To examine the expression pattern of Trc and Fry, immunostaining analyses were performed on dissected larvae using polyclonal antibodies raised against Trc or Fry. Both Trc and Fry are widely expressed including all da neurons in third instar wild-type larvae. In da neurons, Trc and Fry are localized predominantly in the soma but are also detected in axons and dendritic branches. When Trc tagged with FLAG-epitope (FLAG-Trc) was expressed in ddaC neurons by a class IV neuron-specific Gal4 driver (Grueber, 2003b; Ye, 2004), FLAG-Trc was distributed in soma, axon, as well as dendritic branches, similar to the endogenous Trc localization (Emoto, 2004).
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).
In a screen for enhancer trap lines showing expression in dendrite arborization (da) neurons, one insertion line, termed KY319, was found with high expression in da neurons throughout the larval stages. Genomic DNA flaking the P element was isolated by plasmid rescue and the P element was found to be inserted in the first intron of the fry gene. Because the fry mutants were known to have branched bristles and multiple wing hair phenotypes (Cong, 2001 and Adler 2002), fry's potential role in the control of the dendritic morphogenesis of da neurons was explored (Emoto, 2004).
To visualize dendrites in fry mutants, the pickpocket-EGFP reporter, which is specifically expressed in class IV sensory neurons (Grueber, 2003a), was introduced into each fry mutant allele. fry mutant neurons exhibit excessive dendritic branches. In the wild-type third instar larvae, class IV ddaC neurons in the dorsal cluster elaborate highly complex but stereotyped dendritic trees and extend a single axon ventrally -- the ddaC dendrites normally display a consistent number of terminal branches (Grueber, 2003b; Ye, 2004). In the fry null mutant (fry1), both the terminal branch number and the total branch length are increased by a factor of two, whereas the major branch architecture appears normal. This branching phenotype of fry mutants is already apparent in early first instar larvae, and the terminal branch number of the mutants is almost twice that of wild-type in the first, second, and third instar larval stages (Emoto, 2004).
The fry gene functions together with the trc gene to control wing hair and bristle morphology (Geng, 2000; Cong, 2001). In addition, fry homologs show strong genetic interactions with trc homologs in a variety of species including S. cerevisiae (Du, 2002; Nelson, 2003), S. pombe (Hirata, 2002), and C. elegans (Zallen, 2000). To test whether trc is also involved in controlling dendritic branching in neurons, ddaC dendrites of a trc null mutant (trc1) were examined; the trc dendrites display overbranching phenotypes similar to those of fry mutants. These observations suggest that both trc and fry function to regulate dendritic branching of class IV da neurons (Emoto, 2004).
Recent studies show that class IV da neurons exhibit dendritic tiling, presumably by exclusion between the terminal branches (Grueber, 2003a; Sugimura, 2003). Indeed, the terminal branches of the same ddaC neuron typically stop growing or turn away before crossing each other, resulting in minimum overlap. In contrast, the dendritic branches of fry1 and trc1 ddaC neurons often overlapped each other. Approximately 13% of dendritic terminal branches crossed one another in fry1 and trc1 ddaC dendrites, compared to ~1% of crossing in wild-type dendrites. The crossing branches of fry and trc mutants display rigid and straight trajectories, implying an impairment in like-repels-like navigation. Because these terminal branches were sandwiched between the epidermis and muscles, which were typically less than 1 microm apart in both mutant and wild-type larvae, the excessive overlap of mutant dendrites is unlikely to result from abnormal stratification of terminal branches. These findings suggest that trc and fry are involved in regulation of the dendritic tiling in class IV neurons (Emoto, 2004).
Since trc and fry null mutants display dendritic branching and tiling phenotypes, it was asked whether the tiling phenotype in mutants might simply arise from the excessive branching of terminal dendrites. To address this possibility, the number of dendritic crossings was normalized by the branch number and total dendrite length; the normalized dendritic crossings remained significantly increased in fry and trc mutants (Emoto, 2004).
