At 5-6 hours of development (Stage 10), clusters of progenitors of Malphighian tubules as well as cells in the vicinity of anterior and posterior spiracles are labelled with anti-Cut antibodies. In the head region, precursors of antenna-maxillary and other external sensory organs express cut. The cells expressing cut correspond to the basiconical sensilla, the trichord sensillium and the campaniform sensillum, each of them external sensory cells with unique functions. As development proceeds, more cells express cut, some arising from division of cut-expressing cells. Thus cut is expressed in precursor cells of the external sensory organs (Blocklinger, 1990). Specific cells of the CNS, specifically, the ventral midline also express cut and cut mutation interfers with the fate of these cells (Klambt, 1991).

Both numb and cut are required for the proper differentiation of multiple dendritic neurons. cut acts as a selector-type gene and is required to initiate the correct developmental program of a particular type of sensory organ, and numb is responsible for specifying cellular identities of sublineages of neural precursor cells (Brewster, 1995).


Expression of cut in the Drosophila margin of the wing imaginal disc is necessary for the specification of chemosensory and mechanosensory neurons. This expression is driven by a distal enhancer 80 kb upstream of the cut structural gene (Jack, 1991).

In wild-type imaginal discs of third instar larvae cut is expressed in a band four to five cells wide along the entire prospective wing margin. Shortly after pupariation, cut expression is seen in the precursors of the chemosensory bristles, which are slightly recessed from the wing margin. The band of cut expression spanning the prospective wing margin is absent in ct6 mutant discs: however, cut expression in the chemosensory precursors is not affected. ct6 mutants have scalloped wing margins and in addition lack many of the chemosensory and slender mechanosensory and stout mechanosensory neurons, which are reduced 74%, 3% and 23%, respectively. In addition, all noninnervated hairs of the posterior wing margin are absent (Ludlow, 1996).

Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vgnull mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vgnull pupae. In addition, in vgnull adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vgnull flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).

One of the aims of developmental biology is to determine how a given cell population undergoes specific developmental program. This means trying to determine when cell commitment is specified and what are the factors involved. Adepithelial cells were at first considered as a homogenous population that expressed Twi. Adepithelial cells can, however, be considered as two distinct populations. The population, that forms DFMs, expresses a high level of Ct and does not express Vg. The second population forms IFMs and expresses Vg and a low level of Ct. In addition, Ct and Vg levels are stabilized by a repressive feedback loop: overexpression of Ct in all myoblasts using the 1151-GAL4 driver leads to Vg repression and to IFMs-specific apoptotic degeneration; overexpression of Vg in all adepithelial cells using the same driver leads to Ct repression and to DFM degeneration. The data in vgnull flies are consistent with these observations: absence of Vg leads to Ct derepression in adepithelial cells, in myoblasts surrounding DLMs and in developing DLMs. However, Ct overexpression phenotypes in DLMs are slightly different from those observed in vgnull flies. Splitting of the three larval templates (LOMs) into six DLMs occurs normally and at 48 h APF DLMs are morphologically normal in vgnull flies. It is concluded that DLM degeneration is a late event in vgnull mutants. In contrast, Ct overexpression leads to early degeneration of myoblasts and muscle fibers. This suggests that Ct overexpression induces apoptosis independent of Vg repression. Therefore, it would seem that the effect of Ct overexpression using the 1151-GAL4 driver is not equivalent to that observed in the absence of Vg (Bernard, 2003).

Intrinsic control of precise dendritic targeting by an ensemble of transcription factors

Proper information processing in neural circuits requires establishment of specific connections between pre- and postsynaptic neurons. Targeting specificity of neurons is instructed by cell-surface receptors on the growth cones of axons and dendrites, which confer responses to external guidance cues. Expression of cell-surface receptors is in turn regulated by neuron-intrinsic transcriptional programs. In the Drosophila olfactory system, each projection neuron (PN) achieves precise dendritic targeting to one of 50 glomeruli in the antennal lobe. PN dendritic targeting is specified by lineage and birth order, and their initial targeting occurs prior to contact with axons of their presynaptic partners, olfactory receptor neurons. A search was performed for transcription factors (TFs) that control PN-intrinsic mechanisms of dendritic targeting. Two POU-domain TFs, acj6 and drifter have been identified as essential players. After testing 13 additional candidates, four TFs were identified, (LIM-homeodomain TFs islet and lim1, the homeodomain TF cut, and the zinc-finger TF squeeze) and the LIM cofactor Chip, that are required for PN dendritic targeting. These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit (Komiyama, 2007).

For technical simplicity, larval born GH146-Gal4-positive PNs, originating from three neuroblast lineages, anterodorsal (adPNs), lateral (lPNs), and ventral (vPNs), were studied. Out of ~25 classes defined by their glomerular targets, focus was placed on 17 classes whose target glomeruli are reliably recognized across different animals. The MARCM technique allows visualization and genetic manipulation of PNs in neuroblast and single-cell clones in otherwise heterozygous animals, so PN-intrinsic programs can be studied for dendritic targeting. GH146 is expressed only in postmitotic PNs (Komiyama, 2007).

acj6 and drifter have been identified as lineage-specific regulators of PN dendritic targeting. To identify additional transcription factors (TFs) that regulate dendritic targeting of different PN classes, candidates were tested that have been shown to regulate neuronal subtype specification and targeting specificity and have available loss-of-function mutants. The following was tested; (1) the expression of candidate genes in PNs at 18 hr after puparium formation (APF) when PN dendrites are in the process of completing their initial targeting, and/or (2) their requirement in PNs by examining dendritic targeting in homozygous mutant MARCM clones (Komiyama, 2007).

In addition to the eight genes described below, five other TFs were examined that were not pursued because of the lack of expression in GH146-PNs at 18 hr APF (aristaless and pdm-1) or the lack of targeting defects in homozygous mutant PNs (abrupt [abk02807], kruppel [Kr1], and Dichaete [Dichaete87]) (Komiyama, 2007).

LIM-HD factors and PN targeting: LIM-homeodomain (LIM-HD) TFs are involved in multiple events during neuronal development. Most functions of LIM-HD factors require the LIM domain-binding cofactor, which is represented in Drosophila by ubiquitously expressed Chip. Chip antibody revealed ubiquitous expression of Chip in cells around the antennal lobe (AL) including all GH146-PNs at 18 hr APF (Komiyama, 2007).

The requirement of Chip in PN dendritic targeting was tested. Wild-type adPNs, lPNs, and vPNs target stereotyped sets of glomeruli. PNs homozygous for a Chip null allele (Chipe5.5) failed to target most of the correct glomeruli and occupied inappropriate glomeruli. Most adPN and lPN clones (12/13) also mistargeted a fraction of dendrites to the structure ventral to the AL, the suboesophaegeal ganglion (SOG). Thus, Chip is required for targeting specificity of most, if not all, PN classes studied here, and Chip-interacting proteins including LIM-HD factors likely play important roles in PN dendritic targeting (Komiyama, 2007).

Five LIM-HD factors have been characterized in Drosophila: apterous, arrowhead, islet, lim1, and lim3. apterous, arrowhead, or lim3 were not pursued because they are not expressed in GH146-PNs at 18 hr APF (apterous) or they do not have targeting defects in PNs homozygous for null alleles (lim337Bd6 and awh16) (Komiyama, 2007).

Islet antibody detected Islet expression in ~50% adPNs and most lPNs but not in vPNs at 18 hr APF and adult. isl−/− adPNs failed to target many (but not all) of the normal target glomeruli, including VA1lm, VA3, and VM7. In addition, DA1, a lPN target, was often specifically mistargeted. Defects of isl−/− lPNs were very similar to Chip−/− lPN defects. A fraction of dendrites often mistargeted to the SOG. Within the AL, dendrites were diffusely spread, although DA1 and DL3 were always correctly innervated. Targeting of isl−/− vPNs was normal, consistent with their lack of Islet expression (Komiyama, 2007).

Lim1 antibody revealed Lim1 expression in most or all vPNs, but not in adPNs or lPNs in adults. The expression pattern appears similar at 18 hr APF, although vPNs are difficult to identify unambiguously at early stages. lim1−/− adPNs showed no defects, consistent with the lack of Lim1 expression. lim1−/− lPNs rarely showed a cell number decrease, but in clones in which the cell number was normal, lim1−/− lPNs targeted correct glomeruli. In contrast, lim1−/− vPNs showed a specific targeting defect. Wild-type vPNs innervate DA1 and VA1lm densely because of the single vPNs that specifically innervate these glomeruli, in addition to the diffuse innervation all over the AL contributed by the pan-AL vPN. In lim1−/− vPNs, DA1 innervation was greatly reduced and sometimes undetectable. Therefore, lim1 is required for dendritic targeting by a single vPN class, vDA1, despite its general expression in vPNs. lim1 might be redundant with other factors in non-DA1 vPNs. It was note that phenotypes of islet and lim1 combined are only a subset of the Chip phenotype. Additional Chip phenotype may be explained by non-Lim-HD molecules interacting with Chip (Komiyama, 2007).

cut is required for targeting of several lPN and all vPN classes: cut encodes a homeodomain TF that regulates sensory organ identity and dendritic morphogenesis in Drosophila peripheral nervous system. A monoclonal antibody detected Cut in subsets of adPNs and lPNs (~8 for each) and in all vPNs. The expression pattern appeared similar at 18 hr APF. Costaining with Mz19-Gal4 and various single-cell clones with GH146-Gal4 further narrowed down Cut-expressing PNs; Cut-positive adPNs are likely embryonically born and thus not included in the functional analysis, while DM1 and DM2 lPNs express Cut, but DA1, DL3, and DM5 lPNs do not (Komiyama, 2007).

cut−/− adPNs targeted all their normal glomeruli correctly, consistent with their lack of expression. cut−/− lPNs failed to target DM1, DM2, and VA5. cut−/− vPNs were severely affected, with their cell numbers reduced from 4–6 in wild-type to 2–3 in cut−/− clones. cut−/− vPNs failed to elaborate their dendrites correctly in the AL and mistargeted the SOG. In summary, cut is required by a specific subset of lPNs and all vPNs that express Cut (Komiyama, 2007).

cut appears to control global targeting of PNs along mediolateral axis, as indicated by the fact that loss and gain of cut in lPNs causes a lateral and medial shift of dendrites, respectively. adPNs do not show a cut loss-of-function defect, consistent with the lack of expression. Nevertheless, cut misexpression in adPNs shifted their dendrites medially. Interestingly, adPNs misexpressing cut usually avoid DM1 and DM2, suggesting that cut controls global targeting, rather than simply promoting innervation of these glomeruli (Komiyama, 2007).

