USP R7 mutants did not phenocopy babo. In the dorsal cluster of Atonal-positive neurons, Babo-mediated signaling, via an EcR-independent pathway, mediates morphogenesis and axonal extension. The versatility of Activin signaling likely reflects its ability to regulate the expression of different genes in a context-dependent manner. It is speculated that dActivin signaling activates a transcriptional program that not only restricts growth cone motility once R7s are within their target layer but also promotes synaptogenesis. Such a model could explain the observed strong defects in R7-mediated behavior despite the infrequent specific defects in R7 tiling. Identifying the transcriptional targets of Activin signaling in R7s will likely provide insight into these processes (Ting, 2007).

Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. This study reports that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined (Makhijani, 2017).

This research identified Actβ as one of the elusive genes that govern hemocyte proliferation in the hematopoietic sites (HPs) of the Drosophila larva, as was predicted by previous functional studies. Actβ RNA expression is linked to the level of PNS neuronal activity. This model implies that increased expression of Actβ would give rise to higher levels of active Actβ protein, although the formal demonstration awaits development of a suitable tool for the detection of Actβ protein. In the future, it will be interesting to study specific sensory stimuli that trigger hemocyte responses. Sensory neurons of the PNS have a prime function in detecting innocuous and noxious sensory stimuli such as mechanical strain, temperature, chemicals and light, many of which signal potentially harmful conditions that may cause tissue damage. Thus, linking the detection of challenging conditions with the adaptive expansion of the blood cell pool may be an efficient system to elevate the levels of macrophages, to remove and repair damaged tissues, enhancing the overall fitness of the animal. Because Drosophila larval hemocytes persist into the adult stage, the mechanism of sensory neuron-induced blood cell responses may allow adaptation of the animal beyond the larval stage (Makhijani, 2017).

In Drosophila self-renewing hemocytes, Actβ/dSmad2 signalling has diverse effects on proliferation, apoptosis and adhesion. The current ex vivo data indicate that hemocyte proliferation is likely a direct effect, which is consistent with similar roles of babo/dSmad2 in other tissues such as Drosophila imaginal discs and brain and TGf-β family dependent proliferation in vertebrate systems. Echoing the findings of babo-CA driven hemocyte apoptosis, TGF-β family mediated direct or indirect effects on apoptosis have been described in invertebrate and vertebrate systems. Overall, TGF-β family signalling is known for its multifaceted biological roles, depending on the cellular contexts and levels of ligand stimulation, which often translate into qualitatively distinct transcriptional and other cellular responses, that are mediated by both Smad and non-Smad signalling mechanisms. While Drosophila Actβ and possibly related TGF-β family ligands are known to signal through the induction of ecdysone receptor (EcR) in some but not all Drosophila tissues, this study found no indication for a link with EcR expression in hemocytes, suggesting other signalling mechanisms in the regulation of larval blood cell responses. In the studied Drosophila system, it further remains to be seen whether Actβ/dSmad2 signalling has direct or indirect effects on hemocyte adhesion, and which other rate-limiting step/s may contribute to this process. Since hemocyte-autonomous loss of dSmad2 signalling causes a more severe phenotype than Actβ lof, it is speculated that other Act family ligands such as daw and myo, which are expressed in various tissues including surface glia, muscle, fat body, gut, and imaginal discs may partially substitute for Actβ in its absence. Overall, Actβ is likely to be only one player in a more complex regulatory network. Future research will identify other inducible signals from neurons that regulate neuron-blood cell communications. This is predicted from Actβ mutants that only partially block carbachol-induced blood cell responses. Actβ/dSmad2 lof and pathway silencing in hemocytes also reveal an underlying ability of the cells to compensate for the lack of this signalling pathway and the associated impairment in proliferation. Time course experiments with various RNAi lines suggest that the amplitude and temporal occurrence of the compensatory response may be proportional to the severity of the block in dSmad2 signalling. Future investigation will address whether the related BMP/Mad pathway might play a part in this, as silencing of Mad in hemocytes appeared to dampen elevated hemocyte numbers seen in dSmad2 null mutants. Similar observations of dSmad2 lof causing Mad overactivation have been reported in the Drosophila wing disc and neuromuscular junction previously (Makhijani, 2017).

Larval development may comprise distinct sensitive phases for the regulation of hemocyte responses. This is supported by carbachol promoting hemocyte proliferation preferentially in the early-mid 2nd instar larva, that is, at a stage when hemocytes are still tightly localized to the Hematopoietic Pockets (HPs). Likewise, the effects of Actβ lof and pathway silencing in hemocytes are more pronounced in younger larvae, suggesting a possible stronger dependence on the pathway, in addition to the emergence of compensatory mechanisms under lof conditions over time. Moreover, it will be interesting to investigate whether Actβ signalling may not only vary temporally, but also by the ability of cell types to produce active Actβ ligand, thereby influencing signalling outcomes, consistent with the cell type specific processing known for Activins and other ligands of the TGF-β family in both invertebrates and vertebrates (Makhijani, 2017).

Drosophila Actβ has previously been studied for its role in the formation and function of neuromuscular junctions in the Drosophila larva, where Actβ expressing motor neurons project axons from the CNS, reaching from the center of the larva to the muscle layers of the body wall. However, resident hemocytes are shielded from these areas through the muscle layers of the body wall, which also form the base of the HPs, thereby creating an anatomical space between the muscle layers and epidermis where resident hemocytes and Actβ expressing sensory neurons colocalize (i.e., the Hematopoietic Pocket). The model that sensory neurons signal to adjacent hemocytes in the HPs is further supported by the fact that Actβ silencing in motor neurons did not affect resident hemocyte localization and had, by t-test, no significant effect on hemocyte numbers. However, involvement of alternative or additional scenarios cannot be ruled out, for example, that experimental manipulations of PNS activity, which also feed back to the CNS, would in turn trigger a signal to motor neurons that may respond by secreting Actβ and/or another factor/s, thereby influencing hemocytes and/or the PNS itself. Likewise, although the direct effect of Actβ on hemocytes was confirmed ex vivo, and no signs were found of altered sensory neuron morphology under Actβ lof/silencing, it cannot be ruled out that in the larva, Actβ may contribute to molecular changes in the PNS that in turn might contribute to the observed hemocyte effects (Makhijani, 2017).

Sensory neurons of the HPs project axons to the CNS, and the current work shows that hemocytes are closely adjacent to and/or form direct contacts with sensory neurons, likely along the neuron cell bodies and dendrites, suggesting the communication involves non-canonical mechanisms. In Drosophila, as in vertebrates, signal transfer along all neuronal membrane surfaces, including dendritic synapses and dendrodendritic connections, have been described, which may also form the interface in neuron-blood cell communication. The transcriptional induction of Actβ in response to sensory stimuli recalls previous reports of the transcriptional upregulation of Actβ in the formation of long-term memory in both flies and vertebrates. This suggests parallels between the neuronal regulation within the CNS, and PNS-blood cell circuits, which will be an interesting subject for future study. Based on these findings and another recent report demonstrating that transcriptional regulation of the related BMP Decapentaplegic (Dpp) in the Drosophila wing epithelium depends on the K+ channel Irk2, it is proposed that cellular electrochemical potential may be a more general theme in the expression of TGF-β family ligands (Makhijani, 2017).

These findings in the Drosophila model pioneer a new concept that has not been shown in any vertebrate system to date -- the neuronal induction of self-renewing, tissue-resident blood cells. These cells correspond to the broadly distributed system of self-renewing myeloid cells that are present in most vertebrate organs, which by lineage are completely independent from blood cell formation fueled by hematopoietic stem cells. In vertebrates, TGF-β family ligands such as Activin A and TGF-β regulate the activity and immune functions of macrophages, and cellular and humoral immune responses, in multiple ways through autocrine and paracrine signalling. While the autonomic neuronal and glial regulation of hematopoietic stem and progenitor cells in the bone marrow has been recognized, the role of sensory innervation in bone marrow hematopoiesis remains unknown. Even more so, nothing is known about the role of the nervous system in the regulation of the independent, self-renewing myeloid system of tissue macrophages. However, local neurons and sensory innervation of many organs including skin, lung, heart and pancreas and inducible changes in the self-renewal rates of tissue macrophages, suggest that principles of neuronal regulation are likely also at work in vertebrates, providing a link between neuronal sensing and adaptive responses of local blood cell populations (Makhijani, 2017).

Neuroendocrine regulation of Drosophila metamorphosis requires TGFβ/Activin signaling

In insects, initiation of metamorphosis requires a surge in the production of the steroid hormone 20-hydroxyecdysone from the prothoracic gland, the primary endocrine organ of juvenile larvae. This study shows that blocking TGFβ/Activin signaling, specifically in the Drosophila prothoracic gland (PG), results in developmental arrest prior to metamorphosis. The terminal, giant third instar larval phenotype results from a failure to induce the large rise in ecdysteroid titer that triggers metamorphosis. It was further demonstrated that activin signaling regulates competence of the prothoracic gland to receive prothoracicotropic hormone (PTTH) and insulin signals, and that these two pathways act at the mRNA and post-transcriptional levels, respectively, to control ecdysone biosynthetic enzyme expression. This dual regulatory circuitry may provide a cross-check mechanism to ensure that both developmental and nutritional inputs are synchronized before initiating the final genetic program leading to reproductive adult development. As steroid hormone production in C. elegans and mammals is also influenced by TGFβ/Activin signaling, this family of secreted factors may play a general role in regulating developmental transitions across phyla (Gibbons, 2011).