The class IV dendrites of hypomorphic alleles and transheterozygotes of trc and fry were examined; robust tiling phenotypes were still observed in mild mutants with fry and/or trc function reduced to a level that caused no overbranching. For instance, the fry6 hypomorphic allele shows a reduced fry expression due to a P element insertion (Cong, 2001). The pickpocket-EGFP reporter revealed that the fry6 mutant exhibits a clear tiling defect whereas the terminal branch number appears unaffected. Indeed, normalized to the total branch length, the number of dendritic crossings in the fry6 mutant dendrites was slightly higher (~20%) than that in null mutants. Similarly, tiling defect was apparent in larvae transheterozygous for fry1 and fry6 or fry1 and trc1 despite the normal dendritic length and branch points. Taken together, these observations suggest that the tiling phenotype in trc and fry mutants is not secondary to the overbranching phenotype and that trc and fry function together to ensure dendritic tiling of class IV neurons (Emoto, 2004).
To determine whether trc and fry act cell autonomously in neurons, the MARCM (mosaic analysis with a repressive cell marker) system was used to generate mCD8-GFP-labeled trc, fry, or trc fry double mutant clones in heterozygous background. Compared to the wild-type ddaC dendrites, trc and fry mutant ddaC dendrites display a 50% increase in the number of branches. Moreover, mutant clones exhibit a tiling defect. A similar phenotype was observed in two ventral class IV MARCM clones. The phenotypes of mutant clones are less severe than those seen in null mutant animals, presumably due to perdurance of wild-type proteins. The dendritic phenotypes of trc fry double mutant clones are indistinguishable from those of trc or fry single mutant clones. These results indicate that Trc and Fry act cell autonomously to regulate dendritic branching and tiling of class IV neurons (Emoto, 2004).
Similarly, mutant clones of da neurons of class I, class II, and class III also show a 2- to 3-fold increase in the number of terminal dendritic branches. In contrast to the drastic phenotypes in terminal branches, the major dendritic branch architecture including primary, secondary, and tertiary branches, as well as the cell body shape are not obviously affected. Furthermore, the trc, fry, and double mutant clones show no detectable defects in bipolar dendrite neurons, external sensory neurons, or chordotonal neurons. Thus, among sensory neurons, trc and fry specifically control the terminal branching of da neuron dendrites (Emoto, 2004).
Given that trc and fry mutations compromise tiling of terminal branches from the same neuron (iso-neuronal tiling), it was of interest to see whether trc and fry also control tiling of dendrites from different neurons (hetero-neuronal tiling). The dendrites of the three class IV neurons in each abdominal hemisegment, ddaC, v'ada, and vdaB, normally cover the whole epidermis with minimal overlap (Grueber, 2002; Grueber, 2003a; Sugimura, 2003). For example, the adjacent v'ada and vdaB neurons appear to respect the respective dendritic territories and rarely send their dendrites into the dendritic fields of their neighbors. In fry1 and trc1 null mutants, however, the v'ada and vdaB dendrites often invade neighboring fields, resulting in a partial overlap of the dendritic fields. Major branches as well as terminal branches overlapped extensively in both fry and trc mutants. The fry6 hypomorphic mutant also displays clear hetero-neuronal tiling defects. Similar but milder dendritic tiling defects are observed in transheterozygotes for fry1 and fry6 and in transheterozygotes for fry1 and trc1. As observed in ddaC neurons, v'ada and vdaB dendrites also display iso-neuronal tiling defects with 12%-14% crossing in fry and trc mutants, compared to only 1% crossing in the wild-type control. There is a good correlation between the strength of the iso-neuronal and hetero-neuronal tiling phenotypes. These observations suggest that trc and fry regulate both iso-neuronal and hetero-neuronal tiling, presumably through the same mechanisms (Emoto, 2004).
How might trc and fry prevent overlap of like dendrites? A priori, it is conceivable that multiple dendritic branches initially coinnervate the same territory and have extensive crossings; tiling could result from retraction of some of these branches. Alternatively, in the like-repels-like scenario, dendrites interact with one another to avoid overlap and crossing throughout development (Emoto, 2004).
