Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila

The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, principles underlying the organisation of the motor system have been uncovered. Dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. These results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

The analysis began by correlating the positions of motor neuron dendrites with the distribution of their muscle targets in the periphery. Motor neurons were retrogradely labelled in a pairwise fashion and the positions of their dendritic arbors were mapped. Because of an interest in the mechanisms that underlie the assembly of the motor system, focus was placed on stages when each motor neuron first establishes a characteristic domain of arborisation within the neuropile (early stage 17, 15h after egg-laying [AEL]) (Landgraf, 2003).

Motor axons project into the muscle field via two main nerves, the intersegmental (ISN) and the segmental nerve (SN). The transverse nerve (TN) runs along the segment border and has few motor axons. Choice of nerve root is one of several features that divide the motor neurons into two principal sets, the ISN and SN. (1) The cell bodies of SN motor neurons are located in the same segment as the muscles that they innervate, whereas ISN motor neuron somata are located in the segment next anterior (with the exception of the RP2 and two neuromodulatory efferent ventral unpaired median [VUM] neurons. (2) ISN motor neurons innervate internal muscles, which span a segment from anterior to posterior, whereas SN (and the TN) motor neurons innervate external muscles. External muscles are distinct from the internal set in several respects: (1) they are generally transverse; (2) unlike internal muscles, they require wingless (wg) signalling for their specification; (3) external (but not internal) muscles and their innervating motor neurons express the cell adhesion molecule (CAM) Connectin, with the single exception of muscle ventral transverse 1 (VT1) (Landgraf, 2003 and references therein).

In addition, ISN and SN motor neurons elaborate their dendrites in distinct regions of the neuropile. Dendrites of ISN motor neurons occupy a domain extending posteriorly from the posterior part of one neuromere into the anterior part of the next. SN motor neuron dendrites occupy a domain that lies between the domains of ISN motor neuron arbors (Landgraf, 2003).

Thus, the organisation of the body wall muscles into internal and external sets is reflected centrally in patterns of motor neuron arborisations. The innervating motor neurons project their axons through different nerves and elaborate their dendritic fields in distinct regions of the neuropile. Although dendritic arbors become progressively more elaborate and extensive over developmental time, their separate domains remain clearly recognisable and appear to be maintained at least until the motor system is fully functional (18 h AEL) (Landgraf, 2003).

Having established that there is a central representation of the muscle field, the organisation of the motor neuron dendrites was analyzed in greater detail. (1) The set of external muscles and their innervating (SN) motor neurons were examined. Muscles of similar anteroposterior positions, such as the ventral acute muscle (VA3) and the segment border muscle (SBM), are innervated by motor neurons whose dendritic arbors lie in a common region of the neuropile. Conversely, motor neurons supplying the anterior (lateral transverse 1-2 [LT1-LT2]) versus the posterior (SBM) muscles have dendritic arbors that are correspondingly separated in the anteroposterior axis of the CNS (Landgraf, 2003).

To put the idea of a regular map to the test, focus was placed on an unusual external motor neuron-muscle pair. Muscle VT1 is innervated by a TN rather than an SN motor neuron. However, VT1 lies at the same place in the anteroposterior axis as the SBM, although VT1 is ventral and the SBM more dorsal. The VT1 motor neuron dendritic field is found to overlaps with that of the SBM motor neuron. For the external set, it is concluded that differences in target muscle location in the anteroposterior axis are mapped centrally as regular differences in dendritic position, but dorsoventral distinctions are not (Landgraf, 2003).

It was next asked whether there is a similarly regular representation of the internal muscles in the developing CNS. While most external muscles are transverse and have unique anteroposterior locations, the internal muscles span the width of a segment so that positional distinctions between them are solely in the dorsoventral axis. It was found that the set of internal muscles is represented centrally by three dendritic domains. Motor neurons innervating ventral internal muscles elaborate their dendritic arbors in the anterior half of the ISN dendritic domain. Motor neurons with dorsolateral internal muscle targets (lateral longitudinal [LL] 1, dorsal acute [DA] 3, dorsal oblique 3-5 [DO3-DO5]) put their arbors into the posterior part of the ISN dendritic domain. Finally, dorsal muscles are represented by a motor neuron dendritic domain that lies between those representing ventral (anterior) and dorsolateral (posterior) internal muscle groups. Thus, the internal muscles are represented in the neuropile by three domains of dendritic arborisation that reflect their different dorsoventral locations in the periphery. Once again, it is concluded that there is a regular mapping of muscle position in the neuropile: in this case, it is positions in the dorsoventral axis peripherally that are represented centrally as differences in the anteroposterior locations of dendrites (Landgraf, 2003).

To test the idea that dendritic arbor positions relate to the distribution of muscles, an atypical motor neuron-muscle pair was examined. The RP2 motor neuron is reported to innervate dorsal muscle DA2, yet its dendrites span the domains that represent both dorsal and dorsolateral internal muscles. However, on careful analysis it was found that DA2 is, in fact, specifically innervated by a U neuron whose dendrites lie in the dorsal internal domain, whereas the RP2 axon forms endings generally on all dorsolateral and dorsal muscles by 19 h AEL. These seem to correspond to the type 1s boutons found in late larvae. Thus, the RP2 neuron puts its dendrites into a region of the neuropile that does indeed represent its targets, namely the dorsolateral and dorsal internal muscles (Landgraf, 2003).

Like the muscle field itself, the map of motor neuron dendrites is metamerically repeated. However, the boundaries of these two units are out of register with one another, since the dendrites of the motor neurons innervating internal muscles lie in the next anterior neuromere. The anterior border of the dendritic map, as defined by the extent of these anterior dendrites, coincides with the anterior margin of engrailed (en) expression. Thus, while the muscles are segmental in their organisation, the domains occupied by the dendrites of their innervating motor neurons are parasegmental (Landgraf, 2003).

To test whether genes that implement the parasegmental pattern in the epidermis are also required for the formation of the parasegmental organisation of the neuromuscular system, the formation of SN and ISN dendritic fields was studied in embryos singly mutant for the following segment polarity genes: en/invected (Df(en^E)), wg (wg^CX4), naked (nkd²), patched (ptc⁹), hedgehog (hh²¹), and gooseberry (Df2R(gsb)). Every one of the six different mutants that were analysed has partially aberrant patterns of neuroblasts (NBs). Nevertheless, SN and ISN motor neurons still form and can be identified by their characteristic axonal projections into the periphery. In addition, it was found that the fundamental separation between SN and ISN dendritic domains is present despite often severe perturbations in CNS structure. For example, in gsb mutant embryos, both nerve roots are frequently fused so that the SN and ISN share a common CNS exit point. Nevertheless, SN and ISN axons as well as their dendritic fields do not intermingle but remain separate. These results suggest that the subdivision of the neuropile into the principal ISN and SN dendritic domains is a robust feature of the system, which appears to be specified early in development, since the embryo subdivides into parasegmental units (Landgraf, 2003).

It was next asked what mechanisms underlie the formation of the myotopic map. Because ISN and SN motor neurons lie at different positions in the CNS and their axons grow out into the muscle field through different nerves, it is reasonable to suppose that at least the major subdivision of dendritic arborisations into internal and external domains could be a byproduct of the locations at which the motor neurons are generated and the paths taken by their growing axons. This ‘passive mapping' explanation can be excluded by considering a single motor neuron-muscle pair, namely dorsal transverse 1 (DT1) and its innervating motor neuron. DT1 is an external muscle (by position, orientation, wg dependence, and Connectin expression), yet its motor neuron is clustered with the internal muscle innervating set and its axon (uniquely for the external muscles) grows out through the ISN. Despite its packing within the ‘internal motor neuron' set, the DT1 motor neuron makes a long posterior projection through the internal muscle domain of the myotopic map to reach the external domain, where it arborises appropriately, reflecting the orientation and external nature of its target muscle. In contrast, motor neurons derived from the same NB as DT1 innervate neighboring internal muscles DO3-DO5 and put their dendrites in a more anterior region characteristic of the dorsolateral muscles. These findings strongly suggest that the mapping of the muscle field within the CNS is an active process of growth and arborisation that partitions dendrites into subdomains of the neuropile that are appropriate to their function, rather than a passive subdivision of available space by position of origin or axon trajectory (Landgraf, 2003).

Since dendritic arbors form after motor axons have reached their targets, the muscles could be instrumental in dictating the organisation of the central map. To test this idea, the UAS/GAL4 system was used to misexpress an activated form of Notch (Kidd et al. 1998) in the developing mesoderm, suppressing the formation of muscle founder cells while leaving other tissues intact. In such muscleless embryos, the main nerve trunks, SN and ISN, still form and project into the periphery. Retrograde labellings of these nerves show that SN and ISN motor neurons form relatively normal dendritic arbors that consistently conform to the characteristic separation of SN and ISN dendrites. Thus, the neuropile is partitioned into distinct fields of dendritic arborisation independently of the muscles. It is concluded that the mapping process is likely to be an autonomous property of the motor neurons and their neighboring cells (Landgraf, 2003).

It was next asked whether motor neuron dendritic fields could be patterned by the substrates on which they grow. In the Drosophila ventral nerve cord (VNC), motor neuron dendrites form in the dorsal-most region of the neuropile, sandwiched between longitudinal glia above and the underlying scaffold of axons. Glial cells can act as substrates for supporting and guiding axonal growth. To test whether they might also be required for the growth and spatial patterning of dendritic fields, dendritic arbors were analysed in glial cells missing (gcm) mutant embryos, which are defective in glial cell differentiation. Although the structure of the nervous system is disrupted in gcm mutant embryos and the dendritic arbors are abnormal, they continue to form in their characteristic locations and the fundamental distinction between the ISN and SN motor neuron dendritic fields is maintained. Remarkably, even the long posterior dendritic projection of the DT1 motor neuron forms and reaches its target region, the SN external muscle dendritic domain. These results suggest that the patterning of the neuropile into distinct motor neuron dendritic domains is a process that appears to be intrinsic to the motor neurons and their neighboring neurons, but does not require proper glial cell differentiation (Landgraf, 2003).

One likely explanation for the division of dendrites into separate domains is that there is a process of mutual exclusion between the arborisations of neighboring cells. Such a process of dendritic ‘tiling' has so far only been documented between particular classes of sensory neurons, but could also occur in the motor system. The idea of tiling was tested by considering two groups of motor neurons whose axons have a common trajectory, but whose dendritic fields form in adjacent territories. The DO3-DO5 and DT1 motor neurons project their dendrites posteriorly, and at their most-anterior point, these dendrites meet the axons and dendrites of the anterior corner cell (aCC) and U/CQ neurons. To show whether the aCC and U/CQ axons and/or dendrites inhibit the growth of DO3-DO5 and DT1 dendrites anteriorly, these neurons (as well as RP2 and the posterior corner cell [pCC] interneuron) were selectively ablated. Using anti-Even-skipped (Eve) staining as a marker for aCC, RP2, and U/CQs (there are an additional two medially located eve-expressing interneurons, pCC and friend of pCC [fpCC], it was found that these neurons can be selectively ablated before they form dendrites (at approximately 11 h AEL): on average, by 10.5 h AEL all but 0.6 and by 12 h AEL all but 0.06 of the seven medially located eve-expressing neurons have been ablated per half-neuromere. In no instance was a concomitant anterior expansion of the DO3-DO5 and DT1 motor neuron dendrites into the regions vacated by the aCC and U/CQ dendrites observed. It is concluded that, at least in this instance, the initial dendritic territory of one set of motor neurons (DO3-DO5 and DT1) is not defined by a process of tiling, in which they are excluded by neighboring (aCC and U/CQ) dendritic arbors. However, it is possible that the elaboration of motor neuron dendritic arbors during later developmental stages may involve interactions between neighboring dendritic territories, activity-dependent processes, or both (Landgraf, 2003).

Thus, in summary, these results suggest that the mechanisms that subdivide the neuropile into distinct dendritc domains are very robust and refractory to perturbations. They further suggest that the cues that organise the map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

The patterning of the motor neuron dendritic arbors in the Drosophila embryo represents a first layer of organisation in the motor system. This is likely in part to be mirrored by the endings of higher-order neurons of central pattern generating circuits, which converge onto the myotopic map. While motor neuron cell body positions may, as has been proposed for vertebrate systems, relate to the ontogeny of target muscles, the operation of mature muscles is reflected by the allegiance of corresponding motor neuron dendrites to a particular territory in the neuropile. Thus, changes in muscle operation could be accommodated by a change of allegiance of the appropriate motor neuron dendrites from one domain to another (e.g., the DT1 motor neuron-muscle pair) without the need for rewiring the underlying higher-order circuitry. Such a model resolves the apparent discrepancy between the distributions of motor neuron cell bodies centrally and target muscles in the periphery. It also implies a considerable degree of flexibility, particularly at the level of motor output, yet suggests that elements of the underlying motor circuitry may have been highly conserved (Landgraf, 2003).

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 (Cha^l13), which are therefore immobile and unable to hatch. The development of aCC dendritic arborisations was examined in animals homozygous for the null mutation Cha^l13. 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).

Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. In the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. This study shows that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. This map is 'hard-wired'; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. The midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. These results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. It is further proposed that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common 'meeting regions' (Mauss, 2009).

How different dendritic morphologies and territories are generated in a motor system was investigated using the neuromuscular system of the Drosophila embryo as a model. Its principal components are segmentally repeated arrays of body wall muscles (30 per abdominal half segment), each innervated by a specific motorneuron. The motorneuron dendrites are the substrate on which connections with presynaptic cholinergic interneurons form. 180 cells (on average 11.25 for each identified motorneuron and a minimum of five) were labelled, and the dendritic morphologies and territories of the motorneurons that innervate the internal muscles were charted using retrograde labelling with the lipophilic tracer dyes 'DiI'and 'DiD.' This was done in the context of independent landmarks, a set of Fasciclin 2-positive axon bundles, at 18.5 h after egg laying (AEL), when the motor system first becomes robustly functional and the geometry of motorneuron dendritic trees has become sufficiently invariant to permit quantitative comparisons (Mauss, 2009).