Postmitotic expression of a cut transgene only in labeled cut−/− lPNs completely rescued targeting of DM1, DM2, and VA5. There were also gain-of-function phenotypes, and DA1 and DL3 innervation was often lacking in these clones. Thus, cut postmitotically rescues dendritic targeting defects of lPNs that normally express cut, whereas postmitotic misexpression in other lPNs disrupts their targeting fidelity (Komiyama, 2007).

The vPN rescue phenotype was more complex. The cell number decrease was not rescued by postmitotic cut expression. However, the targeting defect was partially rescued. 71% of vPN rescue clones examined sent some dendrites to the AL (the rest completely failed to innervate the AL), and 68% innervated VA1lm. This is markedly better than cut−/−, in which only 51% entered the AL and 23% innervated VA1lm. DA1 targeting was not rescued, raising the possibility that the DA1 vPN was never born or correctly specified in these animals (Komiyama, 2007).

Relationship of cut and lim1 in vPNs: The lim1 phenotype in vPNs is a subset of the cut phenotype. Lim1 immunoreactivity in cut−/− vPNs was either absent or greatly reduced compared to wild-type. Therefore, Cut directly or indirectly controls Lim1 expression (Komiyama, 2007).

If a major function of Cut in vPNs is to upregulate Lim1, then transgenic lim1 expression in cut−/− vPNs might suppress part of the cut−/− phenotype. In cut−/− vPNs expressing a lim1 transgene, the reduction of cell number was not suppressed. However, 67% clones innervated the AL (compared to 51% in cut−/−). VA1lm innervation was also mildly improved (36% in UAS-lim1 versus 23% in cut−/−). Thus, UAS-lim1 expression partially suppresses cut−/− targeting defects, although not quite as well as UAS-cut. In contrast, UAS-lim1 expression in cut−/− lPNs, which normally do not express Lim1, did not suppress the cut−/− targeting defects. Therefore, Cut and Lim1 are not simply interchangeable, and the partial suppression of cut−/− defects by lim1 is specific to vPNs (Komiyama, 2007).

Although postmitotic expression of cut partially rescued the cut−/− vPN phenotypes, it failed to rescue Lim1 expression. In addition, postmitotic misexpression of cut in adPNs or lPNs did not lead to an ectopic expression of Lim1. Therefore, cut is not sufficient to upregulate Lim1 expression in postmitotic neurons. It is proposed that cut functions at two distinct stages of vPN development. First, cut controls the proliferation and/or fate specification of the vPN neuroblast, including Lim1 expression. Second, cut controls dendritic targeting by postmitotic VA1lm vPNs, partially redundantly with lim1. This partial redundancy may explain the observation that lim1−/− vPNs target VA1lm normally. These pre- and postmitotic functions of cut in the same neuronal lineage are reminiscent of its function in peripheral nervous system development (Komiyama, 2007).

If combinations of the TFs identified in this study instruct PN dendritic targeting, then misexpression or swapping of them might cause predictable changes of targeting specificity. This hypothesis was tested by using the DL1 adPN as a model, because this class can be unambiguously identified based on the time of heat shock to induce clones with GH146-Gal4, and GH146-Gal4 is strong enough for single-cell rescue or misexpression experiments (Komiyama, 2007).

DL1 adPN expresses Acj6, an adPN lineage factor, but not Drifter or Cut. acj6−/− DL1 PNs typically have diffuse dendrites that always innervate, but are not limited to, DL1. drifter misexpression alone did not affect their dendritic targeting. However, when loss of acj6 and gain of drifter were combined, the dendrites completely missed DL1 and targeted anterior glomeruli (Komiyama, 2007).

Misexpression of Cut alone caused DL1 PNs to target part of DL1 and the vicinity, similar to acj6−/−. Notably, this diffuse phenotype was directional, because most mistargeted dendrites targeted medially to DL1 (Komiyama, 2007).

cut misexpression combined with loss of acj6 caused severe mistargeting of DL1 adPNs. The dendrites completely missed DL1 and occupied the medial to dorsomedial AL, typically VM2, DM6, and DC1. Interestingly, these glomeruli are all adPN targets near DM1 and DM2, the two glomeruli that most frequently fail to be innervated by cut−/− lPNs. One interpretation is that loss of acj6 made the DL1 adPN more sensitive to the instructive information of cut to target the medial AL, but the remaining lineage information kept the dendrites within the adPN glomeruli in the area. If this were true, adding a lPN lineage factor drifter may bring the dendrites to DM1 or DM2, since this might recreate, based on partial knowledge of the TF code, a code for targeting these glomeruli. Loss of acj6 and misexpression of cut and drifter were combined simultaneously in DL1 adPNs. Under this condition, the dendrites again mostly targeted the medial to dorsomedial AL. However, glomerular preferences were strikingly different: they frequently innervated 1, DM2, and DA2. Notably, DA2 and DM2 are lPN targets (Komiyama, 2007).

These results suggest that cut and drifter have qualitatively different instructive information, with cut controlling global targeting and drifter controlling local glomerular choice according to their lineage (Komiyama, 2007).

These experiments described have identified six TFs and a cofactor required for dendritic targeting of specific subsets of 17 classes of Drosophila olfactory projection neurons. Of the six TFs identified here, at least five are expressed in subsets of PNs. Based on the expression data, it is estimated that expression of these six TFs could define 5–11 unique identities. Although unique combinations of TFs have not been identified for all 17 classes studied, the results suggest that distinct PN classes are at least partially defined by combinatorial expression of TFs that regulate their targeting specificity (Komiyama, 2007).

How many TFs are required to specify the dendritic targeting of 17 PN classes? With a binary combinatorial code, 5 factors could specify 25 (=32) different states. If different levels of single factors carry different information, even fewer factors could be sufficient. However, six TFs have been identified that regulate dendritic targeting specificity of subsets of PN classes, or 'specificity TFs', after testing 14 candidate TFs. Given that there are 694 predicted TFs in the Drosophila genome, it is almost certain that only a small fraction of specificity TFs have been identified. Thus, the number of specificity TFs is likely much larger than the theoretical minimum (Komiyama, 2007).

Redundancy could be a major reason. cut and lim1 in vPNs provide an example. Redundancy could ensure the robustness of wiring, making it tolerant to mutations in specificity TFs. Such tolerance could provide a substrate for evolution, allowing mutations to accumulate without devastating effects on the wiring of preexisting neuronal classes and making it easier for new classes to evolve. Whatever the evolutionary advantages might be, it is suggested that many TFs function redundantly and at different levels in a complex hierarchy that cooperatively define neuronal connection specificity (Komiyama, 2007).

It is found that different TFs regulate different steps of dendritic targeting, some specifying the coarse area (e.g., cut), followed by others controlling local glomerular choice within the area (e.g., drifter and acj6). adPNs and lPNs target highly intercalating but nonoverlapping sets of glomeruli. This could be explained now by acj6 and drifter controlling local glomerular choices, enabling adPNs and lPNs to locally segregate into distinct sets of glomeruli. These findings fit well with recent finding that graded expression of Sema1a cell-autonomously controls the initial and coarse targeting of PN dendrites along the dorsolateral to ventromedial axis. This coarse targeting is likely refined by PN dendrodendritic interactions and ORN-PN interactions. Thus, PN dendrites perform multistep targeting, gradually restricting their dendritic regions. Such multistep targeting could increase the robustness of neuronal wiring, reducing the complexity of decisions at each decision point and minimizing mistakes made by each neuron (Komiyama, 2007).

These results begin to provide insights into the global strategy of how an ensemble of TFs regulates wiring specificity of a large number of neurons constituting a neural circuit. It is envisioned that the properties identified in this tudy, such as a redundant TF code and multistep targeting, are generally applicable to the establishment of wiring specificity of other complex neural circuits in nervous systems (Komiyama, 2007).

Knot/Collier and Cut control different aspects of dendrite cytoskeleton and synergize to define final arbor shape

In a complex nervous system, neuronal functional diversity is reflected in the wide variety of dendritic arbor shapes. Different neuronal classes are defined by class-specific transcription factor combinatorial codes. The combination of the transcription factors Knot and Cut is particular to Drosophila class IV dendritic arborization (da) neurons. Knot and Cut control different aspects of the dendrite cytoskeleton, promoting microtubule- and actin-based dendritic arbors, respectively. Knot delineates class IV arbor morphology by simultaneously synergizing with Cut to promote complexity and repressing Cut-mediated promotion of dendritic filopodia/spikes. Knot increases dendritic arbor outgrowth through promoting the expression of Spastin, a microtubule-severing protein disrupted in autosomal dominant hereditary spastic paraplegia (AD-HSP). Knot and Cut may modulate cellular mechanisms that are conserved between Drosophila and vertebrates. Hence, this study gives significant general insight into how multiple transcription factors combine to control class-specific dendritic arbor morphology through controlling different aspects of the cytoskeleton (Jinushi-Nakao, 2007).