To distinguish between these possibilities, dendritic crossings were examined during development. Within a few hours around the time of hatching (AEL 20-23 hr), the territories of v'ada and vdaB neurons become defined. In wild-type control, no significant overlap was found between dendrites of v'ada and vdaB neurons throughout development, including in newly hatched larvae, in first instar larvae 3 or 6 hr after hatching, or in second or third instar larvae. In contrast, dendritic crossings were already evident in newly hatched fry mutant larvae, and the number of crossings increased continuously during larval development. Thus, consistent with previous studies (Grueber, 2003; Sugimura, 2003), dendrites normally avoid one another as they meet initially, without going through a noticeable period of coinnervation followed by pruning; fry mutations affect the mechanism underlying this avoidance (Emoto, 2004).
To further define the cellular functions of fry and trc in dendritic tiling, the dendrites of live wild-type and fry mutants were examined for 16 hr starting at the early second instar larval stage, when class IV neurons have stabilized their major arbors. Many of the terminal branches remain dynamic, however, from the beginning to the end of the 16 hr period. Nearly 75% of the dynamic branches displayed a net extension, whereas 25% showed a net retraction in both wild-type and fry mutants. In wild-type larvae, 73% of those branches that extended their tips close (< 5 microm) to other branches turned away, hence avoiding crossing, and only 1% extended beyond other branches. The percentage of branches making a turn was significantly reduced by a factor of three in both null and hypomorphic fry mutants. Instead of turning, about 45% of the mutant dendritic branches ran across other branches at many locations. These findings are consistent with the like-repels-like scenario. It appears that in fry mutants, dendrites can grow and retract normally, but their inability to turn in order to avoid like dendrites results in tiling defects (Emoto, 2004).
To investigate the function of the Trc kinase in neurons, wild-type and mutant Trc were expressed in neurons of trc mutants and whether the trc mutant phenotype could be ameliorated was tested. The kinase domains of Trc and its orthologs share 70%-80% amino acid identity (Tamaskovic, 2003b). In addition, Trc has conserved phosphorylation sites at Ser292 and Thr449; phosphorylation at these residues is essential for maximal activation for the human Trc kinase in vitro (Tamaskovic, 2003b). To test whether Trc kinase activity is required for proper dendritic branching and tiling in vivo, a kinase-dead mutant (K122A) was generated and a mutant was generated in which both Ser292 and Thr449 were replaced with alanine (S292AT449A) to prevent phosphorylation. Specific expression of wild-type Trc with a class IV neuron-specific Gal4 driver largely rescued both dendritic branching and tiling defects of the trc1 mutant; however, neither the K122A nor the S292AT449A mutant could rescue these phenotypes. These results suggest that Trc kinase activity in class IV neurons is important for their proper dendritic branching and tiling in vivo; this Trc activity is sufficient even in animals lacking Trc in other cell types (Emoto, 2004).
Interestingly, overexpression of wild-type Trc in class IV neurons of wild-type larvae caused a slight reduction of branch number. Moreover, expression of the K122A in wild-type class IV neurons resulted in a highly penetrant increase in terminal branches of ddaC dendrites as well as tiling defects similar to the phenotypes of trc and fry mutants, indicating that the K112A acts as a dominant-negative mutant. Specific expression of the S292AT449A Trc mutant in wild-type class IV neurons also led to an increase of terminal dendrites, albeit milder than that induced by the K122A mutant, whereas the dendritic tiling defects seen in S292AT449A-expressing dendrites were as obvious as those induced by the K122A mutant. These results strongly suggest that both Trc phosphorylation and Trc kinase activity in neurons play an essential role in dendritic branching and tiling (Emoto, 2004).
