bantam miRNA is expressed at all developmental stages, though at varying levels. To ask whether bantam expression is spatially regulated during development, an assay was developed based on the ability of miRNAs to inactivate genes by RNAi. A transgene was prepared expressing GFP under control of the tubulin promoter and two copies of a perfect bantam target were placed in the 3'UTR. A comparable construct without the bantam target sequences in the 3'UTR was used as a control. Where present, bantam miRNA should reduce expression of the transgene containing the target sequences by RNAi, providing an in vivo sensor for bantam activity. When expression of the two transgenes was compared using the same settings on the confocal microscope, it was apparent that the control transgene was expressed at much higher levels overall. The bantam sensor transgene showed a complex pattern of spatial modulation in the third instar wing disc, being higher in cells near the anteroposterior and dorsoventral boundaries and in patches in the dorsal thorax. The control transgene showed limited spatial modulation (Brennecke, 2003).
The difference in the overall levels of control and bantam sensor transgenes suggested that bantam miRNA is expressed broadly in the wing disc, lowering the expression of the specific sensor transgene. Indeed, removing bantam miRNA in clones of cells homozygous for the bantamΔ1 deletion increased expression of the bantam sensor. The level of sensor expression in the clones was considerably higher than the maximal endogenous level at the DV boundary, indicating that the miRNA is present throughout the disc, though at varying levels. The P element EP(3)3622 is inserted 2.7 kb from the hairpin and has been identified as a hypomorphic allele of bantam (Hipfner, 2002). Clones of cells homozygous mutant for EP(3)3622 also show upregulation of the bantam sensor, demonstrating that this insertion reduces bantam miRNA levels. In this case, the maximal level of sensor expression is similar to the level at the DV boundary. The level of sensor expression is lower in the twin spots, which express two copies of the endogenous bantam gene than in the surrounding cells, which have one wild-type and one mutant copy of the gene. This suggests that elevated bantam activity would reduce sensor expression. Indeed, clones overexpressing bantam do reduce sensor levels. The control sensor was not affected by overexpression of bantam. Taken together, these observations indicate that the sensor is capable of reflecting even subtle increases and decreases in bantam miRNA levels in vivo. In all cases, the effects on the sensor were cell autonomous. The sensor transgene method may provide a generally useful tool to visualize miRNA activity in vivo (Brennecke, 2003).
The bantam locus was identified by several EP element insertions clustered in a region of ~41 kb that lacks predicted genes. EP elements are transposable elements designed to allow inducible expression of sequences flanking the insertion site under control of the yeast transcription factor Gal4. Gal4-dependent expression of the EP elements inserted at the bantam locus causes tissue overgrowth due to an increase in cell number. Conversely, flies homozygous for the bantamΔ1 deletion, which removes ~21 kb flanking the insertion site of EP(3)3622 grow poorly and die as early pupae. Flies heterozygous for the bantamΔ1 deletion and three of the P element inserts survived and were morphologically normal but smaller than normal flies. These observations led to the conclusion that the bantam locus is involved in growth control during development (Hipfner, 2002). In an effort to molecularly define the bantam locus, transgenic flies were produced carrying fragments of genomic DNA overlapping the region where P element inserts clustered. Two fragments rescued the growth defects and pupal lethality of flies homozygous for the bantamΔ1 deletion. The 3.85 kb overlap of these transgenes defines the extent of essential sequences comprising the bantam locus. This region contains an EST, RE64518. Expression of RE64518 under Gal4 control failed to reproduce the overgrowth phenotype caused by the EP elements, indicating that RE64518 does not encode bantam function (Brennecke, 2003).
To verify that the miRNA produced by the predicted hairpin is the functional product of the bantam locus, a series of rescue assays and gain of function overgrowth assays were performed. bantamΔ1 homozygous mutant larvae generally lack some or all imaginal discs and show undergrowth of larval tissues, including the brain. These animals develop slowly, but survive larval development and die shortly after pupation, lacking evidence of imaginal structures. A 6.7 kb rescue fragment, UAS-A, is able to rescue growth of the imaginal discs and allowed the bantamΔ1 homozygous mutant animals to overcome pupal lethality, so that viable adults were produced. Although the construct is in a UAS vector, rescue was independent of GAL4, suggesting that the endogenous regulatory elements needed to produce the bantam miRNA are contained within this fragment. However, the surviving flies often had rough eyes, duplicated bristles, and missing halteres, suggesting that some regulatory elements may be missing. When provided with a weak ubiquitous source of GAL4, the smaller 584 nt fragment contained in UAS-C rescues the growth defect in the imaginal discs. Approximately half of the larvae formed morphologically normal pupae that expressed the GFP transgene from which the miRNA is excised. Many of these animals survive to adulthood. Thus, sequences contained within the 584 nt fragment are sufficient to provide bantam function when expressed. The UAS-B transgene contains the same DNA fragment as UAS-A, except that it lacks 81 nt containing the hairpin. UAS-B is unable to rescue the mutant phenotype, indicating that the deleted residues are essential for bantam function. When expressed under engrailed-GAL4 control, constructs UAS-A, UAS-C, and a shorter construct UAS-D (containing 100 nt including the hairpin) each produced overgrowth of the posterior compartment of the wing and of segments in the larval body. UAS-B did not produce any overexpression phenotype. Together these observations assign bantam function to the region containing the hairpin and suggest that the 21 nt miRNA is the bantam gene product (Brennecke, 2003).