To understand the mechanism by which Knot promotes dendrite outgrowth, attempts were made to identify Knot-regulated genes in da neurons. Knot promotes formation of a microtubule-based dendritic arbor cytoskeleton. Therefore, candidate genes for regulation by Knot were chosen based on annotation in the Gene Ontology database indicating their association with microtubule biogenesis and function. The small GTPases rac1, cdc42, and rho, were also analyzed. Ectopic (classes I-III) or endogenous (class IV) Knot activity promotes da neuron dendritic arbor outgrowth. With this in mind, the relative expression of candidate genes was compared between wild-type and ectopic knot-expressing da neurons. To do this the RluA1-Gal4 line was used that drives UAS-mCD8::GFP expression solely in all da and some ES neurons. Purifying Gfp-positive cells from RluA1-Gal4, UAS-mCD8::GFP embryos gave a highly enriched population of da neurons for analysis. Gfp-positive cells were sorted from control (RluA1-Gal4, UAS-mCD8::GFP) and ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) embryos by Fluorescence-Activated Cell Sorting (FACS). Total RNA was isolated from the purified cells and the relative expression of candidate genes was compared between these populations by Reverse Transcription Polymerase Chain Reaction (RT-PCR). mRNA expression levels of all candidates were normalized against gapdh, and as a positive control analyzed knot levels were analyzed. Between the wild-type and ectopic knot-expressing cells, knot mRNA expression was upregulated by ratio of 2.6. Of the candidates analyzed, only spastin had an altered expression level; it was strongly upregulated by a ratio of 3.3 (Jinushi-Nakao, 2007).

These RT-PCR findings were confirmed by examining upregulation of Spastin protein via Knot ectopic expression. Spastin is a member of the ATPases associated with diverse cellular activities (AAA) family, all of which have a related protein structure. To avoid cross-reactivity between family members, a Spastin-specific antibody, which was additionally preabsorbed against the other AAA family members, was used. Spastin protein levels were examined in western blots of protein extracts from sorted Gfp-positive cells. Ectopic knot-expressing (RluA1-Gal4, UAS-mCD8::GFP, UAS-kn) da neurons showed a very large upregulation in Spastin protein content as compared with those prepared from wild-type (RluA1-Gal4, UAS-mCD8::GFP) da neurons (Jinushi-Nakao, 2007).

If spastin is a bona fide target of Knot in class IV neurons, then spastin expression may be enriched in class IV da neurons versus other da neuron classes. To examine spastin expression whole-mount embryonic spastin mRNA in situ experiments were carried out. The results confirmed those of previous studies that show that spastin is ubiquitously expressed, with a higher-than-background expression level in nervous system tissues. However, the high ubiquitous background expression level made it impossible to compare levels of spastin in specific da neuron classes. To get around this problem, FACS purified a mixed population of all da neurons (RluA1-Gal4, UAS-mCD8::GFP) and a pure population of class IV neurons (ppk-Gal4, UAS-mCD8::GFP) were examined. spastin expression in these two populations was compared. spastin expression levels were clearly enriched (62% more) in the pure population of class IV neurons as compared with the mixed population of da neurons (Jinushi-Nakao, 2007).

Spastin has microtubule-severing activity in cultured cells and in vitro. It was asked if Spastin is also able to alter microtubule structure in the dendritic arbor of da neurons. To do this, spastin was ectopically expressed in class I ddaE neurons (Gal42-21, UAS-mCD8::GF8, UAS-spastin). Then the entire dendritic arbor was visualized at the wandering third-instar larva stage by staining with an anti-Gfp antibody, and examined the microtubule cytoskeleton was simultaneously by staining the arbor with antibodies to detect Futsch. In wild-type class I neurons, Futsch was present throughout the dendritic arbor. When spastin was expressed ectopically in the class I neuron, a loss of Futsch from the dendritic arbor was observed and disruption of arbor morphology. Therefore, high levels of Spastin disrupt microtubule organization within the arbor, a finding consistent with Spastin's microtubule-severing activity (Jinushi-Nakao, 2007).

Next, whether Spastin activity is required to promote class IV dendritic arbor complexity, as would be expected if it is part of the program controlled by Knot activity, was investigated. To do this class IV neurons were marked with ppk-Gal4, UAS-mCD8::GFP in the background of a null spastin5.75 allele. ppk-Gal4 was used to express an RNAi construct directed against spastin (UAS-spastinRNAi) along with UAS-mCD8::GFP to selectively knock down spastin class IV neurons. The morphology of these neurons was examined at the wandering third-instar larva stage (Jinushi-Nakao, 2007).

Reduction of spastin levels in either heterozygous null background or spastin RNAi-mediated knockdown background lead to large gaps both between neighboring class IV dendritic arbors and within the arbor of an individual neuron. Such gaps were not seen in wild-type control larvae, but were also seen in loss- or reduction-of-function knot mutants. To quantify this effect, dendrite coverage was measured by drawing a 34 × 34 grid of 10 μm × 10 μm squares over the central portion of the neuron. The number of squares that did not contain any portion of a dendrite branch was counted. This analysis provides an approximate measure of the amount of area that is not covered by the dendritic arbor. spastin RNAi knockdown had 17% more uncovered area than wild-type ddaC neurons; spastin5.75/+ mutants had 43%, and kn1/knKN2 mutants had 59% (Jinushi-Nakao, 2007).

Spastin is upregulated by Knot in da neurons and is required for class IV neuron dendritic arbor outgrowth. To confirm that Spastin is part of the program by which Knot mediates dendritic arbor outgrowth, the outcome was investigated of spastin RNAi in either a wild-type or an ectopic knot-expressing class I neuron. UAS-spastinRNAi was crossed to Gal42-21, UAS-mCD8::GFP or UAS-kn; Gal42-21, UAS-mCD8::GFP, and class I ddaE dendritic arbor shape was assayed at wandering third-instar larva stage. The spastinRNAi construct had no effect on either branching or dendrite length when expressed in a wild-type class I neuron. However, the UAS-spastinRNAi construct strongly reduced both branching and total dendrite length (by 18% and 19%, respectively) when expressed in an ectopic knot-expressing class I neuron. Therefore, Spastin activity is an essential part of the program by which ectopic knot expression mediates an increase in dendritic arbor complexity (Jinushi-Nakao, 2007).

This study has shown that Knot and Cut act simultaneously in the class IV neuron to promote dendritic arbor outgrowth and branching. However, the loss-of-function phenotypes for Knot and Cut are different, which demonstrates that each transcription factor works through a dissimilar mechanism. Indeed, ectopic expression experiments show that Knot and Cut regulate different aspects of the cytoskeleton. Knot expressed ectopically in the class I neuron promotes arbor extension that is microtubule-positive. Conversely, ectopic expression of Cut in the class I neuron leads to arbor extension that is F-actin-positive but microtubule deficient. When Cut and Knot are expressed together in the class I neuron, they have a synergistic effect on dendritic arbor area and branching. However, the effect of Cut and Knot coexpression on dendritic arbor total length is additive: both the microtubule-positive and microtubule-negative regions of the arbor are increased. This overall arbor organization mimics that of class IV neurons. The majority of the dendritic arbor of the class IV neuron contains microtubules, but the highest-order branches are microtubule deficient (Jinushi-Nakao, 2007).

When Cut levels are increased and Knot levels are reduced in a class IV neuron, its dendritic arbor takes on characteristics similar to those of class III. This transformation from a class IV to a class III shape is not absolute. Hence, it is likely that other factors are also required to fully control all aspects of class IV dendritic arbor morphology versus those of other da neuron classes. Overall, however, the data suggest that class IV-specific Knot expression demarcates arbor shape via multiple mechanisms. Knot synergizes with Cut in promoting dendrite length, branching, and area. Additionally it represses the ability of Cut to mediate filopodia/spike formation. Finally, Knot induces symmetry in the dendritic arbor of the class IV neuron, as opposed to class I–III neurons, which have asymmetric dendritic arbor shapes (Jinushi-Nakao, 2007).

Suppression of Cut-mediated filopodia/spike formation by Knot does not occur through repression of Cut protein levels and therefore acts either downstream of Cut or in parallel. Interestingly, though, the absolute level of Knot protein is controlled by the level of Cut in the cell. Tuning the level of Knot to the level of Cut protein in each neuron could be a mechanism by which Knot acts to repress only specific aspects of the Cut-driven morphogenesis program (Jinushi-Nakao, 2007).

Knot and Cut also interact very differently with Rac1. A major function of Rac1 is to promote reorganization of the actin cytoskeleton. Filopodia/spikes are rich in F-actin and deficient in microtubules, and indeed Rac1 significantly enhances the ability of Cut to promote filopodia/spike formation. Ectopic coexpression of Knot and Rac1 in class I neurons leads to large increases in the length of the short, thorn-shaped projections that are induced by expression of Rac1 alone. Rac1 has been shown to form focal F-actin in the distal edge of axonal growth cones, which acts as a site of microtubule capture during outgrowth. Perhaps similar processes are occurring in the Rac1-mediated thorn-shaped projections. Knot activity could then promote microtubule invasion and outgrowth at these points of Rac1-mediated F-actin reorganization (Jinushi-Nakao, 2007).

Knot promotes microtubule-mediated dendritic arbor outgrowth by inducing Spastin expression. A large amount of the extra arbor outgrowth induced by ectopic Knot expression is suppressed by reducing Spastin function. Therefore, Spastin is a primary component of the mechanism by which Knot promotes arbor outgrowth (Jinushi-Nakao, 2007).