Given the genetic interaction between trc and fry and their evolutionarily conserved function in controlling branching of cellular processes, their functional relationship was investigated by assessing the Trc kinase activity in trc and fry mutants. Trc immunoprecipitates from wild-type, trc, or fry embryos were assayed for kinase activity by using histone H1 as an artificial substrate. Although similar amounts of Trc protein were precipitated from wild-type and fry homogenates, kinase activity of precipitates from fry mutants was significantly reduced, to the level similar to that of trc mutants. Since the Trc protein was undetectable in immunoprecipitates from trc embryos, histone phosphorylation by precipitates from trc mutants is likely due to other kinases coprecipitated with the beads. These results indicate that the Trc kinase is inactive in fry mutants. Most likely, Trc kinase activity requires Fry and is a key component of a signaling pathway primed for restraining growth toward like structures and limiting branching (Emoto, 2004).
During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility, a weak tricornered mutant was found to be hypersensitive to cytochalasin D. The tricornered gene was cloned and found to encode the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation (Geng, 2000).
Mutations in trc result in the splitting of epidermal hairs, the shafts of sensory bristles, larval denticles, and the lateral branches of the arista. Mutations in trc do not however cause dramatic effects on hair or bristle shape or length. Nor is evidence seen for trc delaying prehair initiation or slowing prehair elongation. Based on the similar phenotypes it seems likely that the Trc protein has a similar target(s) in all of these cell types. In comparing the trc-induced phenotypes to the effects of various inhibitors the actin cytoskeleton was identified as a candidate target; the inhibition of actin polymerization with CD or LAT A results in frequent split hairs, bristles, and arista lateral branches. However, the CD- and LAT A-induced phenotypes differed substantially from those found in trc mutants in that the drug treatments had major effects on the shape and length of hairs and bristles. Because of this it is suggested that trc mutations do not inhibit actin polymerization. Consistent with this, trc mutant hairs, bristles, and arista laterals routinely stained strongly for F-actin. It was also found that trc prehairs in clones develop and grow at the same rate as their wild-type neighbors. The data also argue that trc is not needed for the bundling of actin filaments in developing bristles since these looked normal in trc8/Df pupae (Geng, 2000).
The split hairs and bristles that are found in trc mutants appear normal except where they are split. In this way the trc phenotype differs profoundly from the phenotypes found in cells mutant for genes such as f, sn, or ck, which encode proteins that are functional components of the actin cytoskeleton. In these cases the mutant phenotype is displayed all along the length of the bristle or hair. It is suggested that this is because these proteins are part of the machinery that is directly involved in the morphogenesis of hairs and bristles and hence are needed throughout morphogenesis. This suggests the possibility that trc has a regulatory as opposed to structural function (Geng, 2000).
A possible hypothesis is that trc functions to repress the initiation of outgrowths and that splitting is a consequence of ectopic outgrowths. This hypothesis can account for some of the data, but it does not easily accommodate the finding that in splitting bristles no evidence is seen for new ectopic large bundles of actin filaments. Rather, existing neighboring bundles appear to separate as the segments split apart. These observations on trc differ from those on bristles after CD treatment where evidence was seen for ectopic bundles of actin filaments (Geng, 2000).
It is suggested that what is important for maintaining the integrity of hairs and bristles is coordinating the growth of actin filaments and/or other cellular components. When some filaments or bundles grow more rapidly than others splitting might occur. It is suggested that this mechanism contributes to the split hairs and bristles seen after CD and LAT A treatment. If this is the case then developing bristle cells would be expected to monitor the polymerization of actin filaments and try to prevent splitting by slowing down the polymerization of filaments or bundles that are growing more rapidly than others. This could be thought of as being equivalent to a morphogenetic checkpoint. The trc gene could be a component of a signal transduction pathway that mediates the monitor function. Such a monitor function might involve regulating gene expression, which could explain the nuclear location of NDR (Millward, 1995). A hint that such a monitor/checkpoint might exist comes from the finding of a delay in bristle or hair morphogenesis after treatment with low doses of CD. Further experiments will be needed to determine if such a monitoring function exists and if trc encodes a component of it. Several other plausible models exist to explain trc function in maintaining the integrity of cellular extensions. The Trc protein could function in the assembly of the cellular components that organize the outgrowth of these cellular extensions. Defects in properly assembling an organizing center might result in the splitting of the extensions at later stages in elongation. The Trc protein could also function to mechanically strengthen cellular extensions in some way to insure their integrity, e.g., by increasing the degree of cross-linking of the membrane cytoskeleton (Geng, 2000).