Previous work in a number of holometabolous insects, including Drosophila, has highlighted the importance of the PTTH and insulin signaling pathways in stimulating 20E production in the PGs to trigger metamorphosis. This study demonstrates that PG competence to respond to these two essential metamorphic stimuli in Drosophila is crucially dependant on TGFβ/Activin signaling, which controls production of the primary receptors for these two pathways. The ability of activins to act as either direct or indirect permissive signals in the PG is interesting as competence factors have long been thought to play a central role in modulating cellular responses to hormonal signals and one generally recognized mechanism through which they function is by regulating the expression of receptors for a variety of signals. In the mammalian ovary, Activin has been shown to induce the expression of the receptor for follicle stimulating hormone (FSH) in rat granulosa cells, contributing to the stage-specific response of the developing follicles to FSH stimulation. It is also interesting to note in this regard that in Bombyx, the prothoracic gland, has been shown to be refractory to PTTH signals at certain stages and that Torso (the PTTH receptor) levels fluctuate dramatically during the 5th instar stage, potentially accounting for the lack of PTTH response at certain times. At present it is not known whether Torso or InR levels in the PG fluctuate during normal Drosophila larval development, nor is it know whether activins play a role in regulating Torso and InR levels in response to some specific timing or nutritional cue. It is possible that activins simply impart constitutive expression of these receptors in the PG as part of a general developmental program responsible for endowing the PG cells with their steroidogenic capacity. As the general morphological features of the PG cells are not perturbed by Activin signal knockdown, even when using the phantom-Gal4 driver, which is activated in the early embryo, it is not suspected that activins are required for specification of the gland itself. However, this conclusion must be tempered by the fact that RNAi knockdown is probably not complete and the phantom-Gal4 driver may not turn on until after gland formation is largely finished. To fully rule out a role of activins in gland specification, null germline clones for babo or dSmad2, encoding respectively for a receptor and signaling protein of the activin pathway, need to be analyzed, and at this time it is not clear that any such alleles exist for either gene (Gibbons, 2011).

The lack of true null mutations in both babo and dSmad2 may also account for the stronger phenotype produced by PG-specific knockdown of babo or dSmad2 compared with the reported babo genetic loss-of-function phenotype. Previous studies examining the phenotype of babo zygotic mutants revealed a 2-3 day developmental delay in puparium formation, but up to 30% of the mutant larvae do initiate metamorphosis. By contrast, elimination of activin signal reception only in the PG produces a stronger phenotype where virtually 100% of the knockdown larvae arrest development without puparium formation. Although residual Babo function is a possible explanation for this difference, it is pointed out that another explanation exists that highlights the difficulties associated with interpreting tissue-specific knockdown experiments. It is theoretically possible to produce a stronger whole animal phenotype by tissue-specific knockdown than by a genetic null mutation, owing to potential compensatory changes in other mutant tissues in the genetically null animal. This is especially true when the factor potentially regulates multiple systemic signals in different tissues. For example, the ligand Actβ is produced in insulin-producing cells (IPCs) of the brain. If Actβ negatively regulates insulin production or secretion, then, in a babo genetic null background, upregulation of insulin signal upon loss of Actβ signaling in IPCs may compensate for reduced insulin reception capacity in the PGs. This compensatory mechanism may enable a percentage of the babo mutant larvae to undergo puparium formation. By contrast, knockdown of activin signal reception in the PG alone, as in the case of phantom>babo RNAi animals, would probably not lead to this compensatory response and thus results in a stronger developmental arrest phenotype. Consistent with this view, it was found that using a ubiquitous driver, such as daughterless-Gal4, to knockdown dSmad2 in all tissues, including the PG, does not lead to developmental arrest, whereas using a PG specific driver does. These potential complications in evaluating phenotypic differences obtained using genetic mutations and tissue-specific knockdown methods should be borne in mind as they are likely to be observed more frequently with the increasing use of tissue-specific knockdown analyses in numerous model organisms (Gibbons, 2011).

The issue of which activin-like ligand(s) are responsible for providing the competence signal and whether they are regulated by particular developmental or nutritional cues is also important to answer but is problematic because of redundancy concerns. It is suspected that Actβ probably plays a role as it is expressed in numerous neurosecretory cells including the insulin-producing cells (IPCs), which innervate the heart tube and thereby probably provide systemic delivery of this ligand to many tissues. In addition, overexpression of this ligand in the PG causes stage precocious pupation, similar to that produced by expression of activated the activin receptor Babboon (Babo). However, the one available Actβ loss-of-function mutation does not produce substantial developmental delay, and the majority of larvae (>90%) instead undergo slightly precocious puparation consistent with potential upregulation of insulin signaling, as suggested above. Likewise, mutations in daw, a second Activin-like ligand that is produced in the PG, do not elicit major developmental delay when fed a yeast-enriched diet. However, when strong daw alleles are combined with the one available Actβ mutation, then only 25-35% of the larvae are able to initiate pupariation on rich food. This observation suggests a functional redundancy between these ligands for regulating developmental timing, similar to their previously noted redundant roles in regulating neuroblast proliferation in the larval brain (Zhu, 2008). The residual pupation ability of the daw-Actβ double mutants may be accounted for by the compensation mechanism described above or further functional redundancy provided by the two other Activin-like ligands, Myo and Mav. At present, no mutations are available in these genes (Gibbons, 2011).

Although these observations clearly show that loss of ecdysone (E) biosynthetic enzyme expression underlies the dSmad2 developmental arrest phenotype, it cannot be said with certainty that this downturn is due solely to the loss of insulin and PTTH signaling or whether dSmad2 might also participate directly in regulating biosynthetic enzyme gene expression. In addition, whether dSmad2 binds directly to target sequences within Torso and InR regulatory sequences also remains uncertain as no dSmad2 responsive elements have been identified for any gene in Drosophila. It is interesting to note, however, that one other molecular process, SUMOlyation, has recently been implicated in regulating E biosynthetic enzyme expression and localization in the PG (Talamillo, 2008). Knockdown of smt3, the sole SUMO-encoding gene in Drosophila, in the PG results in third instar larval arrest phenotype that is rescuable by feeding the larvae 20E. In addition, these larvae show low levels of Disembodied (Dib) protein accumulation in the PG (Talamillo, 2008). These phenotypes are strikingly similar to those seen in dSmad2 RNAi larvae. Interestingly, previous studies have demonstrated that Medea can be SUMOlyated in vitro, providing a potential link between SUMOlyation and Activin signaling. However, no SUMOylated forms of Medea or dSmad2 have been detected under various signaling conditions in vivo or in S2 cells. Additional studies will be required to determine whether SUMOlyation and Activin signaling are linked in a common pathway or whether they act through independent means to control steroid production in the PG (Gibbons, 2011).

Despite the uncertainty in determining whether one or more ligands act as a PG competence factor, the observation that TGFβ/Activin signaling regulates metamorphosis may highlight an ancient and conserved role for these factors in regulating developmental transitions in many organisms. For example, the nematode worm C. elegans employs the TGFβ pathway to make a nutritionally dependent decision on whether to continue the normal development program into a mature adult or to enter a developmentally arrested stage known as dauer. The activation of the TGFβ pathway promotes normal development whereas its inactivation results in dauer formation (for a review, see Fielenbach, 2008). Interestingly, a key target of the TGFβ pathway is daf-9, which encodes a cytochrome P450 protein. DAF-9 is involved in the synthesis of dafachronic acid, an ecdysone-like steroid hormone that prevents the developmental arrest as a dauer. Likewise, in mammals, Activin signaling probably affects pubertal timing by controlling sex steroid synthesis. Activin signaling can stimulate the production of the sex steroid hormone estradiol by enhancing the activity of CYP 450 aromatase. These observations from C. elegans and mammalian studies, together with the finding that Drosophila Activin signaling also regulates cytochrome P450 expression, although probably by indirect means, strongly indicate that TGFβ/Activin signaling is a common means by which developmental transitions are regulated across species (Gibbons, 2011).

