InteractiveFly: GeneBrief

turtle: Biological Overview | References

BIOLOGICAL OVERVIEW

The dendritic trees of neurons result from specific patterns of growth and branching, and dendrite branches of the same neuron avoid one another to spread over a particular receptive field. Recognition molecules on the surfaces of dendrites influence these patterning and avoidance processes by promoting attractive, repulsive or adhesive responses to specific cues. The Drosophila transmembrane protein Turtle (Tutl) and its orthologs in other species are conserved members of the immunoglobulin superfamily, the in vivo functions of which are unknown. In Drosophila sensory neurons, tutl is required to restrain dendrite branch formation in neurons with simple arbors, and to promote dendrite self-avoidance in neurons with complex arbors. The cytoplasmic tail of Tutl is dispensable for control of dendrite branching, suggesting that Tutl acts as a ligand or co-receptor for an unidentified recognition molecule to influence the architecture of dendrites and their coverage of receptive territories (Long, 2009).

Developing neurons form dendritic trees with cell type-specific patterns of arborization, ranging from simple arbors with few branches to highly elaborate arbors that cover receptive territories with many branches. In neurons with even the most complex trees, dendrite branches growing from the same neuron (isoneuronal branches) avoid one another as they spread over a territory to receive sensory or synaptic inputs. Together, dendrite branching and self-avoidance are crucial for sculpting the particular architecture of a neuron's receptive field. Both processes are thought to be controlled by molecular recognition events that occur between isoneuronal branches, or between dendrites and the substrata along which they grow. However, few of the molecules participating in these recognition events have been described (Long, 2009).

Cell surface recognition molecules that promote dendrite growth and/or branching include cadherins, as well as those mediating responses to neurotrophins, B-type ephrins, and cues that direct dendritic guidance, such as Semaphorins and Netrins. In contrast to these examples, which promote or guide dendrite arborization, recognition molecules that prevent inappropriate or excessive dendrite branching in neurons with simple arbors remain unidentified (Long, 2009).

Recognition mechanisms underlying dendrite self-avoidance have only recently emerged, with findings that the Dscam family of immunoglobulin superfamily (IgSF) proteins promote self-avoidance in Drosophila and mice. It remains to be determined whether other families of cell surface proteins are also required to promote self-avoidance (Long, 2009).

The identification of novel transmembrane proteins required for dendrite branching and self-avoidance is a key step in understanding molecular mechanisms that underlie dendrite patterning. The Drosophila protein Turtle (Tutl) and its mammalian orthologs, Dasm1 (Igsf9) in mice and IGSF9 (KIAA1355) in humans (Doudney, 2002; Shi, 2004b), are type 1 transmembrane proteins with an ectodomain comprising five immunoglobulin (Ig)-like domains and two fibronectin type III repeats. In Drosophila, mutations of the tutl gene impair responses to tactile stimuli and the execution of complex coordinated behaviors (Bodily, 2001), but the causes of these nervous system deficits are unknown. To date, no morphological defects have been reported for tutl mutants, despite the structural similarity of Tutl to the Neogenin, Deleted in Colorectal Carcinoma, Frazzled and Roundabout families of axon guidance receptors (Long, 2009).

In mice, the Tutl ortholog Dasm1 is selectively expressed in the developing hippocampus (Mishra, 2008; Shi, 2004b). Dasm1 knockout mice have no observable defects in dendrite morphogenesis in the developing hippocampus, nor have defects of neuronal differentiation, synaptogenesis or behavior been seen in these mice (Mishra, 2008). Therefore, the role Dasm1 in the mammalian nervous system remains uncertain, and genetic approaches to study Dasm1 function in mice could be complicated by redundancy of Dasm1 with Igsf9b, a closely related protein (Long, 2009).

This study has used genetic approaches to study the effects of tutl mutations on dendritic arborization (da) neurons in the Drosophila peripheral nervous system. Tutl was found to be expressed on dendrites of da neurons and, through loss-of-function and gain-of-function experiments in vivo, it was demonstrate that Tutl cell-autonomously controls dendrite branching and self-avoidance. Tutl restricts branching in neurons with simple arbors and promotes self-avoidance in neurons with highly complex arbors. These results demonstrate that a member of the Tutl/Dasm1/IGSF9 family of proteins can influence dendrite morphogenesis in vivo, and that neurons of different classes employ Tutl as a common molecular component of mechanisms that sculpt dendrite arborization patterns of dramatically different complexity (Long, 2009).

