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).

GENE STRUCTURE

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).

date revised: 15 April 99

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