Gene name - roundabout1
Cytological map position - 58E10+--58E10+
Function - potential receptor and adhesion protein
Symbol - robo1
FlyBase ID: FBgn0005631
Genetic map position - 2-
Classification - transmembrane protein with Ig and FN repeats, roundabout family
Cellular location - cell surface
|Recent literature||Brown, H. E., Reichert, M. C. and Evans, T. A. (2015). Slit binding via the Ig1 domain is essential for midline repulsion by Drosophila Robo1 but dispensable for receptor expression, localization, and regulation in vivo. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26362767
This study examined the in vivo functional importance of the Ig1 domain of the Drosophila Roundabout1 receptor, which controls midline crossing of axons in response to Slit produced by the embryonic midline. Deleting Ig1 from Robo1 disrupts Slit binding in cultured Drosophila cells, and that a Robo1 variant lacking Ig1 (Robo1Ig1) is unable to promote ectopic midline repulsion in gain of function studies in the Drosophila embryonic CNS. The Ig1 domain is not required for proper expression, axonal localization, or Commissureless (Comm)-dependent regulation of Robo1 in vivo, and a genetic rescue assay was used to show that Robo1Ig1 is unable to substitute for full-length Robo1 to properly regulate midline crossing of axons. These results establish a direct link between in vitro biochemical studies of Slit-Robo interactions and in vivo genetic studies of Slit-Robo signaling during midline axon guidance, and distinguish Slit-dependent from Slit-independent aspects of Robo1 expression, regulation, and activity during embryonic development.
|Suzuki, T., Hasegawa, E., Nakai, Y., Kaido, M., Takayama, R. and Sato, M. (2016). Formation of neuronal circuits by interactions between neuronal populations derived from different origins in the Drosophila visual center. Cell Rep [Epub ahead of print]. PubMed ID: 27068458
A wide variety of neurons, including populations derived from different origins, are precisely arranged and correctly connected with their partner to establish a functional neural circuit during brain development. The molecular mechanisms that orchestrate the production and arrangement of these neurons have been obscure. This study demonstrates that cell-cell interactions play an important role in establishing the arrangement of neurons of different origins in the Drosophila visual center. Specific types of neurons born outside the medulla primordium migrate tangentially into the developing medulla cortex. During their tangential migration, these neurons express the repellent ligand Slit, and the two layers that the neurons intercalate between express the receptors Robo2 and Robo3. Genetic analysis suggests that Slit-Robo signaling may control the positioning of the layer cells or their processes to form a path for migration. These results suggest that conserved axon guidance signaling is involved in the interactions between neurons of different origins during brain development.
|Ito, H., Sato, K., Kondo, S., Ueda, R. and Yamamoto, D. (2016). Fruitless represses robo1 transcription to shape male-specific neural morphology and behavior in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 27265393
The Drosophila fruitless (fru) gene is regarded as a master regulator of the formation of male courtship circuitry, yet little is known about its molecular basis of action. This study shows that roundabout 1 (robo1) knockdown in females promotes formation of the male-specific neurite in sexually dimorphic mAL interneurons and that overexpression of the male-specific BM diminishes the expression of Robo1 in the fly brain. Electrophoretic mobility shift and reporter assays identify the 42-bp segment encompassing the palindrome sequence T T C G C T G C G C C G T G A A in the 5' UTR of robo1 exon1 as the FruBM-responsive element. It was also found that ~10-bp deletions in the palindrome sequence induce a loss of the male-specific neurite and disrupt male courtship patterns. These data paves the way for a thorough understanding of the mechanism whereby Fru proteins orchestrate transcription for the formation of courtship circuitry.
|Morin-Poulard, I., Sharma, A., Louradour, I., Vanzo, N., Vincent, A. and Crozatier, M. (2016). Vascular control of the Drosophila haematopoietic microenvironment by Slit/Robo signalling. Nat Commun 7: 11634. PubMed ID: 27193394
Self-renewal and differentiation of mammalian haematopoietic stem cells (HSCs) are controlled by a specialized microenvironment called 'the niche'. In the bone marrow, HSCs receive signals from both the endosteal and vascular niches. The posterior signalling centre (PSC) of the larval Drosophila haematopoietic organ, the lymph gland, regulates blood cell differentiation under normal conditions and also plays a key role in controlling haematopoiesis under immune challenge. This study reports that the Drosophila vascular system also contributes to the lymph gland homoeostasis. Vascular cells produce Slit that activates Robo receptors in the PSC. Robo activation controls proliferation and clustering of PSC cells by regulating Myc, and small GTPase and DE-cadherin activity, respectively. These findings reveal that signals from the vascular system contribute to regulating the rate of blood cell differentiation via the regulation of PSC morphology.
|Reichert, M. C., Brown, H. E. and Evans, T. A. (2016). In vivo functional analysis of Drosophila Robo1 immunoglobulin-like domains. Neural Dev 11: 15. PubMed ID: 27539083
In animals with bilateral symmetry, midline crossing of axons in the developing central nervous system is regulated by Slit ligands and their neuronal Roundabout (Robo) receptors. Multiple structural domains are present in an evolutionarily conserved arrangement in Robo family proteins, but understanding of the functional importance of individual domains for midline repulsive signaling is limited. This study has examined the functional importance of each of the five conserved immunoglobulin-like (Ig) domains within the Drosophila Robo1 receptor. A series of Robo1 variants were generated, each lacking one of the five Ig domains (Ig1-5), and each was tested for its ability to bind Slit when expressed in cultured Drosophila cells. A transgenic approach was used to express each variant in robo1's normal expression pattern in wild-type and robo1 mutant embryos. Individual deletion of Ig domains 2-5 does not interfere with Robo1's ability to bind Slit, while deletion of Ig1 strongly disrupts Slit binding. None of the five Ig domains (Ig1-5) are individually required for proper expression of Robo1 in embryonic neurons, for exclusion from commissural axon segments in wild-type embryos, or for downregulation by Commissureless (Comm), a negative regulator of Slit-Robo repulsion in Drosophila. Each of the Robo1 Ig deletion variants (with the exception of Robo1Ig1) were able to restore midline crossing in robo1 mutant embryos to nearly the same extent as full-length Robo1, indicating that Ig domains 2-5 are individually dispensable for midline repulsive signaling in vivo. These findings indicate that four of the five Ig domains within Drosophila Robo1 are dispensable for its role in midline repulsion, despite their strong evolutionary conservation, and highlight a unique requirement for the Slit-binding Ig1 domain in the regulation of midline crossing.
|Ahmed, Y. A., Yates, E. A., Moss, D. J., Loeven, M. A., Hussain, S. A., Hohenester, E., Turnbull, J. E. and Powell, A. K. (2016). Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity. Mol Biosyst [Epub ahead of print]. PubMed ID: 27502551
Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. This study extended these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.