Dendrite branching and self-avoidance are two important cellular mechanisms that shape the receptive fields of neurons during development. This study investigated the role of Tutl in these processes using the da sensory neurons of Drosophila, an excellent system in which to study dendrite arborization at a single cell level in vivo. Tutl is a member of the Tutl/Dasm1/IGSF9 family of evolutionarily conserved transmembrane proteins. Tutl inhibits excessive branch formation in neurons with simple dendrites (class I), and contributes to the processes that prevent crossing of isoneuronal dendrite branches in neurons with complex arbors (class IV), which demonstrates that Tutl influences the architecture of dendrites and their coverage of receptive territories. In contrast to these results for class I and class IV neurons, MARCM studies found no evidence of a cell-autonomous role for Tutl in class II or class III neurons, despite detectable Tutl expression in their cell bodies. The reasons for a lack of apparent effects on class II or class III da neuron dendrites in tutl MARCM clones remain unclear. Sufficient Tutl protein, inherited from precursors, could have remained in MARCM clones to promote normal outgrowth. Alternatively, there might be no role for Tutl in these cells. Nevertheless, it is clear from these results for class I and class IV da neurons that Tutl is required for the arborization of dendritic trees with dramatically different complexity (Long, 2009).

Tutl cell-autonomously inhibits dendrite branching in vivo, providing a means by which da neurons with the simplest architecture suppress the formation or stabilization of supernumerary dendrite branches during development. A clear increase was observed in the number of second and third order branch points on tutl mutant ddaE neurons. This finding suggests that tutl regulates branching only at certain locations along the growing arbor, perhaps by inhibiting branch additions or promoting branch retractions (Long, 2009).

The tutl phenotype is distinct from that of mutants of Neuroglian (Nrg), which also encodes a cell surface IgSF protein that affects dendrite branching. Loss of Nrg reduces the number of branches on the dendritic arbors of class I da neurons, and increases branching along their axons, suggesting a role for Nrg in correctly distributing neurites but not as a branching inhibitor. The tutl mutant phenotype is also distinct from the dendrite overgrowth phenotype observed in mutants of the IgSF receptor Robo, or of the cadherin Flamingo (also known as starry night. In vertebrate systems, no recognition molecules have yet been shown to inhibit dendrite branching in vivo. However, it is noteworthy that inhibition of axon branching has been demonstrated in the chick visual system, where inappropriate arborization of retinal ganglion cell (RGC) axon terminals is thought to be inhibited by EphA and Ryk receptors. In zebrafish, RGC axons are inhibited from branching by Robo2, an IgSF protein with which Tutl shares homology (Long, 2009).

After Dscam, Tutl is the only cell surface protein that has been shown to be required for dendrite self-avoidance in either invertebrates or vertebrates. As in Dscam mutants, the dendrites of tutl mutant neurons cross one another with increased frequency, leading to uneven coverage of the receptive field. Unlike Dscam, which promotes self-avoidance in all four da neuron classes, Tutl does so only in the highly complex arbors of class IV neurons. No genetic interactions were observed between Dscam and tutl mutations, and no evidence has been found that Dscam and Tutl could act in a common molecular pathway to control dendrite self-avoidance. Future studies could reveal whether and how these seemingly distinct pathways converge but, based on these findings, it is speculated that the molecular mechanisms ensuring dendrite self-avoidance will prove to be more complex than is appreciated currently (Long, 2009).

Neither Tutl nor Dscam affect dendritic tiling among neurons of a similar functional type, illustrating that self-avoidance and tiling are likely to be mediated by distinct recognition molecules on the surfaces of dendrites (Long, 2009).

The full-length form of Tutl is a transmembrane protein with a five Ig/two FnIII ectodomain and a cytoplasmic tail, which suggests that it could act as a signaling receptor. Alternative splicing also gives rise to a membrane-tethered form that lacks the cytoplasmic tail (Bodily, 2001). This suggests that Tutl could also function as a membrane-bound ligand for an unknown receptor or, alternatively, as a co-receptor in a multiprotein receptor complex. These possibilities are not mutually exclusive, because Tutl could conceivably act as a ligand or a co-receptor in one cellular context, and as a signaling receptor in another. The cytoplasmic tail is completely dispensable for the inhibition of dendrite branching in class I da neurons. This is consistent with a model in which Tutl acts as a ligand or a co-receptor in dendrites. By contrast, it was found that the cytoplasmic tail was required to fully rescue viability in tutl mutants, suggesting that Tutl acts as a signaling receptor in this context (Long, 2009).

It is currently unclear how Tutl controls dendrite branching and self-avoidance because these studies have not revealed a connection between Tutl and known regulators of dendrite morphogenesis such as Trc. No evidence was found for genetic interactions between trc and tutl. These results alone cannot exclude the possibility that Trc and Tutl act in a common pathway to govern dendrite branching or self-avoidance, but it is noteworthy that the phenotypes of trc and tutl mutants also show some differences that could suggest they work through independent molecular pathways. Unlike tutl, trc is required for dendritic tiling among different class IV neurons, and tutl mutants do not display the excessive terminal branching in class IV neurons that is characteristic of trc mutants (Long, 2009).

