The leucine rich region (LRR) is a common motif of extracellular proteins, Toll among others. The LRR often functions in adhesion, in protein-protein interactions, and developmentally as a receptor-binding ligand (Rothberg, 1990). The EGF repreat is found in many vertebrate receptors and ligands (Rothberg, 1988). The C-terminal cysteine rich domain is found in agrin, laminin, and perlican (Rothberg, 1992). The domain is involved in mediating interactions among extracellular proteins.

The Drosophila homolog of vertebrate classic cadherins is a transmembrane protein with similarity to vertebrate classic cadherins. DE-cadherin is distinguishable from its vertebrate counterparts by a large insertion with local sequence similarity to Fat, laminin A chain, Slit, and neurexin I at the proximal region of the extracellular domain (Oda, 1994).

C. elegans Slit homologs

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which act together to direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development (Hao, 2001).

The predicted SLT-1 protein consists of 1410 amino acids and shares a common domain structure and high sequence similarity with Drosophila Slit (41% amino acid identity) and all three mammalian Slit family members (39%-41% identity). All Slit proteins contain a putative signal sequence, four tandem arrays of leucine-rich repeats (LRRs), seven to nine EGF repeats, and a cysteine knot. C. elegans Slit has extensive similarity to Drosophila Slit and mammalian Slit2 in LRR-2 and LRR-4 (52%-60% identity); less similarity in LRR-1 and LRR-3 (25%-40% identity), and substantial similarity in all EGF repeats (28%-63% identity). C. elegans Slit and Drosophila Slit possess seven EGF repeats each, whereas nine EGF repeats are present in all three vertebrate Slits. The absence of hydrophobic anchor sequences in all Slits suggests that they encode large secreted proteins (Hao, 2001).

Axon migrations are guided by extracellular cues that can act as repellants or attractants. However, the logic underlying the manner through which attractive and repulsive responses are determined is unclear. Many extracellular guidance cues, and the cellular components that mediate their signals, have been implicated in both types of responses. Genetic analyses indicate that MIG-10/RIAM/lamellipodin, a cytoplasmic adaptor protein, functions downstream of the attractive guidance cue UNC-6/netrin and the repulsive guidance cue SLT-1/slit to direct the ventral migration of the AVM and PVM axons in C. elegans. Furthermore, overexpression of MIG-10 in the absence of UNC-6 and SLT-1 induces a multipolar phenotype with undirected outgrowths. Addition of either UNC-6 or SLT-1 causes the neurons to become monopolar. Moreover, the ability of UNC-6 or SLT-1 to direct the axon ventrally is enhanced by the MIG-10 overexpression. An interaction between MIG-10 and UNC-34, a protein that promotes actin-filament extension, is important in the response to guidance cues and MIG-10 colocalizes with actin in cultured cells, where it can induce the formation of lamellipodia. It is concluded that MIG-10 mediates the guidance of AVM and PVM axons in response to the extracellular UNC-6 and SLT-1 guidance cues. The attractive and repulsive guidance cues orient MIG-10-dependant axon outgrowth to cause a directional response (Quinn, 2006).

A model predicts that MIG-10 activity is involved in the process of polarized axon growth. Although it is difficult to directly measure whether MIG-10 activity is polarized in C. elegans neurons, MIG-10 can induce lamellipodia in cultured cells. The asymmetric formation of lamellipodia has been implicated as an initial step toward polarized axon growth. The observations are also consistent with those described for two vertebrate MIG-10 orthologs, RIAM and lamellipodin. Overexpression of RIAM induces cell spreading and lamellipodia formation, and overexpression of lamellipodin has been shown to increase the velocity of actin-based protrusive activity in fibroblasts. RIAM interacts with Rap1, Ena/vasp proteins, and profilin [14]. Lamellipodin is localized to the tips of filopodia in fibroblasts and neuronal growth cones and can interact with Ena/vasp proteins. Interestingly, lamellipodin can interact with PI(3,4) phosphoinostide, a molecule that is asymmetrically localized in response to chemotactic cues in several types of . The ability of lamellipodin to interact with an asymmetrically localized molecule suggests a potential mechanism that could mediate the polarization of MIG-10 activity in response to guidance cues (Quinn, 2006).

