slit: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - slit

Synonyms -

Cytological map position - 52D

Function - receptor-binding ligand

Keywords - CNS, axon guidance

Symbol - sli

FlyBase ID:FBgn0264089

Genetic map position - 2-77

Classification - EGF-like - leucine-rich repeat motif

Cellular location - extracellular



NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene

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
Summary:
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
Summary:
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.

Chance, R. K. and Bashaw, G. J. (2015). Slit-dependent endocytic trafficking of the Robo receptor is required for Son of Sevenless recruitment and midline axon repulsion. PLoS Genet 11: e1005402. PubMed ID: 26335920
Summary:
Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. This study shows that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. Two conserved motifs were identified in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. These data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.
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
Summary:
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.
Alavi, M., Song, M., King, G.L., Gillis, T., Propst, R., Lamanuzzi, M., Bousum, A., Miller, A., Allen, R. and Kidd, T. (2016). Dscam1 forms a complex with Robo1 and the N-terminal fragment of Slit to promote the growth of longitudinal axons. PLoS Biol 14: e1002560. PubMed ID: 27654876
Summary:
The Slit protein is a major midline repellent for central nervous system (CNS) axons. In vivo, Slit is proteolytically cleaved into N- and C-terminal fragments, but the biological significance of this is unknown. Analysis in the Drosophila ventral nerve cord of a slit allele (slit-UC) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted, particularly across segment boundaries. Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype, suggesting they cooperate in longitudinal axon guidance, and through biochemical approaches, it was found that Dscam1 and Robo1 form a complex dependent on Slit-N. In contrast, Robo1 binding alone shows a preference for full-length Slit, whereas Dscam1 only binds Slit-N. Using a variety of transgenes, it was demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive. These data suggest that the complex is promoting longitudinal axon growth across the segment boundary. The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins.

Ordan, E. and Volk, T. (2016). Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning. Biol Open [Epub ahead of print]. PubMed ID: 27628033
Summary:
Slit cleavage into an N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Previous analysis has indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage, this study performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The Drosophila homolog of Pheromone Convertase 2 (PC2) Amontillado (Amon) was identified as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for Amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos is rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.

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
Summary:
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.
Nagy, P., Szatmari, Z., Sandor, G. O., Lippai, M., Hegedus, K. and Juhasz, G. (2017). Drosophila Atg16 promotes enteroendocrine cell differentiation via regulation of intestinal Slit/Robo signaling. Development 144(21): 3990-4001. PubMed ID: 28982685
Summary:
Genetic variations of Atg16l1, Slit2 and Rab19 predispose to the development of inflammatory bowel disease (IBD), but the relationship between these mutations is unclear. This study shows that in Drosophila guts lacking the WD40 domain of Atg16, pre-enteroendocrine (pre-EE) cells accumulate that fail to differentiate into properly functioning secretory EE cells. Mechanistically, loss of Atg16 or its binding partner Rab19 impairs Slit production, which normally inhibits EE cell generation by activating Robo signaling in stem cells. Importantly, loss of Atg16 or decreased Slit/Robo signaling triggers an intestinal inflammatory response. Surprisingly, analysis of Rab19 and domain-specific Atg16 mutants indicates that their stem cell niche regulatory function is independent of autophagy. These study reveals how mutations in these different genes may contribute to IBD.
BIOLOGICAL OVERVIEW

Extracellular proteins have a unique role in development; they provide a matrix for the attachment and migration of cells. They also serve as ligands for cell receptors, insuring proper communication between cells. Slit is a complex extracellular protein containing at least four different motifs shared with other differentiation factors and receptors, including the vertebrate epidermal growth factor, the Drosophila receptor Toll, and the matrix protein laminin. Slit protein is made by midline glial cells. It provides a matrix for the migration of ventral nerve cord axons and is therefore likely to serve as an axon guidance protein. The actions of Slit are not confined to the nervous system; the roles of slit in gut and heart differentiation await exploration.