The transcription factors Abrupt, Spineless, Knot and Cut each regulate patterns of dendrite branching in keeping with tutl mutations or Tutl overexpression. However, in immunohistochemical studies of loss-of-function mutants for these transcription factors, found Tutl expression to be more likely to be influenced by a regulatory program that is distinct from those involving Abrupt, Spineless, Knot or Cut (Long, 2009).

Tutl remains somewhat enigmatic because as yet no genetic or regulatory connection has been found between tutl and genes with similar mutant phenotypes. Nevertheless, the discovery that Tutl regulates dendrite morphogenesis and the coverage of receptive territories underscores the fact that the molecular mechanisms that underlie dendrite morphogenesis remain incompletely understood. Why tutl mutants have class-specific effects on dendrite morphogenesis, despite Tutl expression in all da neuron classes, is unknown. Perhaps an unidentified Tutl-interacting protein, such as a receptor required for Tutl function, might be differentially expressed among da neurons and could thus account for the specificity of the phenotype. Other explanations may also exist. For example, it is possible that MARCM experiments failed to show cell-autonomous defects in certain da neuron classes (classes II and III) because the requirement for Tutl in these cells was met by perdurance of sufficient Tutl protein inherited from the precursor cells of MARCM clones. Alternatively, Tutl in class II and class III da neurons might function non-cell autonomously to influence neighboring cell types (Long, 2009).

It is intriguing that the two processes of branching and self-avoidance are related by a common requirement for tutl. Both phenotypes are consistent with the idea that Tutl promotes repulsion, perhaps between isoneuronal dendrite branches, or between dendrites and the substrata along which they grow. However, there is no direct evidence at this time for a repulsive role for Tutl. Simultaneous overexpression of Tutl in different da neuron classes was insufficient to induce branch repulsion among their dendrites. Together with rescue experiments showing the dispensability of the cytoplasmic tail for dendrite branching, these data suggest that Tutl could function as a ligand or a co-receptor in complexes with one or more unidentified proteins at the cell surface. Such proteins might not be expressed in all da neurons, which could explain why tutl mutations do not affect da neuron classes II and III, and why Tutl cannot induce repulsion when overexpressed in overlapping neurons of Classes I-III. The tutl mutant phenotypes remain the strongest evidence of a repulsive role for Tutl, and it is likely that direct evidence for repulsion must await the identification of the relevant Tutl-interacting proteins (Long, 2009).

If it is true that Tutl mediates repulsion, it is speculated that the nature or degree of that repulsion could be influenced by the size of the dendritic arbor, leading to class-specific effects. Class I dendrites remain relatively small with Tutl protein distributed along the entire arbor, where Tutl-mediated repulsion could promote the collapse of transient interstitial branches that are known to extend during development (branch inhibition). In large class IV arbors where Tutl is distributed more sparingly, Tutl-mediated repulsion could be one part of a multi-component system to redirect isoneuronal branches away from one another (self-avoidance) and thereby ensure proper distribution of dendrites over receptive territories. In this way, neurons of different classes could employ a common repulsive mechanism involving Tutl to sculpt arborization patterns of dramatically different complexity (Long, 2009).

The findings that Tutl inhibits dendrite branching in Drosophila contrast with initial observations in cultured rodent neurons, in which RNAi-knockdown experiments suggested that the Tutl ortholog Dasm1 was required to promote dendritic outgrowth (Shi, 2004b). However, it was recently argued that these RNAi findings were due to off-target effects (Mishra, 2008). The role of Dasm1 in mammalian dendrite morphogenesis is currently unclear, since Dasm1 knockout mice have no observable dendritic defects (Mishra, 2008). However, the possibility has been raised that Dasm1 function in dendrites is redundant with the function of Igsf9b, a closely related protein that is coexpressed in the developing hippocampus, the expression of which is unaltered in the brains of Dasm1 knockout mice (Mishra, 2008). Loss-of function studies for both Dasm1 and Igsf9b should reveal whether Tutl-like proteins in mammals share with Tutl an evolutionarily conserved role in dendrite morphogenesis (Long, 2009).

The conserved Ig superfamily member Turtle mediates axonal tiling in Drosophila

Restriction of adjacent same-type axons/dendrites to separate single columns for specific neuronal connections is commonly observed in vertebrates and invertebrates, and is necessary for proper processing of sensory information. Columnar restriction is conceptually similar to tiling, a phenomenon referring to the avoidance of neurites from adjacent same-type neurons. The molecular mechanism underlying the establishment of columnar restriction or axonal/dendritic tiling remains largely undefined. This study has identified Turtle (Tutl), a member of the conserved Tutl/Dasm1/IgSF9 subfamily of the Ig superfamily, as a key player in regulating the tiling pattern of R7 photoreceptor terminals in Drosophila. Tutl functions to prevent fusion between two adjacent R7 terminals, and acts in parallel to the Activin pathway. Tutl mediates homophilic cell-cell interactions. It is proposed that extrinsic terminal-terminal recognition mediated by Tutl, acts in concert with intrinsic Activin-dependent control of terminal growth, to restrict the connection made by each R7 axon to a single column (Ferguson, 2009).