Three classes of motorneurons were found based on dendritic arbor morphology and territory with respect to the ventral midline: (1) motorneurons with dendrites in the lateral neuropile (between the lateral and intermediate Fasciclin 2 tracts); (2) in the lateral and intermediate neuropile (between the intermediate and medial Fasciclin 2 tracts), and (3) in the lateral, intermediate plus medial neuropile (posterior commissure) (Mauss, 2009).

Moreover, the medio-lateral positions of motorneuron dendrites correlate with the dorsal to ventral locations of their target muscles in the periphery. Motorneurons with dorsal targets (DA1, DA3, DO1-5) have their dendrites in the lateral neuropile, while those innervating ventral and lateral muscles (LL1, VL2-4, VO1-2) also have dendrites in the intermediate neuropile. Coverage of the medial neuropile is particular to motorneurons innervating the most ventral group of muscles (VO3-6). These dendritic domains are arranged in the medio-lateral axis of the neuropile in such a way that they form a neural, myotopic representation of the distribution of body wall muscles in the periphery. Only a single motorneuron deviates from this clear-cut correlation between dendritic medio-lateral position and target muscle location: MN-DA2 has dendrites not only in the lateral neuropile, like other motorneurons with dorsal targets, but also in the intermediate neuropile (Mauss, 2009).

Previously studies have shown that motorneurons in the Drosophila embryo distribute their dendrites in distinct anterior to posterior domains in the neuropile, forming a central representation of target muscle positions in the periphery. The mechanisms required for the generation of this dendritic myotopic map remain elusive. In this study, dendritic myotopic organisation was characterized in a second dimension, with respect to the ventral midline, and the main molecular mechanism that underlies the formation of this dendritic neural map were identified, namely the combinatorial action of the midline signalling systems Slit/Robo and Netrin/Frazzled (Mauss, 2009).

Neural maps are manifestations of an organisational strategy commonly used by nervous systems to order synaptic connections. The view of these maps has been largely axonocentric and focused on sensory systems, though recent studies have challenged the notion of dendrites as a 'passive' party in arranging the distribution of connections. This study has demonstrated that motorneuron dendrites generate a neural, myotopic map in a motor system and that this manifest regularity can form independently of its presynaptic partner terminals (Mauss, 2009).

An essential feature of neural maps is the spatial segregation of synaptic connections. In the Drosophila embryonic nerve cord, there is some overlap between dendritic domains in the antero-posterior neuropile axis. Overlap of dendritic territories is also evident in the medio-lateral dimension, since all motorneurons have arborisations in the lateral neuropile, though distinctions arise by virtue of dendrites in additional intermediate and medial neuropile regions. The combination of myotopic mapping in both dimensions may serve to maximise the segregation between dendrites of different motorneuron groups. For example, the dendritic domain of motorneurons with dorsal targets differs from the territory innervated by ventrally projecting motorneurons in the antero-posterior location and the medio-lateral extent. Myotopic mapping in two dimensions could also provide a degree of flexibility that could facilitate wiring up in a combinatorial fashion. For instance, muscle LL1 lies at the interface between the dorsal and ventral muscle field; its motorneuron, MN-LL1, has one part of its dendritic arbor in the lateral domain that is characteristic for dorsally projecting motorneurons, while the other part of the dendritic tree innervates the intermediate neuropile precisely where ventrally projecting motorneurons put their dendrites (Mauss, 2009).

Myotopic dendritic maps might constitute a general organisational principle in motor systems. In insects, a comparable system of organisation has now been demonstrated also for the adult motor system of Drosophila (Brierley, 2009; Baek, 2009) and a degree of topographic organisation had previously been suggested for the dendrites of motorneurons that innervate the body wall muscles in the moth Manduca sexta. In vertebrates too, there is evidence that different motor pools elaborate their dendrites in distinct regions of the spinal cord in chick, turtle, and mouse. Moreover, elegant work in the mouse has shown that differences in dendritic territories correlate with and may determine the specificity of proprioceptive afferent inputs (Mauss, 2009 and references therein).

The neural map characterised in this study is composed of three morphological classes of motorneurons with dendrites innervating either (1) the lateral or (2) the lateral and intermediate or (3) the lateral, intermediate, and medial/midline neuropile (Mauss, 2009).

The motorneuron dendrites are targeted to these medio-lateral territories by the combinatorial, cell-autonomous actions of the midline guidance cue receptors Robo and Frazzled. The formation of dendritic territories by directed, targeted growth appears to be an important mechanism that may be more widespread than previously anticipated, though the underlying mechanisms may vary. Global patterning cues have been implicated in the vertebrate cortex (Sema3A). In the zebrafish retina, live imaging has shown that retinal ganglion cells put their dendrites into specific strata of the inner plexiform layer, but the roles of guidance cues and interactions with partner (amacrine) cells have not yet been studied (Mauss, 2009).

Slit/Robo and Netrin/Frazzled mediated gating of dendritic midline crossing has been previously documented in Drosophila embryos and zebrafish. This study demonstrated that dendrites are targeted to distinct medio-lateral territories by the combinatorial, opposing actions of Robo and Frazzled and that this is the main mechanism underlying the formation of the myotopic map. Strikingly, the same signalling pathways also regulate dendritic targeting of adult motorneurons in Drosophila, suggesting this to be a conserved mechanism (Brierley, 2009). Robo gates midline crossing of dendrites and in addition, at progressively higher signalling levels, restricts dendritic targeting to intermediate and lateral territories. Frazzled, on the other hand, is required for targeting dendrites towards the midline into intermediate and medial territories. The data argue that Frazzled is expressed by representatives of all three motorneuron types. Recently, Yang (2009) has shown that expression of frazzled leads to a concomitant transcriptional up-regulation of comm, thus linking Frazzled-mediated attraction to the midline with a decrease in Robo-mediated repulsion. While this has been demonstrated for midline crossing of axons in the Drosophila embryo, this study found that, at least until 18.5 h AEL, expression of UAS-frazzled alone was not sufficient to induce midline crossing of dendrites in MN-LL1 and MN-DA3. It is conceivable that differences in expression levels and/or timing between CQ-GAL4 used in this study and egl-GAL4 used by Yang might account for the differences in axonal and dendritic responses to UAS-frazzled expression. Moreover, the widespread expression of Frazzled in motorneurons and other cells in the CNS may point to additional functions, potentially synaptogenesis, as has been shown in C. elegans (Mauss, 2009).

Strikingly, neither synaptic excitatory activity nor the presynaptic (cholinergic) partner terminals seem to be necessary for the formation of the map. The map is already evident by 15 h AEL, before motorneurons receive synaptic inputs. It also forms in the absence of acetylcholine, the main (and at that stage probably exclusive) neurotransmitter to which motorneurons respond. Moreover, motorneuron dendrites innervate their characteristic dendritic domains when the cholinergic terminals have been displaced to outside the motor neuropile. However, interactions with presynaptic partners seem to contribute to its refinement. First, it was found that dendritic mistargeting phenotypes show a greater degree of penetrance earlier (15 h AEL) than later (18.5 h AEL) in development. Secondly, when interactions with presynaptic partner terminals are reduced or absent, dendritic arbor size increases and the distinction between dendritic territories is less evident than in controls. Fine-tuning of terminal arbors and sets of connections through contact and activity-dependent mechanisms is a well-established feature of neural maps in sensory systems and the current observations suggest that this may also apply to motor systems (Mauss, 2009).

The formation of the myotopic map is the product of dendritic targeting. It is therefore intimately linked with the question of how cell type-specific dendritic morphologies are specified. For instance, changing the balance between the Robo and Frazzled guidance receptors in motorneurons is sufficient to 'convert' dendritic morphologies from one type to another. The importance of target territories for determining dendritic arbor morphology has recently been explored in a study of lobula plate tangential cells in the blowfly, where the distinguishing parameter between the dendritic trees of four functionally defined neurons were not growth or branching characteristics but the regions where neurons put their dendrites (Mauss, 2009).

Because Slit/Robo and Netrin/Frazzled signalling have been reported to affect dendritic and axonal branching as well as axonal growth, respectively, it was asked what the effect was on motorneuron dendrites of altered Robo and Frazzled levels. It was found that in the wild-type different motorneurons generate characteristically different amounts of dendritic length and numbers of branch points (MN-DA1/aCC and MN-VO2/RP1, RP2, MN-DA3 and MN-LL1). In the Drosophila embryo and larva, Slit/Robo interactions have been suggested to promote the formation of dendrites and/or branching events, similar to what has been shown for cultured vertebrate neurons. The current data on embryonic motorneurons are not compatible with this interpretation. First, when altering the levels of Robo (or Frazzled) in individual motorneurons and mistargeting their dendrites, no statistically significant changes were detected in total dendritic length or number of branch points. Instead, for MN-DA3 and MN-LL1, it was observed that dendritic arbors respond to changes in the expression levels of midline cue receptors by altering the amount of dendritic length distributed to the medial, intermediate, and lateral neuropile. Secondly, in nerve cords entirely mutant for the Slit receptor Robo an increase is seen in dendrite branching at the midline. These observations suggest that for Drosophila motorneurons Slit/Robo interactions negatively regulate the establishment and branching of dendrites and thus specify dendritic target territories by defining 'exclusion' zones in the neuropile. The quantitative data from this study suggest that dendritic morphology is the product of two intrinsic, genetically separable programmes: one that specifies the total dendritic length to be generated and the frequency of branching; the other implements the distribution of these dendrites in the target territory, presumably by locally modulating rates of extension, stabilisation, and retraction of branches in response to extrinsic signals (Mauss, 2009).

The question of how neural circuits are generated remains at the heart of developmental neurobiology. At one extreme, one could envisage that every synapse was genetically specified, the product of an exquisitely choreographed sequence of cell-cell interactions. At the other extreme, neural networks might assemble through random cell-cell interactions and feedback processes enabling functional validation. The latter view supposes that neurons inherently generate polarised processes, have a high propensity to form synapses, and arrive at a favourable activity state through homeostatic mechanisms. Current evidence suggests that, at least for most systems, circuits form by a combination of genetic specification and the capacity to self-organise (Mauss, 2009).

This study has demonstrated that the postsynaptic structures of motorneurons, the dendrites, form a neural map. It was also shown that dendrites are closely apposed to cholinergic presynaptic specialisations in their target territories, suggesting that the segregation of dendrites may be a mechanism that facilitates the formation of specific sets of connections. Strikingly, this map of postsynaptic dendrites appears to be 'hard-wired' in that it can form independently of its presynaptic partners and it is generated in response to a third party, the midline guidance cues Slit and Netrin. A comparable example is the Drosophila antennal lobe, where projection neurons form a neural map independently of their presynaptic olfactory receptor neurons, though in this sensory system the nature and source of the cue(s) remain to be determined. This study complements previous work that demonstrated the positioning of presynaptic axon terminals by midline cues, also independently of their synaptic partners. Together, these results suggest that global patterning cues set up the functional architecture of the nervous system by independently directing pre- and postsynaptic partner terminals towards common 'meeting' areas (Mauss, 2009).

Clearly, such global guidance systems deliver a relatively coarse level of specificity and there is ample evidence for the existence of codes of cell-adhesion molecules and local receptor-ligand interactions capable of conferring a high degree of synaptic specificity. Therefore, one has to ask what the contribution is of global partitioning systems in establishing patterns of connections that lead to a functional neural network. A recent study in the Xenopus tadpole spinal cord has addressed this issue. Conducting patch clamp recordings from pairs of neurons, it has been found that the actual pattern of connections in the motor circuit reveals a remarkable lack of specificity. Furthermore, the segregation of axons and dendrites into a few broad domains appears to be sufficient to generate the connections that do form and to enable the emergence of a functional network. The implication is that neurons might be intrinsically promiscuous and that targeting nerve terminals to distinct territories by global patterning cues, as has been shown in this study, is important to restrict this synaptogenic potential and thereby confer a degree of specificity that is necessary for the emergence of network function (Mauss, 2009).

Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

Group A TFs that promote arborization through concerted regulation of outgrowth and branching

A group of transcriptional regulators, group A, controls the size of the dendritic field of class I neurons. RNAi of 19 TFs resulted in reduction of the field size covered by ddaD and ddaE. A reduction of coverage could be the result of a net reduction in dendrite outgrowth, branching, or both. Group A TFs have effects on both primary dendrite growth and secondary dendrite growth. For example, RNAi of the PAS-domain TF trachealess (trh) caused a minor reduction in both primary branch outgrowth and the number of lateral branches and a more marked reduction in the overall length of lateral branches. Consequently, the most distal regions of the dendritic field, especially the regions covered by lateral branches, are not innervated. By contrast, RNAi of genes such as the zinc-finger TF pygopus or the BTB/POZ-domain TF cg1841 caused more severe reduction of primary branch outgrowth as well as lateral branching and lateral branch length, resulting in a more drastic reduction of receptive field. In an extreme case, RNAi of the high mobility group gene hmgD resulted in an almost complete block of primary dendrite extension and lateral branching. In general, the genes with the most severe effects on primary branch outgrowth also have the most severe effects on branching, suggesting that these genes may function to regulate dendritic arborization overall (Parrish, 2006).

Although the genes in this class all caused qualitatively similar defects in arborization, some notable phenotypic differences are suggestive of distinct functions for some of these genes in regulating dendrite arborization. RNAi of the nuclear hormone receptors ultraspiracle (usp) and ecdysone receptor (EcR) significantly reduced primary dendrite outgrowth, but caused only modest reduction of lateral branching and lateral branch outgrowth, suggesting that branching is not absolutely dependent on proper outgrowth. Since the Usp/EcR heterodimer is responsible for ecdysone-responsive activation of transcription, as well as ligand-independent transcriptional repression, it is likely that these genes function together to promote dendrite outgrowth (Parrish, 2006).

RNAi of many group A genes resulted in embryonic lethality at a significantly higher rate than control injections. Thus, many of these genes are likely essential for embryonic development, either due to their involvement in regulating neuronal morphogenesis or due to other aspects of their functions (Parrish, 2006).

