org Interactive Fly, Drosophila

cAMP-dependent protein kinase 1



Structural homeostasis: Compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input

As the nervous system develops, there is an inherent variability in the connections formed between differentiating neurons. Despite this variability, neural circuits form that are functional and remarkably robust. One way in which neurons deal with variability in their inputs is through compensatory, homeostatic changes in their electrical properties. This study shows that neurons also make compensatory adjustments to their structure. The development of dendrites on an identified central neuron (aCC) was studied in the late Drosophila embryo at the stage when it receives its first connections and first becomes electrically active. At the same time, the distribution of presynaptic sites on the developing postsynaptic arbor was charted. Genetic manipulations of the presynaptic partners demonstrate that the postsynaptic dendritic arbor adjusts its growth to compensate for changes in the activity and density of synaptic sites. Blocking the synthesis or evoked release of presynaptic neurotransmitter results in greater dendritic extension. Conversely, an increase in the density of presynaptic release sites induces a reduction in the extent of the dendritic arbor. These growth adjustments occur locally in the arbor and are the result of the promotion or inhibition of growth of neurites in the proximity of presynaptic sites. Evidence is provided that suggest a role for the postsynaptic activity state of protein kinase A in mediating this structural adjustment, which modifies dendritic growth in response to synaptic activity. These findings suggest that the dendritic arbor, at least during early stages of connectivity, behaves as a homeostatic device that adjusts its size and geometry to the level and the distribution of input received. The growing arbor thus counterbalances naturally occurring variations in synaptic density and activity so as to ensure that an appropriate level of input is achieved (Tripodi, 2008).

Since cholinergic neurons provide the only known excitatory input to Drosophila motor neurons in the embryo, the effect of the lack of neurotransmitter (synthesis) in the cholinergic neurons was examined on the development of the aCC dendritic arbor. Acetyl choline is not synthesized in animals mutant for choline acetyl transferase (Chal13), which are therefore immobile and unable to hatch. The development of aCC dendritic arborisations was examined in animals homozygous for the null mutation Chal13. In Cha mutant embryos, it was found that the development of the aCC dendritic arbor proceeds normally until 16 h AEL. However, in the interval between 16 to 18 h AEL, Cha mutants, unlike controls, fail to reduce the rate of dendritic growth. As a result, at 18 h AEL, the extent of the aCC dendritic arbor is increased by about 26% in Cha mutants as compared to controls. It is concluded that in the window of development, when in normal embryos acetyl choline-dependent excitation of aCC first begins and the rate of dendritic growth declines, the absence of neurotransmitter in presynaptic neurons allows postsynaptic growth to continue linearly at an undiminished constant rate. These findings suggest that the aCC dendritic arbor reacts to the loss of synaptic input by increasing in size. A consequence of this increase in overall dendritic length is that it allows the dendritic arbor to explore a larger portion of the neuropile than in normal animals. In fact, it was observed that in Cha mutants, the dendritic arbor of aCC extends into regions of the neuropile that are not normally invaded in control conditions (Tripodi, 2008).

Growth adjustments by the aCC arbor appear to operate sequentially at two levels. In the first instance, the dendritic arbor determines the presence or absence of presynaptic partners. This event is independent of presynaptic activity. It is also a local event that appears to affect primarily neurites receiving presynaptic sites, which act as a local stop-growing signal. The second step depends on the activity of the synapse. In normal conditions, presynaptic sites are able to inhibit growth, both in synaptic neurites as well as neighbouring nonsynaptic sister neurites. Interestingly, the dendritic arbor does not measure the efficacy of synapses (since there is no significant change in arbor size in Ace mutants as compared to wild type), but simply determines whether a synapse is active or not. However, the possibility of there being a compensatory response to reduced efficacy cannot be excluded. This phase of activity-dependent inhibition of dendritic growth is mediated by postsynaptic activation of PKA (Tripodi, 2008).

The morphology of dendrites is likely to be an important determinant of the connectivity state of a nervous system. It is not by chance that even in complex nervous systems, one of the most distinctive feature of different classes of neurons is probably the morphology of their dendritic arbors. Indeed, the anatomical discrimination of different classes of neurons, based on their dendritic morphology, has often anticipated and predicted molecular, electrophysiological, and computational differences (Tripodi, 2008).