The results indicate an essential and specific role for Tutl in the establishment of column-specific R7 connections in the Drosophila visual system. Loss of Tutl causes a failure of R7 terminals to separate from each other leading to R7 tiling defects. Tutl is required in R7 axons but not in medulla target neurons for the restriction of R7 connections to single columns. Genetic interaction and ablation analyses indicate that Tutl functions in parallel to the Activin pathway to control R7 tiling. That Tutl functions in both cell-autonomous and cell-non-autonomous manners to prevent the fusion of adjacent R7 terminals, together with that Tutl mediates homophilic cell-cell interactions in transfected S2 cells, suggest a model in which the Tutl-mediated terminal-terminal recognition functions together with the Activin-mediated intrinsic growth control to restrict the connection of each R7 terminal to a single column (Ferguson, 2009).

Restriction of R7 connections to single columns requires both repulsion between R7 terminals and the control of intrinsic R7 terminal growth. A recent study by Ting (2007) provides convincing evidence that the restriction of intrinsic R7 terminal growth is mediated by the Activin pathway, which functions together with an unknown parallel mechanism that mediates mutual repulsion between adjacent R7 terminals. The requirement of mutual repulsion in R7 columnar restriction is not surprising. R7 terminals show extensive contacts with each other before segregation into separate columns. Given the expression of a number of cell adhesion molecules such as N-Cadherin and Chaoptin on R7 terminals throughout development, it seems clear that a repulsive force or an anti-adhesion mechanism is necessary to overcome the adhesion and thus facilitate the segregation of adjacent R7 terminals. Mutual repulsion has been suggested to be required for columnar restriction of L1 neurons in the Drosophila visual system, which is mediated by Dscam2 (Millard, 2007). Up to now, Dscam2 and its mouse homolog Dscam are only known cell surface transmembrane proteins to be implicated in mediating mutual repulsion for axonal/dendrite tiling. In addition, Dscam and the receptor tyrosine phosphatase HmLAR2 have been shown to be involved in mediating self-avoidance, a phenomenon referring to the avoidance of sister neurites projected from a single neuron, in sensory neurons in Drosophila and the comb cell in Leech, respectively (Ferguson, 2009 and references therein).

That Dscam2 is not required for R7 tiling (Millard, 2007) indicates the involvement of other cell surface proteins in mediating the repulsive interaction between adjacent R7 terminals. The results suggest that Tutl is an essential component of this repulsive pathway that functions in parallel to the Activin pathway to restrict the connections of R7 terminals to single columns. First, in tutl mutants, fusion between neighboring R7 terminals were frequently observed at the R7 recipient layer in the medulla, consistent with a role for Tutl to antagonize the adhesion between adjacent R7 terminals. Second, unlike that the Activin pathway functions cell-autonomously in restricting intrinsic R7 terminal growth, Tutl is required both cell-autonomously and cell-non-autonomously for R7 tiling. In tutl mosaics, loss of tutl in a single R7 terminal could lead to the invasion of a wild-type column by a neighboring mutant R7 terminal, the invasion of a mutant column by a neighboring wild-type R7 terminal, or the fusion of adjacent mutant and wild-type R7 terminals at intercolumnar space. Third, reducing the level of tutl or importin-α3 significantly increased the frequency of tiling defect in importin-α3 or tutl mutants, respectively. And fourth, ablation of neighboring R7 terminals decreased the frequency of tutl tiling phenotype, which is in marked contrast to the enhancement of Activin tiling phenotype with genetic ablation of neighboring R7 terminals. Those results are consistent with that Tutl mediates the recognition between neighboring R7 terminals to restrict their connections to a single column (Ferguson, 2009).

There are several possible mechanisms for the action of Tutl in preventing fusion between neighboring R7 terminals. For instance, like the action of Semaphorin-1a and Plexin-A in motor axon de-fasciculation, Tutl may directly trigger a repulsive response between neighboring R7 terminals, thus forcing the separation of R7 terminals. Alternatively, Tutl may act to interfere with the function of certain cell adhesion molecules, thus downregulating the attraction between neighboring R7 terminals. The current data does not allow distinguishing among these possibilities (Ferguson, 2009).

The results reveal cellular action of the novel and conserved Tutl/Dasm1/IgSF9 family in the developing nervous system. While tutl was previously reported to be required for larval coordinated movement and adult flight behaviors (Bodily, 2001), neural basis of those behavioral phenotypes remains unclear. That tutl mutants do not show detectable morphological defects in the ventral nerve cod and neuromuscular junctions led to the speculation that Tutl may be required for the connectivity of a very small subset of neurons (Bodily, 2001). Consistent with this view, the results show that tutl functions on a fine scale to restrict the connections of R7 terminals to single columns, but it is not required for the targeting of R7 axons from the retina into the R7 recipient layer nor involved in R8 columnar restriction in the medulla. It is proposed that Tutl functions to prevent fusion between adjacent R7 terminals by mediating mutual repulsion or interfering with adhesion. Similar Tutl-mediated cell surface recognition may also be used by other neurons to refine neuronal circuits necessary for controlling larval and adult coordinated movements (Ferguson, 2009).