Group A transcriptional regulators that restrict dendrite arborization

In addition to genes with functions in promoting dendrite arborization, 20 group A genes were identified that regulate dendrite arborization by limiting dendrite growth and/or branching. Consistent with recent reports that loss of function of the BTB/POZ domain TF abrupt (ab) causes an increase in dendritic branching and altered distribution of branches, it was found that ab(RNAi) altered the arborization of class I dendrites. ab(RNAi) caused an increase in the number and length of lateral branches, expanding the coverage field most noticeably along the anteroposterior (AP) axis. In addition to these defects, ab(RNAi) also caused frequent cell death, consistent with the phenotype observed for a hypomorphic allele of ab (Parrish, 2006).

Increased dendritic branching also resulted from RNAi of several genes known to affect nervous system development, including Adh transcription factor 1 (Adf1), the zinc finger TF nervy (nvy), the basic helix–loop–helix (bHLH) TF deadpan (dpn), as well as genes not previously known to affect neuronal function, such as the putative transcription elongation factor Elongin c. Both Adf1 and dpn mutants have defects in larval locomotion and, in light of recent findings suggesting that da neurons may regulate aspects of larval locomotion, it is possible that dendrite defects underlie these behavioral defects. Consistent with its role in class I dendrite development, dpn is expressed in all PNS neurons. Likewise, nervy has been implicated in regulation of axon branching in motorneurons and is apparently expressed in most neurons. Thus, nervy likely regulates multiple aspects of neuronal differentiation. Finally, Elongin C may regulate transcriptional elongation but also likely functions as a component of a multimeric protein complex that includes the von Hippel-Lindau (VHL) tumor suppressor and targets specific proteins for poly-ubiquitination and degradation. Moreover, BTB/POZ domain proteins (such as cg1841 and ab) function as substrate adaptors for cullin E3 ligases. Interestingly, RNAi of a Drosophila homolog (tango) of a known VHL substrate (HIF-1) also affected dendrite arborization. It thus appears that protein degradation pathways regulate dendrite arborization (Parrish, 2006).

RNAi of the Polycomb group (PcG) genes Su(z)12, E(z), esc, or Caf1 similarly caused an increase in branch number and an expansion of the receptive field of class I neurons. Consistent with the similar RNAi phenotypes for these genes, Su(z)12, E(z), esc, and Caf1 are components of the multiprotein esc/E(z) polycomb repressor complex. One critical role for PcG-mediated gene silencing is the regulation of hox gene expression. Therefore, Polycomb-mediated regulation of hox gene expression likely contributes to arborization of class I neurons (Parrish, 2006).

RNAi of several genes affected dendrite arborization primarily by causing an increase in dorsal and lateral dendrite extension without significantly affecting branch number. For example, RNAi of the putative transcriptional repressor cg5684 caused dorsal overextension of the primary dendrite in ddaE and an overall increase in dendritic length in both ddaD and ddaE. In general, RNAi of genes that increased arborization rarely caused dendrites to cross the dorsal midline or segment borders, or increased branching more than twofold as compared with untreated neurons. It thus appears that dendritic outgrowth is further limited by neuronal growth capacity and/or other external constraints (Parrish, 2006).

Group B TFs with opposing actions on dendrite outgrowth and branching shape dendrite arbors

In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

In addition to the effects on primary dendrite extension, RNAi of each of these 18 genes limits the number and length of lateral dendrite branches. RNAi of some genes such as snail or knirps almost completely blocked dendrite branching, whereas RNAi of other genes such l(3)mbt had more modest effects on dendrite branching. In addition, a significant reduction of branching was noticed at the distal tip of the dorsally projected primary dendrite. In control treated stage 17 embryos, branchpoints are distributed along the primary dendrite, with the most distal branchpoint usually located within a few microns of the distal tip of the dendrite. In contrast, branching is rarely observed within 10 microns of the distal dendritic tip following RNAi of these group B genes. In some cases, such as snail(RNAi), knirps(RNAi), or l(3)mbt(RNAi), the most distal branchpoint is located 25 microns or further from the distal tip of the primary dendrite. Therefore, these TFs inhibit primary branch extension but promote lateral branching and lateral branch extension (Parrish, 2006).

In addition to identifying a large class of TFs that inhibit primary branch extension and promote lateral branching, it was also found that TFs promote dendrite extension and limit dendrite branching. RNAi of two genes, glial cells missing 2 (gcm2) and the histone acetyltransferase pcaf, caused an increase in lateral branching and a marked reduction in dorsal extension of ddaE. Thus, transcriptional pathways exist that have opposing effects on primary branch outgrowth and secondary branching, suggesting that these processes may normally antagonize one another (Parrish, 2006).

Group C TFs regulate dendrite routing

Proper dendritic routing is important for primary dendrites of ddaD and ddaE to grow in parallel toward the dorsal midline without crossing each other and for secondary branches of ddaD and ddaE to avoid the space between ddaD and ddaE. Therefore, there must be mechanisms that promote this stereotyped arborization pattern, including signals that promote anterior arborization of ddaD and posterior arborization of ddaE, as well as signals that antagonize posterior arborization of ddaD and anterior arborization of ddaE. Indeed, RNAi of 10 TFs disrupted the dendritic routing patterns of ddaD and ddaE, resulting in aberrantly oriented primary dendrites. RNAi of cg1244, bap55 (brahma associated protein of 55kD), cg9104, cg4328, and cg7417 resulted in inappropriate anterior arborization of ddaE as well as inappropriate posterior arborization of ddaD. Anterior or even ventral displacement of ddaD concomitant with anterior arborization of ddaE was also observed as well as displacement of ddaE arbors concomitant with misrouting of ddaD. Finally, reducing sens function by RNAi or genetic mutation caused extensive mixing of dendritic arbors from ddaD and ddaE, in addition to dorsal overextension of primary dendrites and an overall reduction in the number of class I neurons (Parrish, 2006).

It is also worth noting that RNAi treatment that caused reduced dendritic outgrowth often caused minor routing defects. For example, pyg(RNAi) or cg1841(RNAi) caused inappropriate routing of ddaE. The routing defects seen with these candidates may reflect a disruption of attractive/repulsive signaling that normally regulates dendrite arborization. The source of such signals is currently unknown, although of great interest (Parrish, 2006).

TFs likely exert distinct mitotic and post-mitotic functions to regulate neuron morphogenesis

TFs play critical roles in neurogenesis, and some genes that regulate neurogenesis also affect post-mitotic neuronal differentiation. Because clones of duplicated class I neurons have wild-type dendrite arborization patterns, class I dendritic arbors appear to be insensitive to cell number defects. Indeed, dendrite arborization of class I neurons in embryos carrying the temperature-sensitive neurogenic mutation Notch^ts (N^ts) is unaffected by as much as a fivefold increase in class I neuron number, and is likely insensitive to multiplication of other da neurons as well since N^ts experiments caused increased numbers of other da neurons. Furthermore, in cases where only one of the class I neurons is multiplied, the dendrites of neighboring class I neuron are unaffected. In contrast laser ablation of ddaD or ddaE or the occasional cell loss caused by RNAi of various genes did not generally cause defects in arborization of neighboring class I neurons. Therefore, analysis of class I neurons should allow study of post-mitotic functions of genes that affect neuron number (Parrish, 2006).

RNAi of several genes affected the number of class I neurons as well as morphogenesis of class I dendrites; RNAi of seven genes caused supernumerary cells and RNAi of four genes caused high penetrance cell loss in addition to dendrite defects. For example, RNAi of the zinc finger TF nerfin-1 caused an increase in neurons labeled by Gal4²²¹ with as many as eight neurons visible in some segments. Unlike wild-type class I neurons, neurons from nerfin-1(RNAi)-treated embryos extended mostly unbranched dendrites. In many cases, the routing pattern of the dendrites appeared abnormal, but the cell number defects make it difficult to resolve the projection pattern of individual dendrites or conclusively determine whether each neuron projects the same number of primary dendrites. RNAi of six other genes, including jumeau, a winged-helix TF known to regulate neuroblast cell fate and the number of PNS neurons, similarly caused an increase in neuronal number as well as defects in dendrite morphogenesis (Parrish, 2006).

In addition to the seven genes that function to restrict class I neuron number and control dendrite morphology, three other genes are required to maintain the number of class I neurons. Reduction of their function caused a reduction of class I neurons and defects in dendrite morphogenesis in the remaining neurons. For example, RNAi of the zinc finger TF senseless (sens) reduced the number of class I neurons, consistent with previous findings that sens is required for development of most cells in the PNS. In addition, sens(RNAi) or a sens loss-of-function mutation caused an increase in dendrite outgrowth and mixing of dendrites in segments with both ddaD and ddaE present. Similarly, RNAi of the proneural bHLH TF atonal (ato) reduced the number of class I neurons, consistent with previous findings that chordotonal organs and some md neurons are absent in embryos lacking ato. Consistent with reports that ato functions in neurite arborization in the larval brain, it was also found that ato(RNAi) caused altered arborization patterns of class I dendrites. Thus, it is likely that multiple TFs that regulate neuron number also regulate aspects of post-mitotic neuronal differentiation (Parrish, 2006).

Finally, gene was found that appears to be essential for a subset of class I da neurons. RNAi of cubitus interruptus (ci), a component of the hedgehog signaling pathway, caused a loss of dorsal class I da neurons without affecting the ventral class I neuron vpda. Because ci(RNAi) causes embryonic lethality at high concentrations of dsRNA, the phenotypes in surviving embryos likely represent hypomorphic phenotypes. Loss of ddaD and ddaE is not compensated for by a concomitant increase in other md neurons since ci(RNAi) leads to an overall reduction of dorsal md neurons expressing the pan-da marker Gal4^109(2)80. Therefore, ci likely promotes differentiation or survival of a subset of da neurons, including ddaD and ddaE, but not of the ventral class I neuron vpda, demonstrating that morphologically similar neurons can be molecularly distinct (Parrish, 2006).

Some TFs are continuously required to regulate class I dendrite morphology

As another indication of the hypomorphic nature of many of the alleles and maternal rescue of gene function in mutant embryos, focus was place on dendrite defects that were first apparent during larval stages. For example, a mutant allele of Drosophila Mi-2, which encodes a Hunchback-interacting ATP-dependant chromatin remodeling factor, shows only minor defects in late embryonic stages, but shows an obvious reduction in arborization by 72 h after egg laying. Since Mi-2(RNAi) demonstrates that Mi-2 is required for embryonic dendrite arborization, these findings suggest that Mi-2 is continuously required for class I neurons to maintain proper dendrite arborization patterns. Similarly, the dendritic overbranching associated with a P-element insertion allele of Adf1 was first apparent after embryonic stages, although Adf1(RNAi) caused overbranching in embryos. Class I dendritic arbors of Adf1 mutants are indistinguishable from wild-type neurons until 96 h AEL. By 144 h AEL, ddaE arbors of Adf1 mutants showed a greater than twofold increase in branch number when compared with time-matched wild-type controls. Interestingly, ddaD showed only very minor branching defects in Adf1 mutants, suggesting that ddaD and ddaE might have distinct requirements for Adf1. Similarly, mutant alleles of either E(bx) or Elongin C showed dendrite branching defects only at late larval stages. These findings indicate that Adf1, E(bx), and Elongin C are continuously required to inhibit branching in class I neurons, demonstrating that although class I neurons have very little new branching after embryogenesis, they still retain the capacity to branch (Parrish, 2006).

RNAi of some group A genes that cause reduced dendrite outgrowth and branching is epistatic to mutation of ab or sens

Since group A and B TFs regulate aspects of dendritic growth and branching, potential epistatic relationships among TFs was explored in these phenotypic classes. To do this, RNAi was used to knockdown expression of select TFs in Drosophila embryos carrying a loss-of-function mutation in either the group B/C gene senseless (sens) or the group A gene abrupt (ab). sens mutant class I dendrites overextend dorsally and have reduced lateral branching in addition to routing defects. In sens mutants, RNAi of the group A genes Su(z)12 and ab, which cause increased lateral branching following RNAi in wild-type embryos, led to an increase in lateral branching compared with injected controls. Therefore, Su(z)12 and ab function are still required to limit arborization in sens mutants, and the increased dendritic branching as a result of Su(z)12(RNAi) or ab(RNAi) is epistatic to the increased dorsal extension and reduced lateral branching of sens mutants. In contrast, RNAi of the group A genes cg1244 and cg1841, which caused reduced arborization following RNAi in wild-type embryos, led to a reduction in primary dendrite outgrowth and lateral dendrite branching compared with injected controls. Therefore, at least in the instances described above, loss of group A genes is epistatic to loss of group B genes (Parrish, 2006).

RNAi of group A genes either promoted or antagonized dendrite arborization; therefore, the effect was examined of simultaneously disrupting one group A gene that promoted and one group A gene that antagonized dendrite outgrowth and lateral branching. RNAi or a loss-of-function mutant of the group A gene ab caused increased dendritic branching and extension of class I dendrites. In addition, mutation of ab caused a significant reduction in the number of class I neurons labeled by Gal4²²¹ that was most pronounced in the dorsal cluster of PNS neurons, consistent with the results from RNAi experiments. To facilitate epistasis analysis in ab mutants, dendrite arborization effects in vpda, the ventrally located class I neuron, were assayed. RNAi of the group A gene hmgD, which caused reduced primary dendrite outgrowth and reduced lateral branching when injected into wild-type embryos, caused a striking reduction in the number of dendritic branches and size of the receptive field of vpda in ab mutants. RNAi of the group A gene bap55 had similar effects in ab mutants, demonstrating that, at least in some cases, loss of group A genes that results in reduced arborization is epistatic to loss of group A genes that results in increased arborization. Therefore it is possible that the different classes of group A genes antagonistically regulate a common set of target genes required for dendrite arborization (Parrish, 2006).