Therefore, an appreciation of the logic governing the assembly of a nervous system must include an understanding of how dendritic morphology is acquired. Although many of the distinctive features that differentiate the dendritic morphologies of different classes of neurons are likely to be cell-autonomously and genetically determined, the effect of partner-derived cues, as shown in this study, can have a substantial impact in modulating these features. This investigation was initiated by analysing the effect of presynaptic transmission in shaping the morphology of the postsynaptic dendritic arbor. Previous studies on this issue have not reached a clear agreement on the role of activity in regulating dendritic growth. Even though in some instances it has been reported that activity has no effect on regulating arbor growth, in the majority of studies, activity emerges clearly as an essential modulator of dendritic remodelling. The main issue has been whether the incoming presynaptic input acts as a trophic factor that promotes arbor growth or whether it delivers a stop-growing signal. Unfortunately, with a few notable exceptions, many of these studies were carried out in different animals, in different neural populations, at different developmental stages, and by using different experimental approaches (genetic manipulation or pharmacological treatments), thus making it difficult to find a common theme in the results. One major source of variation that could explain the differences in the results of previous studies is likely to be the developmental stage at which manipulations were applied. For instance, a single class of tectal neurons in Xenopus tadpoles appears to respond in opposite ways to presynaptic input at different developmental stages. In immature neurons, presynaptic input acts as a growth-promoting signal, whereas in mature neurons, it acts as a stop-growing or stabilization signal. In this study, no evidence was found for a growth-promoting effect of synaptic activity. Instead, it was shown that synaptic input inhibits dendritic growth starting from the earliest stages at which neurotransmission occurs (16-18 h AEL). Altering activity before the onset of evoked synaptic transmission causes no dendritic phenotype (14-16 h AEL). It appears that developing Drosophila embryonic motor neurons behave like mature tectal neurons in Xenopus (Tripodi, 2008).

The difference in the effect obtained at different stages in Xenopus tectal neurons nicely correlates with a change in their molecular characteristics. At later stages, when synaptic input acts as a stop-growing signal, the tectal neurons in Xenopus have acquired the ability to respond to local calcium increases via the activation of a calcium-dependent protein kinase CamKII. In Drosophila, aCC appears be sensitive to the activation state of a different protein kinase, PKA (which is also regulated, albeit indirectly, by intracellular calcium levels) throughout the interval of development that was studied (Tripodi, 2008).

A great deal of attention has been given to global changes in postsynaptic activity and how these changes might regulate global dendritic growth. However, although this is an extremely interesting question, it is difficult to imagine how global variations in the state of activation of the whole dendritic arbor or the soma might contribute to fine-tuning the morphology of dendritic arbors. Far more compelling would be a system that was able to calculate and respond to local changes in activity levels. It is well known that calcium levels can be altered locally at the synaptic site following synaptic input. It is also known that changes in dendritic levels of calcium can induce dramatic changes in dendritic morphology. Nevertheless, there have been few investigations of how dendritic morphology might be altered locally by synaptic activity (Tripodi, 2008).

Because the system used allows the study a single identified postsynaptic neuron whose presynaptic input can be altered, this study begins to address the issue of local versus global changes of dendritic morphology induced by synaptic activity. Analyzing the branching pattern of the dendritic arbor with respect to the position of its synaptic inputs highlights some interesting and unexpected features. By simply looking in wild-type animals, it is clear that neurites bearing presynaptic sites branch less than nonsynaptic neurites of the same arbor. Through experimental manipulation, this study has shown that this local inhibition of the growth and branching of synaptic neurites appears to be mediated by contact between pre- and postsynaptic partners and does not require evoked transmission. The lack of this contact-dependent inhibition of dendritic growth is already apparent when the terminals normally presynaptic to aCC are mistargeted before they can form functional synapses at 16 h AEL. Moreover, this effect cannot be attributed to an interference with transmission, since Cha mutants of the same stage (16 h AEL) do not show this dendritic overgrowth phenotype. Loss of neurotransmitter release, on the other hand, can also induce an overgrowth of the postsynaptic dendritic arbor, though only after synapses would have been active for 2 h in normal conditions (i.e., 18 h AEL). It was found that this neurotransmitter-dependent overgrowth is due to increased extension of nonsynaptic segments that are immediately adjacent to neurites receiving presynaptic sites. Thus, neurotransmitter release at the synaptic sites acts on the nonsynaptic sister neurites to inhibit their extension (Tripodi, 2008).

It is concluded that the geometry of the dendritic tree is regulated by two partner-dependent mechanisms. First, contact with presynaptic terminals locally inhibits dendritic growth and branching in an activity-independent fashion. A second inhibitory effect requires evoked presynaptic neurotransmitter release. It extends from these sites to the immediate proximity, affecting nonsynaptic sister branches. This second 'neighbourhood effect' could be mediated by local increases in dendritic calcium levels (Tripodi, 2008).

Neurotransmission-dependent variations in intracellular calcium levels are an attractive mechanism that might implement activity-dependent local rearrangement of the dendritic arbor geometry. Therefore the role of the protein kinase PKA in dendritic remodelling was investigated, since its activity is regulated directly or indirectly by calcium levels. PKA is activated by increases in the intracellular levels of cAMP. It has been shown that levels of cAMP are finely regulated by intracellular levels of calcium, which in turn respond to levels of presynaptic neurotransmitter release (Tripodi, 2008).