The in vivo function of Dasm1 and IgSF9, the mouse and human homologs of Tutl, remains unknown. Previous in vitro studies show that Dasm1-RNAi knockdown impaired dendrite growth and synaptic maturation in cultured hippocampal neurons (Shi, 2004a; Shi, 2004b). However, a recent study shows that Dasm1 null mutants displayed normal dendritic arborization pattern in vivo and in vitro (Mishra, 2008). One possible explanation for normal dendritic growth in Dasm1 null mice is that Dasm1 may be functionally redundant with its close homolog IgSF9b in regulating dendritic patterning in the developing hippocampus. Alternatively or additionally, Dasm1 may function in other brain regions to regulate different developmental events. Thus, it will be of interest to determine whether Dasm1 and IgSF9 also play a similar role in specifying columnar restriction and/or axonal/dendritic tiling in the mammalian nervous system (Ferguson, 2009).

The Drosophila immunoglobulin gene turtle encodes guidance molecules involved in axon pathfinding

Neuronal growth cones follow specific pathways over long distances in order to reach their appropriate targets. Research over the past 15 years has yielded a large body of information concerning the molecules that regulate this process. Some of these molecules, such as the evolutionarily conserved netrin and slit proteins, are expressed in the embryonic midline, an area of extreme importance for early axon pathfinding decisions. A general model has emerged in which netrin attracts commissural axons towards the midline while slit forces them out. However, a large number of commissural axons successfully cross the midline even in the complete absence of netrin signaling, indicating the presence of a yet unidentified midline attractant. The evolutionarily conserved Ig proteins encoded by the turtle/Dasm1 genes are found in Drosophila, C. elegans, and mammals. In Drosophila the turtle gene encodes five proteins, two of which are diffusible, that are expressed in many areas, including the vicinity of the midline. Using both molecular null alleles and transgenic expression of the different isoforms, this study shows that the turtle encoded proteins function as non-cell autonomous axonal attractants that promote midline crossing via a netrin-independent mechanism. turtle mutants also have either stalled or missing axon projections, while overexpression of the different turtle isoforms produces invasive neurons and branching axons that do not respect the histological divisions of the nervous system. These findings indicate that the Turtle proteins function as axon guidance cues that promote midline attraction, axon branching, and axonal invasiveness. The latter two capabilities are required by migrating axons to explore densely packed targets (Al-Anzi, 2009).

The conclusion that the turtle gene encodes a midline attractant is based on the observation that turtle null mutants have a reduction in the number of midline-crossing commissures, while overexpression of diffusible turtle isoforms causes axons that normally do not cross the midline to do so. This conclusion is further supported by the initial expression of turtle isoforms close to the midline during embryonic stages where axons initiate their midline-crossing behavior (Al-Anzi, 2009).

Genetic interactions indicate that turtle attracts commissural axons via a netrin-independent mechanism, and does not stimulate midline attraction by direct inhibition of the slit midline repellent signaling pathway. However, a reduction in the level of abl, a known component of both netrin and slit pathways, does enhance turtle midline defects (Al-Anzi, 2009).

Although most of the data suggest that the Turtle proteins promote axonal midline crossing, in turtle null mutants some FasII-positive tracks do abnormally cross the midline, suggesting that the Turtle signal prevents, rather than encourages, midline crossing in some axons. It is possible, therefore, that Turtle produces different effects on different axons. Indeed, many guidance cues, such as netrin, have opposite effects on different axons, depending largely, though not entirely, upon which type of netrin receptor complex the axons express. However, even mutations in genes that function chiefly to increase midline crossing, such as abl, are known to produce FasII-positive tracks that cross the midline abnormally. Furthermore, the manner in which FasII-positive tracks in turtle null mutants cross the midline is clearly distinct from the midline crossing defects seen in mutations or transgenic manipulations that are known to reduce midline repulsion (Al-Anzi, 2009).

These results also indicate that the turtle gene encodes signaling molecules that promote axon and neuronal cell body invasiveness and axonal branching, and that these molecules are capable of performing those functions via a non-cell autonomous mechanism. Axon branching and invasiveness are both necessary behaviors for growing axons to explore and choose between closely packed targets, and the loss of these activities could explain the inability of turtle mutant axons to make their final connections. Indeed, turtle has been re-isolated in a gain-of-function screen by the Zinn group at Caltech for factors that promote motor axon branching in Drosophila (Kurusu, 2008). It is also worth noting that human Dasm1 is frequently overexpressed in tumors (Doudney, 2002). That turtle can cause both axons and cell bodies to enter abnormal locations suggests that turtle family members may stimulate the loss of tissue organization common in tumors (Al-Anzi, 2009).