|Manavalan, M. A., Jayasinghe, V. R., Grewal, R. and Bhat, K. M. (2017). The glycosylation pathway is required for the secretion of Slit and for the maintenance of the Slit receptor Robo on axons. Sci Signal 10(484). PubMed ID: 28634210
Slit proteins act as repulsive axon guidance cues by activating receptors of the Roundabout (Robo) family. During early neurogenesis in Drosophila melanogaster, Slit prevents the growth cones of longitudinal tract neurons from inappropriately crossing the midline, thus restricting these cells to trajectories parallel to the midline. Slit is expressed in midline glial cells, and Robo is present in longitudinal axon tracts and growth cones. This study shows that the enzyme Mummy (Mmy) controls Slit-Robo signaling through mechanisms that affected both the ligand and the receptor. Mmy was required for the glycosylation of Slit, which was essential for Slit secretion. Mmy was also required for maintaining the abundance and spatial distribution of Robo through an indirect mechanism that was independent of Slit secretion. Moreover, secretion of Slit was required to maintain the fasciculation and position of longitudinal axon tracts, thus maintaining the hardwiring of the nervous system. Thus, Mmy is required for Slit secretion and for maintaining Robo abundance and distribution in the developing nervous system in Drosophila.
|Brown, H. E., Reichert, M. C. and Evans, T. A. (2017). In vivo functional analysis of Drosophila Robo1 fibronectin type III repeats. G3 (Bethesda). PubMed ID: 29217730
The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of axons during development of the embryonic CNS. Slit proteins are produced at the midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates. While previous studies have investigated the roles of Drosophila melanogaster Robo1's five Immunoglobulin-like (Ig) domains, little is known about the importance of the three evolutionarily conserved Fibronectin (Fn) type-III repeats. Each of each of Drosophila Robo1's three Fn repeats were individually deleted, and then these Robo1 variants were tested in vitro to determine their ability to bind Slit in cultured Drosophila cells and in vivo to investigate the requirement for each domain in regulating Robo1's embryonic expression pattern, axonal localization, midline repulsive function, and sensitivity to Commissureless (Comm) downregulation. We demonstrate that the Fn repeats are not required for Robo1 to bind Slit or for proper expression of Robo1 in Drosophila embryonic neurons. When expressed in a robo1 mutant background, these variants are able to restore midline repulsion to an extent equivalent to full-length Robo1. A novel requirement is identified for Fn3 in the exclusion of Robo1 from commissures and downregulation of Robo1 by Comm. These results indicate that each of the Drosophila Robo1 Fn repeats are individually dispensable for the protein's role in midline repulsion, despite the evolutionarily conserved "5+3" protein structure.
|Gorla, M., Santiago, C., Chaudhari, K., Layman, A. A. K., Oliver, P. M. and Bashaw, G. J. (2019). Ndfip proteins target Robo receptors for degradation and allow commissural axons to cross the midline in the developing spinal cord. Cell Rep 26(12): 3298-3312. PubMed ID: 30893602
Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. This switch prevents axons from re-entering the midline. In insects and mammals, negative regulation of Roundabout (Robo) receptors prevents premature response to the midline repellant Slit. In Drosophila, the endosomal protein Commissureless (Comm) prevents Robo1 surface expression before midline crossing by diverting Robo1 into late endosomes. Notably, Comm is not conserved in vertebrates. This study identified two Nedd-4-interacting proteins, Ndfip1 and Ndfip2, that act analogously to Comm to localize Robo1 to endosomes. Ndfip proteins recruit Nedd4 E3 ubiquitin ligases to promote Robo1 ubiquitylation and degradation. Ndfip proteins are expressed in commissural axons in the developing spinal cord and removal of Ndfip proteins results in increased Robo1 expression and reduced midline crossing. The results define a conserved Robo1 intracellular sorting mechanism between flies and mammals to avoid premature responsiveness to Slit.
|Brown, H. E. and Evans, T. A. (2020). Minimal structural elements required for midline repulsive signaling and regulation of Drosophila Robo1. PLoS One 15(10): e0241150. PubMed ID: 33091076
The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin type III (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well as in vitro structural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. This study defined the minimal ectodomain structure required for Slit binding and midline repulsive signaling in vivo by Drosophila Robo1. The majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. A significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, deleting four out of five Robo1 Ig domains (ΔIg2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats (ΔFn1-3 and ΔIg2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1's Fn repeats. These results provide an in vivo perspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observed in vitro for domains other than Ig1 may have limited significance for in vivo signaling in the context of midline repulsion.
|Daiber, T., VanderZwan-Butler, C. J., Bashaw, G. J. and Evans, T. A. (2021). Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3. Genetics 217(3). PubMed ID: 33789352
The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. This study used a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. SAX-3's ability to repel Drosophila axons from the Slit-expressing midline was tested in gain of function assays, and SAX-3's ability to substitute for Drosophila Robo1 during fly embryonic development was tested in genetic rescue experiments. C. elegans SAX-3 was shown to be properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, this study shows that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.
|Guzman-Palma, P., Contreras, E. G., Mora, N., Smith, M., Gonzalez-Ramirez, M. C., Campusano, J. M., Sierralta, J., Hassan, B. A. and Oliva, C. (2021). Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System. Front Cell Dev Biol 9: 612645. PubMed ID: 33968921
Neurogenesis is achieved through a sequence of steps that include specification and differentiation of progenitors into mature neurons. Frequently, precursors migrate to distinct positions before terminal differentiation. The Slit-Robo pathway, formed by the secreted ligand Slit and its membrane bound receptor Robo, was first discovered as a regulator of axonal growth. However, today, it is accepted that this pathway can regulate different cellular processes even outside the nervous system. This study describes the participation of the Slit-Robo pathway in the development of motion sensitive neurons of the Drosophila visual system. Slit and Robo receptors are expressed in different stages during the neurogenesis of motion sensitive neurons. Furthermore, it was found that Slit and Robo regulate multiple aspects of their development including neuronal precursor migration, cell segregation between neural stem cells and daughter cells and formation of their connectivity pattern. Specifically, loss of function of slit or robo receptors in differentiated motion sensitive neurons impairs dendritic targeting, while knocking down robo receptors in migratory progenitors or neural stem cells leads to structural defects in the adult optic lobe neuropil, caused by migration and cell segregation defects during larval development. Thus, this work reveals the co-option of the Slit-Robo signaling pathway in distinct developmental stages of a neural lineage.
The ability of neurons to alter their properties is fundamental to achieving the complex wiring required of nervous systems. At certain stages in development, projecting axons will respond to attractants while at other times the same axons change properties and become refractory to the same attractants that once served to direct their migratory behavior. An important region where these varying responses occurs is the midline of the central nervous system.
