Dendrites of da neurons grow rapidly to establish receptive field coverage then grow in proportion to larval growth to maintain this coverage
Dendrites of class IV da neurons completely and non-redundantly cover the larval body wall early in larval development, a phenomenon referred to as dendritic tiling (Grueber et al., 2002
). 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. In this study we have addressed the question of how late-stage dendrite growth is precisely coordinated with larval growth to maintain proper dendrite coverage of the body wall.
To examine this process, we used the pickpocket
-EGFP) marker (Grueber et al., 2003b
) to monitor class IV dendrite growth before and after establishment of tiling. To quantitatively assess dendrite coverage we have used a metric that we refer 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 (Figure S1
and Experimental Procedures). Dendrite outgrowth of class IV neurons begins at approximately 16 hr After Egg Laying (AEL), with class IV dendrites growing rapidly during late embryonic/early larval stages to tile the body wall between 40-48 hr AEL and subsequently maintaining this coverage until dendrites are pruned during metamorphosis (). Between 48hr 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.
Dendrites of da neurons grow rapidly to establish receptive field coverage then grow in proportion to larval growth to maintain this coverage.
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 (Edgar and Nijhout, 2003
). 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 we used a protein trap line 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, we monitored growth of class IV dendrites and epithelial cells throughout embryonic/larval stages.
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 (). We will refer to this late dendrite growth 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.
Scaling growth of dendrites is a general property of da neurons
To determine whether scaling growth is a general property of da neurons we monitored dendrite growth in class I and class III da neurons, two additional morphologically distinct classes of da neurons (Grueber et al., 2002
) 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 (Figures and S2
). 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.
Based on the fidelity of dendrite coverage in class IV neurons we focused on these neurons for our studies of dendrite scaling. Our 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. We therefore set out to characterize the signaling that underlies dendrite scaling.
Scaling growth of dendrites ensures proper dendrite coverage in larva of diverse sizes and shapes
To test the capacity of dendrite scaling, we examined the effects of mutations that alter the dimensions of larvae at different developmental states on class IV dendrite growth. We chose alleles that survive until at least the 2nd
larval instar, allowing us to monitor dendrite coverage by class IV neurons at a time when they should have already established tiling. Overall, we screened 35 mutant alleles that cause a range of defects in larval size, shape and growth rate, and results for a representative subset of these alleles are shown (, Table S1
). 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
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 2nd
instar larvae or in broad
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.
Screen for mutants that affect scaling growth of dendrites.
ban is required for scaling growth of dendrites
Among the few mutants that had any effect on scaling growth of dendrites, the ban
mutant had the most severe dendrite overgrowth phenotype we observed (), with the first sign of larval growth defects at 72 hr AEL (). We reasoned that ban
might be required for dendrite scaling but not earlier aspects of dendrite development and focused the remainder of our study on the role of ban
in dendrite scaling. Notably, ban
encodes a miRNA (Brennecke et al., 2003
) and might represent a regulatory node for scaling of dendrite growth since miRNAs likely regulate expression of 100 or more target genes (Lim et al., 2005
ban is required for scaling growth of dendrites in a subset of da neurons.
Dendrites of individual class IV neurons occupy a larger proportion of the body wall in ban mutant 3rd instar larvae (Figures , ). 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 2 dendrite branches cross the midline for a given wt class IV neuron, whereas more than 18 dendrite branches cross the midline in ban mutants (). However, although we see a coverage index of >1 for ban mutant, we do not see a significant tiling defect because branches that cross normal boundaries still avoid dendrites of neighboring class IV neurons.
The exuberant growth of dendrites in ban
mutants is manifest throughout the arbor, not just at the boundaries. 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
mutations cause a 100% increase in the number/density of terminal dendrites (Emoto et al., 2004
) without an accompanying increase in coverage index at 96hr AEL (). Likewise, overexpression of the small GTPase Rac drastically increases terminal dendrite branching but reduces receptive field coverage (data not shown).
ban regulates growth-inhibitory signals to ensure dendrite scaling.