Slit has been identified as the midline repellent for the Roundabout (Robo) receptor. Robo has been shown to be a repulsive guidance receptor on growth cones that binds to an unknown midline ligand. In the original large-scale mutant screen for genes controlling midline axon guidance, 8 alleles were recovered of robo, 2 alleles of commissureless, and 13 alleles of slit. At the time, because slit had such a similar axon phenotype to single minded, which controls midline cell fate and survival, and because of the lack of good midline markers, there was some uncertainty as to whether slit like sim might also control midline cell fate and survival. As a result, initial attention was placed on robo and comm, two genes that clearly control midline axon guidance. Nevertheless, there was always the lingering possibility that Slit might directly control axon guidance. Slit is a large extracellular matrix protein expressed almost exclusively by midline cells; some Slit protein is found on axons, and the slit mutant displays a striking axon pathway phenotype. In slit mutants, growth cones enter the midline but never leave it. With the advent of better markers for midline cells it was shown that midline cell fate and differentiation are relatively normal in slit mutant embryos, thus suggesting that Slit might indeed control axon guidance. The key result that led to the insight that Slit is likely to be the Robo ligand came from a further analysis of Comm. Overexpression of Comm produces a robo-like phenotype in which axons freely cross and recross the midline. If the copy number of the comm transgene is increased, a more severe phenotype results in which axons enter the midline but fail to leave it, leading to a midline collapse of the CNS axon scaffold. The strongest comm gain-of-function phenotype is highly reminiscent of the slit loss-of-function phenotype and led to an evaluation of Slit as a candidate Robo ligand (Kidd, 1999 and references).

Dosage-sensitive genetic interactions between slit and robo are a good indicator that the two gene products are functionally related. The CNS was examined in embryos transheterozygous for slit and robo, that is, embryos carrying one mutant and one wild-type copy of each gene. Would Fas II positive fascicles (those stained with the 1D4 mAb) abnormally cross the midline, particularly the most medial pCC pathway? In either slit or robo heterozygotes, few guidance defects were observed in these pathways. However, depending upon the combination of alleles used, 26%-39% of the segments examined in embryos transheterozygous for slit and robo had Fas II-positive axons inappropriately crossing the midline. Such a dosage-dependent, transheterozygous phenotype is a strong indication that Slit and Robo function in the same pathway. Double mutants for slit and robo were prepared. The genetic distance between the two loci predicted recovery of the double mutant chromosomes at a frequency of 1 in 8: when null alleles of both slit and robo are used instead, the recovery rate is 1 in 35, indicating that removal of one copy of each locus decreases viability (Kidd, 1999).

In a late stage wild-type embryo, the cell bodies of the RP neurons are readily visible between the two commissures. In robo mutants, typically one or both RP cell bodies are obscured by the increased number of axons abnormally crossing in the commissures. However, the longitudinal part of the scaffold always remains outside (lateral to) the RP cell bodies. In slit mutants, this is not the case. The effect of removing one copy of slit on the robo phenotype was tested. When the spacing of the longitudinal axons was examined, slit was found to dominantly enhance the robo phenotype, as judged by the presence of segments displaying greater medial constrictions than are ever seen in robo mutants alone. In some instances, an RP cell body could be seen lateral to the axon scaffold. If Slit is the Robo ligand, then the double robo;slit mutant phenotype would be predicted to resemble that of a slit mutant alone (due to slit having the more severe phenotype). Embryos homozygous for a recombinant chromosome carrying null alleles of both slit and robo resemble the slit null phenotype (Kidd, 1999).

The commissureless phenotype produced by high-level overexpression of Robo suggests that Robo responds to a repulsive cue at the CNS midline. Slit is a large extracellular matrix protein secreted by the midline glia. Slit was reported to be transferred to axons (albeit at a low level). The mAb used for Slit detection displays only a very low level of axon staining, making an analysis of putative transfer in robo mutant embryos inconclusive. Robo is primarily localized to growth cones of the longitudinal portion of the axon scaffold. These expression patterns are consistent with Slit being the repulsive ligand for Robo because Robo-positive axons avoid areas of high Slit expression. slit embryos were stained with anti-Robo mAb 13C9 and it was found that Robo-positive growth cones were then present at the midline. Staining of the mature CNS in slit mutants reveals that Robo protein levels are unaffected (unlike in comm gain-of-function embryos), and thus Robo is expressed at high levels along the midline. In wild-type embryos, Slit and Robo both localize to the muscle attachment sites in complementary dorsoventral gradients, further suggesting the possibility of a functional relationship (Kidd, 1999).