PKA signalling in postsynaptic cells has been shown to mediate homeostatic responses. For instance, at the neuromuscular junction, postsynaptic PKA signalling modulates quantal size (Davis, 1998), while centrally, it mediates homeostatic change in the electrical excitability of aCC neurons following alterations to presynaptic input (Baines, 2003). This study has shown that PKA activity is also a potent modulator of dendritic morphology. PKA signalling is probably downstream of presynaptic transmission mediating the inhibition of dendritic growth. Overexpression of a constitutively active form of PKA (PKAact) is able to rescue the dendritic overgrowth phenotype that ensues in the absence of presynaptic transmission in Cha mutants. However, overexpression of PKAact in aCC in control animals has no measurable effect on dendritic development, suggesting that PKA signalling operates to saturation under normal levels of synaptic input. Although neurotransmission is clearly one signal that regulates dendritic development through downstream PKA signalling, it is not necessarily the only one (Tripodi, 2008).

The term homeostasis has classically been used to refer to compensatory variations in the electrical properties of neurons that tend to counterbalance changes in their synaptic input. This paper proposes that neurons might combine the homeostatic regulation of electrical properties with compensatory structural adjustments of their dendritic geometry. It is argued that variations in the morphology of aCC dendritic arbors represent such compensatory adjustment of dendritic growth and branching (Tripodi, 2008).

These experimental observations indicate that eliminating synaptic input induces a compensatory dendritic overgrowth in the postsynaptic neuron, whereas an increase in the density of active synapses induces the opposite effect. Interestingly, increasing the overall level of neurotransmitter released at presynaptic terminals without altering the density of presynaptic sites on the arbor does not induce compensatory adjustments of the postsynaptic arborisation. This suggests that the compensatory changes in dendritic arbor morphology that were observed act to compensate for variations in the density of active synaptic release sites rather than variations in the global state of dendritic or neuronal depolarization. This is in agreement with observations that changes in dendritic morphology in response to changes in presynaptic input or synaptic density appear to be implemented locally rather than across the entire arbor. This structural homeostasis, therefore, seems to work on a local scale, allowing particular regions of the dendritic arbor to compensate for variation in their inputs while leaving other regions of the arbor substantially unchanged. This could be an effective mechanism for neurons that use distinct regions of their dendritic arbor to independently compute different inputs (Tripodi, 2008).

It will be interesting to understand whether electrical homeostasis and what is proposed to be a structural homeostasis operate in concert to shape the postsynaptic response, or whether one or the other is preferentially deployed depending on the circumstances. At the moment, it is not possible to answer this question, and more experiments are required (Tripodi, 2008).

Nonetheless, one can begin to envisage how PKA signalling in the context of electrical homeostasis might be integrated with the structural homeostasis that was shown in this study. As shown by Baines (2003), postsynaptic overexpression of a constitutively active form of PKA induces a change in the electrical properties of aCC, resulting in a decrease in excitability. Overexpression of a PKA inhibitor, on the other hand, does not modulate the excitability of aCC. This study has described a perfect mirror image of this situation, namely that the expression of a PKA inhibitor induces a dendritic overgrowth phenotype, while expression of a constitutively active form of PKA does not. Bringing these two lines of observations together suggests the following model: following a decrease in presynaptic input (and PKA signalling), the postsynaptic neuron expands its receptive field so as to increase the number of presynaptic sites that it contacts. In these circumstances, an increase in postsynaptic excitability would not be required. On the other hand, following an increase in presynaptic input, the postsynaptic neuron decreases its excitability (via increased PKA signalling) without the need to reduce its receptive field (Tripodi, 2008).

The view is favoured that electrical and structural homeostatic mechanisms might indeed be integrated by neurons. This would enable the cells to implement compensatory changes that resulted in adjustments to their electrical characteristics and dendritic geometry, so as to ensure that in an inherently variable environment an adequate pattern and level of connectivity and excitability is achieved (Tripodi, 2008).

Germ cell line

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. A mutation affecting Pka-C1 acts in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of Bicoid and Oskar mRNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition; Oskar RNA fails to accumulate at the posterior, and Bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns previously reported for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells (Lane, 1994).

Pka-C1 is required in fly oogenesis. Intercellular bridges in egg chambers from PKA deficient females are unstable, leading to the formation of multinucleate nurse cells by fusions of adjacent cells. Germline clones of cells homozygous for null mutations of PKA-C1 indicate that PKA acts autonomously in the germline. Highest levels of PKA catalytic subunit protein are associated with germ cell membranes, suggesting that targets of PKA are associated with the membrane or membrane skeleton and contribute to the stabilization of intercellular bridges. The migration of a subset of follicle cells, the border cells, is also disrupted by germline PKA mutations, implying that nurse cell junctions provide an essential path for border cell migrations (Lane, 1995).

