BIOLOGICAL OVERVIEW

Flies that are mutant for commissureless lack all embryonic central nervous system commissures (the tracts of nerve fibers passing from one side to the other of the CNS). To understand the role of comm in generating commissures, it will help first to describe what is known about the ventral midline of the CNS. Organisms as diverse from one another as Drosophila and humans share a nervous system structure based on the midline, a special embryological region around which the future nervous system develops.

The midline in Drosophila is formed during gastrulation by the fusion of mesoectodermal cells lying on either side of the mesoderm. Within each segment (known as a neuromere) of the ventral CNS there develops a scaffold of axon pathways, including a pair of bilaterally symmetric longitudinal axon tracts and the anterior and posterior commissural tracts connecting the two sides of each neuromere. There is, in addition, a pair of nerve roots on either side from which neurons exit to reach the CNS. Special midline cells that separate right and left neurogenic regions are thought to play a key role in the formation of these axon commissures. One of the main tasks of the midline cells is to provide directional cues for incoming axons seeking to cross the midline. The cues provided by midline cells help guide axons along and across the midline. Longitudinal axon fibers form what is termed the intersegmental connective, supporting (ladder-like) the transverse fibers that form the two commissures. During commissure formation, specific midline glial cells actually migrate, migrating over each other and switching positions, so that each has new neighbors, different from the original ones. When the two commissures initially form, they are in close proximity, but soon separation takes place correlating with the migration of a specific glial cell (Klambt, 1991).

The formation of commissures can be divided into four stages.

1. The growth cones that pioneer both commissures appear to be attracted toward the midline from a distance, suggesting a possible role for chemotropism.
2. The growth cones that pioneer the posterior commissure are guided toward medial precursor neurons (MP1) and ventral unpaired medial neurons (VUMs), the neural derivatives of the first and second respectively of the eight cells that serves as midline precursors in each segment. The commissure appears to form in two steps: initially the growth cones extend toward the VUM cells; once reaching them, they then turn anteriorly over the MP1 neurons to cross the midline anterior to the VUM cell nuclei.
3. The growth cones that pioneer the anterior commissure are guided toward the paired anterior midline glia (MGA) and the VUM growth cones. Thus different midline cell types, and possibly different signals, appear to be involved in the pioneering of the anterior and posterior commissures.
4. The last step of axon commissure formation requires the separation of the two commissures. This event involves the posterior migration of the middle midline glia (MGM) over the top of the MGA glia and along the VUM growth cones between the two commissures.

This model predicts that disruptions in any one of these proposed functions should lead to a predictable phenotype (Klambt, 1991).

To better understand the origin of commissures, a mutant screen was carried out for flies with defective commissure formation. commissureless is one of the mutations isolated. Although the midline cells are present and appear to differentiate normally, growth cones that would normally project across the midline remain instead only on their own side. In comm mutants, commissural growth cones initially orient toward the midline, but none actually cross it. Rather, any short medially oriented processes are retracted, and the axons remain exclusively on their own side, producing the commissureless axon guidance phenotype (Seeger, 1993). roundabout (robo) is another mutant that was isolated in the same screen, and leads to the opposite misrouting; some growth cones that normally extend only on their own side project across the midline in robo mutants. The phenotypes of these two genes suggests that they encode components of attractive and repulsive signaling systems at the midline. Mutations in another set of Drosophila genes, the netrins, produce a similar phenotype. Deletion of both NetA and NetB gives rise to thinner and sometimes absent commissures and occasional breaks in the longitudinal tracts. The posterior commissure is more severly affected that the anterior commissure. Netrins do not function as permissive agents to promote axon outgrowth, but rather Netrin localization is required for proper guidance: Netrins function as instructive guidance cues. There is some evidence from phenotypes of ectopic Netrin expression to suggest that in Drosophila Netrins repel axon growth (Harris, 1996, Mitchell, 1996).

In the absence of Commissureless (Comm) function, axons are unable to extend across the central nervous system midline. Comm downregulates levels of Roundabout (Robo), a receptor for the midline repellent Slit, in order to allow axons to cross the midline. comm transcript is expressed at high levels in the midline glia and Comm protein accumulates on axons at the midline. This has led to the hypothesis that Comm moves from the midline glia to the axons, where it can reduce Robo levels. Expression of Comm in the midline cells is unable to rescue the comm phenotype and tagged versions of Comm are not transferred to axons. A re-examination of Comm protein expression and the use of targeted RNA interference reveal that correct midline crossing requires that Comm is expressed in the commissural axons and midline glia. It is suggested that accumulation of Comm protein at the midline spatially limits Comm activity and prevents it from being active on the contralateral side of the central nervous system (Georgiou, 2002). How does Comm limit the expression of Robo? comm is required in commissural neurons for crossing. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway in neurons that cross the midline. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the guidance receptor Robo that in turn depends on the precisely regulated expression of the Comm sorting receptor (Keleman, 2002).