In light of the observation that bantam acts cell autonomously to regulate sensor expression, it was asked whether bantam acts autonomously to control cell proliferation. FLP-induced mitotic recombination was used to produce clones of cells homozygous for the bantamΔ1 deletion and sister clones that were homozygous wild-type. Each pair of clones derives from a single cell division. Consequently, growth rates can be compared by measuring the areas of individual pairs of mutant and wild-type twin clones after a period of time. Clones were generated at the end of second instar and analyzed late in third instar. Mutant clones were on average 1/3 the size of the wild-type twins. Although a few relatively large bantam mutant clones were observed, mutant clones were typically very small. DAPI labeling did not reveal an observable difference in the size or spacing of nuclei in mutant and wild-type tissue, suggesting that cell size was unaffected by the deletion mutant. No obvious increase in apoptosis was observed in these clones. These observations suggest that bantam acts cell autonomously to control cell proliferation (Brennecke, 2003).
The bantam sensor transgene (expressing GFP under control of the tubulin promoter and two copies of the bantam target in the 3'UTR) provides a means to compare bantam activity and cell proliferation in vivo. Cell proliferation was visualized using BrdU incorporation to label cells undergoing DNA synthesis. A striking correlation was observed between bantam activity and cell proliferation in the developing larval brain. Proliferating cells had a lower level of sensor expression, indicating elevated bantam miRNA activity, compared to adjacent nonproliferating cells. This correlation was also observed in the wing disc. Elevated sensor levels coincide with the zone of nonproliferating cells adjacent to the dorsoventral boundary (ZNC), indicating that bantam miRNA levels are reduced in the ZNC (Brennecke, 2003).
To ask whether regulation of bantam miRNA contributes to the exit of ZNC cells from proliferation, bantam was expressed under ptc-Gal4 control. Restoring bantam expression was sufficient to direct cells in the nonproliferating zone to enter S phase. The ZNC depends on the activity of the secreted signaling protein Wingless and on Notch activity. Myc expression is downregulated in the ZNC by Wingless signaling. Forced expression of Myc in the ZNC can drive G1-arrested cells into S phase, but does not affect the G2-arrested cells. Although bantam can drive both G1/S and G2/M transitions when expressed in the wing disc (Hipfner, 2002), bantam expression does not affect Myc protein levels in the ZNC, suggesting that the myc transcript is not a target of bantam regulation (Brennecke, 2003).
bantam mutant animals are smaller than wild type, due to a reduction in cell number but not cell size, and do not have significant disruptions in patterning. Conversely, overexpression of the bantam product using the EP element EP(3)3622 causes overgrowth of wing and eye tissue (see Abdelilah-Seyfried, 2000). Overexpression in clones of cells results in an increased rate of cell proliferation and a matched increase in cellular growth rate, such that the resulting tissue is composed of more cells of a size comparable to wild type. These effects are strikingly similar to those associated with alterations in the activity of the cyclinD-cdk4 complex. However, epistasis and genetic interaction analyses indicate that bantam and cyclinD-cdk4 operate independently. Thus, the bantam locus represents a novel regulator of tissue growth (Hipfner, 2002).
bantam gene function appears to be important for regulation of tissue growth rates. Several EP elements inserted in this locus, most notably EP(3)3622, are capable of promoting substantial tissue overgrowth in the eye and wing in a GAL4-dependent manner. Conversely, ban mutations decrease tissue growth. Mutant phenotypes range from decreased body size to lethality. The strongest available allele is a small deletion that does not remove any known genes. This allele is pupal lethal and causes the absence of detectable imaginal discs. The simplest explanation for the reciprocal nature of gain-of-function and loss-of-function phenotypes is that EP(3)3622 is driving expression of the same transcription unit that is affected by ban mutations. This is further supported by the specific and reciprocal nature of the genetic interaction of gain and loss of ban function with the expanded tumor suppressor gene in the eye. However, this remains to be confirmed by molecular characterization of the locus. Growth regulation appears to be a primary function of ban, since EP(3)3622 expression does not cause significant patterning alterations, and ban mutant flies, although small, are proportioned normally (Hipfner, 2002).