Spastin acts as a microtubule-severing protein and may function by producing new seeds for microtubule polymerization. Maintenance of a population of dynamic microtubules is important for axonal extension, branching, and growth cone guidance. In vivo, Spastin has been shown to be required for growth of synaptic terminals at the Drosophila neuromuscular junction and for axon outgrowth in zebrafish. This study shows that Spastin activity can also destabilize microtubules in the dendritic arbor, and that Spastin is itself required for class IV da dendritic arbor outgrowth (Jinushi-Nakao, 2007).

The human spastin gene (SPG4) is mutated in over 40% of autosomal dominant hereditary spastic paraplegia (AD-HSP) cases. SPG4 mutation usually causes pure spastic paraplegia of the lower limbs due to degeneration of the corticospinal tract axons. However, in some families SPG4 mutation is associated with additional neurological symptoms that cannot be explained by dysfunction of the corticospinal tract axons alone. This study shows that Spastin is also required for complex dendritic arbor development; hence, defects in dendrite as well as axon development and function may be part of the pathology of some AD-HSP cases (Jinushi-Nakao, 2007).

Accumulating evidence suggests that mechanisms of dendritogenesis are closely conserved between Drosophila and other species. For example, actin binding proteins, rac/rho GTPases, and calcium/calmodulin-dependent protein kinase II (CaMKII) all control dendrite branching and filopodia/spine morphogenesis in Drosophila and in vertebrates. knot (Ebf1, 2, and 3) and cut (Cux1 and 2) homologs are expressed in the developing mouse nervous system and may overlap in some subsets of neurons, e.g., in the spinal cord and cerebellum. In vertebrates, it is possible that these genes may also regulate dendrite morphology. Both human CUX1 and mouse Ebf2 can phenocopy cut and knot, respectively, when ectopically expressed in class I neurons. Interestingly, Ebf2 is involved in the migration and differentiation of Purkinje neurons. However, a specific role for Ebf2 in controlling the highly complex dendritic arbor shape of these neurons remains to be assayed (Jinushi-Nakao, 2007).

This study has elucidated mechanisms of transcription factor-mediated control of dendritogenesis. It was found that Knot and Cut function to control Drosophila class IV da sensory neuron dendritic arbor morphogenesis through different aspects of the cytoskeleton. Further analysis of Knot and Cut targets will provide a powerful entry point into understanding dendritic arbor morphogenetic mechanisms that are potentially conserved between Drosophila and vertebrate species (Jinushi-Nakao, 2007).

Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing: Cut is not only required but also sufficient to inhibit Wg target gene expression in boundary cells downstream of Notch

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, this regulatory network was modeled and tested; evidence is presented that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements the results and ensures its biological plausibility (Buceta, 2007).

In silico evidence is presented that refractoriness to the Wg signal in boundary cells provides stability to the gene regulatory network. Boundary cells are characterized by high levels of Notch activity, thus suggesting Notch is responsible for making boundary cells refractory to the Wg signal. The role of Notch in this process was analyzed in the developing wing primordium. Ectopic activation of Notch in non-boundary cells represses Wg target gene expression. Note that Notch, in this case, causes ectopic Wg expression in non-boundary cells, which induces target gene expression only in Wg non-expressing cells. By contrast, ectopic expression of Wg alone induces the expression of target genes in both Wg-expressing and non-expressing cells. When boundary cells lack Notch activity, either by mutation or by expression of a dominant negative form of Delta known to titrate out the Notch receptor, these cells start to express target genes of Wg. It can then be concluded that either Notch activity itself, or one or several of its target genes inhibits the expression of Wg target genes in boundary cells (Buceta, 2007).

High levels of Notch activity induce expression of the homeobox gene cut in boundary cells and Cut has been previously shown to be required to repress Delta and Serrate expression in these cells. Then, whether Cut mediates the activity of Notch in inhibiting the expression of other Wg target genes was examined. In the absence of Cut activity, either in a homozygous mutant background or in clones of mutant cells, boundary cells start expressing genes regulated by the Wg signal, and ectopic Notch activation in non-boundary cells is now unable to repress Wg target gene expression. Note that Notch, in this case, causes ectopic expression of Wg, which induces target gene expression in both Wg-expressing and non-expressing cells. Finally, forced expression of Cut in non-boundary cells represses the expression of Wg target genes. Taken together, these results indicate that Cut is not only required but also sufficient to inhibit Wg target gene expression in boundary cells downstream of Notch (Buceta, 2007).

Cut might exert its function either by blocking the Wg signaling pathway or, alternatively, by inhibiting the expression of every Wg target gene. The Wg signaling pathway is activated by controlling the levels and subcellular localization of the transcriptional co-activator Armadillo (Arm, known as β-catenin in vertebrates). In the absence of Wg signal, Arm levels are kept low through degradation. This degradation depends on the phosphorylation of Arm by the kinase Shaggy/Zeste white-3/Glycogen synthase kinase-3β (GSK-3β). Phosphorylated Arm is recognized rapidly by the proteasome and destroyed. Following Wg ligand binding, this degradation is inhibited, which enables Arm to accumulate, enter the nucleus and activate a transcriptional response. In the Drosophila wing, Arm protein levels are severely reduced in boundary cells, when compared with adjacent cells, even though extracellular Wg protein is available in both types of cells. This observation indicates that the activity of the Wg signaling pathway is repressed in these cells at the level or upstream of Arm. Consistent with this observation, a dominantly activated form of Arm (ArmS10), which lacks the GSK-3ß phosphorylation sites and escapes degradation, induces expression of Wg targets in boundary cells. Overexpression of any other limiting factor of the Wg pathway that acts upstream of Arm is unable to induce Wg target gene expression in these cells (Buceta, 2007).

Cut appears to mediate this type of repression of the Wg signaling pathway. In the absence of Cut activity, Arm protein levels are not reduced in boundary cells, and ectopic expression of Cut in non-boundary cells reduces Arm protein levels and represses the expression of Wg target genes. Moreover, ArmS10 can bypass the effects of ectopic Cut expression and restores Wg target gene expression in non-boundary cells. Co-expression of limiting factors of the Wg pathway acting upstream of Arm does not cause this effect. Taken together, these results indicate that Cut blocks the Wg signaling pathway at the level or upstream of Arm. Cut might exert its function through transcriptional regulation of a gene product involved in regulating the degradation of Arm (Buceta, 2007).

So far in vivo evidence has been provided that Cut is required in boundary cells to repress the Wg signaling pathway and also, by means of in silico experiments, it has been shown that such repression leads to a stable DV boundary formation. In silico implementation of the refractoriness to the Wg signal via Cut leads to stable DV boundary formation. The stationary pattern of gene expression and activity observed in this case is in agreement with in vivo results (Buceta, 2007).

The conclusions can be extended further with regard to the role played by Cut in DV boundary formation. In the absence of refractoriness to the Wg signal (provided by the activity of Cut in boundary cells) an initial increase in Notch activity and Wg expression takes place. This result suggests that Cut is dispensable for the onset of the DV boundary. This and the evolution predicted by modeling are in agreement with the in vivo results. In cut mutant discs, the early activation of Notch at the DV boundary, as shown by the expression of Wg, is comparable to wild-type discs. However, in mature third instar discs Notch activity and Wg expression are not maintained in the mutant background. Taken together, these results indicate that refractoriness of boundary cells to the Wg signal provided by the activity of Cut is required to shape a stationary and stable DV boundary in the developing wing primordium (Buceta, 2007).

This study analyzed the properties of the regulatory network for the establishment and maintenance of the DV organizer in the Drosophila wing imaginal disc. Evidence is provided that that a mathematical model can convert the initial DV asymmetric expression pattern of Notch ligands into the DV symmetric and mutually exclusive domains of active receptor and Notch ligands in boundary and non-boundary cells, respectively. To model the network 'circuitry', and test and verify the proposal, advantage was taken of a combination between in vivo and in silico experiments that has allowed checking of the analytical and predictive capacity of the modeling (Buceta, 2007).

The most striking finding of this research is that a novel property is required in the regulatory network for a robust and stable maintenance of the DV organizer: namely boundary cells must be refractory to the Wg signal. This property is conferred by the activity of Notch through its target gene cut. The role of Cut in repressing the Wg signaling pathway in boundary cells, and Wg in repressing Notch in non-boundary cells, generates two mutually exclusive domains of Notch and Wg activities, corresponding to boundary and non-boundary cells, respectively. Consequently, Notch ligands and receptors are expressed in two distinct non-overlapping cell populations. This helps to restrict the width of the boundary population to few (two-three) cells and contributes to polarizing ligand-receptor signaling towards the boundary and not against it, i.e., flanking ligands signal Notch towards the boundary but not against it since down-regulation of the Notch pathway in non-boundary cells inhibits the receptors' activity in those cells. In addition, light has been shed on several dynamical properties of the network, such as the refinement of Notch activity (Buceta, 2007).

At the time the role of Cut in the repression of Delta and Serrate expression was described, Cut and the concomitant restriction of ligand expression to non-boundary cells were postulated to be essential for the stability of the DV boundary. However, the other negative input of Wg into the Notch pathway through the activity of Dishevelled was not taken into account. In silico results have predicted that a general repression of the Wg pathway is required for stable activity of Notch at the DV boundary. In vivo results indicate that this repression takes place at the level or upstream of Armadillo. In order to be refractory to the inhibitory effect of Dishevelled on Notch, this repression should be taking place close to Dishevelled if not further upstream in the Wg signaling cascade (Buceta, 2007).

Finally, the conclusions are placed into a broader context. Boundary formation between adjacent rhombomeres in vertebrates relies on the same Wnt/Notch-dependent regulatory network. Therefore, it is speculated that boundary cells also need to be refractory to the Wnt signal to generate stable boundaries. To close, it is concluded that the robustness and stability of this network, in which the interconnectivity of the elements is crucial and even more important than the value of the parameters used, might explain its use in boundary formation in other multicellular organisms (Buceta, 2007).