These investigations suggest that Comm expression in the midline cells is not sufficient to allow commissural axons to cross the midline. Furthermore, the Comm protein observed on Comm axons does not originate from the midline. These results suggest that Comm function is necessary in cells other than or in addition to the midline cells. To address directly the question of where comm expression is required, gene expression in specific cell types was disrupted using targeted RNA interference (RNAi). Targeted RNAi uses the same rationale as conventional RNAi in which double stranded RNA from a gene is used to disrupt the function of that gene, but in this modification, gene disruption is targeted to specific cells using the UAS/GAL4 system. To direct the generation of double stranded comm RNA in targeted cells, a construct was generated (UAS-comm-HP) that consists of an inverted repeat of a 375 bp region of comm sequence, with the repeats separated by a linker region, downstream from a GAL4 UAS. GAL4 will drive transcription of this construct resulting in the production of a RNA hairpin loop, thus producing double stranded RNA specific to comm (Georgiou, 2002).

comm-HP was expressed in midline cells and/or neurons to identify if interference with Comm function in these cells affect axon outgrowth in the CNS. In order to look at specific neurons that cross the midline, the Sema2b-taumyc marker was used. This marker labels the cell bodies and axons of two to three neurons per hemisegment within five posterior abdominal segments, A4-8. These laterally positioned neurons normally send their axons immediately across the midline at the anterior margin of the anterior commissure, then turn to project anteriorly within the contralateral longitudinal tract. Thus, in each segment, three Sema2b tracts can be scored: two longitudinal tracts and a commissural tract. Absence of a commissural tract will occur when Sema2b axons from both sides of the CNS fail to cross the midline, whereas a defect in a longitudinal tract will occur when some axons fail to extend across the midline to the contralateral longitudinal pathway. In the wild-type embryo, the Sema2b commissural and longitudinal axon tracts are rarely defective (five tracts affected in 34 embryos; i.e., 0.15 defective tracts per embryo). When comm-HP is expressed, either pan-neurally or at the midline, an increased incidence of failures to form Sema2b tracts is observed. These failures occur as the Sema2b axons now stall and fail to extend across the midline or do not fasciculate with one another appropriately. Counting the number of Sema2b tracts severely affected reveals an increase from an average of 0.15 defective tracts per embryo in wild type to 0.78 tracts affected per embryo when comm-HP is expressed at the midline or 1.96 tracts affected per embryo when comm-HP is expressed in neurons. This phenotype suggests a requirement for Comm in both midline cells and CNS neurons. If the observed defects were due to a limited inhibition of Comm function, it would be expected that the phenotype would be enhanced if Comm levels in the embryo are reduced by removing one copy of the gene. Indeed the phenotype is enhanced when comm-HP is driven in a comm heterozygous background. In these embryos, an average of 2.9 Sema2b tracts are severely affected or absent per embryo, in addition many or all of the Sema2b tracts are reduced in thickness in the embryos. In all cases the same phenotypes are observed using several different UAS-Comm-HP inserts at various sites in the genome. The observed failure of axons to cross the midline and the genetic interaction with comm shows that comm-HP is indeed disrupting comm expression in vivo (Georgiou, 2002).

Rescue and targeted RNAi experiments both suggest a requirement for Comm function not only in the midline cells but also in the commissural neurons. The Comm protein distribution was re-examined in the embryo. A rabbit polyclonal antibody specific for Comm was developed. Using confocal microscopy, it is apparent that Comm is expressed on the commissural tracts and throughout the nerve cord. The staining within the neuronal cell bodies is qualitatively different from that seen at the commissure in that it appears punctate (commissural axons have what appears to be cell surface staining). This punctate staining was previously suggested to result from transfer of Comm to the axons from the midline cells. It is now thought that this transfer does not take place and that the vesicular Comm is produced by the neurons themselves. The data from expression of comm-HP suggests that Comm is required in commissural but not ipsilateral neurons. To identify whether all neurons express Comm, the Sema2b, eg-positive and 15J2 neurons were labelled and colocalisation with Comm was examined. Punctate Comm staining clearly co-localizes with both the Sema2b and eg-positive cell bodies. However no obvious colocalization is apparent when using the 15J2 driver to drive GFP in the vMP2 and dMP2 neurons. It therefore appears, from both the hairpin and co-localization experiments, that these ipsilateral interneurons possess no Comm protein or RNA (Georgiou, 2002).