Previously, it was found that complete loss of PTTH signaling delayed metamorphosis but did not completely block it (McBrayer, 2007). This observation led to a speculation that a second metamorphic signal, perhaps supplied by insulin, eventually facilitated pupation in the PTTH negative larvae. The finding in this study that simultaneous knockdown of both pathways by eliminating dSmad2 in the PG leads to third instar developmental arrest and that resupplying activity in either pathway promotes metamorphosis is consistent with this idea. Even more intriguing is this finding that the two metamorphic signals provided by PTTH and insulin appear to regulate the steroid biosynthetic capacity of the gland in two distinct ways: PTTH at the level of biosynthetic enzyme mRNA accumulation and insulin at the biosynthetic enzyme protein level (see Model for TGFβActivin regulation of metamorphosis). The observation that loss of PTTH signal reception in dSmad2 PG knockdown larvae reduces the steady-state mRNA levels of the E biosynthetic enzymes is consistent with earlier PTTH-neuron ablation results with one exception. In the present studies, downregulation of only dib, spok and nvd was obaserved in response to reduction in Torso expression, whereas in PTTH-neuron ablated larva, transcription of phantom and sad are also reduced (McBrayer, 2007). One possible explanation for this discrepancy is that the five biosynthetic enzymes exhibit different sensitivities to the strength of the PTTH signal. In PTTH-neuron ablation experiments, all signaling is lost, whereas in the dSmad2 knockdown, it is likely that some Torso expression remains and provides enough signal to activate phantom and sad. Consistent with this view, it was found that in the rescued larvae, where PTTH signaling was resupplied in the PG using activated Ras, the mRNA levels of the phantom and sad genes are dramatically upregulated compared with the other three E biosynthetic enzymes, indicating they are very sensitive to the PTTH signal (Gibbons, 2011).

The effects of insulin signaling on protein levels is intriguing and has not been examined previously, although there are two reports to indicate that insulin signaling also affects transcription of at least two biosynthetic enzymes, dib and phantom. However, the reported effects were modest (between 30% to 2 fold) and it was not clear whether the determinations were normalized for differences in body size and/or ring gland size, which are crucial when considering the small reported differences. In the current measurements, ring gland-brain complexes were used that were of similar size and staging, and no differences were detected in transcription of biosynthetic enzymes between the dSmad2 knockdown animals and those in which InR expression was restored to the PG. Instead, significant differences were observed in biosynthetic enzyme protein levels. The known role of insulin in modulating translational capacity of cells is consistent with the idea that these changes in biosynthetic enzyme levels are the result of translational differences. Whether this effect is through Tor, which has been shown to be an important mediator of developmental timing in the PG, and its modulation of S6 kinase, remains to be determined. In addition, effects on protein stability cannot be excluded, and further studies examining protein turnover will also be required to address this issue fully. Overall, this two-tiered regulatory control of biosynthetic enzyme expression may better enable the larva to fine-tune its ecdysone level depending on conditions, or perhaps may serve as a coincidence detector to ensure that both developmental and nutritional conditions are appropriate before triggering a terminal developmental program such as metamorphosis (Gibbons, 2011).

Mg2+ block of Drosophila NMDA receptors is required for long-term memory formation and CREB-dependent gene expression

NMDA receptor (NMDAR) channels allow Ca2+ influx only during correlated activation of both pre- and postsynaptic cells; a Mg2+ block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg2+ block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg2+ block, it was found that Mg2+ block mutants are defective for LTM formation but not associative learning. It was demonstrated that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg2+ block defective NMDARs. Furthermore, it was shown that genetic and pharmacological reduction of Mg2+ block significantly increases expression of a CREB repressor isoform. These results suggest that Mg2+ block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction (Miyashia, 2012).

Although the mechanism through which Mg2+ block restricts NMDAR activity is well known, the cellular and behavioral functions of Mg2+ block have not been extensively studied. In this study, transgenic flies expressing dNR1N63IQ to show that Mg2+ block is important for formation of LTM. Previous studies of hypomorphic mutants have shown that NMDARs are required for both learning and LTM. In contrast, our Mg2+ block mutants do not have learning defects. This suggests that although Ca2+ influx through NMDARs is important for learning, inhibition of influx during uncorrelated activity is not. Notably, elav/dNR1N63IQ flies have slightly enhanced learning. Consistent with this result, NMDAR-dependent induction of hippocampal LTP is enhanced in the absence of external Mg2+. In the current studies, Mg2+-block-defective dNR1 was overexpressed in an otherwise wildtype background, so it cannot be definitively concluded that Mg2+ block is dispensable for learning. However, electrophysiology experiments indicate that Mg2+ block is abolished in the flies at physiological potentials. Furthermore, it was demonstrated that expression of Mg2+-block-defective dNR1 rescues learning defects in dNR1 hypomorphs, consistent with a model in which Mg2+ block is not required for learning. Interestingly, the dNR1N63IQ transgene does not rescue the semilethality of dNR1 hypomorphs, suggesting that Mg2+ block has an essential biological function unrelated to learning (Miyashia, 2012).

The results suggest that Mg2+-block-dependent suppression of NMDAR activity and Ca2+ influx at the resting state is critical for LTM formation. Supporting this idea, chronic reduction of NMDAR-mediated Ca2+ influx at the resting state has been shown to enhance long-term synaptic plasticity. Extending these results, it was found that Mg2+ block is required for CREB-dependent gene expression during LTM formation. A CREB-dependent increase in staufen expression upon spaced training is essential for LTM formation, and this study shows that Mg2+ block is required for this increase. Two other genes, activin and homer, were identified that are expressed upon LTM induction in a CREB-dependent manner. It is proposed that all three genes are maintained in an LTM-inducible state by Mg2+-block-dependent inhibition of CREB repressor, and it was shown that the amount of increase in expression of dCREB2-b in Mg2+ block mutants correlates with the ability of dCREB2-b to suppress LTM. The 4-fold increase in dCREB2-b protein in Mg2+ block mutant flies is comparable to the increase in dCREB2-b in heat-shocked hs-dCREB2-b flies showing equivalent defects in LTM (Miyashia, 2012).

Next the homer gene was further characterized and it was determined to be required specifically for LTM but not for learning or ARM. It was determined that spaced training increases HOMER expression in several brain regions, including the antennal lobes, lateral protocerebrum, protocerebral bridge, and calyx of the MBs. This increase does not occur in the absence of Mg2+ block. Significantly, when Mg2+ block is abolished by dNR1N63IQ expression, specifically in the MBs, increased Homer expression is suppressed in the MBs but not in other regions, including the protocerebral bridge, indicating that Mg2+ block regulates CREB repressor and LTM-associated gene expressions in a cell autonomous manner (Miyashia, 2012).

Electrophyisiological experiments demonstrate that 20 mM Mg2+ is sufficient to block Drosophila NMDAR currents at the resting potential (-80 mV). Although this concentration is higher than the concentrations needed to block mammalian NMDARs, the Mg2+ concentration in Drosophila hemolymph has been shown by various groups to be between 20 and 33 mM, which is correspondingly higher than the Mg2+ concentration reported in mammalian plasma. In mammals, Mg2+ concentration is higher in cerebrospinal fluid than in plasma, further suggesting that the 20 mM Mg2+ concentration used in this study is likely to be within the physiologically relevant range (Miyashia, 2012).

An N/Q substitution at the Mg2+ block site of mammalian NR1 disrupts Mg2+ block and reduces Ca2+ permeability, while a W/L substitution in the TM2 domain of NR2B disrupts Mg2+ block and increases Mg2+ permeability. This raises the possibility that Mg2+-block-independent changes in channel kinetics and Mg2+ permeability may be responsible for the effects observed in the dNR1N63IQ-expressing flies. While this possiblity cannot be completely ruled out, increases were observed in dCREB-2b protein in wild-type neurons in Mg2+-free conditions, indicating that disruption of Mg2+ block, rather than changes in other channel properties, causes increased CREB repressor expression and decreased expression of LTM-associated genes (Miyashia, 2012).

A chronic elevation in extracellular Mg2+ enhances Mg2+ block of NMDARs, leading to upregulation of NMDAR activity and potentiation of NMDA-induced responses at positive membrane potentials (during correlated activity) (Slutsky, 2010). This raised the possibility that the Mg2+ block mutations may cause a downregulation of NMDAR-dependent signaling and decreased NMDA-induced responses at positive membrane potentials. Since this study recorded NMDA-induced responses from various sizes of cells, it was not possible to directly compare amplitudes of NMDA-induced responses between elav/dNR1wt cells and elav/dNR1N63IQ cells. However, training-dependent increases in ERK activity, required for CREB activation, occurred normally in both elav/dNR1wt cells and elav/dNR1N63IQ cells, while it was significantly suppressed in dNR1 hypomorphs. These results suggest that the Mg2+ block mutations do not alter NMDA-induced responses at positive membrane potentials (Miyashia, 2012).

Similar to dNR1 Mg2+ block mutants, dNR1 hypomorphic mutants also have defects in CREB-dependent gene expression upon LTM formation. However, dNR1 hypomorphs and Mg2+ block mutants are likely to have opposing effects on Ca2+ influx. While hypomorphic dNR1 mutants should have decreased Ca2+ influx during spaced training because of a reduction in the number of dNMDARs, elav/dNR1N63IQ flies are unlikely to have this effect. Conversely, while elav/ dNR1N63IQ flies should have increased Ca2+ influx during the resting state when uncorrelated activity is likely to occur, dNR1 hypomorphs should not. Supporting a model in which dNR1 hypomorphs and Mg2+ block mutants inhibit LTM-dependent gene expression through different mechanisms, it was shown that Mg2+ block mutants increase basal expression of dCREB2-b repressor while NMDAR hypomorphs do not. Conversely, the data indicating that NMDAR hypomorphs are defective for training dependent increases in ERK activity, while elav/dNR1N63IQ flies are not. These data fit a model in which there may be two equally important requirements for NMDARs in regulating LTM-dependent transcription. First, during correlated, LTM-inducing stimulation, a large Ca2+ influx through channels, including NMDARs, may be required to activate kinases, including ERK, necessary to activate CREB. dNR1 hypomorphs are defective for this process. However, a second and equally important requirement for NMDARs may be to inhibit low amounts of Ca2+ influx during uncorrelated activity to maintain the intracellular environment in a state conducive to CREB-dependent transcription. Mg2+ block is required for this process (Miyashia, 2012).