The midline of the insect central nervous system acts as a barrier to axon crossing. The trajectory of most growth cones in the nerve cord of insects extends toward the midline at some time during development, and a majority of these cross the midline, propelled by long-range chemoattractants. After crossing, the majority of these growth cones turn to project longitudinally, growing along or near the midline. Interestingly, these axons never cross the midline again. What mechanism prevents them from re-crossing the midline? What changes, if any, are there in neuronal properties as a result of a midline crossing, which then prevent additional crossing events? The existence of a mechanism to prevent subsequent crossing events is indicated by the mutation termed roundabout (robo), in which many growth cones that normally cross the midline only once appear to cross and recross multiple times. robo was isolated in a large-scale screen for mutations in which either too many or too few axons crossed the midline (Seeger, 1993). One possibility is that Robo is a growth cone receptor for a putative midline repellent and that Robo serves as a gatekeeper, preventing midline crossing by acting in a cell autonomous fashion. Alternatively, Robo may code for an unknown longitudinal attractant, ensuring that axons, once they have crossed the midline, never change course, thereby preventing a recrossing of the midline (Kidd, 1998a). Subsequent studies demonstrate that Slit functions as a midline repellent for the Robo receptor (Kidd, 1999).
Analysis by immunostaining of Robo expression on neurons demonstrates an alteration in the presence of Robo during the pathfinding process. Little or no Robo expression is observed on commissural growth cones as they extend toward and across the midline. However, as these growth cones turn to project longitudinally, their levels of Robo expression dramatically increase. Robo is expressed at high levels on all longitudinally projecting growth cones and axons. In contrast, Robo is expressed at nearly undetectable levels on commissural axons. Immunoelectron microscopy was used to examine Robo localization at high resolution. In stage 13 embryos, Robo is expressed at higher levels on growth cones and filopodia in the longitudinal tracts than on the longitudinal axons themselves. This localization is consistent with the model that Robo functions as a guidance receptor. The increased sensitivity of immunoelectron microscopy reveals the presence of very low levels of Robo protein on the surface of commissural axons. In addition, Robo-positive vesicles can be seen inside the commissural axons, possibly representing transport of Robo to the growth cone or internalization of Robo from the commissural axons. By reconstructing the path of single axons, using serial sections, it has been confirmed that Robo expression is greatly up-regulated after individual axons turn from the commissure into a longitudinal tract. The expression of Robo on noncrossing and postcrossing axons and its higher level of expression on growth cones and their filopodia suggest a model where Robo functions as an axon guidance receptor for a repulsive midline cue (Kidd, 1998a).
To test whether Robo functions in a cell-autonomous fashion, Robo was expressed in a subset of CNS neurons, including many of the earliest neurons to be affected by the robo mutation, such as pCC, vMP2, dMP2, and MP1. Expression of robo is sufficient to rescue these identified neurons in the robo mutant. In such rescued embryos the neuron pCC, which in robo mutants heads toward and crosses the midline, now projects ipsilaterally (to the same side) and does not cross the midline. When the same embryos are stained with the anti-Robo antibody, all Robo-positive axons are found not to cross the midline. In this directed expression, Robo was expressed in many of the axons in the pCC pathway, a medial longitudinal fascicle. In robo mutants, this axon fascicle freely crosses and circles the midline, joining with its contralateral pathway. When rescued by directed expression, this pathway largely remains on its own side of the midline, even though occasionally a few axons cross the midline. These experiments support the notion that Robo can function in a cell-autonomous fashion, that is robo directs the behavior of the neurons in which it is expressed (Kidd, 1998a).
While this model of Robo as a midline repellent remains a formal possibility, it is now thought unlikely, for at least two reasons: (1) in robo mutants, growth cones that abnormally cross the midline still tend to extend in the correct direction and project longitudinally the normal distance, and they display their normal patterns of selective fasciculation with the appropriate axons on both sides of the midline. (2) Other mutants that perturb longitudinal extension do not automatically lead growth cones to cross the midline. Crossing the midline is not the default pathway for abnormal longitudinal guidance cues. Rather, the midline appears to represent a repulsive barrier that is not easily penetrated, even when growth cones do not find their normal longitudinal pathways. These arguments lead to a preference for the model in which Robo functions as a guidance receptor for a midline repellent. Regardless of which model is correct, Robo clearly functions as a guidance receptor, and this receptor's major role is as a gatekeeper, controlling crossing and recrossing of the midline. Put succinctly, growth cones expressing high levels of Robo are prevented from crossing the midline; growth cones expressing very low levels of Robo are allowed to cross the midline (Kidd, 1998a).
Of special interest in understanding the function of roundabout are the dosage-sensitive interactions between robo and commissureless. In comm mutant embryos, commissural growth cones initially orient toward the midline but then fail to cross it and instead recoil and extend on their own side. Thus comm mutation has a complementary phenotype to that of robo; that is, too few axons cross the midline (Seeger, 1993).
comm encodes a novel surface protein expressed on midline cells. Comm is thought to be a midline attractant. As commissural growth cones contact and traverse the CNS midline, Comm protein is apparenty transferred from midline cells to commissural axons. The double mutant for robo and comm is indistinguishable from robo. The expression of Robo protein was examined in comm hypomorphic alleles. Normally, Robo is expressed at very low levels on commissural axons and at high levels on longitudinal axons. In mutant embryos, Robo expression in the longitudinal tracts appears as if it might be higher than normal. Interestingly, in comm hypomorphic alleles, the occasional thin commissures express Robo protein at levels that are higher than normally seen in the commissures and closer to what is typically seen in the longitudinal tracts. This result was the first hint that Comm protein might function by suppressing Robo expression on commissural axons, thus allowing axons to cross the midline, an event that occurs as a default state in the absence of both proteins (Kidd, 1998b).
To test the hypothesis that increased expression of comm might lead to a robo-like phenotype comm was expressed pan-neurally. A continuous range of robo-like phenotypes is generated under these conditions. The range of phenotypes reveals the comm-gain-of-function phenotype to be dosage-sensitive, since the severity increases in embryos expressing two copies of comm, as compared to those expressing only one. In wild-type embryos, the pattern of Robo protein expression begins in the neuroepithelium as well as in some lateral epidermal stripes, but is conspicuously absent from the midline region. In comm gain-of-function embryos, Robo expression in the neuroepithelium is greatly reduced or absent, while the epidermal expression outside the nervous system is maintained. This same pattern can be observed around the time when the first growth cones are extending. In wild-type embryos during stages 12 and 13, no Robo is seen at the midline, but there is a high level of Robo expression on ipsilaterally projecting growth cones and a significant level throughout the neuroepithelium. In contrast, in comm gain-of-function embryos, such growth cones lack Robo protein, and the neuroepithelium expresses greatly reduced levels of Robo (Kidd, 1998b).