The bHLH-PAS protein Spineless is necessary for the diversification of dendrite morphology of Drosophila dendritic arborization neurons

Dendrites exhibit a wide range of morphological diversity, and their arborization patterns are critical determinants of proper neural connectivity. How different neurons acquire their distinct dendritic branching patterns during development is not well understood. This study reports that Spineless (Ss), the Drosophila homolog of the mammalian aryl hydrocarbon (dioxin) receptor (Ahr), regulates dendrite diversity in the dendritic arborization (da) sensory neurons. In loss-of-function ss mutants, class I and II da neurons, which are normally characterized by their simple dendrite morphologies, elaborate more complex arbors, whereas the normally complex class III and IV da neurons develop simpler dendritic arbors. Consequently, different classes of da neurons elaborate dendrites with similar morphologies. In its control of dendritic diversity among da neurons, ss likely acts independently of its known cofactor tango and through a regulatory program distinct from those involving cut and abrupt. These findings suggest that one evolutionarily conserved role for Ahr in neuronal development concerns the diversification of dendrite morphology (Kim, 2006).

The ss protein is present at nearly the same level in all da neurons and acts cell-autonomously to dictate their dendritic complexity, while different da neurons exhibit different sensitivity to the level of Ss, and even the bipolar td neuron can respond to elevated ss activity by increasing dendritic complexity (Kim, 2006).

Previous studies in C. elegans have demonstrated essential roles for invertebrate homologs of Ahr in neuronal cell fate determination. For example, ahr-1 regulates the differentiation program of a subclass of neurons that contact the pseudocoelomic fluid, and both ahr-1 and aha-1 specify GABAergic neuron cell fate in C. elegans. The dramatic changes in the dendrite morphologies of the da neurons, however, are not due to an all-or-nothing change in cell fate because the da neurons in ss mutants displayed normal class-specific expression patterns of the molecular markers Ab and Cut and normal axon projection patterns characteristic of individual da neurons. However, this also does not assume that a partial cell fate change has not occurred. One reflection of the ss function as a transcription factor is the altered expression levels of GFP in the class I Gal4221 reporter, with increased levels of expression in all class IV neurons and essentially no expression in the dorsal class I neuron ddaD and the ventral class I neuron vpda in ss mutants. It will be of interest to further characterize the genetic basis for this Gal4 reporter, to determine whether this regulation constitutes a partial cell fate alteration or transcriptional regulation of genes downstream from ss in the execution of adjustment of dendritic complexity (Kim, 2006).

There is an emerging theme that ss functions to diversify neuronal differentiation by expanding the photopigment repertoire of R7 photoreceptors in the Drosophila eye and by diversifying da neuron dendritic morphologies. Recent studies have demonstrated that the entire retinal mosaic pattern required for color vision in Drosophila is regulated by ss. In the Drosophila retina, two types of ommatidia form the wild-type retinal mosaic: 'pale' and 'yellow.' In ss^D115.7 mutants, the yellow ommatidial subtype is lost and normally yellow R7 cells are misspecified into the pale subtype. As a result, nearly all R7 cells adopt the pale subtype, leading to loss of the retinal mosaic pattern. Thus, the pale R7 subtype represents the R7 'default state' (Kim, 2006 and references therein).

The overall lack of dendritic diversity in the da neurons in ss mutants is suggestive of the hypothesis that ss, an ancient, evolutionarily conserved gene, may act to convert a primordial dendrite pattern (perhaps a default state) to different complexities for different neurons in the peripheral nervous system. The loss-of-function ss phenotype in the da neuron dendrites might reflect such a primordial pattern as the dendrites in the mutant are devoid of specific morphological features that define distinct neuronal subclasses. In support of this notion, dendrites of the different classes of da neurons share similar morphological characteristics and elaborate similar numbers of total branches in ss mutants. The ability of ss to regulate the complexity and diversity of this dendrite pattern, by limiting dendritic branching to shape the simpler arbors of the class I and class II neurons and by promoting class-specific terminal branching to shape the more complex arbors of the class III and class IV neurons, is quite unique. Of the many mutants that affect multiple classes of da neurons, the great majority affect da neurons with simple or complex dendritic arbors the same way; that is, causing them to all become simpler or more complex. The ss phenotype of making simple dendritic arbors more complex and complex arbors simpler is very unusual among the many mutants affecting dendrite complexity. It thus seems likely that the distinct dendritic patterns rely not only on a cohort of gene activities specifying the mechanics of dendrite outgrowth and branching, but also a genetic program that diverts the generic primordial mode of dendritic formation to a diverse range of dendritic patterns (Kim, 2006).

How might spineless exert its functions? Unlike the homeodomain protein Cut, which promotes dendritic complexity in a specific direction, ss functions in an opposing manner in different cell types to regulate dendritic diversity. How might ss function differently in different neuronal cell types? One possibility is that ss is activated by different ligands in different neurons. ss is incapable of binding dioxin and other related compounds, suggesting that other, as yet unidentified ligands are required for its activation. Previous reports have suggested that ss and other invertebrate homologs of Ahr are activated by an endogenous ligand or that no ligand is required at all. Recent studies have shown that Ahr can accumulate in the nucleus upon activation by the second messenger cyclic AMP (cAMP), although it is not yet known whether cAMP signaling can activate ss in Drosophila. Thus, it is conceivable that ss is activated by different upstream factors in different cell types. It will be of interest to test in future studies whether in different neuronal cell types ss is activated by different ligands or upstream second messengers and whether ss acts in concert with regulatory programs for cell fate specification to dictate dendritic complexity (Kim, 2006).

In the canonical Ahr signaling pathway, Ahr requires the appropriate cofactor for its proper function. Members of the bHLH-PAS protein family are able to heterodimerize with other bHLH-PAS proteins. Previous studies have shown that, upon ligand binding, Ahr is translocated to the nucleus, where it heterodimerizes with Arnt to form a transcriptionally active complex. However, tango, the Drosophila homolog of Arnt, is likely not required for the regulation of dendritic morphogenesis, indicating that ss is probably not acting through its canonical signaling pathway to specify dendritic complexity. In Sf9 cells, ss can act independently of tgo to enhance expression of a reporter in the absence of a ligand. Furthermore, Ahr is unable to interact with Arnt upon activation by cAMP. Although Ahr, Arnt, and the Arnt homolog Arnt2 are widely distributed throughout the rat brain, Ahr does not preferentially colocalize with either Arnt or Arnt2. Ahr is also expressed in specific regions of the rat brain where neither Arnt nor Arnt2 is expressed. These studies support the notion that ss can act independently of tgo in certain developmental contexts. Tgo can heterodimerize with other bHLH-PAS proteins in addition to ss. It is conceivable that ss may act with different heterodimerization partners to mediate its different functions in different cell types (Kim, 2006).

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, an E2/E3 ubiquitinating enzyme mutant screen was conducted, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme encoding Effete, resulted in retention of C4da neuron dendrites during metamorphosis. Further, UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling (Kuo, 2006).

The ubiquitin-proteasome system (UPS), evolutionarily conserved for the regulation of protein turnover, targets proteins for degradation via a complex, temporally regulated process that results in proteasome-mediated destruction of polyubiquitinated proteins. There are two distinct steps involved: first, the covalent conjugation of ubiquitin polypeptide to the protein substrates, and second, the destruction of tagged proteins in the proteasome complex. The transfer of ubiquitin to a target molecule slated for degradation involves at least three enzymatic modifications: ubiquitin is first activated by the ubiquitin-activating enzyme E1; ubiquitin is then transferred to a carrier protein, a ubiquitin-conjugating enzyme E2, and finally, ubiquitin is transferred to a protein substrate bound by a ubiquitin ligase E3. There are minor variations to this enzymatic cascade, but overall, these highly specific protein-protein interactions ensure ubiquitin targeting specificity and regulate many aspects of housekeeping protein turnover and cellular maintenance. However, with the multiple regulatory layers, different parts of this complex machinery can break down. Mutations in the UPS pathway causing accumulation of nondegraded proteins have been implicated in a variety of human diseases (Kuo, 2006).

In the nervous system, aberrations in the UPS pathway have been implicated in disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. One of the common pathological features of neurodegenerative diseases, besides neuronal loss, is local axon degeneration. For example, in the case of Wallerian degeneration in vertebrates, distal parts of a severed axon remain viable and conduct action potentials in vivo for some time before a rapid dismantling of cytoskeletal proteins and axon degeneration, and the initiation of this rapid axon degeneration involves the UPS pathway. It is thought that UPS activation can lead to microtubule depolymerization and subsequent neurofilament degradation, possibly acting in conjunction with the Ca²⁺-dependent protease calpain. Moreover, inhibiting UPS activity in neurons prior to severing their axons can dramatically retard degradation of the severed axons. These results suggest that a cell-intrinsic UPS pathway regulates axon stability and that pharmaceutical inactivation of the UPS may prevent axonal degeneration in disease states (Kuo, 2006 and references therein).

In Drosophila, the remodeling of neuronal processes during normal development closely resembles the pathological phenotypes in Wallerian degeneration. In the mushroom body γ neurons, extensive pruning of larval axons occurs during metamorphosis in a process regulated by glia engulfment and neuron-intrinsic UPS activity. Similarly, in the fly peripheral nervous system, the class IV dendritic arborization (C4da) neurons undergo complete pruning of their extensive larval dendrites during metamorphosis, in a process that is also regulated by UPS activity (Kuo, 2005). In both of these examples, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration, all phenotypes resembling Wallerian degeneration. Therefore, these fly neurons represent excellent systems in which to understand the roles of the UPS in regulating neuronal axon/dendrite integrity, given the rather limited knowledge of how the UPS participates in the degradation of neuronal processes. It is not known which specific E2 ubiquitin-conjugating enzyme(s) and E3 ubiquitin ligase(s) are involved in UPS-mediated remodeling/degradation of neuronal processes, or their specific downstream target(s) (Kuo, 2006).

It has been shown that mutations in the fly ubiquitin activation enzyme (uba1) and the proteasome complex (mov34) can prevent efficient pruning of C4da neuron larval dendrites during metamorphosis (Kuo, 2005). To further investigate the role of UPS in C4da neuron dendrite remodeling, a candidate gene screen was conducted to identify the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase required for this process. Analysis of genetic mutants showed that UPS activation in C4da neurons likely results in UbcD1 (an E2 ubiquitin-conjugating enzyme) mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1), an E3 ligase that antagonizes caspase activity. Degradation of DIAP1 leads to activation of caspase Dronc, which results in local caspase activation and cleavage of proximal dendrites in C4da neurons during metamorphosis. In addition to the identification of a set of E2/E3 ubiquitinating enzymes for C4da neuron dendrite remodeling—with the surprising finding that the UPS mediates degradation of the potent protease inhibitor DIAP1—this study also establishes a mechanistic link between the UPS and caspase pathways in regulating C4da neuron dendrite pruning (Kuo, 2006).

To identify the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase mediating dendrite pruning of C4da neurons during metamorphosis, a candidate gene approach was taken to systematically test the roles of known E2/E3 ubiquitinating enzymes in Drosophila. A set of putative E2/E3 ubiquitinating enzyme mutations was assembled, and live imaging was used to visualize C4da neurons carrying the E2/E3 mutation via the pickpocket(ppk)-EGFP marker, which specifically labels C4da neurons during Drosophila development. Those mutants with an early lethal phase were characterized by generating mosaic analysis with a repressible cell marker (MARCM) mutant neuronal clones. Since wild-type (wt) C4da neurons during metamorphosis do not retain any larval dendrites following head eversion, as imaged 18–20 hr after puparium formation (APF), mutations that caused larval dendrite retention in C4da neurons at this stage were sought. The candidate genes tested mostly showed no defects in dendrite pruning or neuronal cell death. However, one candidate, ubcD1, showed a modest level of larval dendrite retention at 18 hr APF (Kuo, 2006).

Live imaging of wt C4da neuron MARCM clones at the start of pupariation (white pupae, WP) showed primary and secondary dendritic branching patterns typical of C4da neurons. Consistent with previous reports (Kuo, 2005; Williams, 2005), wt C4da neurons sever their larval dendrites during early metamorphosis and by 18 hr APF are devoid of any dendrites. The ubcD1 mutant C4da MARCM clones showed similar dendritic morphology to the wt clones at the onset of metamorphosis. However, at 18 hr APF, the mutant clones consistently retained intact, nonsevered larval dendrites. Thus, the UbcD1 E2 ubiquitin-conjugating enzyme is required for proper UPS-mediated dendrite pruning in C4da neurons during metamorphosis (Kuo, 2006).

UbcD1, encoded by the gene effete, regulates UPS-mediated degradation of the antiapoptotic protein DIAP1 (Treier, 1992; Wang, 1999; Ryoo, 2002). In protecting cells from apoptosis, the DIAP1 E3 ubiquitin ligase antagonizes Dronc caspase activity by regulating ubiquination and degradation of the Dronc protein. Following apoptotic stimuli, UbcD1 mediates self-ubiquination and degradation of DIAP1, allowing for subsequent Dronc caspase activation. The biochemical and genetic interactions between these molecules are well established. The baculovirus p35, which is commonly used to inhibit caspase activity in Drosophila, and does not block C4da neuron dendrite pruning (Kuo, 2005). This may seem to make the involvement of caspases in this process unlikely; however, p35 has only limited activity against the caspase Dronc. To study the effects of dronc mutation on C4da neuron dendrite pruning, two null alleles of Dronc, dronc⁵¹ and dronc¹¹, were used. MARCM analysis of dronc mutant clones revealed that the dendrites of mutant C4da neurons appeared normal at larval stages. However, unlike wt clones, without Dronc these neurons failed to properly prune their larval dendrites during metamorphosis, and most showed relatively intact primary and secondary larval dendritic arbors at 18 hr APF. These results show that severing of primary larval dendrites from C4da neurons during early metamorphosis requires the Dronc caspase (Kuo, 2006).