Although it is unclear what types of uncorrelated activity are suppressed by Mg2+ block, one type may be spontaneous, action potential (AP)-independent, single vesicle release events (referred to as 'minis'). Supporting this idea, an increase in dCREB2-b was observed in cultured wild-type brains in Mg2+-free medium in the presence of TTX, which suppresses AP-dependent vesicle releases but does not affect minis. In addition, a significant increase was observed in cytosolic Ca2+, [Ca2+]i, in response to 1 mM NMDA in the presence of extracellular Mg2+ in neurons from elav/dNR1N63IQ pupae. In neurons from transgenic control and wild-type pupae, which have an intact Mg2+ block mechanism, 1 mM NMDA does not cause Ca2+ influx and membrane depolarization. The concentration of glutamate released by minis is on the order of 1 mM at the synaptic cleft, suggesting that an increase in frequency of mini-induced Ca2+ influx due to decreased Mg2+ block may contribute to the increase in dCREB2-b in elav/ dNR1N63IQ flies (Miyashia, 2012).

Correlated, AP-mediated NMDAR activity has been proposed to facilitate dCREB2-dependent gene expression by increasing activity of a dCREB2 activator. The present study suggests that, conversely, Mg2+ block functions to inhibit uncorrelated activity, including mini-dependent Ca2+ influx through NMDARs, which would otherwise cause increased dCREB2-b expression and decreased LTM. Other studies have also suggested opposing roles of AP-mediated transmitter release and minis. For activity-dependent dendritic protein synthesis, local protein synthesis is stimulated by AP-mediated activity and inhibited by mini activity. In the case of NMDARs, the opposing role of low Ca2+ influx in inhibiting CREB activity must be suppressed by Mg2+ block for proper LTM formation (Miyashia, 2012).

Anterograde Activin signaling regulates postsynaptic membrane potential and GluRIIA/B abundance at the Drosophila neuromuscular junction

Members of the TGF-beta superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. This study analyzed whether the Activin branch of the TGF-beta superfamily also contributes to NMJ development and function. Elimination of the Activin/TGF-beta type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. These results demonstrate that anterograde Activin/TGF-beta signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling via Glass bottom boat and Wishful thinking, these findings indicate that the two branches of the TGF-beta superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function (Kim, 2014).

Numerous reports have now implicated the Activin/TGF-β and BMP branches of the TGF-β superfamily in regulating neuronal development, synaptic plasticity and cognitive behavior. Accordingly, members from both subfamilies are widely expressed in the nervous system and are co-expressed in multiple regions of vertebrate and invertebrate brains. It is therefore quite likely that ligands of both subfamilies co-exist within the extracellular space and in some cases, act on the same neurons. Lending support to this idea, pyramidal neurons in the CA3 region of the rat hippocampus are known to accumulate both phosphorylated Smad2 and Smad1/5/8, transcriptional transducers of the canonical Activin/TGF-β and BMP-type signaling, respectively. The activation of these two closely-related signaling pathways in common sets of neurons, or different cells of a common neuronal circuit raises the intriguing question of whether the two pathways play different or redundant roles during neuronal development and function (Kim, 2014).

This study utilized the Drosophila neuromuscular junction to address this issue since ligands of both Activin/TGF-β and BMP families are expressed in both muscle and motor neurons. The data, together with previous studies on the role of BMP signaling at the NMJ, clearly demonstrate that the two pathways influence NMJ synaptogenesis in different ways. The Activin/TGF-β pathway is necessary for achieving the proper densities of GluRIIA, GluRIIB and Dlg in postsynaptic muscle membrane, while the BMP pathway has a smaller effect on the distribution of these postsynaptic proteins. In addition, the Activin/TGF-β pathway was dispensable for maintaining overall synaptic growth and homeostasis, both of which are strongly affected by mutations in the BMP pathway. In addition, tissue-specific rescue experiments indicate that the postsynaptic reception of Activin/TGF-β signaling is important in regulating synaptic GluR abundance, whereas BMP signal reception is known to act in the presynaptic motor neurons to promote synaptic growth. These observations suggest that each pathway influences NMJ synapse development and function by acting mainly in either the pre- or postsynaptic cell (Kim, 2014).

Interestingly, the BMP and Activin/TGF-β pathways have also been recently found to control different aspects of the Drosophila innate immune response (Clark, 2011). In this case BMP signaling suppresses the expression of multiple antimicrobial peptide genes following wounding, whereas the Activin/TGF-β pathway limits melanization after bacterial infection in adult flies. Therefore, it appears that the division of labor between these subpathways is not limited to just the nervous system, rather it may be the norm when these related signaling pathways act in concert to regulate a common biological process (Kim, 2014).

The fact that the pathways actually differ in how they affect a complex biological process is not surprising given that the different R-Smads are likely to have different selectivity in gene activation. Within motor neurons, BMP signaling promotes microtubule formation in axons and directly regulates expression of trio, a Rac GEF, that acts as a major regulator of actin cytoskeleton in many types of cells. Thus, it is likely that BMP signaling modulates synaptic growth, in part, by changing the structure and dynamics of the actin and microtubule cytoskeleton within motor neurons. BMP signaling also regulates the transcription of twit, a gene encoding a L-6 neurotoxin-like molecule that controls the frequency of presynaptic spontaneous vesicle release (Kim, 2012; Kim, 2014 and references therein).

Targets of Drosophila Activin/TGF-β signaling in any tissue are less well characterized. Within the central brain, glial-derived Myo signals through Smox to control expression of the Ecdysone B1 receptors in remodeling mushroom body neurons. However, it is not clear if EcR-B1 is a direct or indirect target of smox transcriptional regulation. It is also unclear if Ecdysone signaling plays a role in regulating synaptogenesis at the NMJ, although it may play a role during metamorphic remodeling of the NMJ as it does for the mushroom body neurons. The only other known targets of Smox are InR, Pi3K and Akt, all of which are Insulin signaling components and are reduced in the Drosophila prothoracic gland in the absence of Activin/TGF-β signaling. Once again the effect may be indirect, but this finding is interesting since Insulin signaling components have been shown to control synaptic clustering of GluRs (Kim, 2014).

The clustering of GluRs and Dlg at the NMJ have been shown to be regulated by both transcriptional and post-transcriptional mechanisms. For example, a recent genetic screen identified longitudinals lacking (lola), a BTN-Zn finger transcription factor, as an essential regulator of GluR and dPak expression in muscles. In contrast, the current studies on Activin/TGF-β signaling suggest, at least for GluRIIA, that this pathway functions at the post-transcriptional level since this study found that overexpression of glurIIA-gfp using an exogenous promotor and transcriptional activator does not lead to an enrichment of GluRIIAGFP at synaptic sites of Activin/TGF-β pathway mutants. This phenotype is reminiscent of that found for certain mutants in the NF-κB signaling system. Loss of Dorsal (an NF-κB homolog), Cactus (an IκB related factor), or Pelle (an IRAK kinase) leads to a substantial reduction of GluRIIA and a slight reduction of Dlg postsynaptic localization at the NMJ and a concomitant reduction in mEJP size. In addition, as was found for loss of Activin/TGF-β signaling, exogenously-expressed GluRIIA-myc did not reach the synaptic surface in NF-κB signaling mutants consistent with a possible role of Activin/TGF-β signaling in regulating NF-κB signaling. However, even if future studies show that the relationship is true, the Activin/TGF-β pathway likely regulates additional factors since its loss also affects GluRIIB levels and muscle resting potential, neither of which is altered in NF-κB pathway mutants. Interestingly, the regulation of GluRIIB levels by Activin/TGF-β signaling does appear to be at the level of transcription, indicating that this signaling pathway likely affects GluR clustering at the NMJ via both transcriptional and post-transcriptional mechanisms (Kim, 2014).