The effect of high-level overexpression of slit in all postmitotic neurons was examined. The resulting phenotype resembles the robo loss-of-function phenotype. However, when individual axon fascicles are examined, the slit overexpression phenotype appears stronger than the robo loss-of-function phenotype. In addition to aberrant midline crossing by axons in the innermost pCC pathway as seen in robo mutants, the medial and lateral pathways are also disrupted, sometimes crossing the midline. These results suggest that when Slit is panneurally expressed throughout the CNS, growth cones are impaired in their ability to respond to Slit at the midline. A similar effect is seen when Netrins are expressed panneurally: the panneural overexpression phenotype resembles the loss-of-function phenotype. In both cases (Slit and Netrins), these results support the notion that the localized distribution of the guidance signal is of crucial importance and that approximating an even distribution throughout the CNS is equivalent to no expression at all (Kidd, 1999 and references).

Slit was ectopically expressed on muscles; the guidance and connectivity of motor axons was then examined. The ISNb motor axons normally innervate muscles 6, 7, 12, and 13. When their muscle targets abnormally express Slit, their innervation is greatly perturbed. Most of these motor growth cones stall in the vicinity of these muscles and fail to innervate them. This lack of innervation is reminiscent of what is observed when the chemorepellent Semaphorin II is ectopically expressed by the same muscles. The morphology of muscles 6, 7, 12, and 13 ectopically expressing Slit was examined and they are normal in attachment sites, size, and position relative to one another and to the epidermis. The motor axon phenotype is not suppressed by removal of robo activity, providing further evidence that there is more than one Slit receptor. Robo2 is a potential candidate for mediating the motor axon response to ectopic expression of Slit (Kidd, 1999).

After gastrulation in Drosophila, many myoblasts migrate laterally at least five to six cell body diameters away from the ventral midline. This migration occurs over the dorsal surface of the neuroepithelium. Later, some ventral body wall muscles extend back toward the midline ventrally under the developing CNS, normally attaching to the epidermis underneath the CNS at some distance from the midline. In contrast, in slit mutant embryos many developing muscles are found near and at the midline, stretching across the midline dorsally over the CNS. This defect is not seen in robo embryos, although very rarely a single muscle can be seen extending inappropriately dorsally across the CNS, suggesting that Robo participates in this process in conjunction with at least one other receptor (possibly Robo2). However, in robo mutant embryos the ventral muscles are frequently found attached closer to the midline than in wild type, suggesting that Robo may in part prevent muscles from extending too close to the midline. When slit mutant embryos are rescued by slit-GAL4 driving UAS-slit, the ventral muscle pattern is restored to near wild type, confirming that Slit expression at the midline is required for migration of muscle precursors away from the midline (Kidd, 1999).

The axon guidance defects seen in robo mutant embryos in Drosophila suggest that the primary function of Slit in controlling Robo-mediated midline guidance is as a short-range repellent. Growth cones that express high levels of Robo do not extend away from the midline, but rather they avoid entering and crossing the midline. For example, the pCC growth cone expresses high levels of Robo, and it extends anteriorly near the edge of the midline. In a robo mutant, the pCC growth cone freely crosses and recrosses the midline; in a slit mutant, the pCC growth cone enters the midline and does not leave it. Although it is possible that Slit might also function as a long-range chemorepellent during axon guidance in Drosophila, causing some growth cones to extend some distance away from the midline, at present the strongest genetic evidence in Drosophila is for a short-range function. This is in contrast to its function during mesoderm migration and muscle formation. After gastrulation in Drosophila, many myoblasts migrate laterally away from the ventral midline. The ventral body wall muscles normally attach to the epidermis underneath the CNS but stay some distance from and do not cross the midline. In contrast, in slit mutant embryos, many developing muscles are found near the midline, stretching across the midline dorsally over the CNS. The slit mutant muscle defects are nearly identical to those seen in single minded mutant embryos in which the midline cells are missing (Lewis, 1994). In contrast, in slit mutants, the midline cells are present but do not secrete Slit into the extracellular environment (Kidd, 1999).