What conclusions can be drawn from the results of comm overexpression? These are indictions that the normal function of comm is to down-regulate the low level of Robo expression present on commissural axons, thereby allowing them to cross the midline. Increasing levels of Comm in the CNS lead to more severe robo -like phenotypes, indicating a dosage sensitivity. This sensitivity to dosage is also reflected in the behavior of axons in robo heterozygotes, thus showing a parallel dosage sensitivity by either decreasing Robo or increasing Comm. Whereever Comm is high, Robo is low, and vice versa. Comm is expressed at the midline, and Robo expression is very low on commissural axons crossing the midline. In comm hypomorphic mutants, those few axons that do cross the midline now express higher levels of Robo protein. In comm gain-of function embryos, the overall levels of Robo are dramatically decreased wherever increased Comm expression coincides with Robo expression. Futhermore, once Comm disappears in older embryos, Robo protein expression begins to increase toward its normal levels. Thus, Comm appears to down-regulate Robo expression in a very tight fashion. It is suspected that it may do so by acting locally through cell-cell contact (Kidd, 1998b).
Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. A combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 (bearing the official FlyBase designation of leak) or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region) (Simpson, 2000b).
Robo and Robo2 together play an early function in the control of midline crossing. robo continues to be expressed by all neurons, and Robo protein appears at high levels on axons either after they cross the midline, or from the outset if they never cross the midline. Robo2 is more dynamic in its pattern of expression. Initially, it is expressed by a wide range of neurons, including all of the early pioneer neurons whose axons do not cross the midline (e.g., pCC, MP1, dMP2, and vMP2). But during the period around late stage 13 in which these axons selectively defasciculate to form the medial pCC pathway and the intermediate MP1 pathway, the expression of Robo2 declines in many of these neurons. It is during this same period (late stage 13 to stage 14) that Robo3 begins to be expressed by a subset of neurons (Simpson, 2000b).
From stage 14 onward, as multiple longitudinal pathways form, all three Robos are expressed on some or all longitudinal axon tracts and are excluded from commissural axon tracts. Within the longitudinal tracts, their expression patterns differ dramatically. Robo is found on all longitudinal axon pathways. The second phase of Robo2 expression, and the only phase of Robo3 expression, have a common quality. Both are expressed on a subset of axons that extend in specific lateral positions of the developing CNS. Robo3 is expressed only on axons that extend in the outer two-thirds of the longitudinal pathways (the intermediate and lateral regions). A high level of Robo2 expression is restricted to axons that extend in the outer third of the longitudinal pathways (the lateral region), farthest from the midline and thus farthest from the source (Simpson, 2000b).
All three Robos show relatively tight boundaries. All three are absent from the commissures, and Robo3 and Robo2 are restricted to certain regions of the longitudinal pathways. The expression of Robo3 and Robo2 is not graded, but rather appears to form regional boundaries. While the high level of Robo2 is restricted to the lateral pathways, a lower level of Robo2 expression is detected on some of the intermediate pathways (the more lateral ones). The low level of Robo2 expression begins right in the middle of the intermediate Fas II pathway. This step-wise expression of Robo2 (from none on the medial portion of the intermediate pathways, to a low level on the lateral portion of the intermediate pathways, to a high level laterally) reveals further regional subdivisions of the longitudinal pathways (Simpson, 2000b).
The pattern of expression of Robo3 and Robo2 in individual identified neurons is consistent with their overall patterns of expression. For example, robo3 RNA is expressed in the MP1 neuron whose axon pioneers the intermediate Fas II pathway. robo3 is largely absent from pCC and other neurons whose axons pioneer the medial Fas II pathway. All of these neurons (e.g., MP1, pCC) transiently express robo2 when they are making the earlier decision not to cross the midline, but such expression declines by the time the medial and intermediate Fas II longitudinal pathways separate from one another. This is consistent with the presence of Robo2 only on lateral axons during later stages of development (Simpson, 2000b).
All three Robos are expressed on specific growth cones and filopodia. Commissural growth cones and axons are devoid of all three Robos. All ~150 longitudinal axons express Robo. The ~50 intermediate axons and the ~50 lateral axons express Robo3 (with one exception). The ~50 lateral axons express Robo2. Most lateral axons express Robo2 and Robo3, with one exception; the most lateral axon bundle of ~10 axons expresses high levels of Robo2, but is largely or completely devoid of Robo3 (Simpson, 2000b).
The mutant phenotypes of robo, robo2, and robo3 support the hypothesis that the Robos specify lateral position with respect to the midline. robo mutants show axons ectopically crossing and recrossing the midline. These axons are predominantly those of the innermost part of the longitudinal scaffold. When a robo mutant is examined with anti-Fas II (mAb 1D4), only the medial Fas II pathway crosses the midline. The intermediate and lateral Fas II pathways stay on their own side. One possible interpretation is that the intermediate and lateral expression of Robo3 and Robo2 keeps these axons from crossing the midline in a robo mutant. In a robo, robo2 double mutant, all axons go to the midline and do not leave it (and thus it looks like a slit mutant) (Simpson, 2000b).
robo2 loss-of-function mutations show occasional ectopic midline crossing, but, more prominently, they show abnormalities in lateral positioning. The most common phenotype as revealed with anti-Fas II staining is crossovers and 'braiding' between the intermediate and lateral Fas II pathways (and sometimes between the medial and intermediate Fas II pathways). Although superficially the axon scaffold looks relatively normal in a robo2 mutant (when visualized with mAb BP102, which labels all axons), the lateral positions of the longitudinal pathways are altered in the absence of Robo2 (Simpson, 2000b).
The role of Robo3 in lateral positioning was examined using RNA interference (RNAi). Injection of robo3 dsRNA causes the stage 16 embryo to have two Fas II longitudinal pathways instead of three. The intermediate pathway is missing, and the medial pathway is larger than normal. The lateral (normally Robo2 expressing) Fas II pathway appears normal. In the absence of Robo3, the medial and intermediate Fas II pathways fail to separate, and the intermediate (normally Robo3 expressing) pathway does not properly form (Simpson, 2000b).
The robo2, robo3 double mutant, generated by injecting robo3 dsRNA into a robo2 mutant, contains a large single Fas II longitudinal pathway. This single fascicle is thicker than wild-type Fas II pathways and is close to the midline in the normal location of the medial Fas II pathway. In the absence of both Robo3 and Robo2, it appears as if most (and in some cases all) of the Fas II axons selectively fasciculate into one Fas II pathway in the medial position. This suggests that Robo3 and Robo2 are required for the normal formation of the Robo3 expressing intermediate Fas II pathway and the Robo2/Robo3 expressing lateral Fas II pathway (Simpson, 2000b).