Comm is one of the few molecules to have been identified as having an important role to regulate the levels of a receptor protein necessary for axon guidance at the midline. It is able to downregulate levels of the Robo protein, which acts as a receptor for the midline-derived axon outgrowth inhibitor molecule Slit. Downregulation of Robo is necessary for axons to cross the midline; however, very little is known about how or in which cells Comm functions. This study shows that Comm is expressed and required in both commissural neurons and midline cells for correct midline crossing (Georgiou, 2002).

To allow commissural axons to cross the midline, Comm specifically downregulates Robo on these axons. Once across the midline, Robo levels increase, suggesting Comm acts only at the midline. It has been suggested previously that Comm acts specifically at the midline via a mechanism whereby Comm is supplied to axons by the midline cells. In this model, Comm transfers from the surface of midline cells to the commissural axons during cell/cell contact. If Comm were then unable to extend far along the axon it could act locally to downregulate Robo in the commissures and allow extension across the midline. This model predicts that Comm can transfer from midline cells to the commissural axons and that Comm expression is required only at the midline. This study has shown that the expression of Comm protein at the midline alone is not sufficient for commissure formation. No rescue of midline crossing is generated when re-expressing wild-type Comm at the midline in comm-null mutant embryos. However, a mild rescue is observed when Comm is re-expressed at the midline in comm^A490 mutants. Since no rescue is seen in null embryos and only partial rescue is seen in comm^A490 embryos, Comm is necessary in other CNS cells and this study shows that Comm is also necessary within the commissural axons themselves. Thus, in the comm^A490 embryos, the truncated protein is expressed in commissural axons in addition to the midline cells and it retains some weak function. The re-expression of Comm protein adds function to the midline cells, thereby slightly increasing the number of axons able to cross the midline. Unfortunately the comm phenotype could not be rescued by overexpression of Comm in all neurons since this results in the efficient downregulation of Robo and promiscuous midline crossing. However a requirement for Comm was revealed in neurons using a targeted RNAi approach (Georgiou, 2002).

Comm transfer from the midline cells was examined using a C terminal GFP-tagged Comm protein, Comm-GFP. The addition of GFP to the Comm protein allowed the re-expressed Comm to be differentiated from that expressed endogenously and to identify whether Comm could move from one cell to another in the embryo. Comm-GFP retains full function, as revealed by its ability to phenocopy the robo phenotype when overexpressed in all neurons. Yet when Comm-GFP was expressed at the midline, no transfer into commissural axons was observed. The Comm-GFP does not appear to be tightly associated with any cellular component, since the protein is clearly able to move within the cells. Comm-GFP was visible within the axons of the neurons derived from the midline cells, e.g. MP1 and the VUMs. Despite clear labelling of these axons, no staining of commissural axons was visible. Thus, Comm is unable to move from the midline cells to the commissural neurons, confirming that Comm must function in cells other than or in addition to the midline cells to regulate commissural axon extension at the midline. Interestingly when Comm is driven in MP1 Comm does not affect MP1 axon guidance, even though Robo is normally required in this neuron to keep it away from the midline (Georgiou, 2002).

To identify which cells in the CNS require Comm function, use was made of the technique of targeted RNAi. This method inhibits the production of Comm by expressing double-stranded RNA, in the form of a hairpin-loop (comm-HP). Used was made of a Drosophila S2 cell assay to test the ability of comm-HP to inhibit comm expression. Here, comm-HP causes a marked reduction in Comm expression, but does not affect the expression of Robo. Expression of comm-HP does not lead to general, nonspecific axon guidance defects on PNS axons. Furthermore, when comm-HP is driven in specific ipsilateral neurons, guidance of these neurons is unaffected. However, expression of comm-HP in commissural neurons does lead to midline crossing defects and these defects are enhanced when one copy of comm is removed. This suggests that comm-HP specifically inhibits Comm expression in vivo (Georgiou, 2002).

Several conclusions can be drawn from the targeted RNAi experiments. (1) Midline crossing errors are observed when Comm expression is inhibited specifically in neurons. Thus, comm transcript and Comm activity are present in neurons. (2) An identical yet milder phenotype is observed when driving the hairpin specifically at the midline. Comm function is therefore required in both neurons and the midline glia. The identical nature of the neuronal and glial phenotypes suggests that comm within both of these cell types is required for the same process, i.e. allowing a growth cone to cross the midline. (3) The lack of any guidance errors in ipsilateral neurons, when driving the hairpin either throughout the nervous system or in specific neurons, suggests that Comm activity is not required in these ipsilateral neurons (Georgiou, 2002).