Genetic analysis of sim (Lewis, 1994) shows that after gastrulation the midline cells are required for the migration of muscle precursor cells away from the midline. Many of these mesodermal cells normally migrate at least five to six cell body diameters away from the midline. In the sim mutant, the precursors do not migrate away from the midline, presumably due to the absence of a midline-derived long-range chemorepellent. Moreover, in the sim mutant the muscle precursors that extend ventrally toward the midline are not prevented from crossing the midline, presumably due to the absence of a midline-derived short-range repellent. Rather, when these misplaced muscle precursor cells undergo myogenesis, they form abnormal contacts with each other that freely extend across the dorsal midline of the CNS. slit mutant embryos display the exact same midline mesoderm phenotypes as do sim mutant embryos. This suggests that Slit is both the long-range chemorepellent controlling mesoderm migration away from the midline and the short-range repellent preventing muscles from crossing the midline. The Robo receptor appears to play only a minor role in the ability of Slit to direct the long-range migration of muscle precursors away from the midline. Either Robo2 or some other Slit receptor must function as the major muscle receptor for Slit-mediated long-range chemorepulsion (Kidd, 1999 and references).

If commissural growth cones are so attracted to Netrin, if the highest concentration of Netrin is at the midline, and if when growth cones arrive at the midline they meet their homologs from the other side for which they have a high affinity, why do these growth cones ever leave the midline? Although the mechanism is not fully understood, the answer to this question has something to do with the qualitatively different ways in which growth cones respond to Slit. For growth cones near the midline that do not cross it, Slit forms a strong repulsive barrier. But for growth cones that do cross the midline, Slit cannot be such a strong repellent, rather functioning in a more subtle fashion, somehow preventing them from lingering at the midline and driving them across. In the absence of Slit, growth cones enter the midline but do not leave it, extending in a single fused longitudinal tract at the midline. Thus, Slit must be part of the anti-linger mechanism. One thing is certain: the ability of Slit to form a repulsive barrier requires the Robo receptor. Any growth cone that expresses high levels of Robo cannot cross the midline. So in a robo mutant, growth cones freely cross and recross the midline, but they do not stay at the midline. Two inferences follow from these observations: (1) there must be at least one additional Slit receptor that controls midline guidance, and at present Robo2 is the best candidate; (2) because Slit appears to have two different functions (one as a midline repulsive barrier and the second as a midline anti-linger signal), it follows that either Robo2 signals differently from Robo, or alternatively, that the low levels of Robo2 alone (or Robo2 and Robo together) on growth cones crossing the midline give rise to a qualitatively different response as compared to high levels of Robo. Whether these are two qualitatively different negative responses, or alternatively, quantitative differences in a common repulsive mechanism, is not yet clear. Teasing this mystery apart in the future should shed some light on how growth cones make stereotyped and divergent decisions at complex choice points (Kidd, 1999).

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea

Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. The tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. It is proposed that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface (Englund, 2002).