The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. This study shows that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. It is concluded that mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance (Chang, 2006).

Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. This study shown with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, axons can be cell specifically rerouted, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. These experiments provide genetic in vivo evidence for the 'HS code' hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions (Bülow, 2008).

MIG-10 functions with ABI-1 to mediate the UNC-6 and SLT-1 axon guidance signaling pathways

Extracellular guidance cues steer axons towards their targets by eliciting morphological changes in the growth cone. A key part of this process is the asymmetric recruitment of the cytoplasmic scaffolding protein MIG-10 (lamellipodin). MIG-10 is thought to asymmetrically promote outgrowth by inducing actin polymerization. However, the mechanism that links MIG-10 to actin polymerization is not known. This study identified the actin regulatory protein ABI-1 as a partner for MIG-10 that can mediate its outgrowth-promoting activity. The SH3 domain of ABI-1 binds to MIG-10, and loss of function of either of these proteins causes similar axon guidance defects. Like MIG-10, ABI-1 functions in both the attractive UNC-6 (netrin) pathway and the repulsive SLT-1 (slit) pathway. Dosage sensitive genetic interactions indicate that MIG-10 functions with ABI-1 and WVE-1 to mediate axon guidance. Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations suggest that MIG-10 interacts with ABI-1 and WVE-1 to mediate the UNC-6 and SLT-1 guidance pathways (Xu, 2012).

Zebrafish Slit homologs

cDNA fragments have been isolated of the zebrafish slit2 and slit3 homologs. Both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system: both in the axial mesoderm; slit2 in the anterior margin of the neural plate, and slit3 in the polster. This gives rise to the hatching gland cells later in development, at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, Slit2 was overexpressed in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos, which carry the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impairs the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and results in cyclopia. These results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation (Yeo, 2001).

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. A small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial 'bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expands the glial bridges and causes defasciculation of the POC, consistent with a 'channeling' role for these repellent molecules. By contrast, reducing Slit1a function leads to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescues POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. Hh signaling appears to affect axon guidance indirectly through its role in patterning of the midline, including the formation of the glial bridge and the regulation of axon guidance-molecule expression. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain (Barresi, 2005).

Members of the Slit family of secreted ligands interact with Roundabout (Robo) receptors to provide guidance cues for many cell types. For example, Slit/Robo signaling elicits repulsion of axons during neural development, whereas in endothelial cells this pathway inhibits or promotes angiogenesis depending on the cellular context. This study shows that duplicated miR-218 genes are intronically encoded in slit2 and slit3 and that the two mir-218 microRNAs suppresses Robo1 and Robo2 expression. The data indicate that miR-218 and multiple Slit/Robo signaling components are required for heart tube formation in zebrafish and that this network modulates the previously unappreciated function of Vegf signaling in this process. These findings suggest a new paradigm for microRNA-based control of ligand-receptor interactions and provide evidence for a novel signaling pathway regulating vertebrate heart tube assembly (Fish, 2011).

Slit homologs in Xenopus

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. This study shows that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. These findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion (Piper, 2006).

The activation of selected MAPK proteins was monitored. To do this, Slit2-CM was applied to retinal cultures for 5 min, then active (phosphorylated) MAPK proteins were labeled with phospho-specific antibodies and digital quantitation of immunofluorescence was used to monitor relative changes in signal intensity. Five minutes was selected because a collapse assay time course showed that 40.3% of growth cones collapsed 5 min after Slit2-CM addition. Thus, it was reasoned that this time point would be optimal for assessing the signaling events leading to collapse, prior to the occurrence of overt collapse. Using a phosphospecific p38 antibody (p38-P), it was found that Slit2-CM stimulated an 80% increase in signal intensity compared to controls. The MAPK p42/p44 can be directly phosphorylated by MEK1/MEK2. After Slit2-CM application, a 2-fold rise was seen in phospho-p42/p44 fluorescent intensity. Slit2-CM did not have a significant effect on the fluorescent intensity of either p38 or p42/p44 (Piper, 2006).