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. This study presents evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and the Actin regulator Enabled was identified as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary (Becam, 2011).

Cell divisions lead to cell rearrangements that may challenge straight and sharp compartment boundaries. The DV boundary of mid- and late third instar wing primordia is characterized by a reduced rate of cell proliferation which defines the zone of non-proliferating cells (ZNC). The contribution of the ZNC to the maintenance of the DV affinity boundary was proposed many years ago but this notion was subsequently questioned. This study provides evidence that the ZNC does indeed play a role in boundary formation. bantam miRNA positively modulates the activity of the E2F transcription factor and drives G1-S transition in Drosophila tissues. Notch-mediated downregulation of bantam miRNA defines the ZNC and contributes to maintain a stable DV affinity boundary. Induction of proliferation in boundary cells by the ectopic expression of bantam, the cell cycle regulators Cyclin E and String, or the proto-oncogene dMyc, which is known to drive G1-S transition, compromises the formation of a smooth DV affinity boundary. A similar reduction in proliferation rates is observed at the rhombomere boundaries in the developing hindbrain, suggesting that reduced rate of cell proliferation might often be used in compartment boundary formation (Becam, 2011).

Notch-mediated downregulation of bantam activity is not only required to define the ZNC but also to establish the actomyosin cables observed at the interface between boundary and non-boundary cells. Ena, a regulator of Actin elongation, was identified as a direct target of bantam that is involved in DV boundary formation. The multiple roles of bantam in promoting G1-S transition and tissue growth, blocking apoptosis and regulating Actin dynamics unveil a new molecular connection between these three processes that might have relevance in growth control and tumorigenesis (Becam, 2011).

Intriguingly, bantam miRNA has no major role in the maintenance of the anterior-posterior compartment boundary of the developing wing and this boundary is not affected upon depletion of Ena protein levels. Thus, different regulators of actin elongation might be at work to regulate the actomyosin cytoskeleton and direct cell sorting in diverse developmental contexts. Whether reduced levels of bantam miRNA and increased levels of Ena protein are required to maintain differential cell sorting in the embryonic ectoderm or other imaginal tissues remains to be elucidated (Becam, 2011).

Cut is a late target of Notch that is expressed in boundary cells and is required to induce a stable Notch signaling center. This study demonstrate that Cut activity has also a specific function in reducing Ena mRNA and protein levels in boundary cells. Although depletion of Cut compromises the formation of the actomyosin cables at the interface of boundary and non-boundary cells and the maintenance of a stable DV affinity boundary, cell lineage and clonal analysis of wild-type and cut mutant cells reveal that Cut plays a major role in sorting boundary from non-boundary cells. The finding that the Notch signaling pathway defines, through Cut, a distinct population of boundary cells at the DV interface reinforces the mechanistic similarities in the maintenance of compartment boundaries within the vertebrate hindbrain and the Drosophila wing. In both developmental contexts, Notch defines a distinct population of boundary cells and contributes to segregating boundary from non-boundary cells. Although Cut mediates the role of Notch in the Drosophila wing, the molecular effectors mediating the role of vertebrate Notch in boundary formation remain uncharacterized. The data indicate that the later subdivision into boundary and non-boundary cells contributes to the maintenance of a stable DV affinity barrier in the mature wing primordium (Becam, 2011).

The RhoGEF trio functions in sculpting class specific dendrite morphogenesis in Drosophila sensory neurons

As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. This study demonstrates that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, it was shown that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. Collectively, these analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation (Iyer, 2012).

This analysis demonstrates that Trio functions in promoting and refining class specific dendritic arborization patterns via GEF1- and GEF2-dependent interactions with Rac1 and Rho1, respectively. It was also demonstrated that Trio is required in mediating Cut induced effects on dendritic branching and filopodia formation suggesting that these molecules may operate in a common pathway to direct dendritic morphogenesis. Giniger and colleagues (NINDS/NIH) have likewise been investigating Trio function in da neurons via a non-overlapping, complementary experimental approach, and that they arrived at conclusions regarding Trio function largely consistent with those reported in this study (Iyer, 2012).

Previous studies have demonstrated that Trio functions via its GEF1 domain in mediating the regulation of axon morphogenesis by modulating Rac1 activity, however much less is known regarding the potential in vivo functional role(s) of the Trio GEF2 domain. Intriguingly, a previous study demonstrated that trio mutant neuroblast clones display a neurite overextension phenotype from the dendritic calyx region of the mushroom body which strongly resembled the dendrite-specific overextension phenotype observed in RhoA mutant mushroom body clones suggesting that RhoA/Rho1 activation may be required for restricting dendritic extension. In Drosophila da neurons, trio loss-of-function analyses reveal a reduction in dendritic branching in three distinct da neuron subclasses (class I, III, and IV), indicating a functional role for Trio in promoting dendritic branching. However, class specific differences are observed with Trio gain-of-function studies in which Trio overexpression in class I neurons increases dendritic branching, whereas in class III neurons there is no change in overall dendritic branching, but rather a redistribution of branches, and in class IV there is a reduction in overall dendritic branching. The basis for these differences appear to lie in the observation that refinement of dendritic branching in da neurons is subject to the opposing roles of Rac1 and Rho1 activation via Trio-GEF1 and Trio-GEF2, respectively, where Trio-GEF1 activity promotes higher order dendritic branching, whereas Trio-GEF2 activity restricts higher order branching and also limits overall dendritic length/extension (Iyer, 2012).

One of the key distinctions between class I versus class III and IV neurons relates to inherent differences in normal dendritic branching complexity and the relative roles of dynamic actin cytoskeletal based processes in these neurons which are known to mediate higher order branching including the dendritic filopodia of class III neurons and fine terminal branching in class IV neurons, whereas the class I neurons do not normally exhibit this degree of higher order branching and are predominantly populated by stable, microtubule-based primary and secondary branches. As such, Trio overexpression in these distinct subclasses may yield different effects on overall dendritic branching morphology based upon the normal distribution of actin cytoskeleton within these subclasses leading to unique effects on class specific dendritic architecture. Both loss-of-function and gain-of-function results support this hypothesis as the predominant effects are restricted to actin-rich higher order branching, whereas the primary branches populated by microtubles are relatively unaffected. This is further supported by the demonstration that trio knockdown suppresses Cut induced formation of actin-rich dendritic filopodia. Moreover, phenotypic analyses revealed that co-expression of Cut and Trio-GEF1 synergistically enhance dendritic branching in class I neurons likely due to increased activation of Rac1, whereas co-expression of Cut and Trio-GEF2 lead primarily to increased dendritic extension likely due to increased activation of Rho1. Thus, Trio mediated regulation of Rac1 and/or Rho1 signaling has the potential for sculpting dendritic branching and outgrowth/extension depending upon the combinatorial and opposing effects of Rac1 and Rho1 (Iyer, 2012).

In contrast to Cut, which has been shown to be differentially expressed in da neuron subclasses and exert distinct effects on class specific dendritic arborization, this study has demonstrated that Trio is expressed in all da neuron subclasses and can exert distinct effects on class specific dendritic branching. For example, in all subclasses examined, loss-of-function analyses indicate Trio is required to promote dendritic branching and yet individual subclasses exhibit strikingly distinct dendritic morphologies. These results suggest that Trio is generally required in each of these subclasses to regulate branching, however alone is insufficient to drive these class specific morphologies solely via activation of Rac1 and/or Rho1 signaling. One logical hypothesis is that differential expression of RhoGAP family members in distinct da neuron subclasses may work in concert with Trio to refine class specific morphologies. The potential for combinatorial activity between Trio and various RhoGAPs is significant given that 20 RhoGAPs have been defined in the Drosophila genome. For example, given that class I da neurons exhibit a simple branching morphology which becomes more complex when Trio or Trio-GEF1 domains are overexpressed, perhaps there is higher expression of Rac-inactivating GAPs in class I neurons that function in limiting dendritic branching, whereas in the more complex class III or IV da neurons, there may be lower expression of RacGAPs. Since overexpression of Trio-GEF2 reduces dendritic branching complexity in all three da neuron subclasses analyzed, it might be predicted that Rho1 activation limits dendritic branching and that therefore the expression of RhoGAPs may be modulated to facilitate branching in class III and IV neurons relative to class I neurons. In concert, differential expression of RacGAPs and RhoGAPs together with the uniform expression of Trio in all da neuron subclasses could potentially account for differential levels of activation/inactivation of Rac1 and/or Rho1 in individual subclasses and thereby influence overall class specific dendritic architecture (Iyer, 2012).

In support of this hypothesis, class-specific microarray analyses conducted in class I, III, and IV da neurons indeed reveal differential gene expression levels for most of the 20 known RhoGAP family members at a class-specific level. These expression analyses reveal one trend whereby select RhoGAP encoding genes are upregulated in the more complex class III and IV da neurons relative to the simple class I da neurons, whereas select RacGAP encoding genes are downregulated in complex neurons relative to simple neurons. Moreover, it is known that individual RhoGAPs display differential specificities for Rac, Rho and Cdc42 in vivo, such that a given RhoGAP may function in activating one or more of these small G proteins thereby increasing the potential for fine-tuning activation levels of a particular G protein at a class specific level. Furthermore recent studies provide direct evidence of the importance of RhoGAP family members in regulating da neuron dendritic morphogenesis. Analyses of the tumbleweed (tum) gene, which encodes the GTPase activating protein RacGAP50C, demonstrate that tum mutants display excessive da neuron dendritic branching. The dendritic phenotype observed in tum mutant da neurons is strikingly similar to that observed with Trio-GEF1 overexpression which also leads to excessive dendritic branching. Together these data suggest that Trio-GEF1 functions in activating Rac1 to promote dendritic branching whereas Tum/RacGAP50C function in inactivating Rac1 via its GTPase activity and thereby limit dendritic branching. In contrast, mutant analyses of the RhoGAP encoding gene, crossveinless-c, whose target in da neurons is the Rho1 small G protein, reveal defects in directional growth of da neuron dendrites. These results indicate that Crossveinless-C is required to inactivate Rho1 in order to promote directional dendritic growth and further suggest that a failure to inactivate Rho1 leads to restricted dendritic growth consistent with the phenotypes observed with Trio-GEF2 overexpression in all da neuron subclasses examined. These results, together with those presented herein, suggest that potential combinatorial activity of Trio and RhoGAP family proteins may converge in shaping the class specific dendritic architecture. Ultimately, future functional studies will be required to validate this hypothesis (Iyer, 2012).