During apoptosis, Dronc activation requires the degradation of the antiapoptotic/anticaspase protein DIAP1, which is downstream of UbcD1. The requirement of UbcD1 for C4da neuron larval dendrite pruning during metamorphosis, together with the finding that Dronc caspase activity is also essential, raised the question of whether UPS-mediated DIAP1 degradation is a key step that allows for the severing of larval dendrites. Because loss of DIAP1 function causes C4da neuron cell death prior to the onset of metamorphosis, this question was approached using a gain-of-function allele of diap1, diap1^6-3s, which has a single amino acid mutation that makes DIAP1 an inefficient substrate for UPS-mediated degradation. ppk-EGFP was crossed into the gain-of-function mutant background and live imaging was used to follow C4da neuron dendrite pruning during metamorphosis. The diap1^6-3s mutation did not significantly affect the ability of C4da neurons to elaborate larval dendrites. However, unlike wt C4da neurons that completely pruned their larval dendrites by 18 hr APF, C4da neurons in the diap1^6-3s gain-of-function mutants failed to efficiently sever larval dendrites at 18 hr APF. These results suggest that the degradation of DIAP1 during early metamorphosis is required for proper C4da neuron larval dendrite pruning. Quantitatively, mutations in the UPS pathway that modulate Dronc activity (diap1^6-3s and ubcD1) resulted in less severe dendrite pruning defects than dronc mutants, both in terms of total number of large dendrites attached to soma and in the length of the longest attached dendrite at 18 hr APF (Kuo, 2006).

The UbcD1-DIAP1-Dronc pathway in apoptosis is well established. Thus, it may be necessary for C4da neurons to restrict the action of this pathway to specific cellular locations in order to prune unwanted dendrites without triggering apoptosis. To address this possibility, the subcellular distribution of DIAP1 and Dronc proteins was examined in ppk-EGFP C4da neurons. During the transition from third instar larvae to white pupae at the onset of metamorphosis, as well as 2 hr APF, there was a consistent induction of nuclear DIAP1 in GFP-labeled C4da neurons. During the same period a concurrent decrease was detected in Dronc staining in the soma of C4da neurons, unlike those from the neighboring cells at 2 hr APF. These results are consistent with previous observations that C4da neurons survive through this stage of metamorphosis. However, the level of antibody staining made it difficult to monitor the distribution of DIAP1 and Dronc within the dendritic structures of the C4da neurons. Because overexpression of Dronc caused C4da neuron to undergo apoptosis prior to metamorphosis, it was not possible to use GFP-tagged Dronc to examine its distribution in these neurons during pupariation. It was therefore necessary to search for alternative means to visualize activated Dronc or its downstream caspases (Kuo, 2006).

An antibody generated against activated mammalian caspase 3 has been shown to be effective in recognizing activated caspases in Drosophila. Whereas this antibody reportedly recognizes the Drosophila effector caspase Drice, it may also cross react with other activated Drosophila caspases such as Dronc during tissue staining, because of similarities in the sequences of these caspases in the region corresponding to the peptide used to generate this antibody. Therefore this antibody was used to determine whether activated caspase is localized to the dendrites of C4da neurons during the initial severing event. At 4 hr APF, just prior to dendrite severing, antibody staining for activated caspase was consistently observed within the proximal larval dendrites of C4da neurons, especially within dendritic swellings. In the diap1^6-3s gain-of-function mutant that inhibits Dronc activity, as well as in ubcD1 and dronc mutant MARCM clones, C4da neurons did not show dendritic swellings or activated caspase staining in dendrites during early metamorphosis. Consistent with previous observation that C4da neurons do not remodel their axons during concurrent dendrite pruning, no activated caspase staining was seen within the axons of C4da neurons during dendrite severing. Since overexpression of p35 in these neurons did not block dendrite pruning (Kuo, 2005), it is believed this antibody staining likely recognizes activated Dronc directly or recognizes a p35-resistant caspase that is activated by Dronc. These results show that, concurrent with the nuclear upregulation of DIAP1 in C4da neurons that prevents apoptosis, there is a local activation of caspases in the dendrites, likely as a result of UPS-mediated degradation of DIAP1. The spatially restricted activation of caspases then allows the severing of proximal larval dendrites from the soma (Kuo, 2006).

This study has shown that the UPS regulates pruning of larval dendrites from C4da neurons in a cell-intrinsic manner. To better understand the molecular pathways regulating UPS-mediated pruning, a candidate E2/E3 ubiquitinating enzyme screen was conducted. In this screen an E2 ubiquitin-conjugating enzyme mutation in was uncovered ubcD1, causing dendrite pruning defects. Taken together with the extensive biochemical characterization of interactions between UbcD1, DIAP1, and Dronc, this study suggests that in C4da neurons, UPS activation leads to UbcD1-mediated degradation of E3 ubiquitin ligase DIAP1, thereby allowing Dronc caspase activation and the subsequent cleavage of larval dendrites. This work not only identifies a set of E2/E3 ubiquitinating enzymes regulating neuronal process remodeling, it also links the UPS to a hitherto unappreciated mechanism for local caspase activation in dendrites during Drosophila metamorphosis (Kuo, 2006).

The mechanistic link between the UPS and caspase activity in regulating C4da neuron dendrite pruning is unexpected. Although the UPS is known to regulate remodeling and degradation of neuronal processes, it is generally believed that this process is accomplished by degradation of cellular proteins (such as microtubules and neurofilaments) that are required to keep dendrites and axons intact. However, it was found that the UPS in C4da neurons is in fact causing the degradation of an E3 ligase, DIAP1, thereby allowing for subsequent dendrite pruning. In this case, UPS-mediated degradation of a protein does not in and of itself lead to a structural compromise in dendrites, but rather it leads to the activation of another protease that executes dendrite pruning. This two-step activation cascade, which involves both the UPS and the apoptotic machinery, may provide an additional level of control and flexibility that would not be possible if UPS alone regulated the pruning program. After all, these C4da neurons, which are specified during fly embryogenesis, maintain a highly elaborate dendritic field to receive sensory inputs throughout larval development, which lasts for several days. The maintenance of these dendrites over time requires a network of finely tuned cell-intrinsic and -extrinsic pathways. Just as important, the dendritic pruning program enables dramatic neuronal remodeling in response to profound environmental changes during metamorphosis. It is conceivable that C4da neurons evolved this dual control mechanism to prevent any accidental triggering of dendrite pruning prior to metamorphosis. Initiation of C4da neuron dendrite pruning requires cell-intrinsic ecdysone signaling, and ecdysone receptors have been shown to regulate Dronc expression. It will be of interest to determine how this UPS/caspase dendritic pruning pathway is related to the ecdysone signaling cascade (Kuo, 2006).

During metamorphosis, C4da neurons upregulate DIAP1 expression in the nucleus, which is consistent with this class of neurons surviving early stages of the metamorphosis (only one of the three C4da neurons per hemisegment, the ventral neuron, is lost at a later stage of pupariation). Remarkably, there are activated caspases within the dendrites prior to severing, and a gain-of-function diap1 mutation can block dendrite pruning, strongly implicating a local dendritic program that can activate caspases without causing apoptosis of the neuron. Although mutations in both the Dronc caspase and the UPS pathway that modulate Dronc activity (UbcD1 and DIAP1) result in retention of larval dendrites, their dendrite pruning defects differed somewhat quantitatively. Compared to dronc mutants, diap1 gain-of-function and especially ubcD1 mutants showed less retention of larval dendrites during metamorphosis. This is not surprising for diap1 gain-of-function, as it is an effective Dronc inhibitor but unlikely to be 100% efficient. UbcD1, as an E2 ubiquitin-conjugating enzyme, has wider substrate specificity than E3 ligases. Previous study showed that UbcD1 is involved in mushroom body neuroblast proliferation, so it may be involved in other UPS-mediated pathways during dendrite pruning. It is also conceivable that in the absence of UbcD1 another E2 may trigger a low level of DIAP1 degradation, allowing residual Dronc activation which results in a milder dendrite pruning phenotype in ubcD1 mutants. It is currently unclear whether UbcD1 is also required during DIAP1-mediated degradation of Dronc. However, pruning defects in the ubcD1 mutants suggest that it may not be absolutely required, since undegraded DIAP1 continues to inhibit Dronc, presumably via interaction with another E2 protein (Kuo, 2006).

How is the specificity of dendrite pruning achieved? Several possible mechanisms are proposed: first, C4da neurons do not change their axonal projections during dendrite pruning, so there could be dendrite-specific trafficking of components of the UPS, such as UbcD1, and/or the caspase Dronc. Of the known proteins that are preferentially trafficked to dendrites, these molecules have not been implicated but warrant further investigation. Second, it is also possible that activated Dronc, or another p35-resistant protease activated by Dronc, could cleave a dendrite-specific substrate. Examples are now emerging from other cellular systems, such as in sperm formation and border cell migration, in which caspases can participate in cleavage of proteins not resulting in apoptosis. Third, the dendritic pruning program takes place during drastic environmental changes that include concurrent degradation and regrowth of the overlying epidermis, activation of extracellular matrix metalloproteases, and blood phagocytes. These environmental cues likely complement the neuronal intrinsic pruning programs, but their exact relationships are not known. Experiments addressing these and other possible mechanisms should provide a greater insight into how the large-scale remodeling of C4da neuron dendrites is achieved (Kuo, 2006).

In vertebrates, the UPS pathway has been implicated in Wallerian degeneration of severed axons. In the fly, mushroom body γ neurons undergo extensive remodeling of their processes during metamorphosis. The initial stages of axon pruning in these mushroom body neurons closely resemble Wallerian degeneration, and the UPS again plays a critical role. To date, the specific ubiquitin-conjugating enzymes and ligases that mediate target protein degradation have not been identified in these systems. It will be interesting to see whether the UbcD1-DIAP1-Dronc pathway implicated in C4da neuron dendrite pruning also participates in remodeling/degradation of neuronal processes in other systems. It seems likely that more than one pathway would be employed in remodeling different neurons; a previous study excluded UbcD1 as a possible ubiquitin-conjugating enzyme regulating mushroom body γ neuron remodeling, and normal remodeling of mushroom body neuron processes in is seen dronc mutant MARCM clones during metamorphosis (Kuo, 2006).

A multilayered regulatory machinery for remodeling neurons, as uncovered in this study for C4da neurons, offers versatility and flexibility. It is conceivable that another ubiquitin ligase/caspase pair may function in an analogous UPS pathway during mushroom body neuron remodeling, potentially affording differential regulation of neuronal remodeling. Although pharmacological inhibition of mammalian caspases showed no effect on Wallerian degeneration, it would be important to assess the in vivo effectiveness of the inhibitors against a comprehensive panel of caspases. Moreover, a dual control mechanism, similar to what is proposed for C4da neuron remodeling, may coordinately regulate UPS and another protease that executes axon degradation. Conceivably, instead of having the target of the UPS directly involved in maintaining dendrite/axon stability, the executor of neuronal process degradation may involve a different protease: in the case of C4da neurons it is the caspase Dronc, and in Wallerian degeneration the relevant protease might be the Ca²⁺-responsive calpain. Future experiments along these lines of thinking may accelerate the identification of specific ubiquitinating enzymes involved in other areas of developmental neuronal remodeling and in diseases where the UPS pathway has been implicated. As target-specific E3 ligases are excellent candidates for pharmaceutical intervention, this approach may also help to find effective treatments for developmental and neurodegenerative diseases that involve degeneration of neuronal processes (Kuo, 2006).

The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance

Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).

Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).

Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).

In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).

In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).

In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).

To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).

The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).

The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).

Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).

Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).

To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).

Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of ³²P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).

The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons

In addition to establishing dendritic coverage of the receptive field, neurons need to adjust their dendritic arbors to match changes of the receptive field. This study shows that dendrite arborization (da) sensory neurons establish dendritic coverage of the body wall early in Drosophila larval development and then grow in precise proportion to their substrate, the underlying body wall epithelium, as the larva more than triples in length. This phenomenon, referred to as scaling growth of dendrites, requires the function of the microRNA (miRNA) bantam (ban) in the epithelial cells rather than the da neurons themselves. ban in epithelial cells dampens Akt kinase activity in adjacent neurons to influence dendrite growth. This signaling between epithelial cells and neurons receiving sensory input from the body wall synchronizes their growth to ensure proper dendritic coverage of the receptive field (Parrish, 2009).

Dendrites of class IV da neurons completely and nonredundantly cover the larval body wall early in larval development, a phenomenon referred to as dendritic tiling. Once field coverage is established, dendrites continue to branch and lengthen to maintain tiling as larvae grow, providing a sensitive system for analysis of how neurons first establish and later maintain coverage of the receptive field. This study addressed the question of how late-stage dendrite growth is precisely coordinated with larval growth to maintain proper dendrite coverage of the body wall (Parrish, 2009).

To examine this process, the pickpocket-EGFP (ppk-EGFP) marker was used to monitor class IV dendrite growth before and after establishment of tiling. To quantitatively assess dendrite coverage, a metric was used that is referred to as the coverage index, the ratio of the territory covered by dendrites of a given da neuron, such as the class IV neuron ddaC, to the area of a hemisegment that harbors the da neurons. Dendrite outgrowth of class IV neurons begins at ~16 hr After Egg Laying (AEL), with class IV dendrites growing rapidly during late embryonic/early larval stages to tile the body wall between 40 and 48 hr AEL and subsequently maintaining this coverage until dendrites are pruned during metamorphosis. Between 48 hr AEL and 120 hr AEL (just prior to metamorphosis), larvae grow nearly 3-fold in length and the dorsal area of class IV receptive fields expands by more than 6-fold. Therefore, class IV dendrites grow extensively and this dendrite growth must be precisely coordinated with larval growth in order to maintain proper coverage of the receptive field (Parrish, 2009).

Class IV dendrites are located between muscle and epithelial cells. Cell divisions that give rise to larval cells are complete by mid-embryogenesis, and larval growth is achieved by increasing cell size rather than additional proliferation. Thus, all the cells that will comprise the larval body wall musculature and epithelia are in place when dendrite outgrowth begins. To simultaneously visualize growth of class IV dendrites and epithelial cells, a protein trap line was used that directs GFP expression in epithelial cells and outlines their borders (Armadillo::GFP, adherens junctions, or Neuroglian::GFP, septate junctions) in combination with ppk-GAL4 driving expression of mCD8-RFP in class IV neurons. Using these markers, growth of class IV dendrites and epithelial cells was monitored throughout embryonic/larval stages (Parrish, 2009).