Analysis of Activin/TGF-β signaling at the NMJ, coupled with previous studies on BMP signaling and the novel ligand Maverick, indicates that TGF-β ligands are produced in, and act upon, all three cell types that contribute to NMJ function, specifically the motor neuron, wrapping glia, and muscle (see Model of controlling NMJ development and function by Activin/TGF-β and BMP pathways). This leads to the important issue of how directionality of TGF-β signaling at the NMJ is regulated. One possibility is that ligands are sequestered, either inside the secreting cells or on their surfaces, so that they have limited access to receptors on the opposing pre or postsynaptic membrane. For example, Gbb is produced both in muscle and motor neurons, leading to the issue of how directional signaling from muscle to motor neurons is achieved. On the postsynaptic muscle, Gbb release is potentiated by dRich (Rho GTPase activating protein at 92B), a Cdc42 selective Gap while in the presynaptic neuron Crimpy, a Drosophila homolog of the vertebrate Crim1 gene, has been shown to bind to a precursor form of Gbb. The Gbb/Crimpy complex is thought to either interfere with secretion or activation of motor neuron-derived Gbb thus ensuring that only muscle-derived Gbb activates the retrograde BMP signal at the NMJ. Since there are a large number of characterized TGF-β superfamily binding proteins, Drosophila homologs of some of these factors such as the BMP binding proteins Cv-2, Sog, Tsg and Dally, or the Activin-binding protein Follistatin, may sequester and regulate levels of active ligands within the NMJ. Sequestering mechanisms may also provide direction control by facilitating autocrine as opposed to juxtacrine signaling. If ligand-binding proteins are associated with the membrane surface of the ligand-producing cell, they may facilitate delivery of the ligand to receptors on the producing cell, thus enhancing autocrine signaling. It is interesting in this regard that in the developing Drosophila retina, Actβ appears to signal in an autocrine fashion to control photoreceptor connectivity in the brain (Kim, 2014).

Activin-type ligands are secreted from glia, motor neuron and muscle. The Activin-type ligands induce Babo-mediated phosphorylation of Smox that facilitates association with Med. In the muscle, the phospho-Smox/Med complexes activate the transcription of glurIIB and an unknown factor controlling post-transcriptional process or stability of glurIIA mRNA. In the motor neuron, the phospo-Smox/Med complex controls spontaneous release of synaptic vesicles via unknown mechanism(s). On the other hand, glia-secreted Mav stimulates Mad phosphorylation in the muscle resulting in increased gbb transcription. Gbb protein is released from the muscle and binds Tkv/Sax and Wit complex on the motor neuron leading to an accumulation of phospho-Mad in the nuclei by an unknown mechanism. The resultant phospho-Mad/Med complex activates the transcription of trio whose product promotes synaptic bouton formation (Kim, 2014).

Another important mechanism to control signal direction is likely to be tissue-specific receptor expression. For example, Wit is highly enriched in motor neurons compared to muscle, and this may help ensure that Gbb released from the postsynaptic muscle signals to the presynaptic motor neuron. Type I receptor diversity may be even more important in controlling directionality since at least 2 isoforms of Tkv and three isoforms of Babo have been identified. In the case of Babo, Activin-like ligands have a clear preference for signaling through different receptor isoforms, and these isoforms show differential tissue expression (Kim, 2014).

An additional factor to be considered in understanding TGF-β superfamily signal integration within different NMJ cell types is the possibility of canonical versus non-canonical and/or cross-pathway signaling. For example, in mushroom body neurons Babo can signal in a non-Smad dependent manner through Rho1, Rac and LIM kinase1 (LIMK1) to regulate axon growth and target recognition. Whether this mechanism, or another non-canonical pathway is operative at the NMJ is unclear. Cross-pathway signaling has also recently been identified in Drosophila. In this example, loss of Smox protein in the wing disc has been shown to up-regulate Mad activity in a Babo-dependent manner. Double mutants of babo and smox suppress the cross-pathway signal. As is described in this study, smox protein null mutations lead to significantly more severe GluR and mEJP defects than strong babo mutations alone, and this phenotype is suppressed in double mutants. Thus, as in wing discs, loss of Smox protein likely leads to ectopic Mad activity in muscles that further decrease GluR expression and/or localization at the NMJ. Consistent with this view, this study found that loss of Mad actually increases GluRIIB localization, suggesting that Mad acts negatively to regulate GluRIIB in muscle. One possible model to explain the Smox/Mad data is that normally the Babo/Smox signal inhibits Mad signaling which is itself a repressive signal for GluR accumulation. Thus, in babo mutants, total GluR levels decrease due to the loss of smox and therefore an increase in the repressive Mad signal. In the smox protein null mutant even more repressive Mad signal is generated by Babo further hyperactivating Mad activity leading to even lower levels of GluR accumulation. In medea mutants the activity of both pathways is reduced thereby returning the level of GluR levels close to normal. Additional experiments employing various single and double mutants, together with tissue-specific expression of various ligands, receptor isoforms and ligand-binding proteins will be needed to fully elucidate how vectorial TGF-β signaling is accomplished at the NMJ. Likewise, the identifcation of directly responding target genes and how they are influenced by both Smox and Mad signals is needed to fully appreciate how these two TGF-β signaling branches regulates NMJ functional activity (Kim, 2014).

TGF-ß signaling activates steroid hormone receptor expression during neuronal remodeling in the Drosophila brain

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-β (dActivin) (Kutty, 1998) 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).

It was of interest to identify possible ligands that participate in the remodeling process. Seven TGF-β type ligands are present in the Drosophila genome. Three of these, dpp, scw, and gbb, are clearly of the BMP family. The remaining, maverick (mav) (Nguyen, 2000), myoglianin (myo), dActivin (dAct), and activin-like-protein (alp), have not been assigned either genetically or biochemically to a particular family or signaling pathway. Phylogenetic considerations place dAct clearly within the Activin subfamily, while Myo is most similar to BMP-11 and GDF-8, and Mav and Alp are equidistant from both the BMP and TGF-β/Activin subgroups. Therefore, possible involvement of dAct in the Babo signaling was examined (Zheng, 2003).

First, in situ hybridization revealed that dAct is widely expressed in larval brain. Next, when conditioned media from cells expressing dAct was added to S2 cells transfected with Smox, it was found that this ligand is able to stimulate phosphorylation of Smox, while the prototypical BMP ligand Dpp is not. Finally, attempts were made to knock out dAct activity using two independent approaches and dAct, like Babo, was found to be essential for both optic lobe development and EcR-B1 expression in larval brains. Since dAct mutations are currently unavailable, attempts were made to produce a partial loss-of-function condition by overexpression of a dominant-negative form of the protein or RNAi. All TGF-β type ligands that have been examined dimerize and are processed prior to secretion. Previous studies have shown that overexpression of a cleavage-defective form of a particular ligand can interfere with processing and secretion of endogenous ligand. Therefore, a cleavage defective form of dAct (CMdAct) was expressed using either a general GAL4 driver (tubP-GAL4) or an MB-specific driver (GAL4-OK107). CNS development was observed to be retarded only when the CMdAct is ubiquitously expressed and not when it is expressed in MBs. This suggests that dAct does not function within MBs in an autocrine-like fashion. Poor development of the optic lobes is apparent in the tubP-GAL4>CMdAct larval brains, similar to that observed in babo and punt mutant larvae. More importantly, EcR-B1 expression is largely absent in γ neurons of animals that ubiquitously express CMdAct, similar to what is observed in babo mutants. Hs-GAL4-mediated transient expression of CMdAct around the mid-third instar stage also blocks both optic lobe development and EcR-B1 expression (53%). Consistent results are obtained after induction of RNAi using a hairpin-loop dAct construct (UAS-HLdAct). For instance, no EcR-B1 expression was detected in 65% of the late third instar larval brains that were heat shocked to express UAS-HLdAct transiently around the mid-third instar stage. Again, absence of EcR-B1 expression is tightly associated with poor optic lobe development. Similar treatments yield no detectable phenotypes when UAS-CMdAct/UAS-HLdAct is absent or replaced with other UAS-transgenes, such as UAS-mCD8-GFP and UAS-antisense dActivin. In addition, punt mutants, despite having small brains, continue to show EcR-B1 expression. Taken together, these results suggest that dAct, like Babo and dSmox, is indispensable for EcR-B1 expression in the CNS of wandering larvae (Zheng, 2003).

Therefore, from forward genetic mosaic screens, it was found that the Babo TGF-β/Activin type I receptor and a well-known TGF-β/Activin receptor downstream effector, Smox, are both cell autonomously required for remodeling of MB neurons during metamorphosis, providing definitive evidence for involvement of TGF-β/Activin signaling in neuronal plasticity. No evidence exists for any cell fate change in babo mutant MB neurons. For instance, expression of multiple cell type-specific markers remains normal, and mutant γ neurons, unlike wild-type α'/β' neurons, consistently acquire mature dendritic morphological features before metamorphosis. In addition, MB γ neurons that are born at various stages all commit to expressing EcR-B1 in response to TGF-β signaling at the same time and after they all develop into morphologically mature neurons. Therefore, TGF-β signaling probably plays a direct role in programming neuronal plasticity and is not required for cell specification (Zheng, 2003).

TGF-β signaling is implicated in regulating neuronal plasticity in diverse organisms. For instance, environmental cues regulate synthesis of a TGF-β-related ligand (DAF-7) in a pair of chemosensory neurons in C. elegans to direct entry into and exit from an alternative third larval stage called the dauer larva. Dauer formation involves arrest of all postembryonic cell divisions and remodeling of various tissues throughout the body. Given that the DAF-7 TGF-β ligand is primarily sensed by neurons expressing appropriate TGF-β receptors and Smads, it is likely that changes in TGF-β activities directly mediate neuronal remodeling and in turn orchestrate diverse dauer entry/exit responses outside the nervous system. However, it remains to be elucidated whether and how TGF-β signaling regulates neuronal projections and connections in individual remodeling neurons during the entry into and exit from the dauer stage (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).