Unlike robo mutants alone, robo, robo3 embryos (robo mutants with robo3 dsRNA) exhibit ectopic crossing of the intermediate Fas II pathway as well as the medial one. This resembles the addition of the two individual phenotypes: lack of Robo3 causes the intermediate pathway to join the medial pathway, and the lack of Robo allows this fused Fas II pathway to weave back and forth across the midline. In the robo, robo3 double mutant, the outer Fas II pathway remains on its appropriate side, presumably due to the presence of Robo2 (Simpson, 2000b).
These phenotypes support the model that Robo is the most important contributor to maintaining the Fas II pathways on the appropriate side of the midline, but that Robo2 and Robo3 determine the lateral position of these and other longitudinal pathways. Robo3 specifies the intermediate region and its pathways, while Robo2 specifies the lateral region and its pathways (Simpson, 2000b).
Overexpression of Robo2 supports the model that Robo2 levels contribute to the lateral position of axons. Overexpression of UAS-robo2 in all CNS axons using the elav-GAL4 driver results in a commissureless-like phenotype (i.e., appearing like the commissureless mutant). There are a number of other genetic combinations that result in a commissureless-like phenotype. These all look the same when examined with mAb BP102 that stains all axons: the commissures are missing. But the appearance of the three Fas II pathways differs, depending upon the genetic makeup of the embryo (Simpson, 2000b).
When Robo expression is increased on all axons, by either directly driving more Robo or in a comm mutant (in which Comm no longer downregulates Robo), three distinct Fas II pathways are still detected. This is true even when the Robo Y-F 'hyperactive' receptor is transgenically expressed on all axons. Under these various conditions, there is disorganization of longitudinal pathways, but the three Fas II pathways can generally be detected (Simpson, 2000b).
But when Robo2 is ectopically expressed on all axons, the lateral position of the pathways is disrupted, and as a result, all three Fas II pathways are bundled together into a single, thick tract. Ectopic Robo2, but not ectopic Robo or loss of Comm, is sufficient to override the endogenous positional information that specifies the locations of the three Fas II pathways (Simpson, 2000b).
Overexpression of Robo3 in all CNS axons (using the elav-GAL4 driver) results in a weakly commissureless-like phenotype. As with UAS-robo and UAS-robo2, the gain-of-function commissureless phenotype of Robo3 requires two copies of the UAS-robo3 reporter to generate commissureless segments. Some of the Fas II bundles are fused, but at least two distinct fascicles are still visible at this level of overexpression (Simpson, 2000b).
Expressing Robo2 or Robo3 in subsets of neurons whose axons normally extend in medial longitudinal pathways can drive these axons to assume more lateral positions. Although both can drive medial axons further laterally, Robo2 and Robo3 are not identical: when tested on the same axons, Robo2 drives medial axons further laterally than does Robo3 (Simpson, 2000b).
For example, in each abdominal hemisegment, three neurons express the transcription factor Apterous (Ap). These neurons normally extend their axons toward the midline, and then turn anteriorly on their own side close to the midline. These axons turn anteriorly in the medial region. When viewed at the light level with confocal microscopy, the Ap axons sometimes look like they are running just at the lateral edge of the medial Fas II bundle, and sometimes there is a little space between them (suggesting another axon or two interposed. This staining pattern was used to infer that the Ap axons run in a medial axon pathway just lateral to the medial Fas II tract (Simpson, 2000b).
When these axons ectopically express Robo2 under control of Apterous-GAL4 (Ap-GAL4), they move laterally and extend anteriorly in a specific location between the intermediate and the lateral Fas II pathways. In fact, they extend in the medial-most region of the endogenous Robo2 expression zone. The Ap axons from neighboring segments appear to pick the same lateral pathway, and to fasciculate together as they extend anteriorly from segment to segment. Two different GAL4 reporters were tested. Both drive different levels of Robo2 expression as indicated by their different strengths of pan-neural gain-of-function phenotypes. Nevertheless, both drive the Ap axons to the same lateral location. There is a second argument that supports this same conclusion. It is well known that the GAL4 expression system drives different levels of expression from a UAS transgene in different cells and segments of the same embryo. Yet, from cell to cell, segment to segment, and embryo to embryo, the Ap axons (expressing what is presumed to be variable levels of Robo2) always turn anteriorly in the same lateral location between the intermediate and lateral Fas II pathways (Simpson, 2000b).
This suggests that Robo2 reading of the Slit gradient drives axons to a rough lateral position, regardless of the precise level of Robo2, after which local cues determine which specific pathway a particular axon joins. Thus, Robo2 drives the Apterous axons to the lateral third of the scaffold, and regardless of the precise level of Robo2, this is sufficient to allow their final pathway choice to be precisely and uniformly dictated by some unknown but specific local cue. All of these experiments are done at different Robo2 levels within a relatively narrow range. It is conceivable that much higher levels of Robo2, or Robo3, might drive axons further laterally, even into more lateral zones (Simpson, 2000b).
Using the same Ap-GAL4 transgene to drive overexpression of Robo3 in the Ap axons leads to a different alteration in lateral position. Whereas ectopic Robo2 drives these axons quite far laterally to a position between the intermediate and lateral Fas II pathways, the ectopic expression of Robo3 drives them to an intermediate position, just medial to the intermediate Fas II pathway. Thus, Robo3 and Robo2 can both drive the medial Ap axons to more lateral positions, but they do so to different extents, as might be expected by their normal patterns of expression. Robo2 drives axons further laterally than does Robo3, relative to the intermediate Fas II bundle (Simpson, 2000b).
Using the same Ap-GAL4 transgene to drive overexpression of either Robo or the hyperactive Robo Y-F in the Ap axons leads to no alteration in lateral position. Even with more Robo, these axons still continue to extend in their normal medial location. These axons, and for that matter all longitudinal axons, normally express Robo, and it is inferred from this result that increasing the level of Robo does not alter the choice of lateral position by typical follower growth cones. The choice of lateral position by these axons is exquisitely sensitive to the presence of Robo2 and Robo3, but apparently not to the level of Robo. The ability of Robo2 to lateralize these axons does not depend on the presence of Robo: lateralization occurs in neurons that coexpress transgenic Robo2 and a Robo dominant negative receptor, or when Robo2 is expressed ectopically in a robo null mutant background (Simpson, 2000b).