The directed expression of a dominant negative Trc protein provides a sensitized system for identifying interacting genes (He, 2005a). Deficiencies for each of the fly mob genes (see mats) enhances the wing hair phenotype that results from driving expression of UAS-trcT453A using either ap-GAL4 or ptc-gal4. The strongest enhancement is seen with deficiencies for mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). These results suggested the possibility that all 4 Dmobs can redundantly interact with Trc, although it is possible that the interactions could be indirect or due to other genes in deleted regions. It is worth noting that such interactions are not common. When the Drosophila deficiency collection was screened for enhancement or suppression of ap-GAL4 UAS-trcT453A, <10% of the Dfs showed an interaction (He, 2005b).
To confirm that the genetic interaction between trc and Df (mats) was due to the reduction in mats dose, two independent alleles were used. One was the null allele described by Lai (2005) (matse235), which is deleted for almost the entire coding region, and the other was a lethal PiggyBac insertion allele of mats [PBac{RB}CG13852e03077] (this allele is referred to as matsPB). Because this later allele has not been well characterized, the insertion was determined to be lethal over a deficiency for the region (Df(3R)Exel6191), and it failed to complement the recessive lethality of matse235 consistent with the lethality being due to the PB insertion. This mutation could be reverted using a source of PiggyBac transposase. It was found that both mats alleles dominantly enhance the trc dominant negative phenotype and this enhancement is lost in the PB revertant. It was also found that over expression of mats from a UAS-mats transgene (Lai, 2005; driven by ptc-Gal4) partially suppresses the multiple hair cell phenotype that results from driving expression of Trc-DN using ptc-Gal4. These dose responses argue that Mats activates Trc. Interestingly, it was found that heterozygosity for a wts mutation also enhances the Trc dominant negative phenotype, although somewhat less strongly (He, 2005b).
Evidence was also obtained for mats functioning with trc and fry using simple loss of function mutations. Wild-type flies or flies heterozygous for either trc, fry, or mats appear normal and only rarely (on fewer than 5% of wings) is even a single multiple hair cell seen. Flies that were heterozygous for two of these genes showed a slightly higher frequency of wings with one or a couple of multiple hair cells (often ~10%) but the increase was not routinely significant. However, almost half of the wings from flies that are heterozygous for all three genes (e.g., fry2 trc1+/+ + matsPB) show a weak multiple hair cell phenotype, a significant increase. This genetic interaction is further support for the hypothesis that trc, fry, and mats function together in regulating wing hair development. In this assay no equivalent interaction with wts3-17 was seen (He, 2005b).
Previous studies established that trc also has a larval denticle phenotype (Geng, 2000). In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of matsPB/matsPB homozygous larvae is also disorganized and contains many split denticles. The number of split denticles is similar in matsPB/Df larvae, suggesting that for this phenotype, matsPB is a strong, near phenotypic null allele. The matse235 also showed a similar denticle phenotype. The phenotype of matsPB homozygotes is slightly less severe than that of trcP/trcP larvae. Notably, trcP matsPB/trcP matsPB double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development (He, 2005b).
Both mats alleles are larval lethals with death typically in the second or early third instar. To examine the phenotype of mats in wing cells, mosaics were generated using FLP/FRT. mats clones on the wing, leg, thorax, and head display two types of phenotype. The most notable is indistinguishable from those produced by clones of wts, suggesting that mats also functions with wts as has been shown by Lai (2005). On the wing, small clones produced bulges that can be seen at low magnification with a stereomicroscope. In mounted wings individual cell outlines are visible in the cuticle and the cells appear to have a bulging apical surface. The hairs are located on an elevated pedestal, a phenotype that is indistinguishable from those seen in wts clones. The hairs were often broader than normal. Particularly in other body regions clones were abnormally pigmented (either darker or lighter than normal, and there were outgrowths of clone tissue. In highly abnormal wing clones, evidence of clustered and split hairs were often seen that were typical of trc mutant clones. Some multiple hair cells were seen in very abnormal wts clones but this phenotype appears less severe (e.g., number of hairs per cell) than that seen with mats or trc. These observations suggest that mats functions with both Trc and Wts (He, 2005b).