The tracheal system develops from 20 clusters of ectodermal cells, each containing about 80 cells. After invagination and without further cell division, each epithelial cluster sequentially extends primary, secondary, fusion and terminal branches to generate the tubular network that facilitates larval respiration. The regular outgrowth pattern of the primary branches is determined by the localized expression of signaling factors in the surrounding tissues. Among these signals, Branchless (Bnl), a member of the Fibroblast Growth Factor family, first directs the outgrowth of multicellular branches to its site of expression, and it then induces the activation of a set of terminal branching genes in the leading cells of the primary branches. Single terminal cells then form a unicellular branch, migrate over substantial distances and finally stretch and bind to distinct parts of the target tissue to facilitate respiration. A single terminal cell of each ganglionic branch (GB), for example, targets each hemisegment of the embryonic ventral nerve cord (VNC). A cluster of bnl-expressing cells just outside the CNS attracts the GB toward the CNS. The GB cells migrate ventrally along the intersegmental nerve (ISN), but just before reaching the entry point into the CNS, they break their contact with ISN and turn posteriorly to associate with the segmental nerve (SN). This substrate switch is promoted by the expression of adrift (aft), a bnl-induced gene required in the trachea for efficient entry into the CNS. Inside the CNS, the GB1 cell extends over a distance of about 50 µm, from the entry point into the CNS via four different neural and glial substrata to its target on the dorsal side of the neuropil. During the first 20 µm of its journey inside the CNS, the GB1 cell moves its cell body and nucleus along the exit glia, the SN and ventral longitudinal glia towards the midline. The rest of the path is covered by a long cytoplasmic projection that turns dorsally at the midline and reaches the dorsal part of the neuropil by the end of embryogenesis. The signals that guide GB1 migration inside the CNS are not known but the substrata that the GB contacts along its path could potentially provide important guidance cues (Englund, 2002).

The importance of glial substrata in guiding the GB1 inside the CNS was investigated. By genetic ablation experiments, it has been shown that different glial cells provide distinct positional cues to the trachea. Longitudinal glia are first required for GB1 migration towards the midline, whereas midline and channel glia are necessary for inhibiting it from crossing the midline and to make it migrate dorsally through the neuropil. Slit signaling plays a major role in the migration of the GB1 cell. Slit is produced by midline cells and prevents GBs from crossing the midline of the VNC. Slit is also required as an attractant for the outgrowth of the primary, dorsal and visceral branches. The Slit receptors Roundabout (Robo) and Roundabout 2 (Robo2) are both required in the trachea independently of their function in axonal migration. The analysis of the tracheal robo and robo2 mutant phenotypes suggests that they may mediate different responses to the Slit signal. These results provide a first insight into the signaling mechanisms that guide the GB in the CNS, and identify an in vivo system for the study of the bi-functional role of Slit in epithelial cell guidance at the level of single cells (Englund, 2002).

A major determinant of axonal pathways inside the CNS is the repellent signal Slit. Midline cells express Slit, a large extracellular matrix protein that functions both as a short- and long-range repellent, controlling axon crossing at the midline and mesodermal cell migration away from the midline. In axon guidance, the Slit repulsive signal is mediated by the Roundabout (Robo) receptors. Different axons express different combinations of the three receptors, which determine the distance of their projections from the midline along the longitudinal fascicles. The midline crossing phenotypes of GBs in embryos expressing Ricin A in the midline glia (thus ablating these cells) suggests that Slit signaling may also guide GB1 in its turn away from the midline. Embryos expressing GFP under the control of the pan-tracheal btl-GAL4 driver, which drives expression of GAL4 in all tracheal cells from stage11, were double stained with antibodies against GFP and Slit or its receptors, and their expression was analyzed by confocal microscopy. The GB1 cell comes close to the midline source of Slit at early stage 16 but it then turns dorsally and posteriorly at the midline. Slit is also expressed in several other tissues close to the migrating tracheal branches. At early stage 14 in the dorsal side of the embryo, two rows of migrating mesodermal cells that will form the larval heart express Slit. These cardioblasts are in close proximity to the two leading cells of the tracheal dorsal branches (DBs), which also migrate towards the dorsal midline and give rise to the dorsal anastomosis (DB2) and the dorsal terminal branch (DB1). Slit expression is also detected from stage 13 on the surface of the midgut, at the sites of contact of the growing tracheal visceral branches (VBs). Finally, Slit is detected in lateral stripes of epidermal cells adjacent to the growing dorsal trunk (DT) and dorsal branches from stage 13. Are the Slit receptors expressed at this time in the trachea? Robo staining can be detected in all tracheal cells as they invaginate from the epidermis already at stage 11. Its tracheal expression is decreased by stage 13, when it is only weakly expressed in the dorsal trunk. No convincing expression of Robo was detected in the trachea after stage 14, even when serial optical sections of the GB1 cell were analyzed along its path in the CNS. Robo2 is also expressed in all tracheal cells from stage 11 and it then becomes restricted to the dorsal trunk and dorsal and visceral branches by stage 13. In contrast to Robo, which becomes undetectable in the trachea by stage 14, Robo2 expression is stronger and is maintained as late as at stage 16 in the DB1 and DB2 cells at the dorsal midline. Robo3 expression could not be detected in the trachea. The expression of Slit in tissues surrounding the developing trachea and the dynamic expression of its two receptors in different tracheal branches suggests a role for Slit signaling in tracheal branch outgrowth towards their target tissues (Englund, 2002).