Epithelial cells grow at a nearly constant rate over the time course. Likewise, the class IV neuron soma grows at a relatively constant rate. In contrast, the dendrite growth is biphasic. Initially, class IV dendrite growth outpaces growth of epithelial cells and the larva as a whole between 16 hr and 48 hr AEL, the timeframe in which class IV dendrites establish tiling. Dendrite growth slows as class IV dendrite arbors achieve complete body wall coverage, and from 48 hr to 120 hr AEL class IV dendrites grow in proportion to larval growth at a rate comparable to that of epithelial cells. This late dendrite growth will be referred to as scaling growth of dendrites (a phenomenon unrelated to synaptic scaling) to reflect the physical scaling of dendrite arbors as they grow precisely in proportion to surrounding cells and the larva as a whole in order to maintain proper coverage of the receptive field (Parrish, 2009).

To determine whether scaling growth is a general property of da neurons, dendrite growth was monitored in class I and class III da neurons, two additional morphologically distinct classes of da neurons, using the coverage index metric introduced above. Like class IV neurons, dendrites of class I and III neurons rapidly establish coverage of a characteristic region of the body wall and subsequently maintain their coverage by expanding their dendrite arbors in precise proportion to larval growth. Class III neurons cover their territory in the same timeframe as class IV neurons, first establishing receptive field coverage at about 48 hr AEL. In contrast, class I neurons covered their characteristic territory by 24 hr AEL. Thus, temporally distinct signals may regulate scaling of dendrite growth in class I and class III/IV neurons. Nevertheless, scaling growth of dendrites seems to be a general feature of da neuron development (Parrish, 2009).

Based on the fidelity of dendrite coverage in class IV neurons, a focus was placed on these neurons for studies of dendrite scaling. The finding that class IV dendrites have a rapid growth phase during establishment of tiling and a scaling phase with slower dendrite growth to maintain tiling suggests that some signal(s) attenuate dendrite growth following establishment of tiling, synchronizing growth of class IV dendritic arbors with growth of surrounding tissue. Attempts were therefore made to characterize the signaling that underlies dendrite scaling (Parrish, 2009).

To test the capacity of dendrite scaling, the effects were examined of mutations that alter the dimensions of larvae at different developmental states on class IV dendrite growth. Alleles were chosen that survive until at least the second larval instar, allowing monitoring of dendrite coverage by class IV neurons at a time when they should have already established tiling. Overall, 35 mutant alleles were screened that cause a range of defects in larval size, shape, and growth rate. Notably, class IV dendrites properly covered the receptive field in nearly all of these mutants, accommodating a broad range of receptive field areas (ranging from 10% of wild-type [WT] in chico mutants to 120% of WT in giant [gt] mutants) and shapes. Dendrites also scaled properly in mutants defective in developmental rate, for example maintaining proper receptive field coverage in b6-22 mutants that develop slowly and persist as second instar larvae or in broad (br) mutants that persist as third instar larvae for days or even weeks. Taken together, these results demonstrate the robustness of dendrite scaling growth in class IV neurons (Parrish, 2009).

Among the few mutants that had any effect on scaling growth of dendrites, the ban mutant had the most severe dendrite overgrowth phenotype observed, with the first sign of larval growth defects at 72 hr AEL. It was reasoned that ban might be required for dendrite scaling but not earlier aspects of dendrite development, and the remainder of the study focused on the role of ban in dendrite scaling. Notably, ban encodes a miRNA and might represent a regulatory node for scaling of dendrite growth since miRNAs likely regulate expression of 100 or more target genes (Parrish, 2009).

Dendrites of individual class IV neurons occupy a larger proportion of the body wall in ban mutant third instar larvae. At 96 hr AEL, ddaC class IV neurons in ban mutants have a mean coverage index of 1.22, meaning that the receptive field of the average ddaC dendrite in ban mutant larvae is 122% of the size of the dorsal hemisegment that harbors the neuron. Thus, dendrites in ban mutants promiscuously cross boundaries that are observed by dendrites of WT neurons. For example, fewer than two dendrite branches cross the midline for a given WT class IV neuron, whereas more than 18 dendrite branches cross the midline in ban mutants. The exuberant growth of dendrites in ban mutants is manifest throughout the arbor, not just at the boundaries. However, although a coverage index of >1 is seen for ban mutant, no significant tiling defect is seen because branches that cross normal boundaries still avoid dendrites of neighboring class IV neurons. In addition to these defects in dendrite coverage, class IV neurons in ban mutants show significant increases in the number of dendrites, the density of dendrites, and overall dendrite length (data not shown). However, increased terminal dendrite branching is not sufficient to increase receptive field coverage. Several other mutants have been described that increase terminal dendrite branching in class IV neurons, and none of these mutants cause an overall increase in the size of the dendritic field. For example, furry (fry) mutations cause a 100% increase in the number/density of terminal dendrites without an accompanying increase in coverage index at 96 hr AEL. Likewise, overexpression of the small GTPase Rac drastically increases terminal dendrite branching but reduces receptive field coverage (Parrish, 2009).

The dendrite growth defects in ban mutants could reflect increased dendrite growth from early stages of development or defects specific to the scaling phase of dendrite growth. To distinguish between these possibilities, dendrite growth was monitored over a developmental time course, focusing on the coverage index and midline crossing events as metrics for growth of the dendrite arbor as a whole. Importantly, class IV dendrites in ban mutants are indistinguishable from WT during the early, rapid growth phase (through 48 hr AEL) as measured by coverage index, midline crossing events, and total dendrite branch number. However, beginning at 72 hr AEL, progressively more severe defects are noted in the coverage index and a greater number of midline crossing events in ban mutants. This late-onset exuberant dendrite growth demonstrates that ban is not causing a general growth defect since ban is dispensable for establishment of dendrite coverage. Whereas a generalized defect in dendrite growth, as seen in dendritic arbor reduction (dar; mutations that lead to defective dendritic arbors but normal axonal projections), would affect both the early (isometric) and late (scaling) phases of growth, mutations that specifically affect the scaling growth of dendrites would be dispensable for the early, rapid growth of dendritic fields. This is precisely what is seen for ban mutants. Therefore, ban is specifically required for scaling of dendrite arbors, potentially by affecting growth-inhibitory signals that normally restrict dendrite growth (Parrish, 2009).

To confirm that loss of ban causes these phenotypes, the following experiments were conducted. First, whereas heterozygosity for a ban null allele or deficiencies that span the ban locus show no obvious defects in dendrite scaling, placing ban mutations in trans to a deficiency that spans the locus, but not a nearby deficiency that does not span the ban locus, recapitulates the dendrite defects described above. Second, the ban mutant dendrite defects can be fully rescued by a ban genomic rescue transgene but not a genomic transgene in which the ban locus has been deleted. Therefore, disrupting ban function is sufficient to cause defects in scaling growth of dendrites (Parrish, 2009).

Next whether ban is required for scaling growth of dendrites was tested in other classes of da neurons. Both class I and class III neurons establish proper dendrite coverage in ban mutants. However, class III dendrites are defective in scaling of dendrite growth in ban mutants, showing a significant increase in dendrite coverage after 48 hr AEL. In contrast, larval class I dendrites show no obvious defects in dendrite coverage in ban mutants, demonstrating that ban is not required for scaling in class I neurons. The onset of scaling growth of dendrites differs by 24 hr in class I and class III/IV neurons, thus different scaling signals may operate at the two time points with ban required for the scaling growth signal for class III/IV neurons that tile (Parrish, 2009).

Next, time-lapse microscopy of single neurons was conducted to characterize the cellular basis of the ban mutant phenotype. Single class IV neurons were imaged from time-matched WT or ban mutant larvae at 24 hr intervals beginning at 72 hr AEL, just after the ban phenotype is first apparent. Dynamics were monitored of every terminal dendrite that could be unambiguously followed through the time course and dendrite growth, initiation of new dendrites, dendrite retraction, and branch loss were measured. For each of these categories, ban mutants differed from WT controls, exhibiting significantly more dendrite growth and branch initiation and significantly less dendrite retraction and branch loss. Therefore, stabilization of existing dendrites, increased dendrite growth, and increased addition of new dendrites all contribute to the defect in dendrite scaling growth of the ban mutant (Parrish, 2009).

Time-lapse studies suggest that signals normally restricting dendrite growth are largely absent in ban mutants. Attempts were made to verify this hypothesis using laser ablation assays. Previous studies showed that, following embryonic ablation of a class IV neuron, dendrites of neighboring neurons grow exuberantly to invade the unoccupied territory of the ablated neuron, with the ability of dendrites to invade unoccupied territory progressively restricted in older larvae. It was therefore important to determine whether the timing of this restricted growth potential correlates with the onset of scaling of dendrite growth and whether ban is required for restriction of the dendrite growth potential (Parrish, 2009).

Consistent with prior reports, ablating a class IV neuron at 24 hr AEL led to extensive invasion by dendrites of neighboring neurons, with 55% of the unoccupied territory covered by neighboring neurons 48 hr postablation. This ability of dendrites to grow into unoccupied territory was severely attenuated 1 day later, with dendrites of neighboring neurons invading only 23% of the unoccupied territory after ablation of a class IV neuron at 48 hr AEL . The extent of invasion was even further reduced when neurons were ablated at 72 hr AEL. Therefore, the ability of dendrites to grow beyond their normal boundaries to invade unoccupied territory is severely restricted during larval development at a time coincident with the onset of scaling of dendrite growth (Parrish, 2009).

If the restriction of dendrite growth potential in larvae is caused by scaling signals that limit dendrites to growth in proportion to body wall growth, the majority of invading activity by neighboring dendrites should be present before scaling growth ensues at 48 hr AEL. To test this prediction, class IV neurons were ablated at 24 hr AEL and invasion activity was monitored at 24 hr intervals over the next 72 hr. By 48 hr AEL, dendrites of neighboring neurons had invaded unoccupied territory, and the extent of invasion was not noticeably increased at later time points. Instead, the entire dendrite arbor of class IV neurons, including the portion that invaded unoccupied territory, scaled with larval growth after 48 hr AEL. Thus, the receptive field that is established by 48 hr AEL is maintained by scaling of dendrite growth, even in cases in which dendrites establish aberrant body wall coverage. The signals responsible for dendrite scaling growth are likely distinct from the homotypic repulsion required to establish tiling as ablation of all neighboring same-type neurons does not potentiate the ability of a class IV neuron to invade unoccupied territory. Additionally, dendrites of class I da neurons, which do not rely on homotypic repulsion to establish their coverage, also exhibit scaling growth (Parrish, 2009).

As described above, dendrite coverage is properly established in ban mutants. Importantly, unlike WT controls, following ablation at 48 hr AEL, dendrites in ban mutants extensively fill unoccupied space, with dendrites in ban mutants invading unoccupied territory just as efficiently as dendrites in WT controls ablated at 24 hr AEL. Therefore, the receptive field boundaries of class IV neurons have not been fixed in ban mutants at 48 hr AEL. Dendrites in ban mutants invade unoccupied territory more efficiently than WT controls at later time points as well. Thus, either the growth-inhibitory scaling signal is lost or dendrites are refractory to the signal in ban mutants (Parrish, 2009).

To test whether machineries for dendritic tiling contribute to the progressive reduction of a dendrite's ability to invade vacant territories, mutations of fry, which encodes a gene required for establishment of dendritic tiling and of extra sex combs (esc) and salvador (sav), which function in a common pathway to regulate stability of terminal dendrites and, consequently, maintenance of dendrite coverage, were examined for effects on dendrite invasion following neuron ablation. Unlike mutations in ban, mutations in fry, esc, or sav had no effect on the ability of dendrites to invade unoccupied territory. Moreover, consistent with the scaling signal functioning in a distinct pathway, double-mutant combinations of ban with fry or esc showed additive phenotypes. Thus, ban exerts its effects on scaling of dendrite growth independently of known pathways for establishment and maintenance of dendrite coverage (Parrish, 2009).

To further characterize the signaling required for scaling growth of dendrites, it was of interest to determine where ban functions to regulate scaling. First, whether ban is expressed in neurons, surrounding cells, or both, was examined by using a miRNA activity sensor as a reporter for ban expression in third instar larvae. A control sensor directs ubiquitous expression of GFP, including robust GFP expression in muscle, epithelial cells, and sensory neurons. The ban sensor contains two ban binding sites in the 3'UTR of the transgene, hence GFP expression is attenuated in cells that express ban. Unlike the control sensor, very little, if any, GFP expression was detected in third instar muscle cells, epithelial cells, or sensory neurons using several independent transgenic fly lines with distinct insertions of the ban sensor. Significant attenuation of the ban sensor was first observ ed in larval muscle, epithelium, and PNS neurons between 48 and 72 hr AEL, precisely at the time when dendrite defects were first observed in ban mutants, suggesting that ban activity is more pronounced during this period than at earlier time points. Notably, the attenuation of the ban sensor was dependent on ban activity, as shown by the persistent, ubiquitous expression of the sensor in ban mutant larvae. Thus, ban is likely expressed in the muscle, epithelium, and PNS neurons and may be required in any of these cell types for scaling of dendrite growth (Parrish, 2009).

To determine whether ban is required cell-autonomously for dendrite scaling, MARCM was used to generate single neuron clones homozygous for a ban mutation in a heterozygous background. ban activity was effectively dampened in MARCM clones, as indicated by derepression of the ban sensor in the clones. However, loss of ban function had no significant effect on dendrite coverage of class IV neurons. Time-lapse analysis of ban mutant class IV MARCM clones revealed no defects in dendrite coverage at any time during larval development. Furthermore, ban is dispensable in other da neurons for dendrite scaling growth. Thus, ban function in sensory neurons is dispensable for scaling growth of dendrites (Parrish, 2009).