In all tissues in which turtle function was examined, the diffusible isoforms were found to be more potent both in rescuing the mutant phenotype and in producing a gain-of-function phenotype when ectopically expressed. This point is further illustrated by the fact that the embryonic lethality in tutl^ex383 mutants, which carry a deletion that affects all isoforms, can be rescued to the level of adult viability when only the diffusible isoforms are expressed. In contrast, the membrane isoforms are only capable of reducing the severity of some aspects of the tutl^ex383 phenotypes. The importance of these diffusible isoforms is further supported by the high degree of homology in the extracellular domains, but not the cytoplasmic domains, of Turtle/Dasm1 family members. However, it is possible that this increase in potency is due to protein domains shared between those two isoforms that are unique to them (Al-Anzi, 2009).

The turtle/Dasm1 mammalian homologue is expressed in a pattern that is suggestive of a potential role in axon pathfinding (Doudney, 2002). However, to date, the role of mammalian and C. elegans homologues in axon pathfinding has not been examined, nor has their role in vertebrate tumorigenesis been much explored. It is hoped that these results will draw attention to these novel signaling molecules and lead to further investigation of the downstream pathways triggered by turtle signaling (Al-Anzi, 2009).

Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless

Establishment of synaptic connections in the neuropils of the developing nervous system requires the coordination of specific neurite-neurite interactions (i.e., axon-axon, dendrite-dendrite and axon-dendrite interactions). The molecular mechanisms underlying coordination of neurite-neurite interactions for circuit assembly are incompletely understood. This study identified a novel Ig superfamily transmembrane protein that was named Borderless (Bdl), as a novel regulator of neurite-neurite interactions in Drosophila. Bdl induces homotypic cell-cell adhesion in vitro and mediates neurite-neurite interactions in the developing visual system. Bdl interacts physically and genetically with the Ig transmembrane protein Turtle, a key regulator of axonal tiling. These results also show that the receptor tyrosine phosphatase leukocyte common antigen-related protein (LAR) negatively regulates Bdl to control synaptic-layer selection. It is proposed that precise regulation of Bdl action coordinates neurite-neurite interactions for circuit formation in Drosophila (Cameron, 2013).

The presence of numerous axons and dendrites in the neuropils of the developing CNS makes it a daunting task for establishing specific synaptic connections. Studies over the last two decades have identified a number of cell-surface recognition molecules that mediate specific neurite-neurite interactions for circuit assembly. That many cell-surface recognition molecules are present broadly in developing neuropils throughout embryonic development, however, raises the question how the action of cell-surface recognition molecules is modulated temporally to ensure accuracy in circuit formation (Cameron, 2013).

The assembly of visual circuits in Drosophila is an attractive model for understanding the general mechanisms underlying spatiotemporal control of neurite-neurite interactions. The Drosophila adult visual system is comprised of the compound eye and the optic lobe. The compound eye consists of ∼800 ommatidia, each containing six outer photoreceptor neurons (R1-R6) for processing motion and two inner photoreceptor neurons (R7 and R8) for processing color. R1-R6 axons form synaptic connections in the superficial lamina layer, and R7 and R8 axons project through the lamina into the deeper medulla layer, where they are organized into ∼800 regularly spaced columns. Each R7 and R8 axon from the same ommatidium terminate in a topographic manner in two synaptic layers within the same column. The R8 axon terminates within the M3 layer, and the R7 axon terminates in the deeper M6 layer (Cameron, 2013).

Visual circuit assembly in Drosophila involves complex neurite-neurite interactions. Specific recognition between R-cell axons and their target layers in the optic lobe have been shown to be required for synaptic-layer selection. Visual circuit assembly also requires the interactions among R-cell axons. Selection of postsynaptic targets by R1-R6 axons in the lamina requires specific axon-axon interactions. The assembly of medulla columns requires modulation of both heterotypic and homotypic axon-axon adhesion. For instance, receptor tyrosine phosphatases LAR and protein tyrosine phosphatase 69D (PTP69D) are reported to be involved in negatively regulating the adhesion between R7 and R8 axons for facilitating R7 synaptic-layer selection. And Ig-superfamily transmembrane proteins Dscam2 and Turtle (Tutl) prevent homotypic axon-axon terminal adhesion for tiling L1 and R7 axons, respectively. The exact mechanisms by which those cell-surface recognition molecules negatively regulate axon-axon adhesion, however, remain unknown (Cameron, 2013).

The role of a novel Ig-superfamily transmembrane protein Borderless (Bdl) in Drosophila was investigated in this study. Bdl is expressed in the developing visual system, and functions as a cell-surface recognition molecule to mediate neurite-neurite interactions. The receptor tyrosine phosphatase LAR and the Ig-superfamily transmembrane protein Tutl are key regulators of Bdl-mediated axon-axon interactions in controlling synaptic-layer selection and axonal tiling, respectively. The results shed new light on spatiotemporal control of cell-surface recognition molecules for coordinating circuit assembly (Cameron, 2013).