The finding that cofilin immunoreactivity markedly increases in growth cones in response to both Slit2-CM and Sema3A suggests the intriguing idea that the local synthesis of actin-depolymerizing proteins contributes to repellent-induced collapse. Cofilin, a member of the ADF/cofilin family of actin-depolymerizing molecules, is a small protein of 19 kDa, and its mRNA and protein have been detected in a variety of neuronal axons and growth cones. Importantly, in vitro experiments have implicated ADF/cofilin in the control of chick retinal growth cone filopodial dynamics in response to brain-derived neurotrophic factor, and moreover, axons of cultured rat DRG axons have been shown to synthesize cofilin 1. However, the data provide a potential link between cue-induced growth cone behavior and local cofilin translation. Two Xenopus cofilin proteins have been reported, and their high level of identity at the amino acid level suggests that they are allelic variants, as Xenopus has a tetraploid genome. Locally controlling their synthesis may provide one means of influencing the balance of actin stability; in the case of Slit, perhaps resulting in an increase in growth cone cofilin and a subsequent shift toward actin depolymerization and hence growth cone collapse. This may also be consistent with respect to recent reports linking cofilin activity to shrinkage of dendritic spines in long-term synaptic depression and axon growth and neuronal morphogenesis. The idea that the mRNAs of cytoskeletal regulators are key targets for cue-induced translation is in line with recent evidence demonstrating that local synthesis of the small GTPase RhoA is necessary for Sema3A-induced collapse in mammalian sensory growth cones (Piper, 2006).

The data also suggest that regulation of cofilin in response to Slit also occurs at a posttranslational level; Slit2-CM caused a significant decrease in the growth cone signal intensity of phosphorylated cofilin. A similar phenomenon has been reported for Sema3A, which induces a rapid (1 min) increase in phospho-cofilin in embryonic murine DRG neurons, followed by a dramatic decrease after 5 min. This suggests that a cycle of inactivation/activation of cofilin, regulated by kinases such as LIM-kinase and phosphatases such as Slingshot may play a role in collapse. This may also be related to calcium (Ca2+) signaling, as high levels of focally released caged Ca2+ activate calcium-calmodulin-dependent protein kinase II (CaMKII) to induce growth cone attraction, while lower levels cause repulsion via a mechanism involving phosphatases such as calcineurin (CaN). Furthermore, in vitro cell culture studies have shown that the phosphatase responsible for dephosphorylating cofilin, Slingshot, is activated in a Ca2+/CaN-dependent fashion. Thus, Slit may increase the local actin-severing activity of cofilin in growth cones by triggering two parallel mechanisms -- local synthesis of new cofilin and dephosphorylation of existing cofilin -- that act in concert to promote changes in the cytoskeleton (Piper, 2006).

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon's cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). This study assesses and contrasts the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. In vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, it is proposed that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites (Hocking, 2010).

Identification of mammalian Slit homologs

Mammalian homologs of the slit gene (human Slit-1, Slit-2, Slit-3, and rat Slit-1) have been identified. Each Slit gene encodes a putative secreted protein, which contains conserved protein-protein interaction domains including leucine-rich repeats (LRR) and epidermal growth factor (EGF)-like motifs, like those of the Drosophila protein. Northern blot analysis has revealed that the human Slit-1, -2, and -3 mRNAs are exclusively expressed in the brain, spinal cord, and thyroid, respectively. In situ hybridization studies indicate that the rat Slit-1 mRNA is specifically expressed in the neurons of fetal and adult forebrains. These data suggest that Slit genes form an evolutionarily conserved group in vertebrates and invertebrates, and that the mammalian Slit proteins may participate in the formation and maintenance of the nervous and endocrine systems by protein-protein interactions (Itoh, 1998).