Although scaling of dendrite growth proceeds normally, there is some reduction of overall dendrite length and the number of dendrite branches in ban mutant class IV clones. Therefore, ban likely acts cell-autonomously to promote dendrite growth and nonautonomously to limit dendrite Taking advantage and ensure proper scaling (Parrish, 2009).

A genetic rescue assay was used to test the ability of transgenic expression of ban in different tissues to rescue the dendrite growth defects of ban mutants. Consistent with MARCM results, neuronal expression of ban, using either panneuronal or PNS-specific Gal4 drivers, was not sufficient to rescue the scaling growth defect of ban mutants. Thus, ban likely functions nonautonomously in nonneuronal cells to regulate scaling of da neuron dendrite growth. Moreover, expression of ban in muscle alone could not ameliorate the dendrite defects of ban mutants. Remarkably, every time ban expression was rescued in epithelial cells, significant suppression of the exuberant dendrite growth of ban mutants was found. The three epithelial Gal4 driver lines caused reductions of dendrite growth that correlated with Gal4 expression levels in epithelial cells: arm-Gal4 caused the greatest reduction in dendrite growth and had the strongest epithelial expression, whereas twi-Gal4 displayed the lowest activity and drove epithelial Gal4 expression at the lowest level. Taking advantage of the temperature-sensitive nature of Gal4 activity, rescue activity of each epithelial Gal4 line was monitored over a graded temperature series (18°C to 29°C) and it was found that, for each driver, rescue activity was directly proportional to expression level. Therefore, epithelial ban expression is sufficient to suppress the exuberant dendrite growth of ban mutants, and the extent of dendrite growth inhibition varies with the level of ban expression in epithelial cells (Parrish, 2009).

Epithelial expression of twi-Gal4 was first apparent in larval stages, suggesting that postembryonic expression of ban in epithelia is sufficient for proper scaling of dendrite growth. Given that dendrite defects in ban mutants first appear after 48 hr AEL, it was asked whether late expression of ban would suffice for dendrite scaling. To examine the temporal requirement for ban function, a heat-shock-inducible Gal4 driver was used to express ban during larval development. Indeed, inducing ban expression at 48 hr AEL was sufficient to rescue the dendrite defects of ban mutants. These findings reinforce the notion that ban is dispensable for early aspects of dendrite development (Parrish, 2009).

Resupplying ban in tissues known to regulate larval growth, such as the fat body, prothoracic (PTTH) gland, or insulin-producing cells (IPCs), had no measurable effect on dendrite growth in ban mutants. Moreover, ablation of each of these tissues mediated by a reaper transgene caused larval growth defects without obvious dendrite growth defects. Thus, ban function in the fat body, PTTH gland, or IPCs is not sufficient to modulate scaling of dendrite growth. Altogether, these results suggest that epithelial cells are likely the major functional sites for ban in regulation of PNS dendrite scaling (Parrish, 2009).

Because ban expression in epithelial cells affects scaling growth of dendrites in a dose-dependent fashion via a mechanism that likely involves growth-inhibitory signals, it was of interest to see whether ectopic epithelial expression of ban in a WT background could further inhibit dendrite growth and thus disrupt scaling of dendrite arbors. Indeed, overexpression of ban in epithelial cells resulted in a severe reduction in dendrite growth and induced striking defects in the pattern of dendrite growth over epithelial cells, with terminal dendrites appearing to wrap around epithelial cells. Consistent with ban dosage in epithelial cells regulating the strength of dendrite growth-inhibitory signals, epithelial overexpression of ban induced more robust inhibition of dendrite growth at higher temperatures (which lead to higher levels of transgene expression) (Parrish, 2009).

Since ban expression in epithelial cells is sufficient to ensure proper scaling, it was of interest to address whether ban function in epithelial cells is necessary for scaling of dendrite growth. To this end, MARCM was used to generate ban mutant epithelial cell clones. Although it was not possible to address the contribution of epithelial ban to scaling of the entire dendrite arbor using this approach (it was only possible to generate one to four cell epithelial clones), the pattern was monitored of dendrite growth over ban mutant or WT control epithelial clones. Class IV dendrites grow extensively over epithelial cells, with multiple dendrite branches often coursing over a single epithelial cell. The epithelial nucleus was used as a landmark and dendrite growth was monitored over the epithelial cell surface shadowed by the nucleus. Although the gross morphology of epithelial cells was not obviously affected in ban mutant clones, the propensity of class IV dendrites to grow into the region shadowed by the epithelial nucleus was significantly increased for ban mutant epithelial clones when compared to WT controls or ban heterozygous epithelial cells. Therefore, ban is required in epithelial cells to ensure proper dendrite growth and placement over epithelial cells (Parrish, 2009).

To gain insight into the molecular mechanism underlying dendrite scaling, a platform was developed for microarray-based expression profiling of dissociated, FACS-isolated PNS neurons or epithelial cells. Akt and numerous other candidate genes were identified that were deregulated in PNS neurons and/or epithelial cells of ban mutant larvae. Because Akt is a well-established regulator of growth, including dendrite growth in mammalian hippocampal neurons, whether ban regulates Akt as part of the scaling program was investigated (Parrish, 2009).

Microarray experiments found that Akt expression was increased in neurons but reduced in epithelial cells of ban mutants relative to WT controls. By monitoring Akt levels in lysates of larval fillets composed mostly of muscle and epithelial cells, it was found that in the absence of ban function Akt protein levels were substantially reduced. Furthermore, Akt activity was substantially reduced as shown by reductions in active, phosphorylated Akt and phosphorylated S6K, a downstream reporter of Akt activity. Therefore, Akt expression and activity are substantially reduced in ban mutant larval lysates, likely reflecting reduced Akt function in muscle/epithelia (Parrish, 2009).

Next larval fillets were immunostained to determine whether ban influences Akt protein levels in the PNS. In WT controls, Akt is detectible only at low levels in the soma or dendrites of the PNS. By contrast, in ban mutants Akt is highly expressed in the PNS and is detectible in axons, the soma, and dendrites. Similarly, phosphorylated Akt is barely detectible in the larval PNS of WT controls but is present at high levels in the PNS of ban mutants. Therefore, ban regulates Akt expression and activity in the larval PNS (Parrish, 2009).

To test whether this effect on Akt levels reflects a neuronal requirement for ban, Akt expression levels were monitored in ban mutants in which ban expression is resupplied under the control of twist-Gal4, an experimental condition that rescues both the dendrite scaling defect and larval size defect of ban mutants. It was found that ban nonautonomously regulates Akt levels in da neurons since nonneuronal expression of ban (twist-Gal4) is sufficient to dampen the ectopic neuronal Akt expression normally seen in ban mutants. Therefore, ban likely functions in epithelia to regulate signals that influence Akt expression and activity in neurons (Parrish, 2009).

Finally, it was of interest to determine whether Akt function in class IV neurons is important for scaling of dendrite growth. Based on expression data, it was predicted that increasing Akt expression/activity in class IV neurons should cause a scaling defect similar to what is seen in ban mutants. Indeed, ectopic expression of Akt, or a constitutively active form of PI3 kinase (PI3k) that leads to activation of Akt, caused a significant increase in dendrite coverage, similar to ban mutants. Conversely, antagonizing Akt activity in class IV neurons by overexpressing Pten, a PIP3 phosphatase that functions as an inhibitor of Akt activity, by knocking down Akt expression via RNAi in class IV neurons, or by generating Akt null mutant class IV neuron MARCM clones caused a significant reduction in dendrite coverage. Therefore, Akt plays a critical role in regulating dendrite coverage (Parrish, 2009).

If increased neuronal Akt activity underlies the dendrite defects in ban mutants, then antagonizing neuronal Akt activity should suppress the dendrite overgrowth in ban mutants. This hypothesis was tested with the following three experiments. First, RNAi was used to knock down Akt expression in class IV neurons of ban mutant larvae. On its own, Akt(RNAi) causes a reduction in dendrite growth and overall coverage of the receptive field, and this phenotype is epistatic to the dendrite overgrowth seen in ban mutants. Similarly, Pten was overexpressed in class IV neurons of ban mutant larvae and it was found that the Pten-mediated reduction in dendrite coverage is epistatic to the dendrite overgrowth seen in ban mutants. Finally, class IV neurons was ablated in ban mutants in the absence or presence of neuron-specific Akt RNAi and it was found that reducing neuronal Akt expression blocks the exuberant dendrite invasion activity of ban mutants. Altogether, these results strongly suggest that ban functions in epithelial cells to regulate neuronal expression/activation of Akt, and deregulation of Akt leads to the dendrite growth defects of ban mutants (Parrish, 2009).

This study has shown that nonautonomous signals coordinate growth of dendrites with the growth of their substrate and the body as a whole. Dendrites of many types of neurons cover characteristic receptive fields, and growth of the dendrite arbor in synchrony with the receptive field in a process, referred to as scaling growth of dendrites, allows a neuron to maintain proper dendrite coverage of the receptive field. Thus, scaling growth of dendrites is likely a general mechanism to ensure fidelity of dendrite coverage (Parrish, 2009).

Dendrites of class IV neurons cover their receptive field before larval growth is complete and must maintain this coverage as the larva grows. Two properties distinguish the scaling phase of dendrite growth from the early dendrite growth when the neuron establishes receptive field coverage. During the scaling phase of growth, the dendrite arbor grows precisely in proportion to receptive field expansion (which is often achieved by animal growth). Moreover, dendrite growth is constrained by boundaries delineated when the dendrite arbor first covers the receptive field. Thus, although dendrites continue to grow, growth occurs only to maintain proportional coverage of the receptive field (Parrish, 2009).

Dendrites of Drosophila da neurons exhibit a biphasic growth profile: dendrites establish coverage of their receptive field via an early, rapid growth phase and maintain this coverage via a late scaling growth phase in which dendrites grow in proportion with epithelial cells and the animal as a whole. The miRNA ban acts in the second phase to enable scaling of dendrite growth in da neurons, ensuring that dendrites maintain proper body wall coverage. Loss of ban disrupts epithelial-derived signaling that normally modulates dendrite growth, and, as a result, dendrites remain in the 'rapid growth' phase, extending beyond their normal territories. This phenotype is reminiscent of the heterochronic phenotypes of C. elegans lin-4 and let-7 mutants in that an early developmental phase is inappropriately reiterated during a later phase (Parrish, 2009).

How broadly do miRNAs regulate developmental progressions in the nervous system? Although the developmental roles of vertebrate miRNAs have been somewhat elusive because of the vast number of miRNA-encoding genes, several studies suggest that miRNAs may serve highly specialized roles in regulating developmental transitions in neuron morphogenesis. For example, completely abrogating miRNA function causes robust defects in neuron morphogenesis but not specification in zebrafish, consistent with miRNAS regulating late aspects of neuronal differentiation. Likewise, miRNAs miR9a^* and miR-124 regulate the switch in subunit composition of chromatin remodeling complexes as neural progenitors differentiate into neurons in mice. Additionally, a number of miRNAs function primarily at a very late step of neuron development to regulate activity-dependent dendrite growth and synaptic plasticity. For example, neuronal activity antagonizes miR-134, which normally inhibits growth of dendritic spines, and promotes expression of mir-132, which promotes dendritic plasticity (Parrish, 2009).

Although ban is known to regulate growth in proliferating tissues in Drosophila, ban-mediated regulation of dendrite growth likely represents a distinct mode of growth control by ban for the following reasons. First, previous studies focused on autonomous regulation of tissue growth by ban. In contrast, ban acts nonautonomously to regulate scaling of dendrite growth. Second, prior studies of ban function focused on imaginal discs where growth is achieved by increasing cell number rather than cell size. By contrast, dendrite scaling involves ban-mediated regulation of growth in differentiated, postmitotic cells. Likewise, postmitotic expression of ban in the larval eye disc can also regulate cell size. Third, ban functions downstream of the tumor suppressor kinase Hippo to control proliferation, with Hippo activating the transcription factor Yorkie, which in turn activates ban expression. Whereas hippo is required cell-autonomously for establishment and maintenance of dendrite tiling, yorkie is dispensable for dendrite growth. As to the cell-nonautonomous function of ban in dendrite scaling, Hippo is not required. Although these findings suggest that ban regulates growth of proliferating and differentiated tissues by different means, it is possible that in both scenarios ban is antagonizing expression of growth-inhibitory factors, possibly even the same factors, and removing growth inhibition has different consequences on proliferative and differentiated tissues (Parrish, 2009).

It is proposed that ban positively regulates an epithelial-derived signal that modulates neuronal Akt expression and activity to influence dendrite growth. Several observations suggest that the signal acts over a short range, possibly even via direct adhesive interactions between dendrites and epithelia or the underlying matrix. First, ban overexpression in epithelial cells but not in muscle influences growth of dendrites. Second, removing ban function from epithelial cell clones influences the distribution of dendrites over the clone but not over adjacent WT epithelial cells. Third, in addition to inhibiting the overall rate of dendrite growth, overexpressing ban in epithelial cells induces exaggerated “wrapping” of epithelial cells by terminal dendrites. Since these signals appear to preferentially regulate dendrite growth during the scaling phase, ban may modulate dendrite/epithelial adhesion during the scaling phase of dendrite development (Parrish, 2009).

Morphologically distinct classes of da neurons establish type-specific dendritic coverage of the body wall and maintain this coverage by means of dendrite scaling as larvae grow. However, arbors of different da neurons develop at different rates, with class I dendrites establishing their coverage 1 day earlier than class III and class IV dendrites. Mutations in ban disrupt scaling of dendrite growth in class III and class IV but not class I neurons, suggesting that different signals regulate dendrite scaling in distinct types of neurons. Thus, temporally distinct signals or temporally restricted sensitivity to the signals may ensure that different neurons maintain appropriate coverage of their receptive field (Parrish, 2009).

Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling

The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).

This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).

Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of K_ir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).

Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca²⁺, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).

This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).

In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).

Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. This fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. The medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections (Brierley, 2009).