Tiling and self-avoidance, two cellular mechanisms discovered in the early 1980s, are important for patterning neuronal circuitry. Previous studies have identified several cell-surface recognition molecules, such as Dscam, Tutl, Protocadherins, MEGF10, and MEGF11, that mediate homotypic neurite-neurite interactions in tiling and self-avoidance. These cell-surface recognition molecules may act by mediating homotypic repulsion or de-adhesion between adjacent same-type neurites. For instance, molecular and genetic analyses of fly Dscam1 support a role for Dscam1 in mediating homotypic repulsion in dendritic self-avoidance, whereas mammalian Dscams appear to mediate de-adhesion by interfering with some unknown cell-type-specific cell adhesion molecules. The exact mechanisms by which these cell-surface recognition molecules mediate homotypic repulsion or de-adhesion, however, remains elusive (Cameron, 2013).

Several lines of evidence implicate Bdl as a target of Tutl in regulating R7 axonal tiling. First, overexpression of Bdl induced an R7 tiling phenotype similar to that in tutl mutants. Second, Tutl associates with Bdl in cultured cells. And third, loss of bdl rescued the tiling phenotype in tutl mutants. It is proposed that Tutl-mediated surface recognition counteracts the affinity between adjacent R7 axonal terminals by interacting with Bdl. The association of Tutl with Bdl may downregulate the level and/or adhesive activity of Bdl, thus allowing the separation of adjacent R7 axonal terminals. Since co-overexpression of Tutl and Bdl did not affect Bdl-mediated cell-cell aggregation in culture nor the Bdl-overexpression-induced tiling phenotype in flies, it is speculated that the regulation of Bdl by Tutl requires the involvement of additional regulatory molecules. Future studies are needed to determine the exact mechanism by which Tutl downregulates the function of Bdl. It will also be of interest to determine whether other cell-surface recognition molecules implicated in tiling and self-avoidance (e.g., Dscam and Protocadherins), function similarly to modulate certain cell adhesion molecules (Cameron, 2013).

The receptor tyrosine phosphatase LAR and its mammalian homologs have been shown to play important roles in axon guidance, neuronal target selection, and presynaptic development. In the developing Drosophila visual system, LAR is required for target selection of R1-R6 axons in the lamina, and synaptic-layer selection of R7 axons in the medulla. The action of LAR in R7 synaptic-layer selection reportedly involves both stabilization of axon-target interactions and down-regulation of adhesion between R7 and R8 axons. LAR-mediated axon-target interactions may involve the binding between LAR on R7 axons and an unknown ligand in the target layer, which in turn modulates the interaction between LAR and its cytoplasmic domain-binding partner Liprin to stabilize axon-target interactions. It is also reported that LAR negatively regulates an unknown cell adhesion molecule to decrease adhesion between R7 and R8 axons for facilitating synaptic-layer selection of R7 axons (Cameron, 2013).

The current results suggest strongly that LAR downregulates adhesion between R7 and R8 axons by negatively regulating Bdl. That LAR inhibited Bdl-mediated cell-cell adhesion without affecting the level of Bdl suggests that LAR inhibits adhesive activity of Bdl. Although the role of LAR in mediating axon-target interactions requires its binding to Liprin via the cytoplasmic domain, negative regulation of Bdl by LAR appears to involve a Liprin-independent mechanism. This is supported by in vitro analysis showing that a LAR mutant lacking the cytoplasmic domain also inhibited Bdl-mediated adhesion. Consistently, a previous study showed that R8-specific expression of a truncated LAR mutant lacking the cytoplasmic domain in LAR mutants could partially rescue the R7 mistargeting phenotype. LAR may directly modulate Bdl to downregulate R7-R8 adhesion, or act indirectly by interacting with other proteins. Future studies are needed to distinguish between these possibilities (Cameron, 2013).

Although negative regulation of Bdl-mediated axon-axon interactions is necessary for R7 synaptic-layer selection and tiling, it remains unclear how the presence of Bdl contributes to the formation of the R-cell axonal projection pattern in the fly visual system. Cell adhesion molecules, such as NCAM/FasII and L1-CAM/Neuroglian, have been shown to mediate selective fasciculation in axonal pathfinding. Similarly, Bdl-mediated axon-axon interactions may facilitate the projections of R7 and/or R1-R6 axons along the pioneer R8 axon. That the R-cell projection pattern remained normal in bdl mutants may be due to the presence of redundant genes. Functional redundancy among different cell adhesion molecules seems to be common in the developing nervous system, which may account for no or subtle phenotypes in mutants defective in a number of cell adhesion molecules (Cameron, 2013).