Changes in gene expression are believed to mediate most TGF-β-dependent biological processes. The observation that restoration of EcR-B1 expression significantly rescues remodeling defects in babo mutant neurons supports the model that the Babo/dSmox-mediated TGF-β signaling mediates neuronal remodeling via upregulation of the EcR-B1 expression. Interestingly, ecdysone has also been implicated in regulating synaptic efficacy at the Drosophila NMJ, as has BMP signaling. However, as yet no connection between TGF-β signaling and the ecdysone pathway has been established in this system. In C. elegans, the DAF-7 TGF-β ligand as well as the DAF-12 nuclear hormone receptor are involved in dauer formation. In response to hormonal signals, DAF-12 and EcR coordinate changes in diverse tissues during dauer formation and metamorphosis, respectively. Therefore it might be a common theme that TGF-β signaling patterns tissue-specific responses to steroid hormones in diverse organisms by regulating expression and/or activities of specific steroid hormone receptors (Zheng, 2003).

Several lines of evidence support the model that patterned EcR-B1 expression in the late third instar larval CNS is likely established as a consequence of stage-regulated, cell type-specific responses to TGF-β signaling: (1) dActivin is broadly expressed in the CNS, while expression of EcR-B1 is selectively restricted; (2) despite the persistent presence of dActivin expression during all developmental stages and the fact that γ neurons are born at different times, programming of EcR-B1 expression in γ neurons does not occur until the mid-third instar stage; (3) ubiquitous expression of activated Babo fails to activate EcR-B1 expression ectopically. Determining how TGF-β signaling induces such stage-specific, cell type-dependent responses will provide mechanistic cues for how EcR-B1 is differentially expressed to pattern metamorphosis of the CNS. Possible models might include differential expression of a Smox cofactor or the requirement for a second signal that cooperates with the dActivin signal. It will be important to determine how dActivin reaches its target MB neurons. As has been recently suggested for BMP signaling at the NMJ, this might involve retrograde signaling from the MB synapse or it may occur via a juxtacrine mechanism from nearby cells (Zheng, 2003).

No direct connection has been shown between TGF-β signaling and regulation of cytoskeleton dynamics. Consistent with vital roles of TGF-β pathways in regulating gene expression, the results suggest that Babo/Smox signaling might simply lead to activation of EcR-B1 expression to capacitate MB neuronal remodeling during metamorphosis. Although this study provides novel insights into how differential expression of EcR isoforms is achieved, the challenge now is how ecdysone-induced transcriptional hierarchies mediate complex cytoskeletal changes in remodeling neurons. Identifying mutations that block various aspects of the MB neuronal remodeling in mosaic organisms will continue to shed new light on the molecular mechanisms underlying neuronal plasticity (Zheng, 2003).

Drosophila Follistatin exhibits unique structural modifications and interacts with several TGF-beta family members

Follistatin (FS) is one of several secreted proteins that modulate the activity of TGF-β family members during development. The structural and functional analysis of Drosophila Follistatin (dFS) reveals important differences between dFS and its vertebrate orthologues: it is larger, more positively charged, and proteolytically processed. dFS primarily inhibits signaling of Drosophila Activin (dACT) but can also inhibit other ligands like Decapentaplegic (DPP). In contrast, the presence of dFS enhances signaling of the Activin-like protein Dawdle (DAW), indicating that dFS exhibits a dual function in promoting and inhibiting signaling of TGF-β ligands. In addition, FS proteins may also function in facilitating ligand diffusion. Mutants of daw are rescued in significant numbers by expression of vertebrate FS proteins. Since two PiggyBac insertions in dfs are not lethal, it appears that the function of dFS is non-essential or functionally redundant (Bickel, 2008).

Polypeptide cytokines of the transforming growth factor β (TGF-β) family control a wide range of developmental and physiological functions in higher eukaryotes. This diverse group of signaling molecules provides positional information required for axis formation and tissue specification, controls various processes such as tissue growth, cell death, and pathfinding of axons in the nervous system, and prevents differentiation of embryonic stem cells. Many components of this pathway have been linked to tumor formation in humans. The highest degree of sequence conservation between various family members is found within the C-terminal domains, which are released as dimers by proteolytic processing. Similarities in sequence and biological activities allow these factors to be divided into at least two distinct subgroups: Bone Morphogenetic Proteins (BMPs) and Activins/Inhibins/TGF-βs. The latter group exhibits an additional intramolecular disulfide bond at the N-terminus after processing. In Drosophila, there are four Activins/TGF-βs, Drosophila Activin (dACT), Dawdle (DAW, also known as Activin-like protein at 23, ALP23, and Anti-Activin, AACT), Myoglianin (MYO), and Maverick (MAV), and three BMP-type ligands, Decapentaplegic (DPP), Screw (SCW), and Glass Bottom Boat (GBB). Each ligand dimer forms a complex with two type II and two type I receptor serine/threonine kinases that phosphorylate SMAD transcription factors. BMP-type ligands signal primarily through the type I receptors thick veins (TKV) and Saxophone (SAX) and activate Mothers against DPP (MAD). Activins/TGF-β-type ligands are believed to signal through the type I receptor Baboon (BABO), which in turn activates primarily dSMAD2 but to a minor extent also MAD (Bickel, 2008)

TGF-β signaling is regulated by various extracellular proteins. Antagonists like Follistatin (FS), Noggin, Chordin/Short Gastrulation, and DAN/Cerberus bind ligands and prevent interactions with receptors and signaling. In some species, they exhibit overlapping and redundant functions. Recently, it was shown that the simultaneous depletion of FS, Noggin, and Chordin in Xenopus tropicalis results in transformation of ventral into dorsal tissue during embryogenesis (Bickel, 2008).

Follistatin was first identified as an inhibitor of Activin in vertebrates. Subsequent studies showed that it also binds other ligands with lower affinities including BMP 2, 4, 6, 7, and Myostatin. Knockout mice of fs die shortly after birth. They are smaller and exhibit defects in skeletal and muscle development. Recently, the crystal structure of the human FS-Activin complex was resolved. It provides valuable insight into the function of the different FS domains and a basis to explain the mechanism of ligand inhibition (Bickel, 2008 and references therein)

This study has analyzed the function of Drosophila Follistatin (dFS). Like vertebrate FS proteins, dFS is subdivided into a N-terminal domain (N) and three FS domains (FS1-3). However, dFS is substantially larger than its vertebrate homologues due to a large basic insertion into FS1. Interestingly, dFS is proteolytically processed, and small processed forms of dFS are able to bind to ligands like dACT. This result suggests a possible different inhibitory mechanism: ligands bound to processed dFS can bind to type II receptors but cannot recruit type I receptors. Consequently, processed dFS might not only sequester ligands but also prevent unbound ligands from interacting with receptor complexes. Among the seven Drosophila TGF-β ligands, dFS primarily inhibits dACT but can also inhibit signaling of other ligands like DPP. In contrast, dFS can augment signaling of the TGF-β member DAW. These results suggest that dFS might exhibit dual functions in facilitating and inhibiting TGF-β signaling. Analysis of two PiggyBac insertions in dFS reveals that they affect dfs expression. Since homozygous animals of these lines are viable and phenotypically wild type, it is assumed that the function of dFS is non-essential or functionally redundant. Taken together, this study reveals interesting differences between the mechanisms of modulating TGF-β signaling by dFS and its vertebrate orthologues (Bickel, 2008)

The primary structure of dFS shows both similarities and differences compared to its vertebrate orthologues. dFS is divided into a N-terminal domain (N) and three FS domains (FS1-3) that can be further subdivided into EGF-like and Kazal protease inhibitor-like domains. Compared to its vertebrate orthologues, dFS is substantially larger due to an insertion of 260 amino acids. The insertion contains 55 positively charged amino acids (pI > 10) and is located after the heparin binding site in FS1. In contrast to Drosophila, vertebrates produce a second, long isoform of FS by alternative splicing (FS-315). This form contains a C-terminal extension with many negatively charged amino acids that reduce adhesion to sulfates of proteoglycans at the cell surface. FS-315 is the major mammalian circulating form (Bickel, 2008)

To analyze the function of dFS, dfs cDNA was cloned into expression vectors for tissue culture cells and transgenic flies. From comparison to vertebrate orthologues, the start site was assumed to be at nucleotide 577. At this position, the SignalP server website predicts a signal sequence (MALLIGLLLLNFRLTAA-GTCW) that is cleaved equivalently to the vertebrate FS proteins. However, expression of a construct that contains this predicted signal sequence but lacks the first 279 nucleotides does not result in a translated protein in culture cells and growth inhibition in transgenic flies. In contrast, the complete cDNA is translated in tissue culture cells and inhibits growth in vivo. There are three potential start codons upstream of the predicted signal peptide sequence. Based on the consensus for Drosophila start sites, the most likely start codon is at nucleotide 181-183 (CAACA-ATG). It is noted that there is a full-length dfs cDNA that is 671 nucleotides longer, GH04473 (NM_144119). This cDNA extends the coding sequence by 62 additional amino acids. Although conserved between different Drosophila species, the absence of this additional sequence in the shorter cDNA does not prevent translation and secretion of a growth inhibitory protein. Interestingly, the full-length cDNA does not encode an N-terminal signal peptide sequence either. Instead, several protein analysis programs predict three potential transmembrane domains within the 211 amino acid leader. Thus, in contrast to vertebrate FS proteins, the secretion of dFS might involve an initial membrane-anchoring step (Bickel, 2008)