The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).

Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptcmat-zyg+ and smomat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).

As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptcmat- (and to a lesser extent pkamat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).


Seventy-six genes have been identified that are strongly expressed in the Drosophila ring gland during development. For nine of these, further studies of expression pattern, mutant phenotype and molecular nature identify the genes as strong candidates to carry out an important role in endocrine functions controlling development. Two of the genes identified encode products that have already been implicated in the functioning of prothoracic glands in other insects. The Calmodulin gene is expressed exclusively and at high levels in the ring gland of third-instar larvae, suggesting an important, presumably endocrine function for calmodulin in that tissue, as has already been suggested for lepidopterans. Calmodulin and other Ca2+-binding proteins are integral to the transduction of a wide range of Ca2+-dependent signals; there is clear evidence for the Ca2+ dependence of ecdysteroid molting hormone (EC) production in the Manduca larval prothoracic gland (PTG), at least for the commitment peak early in the last larval instar. It is known that Ca2+ activates prothoracic gland adenylate cyclase both directly and as a complex when bound to calmodulin. Since cAMP phosphodiesterase activity is low at this stage, cAMP is expected to accumulate. Both large and small PTTHs (see Bombyx and Manduca prothoracicotropic hormone) stimulate increased cAMP levels in PTG; a rise in cAMP levels occurs with PTTH-stimulated EC production in early last-instar PTG (Harvie, 1998 and references).

The catalytic subunit of protein kinase A (PKA or cAMP-PK) is also expressed in the Drosophila ring gland. This protein probably functions downstream of cAMP in the Ca2+-cAMP-dependent signaling pathway. PKA is activated in M. sexta PTGs by PTTH immediately prior to EC production. This is consistent with the idea that activation of the Ca2+-cAMP-dependent signaling pathway by PTTH leads to PKA-dependent phosphorylation of key proteins, including ribosomal protein S6, and that this causes changes in selective translation leading to increased EC production (Harvie, 1998 and references).

The catalytic subunit of cyclic AMP-dependent protein kinase A is required for the correct spatial regulation of dpp expression during eye development. Loss of Pka-C1 function is sufficient to produce an ectopic morphogenetic wave, marked by premature ectopic photoreceptor differentiation and non-autonomous propagation of dpp expression. Pka-C1 lies in a signaling pathway that controls the orderly temporal progression of differentiation across the eye imaginal disc (Strutt, 1995).

Unlike the thoracic discs, the anterior and posterior compartmental organization of the genital imaginal disc is compound, consisting of three primordia Ð the female genital, male genital, and anal primordia. Each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in a compartment-specific manner in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. en and inv are required in the posterior comparment of the genital disc to repress dpp and activate hh. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments (Chen, 1997).

cAMP-dependent protein kinase A and engrailed-invected are genes known to play compartment-specific functions in other discs. The anterior/posterior patterning functions of these genes are conserved in the genital disc. en-inv mutant clones cause posterior to anterior transformations in adult terminalia. Pka is required to repress ptc, dpp and wg expression in the anterior compartment of the genital disc. Pka mutant clones result in pattern duplications in adult terminalia (Chen, 1997).

The adult clonal phenotypes of protein kinase A and engrailed-invected mutants provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. A new model has been proposed to describe the anterior and posterior compartmental organization of the genital disc. Each of the three primordia (female, male and anal) is composed of its own anterior and posterior compartments. Each primordium has a larger anterior compartment and a smaller posterior compartment. Each genital disc is divided into anterior and posterior compartment (Chen, 1997).

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, the formation of new boutons was investigated in NMJs lacking synapsin [Syn-], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. Budding of new boutons at Syn- NMJs was significantly diminished, and new boutons in Syn- preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway was assayed. Preparations were pretreated with forskolin to raise cAMP levels; this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn- preparations. To examine the trafficking of synapsin during synaptic growth, transgenic animals were generated expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway (Vasin, 2014).


Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, two mutant phenotypes were undertaken: (1) the batone mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map (batone has not yet been cloned); (2) ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic 'tumors'. Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. It is proposed that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. It is proposed that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing (Kiger, 2001).