While previous studies have revealed Trio acts in concert with Abl and Ena in coordinately regulating axon guidance, the same regulatory relationship does not appear to operate in da neuron dendrites as Abl has been shown to function in limiting dendritic branching and the formation of dendritic filopoda, whereas both Ena functions in promoting dendritic branching. This study demonstrates that Trio functions in promoting dendritic branching, consistent with Ena activity, but in da neuron dendrites works in an opposite direction to Abl. These findings suggest that, at least in da neuron dendrites, Trio may operate in either an Abl-independent pathway or that Trio and Abl may exhibit a context dependent regulatory interaction that is distinctly different in dendrites versus axons (Iyer, 2012).


cut is expressed in the precursors of adult sensory organs. cut is expressed in cells of the prospective wing margin and correlate the wing margin phenotype caused by two cut mutations with altered cut expression patterns. Finally, there are cut-expressing cells in other adult tissues, including Malpighian tubules, muscles, the central nervous system and ovarian follicle cells (Blochlinger, 1993).

Effects of Mutation or Deletion

Extreme cut alleles result in short, dark and crumpled wings that are cut and scalloped. Abdominal bands are warped; antennae are flattened and embedded; aristae are concave forward; eyes are smaller and kidney shapped; and vibrissae are gone. There is more extreme expression in females than in males, and females have much poorer viability. Loss of cut activity results in a change in neural identity in the peripheral nervous system so that neurons and support cells of external sensory organs are transformed into those of internal chordotonal organs (Blockinger, 1989).

Ubiquitous cut expression in embryos results specifically in the morphological and antigenic transformation of chordotonal sense organs into external sensory organs. cut is necessary and sufficient for the specification of external sensory organ identity in the sensory organ precursor cells and their progeny. Specificity also involves the AS-C and daughterless genes (Blochlinger, 1991).

Flies bearing a temperature sensitive allele of strawberry notch show a modest loss of wing margin tissue when raised at 23 degrees C. When such flies are also made heterozygous for a single copy loss of wingless, extensive loss of wing margin tissue is observed, suggesting a dominant synergistic interaction between wg and sno. In similar experiments, temperature sno combined with a single copy loss of vestigial also results in a dominant enhancement of wing margin defects; mutants exhibit extensive loss of wing margin tissue. Genetic combination of a weak allele of cut with a sno mutation shows extensive loss of wing margin tissue, suggesting a synergistic interaction between sno and cut. These results are reminiscent of the interaction of Notch with wg, vg and ct and further extablish that sno, like Notch, has a crucial role in the establishment of D/V boundary fate by participating in a common genetic pathway that regulates wing margin-specific genes. In addition to the wing margin defects, sno mutants also exhibit thickening of wing veins. This is likely to be a secondary consequence of defective wing pouch development caused by improper D/V boundary specification. This same phenotype can also be seen in some of the other D/V boundary genes, such as vestigial and Serrate (Majumdar, 1997).

A soma-to-germline signaling pathway that requires the activity of the cut gene has been identified. In the ovary, CUT mRNA and protein expression are restricted to the follicle cells; moreover, cut mutant germline clones are phenotypically normal. When cut function is lost in the follicle cells, however, germline-derived cysts are mispackaged into egg chambers with abnormal numbers of cells, and the structural organization of oocyte-nurse cell complexes disintegrates, generating binucleate germline-derived cells. To date, cut is the only gene known to be required in the follicle cells that when mutated results in binucleate cells. The assembly of egg chambers and the maintenance of germline cell morphology therefore requires the activity of the cut gene in the soma, revealing a signaling pathway that influences the morphology and function of the germline-derived cells. In support of this conclusion, cut interacts genetically during oogenesis with two genes that influence intercellular communication, Notch and Pka-C1 (Jackson, 1999).

To understand the mechanism by which cut expression influences germline cell morphology, it had to be determined whether binucleate cells form by defective cytokinesis or by fusion of adjacent cells. Egg chambers produced by cut, cappuccino, and chickadee mutants contain binucleate cells in which ring canal remnants stain with antibodies against Hu li tai shao and Kelch, two proteins that are added to ring canals after cytokinesis is complete. In addition, defects in egg chamber morphology are observed only in middle to late stages of oogenesis, suggesting that germline cell cytokineses are normal in these mutants. The evidence suggests therefore that binucleate cells are generated by cell fusion. cut exhibits dose-sensitive genetic interactions with cappuccino but not with chickadee or other genes that regulate cytoskeletal function, including armadillo, spaghetti squash, quail, spire, Src64B, and Tec29A. Genomic regions containing genes that cooperate with cut were identified by performing a second-site noncomplementing screen, using a collection of chromosomal deficiencies. Sixteen regions that interact with cut during oogenesis and eight regions that interact during the development of other tissues were identified. Genetic interactions between cut and the ovarian tumor gene were identified as a result of the screen. In addition, the gene agnostic(agu) was found to be required during oogenesis, and genetic interactions between cut and agnostic were revealed. These results demonstrate that a signaling pathway regulating the morphology of germline cells is sensitive to genetic doses of cut and the genes cappuccino, ovarian tumor, and agnostic. Since these genes regulate cytoskeletal function and cAMP metabolism, the cut-mediated pathway functionally links these elements to preserve the cytoarchitecture of the germline cells (Jackson, 1999).

Because cut is expressed and required only in the follicle cells, its influence on germline cell morphology must be mediated across the soma-germline boundary. The results demonstrate that capu and otu, which are both required in the germline, interact genetically with cut and may facilitate cut-mediated events originating in the soma. Although cut is a transcription factor, the clear separation of cell types in which these genes are expressed suggests that cut does not regulate the transcription of capu or otu directly by binding to their promoters and/or enhancers. Rather, a multistep model is proposed in which cut activity in the follicle cells first directs expression of a gene or set of genes that regulates adhesion or signaling between the somatic and germline cells. This soma-to-germline interaction then influences cAMP-dependent function in the germline cells. The activity of Capu and Otu is in turn regulated by these cAMP-mediated events, perhaps by post-translational modifications or by alterations in the subcellular localization of one or both of these proteins. Finally, the regulation of Capu and Otu by cAMP results in altered cytoskeletal function. This hypothesis makes several testable predictions that are currently under investigation. Since it is not yet known if agn is required in the germline cells or follicle cells, the possibility that cut influences agn levels directly by regulating agn transcription in the follicle cells cannot be ruled out. A less favorable model is that capu, otu, and/or agn may function genetically upstream of cut, and loss of germline function of these genes influences cut activity in the follicle cells. At some point in this model, however, cut activity in the follicle cells must influence the function of the germline cytoskeleton, since loss of cut function in the follicle cells is sufficient to produce binucleate germline cells (Jackson, 1999).

The observed genetic interactions between cut and otu are consistent with the model that cut-mediated events disrupt the function of the germline cytoskeleton. Actin cytoskeleton function is known to be disrupted in otu mutants; it was hypothesized that this defect was the underlying cause for the various otu phenotypes. Although otu has been cloned and antibodies have been raised against the protein, the gene's sequence and the uniform distribution of Otu protein within the germplasm give no clue as to how Otu affects the function of the cytoskeleton. Nevertheless, the findings suggest that otu regulates cytoskeleton function in response to signaling events that occur after the cystoblast cell divisions are completed and egg chambers leave the germarium. Finally, one of the otu mutant phenotypes is the production of tumorous egg chambers filled with extra germline-derived cells. Egg chambers that were tumorous or that contained extra germline cell nuclei are not observe in cut/otu double heterozygotes, suggesting that cut and otu do not interact in the germarium to regulate germline cell divisions (Jackson, 1999).

There is now clear evidence that agnostic is required during oogenesis. Loss of agnostic function affects the morphology of the follicle cell epithelium and, because follicle cells are missing in late-stage egg chambers, may influence the survival of the follicle cells. In addition, loss of agnostic affects the morphology of the germline-derived cells. It is not known whether agnostic is required in the follicle cells, germline cells, or both. Signaling between these two cell layers occurs throughout oogenesis and models can be hypothesized in which loss of agnostic function in one cell type affects the function and morphology of the other cell type. Nevertheless, agnostic is thought to be involved in cAMP metabolism by coding for a protein that regulates calmodulin activity. Since the requirement for Protein kinase A is restricted to the germline cells, it is tempting to speculate that minimally, agnostic is required in the germline cells (Jackson, 1999).