In conclusion, this study study identifies Bdl as a novel and important regulator of neurite-neurite interactions in the developing visual system. Tuning of Bdl-mediated axon-axon interactions in axonal tiling and synaptic-layer selection presents an excellent example for modulating the action of cell adhesion molecules in ensuring accuracy in circuit assembly. It is highly likely that similar mechanisms are employed for circuit assembly in mammalian nervous systems (Cameron, 2013).

A novel member of the Ig superfamily, turtle, is a CNS-specific protein required for coordinated motor control

This study describes the cloning and functional characterization of a neural-specific novel member of the Ig superfamily, turtle (tutl), with a structure of five Ig C2-type domains, two fibronectin type III domains, and one transmembrane region. Alternative splicing of the tutl gene produces at least four Tutl isoforms, including two transmembrane proteins and two secreted proteins, with primary structures closely related to a human brain protein (KIAA1355), the Deleted in Colorectal Cancer/Neogenin/Frazzled receptor family, and the Roundabout/Dutt1 receptor family. An allelic series of tutl gene mutations results in recessive lethality to semilethality, indicating that the gene is essential. In contrast to other family members, tutl does not play a detectable role in axon pathfinding or nervous system morphogenesis. Likewise, basal synaptic transmission and locomotory movement are unaffected. However, tutl mutations cause striking movement defects exhibited in specific types of highly coordinated behavior. Specifically, tutl mutants display an abnormal response to tactile stimulation, the inability to regain an upright position from an inverted position (hence, ;turtle'), and the inability to fly in adulthood. These phenotypes demonstrate that tutl plays an essential role in establishing a nervous system capable of executing coordinated motor output in complex behaviors (Bodily, 2001. Full text of article).

Search PubMed for articles about Drosophila Turtle

Al-Anzi, B. and Wyman, R. J. (2009). The Drosophila immunoglobulin gene turtle encodes guidance molecules involved in axon pathfinding. Neural Dev. 4: 31. PubMed ID: 19686588

Bodily, K. D., Morrison, C. M., Renden, R. B. and Broadie, K. (2001). A novel member of the Ig superfamily, turtle, is a CNS-specific protein required for coordinated motor control. J. Neurosci. 21(9): 3113-25. PubMed ID: 11312296

Cameron, S., Chang, W. T., Chen, Y., Zhou, Y., Taran, S. and Rao, Y. (2013). Visual circuit assembly requires fine tuning of the novel Ig transmembrane protein Borderless. J Neurosci 33: 17413-17421. PubMed ID: 24174674

Doudney, K., Murdoch, J. N., Braybrook, C., Paternotte, C., Bentley, L., Copp, A. J. and Stanier, P. (2002). Cloning and characterization of Igsf9 in mouse and human: a new member of the immunoglobulin superfamily expressed in the developing nervous system. Genomics 79: 663-670. PubMed ID: 11991715

Ferguson, K., Long, H., Cameron, S., Chang, W. T. and Rao, Y. (2009). The conserved Ig superfamily member Turtle mediates axonal tiling in Drosophila. J. Neurosci. 29(45): 14151-9. PubMed ID: 19906964

Kurusu, M., Cording, A., Taniguchi, M., Menon, K., Suzuki, E. and Zinn, K. (2008). A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection. Neuron 59: 972-985. PubMed ID: 18817735

Long, H., Ou, Y., Rao, Y. and van Meyel, D. J. (2009). Dendrite branching and self-avoidance are controlled by Turtle, a conserved IgSF protein in Drosophila. Development 136(20): 3475-84. PubMed ID: 19783736

Millard, S. S., Flanagan, J. J., Pappu, K. S., Wu, W. and Zipursky. S. L. (2007). Dscam2 mediates axonal tiling in the Drosophila visual system. Nature 447: 720-724. PubMed ID: 17554308

Mishra, A., Knerr, B., Paixao, S., Kramer, E. R. and Klein, R. (2008). Dendrite arborization and synapse maturation (Dasm)-1 is dispensable for dendrite arborization. Mol. Cell. Biol. 28: 2782-2791. PubMed ID: 18268009

Shi, S. H., Cheng, T., Jan, L. Y. and Jan, Y. N. (2004a). The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation. Proc. Natl. Acad. Sci. 101: 13346-13351. PubMed ID: 15340156

Shi, S. H., Cox, D. N., Wang, D., Jan, L. Y. and Jan, Y. N. (2004b). Control of dendrite arborization by an Ig family member, dendrite arborization and synapse maturation 1 (Dasm1). Proc. Natl. Acad. Sci. 101: 13341-13345. PubMed ID: 15340157

Ting, C. Y., Herman, T., Yonekura, S., Gao, S., Wang, J., Serpe, M., O'Connor, M. B., Zipursky, S. L. and Lee, C. H. (2007). Tiling of r7 axons in the Drosophila visual system is mediated both by transduction of an activin signal to the nucleus and by mutual repulsion. Neuron 56: 793-806. PubMed ID: 18054857

date revised: 20 December 2013

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