There are several differences in the structure of dFS and its vertebrate orthologues. One unique characteristic of dFS is the unusual signal trailer that contains three potential transmembrane domains. The results suggest that cleavage likely occurs after the third transmembrane domain at the equivalent position of vertebrate FS proteins. No evidence was found that this feature affects secretion or alters the function of dFS (Bickel, 2008)

A major difference between dFS and its vertebrate orthologues is the large basic insertion. This modification is likely consequential and may alter the activity of dFS. Based on protein folding prediction and the structure of the human FS-Activin complex, the insertion is not well structured and is located after the heparin binding site within the EGF sub-domain of FS1. Since this area projects away from Activin, the positively charged amino acids likely increase the affinity of dFS to heparin sulfate proteoglycans on the cell surface and do not contribute to Activin binding. It is conceivable that this feature leads to a reduction of diffusion and an increase in local concentration of dFS. In addition, it may also reduce diffusion of ligands and enhance the stability of ligand-receptor complexes. Vertebrates have adopted an opposite strategy. They generate a long form of FS by alternative splicing, which contains a negatively charged C-terminal tail that reduces interactions with heparin sulfate. The long form is the major endocrine form in humans. Since it does not efficiently interact with the cell surface, it is not clear whether this form primarily inhibits Activin signaling or actually enhances Activin circulation by preventing unwanted interactions with neighboring cells (Bickel, 2008)

The FS-Activin complex shows that the N domain binds to the wrist region of Activin blocking interaction with the type I receptor. The FS2 domain binds to the type II receptor binding site of Activin. In humans, two FS proteins can encircle an Activin dimer preventing interactions with both types of receptors. It was found experimentally that Activin bound to FS still can interact with the type II receptor. This result is explained if only one molecule of FS is bound to an Activin dimer leaving the second Activin monomer free to bind to receptors. Unlike vertebrate FS proteins, this study found that dFS is proteolytically processed at the C-terminus into two major forms. Based on the migration of epitope tagged forms of dFS on Western blots, the small form contains at least the N and FS1 domains and lacks FS3 and probably part of FS2. Since the small form is immunoprecipitated by dACT, it appears that it can bind to dACT. What is the possible role of this small processed form? The following model suggests that the small form could potentially be a stronger inhibitor: if two small processed dFS proteins without FS2 bind to dACT monomers, they prevent interactions with type I but not type II receptors. A dFS-dACT-PUT complex is inactive, since dACT cannot recruit the type I receptor BABO. In this scenario, the small dFS forms would not only inactivate bound dACT but also reduce interactions of free dACT with type II receptors. If type II receptors were limiting, the small dFS forms would be able to reduce dACT signaling more efficiently than the full-length protein. This hypothesis can be tested by expressing transgenes that encode the small form of dFS. To perform these experiments, the structures of the small and large forms of dFS are currently being determined (Bickel, 2008)

In most experiments, dFS was seen to function as an inhibitor of TGF-β signaling. As a major exception, dFS enhances rather than inhibits DAW signaling in tissue culture assays. Interestingly, unlike other ligands, DAW does not increase but reduces the viability of animals that over-express dFS. This observation suggests that over-expression of dFS may not only be lethal due to reducing the activities of various TGF-β family members but also due to increased signaling of ligands like DAW. If DAW and dFS physically interact, the question arises how dFS can increase ligand signaling. In the early embryo, it has been shown that Short Gastrulation (SOG), the Drosophila orthologues of the vertebrate Chordin protein, not only inhibits signaling of DPP but also facilitates diffusion, which is necessary for the formation of the peak levels of the DPP gradient. The formation of the DPP gradient depends on the equilibrium between bound and unbound DPP by SOG. Similarly, it is conceivable that ligand binding by the positively charged dFS can reduce ligand diffusion. If the concentration or the affinity of dFS for a ligand is high, the ligand is not released or bound again, and signaling is inhibited. Alternatively, if the concentration of dFS or its affinity for a ligand is low, dFS binding can locally increase ligand concentration, while subsequent ligand release will enhance signal transduction. Consistent with such a model, in tissue culture assays dFS rather increases than decreases the level of MAD activation by DPP, while presumably higher levels of dFS inhibit DPP signaling in the wing. Such a dual function could probably be at work during dorsal-ventral axis formation. While dact is not expressed in the early embryo, dfs is present in a dorsal stripe. This pattern corresponds with the highest levels of DPP signaling. Expression of dfs is rather late in the formation of the DPP gradient and is potentially regulated by DPP. It is conceivable that initial low levels of dFS could, like SOG, function to increase the local concentration of DPP and contribute to the formation and maintenance of the dorsal peak activity of DPP signaling. In contrast, higher levels of dFS protein that accumulate by the end of the dorsal-ventral axis formation process may inhibit DPP and contribute to terminating the dorsal DPP signal. Recently, a computational model that described ligand distribution and signaling in the presence of a cell surface BMP-binding protein was developed that supports this idea. A dual function of FS proteins in facilitating and inhibiting TGF-β signaling is also supported by the finding that fs mutants in mice exhibit overlapping phenotypes with activin knock out animals. Consequently, FS was originally described as enhancer of Activin signaling. Finally, facilitating diffusion and redistribution of dACT and potentially other ligands is probably the best mechanism to explain why dFS and vertebrate FS proteins from frogs and humans can partially compensate for the lack of DAW. It is speculated that the vertebrate FS proteins exhibit lower affinities for Drosophila ligands like dACT and diffuse better since they lack the basic insertion of dFS. Most likely, these differences could explain why expression of the vertebrate but not dFS can rescue small but significant numbers of daw mutant animals. Taken together, it is possible that distinct affinities for dFS account for the different interactions seen with various ligands. Further studies are necessary to investigate such a possible dual function of dfs in early embryos and during development (Bickel, 2008).

Two PiggyBac insertions affect dfs transcription. Interestingly, both lines are homozygous viable and do not show any obvious pattern defects. These results suggest that the function of dFS not essential for viability. In mice, a mutation in fs results in various defects with lethal consequences. In contrast, FS, Chordin, and Noggin exhibit overlapping functions in X. tropicalis. It is necessary to reduce all three inhibitors to transform ventral into dorsal tissue during embryogenesis of this species. Although there is no Noggin-like protein in Drosophila, it is possible that dFS also shares overlapping functions with SOG. Expression of sog overlaps with dfs in many tissues, and it is possible that SOG can substitute for dFS in its absence (Bickel, 2008)

The analysis of dfs RNA levels in homozygous f00897 flies shows that they are substantially reduced. Thus, this insertion can be regarded as a hypomorphic allele. It is conceivable that the amount of protein synthesized in this mutant is sufficient for normal development. However, further reduction of dFS by combining f00897 with the deficiency Df(2R)Exel7135 that entirely removes dfs does not affect viability either. In contrast to f00897, the PiggyBac insertion in e03941 clearly disrupts transcription of a full-length mRNA. Based on the location of the insertion, the truncated mRNA likely encodes an altered protein that still contains the N, FS1, and FS2 domains. It lacks the entire FS3 domain and likely contains additional C-terminal amino acids due to altered or absence of splicing. Since the small form is still able to bind dACT, a partially functional dFS protein could still be present in e03941 flies, if proper processing would occur. However, it is unlikely that such an altered protein is processed correctly into the small form of dFS. Since no homozygous lethal lines were obtained in the FRT-mediated deletion screen, it appears that dfs is not lethal. Taken together, the lack of any obvious phenotypes in homozygous f00897 and e03941 lines suggests that dFS is not essential for normal development or is redundant like in some vertebrate species (Bickel, 2008)

Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain

The Drosophila Activin-like ligands Activin-β and Dawdle control several aspects of neuronal morphogenesis, including mushroom body remodeling, dorsal neuron morphogenesis and motoneuron axon guidance. This study shows that the same two ligands act redundantly through the Activin receptor Babo and its transcriptional mediator Smad2 (Smox), to regulate neuroblast numbers and proliferation rates in the developing larval brain. Blocking this pathway results in the development of larvae with small brains and aberrant photoreceptor axon targeting, and restoring babo function in neuroblasts rescued these mutant phenotypes. These results suggest that the Activin signaling pathway is required for producing the proper number of neurons to enable normal connection of incoming photoreceptor axons to their targets. Furthermore, as the Activin pathway plays a key role in regulating propagation of mouse and human embryonic stem cells, the observation that it also regulates neuroblast numbers and proliferation in Drosophila suggests that involvement of Activins in controlling stem cell propagation may be a common regulatory feature of this family of TGF-β-type ligands (Zhu, 2008).