Irrespective of the cell type in which agnostic is required, both adenylyl cyclase and phosphodiesterase enzymatic activity are altered in agnostic mutants. These results raise the possibility that cut function impinges on the activity of either or both of these enzymes. Interestingly, some dunce alleles are female sterile, revealing a role for dunce during oogenesis. Deficiencies uncovering dunce and rutabaga fail to interact with cut in the genetic screen, however, and no morphological defects are observed in the ovaries of double heterozygotes of cut and specific mutant alleles of dunce or rutabaga. Thus, cut does not appear to interact individually with either dunce or rutabaga in the same dose-sensitive manner as agnostic. There are three other adenylyl cyclase genes identified in Drosophila that may be regulated by agnostic and/or cut. Finally, it is interesting to note that both adenylyl cyclase and phosphodiesterase activities are increased in agnostic mutants, suggesting that the role of this gene in regulating cAMP levels is complex (Jackson, 1999).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

The transcription factors encoded by the genes Kr and cut are required for the development of the Malpighian tubules. Kr is required for the Malpighian tubule expression of many genes including cut, which controls a subset of the genes that are expressed specifically in the Malpighian tubules. Other transcription factors downstream of Kr must control genes that are specifically expressed in the tubules but that do not require ct activity. Genes downstream of these transcriptional regulators encode the proteins that control and execute the morphogenetic events that form the Malpighian tubules. Some genes required for the morphogenesis of the tubules have been identified. The control of cell proliferation in the tubules is one aspect of morphogenesis. In addition, wingless is required to establish the appropriate number of tubules, and the genes rib and raw are required for proper shaping of the tubules. Although the Kr transcription factor activates cut in the Malpighian tubules, it may act through other factors. However, no mutation examined, regardless of the effect on Malpighian tubule development, blocks the expression of Cut protein in the tubules. Furthermore, a survey of deletions that together cover 70% of the genome found no deletion that causes the loss of ct activity in the tubules although one of the deletions blocks morphogenesis completely (Jack, 1999).

The flight muscles of Drosophila derive from myoblasts found on the third instar disc. These myoblasts already show distinctive properties: how this diversity is generated was examined. In the late larva, Vestigial and low levels of Cut are expressed in myoblasts that will contribute to the indirect flight muscles. Other myoblasts, which express high levels of Cut but no Vestigial, are required for the formation of the direct flight muscles. Vestigial and Cut expression are stabilized by a mutually repressive feedback loop. Vestigial expression begins in the embryo in a subset of adult myoblasts, and Wingless signaling is required later to maintain this expression. Thus, myoblasts are divided into identifiable populations, consistent with their allocation to different muscles, and ectodermal signals act to maintain these differences (Sudarsan, 2001).

The indirect flight muscles (IFMs) are divided into two classes on the basis of their location; the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). These, together with the direct flight muscles (DFMs), constitute the dorsal muscles of the adult thorax and derive from myoblasts that lie over the notal region of the developing wing disc epithelium. They have been considered to be a uniform population of cells, capable of contributing to a variety of muscles. Consistent with this view, they uniformly express the transcription factors encoded by twist (twi) and Dmef2 (Sudarsan, 2001).

These myoblasts are segregated into two distinct groups well before the onset of myoblast fusion. In late third instar discs, a large group of proximally situated myoblasts expresses Vestigial (Vg), while a more distal group located around the hinge region does not express this gene. These two groups of myoblasts also differ in their levels of Cut expression; Vg-expressing cells show low levels of Cut while cells not expressing Vg are marked by high levels of Cut (Sudarsan, 2001).

It was asked whether the segregation of these two groups of myoblasts, expressing different combinations of transcription factors, predicts a distinctive contribution to the development of specific adult flight muscles. The development of the IFMs was examined in flies bearing adult viable alleles of vg. In two strong alleles, vg1 and vg83b27-R, the DLMs are severely reduced and the DVMs are minimal or completely missing. To assess the gain-of-function phenotype, a UAS-vg transgene was expressed in all wing disc-associated myoblasts using the Gal4 driver 1151. This leads to alterations in the development of the DFMs, so that muscles 51 and 52 are always missing. In contrast, the IFMs develop normally. Viable hypomorphic allelic combinations of cut show no muscle phenotype and therefore do not allow the examination of effects of loss of function on adult muscle development. To assess the effects of cut gain of function, uniformly high levels were expressed in all the myoblasts, which results in the virtual complete loss of IFMs, whereas the DFMs are unaffected (Sudarsan, 2001).

Thus, while the overexpression of Vg or Cut leads to the reduction or loss of one class of muscle, it is striking that the alternative class is not expanded, as would be expected if ectopically expressing cells switched their fate and executed the newly specified myogenic program. The alternative possibility, that cells forced into a new developmental program die was investigated. Pupal wing discs in which Cut had been overexpressed in all the myoblasts were stained for the general myoblast marker, Twi, and for acridine orange. Twi staining reveals an approximately 10-fold reduction in the number of myoblasts overlying the IFM larval templates, and acridine orange shows a greatly increased incidence of cell death. Driving maximal levels of Cut (at 29°C) results in a dramatic reduction in disc-associated myoblasts even earlier, by the end of the larval period. Together these results indicate that alterations in the pattern of gene expression in myoblasts leads to cell death (Sudarsan, 2001).

The location of the two populations of myoblasts on the notum, together with phenotypes for the gain and loss of function for vg and for cut gain of function, suggests that the IFMs derive from proximal, Vg-expressing, low Cut myoblasts and the DFMs from more distal, Vg-negative, high Cut myoblasts. Since these distinctive patterns of gene expression are observed in the third instar, before myoblast fusion, this hypothesis can be tested by following the fate of Vg-expressing cells through pupal development. Indeed, myoblasts expressing Vg contribute to the developing IFMs. Vg-expressing myoblasts overlie the larval templates on which the DLMs develop and in the muscles below after myoblast fusion. After the larval templates have split, the DFMs have many Vg-expressing nuclei. In contrast, the vast majority of myoblasts that contribute to DFMs do not express Vg, either during fusion or later (Sudarsan, 2001).

Together these results show that the third instar myoblasts are partitioned into two populations from which cells contribute to the IFMs or DFMs and that the larval patterns of vg and cut expression play an important role in specifying these populations (Sudarsan, 2001).

Although embryonic myoblasts are allotted to sets that express Vg and those that do not, the other transcription factor, Cut, whose level of expression also distinguishes myoblast groups, is initiated only later, in mid third instar larvae. During the third instar, all adult myoblasts express Cut, but they are in two distinct groups: a distal group that expresses high levels of Cut and a proximal group, also distinguished by the expression of Vg, that expresses Cut weakly (Sudarsan, 2001).

In order to compare the levels of Cut in wild-type and genetically manipulated cells within single populations of myoblasts, mitotic clones were generated of myoblasts carrying a mutation in dishevelled (dsh) and were therefore unable to transduce Wg signaling. As expected, dsh clones lose Vg expression, but they also show an increase in the level of Cut expression relative to neighboring wild-type myoblasts. This indicates that, in addition to its role in maintaining Vg expression, Wg signaling is also required, directly or indirectly, to modulate myoblast expression of Cut. Interestingly, changes in gene expression are confined to the mutant cells, indicating a cell-autonomous response; the induction of secondary signaling by Wg-activated cells is not involved (Sudarsan, 2001).

Since high levels of Cut are found only in the absence of Vg and Vg-expressing cells that show depressed levels of Cut, the relationship between Vg and Cut expression was examined by inducing overexpression of each gene in all the notum myoblasts. A general expression of Vg results in uniformly low levels of Cut expression, while the induction of uniformly high Cut virtually abolishes Vg expression. These results reveal a negative regulatory loop between these two gene products, which from the third instar will act to maintain the distinction between the two groups of notum myoblasts, namely, those expressing Vg, that will contribute to the IFMs, and those expressing Cut at high levels that contribute to the DFMs (Sudarsan, 2001).

A P-element line (P0997) of Drosophila in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut (ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway (Thumm, 2001).

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. It is proposed that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures (Emerald, 2003).

The relationship between ss and Cut was examined. Ectopic expression of ss using ptcGal4 causes ectopic expression of Dan and repression of Cut expression. Repression of Cut is stronger on one side of the disc and is associated with antenna duplication. Ectopic expression of Dan or Danr has no effect on Cut expression, suggesting that ss may act directly to regulate Cut. The ability of ss to repress Cut, contrasts with the observation that cut expression is normal in ss mutants. It is possible that there are redundant mechanisms for Cut repression, one of which is mediated by ss (Emerald, 2003).

To ask whether Cut regulates Dan and Danr, an examination was made of expression clones of cells lacking cut activity, generated using the null allele cut145 and the FLP-FRT system. cut145 mutant clones do not cause ectopic expression of Dan in proximal regions of the antenna disc, but rather show limited expansion of the Dan domain in the region where Dll is expressed. Comparable effects on Danr expression were seen in cut mutant clones. Ectopic expression of Cut in the Dan domain using Act>CD2>Gal4 causes repression of Dan (Emerald, 2003).

Taken together these results suggest that distal expression of ss limits the expression domain of Cut to the proximal antenna. ss is required for Dan and Danr expression in distal antenna. At present it is not possible to determine whether expression of Dan and Danr in response to ss is mediated directly or indirectly by repression of Cut. However, the view that it is direct is favored because removal of Cut does not cause extensive ectopic expression of Dan (Emerald, 2003).

Different levels of Cut regulate distinct dendrite branching patterns of multidendritic neurons

Functionally similar neurons can share common dendrite morphology, but how different neurons are directed into similar forms is not understood. In embryonic and larval development the level of Cut immunoreactivity in individual dendritic arborization (da) sensory neurons is shown to correlate with distinct patterns of terminal dendrites: high Cut in neurons with extensive unbranched terminal protrusions (dendritic spikes), medium levels in neurons with expansive and complex arbors, and low or nondetectable Cut in neurons with simple dendrites. Loss of Cut reduces dendrite growth and class-specific terminal branching, whereas overexpression of Cut or a mammalian homolog in lower-level neurons results in transformations toward the branch morphology of high-Cut neurons. Thus, different levels of a homeoprotein can regulate distinct patterns of dendrite branching (Grueber, 2003).