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 we monitored dendrite growth 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 (data not shown). However, beginning at 72 hr AEL we noted progressively more severe defects 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 dar
mutants (Ye et al., 2007
), 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 we see for ban
mutants. Therefore, ban
is specifically required for scaling of dendrite arbors, potentially by affecting growth-inhibitory signals that normally restrict dendrite growth.
To confirm that loss of ban
causes these phenotypes, we conducted the following experiments. 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 does not span the ban
locus, recapitulates the dendrite defects described above (Figure S3
). 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 (Figure S3
). Therefore, disrupting ban
function is sufficient to cause defects in scaling growth of dendrites.
We next tested whether ban
is required for scaling growth of dendrites in other classes of da neurons. Both class I and class III neurons establish proper dendrite coverage in ban
mutants (data not shown). 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 (Figures and S4
). 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 (Figures and S4
). The onset of scaling growth of dendrites differs by 24 hours 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.
Next, we conducted time-lapse microscopy of single neurons to characterize the cellular basis of the ban
mutant phenotype. We imaged single class IV neurons 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 (Figure S5
). We monitored dynamics of every terminal dendrite that could be unambiguously followed through the time course and measured dendrite growth, initiation of new dendrites, dendrite retraction, and branch loss. 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 (Figures and S5
). 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
Growth inhibitory signals regulate dendrite scaling
Our time-lapse studies suggest that signals normally restricting dendrite growth are largely absent in ban
mutants. We set out 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 (Grueber et al., 2003b
; Sugimura et al., 2003
), with the ability of dendrites to invade unoccupied territory progressively restricted in older larvae (Sugimura et al., 2003
). We therefore wanted 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.
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 post-ablation (). This ability of dendrites to grow into unoccupied territory was severely attenuated one 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.
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 we ablated class IV neurons at 24 hr AEL and monitored invasion activity 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 (data not shown). Additionally, dendrites of class I da neurons, which do not rely on homotypic repulsion to establish their coverage, also exhibit scaling growth.
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.
To test whether machineries for dendritic tiling contribute to the progressive reduction of a dendrite’s ability to invade vacant territories, we examined mutations of furry
), which encodes a gene required for establishment of dendritic tiling (Emoto et al., 2004
), and extra sex combs
) and salvador
), which function in a common pathway to regulate stability of terminal dendrites and, consequently, maintenance of dendrite coverage (Emoto et al., 2006
; Parrish et al., 2007
), for effects on dendrite invasion following neuron ablation. Unlike mutations in ban
, mutations in fry
, 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
showed additive phenotypes (Figure S6
). Thus, ban
exerts its effects on scaling of dendrite growth independently of known pathways for establishment and maintenance of dendrite coverage.
ban functions non-autonomously to regulate scaling growth of dendrites
To further characterize the signaling required for scaling growth of dendrites, we wanted to determine where ban
functions to regulate scaling. First, we investigated whether ban
is expressed in neurons, surrounding cells, or both, by using a miRNA activity sensor as a reporter for ban
expression in 3rd
instar larvae (Brennecke et al., 2003
). 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, we detected very little, if any, GFP expression in 3rd
instar muscle cells, epithelial cells, or sensory neurons using several independent transgenic fly lines with distinct insertions of the ban
sensor (). We first observed significant attenuation of the ban
sensor in larval muscle, epithelium and PNS neurons between 48 and 72 hr AEL (), precisely at the time when we first observed dendrite defects 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.
ban activity is broadly distributed in late larval stages but is dispensible in neurons for dendrite scaling.
To determine whether ban
is required cell-autonomously for dendrite scaling we used MARCM to generate single neuron clones homozygous for a ban
mutation in a heterozygous background (Lee and Luo, 1999
activity was effectively dampened in MARCM clones as indicated by de-repression 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 (data not shown). Furthermore, ban
is dispensable in other da neurons for dendrite scaling growth (, data not shown). Thus, ban
function in sensory neurons is dispensable for scaling growth of dendrites.