The role of the Robo code in determining lateral position, and the potential interplay of this long-range guidance system were further analyzed using the presumptive Slit gradient with other local cues. Are all medial axons driven to the same lateral positions by Robo3 and Robo2? Or, alternatively, are they driven to cell-specific lateral positions? If the latter is the case, are their other cues that help predict the specific location? To this end, other GAL4 lines were used to drive the expression of various Robo family members in other subsets of axons (Simpson, 2000b).
The 15J2-GAL4 line drives expression in the dMP2 and vMP2 neurons (and variably in a few other neurons). These two neurons normally express Fas II, and normally extend in the medial Fas II pathway. Ectopic expression of Robo2 in these neurons leads to a bimodal phenotype. The dMP2 and vMP2 axons always appear to extend in a Fas II pathway, but they now pick either the intermediate or lateral Fas II pathways. These axons are never found medially, are often found in the intermediate Fas II pathway, and occasionally are found in the lateral Fas II pathway. These are distinctly different locations from where the Ap neurons are driven by Robo2 expression. It is sometimes difficult to determine which pathway the dMP2 and vMP2 axons are in (i.e., intermediate vs. lateral) because the two pathways intertwine in these gain-of-function embryos. Comparing the two experiments, Robo2 drives the Ap neurons to a non-Fas II pathway, while it drives dMP2 and vMP2 (which normally follow the medial Fas II pathway) into either the intermediate or lateral Fas II pathway (Simpson, 2000b).
It is concluded that precise topography requires more than just the Robo code. Robo3 and Robo2 expression define specific lateral regions. Ectopic Robo3 drives axons into the intermediate region, while ectopic Robo2 drives them even further laterally. But axons respond in a cell-specific fashion. Ectopic Robo2 drives the three Ap axons to between the intermediate and lateral Fas II pathways, while it drives the dMP2 and vMP2 axons into either the intermediate or lateral Fas II pathway. The control of location appears irrespective of level, since the result is consistent in spite of the different levels of expression generated by different Robo2 reporter lines, and by the variability in expression as driven with the GAL4 system. How can this precision be explained (Simpson, 2000b)?
Many models for topographic specificity involve the notion of two opposing gradients, either both of the same sign (i.e., both either attractive or repulsive) in the opposite orientation, or both of different signs in the same orientation. Such models are very attractive to explain certain aspects of sensory maps in the brain. However, such models need not apply to all topographic projections. Thus far, no evidence has been found for a second gradient working in concert with the repulsive Slit gradient. The most parsimonious model is that precise topography in the medial-lateral axis of the Drosophila CNS requires two opposing forces: long-range repulsion and short-range attraction. Cues might exist, for example, that mark the boundary of the neuropil. But in terms of location with the neuropil, all that is required is an opposing force to the Slit gradient -- it need not be a long-range gradient itself. Discrete local cues would be sufficient. Clearly, the long-range repulsion is controlled by the Slit gradient and the Robo code. It is proposed that the opposing force is short-range attraction as controlled by discrete local cues, one of which is Fasciclin II. In this way, the Robo code specifies the lateral region, while local cues specify precise location within that region (Simpson, 2000b).
The strongest support of this model involves the specification of the three major Fas II pathways. Fas II is a homophilic cell adhesion molecule expressed on axons that fasciculate together in three major longitudinal pathways: one medial, one intermediate, and one lateral. Growth cones expressing Fas II and Robo pick the medial Fas II pathway. Growth cones expressing Fas II, Robo3, and Robo pick the intermediate Fas II pathway. Presumably, the attraction of the medial Fas II pathway is insufficient to balance the repulsion mediated by Robo3. Growth cones expressing Fas II, Robo2, Robo3, and Robo pick the lateral Fas II pathway. In this case, it is not until they contact the lateral Fas II pathway that the Fas II-mediated attraction is stronger than the Robo2-mediated repulsion. Removal of Robo3 leads to only two Fas II pathways in which the intermediate pathway is missing, and instead the medial pathway is twice as thick. Ectopic expression of Robo2 in the dMP2 and vMP2 neurons, which normally extend in the medial Fas II pathway, drives their axons into either the intermediate or lateral Fas II pathway. Specificity is determined by the combination of Fas II and the particular Robo family members (Simpson, 2000b).
It is proposed that other pathways are specified by other pathway labels. For example, two pathways -- one medial and the other lateral -- express Connectin, another homophilic cell adhesion molecule. Growth cones expressing Connectin and Robo pick the medial Connectin pathway, while growth cones expressing Connectin and Robo2 (and presumably Robo and Robo3) pick the lateral Connectin pathway. Removal of Robo2 leads to only one fused medial Connectin pathway (Simpson, 2000b).
How do Robos read and respond to the Slit gradient? How are Robo3 and Robo2 different from Robo? How do Robo3 and Robo2 specify lateral position? Why does Robo3 drive axons into the intermediate region, while Robo2 drives them into the lateral region? Robo3 and Robo2 must differ from one another in either their ectodomains (and thus have different abilities to read the Slit gradient), or in their cytoplasmic domains (and thus have different abilities to signal), or both. What are the key differences that allow them to drive axons to different lateral regions? Both of these receptors (Robo3 and Robo2) differ from Robo in some quality of their signaling, either having some additional output or missing some output found in Robo. Their cytoplasmic domains are quite different from Robo, but what differences are key for determining lateral position (Simpson, 2000b)?
It will be of interest to determine to what extent different chimeric receptors and mutated receptors can drive lateralization. Preliminary collaborative results suggests that it should be possible to separate the functions of the various ectodomains and cytoplasmic domains (Simpson, 2000b).
Slit/Roundabout (Robo) signaling controls midline repulsive axon guidance. However, proteins that interact with Slit/Robo at the cell surface remain largely uncharacterized. This study reports that the Drosophila transmembrane septate junction-specific protein Neurexin IV (Nrx IV) functions in midline repulsive axon guidance. Nrx IV is expressed in the neurons of the developing ventral nerve cord, and nrx IV mutants show crossing and circling of ipsilateral axons and fused commissures. Interestingly, the axon guidance defects observed in nrx IV mutants seem independent of its other binding partners, such as Contactin and Neuroglian and the midline glia protein Wrapper, which interacts in trans with Nrx IV. nrx IV mutants show diffuse Robo localization, and dose-dependent genetic interactions between nrx IV/robo and nrx IV/slit indicate that they function in a common pathway. In vivo biochemical studies reveal that Nrx IV associates with Robo, Slit, and Syndecan, and interactions between Robo and Slit, or Nrx IV and Slit, are affected in nrx IV and robo mutants, respectively. Coexpression of Nrx IV and Robo in mammalian cells confirms that these proteins retain the ability to interact in a heterologous system. Furthermore, the extracellular region of Nrx IV was shown to be sufficient to rescue Robo localization and axon guidance phenotypes in nrx IV mutants. Together, these studies establish that Nrx IV is essential for proper Robo localization and identify Nrx IV as a novel interacting partner of the Slit/Robo signaling pathway (Banerjee, 2010).