This is the first report of hemocytes in the wings of newly eclosed flies. Their presence must have been overlooked because of the debris created by death of the wing epithelium. The power of the GAL4/UAS system to express GFP specifically in hemocytes has now enabled their detection. An apposition of dorsal and ventral wing surfaces occurs after eclosion. Two earlier appositions, followed by separations, of dorsal and ventral wing epithelia have occurred during pupal development. During each of these appositions, hemocytes are believed to secrete extracellular matrix (ECM) that binds the epithelia together. Subsequent separations are believed to be caused by proteolysis and phagocytosis of the ECM by hemocytes. Evidence from Drosophila and from Manduca sexta indicates that components of the ECM are found in hemocytes during pupal development. Therefore, it is reasonable to propose that hemocytes persist between the wing surfaces after eclosion where they phagocytose apoptotic epithelial cells and secrete an ECM that binds dorsal and ventral wing blades together. As a result of the destruction of epithelial cells, this ECM would have to bind directly to the cuticle of the wing surfaces and may contain a protein with chitin-binding domains. Thus, it is likely that the death of the wing epithelia is apoptotic (Kiger, 2001).

The identification of the fluorescent cells in normal wings as hemocytes is an inference based on studies of pupal development and on the detached fluorescent cells observed in wings of flies expressing PKAc, Ricin A, or PanDeltaN, a dominant-negative pangolin transgene. The latter cells fit previous descriptions of hemocytes. The association of PKAc expression with melanotic tumors (known to be caused by hemocytes) in various parts of the body strengthens this identification. The fluorescent cells in normal wings are tightly bound in a strikingly precise array that makes them an integral part of the wing, as might be expected if their role is to secrete ECM. As such, they do not exhibit characteristics that readily identify them as hemocytes. However, PKAc, Ricin A, or PanDeltaN expression disrupts this cellular array and prevents bonding of dorsal and ventral cuticular wing blades without affecting synthesis of the cuticle that forms the wing, demonstrating that the fluorescent cells in the wing are distinct from wing epithelial cells (Kiger, 2001).

Fate mapping places the focus of bae gene activity in the anterior neuroectoderm, a location that could become part of either the brain or the ring gland and distinct from the mesodermal origin of hemocytes. A striking feature of the data is that gynandromorphs either have both wings fully normal or fully mutant. This observation is consistent with a bilateral pair of nervous system primordia that interact in a submissive manner to establish the mutant wing phenotype in a nonautonomous manner. Thus, the role of bae could be to control wing maturation by the release of a hormone that increases blood pressure, causing wing unfolding, and that activates hemocytes to perform their roles of phagocytosis and ECM synthesis. Wing inflation has been ascribed to an unidentified neuroendocrine factor different from the eclosion hormone. A phenotype very similar to that of bae is produced by ectopic expression of UAS-dCBP(nej+) using GAL4 strains expressed in specific central nervous system cells (Kiger, 2001).

Comparison of the effects of Ricin A and of PKAc on wing maturation indicates that ectopic PKAc does not simply inactivate hemocytes. Instead, it appears to substitute one normal function of hemocytes for another. Rather than carry out phagocytosis and ECM synthesis, hemocytes enter into an innate immune response in which lamellocytes are differentiated and crystal cells melanize target cells. Evidently, aggregation of lamellocytes within the wing blade interferes with wing expansion, and loss of normal hemocyte function interferes with bonding of dorsal and ventral surfaces. The observation that the effect of ectopic PKAc on the wing is suppressed by overexpression of Pan, the Drosophila homolog of mammalian blood cell transcription factors (lymphocyte enhancer-binding factor 1 and T cell factor), suggests that ectopic PKAc inhibits, or represses synthesis of, Pan, which in turn inhibits Wingless target gene expression. This conclusion is strengthened by the observation that ectopic expression of UAS-dCBP(nej+) using GAL4-30A produces phenotypes similar to those caused by ectopic PKAc. Pan is bound and its transcriptional activity inhibited by dCBP. Expression of PanDeltaN, a dominant-negative inhibitor of Wingless target gene expression, elicits what seems to be a massive induction of the cellular innate immune response. Thus, the Wingless signal transduction pathway may be involved in regulating a choice between the innate immune response and the apoptotic/ECM response (Kiger, 2001).

The dominant-lethal interaction between ectopic PKAc and bae is intriguing. When and how death occurs needs closer examination, as does the cellular focus of bae activity. What role PKAc normally plays in regulating hemocyte behavior remains to be investigated. The association of a wing phenotype with altered hemocyte behavior should provide a means of identifying additional genes involved in hemocyte function during wing maturation (Kiger, 2001).


Involvement of the cAMP cascade in olfactory learning and memory in Drosophila is suggested by the aberrant behavioral phenotypes of the mutants dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase). PKA-C1 is preferentially expressed in the mushroom bodies. Mutants produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing double mutant heteroallelic viable animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes (Skoulakis, 1993).

dunce, rutabaga and Pka-C1 are expressed preferentially in mushroom bodies, neuroanatomical sites that mediate olfactory learning in Drosophila. Interestingly, the PDE (Dunce) and the catalytic subunit of PKA are found primarily in axonal and dendritic compartments of the mushroom body cells, whereas the adenyl cyclase (Rutabaga) is found primarily in the axonal compartment. The reason for this differential compartmentalization is unclear, although the hypothetical role of adenyl cyclase as coincidence detector would predict that conditioned stimuli and unconditioned stimuli (See Dunce site for definition) are integrated in the axonal compartment (Davis, 1995).