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. The architecture of dendrites and the role they play in establishing connectivity within maps has been somewhat overlooked. Classic cell-labelling studies in the moth Manduca sexta revealed that the dendrites of motoneurons are topographically organized to reflect their site of innervation in the bodywall. More recent work by Landgraf and colleagues has demonstrated that motoneurons in Drosophila embryos generate a detailed dendritic (myotopic) map of body wall muscles within the CNS. Alongside these data, studies on the architecture of the spinal cord also suggest that similar design principles may play a role in organizing information in vertebrate motor systems. How such dendritic maps are built is still largely unknown. This study describes the role dendritic targeting plays in constructing a myotopic map and the molecular mechanisms that control it (Brierley, 2009).

The majority of leg motoneurons in a fly are born postembryonically and most of those are derived from a single neuroblast lineage, termed lineage 15. Perhaps the most striking feature of this lineage is its birth-order-based pattern of innervation along the proximo-distal axis of the leg. Using mosaic analysis, the sequential production was observed of four neuronal subtypes during larval life, each elaborating stereotyped axonal and dendritic projections in the adult. The axon of the first-born neuron innervates a muscle in the body wall and subsequent neurons innervate more distal targets in the leg. This organization has also been reported by Baek (2009) (Brierley, 2009).

This birth-order-based peripheral pattern of lineage 15 is mirrored in the CNS, where dendrites generate a stereotyped anatomical organization. Dendrites of early-born cells span medial to lateral territories, whereas late-born cells elaborate dendrites in the lateral neuropil and cells born between these times occupy intermediate territories. The sequential production of neuronal subtypes by neural precursor cells is a common mechanism for generating a diversity of circuit components. A similar birth-order-based specification of axonal and dendritic projection patterns has previously been described for projection neurons in the fly's olfactory system (Brierley, 2009).

The data reveal the existence of a myotopic map in the adult fly and supports the proposition that dendritic maps are a common organizing principle of all motor systems. Mauss (2009) also reveal a map in the embryonic CNS of Drosophila, where the dendrites of motoneurons are organized along the medio-lateral axis of the neuropil representing dorsoventral patterns of innervation in the body wall muscles (Brierley, 2009).

How are dendritic maps built? The myotopic map seen in the leg neuropil could be generated by two distinctly different mechanisms. Neurons could elaborate their dendrites profusely across a wide field and then remove branches from inappropriate regions or, alternatively, they could target the growth of dendrites into a distinct region of neuropil throughout development. Both mechanisms can generate cell-type-specific projection patterns as seen in the vertebrate retina. To reveal which mechanism is deployed in the leg motor system of Drosophila, single-cell clones of motoneuron subtypes generated by heatshocks at 48 and 96 h AH were imaged, since their final dendritic arborizations cover clearly distinct territories within the map. The dendrites of both elaborate branches only in territories where the mature arborizations eventually reside, which strongly supports the notion that this myotopic map is generated by targeting and not large-scale branch elimination. Importantly, this developmental timeline also revealed that the motoneurons elaborate their dendrites synchronously, regardless of the birth date of the cell. This observation suggests that a 'space-filling/occupancy based' model, where later-born neurons are excluded from medial territories by competitive interactions is unlikely. Similarly, heterochronic mechanisms where different members of the lineage experience different signalling landscapes due to differences in the timing of outgrowth are not likely either. With synchronous outgrowth dendrites experience the same set of extracellular signals, suggesting that the intrinsic properties of cells, defined by their birth order, may be more important for the generation of subtype-specific projections. Such intrinsic properties could include cell-cell recognition systems such as adhesion molecules, e.g., Dscams or classical guidance receptors, that could interpret extracellular signals. In the Drosophila embryo motoneurons also use dendritic targeting to generate a myotopic map (Brierley, 2009).

It is emerging that dendrites are guided by the same molecules that control axon pathfinding. The medio-lateral organization of leg motoneuron dendrites within the leg neuropil prompted an investigation as to whether the midline signalling molecules Slit and Netrin and their respective receptors Roundabout and Frazzled could be involved in targeting growth to specific territories (Brierley, 2009).

Using mosaic analysis it was found that both the 48 and 96 h AH motoneuron subtypes require Robo to generate their appropriate shape and position within the medio-lateral axis. When Robo was removed from the 48 h AH subtype the mean centre of arbor mass was shifted toward the midline. The dendrites of 96 h AH neurons showed a shift in distribution in the absence of Robo but still failed to reach the midline, suggesting that only part of this cell's targeting is due to repulsive cues mediated by the Robo receptor. It was predicted that if Robo levels played an instructive role in dendrite targeting it would be possible to shift dendrites laterally by cell autonomously increasing Robo. This was found to be the case in both subtypes. Taken together these data suggest that differences in the level of Robo signalling may provide a mechanism by which Slit could be differentially interpreted to allow subtype-specific targeting along the medio-lateral axis (Brierley, 2009).

The Robo receptor is part of a larger family of receptors that includes Robo2 and Robo3. This family of receptors have been found to be important for targeting axons to the appropriate longitudinal pathway in the embryonic CNS. Comm plays a key role in allowing contralaterally projecting neurons to cross the midline, and its ectopic expression (CommGOF) is known to robustly knock down Robo and Robo2 and 3. Comm was cell autonomously expressed in both lineage 15 subtypes and shifts to the midline were found in both 48 and 96 h AH neurons. For the 48 h AH neurons, Robo LOF data and CommGOF data are comparable, suggesting that Robo alone plays a major role in the positioning dendrites of these cells. In contrast, in the 96 h AH subtype RoboLOF and CommGOF effects were found to be significantly different, suggesting that the 96 h AH subtype may not only use the Robo receptor but additional Robos as well. Knockdown of Slit also supports this idea, as the branches of late-born neurons were occasionally found reaching the midline, something that was never see in RoboLOF clones. Thus, one way of establishing differences in the medio-lateral position could be through a dendritic “Robo code” where early-born cells express Robo and late-born cell express multiple Robo receptors (Brierley, 2009).

With Netrin being expressed in the midline cells during the pupal-adult transition it was asked whether attractive Netrin-Fra signalling could also contribute to positioning dendrites in the leg neuropil. When Fra was removed from the 48 h AH subtype it was found that the arborization was shifted laterally, whereas removing it from the 96 h AH subtype had little effect, and neither did the removal of Netrin A and B from the midline, suggesting that Netrin-Fra signalling may not play a role in dendritic targeting in the later-born cell. It may be that Fra is expressed in early-born cells within the lineage and then down-regulated, although it cannot be excluded that Netrin-Fra signalling was masked by the repulsion from Slit-Robo signalling. These data are consistent with Fra being a major player in targeting the dendrites of the 48 h AH cell. The fact that both Fra and Robo are required for normal morphogenesis of 48 h AH neurons raises the possibility that members of lineage 15 could use a 'push-pull' mechanism for positioning their dendrites, where the blend of receptors within a cell dictates the territory within the map that they will innervate (Brierley, 2009).

How could such subtype-specific blends of receptors be established? A number of studies have revealed that spatial codes of transcription factors are important for specifying the identity of motoneuron populations. Within lineage 15 it is possible that temporal, rather than spatial, transcription factor codes are important for regulating the blend of guidance receptors. A number of molecules have been identified that control the sequential generation of cell types within neuroblast lineages. Chief amongst these are a series of transcription factors that include Hunchback, Krüppel, Pdm, Castor and Seven-up. These temporal transcription factors are transiently expressed within neuroblasts and endow daughter neurons with distinct “temporal identities”. Castor and Seven-up are known to schedule transitions in postembryonic lineages, regulating the neuronal expression of BTB-POZ transcription factors Chinmo and Broad. It is possible that the temporal transcription factors Broad and Chinmo could control the subtype-specific expression of different Robo receptors or the Netrin receptor Frazzled in leg motoneurons. There is a precedent for this in the Drosophila embryo, where motoneuron axon guidance decisions to distal (dorsal) versus proximal (ventral) targets are orchestrated by Even-Skipped, a homeobox transcription factor, which in turn controls the expression of distinct Netrin receptor combinations (Brierley, 2009).

Studies focusing on the growth of olfactory projection neuron dendrites in Drosophila reveal that they elaborate a glomerular protomap prior to the arrival of olfactory receptor neurons suggesting that target/partner-derived factors may not be necessary for establishing coarse patterning of synaptic specificity. The global nature of the signals describe in this study and their origin in a third-party tissue is a fundamentally different situation to that where target-derived factors instruct partner cells, such as presynaptic amacrine cells signalling to retinal ganglion cell dendrites in the zebrafish retina. Furthermore, although this study shows that Slit and Netrin control the positioning of dendrites across the medio-lateral axis of the CNS, it may be that other similar guidance signals are important for patterning dendrites in other axes. There is a striking conservation of the molecular mechanisms that build myotopic maps in the embryo and pupae. Understanding the similarities and differences between these myotopic maps, from an anatomical, developmental, and functional perspective, may give insight into the evolution of motor systems and neural networks in general (Brierley, 2009).

This study found that individual leg motoneurons that lacked Robo signalling appeared to have more complex dendritic arborizations. The working hypothesis, that dendrites invaded medial territories because of a failure of Slit-Robo guidance function, did not take into account the possibility that cells may generate more dendrites due to a change in a cell-intrinsic growth program. Thus the changes seen in dendrite distribution relative to the midline could formally be a result of 'spill-over' from that increase in cell size/mass. To determine whether this was the case larger cells were generated by activating the insulin pathway in single motoneurons. It was found the dendrites of these 'large cells' remained within their normal neuropil territory, supporting the idea that the removal of Robo-Slit signalling results in a disruption in guidance, not growth. These data underline the fundamental importance of midline signals in controlling the spatial coordinates that these motoneuron dendrites occupy, i.e., that a neuron twice the size/mass of a wild-type cell is still marshalled into the same volume of neuropil (Brierley, 2009).

When the image stacks were reconstructed to look at the distribution of the dendrites in the dorso-ventral axis, it was found that the apparent increase in size was in fact a redistribution of the dendrites from ventral territories into more dorsal medial domains. This was unexpected and suggests that changes in midline signalling can also impact the organization of dendrites in the dorso-ventral axis. So CommGOF 96 h AH neurons may not only encounter novel synaptic inputs by projecting into medial territories, but they may also lose inputs from the ventral domains of neuropil they have vacated. These observations suggest that motoneurons within lineage 15 have a fixed quota of dendrites and where it is distributed in space depends on cell-intrinsic blends of guidance receptors. Taken together these data support the idea that growth and guidance mechanisms are genetically separable programs. In identified embryonic motoneurons where Slit-Robo and Netrin-Fra signalling has been disrupted, quantitative analysis reveals dendrites also show no measurable difference in their total number of branch tips or length (Mauss, 2009). Moreover, recent computational studies in larger flies reveal that dendritic arborizations generated by the same branching programs can generate very different shapes depending on how their 'dendritic span' restricted within the neuropil. Previous work in both vertebrates and Drosophila has shown that a loss of Slit-Robo signalling results in a reduction in dendrite growth and complexity, but this study found no evidence to support this (Brierley, 2009).

Neural maps and synaptic laminae are universal features of nervous system design and are essential for organizing and presenting synaptic information. How the appropriate pre- and postsynaptic elements within such structures are brought together remains a major unanswered question in neurobiology. Studies in recent years have shown that neural network development involves both hardwired molecular guidance mechanisms and activity-dependent processes; the relative contribution that each makes is still unclear. Work on the spinal cord network of Xenopus embryos revealed that seven identifiable neuron subtypes can establish connections with one another and that the key predictor of connectivity was their anatomical overlap. This could be interpreted to mean that connectivity is promiscuous and that the major requirement for the generation of synaptic specificity is the proximity of axons and dendrites. This is particularly interesting in light of the current dendrite targeting data and the observation that both sensory neurons and interneurons in Drosophila use the same midline cues to position their pre-synaptic terminals in the CNS. Moreover, a recent study has shown that Semaphorins control the positioning of axons within the dorso-ventral axis. Taken together these observations suggest that during development the coordinated targeting of both pre- and postsynaptic elements into the same space using global, third-party guidance signals could provide a simple way of establishing the specificity of synaptic connections within neural networks. This idea is akin to 'meeting places' such as the traditional rendezvous underneath the four-sided clock at Waterloo railway station where two interested parties organize to meet. Understanding how morphogenetic programs contribute to the generation of synaptic specificity is likely to be key to solving the problem of neural network formation (Brierley, 2009).

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this system was used to understand how cells can maintain dynamic arrays of polarized microtubules. It was found that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, it was shown that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is proposed that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn toward the cell body at branch points and that they track stable microtubules through branches. Kinesin-2, EB1, and APC are all required for maintaining microtubule polarity and are linked in an interaction network (Mattie, 2010).

On the basis of these results, a model for directed growth of microtubules in dendrites is proposed. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2 and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, therefore it is proposed that a growing microtubule plus end coated with EB1 is transiently linked, through the interaction between Apc and the EB1 tail, to kinesin-2 as it passes through the branch. SxIP motifs in Apc and Klp68D could also contribute to this interaction. Because both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule toward the plus end and cell body (Mattie, 2010).

Even a very brief application of force pulling the growing microtubule toward the cell body should be sufficient to steer growth toward the cell body. Once the tip of the microtubule turns, growth should be constrained by the dendrite walls. The association of the growing plus end with a stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1 and that individual EB1 interactions with the microtubule plus end persist for less than a second (Mattie, 2010).

Observations of plus-end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved; frequently plus ends are seen turning sharply. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead, they probably represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also probably necessary to establish uniform microtubule polarity in branched dendrites, but it probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity, and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternatively, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

Because kinesin-2, APC, EB1, and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

Kinesin-2 has previously been shown to be enriched in the tips of growing axons in cultured mammalian neurons, and it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low-actin conditions. Thus, directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be involved in aligning microtubules in many circumstances (Mattie, 2010).

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date revised: 15 August 2011

Genes involved in organ development

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