The CNS defects produced by the expression of Comm-HP in the CNS are fairly mild when observed with BP102, a reduction in the size of the commissural tracts and some longitudinal breaks can be observed. These phenotypes are more obvious when the outgrowth of a smaller population of neurons, the Sema-2b neurons, are examined. Here, there are clear defects in extension across the midline and stalling of some commissural axons. These axons also appear to fail to extend in the longitudinals. This affect is likely to be specific to Comm, since removal of one copy of comm enhances this phenotype. However, it is apparent that comm-HP is unable to fully inhibit comm in all cells. This might explain why the phenotype differs from that seen in comm loss-of-function embryos. In comm mutants, when all cells lose Comm function and all axons do not cross the midline, the default is to extend in the longitudinals. In the comm-HP mutants, a subset of commissural axons fail to extend and thus stall. This may affect the extension of neighboring axons producing the longitudinal defects seen with BP102. Expression of comm-HP in neurons or midline cells gives rise to the same phenotype, suggesting that a slight reduction in Comm levels at the midline also leads to crossing failures. By contrast, driving large amounts of fully functional Comm-GFP at the midline has no effect on guidance. This would suggest that a certain threshold level of Comm is required at the midline (Georgiou, 2002).

These results suggest that the Comm protein identifiable in neuronal cell bodies is not the result of transfer from midline cells. This punctate vesicular neuronal staining is restricted to commissural neurons, with no protein observed within ipsilateral neurons. Thus, the targeted RNA interference and protein localization results suggest that Comm is not present in ipsilateral neurons. It is therefore possible that the presence or absence of neuronal Comm determines whether or not an axon crosses the midline. Indeed expression of Comm in the normally ipsilateral projecting Ap neurons using an Ap-GAL4 driver causes all the Ap neurons to cross the midline. Thus, individual ipsilateral neurons can be converted to midline crossing neurons by the introduction of comm activity, suggesting that the presence of neuronal Comm dictates axon pathway choice (Georgiou, 2002).

Comm protein accumulates on the commissural portion of the contralaterally projecting axons. The appearance of this staining is very different to the vesicular localization seen in the cell body. Rather it appears that Comm protein can accumulate at the cell surface within the commissure. Perhaps Comm protein is transported from the cell bodies within the vesicles to be presented at the cell surface during commissure formation. Furthermore, Comm protein does not seem to extend beyond the midline region onto the contralateral segments of the axon. Comm thus appears to be targeted to the commissural tract or somehow sequestered in this region. This may either concentrate its activity in this region and/or serve as a device for preventing Comm activity spreading further along the axon. In this way Comm could act to prevent Robo-mediated sensitivity to the midline Slit signal in commissural axons prior to crossing, but be unable to act on Robo once the axon has extended beyond the midline. It is possible that an interaction between Comm at the midline and Comm in the neuron provides the means to trap or concentrate Comm at the commissure. Evidence also exists of the ability of Comm to bind itself through its extracellular domain (Georgiou, 2002).

In summary, it is suggested that the presence of Comm in the commissural neurons may encourage midline crossing. This tendency is promoted by Comm activity in the midline cells. The combined action is predicted to allow inhibition of Robo activity specifically in the commissural neurons, allowing growth across the midline. Comm protein accumulates at the axon surface within the commissural region, using a mechanism that is likely to involve Comm in the midline glia. However, Comm activity does not extend beyond the midline, allowing Robo levels to increase at the growth cone surface and initiate a sensitivity to the midline inhibitor Slit that encourages extension away from the midline and prevents re-crossing (Georgiou, 2002).

GENE STRUCTURE

Exons - 2

PROTEIN STRUCTURE

Amino Acids - 370

Structural Domains

Hydropathy analysis of the Comm amino acid sequence indicates a single membrane spanning domain of 29 amino acids. This tranmembrane domain is flanked by charged amino acids on both the N- and the C-terminal sides. While most transmembrane domains are alpha helical, this region of Comm is predicted to form a ß-sheet structure. There is no indication of a cleavable signal sequence at the N-terminus of Comm. The only other hydrophobic stretch in Comm is found at the extreme C-terminus. The presence of a transmembrane domain without an N-terminal signal sequence raises questions about the orientation of the Comm protein within the membrane. The distribution of charged amino acids immediately preceding and following the transmembrane, however, gives clues as to the orientation of the protein. The 15 amino acids preceding the TM domain of Comm have a net negative charge, while the 15 amino acids following the transmembrane domain have a net positive charge. This indicates that the Comm protein almost surely adopts a type I orientation. Comm's relatively large cytoplasmic domain contains a short Adaptin (AP-2) recognition sequence commonly found among endocytosing molecules. Besides these features, the Comm sequence is novel (Tear, 1996).

date revised: 19 June 2024 

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