In both Drosophila and the honeybee Apis mellifera, cyclic adenosine monophosphate (cAMP)-dependent processes have been implicated in mechanisms of learning. This study characterizes the major target of cAMP in adult animals: the type II cAMP-dependent protein kinase (PKAII). In both species, PKAII (composed of Pka-R2 and Pka-C1 subunits) is restricted to neuronal tissue, where it accounts for more than 90% of total PKA activity. Although the intensity of PKAII immunoreactivity differs between distinct brain regions, labeling is detectable in all neuropiles and most somata. While the visual neuropiles, the antennal lobes, and structures of the central brain exhibit intermediate immunostaining, the mushroom bodies show high labeling and contain a three- to four-fold higher PKA activity, as compared to other neuropiles. Since the mushroom bodies are central sites of olfactory learning mediated via cAMP-dependent signaling, the modulatory functions of transmitters on PKA activity were tested using Kenyon cells from the honeybee. Agents that elevate cytoplasmic Ca2+ levels have no effects on PKA activity in cultured Kenyon cells. Dopamine, serotonin, and octopamine, however, cause an increase in PKA activity in Kenyon cells. The modulation of PKA activity by octopamine, the putative transmitter of the unconditioned stimulus in associative olfactory learning in the honeybee, together with the findings on the central role of the cAMP cascade in Drosophila mushroom bodies, suggests a major implication of PKAII-mediated phosphorylation in learning and memory in both Drosophila and Apis (Muller, 1997).

Axonal injury and regeneration in the adult brain of Drosophila

Drosophila is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the CNS. A versatile and physiologically robust preparation has been developed for the long-term culture of the whole Drosophila brain. This method was used to develop a novel Drosophila model for CNS axonal injury and regeneration. Similar to mammalian CNS axons, injured adult wild-type fly CNS axons fail to regenerate, whereas adult-specific enhancement of protein kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms after injury. This model was developed to explore pathways that induce robust regeneration; adult-specific activation of c-Jun N-terminal protein kinase signaling was found to be sufficient for de novo CNS axonal regeneration injury, including the growth of new axons past the lesion site and into the normal target area (Ayaz, 2008).

The first models for axonal severing in the CNS of the fly have been developed and have unveiled a marked conservation in the molecular mechanisms underlying neuronal responses to injury in flies and mammals (Leyssen, 2007). Although these paradigms hold promise to significantly enhance insights into axonal degeneration, they do not allow the evaluation of axonal regeneration. Increased insight into the process of regeneration is crucial from a therapeutic point of view that aims at stimulating axons to reconnect to their postsynaptic target. This work describes a protocol that allows, in addition to a versatile series of manipulations, the precise and reliable severing of sLNv axons in explanted adult Drosophila brains. This technique provides the opportunity to study axonal regeneration in the Drosophila CNS and use genetic screens to identify factors that promote axonal regeneration (Ayaz, 2008).

The regenerative responses of injured axons in Drosophila brain explants show remarkable similarities to those seen in mammals. First, the distal, severed ends of the axons became fragmented in a manner that is reminiscent of Wallerian degeneration in mammals, as has been described also in living Drosophila. In both cases, unconnected axons rapidly split into small vesicles, which then disappear. A major advantage of the explant model is that it also allows the study of the proximal, injured axon. One day after injury, the proximal axonal stump forms a bulbar structure that shows morphological similarities to the dystrophic retraction bulbs seen in injured mammalian CNS axons. This structure is extensively remodeled and develops small, and occasionally longer, thin axonal sprouts. Similar morphological changes have been described recently in severed, GFP-marked axons of the mouse spinal cord (Ayaz, 2008).