The morphology of GB1 allows the separation of its tour in the CNS in two parts. In the first part, starting at the entry point into the CNS, GB1 extends broad filopodial projections and moves its cell body and nucleus ~20 µm towards the ventral longitudinal glia. In the second part, the position of the nucleus remains fixed and the tracheal cell sends a 30 µm long extension that navigates first towards the midline and then turns dorsally through a channel towards the dorsal longitudinal glia. GB1 contacts different groups of glial cells during its migration through the ventral nerve cord. The results from genetic ablation of different glial landmarks provide evidence for an instructive role of these substrates in steering GB1 migration and extension. In particular, the GB1 midline crossing phenotype observed after the ablation of midline glia argues for a repulsive signaling mechanism that redirects the cell from its route towards the midline (Englund, 2002).

The elegant analysis of axonal guidance at the midline of the fly CNS establishes the Slit repellent signal as a major determinant of axonal pathways. A gradient of Slit emanating from the midline prevents axons from crossing the midline through the activation of Robo receptors but it also functions as a long range repellent to position axons in distinct lateral fascicles. This later function is mediated by the expression of different combinations of Robo, Robo2 and Robo3 on axons that take distinct positions along the longitudinal tracts (Englund, 2002).

Mammalian Slit can also function as a positive regulator of axonal elongation and branching of sensory axons from the rat dorsal root ganglia and Slit plays an attractive role for muscles during their extension to muscle attachment sites on the Drosophila epidermis. The molecular mechanism behind the different responses to Slit remains unknown. Repulsion versus attraction could reflect a difference in receptor subunit composition or variations in the cytoplasmic signal transduction machinery of the responsive cells. The complex expression pattern of Slit on several tissues close to the growing tracheal branches, together with the tracheal migration defects in slit mutants, indicates that Slit plays an important role in epithelial cell guidance. Lack of Slit affects the oriented outgrowth of the dorsal, visceral and ganglionic primary branches, the cells of these branches either stall their migration towards the Slit expressing target or they become misrouted. Overexpression of Slit with a mesodermal GAL4 driver is sufficient to attract new branches towards the gut and overexpression of Slit on epidermal stripes running along the dorsoventral axis of the embryo redirects the anteroposterior migration of the dorsal trunk branches along the new sites of Slit expression. This re-orientation phenotype becomes stronger in slit mutants indicating that endogenous slit provides a migration cue for these branches. The analysis of loss-of-function and overexpression phenotypes indicates that Slit is a chemoattractant for the outgrowth of several primary tracheal branches towards their targets (Englund, 2002).

The analysis of GB1 phenotypes in slit mutants argues for a repellent function at the midline. In the absence of functional Slit from the CNS midline, 37% of the GB1 cells cross the midline barrier and ectopic expression of slit on the longitudinal glia causes GB1 to stall or turn prematurely when it approaches the longitudinal tracts. Thus, Slit functions as a bi-functional guidance signal in the trachea. The tracheal phenotypes of slit in primary and secondary branches are not fully penetrant, emphasizing the importance of other signals in guiding the tracheal branches to their targets (Englund, 2002).