A putative human sli homolog, SLIT1, has been identified by EST database scanning. A second human sli homolog, SLIT2, and its murine homolog Slit2 have now been identified. Both SLIT1 and SLIT2 proteins show approximately 40% amino acid identity to Drosophila Slit and 60% identity to each other. In mice, both genes are expressed during CNS development in the floor plate, roof plate and developing motor neurons. Since floor plate represents the vertebrate equivalent to the midline glial cells, a conservation of function is predicted for these vertebrate homologs. Each gene shows additional but distinct sites of expression outside the CNS suggesting a variety of functions for these proteins (Holmes, 1998).

It is well established that leucine-rich repeat (LRR) proteins such as Connectin, Slit, Chaoptin, and Toll have pivotal roles in neuronal development in Drosophila as cell adhesion molecules. However, to date, little information concerning mammalian LRR proteins has been reported. LRR proteins of the mouse brain were sought, based on the assumption that fundamental mechanisms are conserved between different species. A neonatal mouse brain cDNA library was probed with a human partial cDNA encoding LRR protein as a probe. Two independent cDNAs encoding LRR proteins, designated NLRR-1 and NLRR-2 (neuronal leucine-rich repeat proteins) were obtained. The whole sequence of NLRR-1 and partial sequence of NLRR-2 were examined. Sequence analysis shows that these two clones are about 60% homologous to each other, and that NLRR-1 protein is a transmembrane protein. Northern blot analysis and in situ hybridization histochemistry shows that both NLRR-1 and NLRR-2 mRNAs are expressed primarily in the central nervous system (CNS); NLRR-1 mRNA is also detected in the non-neuronal tissues such as cartilage, while NLRR-2 mRNA expression is confined to the CNS at all developmental stages. These results suggest that there is at least one LRR protein family in the mouse and that these molecules may play significant but distinct roles in neural development and in the adult nervous system (Taguchi, 1996).

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, at least two proteins have been isolated from rat brain that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction. These probes enabled the isolation from a rat brain cDNA library of a 4.1-kilobase clone that encodes two of the peptide sequences and represents the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. These peptides were derived from proteins that are members of the Slit/MEGF protein family: they share a number of structural features, such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs. In Drosophila, Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and the peptide sequences in this study correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using the affinity matrix. Northern analysis demonstrates the presence of two mRNA species of 8.6 and 7.5 kilobase pairs, using probes based on both N- and C-terminal sequences; in situ hybridization histochemistry shows that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where the presence of glypican-1 mRNA and immunoreactivity has been demonstrated. Therefore, these results indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis (Liang, 1999).

The amino acid sequence of the Slit proteins can be divided into four domains. These consist of four N-terminal LRRs, seven to nine epidermal growth factor-like repeats (seven in Drosophila and nine in vertebrates), a motif with a high degree of identity to agrin, laminin, and perlecan (designated the ALPS domain), and a C-terminal cysteine-rich domain. LRRs are found in a number of intracellular and extracellular proteins and contribute to protein-protein interactions and cell adhesion. The epidermal growth factor-like motif has been identified as an extracellular binding domain involved in cell adhesion and receptor-ligand interactions; the ALPS domain is responsible for protein-protein interactions and self-aggregation of agrin, laminin, and perlecan, and the cysteine-rich domain is considered to be essential for dimerization of proteins such as von Willebrand factor. It is likely that the Slit proteins function to link multiple ligands and thereby mediate cell interactions. In view of the complex domain structure of the Slit proteins and their heparitinase-sensitive interactions with glypican-1, it will be important to identify which protein domain(s) may also be involved in this binding. Recent genetic and biochemical studies of Drosophila and mammals have demonstrated that Slit proteins bind Robo, a repulsive guidance receptor on growth cones. The identification of interactions between glypican-1 and Slit proteins therefore not only provides the most direct evidence yet available for an involvement of glypican-1 in nervous tissue development, but also suggests a possible regulatory mechanism underlying the dual functionality of Slit proteins with respect to axon guidance and cell differentiation (Liang, 1999 and references).

Mammalian Slit homologs and axonogenesis

Continued: slit Evolutionary homologs part 2/2

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

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