The lack of efficient regeneration of injured Drosophila axons in brain explants is not attributable to the experimental setup. (1) sLNv neurons in the preparation appear healthy both morphologically and functionally. (2) Axons in explanted larval brains do form elaborate growth cones and grow large distances over the course of a few days. (3) The proximal axonal stumps are clearly dynamic and capable of sending out axonal sprouts. It is therefore suggested that the lack of efficient sLNv axonal regeneration is an intrinsic feature of the adult fly brain. Indeed, the adult-specific, cell-autonomous manipulation of the catalytic activity of PKA can stimulate a generally dormant regrowth capacity. A significantly larger proportion of lesioned brains become capable of extending new axons at the cut tip. This is very similar to the mammalian cAMP-based enhancement of regrowth. In both cases, regrowth is achieved by increasing PKA catalytic activity, either reducing the negative regulation of PKAc through adding of cAMP in the mammalian studies or overexpressing a catalytic mutant version of PKA that is free from endogenous inhibition. In summary, the data presented in this study support the concept that the fly CNS is closer in its response to injury to the mammalian CNS than the mammalian PNS. The mammalian PNS regenerates spontaneously, whereas, in contrast, both the fly and mammalian CNS fail to regenerate except when additional signals are activated. In the case of PKA, the same signal enhances regeneration in both models. This, in addition to several other details, such as the anatomy of the retracting injured axons and the appearance of a lesion gap, support the conservation of key injury response mechanisms between the two CNS systems (Ayaz, 2008).

In mammalian models for CNS regeneration, several modes of neuronal repair are proposed. These neuronal recoveries can be accomplished directly by either the transected axons itself or by sprouts from unlesioned axons in the neighborhood into the denervated target area. However, what is defined as true axonal regeneration is the reentry of the cut axons themselves onto the denervated target area. Axonal regeneration encompasses several types of neuronal responses that can be grossly divided into the following steps: (1) regrowth of the cut axons, (2) guidance of the newly growing axons toward the normal area of innervation, and finally (3) reinnervation of the denervated target area (Ayaz, 2008).

Despite the promising results of PKA manipulation, a constitutively active form of PKA is not sufficient to produce axons that can extend into the original target area. Therefore the hypothesis was tested that JNK signaling might support better regeneration. The data suggest that JNK is likely to be a key pathway in CNS axonal regeneration after injury. Inhibiting JNK activity results in a high tendency to inhibit the length of the newly grown axons compared with control brains, whereas its constitutive activation results in a massive increase in the penetrance and extent of regeneration, with one-third of all brains showing regenerated axons reentering the target area. It is surprising that activation of only a single pathway is sufficient to enhance the three major anatomical criteria of regeneration. This may be explained by at least two alternative models. It may be that LNv neurons, and presumably other CNS neurons as well, retain their proper guidance and targeting signals well into adult life, and as such genetic manipulations stimulating the frequent growth of lengthy axons can result in axons reaching the correct target area. Alternatively, JNK activation may act as a reprogramming signal allowing neurons to reactivate their specific developmental pathways. Although a role for JNK in axonal navigation cannot be excluded, previous work shows that JNK can stimulate different cell types to grow longer axons, without influencing their guidance properties. In this context, it may seem surprising that the induction of JNK signaling observed during axonal injury is not sufficient to induce regeneration. However, it is in fact likely that the transient upregulation of JNK does play a role in stimulating growth after injury because, when injured axons express dominant-negative forms of JNK, they show a strongly reduced regrowth tendency compared with wild-type brains, although this reduction remained just below statistical significance. The reason why activated JNK signaling has such a dramatic influence on regeneration is twofold. First, an activated form of the protein is no longer subject to endogenous negative regulation. Second, this form is expressed as a UAS transgene, which means that its expression is permanent and not transient, in contrast to what was observed under wild-type injury conditions (Ayaz, 2008).

Control of lipid metabolism by Tachykinin in Drosophila

The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions (Song, 2014).

Previous studies in mammals have indicated that a few gut secretory hormones, like GLP1 and GLP2, are involved in intestinal lipid metabolism. However, due to gene and functional redundancy, mammalian genetic models for gut hormones and/or their receptors with severe metabolic defects are not available. This study has establish that Drosophila TKs produced from EEs coordinate midgut lipid metabolic processes. The studies clarify the roles of TK hormones in intestinal lipogenesis and establish Drosophila as a genetic model to study the regulation of lipid metabolism by gut hormones (Song, 2014).

Six mature TKs, TK1-TK6, are processed and secreted from TK EEs in both the brain and midgut (Reiher, 2011). Using a specific Gal4 driver line, gene expression in TK EEs was specifically manipulated, leading to the demonstration that loss of gut TKs results in an increase in midgut lipid production. Further, this study showed that TKs regulate intestinal lipid metabolism associated with TKR99D, but not TKR86C, which is consistent with the expression of these receptors. Consistent with previous reports that TK/TKR99D signaling regulates cAMP level and PKA activation, loss of gut TKs is associated with a reduction in PKA activity in ECs, and overexpression of a PKA catalytic subunit was able to reverse the increased intestinal lipid production associated with loss of TKR99D. In addition, the transcription factor SREBP that triggers lipogenesis was controlled by TK/TKR99D/PKA signaling. Taken together, these results suggest that TKs produced from EEs regulate midgut lipid metabolism via TKR99D/PKA signaling and regulation of, at least, SREBP-induced lipogenesis in ECs (Song, 2014).