Recent studies have established strong expression of Nrx IV at the interface between neurons and midline glie (MG) that underlies the adhesive interactions between these cells in maintaining ML cytoarchitecture. This function of Nrx IV is Wrapper-dependent. It is believed that the axon guidance function of Nrx IV is Wrapper independent based on the following observations. First, the juxtaposition of strong neuronal Nrx IV and glial Wrapper in the ML, together with the absence of axon guidance phenotype in wrapper mutants strongly suggest that Nrx IV localization in ML neurons does not contribute to the axon guidance function of Nrx IV. This is further supported by the failure to rescue the axon guidance phenotypes in nrx IV mutant by expressing Nrx IV in all ML neurons and glia using sim-Gal4::UAS-nrx IV. Therefore, a significant contribution of the axon guidance phenotype seen in nrx IV mutants is unlikely to come from ML neurons or glia. Interestingly, the MG ensheathment defects seen in nrx IV mutants are rescued by expression of Nrx IV in neurons alone and not MG. Furthermore, no changes in Robo localization, or crossing of Robo positive axons were seen in wrapper mutants, thus providing additional evidence that alterations in MG/neuronal architecture in wrapper mutants does not significantly contribute to the axon guidance phenotype. Of note, older stage 16 robo and slit mutants show considerable disorganization of MG, as revealed by immunostaining with anti-Wrapper antibodies; however, these glial phenotypes are thought to be secondary to their axon guidance phenotypes. In addition, Contactin (Cont) and Nrg, two well-established binding partners of Nrx IV at SJs, do not contribute to Nrx IV in its CNS axon guidance function, as revealed by Fas II immunostaining of cont and nrg mutants. Based on these observations, it is believed that Nrx IV is a multifunctional protein that functions in a cell type specific manner. Therefore, the axon guidance function of Nrx IV is to ensure proper localization and stability of Robo in the lateral CNS soma and axons during ML axon repulsion (Banerjee, 2010).
Additional support for tissue specific functions of Nrx IV comes from recent reports that Drosophila cardiac development uses a non-canonical role of Nrx IV to maintain cardiac integrity, by coupling with G-protein signaling. Both Robo and Slit have previously been shown to control cardiac cell polarity and morphogenesis. Since the embryonic heart cells lack SJs, these findings further underscore the fact that Nrx IV and Robo/Slit coordinate diverse roles in different tissues involving multiple molecular partners. Thus, Nrx IV functions in Slit/Robo axon guidance pathway independently of its other known partners, such as Cont, Nrg and Wrapper (Banerjee, 2010).
The findings strongly support the existence of Nrx IV, Robo and Slit as a molecular complex. Although Nrx IV function appears interwoven with Robo and Slit, the phenotypes displayed by nrx IV mutants do not completely phenocopy either slit or robo mutants, suggesting that Nrx IV plays a modulatory role in Slit/Robo signaling. The biochemical analyses suggest that Robo is required for Nrx IV stability, as the levels of Nrx IV are significantly reduced in robo mutants. Slit, on the other hand, showed a modest stimulatory effect on Robo and Nrx IV association and expression levels, further confirming that these three proteins are functionally interlinked. Furthermore, the Slit/Robo complex is less efficiently immunoprecipitated from nrx IV mutant. Thus, while loss of Nrx IV does not abolish interactions between Robo and Slit, it could potentially affect proper functioning of the Robo/Slit signaling complex. Similarly, reduced association of Slit and Nrx IV in robo mutants suggests that Robo is also important for efficient complex formation between these three proteins (Banerjee, 2010).
Both Nrx IV and Robo are transmembrane proteins that colocalize in longitudinal axons. Most of the known Nrx IV interacting proteins, such as Cont, Nrg and Wrapper belong to Ig superfamily of cell adhesion molecules (CAM). Therefore, it is conceivable that Nrx IV associates with Robo (an Ig CAM) in neurons which do not express detectable levels of Cont or Wrap. Furthermore, nrx IV mutant phenotypes resemble those of robo mutant, and Nrx IV interacts with Robo/Slit, suggesting that Nrx IV functions in the Robo/Slit pathway (Banerjee, 2010).
Recent studies support a model where Slit stimulation recruits cytoplasmic Sos to Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during repulsion. This study shows that Nrx IV and Robo retain their ability to colocalize and interact when co-expressed in a heterologous system, and indicate that Slit is dispensable for their interaction. In addition, slit RNAi experiments in S2 cells reveal that Nrx IV and Robo associate in the absence of Slit. However, Slit stimulation of nrx IV/robo cotransfected CHO cells caused enhanced colocalization of Nrx IV and Robo in intracellular compartments and membrane ruffles, further supporting a functional relationship between Nrx IV and Robo/Slit. Together, these in vivo and in vitro findings indicate that Nrx IV and Robo interact in the absence of Slit, and in the presence of Slit ligand the molecular interactions between Nrx IV and Robo are strengthened. Formation of this larger molecular complex at the axonal surface thus ensures proper ML axon guidance (Banerjee, 2010).
The phenotypic similarities, dose-dependent genetic interactions and the in vivo biochemical data suggest that Nrx IV acts as a modulator in Slit/Robo signaling pathway. One of the key reasons for this conclusion is the fact that the axon guidance phenotypes in nrx IV mutants is rescued by the expression of the extracellular region of Nrx IV (Nrx IVmycΔCT), where as the phenotype is not rescued by the intracellular region of Nrx IV (Nrx IVmycΔNT). For Nrx IV to act as an independent signal transducer, it would need an intact cytoplasmic region. Since the axon guidance phenotypes and Robo localization are both rescued by Nrx IVmycΔCT, the downstream signaling controlling axon repulsion is controlled by Robo or an as yet unidentified protein. Therefore, the data support a role for Nrx IV in the proper localization and stabilization of Robo at axonal membrane, where it interacts with Slit, to regulate downstream axon guidance signaling. Although the exact domains regulating Nrx IV-Robo interactions are unknown at this point, it is predicted that they occur via the Ig or FNIII domains, as these domains regulate Nrx IV-Cont interactions. The data rule out the possibility that Nrx IV interactions with Robo occur via a large cytoskeletal scaffolding complex, as Nrx IVmycΔCT lacks the cytoplasmic region. This led to the conclusion that Nrx IV and Robo interact in cis through their extracellular regions, and therefore eliminate the possibility of a parallel Nrx IV signaling pathway (Banerjee, 2010).