It is not entirely clear how Activin signaling in neuroblast lineages maintains the wild-type number of brain neuroblasts. Approximately 100 neuroblasts per brain lobe are formed during embryogenesis, and most go quiescent at the embryonic/larval transition. From L1-L3 they progressively re-enter the cell cycle to resume their cell lineages. Perhaps the slower cell cycle of Activin pathway mutants inhibits exit from quiescence and promotes premature differentiation. Mutations in trol, which encodes a heparin sulfate proteoglycan Perlecan, prevent neuroblast reactivation and lead to a severe reduction in neuroblast numbers and brain size. Many TGF-β ligands bind to heparin sulfate proteoglycans, and thus part of the effect of Activin signaling on brain size might be mediated by Perlecan or other proteoglycans such as the glypican Dally (Zhu, 2008). The small brain size is not just caused by a reduced number of neuroblasts, however; babo mutant clones that contained a single neuroblast produced fewer daughter cells in a given time window than did wild-type neuroblasts, presumably due to the increased expression of Cyclin A and the delay in metaphase exit. One additional possibility is that the Activin signal may affect neuroblast temporal identity progression in larval neuroblast lineages, similar to the effect of temporal identity mutations on embryonic neuroblast lineages, leading to the failure to produce early, mid or late subsets of larval lineages. Testing this hypothesis awaits the development of markers for different neurons within optic lobe neuroblast lineages (Zhu, 2008).

The importance of Activin in regulating neuroblast proliferation is reminiscent of the positive role that Activin/Nodal signaling plays in regulating the cell cycle of mouse and human ES cells. In those cells, as in Drosophila neuroblasts, Activin/Nodal signaling enhances, but is not absolutely required for, cell proliferation. Another point of potential similarity is that the mES cells endogenously produce an Activin/Nodal signal leading to an autocrine/paracrine regulation of proliferation. While in situ data are not of sufficient resolution to unambiguously assign expression of actβ and daw to particular cell types, both are expressed in the optic proliferation zones where neuroblasts are highly concentrated. It is also possible that some ligand may be supplied by the innervating photoreceptors. Activin is strongly expressed in R7 and 8 and like hh and spitz may provide a tropic signal that simulates proliferation in the target tissue (Zhu, 2008).

Lastly, it is interesting to note that Activins are not the only TGF-β-like factors required for proliferation of Drosophila neuroblasts. The BMP family member Dpp is expressed in four regions in each brain lobe. Two lie in the dorsal and ventral margins of the posterior optic zone neuroepithelium near what has been termed the lamina glial precursor region, whereas the other two smaller zones are more interior at the base of the inner proliferation zone. In the brain, the dpp loss-of-function phenotype is remarkably similar to that seen in babo mutants. A potential trivial explanation for the similarity in phenotypes might be that Activin signaling is required for dpp expression, or vice versa. However, dpp is still expressed in babo mutants, and daw and actβ are both still expressed in dpp mutants, although it is difficult to know in each case whether the levels are equivalent. Thus, both Dpp and Activin signaling appear to be required to stimulate brain neuroblast proliferation (Zhu, 2008).

In addition to regulating proliferation in the brain, Dpp signaling plays a major role in regulating proliferation in other tissues, including the imaginal discs. Once again, Activins may collaborate with BMPs in regulating proliferation in this tissue. In particular, it is noted that babo mutants show ectopic P-H3 staining within the morphogenetic furrow of the eye disc, which is also observed in loss-of-function mutants in the Dpp receptor Tkv. Furthermore, babo mutant wing disc clones can grow large in contrast to clones mutant in dpp signaling components, although the overall sizes of babo mutant discs are not affected proportionally as much as is the brain. Therefore, the way in which BMP and Activin inputs regulate the cell cycle might be different in discs versus the brain, or the two tissues might exhibit different sensitivities to common inputs (Zhu, 2008).

How Activins and BMPs affect the cell cycle is not entirely clear. In the wing disc, Dpp signaling through Tkv/Mad has been shown to promote the G1-S transition. In the brain, babo mutants exhibit a decrease in the M/S ratio, which could be due to a decrease in cells at the G2/M phase of the cell cycle. Consistent with this view, it was found that Cyclin A levels are enhanced in babo mutants and that heterozygosity for a Cyclin A mutation suppresses the babo phenotype. This is very similar to that seen in dally mutants, which also affect brain development by causing a delay in the G2-M transition within the outer proliferation centers. Just as was found for babo mutants, heterozygosity for Cyclin A suppresses the dally cell cycle defect (Zhu, 2008).

One attractive model for how both BMPs and Activins contribute to cell cycle progression is that they regulate the cycle at different points: Activins at G2-M and BMPs at G1-S. Alternatively, since previous work has suggested that Cyclin A probably has roles in regulating both G2-M and G1-S transitions in Drosophila and since Smads can form heterotrimers, it may be that a composite signal composed of a Smad2/Mad/Medea heterotrimer acts at several points in the cell cycle. Interestingly, several potential target genes that are regulated by both Activin and BMP signals in the larval brain have been identified by microarray studies using activated receptors, but no obvious candidates for genes that might influence proliferation are evident within the list (Zhu, 2008).

Lastly, it is noted that daw plays a role in motoneuron axon guidance in the embryo, while actβ has been implicated in mushroom body remodeling and in regulating the terminal steps in photoreceptor R8 targeting during pupal stages. Since both ligands are expressed in each of these tissues, it is possible that there may be functional redundancy that limits the severity of the previously observed phenotypes. Consistent with this view, both actβ and daw modulate neurotransmission at the neuromuscular junction, and the double mutant phenotypes are more severe than those seen in the single mutants, similar to their redundant function in regulating neuroblast proliferation. In conclusion, all available data suggest that Activin signaling plays at least two important roles in Drosophila nervous system development. First, it ensures that the proper numbers of cells are produced in the CNS; and second, it helps establish correct functional connections between neurons and their synaptic partners (Zhu, 2008).


Functions of Activin orthologs in other species

Combinatorial actions of Tgfβ and Activin ligands promote oligodendrocyte development and CNS myelination

In the embryonic CNS, development of myelin-forming oligodendrocytes is limited by bone morphogenetic proteins, which constitute one arm of the transforming growth factor-beta (Tgfβ) family and signal canonically via Smads 1/5/8. Tgfβ ligands and Activins comprise the other arm and signal via Smads 2/3, but their roles in oligodendrocyte development are incompletely characterized. This study reports that Tgfβ ligands and activin B (ActB) act in concert in the mammalian spinal cord to promote oligodendrocyte generation and myelination. In mouse neural tube, newly specified oligodendrocyte progenitors (OLPs) are first exposed to Tgfβ ligands in isolation, then later in combination with ActB during maturation. In primary OLP cultures, Tgfβ1 and ActB differentially activate canonical Smad3 and non-canonical MAP kinase signaling. Both ligands enhance viability, and Tgfβ1 promotes proliferation while ActB supports maturation. Importantly, co-treatment strongly activates both signaling pathways, producing an additive effect on viability and enhancing both proliferation and differentiation such that mature oligodendrocyte numbers are substantially increased. Co-treatment promotes myelination in OLP-neuron co-cultures, and maturing oligodendrocytes in spinal cord white matter display strong Smad3 and MAP kinase activation. In spinal cords of ActB-deficient inhibin Inhbb-/- embryos, apoptosis in the oligodendrocyte lineage is increased and OLP numbers transiently reduced, but numbers, maturation and myelination recover during the first postnatal week. Smad3-/- mice display a more severe phenotype, including diminished viability and proliferation, persistently reduced mature and immature cell numbers, and delayed myelination. Collectively, these findings suggest that, in mammalian spinal cord, Tgfβ ligands and ActB together support oligodendrocyte development and myelin formation (Dutta, 2014).

Age-dependent neuroendocrine signaling from sensory neurons modulates the effect of dietary restriction on rongevity of Caenorhabditis elegans

Dietary restriction extends lifespan in evolutionarily diverse animals. Little is known about how neuroendocrine signals influence the effects of dietary restriction on longevity. This study shows that DAF-7/TGFbeta (see Drosophila Daw and Activin-β), which is secreted from the C. elegans amphid, promotes lifespan extension in response to dietary restriction in C. elegans. DAF-7 produced by the ASI pair of sensory neurons acts on DAF-1/TGFbeta receptors (see Drosophila Thickveins) expressed on interneurons to inhibit the co-SMAD DAF-3 (see Drosophila Medea). Increased activity of DAF-3 in the presence of diminished or deleted DAF-7 activity abrogates lifespan extension conferred by dietary restriction. It was also observe that DAF-7 expression is dynamic during the lifespan of C. elegans, with a marked decrease in DAF-7 levels as animals age during adulthood. This age-dependent diminished expression contributes to the reduced sensitivity of aging animals to the effects of dietary restriction. These studies establish a molecular link between the neuroendocrine physiology of C. elegans and the process by which dietary restriction can extend lifespan (Fletcher, 2017).


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Biological Overview

date revised: 15 March 2017

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