Interestingly, this study reveals that TKs derived from either the brain or gut exhibit distinct functions: TKs derived from gut control intestinal lipid metabolism, whereas TKs derived from brain control behavior. This is reminiscent of the distinct functions of mammalian secreted regulatory peptides, where different spatial expressions or deliveries of peptides like Ghrelin can result in distinct physiological functions. In addition, some prohormones encode multiple mature peptides that can have multiple functions. For example, processing of proglucagon in the pancreas α cells preferentially gives rise to glucagon, which antagonizes the effect of insulin. In intestine L cells, however, proglucagon is mostly processed into GLP1 to promote insulin release. These studies of TKs exemplify how secreted regulatory peptides derived from different tissues can be associated with fundamentally diverse physiological functions. Clearly, additional studies examining the function of secreted peptides in a cell-type- and tissue-specific manner are needed to fully appreciate and unravel their complex roles both in flies and mammals (Song, 2014).

There is a growing body of studies emphasizing that intestinal lipid metabolism is key to the control of systemic lipid homeostasis. For example, chemicals such as orlistat, designed to inhibit dietary lipid digestion/absorption in the intestine, efficiently reduce obesity. In addition, mammalian inositol-requiring enzyme 1β deficiency-induced abnormal chylomicron assembly in the small intestine results in hyperlipidemia. Similarly, in Drosophila, dysfunction of intestinal lipid digestion/absorption caused by Magro/LipA deficiency eventually decreases whole-body lipid storage and starvation resistance in Drosophila. Further, intestinal lipid transport, controlled by lipoproteins, is essential for systemic lipid distribution and energy supply in other tissues. Consistent with these observations, this study demonstrates that increased midgut lipid synthesis associated with gut TK deficiency is sufficient to elevate systemic lipid storage. Although TK ligands and TK receptors show high homologies between mammals and fruit flies, whether mammalian TK signaling plays a similar role in intestinal lipid metabolism is largely unknown. Future studies will reveal whether mammalian TK signaling affects intestinal lipid metabolism as in Drosophila. If this is the case, it may provide a therapeutic opportunity for the treatment of intestinal lipid metabolic disorder and obesity (Song, 2014).

Production and secretion of gut hormones are precisely regulated under various physiological conditions. Similar to previous observations that starvation induces gut TK secretion in other insects, this study found that nutrient deprivation promotes TK production in EEs. Interestingly, feeding of amino-acid-enriched yeast, but not coconut oil or sucrose, potently suppressed gut TK levels, indicating that amino acids may act directly on TK production in EEs. It has been reported that dietary nutrients regulate gut hormone production through certain receptors located on the cell membrane of EEs in mammals. Future studies will be necessary to elucidate the detailed mechanism by which nutrients regulate TK production from EEs (Song, 2014).

The Octopamine receptor Octβ2R regulates ovulation in Drosophila melanogaster

Oviposition is induced upon mating in most insects. Ovulation is a primary step in oviposition, representing an important target to control insect pests and vectors, but limited information is available on the underlying mechanism. This study reports that the beta adrenergic-like octopamine receptor Octβ2R serves as a key signaling molecule for ovulation and recruits Protein kinase A and Ca2+/calmodulin-sensitive kinase II as downstream effectors for this activity. The octβ2r homozygous mutant females are sterile. They displayed normal courtship, copulation, sperm storage and post-mating rejection behavior but are unable to lay eggs. It has been shown previously that octopamine neurons in the abdominal ganglion innervate the oviduct epithelium. Consistently, restored expression of Octβ2R in oviduct epithelial cells is sufficient to reinstate ovulation and full fecundity in the octβ2r mutant females, demonstrating that the oviduct epithelium is a major site of Octβ2R's function in oviposition. It was also found that overexpression of the protein kinase A catalytic subunit or Ca2+/calmodulin-sensitive protein kinase II leads to partial rescue of octβ2r's sterility. This suggests that Octβ2R activates cAMP as well as additional effectors including Ca2+/calmodulin-sensitive protein kinase II for oviposition. All three known β adrenergic-like octopamine receptors stimulate cAMP production in vitro. Octβ1R, when ectopically expressed in the octβ2r's oviduct epithelium, fully reinstated ovulation and fecundity. Ectopically expressed Octβ3R, on the other hand, partly restores ovulation and fecundity while OAMB-K3 and OAMB-AS that increase Ca2+ levels yielded partial rescue of ovulation but not fecundity deficit. These observations suggest that Octβ2R have distinct signaling capacities in vivo and activate multiple signaling pathways to induce egg laying. The findings reported in this study narrow the knowledge gap and offer insight into novel strategies for insect control (Lim, 2014; PubMed).

cAMP-dependent protein kinase 1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Effects of Mutation | References

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