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 (Figure S7
). Therefore, ban
likely acts cell-autonomously to promote dendrite growth and non-autonomously to limit dendrite growth and ensure proper scaling.
ban functions in epithelial cells to regulate scaling growth of dendrites
We turned to a genetic rescue assay to test the ability of transgenic expression of ban
in different tissues to rescue the dendrite growth defects of ban
mutants. Consistent with our MARCM results, neuronal expression of ban
, using either pan-neuronal or PNS-specific Gal4 drivers, was not sufficient to rescue the scaling growth defect of ban
mutants (Figures and S8
). Thus, ban
likely functions non-autonomously in non-neuronal 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 we rescued ban
expression in epithelial cells, we found significant suppression of the exuberant dendrite growth of ban
mutants. 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, we monitored rescue activity of each epithelial Gal4 line over a graded temperature series (18°C to 29°C) and found that for each driver, rescue activity was directly proportional to expression level (data not shown). 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.
ban expression in epithelial cells is sufficient for scaling growth of dendrites.
Epithelial expression of twi
-Gal4 was first apparent in larval stages, suggesting that post-embryonic 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, we asked whether late expression of ban
would suffice for dendrite scaling. To examine the temporal requirement for ban
function we used a heat shock-inducible Gal4 driver to express ban
during larval development. Indeed, inducing ban
expression at 48 hr AEL was sufficient to rescue the dendrite defects of ban
mutants (Figures and S8
). These findings reinforce the notion that ban
is dispensable for early aspects of dendrite development.
in tissues known to regulate larval growth such as the fat body (Colombani et al., 2003
), prothoracic (PTTH) gland (Layalle et al., 2008
; McBrayer et al., 2007
), or insulin-producing cells (IPCs) (Rulifson et al., 2002
) had no measurable effect on dendrite growth in ban
mutants (, data not shown). Moreover, ablation of each of these tissues mediated by a reaper
transgene caused larval growth defects without obvious dendrite growth defects (data not shown). 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.
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, we wondered 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; ).
Since ban expression in epithelial cells is sufficient to ensure proper scaling, we wanted to address whether ban function in epithelial cells is necessary for scaling of dendrite growth. To this end, we used MARCM 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 (we were only able to generate 1-4 cell epithelial clones), we monitored the pattern 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. We used the epithelial nucleus as a landmark and monitored dendrite growth 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.
ban influences dendrite distribution over the body wall epithelium.
ban regulates neuronal Akt signaling to achieve proper scaling growth of dendrites
To gain insight into the molecular mechanism underlying dendrite scaling we developed a platform for microarray-based expression profiling of dissociated, FACS-isolated PNS neurons or epithelial cells (Parrish, Kim, DeRisi, and Jan, unpublished data). We identified Akt and numerous other candidate genes that are deregulated in PNS neurons and/or epithelial cells of ban
mutant larvae. Because Akt is a well-established regulator of growth (Edgar, 2006
), including dendrite growth in mammalian hippocampal neurons (Jaworski et al., 2005
; Kumar et al., 2005
), we investigated whether ban
regulates Akt as part of the scaling program.
In our microarray experiments, we found that Akt expression was increased in neurons but reduced in epithelial cells of ban mutants relative to wt controls (data not shown). By monitoring Akt levels in lysates of larval fillets composed mostly of muscle and epithelial cells, we found that without 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.
ban regulates neuronal Akt to ensure dendrite scaling.
Next we immunostained larval fillets 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.
To test whether this effect on Akt levels reflects a neuronal requirement for ban we monitored Akt expression levels 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 (). We found that ban non-autonomously regulates Akt levels in da neurons since non-neuronal 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.
Finally, we wanted to determine whether Akt function in class IV neurons is important for scaling of dendrite growth. Based on our expression data, we 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.
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. We tested this hypothesis with the following three experiments. First, we used RNAi 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, we overexpressed Pten in class IV neurons of ban mutant larvae and found that the Pten-mediated reduction in dendrite coverage is epistatic to the dendrite overgrowth seen in ban mutants (). Finally, we ablated class IV neurons in ban mutants in the absence or presence of neuron-specific Akt RNAi and 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.