matsPB and matse235 clones in pupal wings were examined. Mutant mats cells are able to outcompete their neighbors and end up comprising most of the wing when clones are induced early. As was expected from the morphology of clones in adult wings, the pupal clones produce bulges in the wing and individual cells also often appear bulged. Clone cells stain more brightly for F-actin. This is true both in developing hairs and in the general apical cortex. This phenotype is clear-cut enough that it could be used as a convenient marker of mats mutant cells. These phenotypes were seen with both mats alleles tested. A similar, increase in actin staining was seen in wts clones. A similar, but perhaps less severe increase in staining, is seen in trc clones (He, 2005a). In some, but not all mats clones, large numbers of multiple hair cells can be seen. At later stages a circular pedestal of actin staining could be seen surrounding the base of the hair in mutant cells but not in surrounding wild-type cells. At still later stages the wild-type cells also had a circular pedestal of actin staining, suggesting that the mutant cells might be developmentally more advanced. Consistent with this possibility, in many clones hair initiation and outgrowth appear to be advanced in mats mutant cells compared with neighbors. This is also the case for wts clones, but it is the opposite of trc clones, in which hair development is often delayed (He, 2005a). Cells in mats clones have a smaller cross section so that the array of hairs appears denser, which is also the opposite of what is seen in trc clones, in which there is an increase in cross-sectional area (He, 2005a). Once again the phenotype of the wts clone cells resembles that seen for mats cells. Thus, for several wing phenotypes mats mutant cells resembled wts and not trc cells. Indeed, the mats phenotype is the opposite of trc for both cell area and the timing of hair morphogenesis (He, 2005b).
It has been found that the accumulation of Fry in wing cells is subject to feedback control that is dependent on Trc activity. Hence, in a trc mutant, increased Fry accumulation is found (He, 2005b). Several of the observations described above suggest the hypothesis that mats functions along with trc and is important for Trc activation. From this it is predicted that Fry accumulation would also be elevated in Dmob1 clones. Increased levels of Fry immunostaining were found in Dmob1 clone cells; this is consistent with the hypothesis. This is also seen in wts mutant clones, although the increase appears less dramatic (He, 2005b).
Tumorous overgrowth phenotypes are a consequence of mutations in a number of Drosophila genes. In several cases, such as lethal giant discs overgrown imaginal discs are found in late third instar larvae. To determine whether that is also the case for mats, mats/Df mutant larvae were examined. These larvae grow slowly and after 5 d of growth, when wild-type larvae begin to pupate, mats/Df larvae are the size of early third instar larvae. These larvae routinely die without growing substantially larger. When 5.5-5-d-old mats larvae were dissected, not evidence of tumors or overgrowth of imaginal or other tissues was found. Rather, the imaginal discs were approximately the size of those seen in 4-d larvae. However, the mats homozygous discs did not appear normal, since they were abnormally shaped and more folded than normal discs of this size (He, 2005b).
In a number of experiments involving mats or wts, what appeared to be spontaneous tumors or clones was observed. This was seen most often in flies that also carried reduced doses of Dmob3 and Dmob4. It is thought that these overgrowths are due to spontaneous mitotic recombination, because when the flies were also mutant for trc or fry, evidence of trc or fry clones was seen. The trc and fry clones were seen less frequently. This could be due to these genes being located more proximally on the chromosome than mats or wts, but it might also be due to the competitive advantage of mats and wts clones, resulting in these clones being larger and easier to detect. The basis for these clones is unclear but suggests genomic instability in mats and/or wts mutants (He, 2005b).
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date revised: 27 December 2005
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