Two of the ban EP insertions were identified initially in a genetic interaction screen as suppressors of the phenotype caused by overexpression of the expanded tumor suppressor gene. When misexpressed in the eye under the control of the sevGAL4 driver, expanded causes a reduction in eye size relative to wild type and external roughening and blistering. Coexpression of EP(3)3622 almost completely suppresses this phenotype, restoring the eye to nearly wild-type size and appearance. Reducing ban function has the opposite effect. Introducing one copy of the banDelta allele noticeably reduces the overall eye size and increases the blistering in the central and anterior regions of the eye. In contrast, alterations in cycD-cdk4 activity does not alter the expanded overexpression phenotype. Coexpression of cycD-cdk4 with expanded increases the overall size of the eye, but has little effect on the roughness and blistering. Removing one copy of cdk4 has no effect. The lack of a strong genetic interaction between expanded and cycD-cdk4 provides additional evidence that bantam is acting independently of this complex to promote coordinated cell growth and cell cycle progression (Hipfner, 2002).
The results suggest that ban regulates tissue growth by a mechanism that involves coordinated stimulation of cell growth and cell division. ban alters tissue growth through effects on cell number rather than cell size. Decreased ban function causes a reduction in cell number in the adult wing, but the surviving cells are of wild-type size, suggesting a coordinated decrease in the rate of cell growth and division. Activation of EP(3)3622 has the opposite effect on cell number, causing an increase in the rate at which imaginal disc cells proliferate. Despite this increased proliferation rate, cell sizes are little changed. These observations suggest that the rate of increase in cell division is coordinated with the rate of increase of cell mass when ban is overexpressed. The effects of ban on growth and fertility are remarkably similar to those of cycD-cdk4. However, no evidence was found of a direct connection between cycD-cdk4 and ban. It seems unlikely that ban regulates growth by controlling the activity of cycD-cdk4, because ban-driven overgrowth is unaffected in the absence of cdk4. Similarly, cycD-cdk4-driven growth is unaffected by reduction of ban, indicating that ban is unlikely to be a downstream effector. The view is favored that ban and cycD-cdk4 act independently. The similarity in their growth phenotypes suggests that they may have some targets in common. However, as attested to by the differences in their interactions with expanded, they clearly can act differently as well (Hipfner, 2002).
The imaginal discs are patterned while they grow. The secreted signaling proteins Decapentaplegic (Dpp) and Wingless pattern the wing and leg discs along their main axes. Dpp and Wingless signaling are also required in some way for disc growth. The parts of the discs that produce the appendages are very small in flies lacking either signal. Cells unable to transduce the Dpp or Wingless signals display cell-autonomous defects in proliferation and are lost from the disc. To date it has not been reported whether loss of cells under these conditions is due to reduced proliferation or to reduced survival. However, recent studies suggest that Dpp signaling may directly influence cell proliferation in the wing disc. Wingless signaling has been shown in one situation to repress growth at late stages of wing development, in part by negative regulation of dmyc expression. If Dpp and Wingless act directly to regulate tissue growth, it would be expected that they coordinately regulate cell growth and cell division rates. It will be of interest to learn whether ban and/or cycD-cdk4 mediate the growth effects of these signaling molecules (Hipfner, 2002).
Altering cell division rates does not alter compartment size, but can increase or decrease the number of cells per compartment. This is consistent with the effects of Minute mutations that vary the proportion of a compartment that can be contributed by the progeny of a single cell, without affecting compartment size or shape. However, it is possible to alter the size of one compartment relative to another by manipulating activity of the insulin/PI3K pathway. PI3K-induced overgrowth requires that the pathway be activated in all cells of the compartment. Clones of overgrowing cells do not affect the size of the compartment. Thus a mechanism must exist that allows a population of cells to measure the size of the compartment. Interestingly, it has been found that altering the size of the compartment feeds back by an unknown mechanism to alter the shape of the Dpp morphogen gradient (Hipfner, 2002).
Overexpression of ban with enGAL4 promotes significant overgrowth of the posterior compartment. Posterior compartment overgrowth is compensated for by a nonautonomous reduction in the final size of the anterior compartment in most cases. This compensation suggests that total disc size may also be regulated to some extent during development. Only in the case of the strongest EP element, EP(3)3622, are total disc and wing size increased (Hipfner, 2002).
These observations suggest that there may be multiple layers of size control operating during imaginal disc development. Morphogen gradients influence tissue growth. Tissue growth rates influence compartment size and morphogen gradient shape. Finally, size compensation mechanisms exist to control both compartment and disc size. At present, little is known about the size-sensing mechanisms, except that they can be overridden by stimulating cell and tissue growth rates by various experimental means. Identifying how size is measured during tissue growth poses a significant challenge (Hipfner, 2002).
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).
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date revised: 15 December 2011
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