In CNS and muscle development Slit function is mediated by the Robo receptors. robo and robo2 are expressed in the trachea; the tracheal phenotypes of robo; robo2 double mutant embryos are very similar to the phenotypes of slit mutants, indicating that the tracheal responses to Slit are mediated by Robo and Robo2. Robo and Robo2 receptors can form homo- and hetero-dimers in vitro and the differences in their expression patterns suggests that they might mediate different responses to Slit. Indeed, the comparison of the phenotypes between the mutants for either of the two receptor genes reveals some intriguing differences. In robo embryos, the GBs erroneously cross the midline, suggesting that slit signaling via robo mediates repulsion away from the midline. In contrast, in robo2 mutants GBs fail to enter the CNS, suggesting that Robo2 may mediate an attractive response to Slit. In addition, the stalls in the migration of the dorsal branches detected in slit embryos were only found in robo2 mutants; no stalling phenotypes were detected in the tracheal branches that did not target the CNS in robo mutants. There is also a difference between the phenotypes generated by overexpression of robo and robo2. Overexpression of Robo in GB1 causes most of the branches to turn away from the midline prematurely. This phenotype is much weaker in embryos overexpressing Robo2, indicating that Robo is a more potent repulsive receptor in the GB. In addition, tracheal overexpression of Robo2 cannot rescue the robo mutant GB phenotype, even though this is possible via the tracheal expression of Robo. This result further indicates that Robo and Robo2 are not identical in their output and they cannot simply substitute for one another (Englund, 2002).

To further investigate whether different receptor complexes may mediate different responses to Slit, advantage was taken of the phenotypes caused by overexpression of Slit in the gut. In wild-type embryos, ectopic Slit can attract new visceral branches to its site of expression. This attractive function of Slit requires Robo2, as evidenced by the observation that overexpression of Slit with the same driver does not induce branch outgrowth in robo2 mutants. Robo alone cannot mediate the attractive response to Slit in the visceral branches -- instead it appears to function as an antagonist of the attractive signal mediated by Slit and Robo 2 in the visceral branches, because the number of new branches induced by Slit in robo mutants is three times higher than the number of branches induced under the same conditions in wild-type embryos (Englund, 2002).

Taken together these results suggest that there are qualitative differences between the cellular responses to Robo and Robo2 activation and that each receptor plays a unique role in the control of tracheal cell migration (Englund, 2002).


GENE STRUCTURE

Gene size - 20 kb

cDNA clone length - 4.4 kb

Exons - nine


PROTEIN STRUCTURE

Amino Acids

There are two splice variants, one with1469 amino acids and a second with 1480. The alternating segment is near the C-terminal end.

Structural Domains

The N terminal contains a signal peptide. This is followed by a four fold duplicated region each one consisting of an amino flanking region, four leucine rich repeats and a carboxyl flanking region (flank-LRR-flank domain). Slit also has seven copies of the EGF motif that participates in extracellular protein-protein interactions. The last EGF repeat is subject to alternative splicing (Rothberg, 1990). The C terminal contains a cysteine rich domain found in other secreted proteins (Rothberg, 1992). An additional laminin related sequence is found separating the sixth and seventh EGF-like domains (Patthy, 1992). The laminin related sequence between the sixth and seventh EGF repeats of Slit are also found in Drosophila proteins Crumbs and Fat (Patthy, 1992).

A slit cDNA was cloned encoding the complete open reading frame (ORF) from the LD 0-22 hr embryonic library. The ORF was sequenced and an additional leucine-rich repeat (LRR) was identified that is absent from the cDNA previously published (Rothberg, 1990). This additional LRR is between the second and third repeats in the first set of tandem LRR arrays. This LRR is present in vertebrate homologs of slit (Brose, 1999). In addition to the extra LRR, eight amino acid differences were identified. All of the substitutions are in LRR regions, but none occur in highly conserved residues of the motifs (Kidd, 1999).


slit: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 April 99

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.