One of the interesting in vivo observations is the association between Nrx IV and Slit still occurs in robo null mutant embryos, indicating that Slit/Nrx IV can interact in the absence of Robo. Although it remains to be seen if Nrx IV and Slit can associate in the absence of all Drosophila proteins in a heterologous system, based on the existing findings it is tempting to speculate that Nrx IV may function as a co-receptor for Slit, and together with Robo, they stabilize the complex to ensure proper presentation or retention at the axonal surface. A similar role has been assigned to Sdc, which is thought to be critical for the fidelity of Slit repellent signaling, as sdc mutants exhibit consistent defects in ML axon guidance. Thus, a multitude of mechanisms seem to operate at the axonal surface and growth cones to ensure that axons reach their correct targets. The Slit-independent interactions between Nrx IV and Robo, but seemingly enhanced colocalization and interactions in the presence of Slit, point to an interesting mechanism where signaling molecules use accessory proteins to ensure their proper localization and stability. This mechanism ensures checks and balances at several molecular levels to allow navigating axons to reach their final destinations. Very few proteins have been implicated in Slit/Robo signaling at the axonal surface, and additional yet unidentified proteins may be involved. With the identification of Nrx IV as an essential component of the Slit/Robo complex, new insights into this highly sophisticated molecular pathway are opened, and may allow for future studies aimed at identifying the modulatory proteins that coordinate and/or control axon guidance (Banerjee, 2010).
Exons - 18+
robo encodes an axon guidance receptor that defines a novel subfamily of immunoglobulin superfamily proteins, which have been found to be highly conserved from fruit flies to mammals. Robo protein is a member of the Ig superfamily; Robo's ectodomain contains five Ig-like repeats followed by three fibronectin (FN) type-III repeats. The protein also begins with a putative signal sequence; in the middle it contains a transmembrane domain, and it ends with a large, 457 amino acid cytoplasmic domain (Kidd, 1998a).
The presence of five Ig and three FN domains, a transmembrane domain, and a long (457 amino acid) cytoplasmic region suggests that Robo may be a receptor and signaling molecule. The netrin receptor DCC/Frazzled/UNC-40 has a related domain structure, with 6 Ig and 4 FN domains and a similarly long cytoplasmic region. Another protein with a "5 + 3" organization is CDO (Kang, 1997). However, CDO is only distantly related to Robo (15%-33% amino acid identity between corresponding Ig and FN domains) (Kidd, 1998a).
The alignment of the Robo family proteins reveals that the first and second Ig domains are the most highly conserved portion of the extracellular domain. The cytoplasmic domains are highly divergent except for the presence of three highly conserved motifs. The consensus for the first motif is PtPYATTxhh, where x is any amino acid and h is I, L, or V. The presence of a tyrosine in the center of the motif suggests that it could be a site for phosphorylation. The other two motifs consist of runs of prolines separated by one or two amino acids. All three of these conserved sites could function as the binding sites for cytoplasmic adapter proteins, which would transmit signals generated by ligand binding (Kidd, 1998a).
The predicted Robo2 and Robo3 proteins consist of 1406 and 1342 amino acids, respectively. Robo family receptors identified to date in diverse species are characterized by an extracellular domain consisting of 5 immunoglobulin and 3 fibronectin type III repeats, and a cytoplasmic domain without any known catalytic activity but containing four short conserved motifs called CC0, CC1, CC2, and CC3. The extracellular domains of Robo2 and Robo3 are typical for the family, being 37% and 33% identical to Robo, respectively, and 49% identical to each other. In contrast, the cytoplasmic domains of Robo2 and Robo3 are unusual in that they lack both the CC2 and CC3 motifs. In Robo, these two motifs are required to prevent inappropriate midline crossing. They are thought to constitute binding sites for various cytoplasmic signaling molecules, including Enabled (Ena) and the Abl tyrosine kinase. Ena interacts primarily with the CC2 motif, and genetic data suggest that it contributes positively to Robo signaling. Abl, on the other hand, binds strongly to CC3 and negatively regulates Robo function. In vitro, Abl phosphorylates Robo on three tyrosine residues, including one in each of the CC0 and CC1 motifs. Both of these tyrosine residues are conserved in Robo2 and Robo3 (Rajagopalan, 2000).
robo2 and robo3 were initially detected by searching the Drosophila genomic database for sequences similar to robo. Homology and protein prediction programs identified two paralogs of robo. The robo2 and robo3 genes are quite close to each other and face in opposite directions on the left arm of chromosome 2 at location 22A; robo is located on the right arm of chromosome 2 at location 58F (Simpson, 2000a).
The robo2 cDNA encodes a 1540 amino acid protein with the same domain structure as Robo. The extracellular region contains five immunoglobulin-like (Ig) domains and three fibronectin type III (Fn) domains, followed by a single-pass transmembrane domain and a 450 amino acid cytoplasmic domain. The homology between Robo and Robo2 is highest in the extracellular region, ranging from 52%-53% (in the first two Ig domains) to 29% and dropping to less than 23% in the cytoplasmic region. Robo2 lacks two of the four conserved cytoplasmic motifs that Robo shares with its orthologs in other species. The first two of these motifs (CCO and CC1), which are tyrosine phosphorylation sites, are maintained, but the second two motifs (CC2 and CC3), a proline-rich Enabled binding motif and another polyproline stretch, are missing. In Robo2, the proline-rich Enabled binding motif (CC2) is replaced by a polyglutamine repeat. Interestingly, although several of the mammalian Robo orthologs contain all four conserved cytoplasmic motifs, a more divergent Robo family member, Rig-1, appears to lack CC1 (Simpson, 2000a).
Robo3 resembles Robo2 more closely than it does Robo; it too lacks two of the four cytoplasmic motifs. The genomic organization of robo2 and robo3 is very similar. Both have large first introns (23 and 17 kb, respectively), while the first intron in robo is less than 1 kb. The intron and exon sizes are similar, and which exons code for particular domains is also conserved, suggesting that robo2 and robo3 may be the result of a recent duplication (Simpson, 2000a).
Examination of the vertebrate databases shows that the identified homologs have cytoplasmic domain organization more like Drosophila Robo than like Robo2 or Robo3, indicating that Robo is closer to the common ancestor with vertebrates. Robo2 and 3 resemble each other and Robo more closely than they resemble any of the mammalian Robos or the Caenorhabditis elegans Sax3. Examination of the exon-intron boundaries within the coding regions of the three robo genes suggests that robo2 and robo3 may be the result of a recent duplication event. This phylogeny holds when the complete protein sequences of the homologs are compared, as well as when the extracellular domains are aligned and when only the most highly conserved first Ig domain is used. The Robo receptor family is related to other neural adhesion and guidance molecules like DCC/Frazzled and Neuroglian but is a distinct subgroup of the Ig superfamily (Simpson, 2000a).
date revised: 2 December 2000
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