Drosophila sensory neurons can be divided into three major types: external sensory (es), chordotonal (ch) (both with single dendrites), and multidendritic (md) neurons. The md neurons are further subdivided into the dendritic arborization (da) neurons, the tracheal dendrite (td) neurons, and the bipolar dendrite (bd) neurons. The da neurons differ from these other types by having extensively branched dendrites that innervate the epidermis. All da neurons are born by 9 hrs after egg laying (AEL and soon thereafter begin to project axons toward the ventral nerve cord. Peripheral dendritogenesis is initiated several hours later, at around 13 hr AEL. The extension and branching of these nascent dendrites can be followed in living embryos using the Gal4-UAS binary expression system to express GFP (and variants such as mCD8::GFP) in all or specific subsets of da neurons. In this study, either elav-Gal4 or Gal4109(2)80 was used to label all da neurons, and Gal4221 and Gal4477, which were identified in an enhancer trap screen was used to label subsets of da neurons beginning late in embryogenesis (Grueber, 2003).

A characterization of the peripheral dendrite morphologies of larval da neurons using the MARCM system (for mosaic analysis with a repressible cell marker) has prompted a segregation of the 15 neurons in each abdominal hemisegment into four morphological classes (classes I-IV). To characterize the proneural gene requirements of these neurons, the scB57 deficiency was introduced into a Gal4109(2)80 background. Loss of proneural genes in the achaete-scute complex (ASC) eliminates class II, III, and IV da neurons, and at least one of the dorsal class I da neurons (likely ddaD). The atonal-dependent vpda neuron is unaffected. Remaining in the dorsal cluster were the amos-dependent dorsal bipolar dendrite neuron, an as yet unidentified dorsal neuron, and an amos-dependent dorsal neuron (henceforth referred to as dmd1) that sends its dendrites to an internal nerve (likely the 'td' neuron). These results indicate that ASC-dependent da neurons are distributed in all four morphological classes (Grueber, 2003).

The selector gene cut acts in ASC-dependent lineages to specify cell identity and is expressed at different levels in various embryonic da neurons. cut expression was examined in embryonic stages to determine the relationship, if any, between these levels and the morphological categories proposed for the da neurons. Cut antibodies directed against a C-terminal protein were used to stain embryos of the enhancer line E7-2-36 (which bestows all md neurons with lacZ expression). During embryonic stages, most da neurons express Cut but at different levels. These levels are serially repeated in each abdominal segment (thoracic segments were not examined) and show a close correlation with morphological class. Cut immunoreactivity is high in class III neurons, which show many short dendritic spikes; medium in the class IV neurons, which have expansive and highly complex arbors, and in the sparsely branching class II neurons; and below detection in the simple class I neurons (Grueber, 2003).

Cut is expressed in sense organ precursors and their progeny, consistent with a role in identity specification. However, an intriguing aspect of cut expression is that it persists in postmitotic cells. The relative levels of Cut expression seen in larval neurons are the same as in embryos, although at these later stages, staining in class IV neurons appeared stronger than in the class II neurons. Thus, neurons showing the highest Cut levels throughout embryonic and larval development match those that exhibit short dendritic spikes along their main trunks, while relatively lower levels were associated with class IV and class II morphologies. These results suggest that Cut might be a good candidate for a regulator of class-specific properties of dendrites (Grueber, 2003).

The highest levels of Cut immunoreactivity are consistently shown by neurons sharing a common morphological property, namely short spikes extending from main dendritic trunks. Other classes of neurons showed either medium to low levels of Cut (class IV and class II, respectively) or levels that were below the limit of detection (class I). These conclusions regarding Cut levels are based on multiple criteria, including matching results with antibodies directed against either N-terminal or C-terminal regions of Cut, a reproducible staining pattern in multiple body segments, and consistent levels of intensity within independently-derived morphological classes of neurons. Thus, although levels of immunoreactivity might not provide a linear readout of protein levels and do not necessarily reflect differences in activity, these considerations support the notion that different levels of Cut might be important for establishing morphological distinctions between da neurons (Grueber, 2003).

Given the expression patterns of Cut described above, it was hypothesized that high Cut directs the production of more complex arbors, perhaps specifically the dendritic spikes shown by the class III neurons, and that low levels lead to simpler dendrites. Results from loss-of-function and gain-of-function manipulations provide strong support for this scenario and suggest that different levels of Cut direct different patterns of dendrite morphogenesis. First, in cut- clones, class III neurons show severe reductions in their class-specific spikes and resemble simpler skeletons of their normal arbor. Conversely, ectopic expression of Cut in nonexpressing neurons and in lower-level neurons initiates the branching of numerous spiked structures from their main dendritic trunks, causing these dendrites to take the appearance of class III arbors. Thus, these classes of neurons are capable of extending class III-like spiked dendrites, but for the class II and class IV neurons, the levels of Cut that direct their proper morphogenesis apparently fall below a required threshold. Considering that Cut is also required for proper dendritogenesis in class IV and class II neurons, it might regulate class-specific traits not only in class III neurons, but also in other neurons when expressed at lower levels (Grueber, 2003).

These results raise potential parallels for Cut action in es organs and da neurons. Merritt (1997) suggested that several distinctions between es and ch sensory organs could result from changes to the program specifying dendritic growth, in particular an enhanced growth of the outer dendritic segment of Cut-positive es neurons relative to Cut-negative ch neurons. Differential growth of these dendrites would likely, in turn, influence the shape and position of support cells and cuticular structures (Merritt, 1997). In the da neurons, several of the morphological differences between neurons likely entail changes in the relative extent of dendrite elongation and branching and/or branch stabilization-programs that, according to gain-of-function and loss-of-function results, appear to be regulated by Cut. Thus, cut may provide a promising nodal point for examining how diverse dendrite morphologies are established among different groups of Drosophila sensory neurons (Grueber, 2003).

Does Cut specify cell identity among the da neurons or, rather, selectively control dendrite morphogenesis? Homeodomain transcription factors, including Cut, have well-established roles in cell fate specification. However, transcription factors expressed at later stages of differentiation can selectively control fine aspects of morphogenesis. For example, in Drosophila photoreceptor formation, the spalt gene complex controls rhabdomere morphology and opsin gene expression, but not axon projection pattern. Conversely, the runt transcription factor controls a subset of axon projection patterns without altering the expression of numerous markers of cell identity. The expression of Cut in both sensory precursors and differentiated neurons, together with studies of cut function in the PNS, suggest that it might control both the identity and later development of da neurons. If Cut does generally specify da neuron fate, then the ability to transform dendrite morphology by postmitotic expression of Cut argues that this aspect of identity is not established in an all-or-nothing fashion at the time of neuronal birth (Grueber, 2003).

Because the Cut protein contains several DNA binding motifs (including a homeodomain and three Cut repeats and is localized to the nucleus, it likely controls dendrite morphology through transcriptional regulation. Such a mechanism of action is supported by the ability of mammalian cut homolog with conserved DNA binding domains to partially rescue Cut loss-of-function and induce growth of da neuron dendritic arbors. Although level-dependent regulation of dendrite morphology by homeoproteins has not previously been demonstrated, level-dependent control of cell identity by homeoproteins has been shown in both nonneuronal and neuronal systems. Bicoid, for example, is distributed in a morphogen gradient along the anterior-posterior axis of the syncytial Drosophila embryo and can exert threshold-dependent effects on gene expression by cooperative DNA binding, which may be an important mechanism of target gene activation as the embryonic body pattern is organized. The identities of transcriptional targets of Cut in the embryonic and larval PNS are not yet known. Based on the present results, Cut could control the expression of key regulators of dendrite branching (Grueber, 2003).

cut homologs have been identified in several vertebrates, including human, dog, chick, and mouse. The ability of a mammalian cut homolog to regulate dendrite development in Drosophila raises the possibility of conserved roles in regulating neuronal morphogenesis. Indeed, of the two murine cut homologs, Cux-1 is expressed in many adult tissues including the nervous system, while Cux-2 is expressed solely in the developing and adult nervous system. In the adult, strong expression is observed in the thalamus, pyramidal neurons of the hippocampus, the granule cell layer of the dentate gyrus, and amygdala, while lower levels are seen in the cerebral cortex. Based on such widespread distribution in the mammalian central and peripheral nervous system, it will be interesting to examine potential roles for Cux genes in cell identity specification and neuronal morphogenesis in vertebrates. Perhaps further studies of how Cut acts to regulate dendrite morphology in Drosophila will prove useful for dissecting this potentially important mechanism by which homeoproteins contribute to cellular diversity (Grueber, 2003).

The homeobox transcription factor cut coordinates patterning and growth during Drosophila airway remodeling

A fundamental question in developmental biology is how tissue growth and patterning are coordinately regulated to generate complex organs with characteristic shapes and sizes. This study shows that in the developing primordium that produces the Drosophila adult trachea, the homeobox transcription factor Cut regulates both growth and patterning, and its effects depend on its abundance. Quantification of the abundance of Cut in the developing airway progenitors during late larval stage 3 revealed that the cells of the developing trachea had different amounts of Cut, with the most proliferative region having an intermediate amount of Cut and the region lacking Cut exhibiting differentiation. By manipulating Cut abundance, it was shown that Cut functioned in different regions to regulate proliferation or patterning. Transcriptional profiling of progenitor populations with different amounts of Cut revealed the Wingless (known as Wnt in vertebrates) and Notch signaling pathways as positive and negative regulators of cut expression, respectively. Furthermore, the gene encoding the receptor Breathless (Btl, known as fibroblast growth factor receptor in vertebrates) was identified as a transcriptional target of Cut. Cut inhibited btl expression and tracheal differentiation to maintain the developing airway cells in a progenitor state. Thus, Cut functions in the integration of patterning and growth in a developing epithelial tissue (Pitsouli, 2013).


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cut: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation

date revised: 15 July 2015

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