Effects of Mutation or Deletion

While commissural axons pathways are absent in comm mutants, other aspects of CNS development appear normal, including formation of longitudinal axon pathways, nerve roots, peripheral axon pathways, and peripheral sensory neurons. The mutant phenotype appears to be quite specific to the midline of the CNS, since the pattern for cuticle, segmentation, and muscles are normal. The normal axon projection of the ventral unpaired medial neurons (VUMs) is particularly interesting because the VUM cell bodies are located at the midline and extend growth cones that bifurcate and project away from the midline and toward the intersegmental nerve root. The fact that this happens in a relatively normal fashion in comm mutants suggests that there is no physical barrier preventing growth cones from extending near the midline. Later in embryogenesis, in the absence of commissural axon pathways and their surrounding nonneuronal cells holding the two sides of the CNS together, the CNS starts to unzip as it splits down the middle into two halves. As the CNS splits, the VUM axons stretch and then are severed on one side or the other (Seeger, 1993).

In comm mutant embryos, some commissural axons initally extend a short distance and then stop. These medially directed axons appear to be quickly retracted, as the axons from these and the rest of what are normally commissural neurons instead turn and extend rostrocaudally in longitudinal axon pathways on their own side. In this way, commissures across the midline do not form. Instead, axons of identified neurons that normally cross the midline make ipsilateral projections that appear relatively normal except for their failure to cross the midline (Seeger, 1993).

Spitz group genes and commissureless are involved in development of the brain commissure that interconnects the fly's two brain hemispheres and longitudinal pathways that connect the brain to the ventral nerve cord. Early in neurogenesis two bilaterally symmetrical cephalic neurogenic regions form. Initially, they are separated from each other and from the ventral nerve cord. Axons that project towards the midline in close association with an interhemispheric cellular bridge pioneer the commissure. A chain of longitudinal glial cells pioneer the descending pathway to the subesophageal ganglion. Both the commissure and descending pathway are dependent on cells of the ventral (or CNS) midline. Knock out mutations of the commissureless gene result in a marked reduction of the brain commissure. Mutation of the single-minded gene and other spitz group genes result in the absence or aberrant projection of longitudinal pathways (Therianos, 1995). For additional information, see axonogenesis.

Cell death was examined in the midline of Drosophila embryos. Approximately 50% of cells within the anterior, middle and posterior midline glial (MGA, MGM and MGP) lineages die by apoptosis after separation of the commissural axon tracts. Glial apoptosis is blocked in embryos deficient for reaper, where greater than wild-type numbers of midline glia (MG) are present after stage 12. Quantitative studies reveal that MG death follows a consistent temporal pattern during embryogenesis. Apoptotic MG are expelled from the central nervous system and are subsequently engulfed by phagocytic hemocytes. MGA and MGM survival is apparently dependent upon proper axonal contact. In embryos mutant for the commissureless gene, a decrease in axon-glia contact correlates with a decrease in MGA and MGM survival and accelerates the time course of MG death (Sonnenfeld, 1995).

Of special interest in understanding the function of roundabout are the dosage-sensitive interactions between robo and commissureless. In comm mutant embryos, commissural growth cones initially orient toward the midline but then fail to cross it and instead recoil and extend on their own side. Thus comm mutation has a complementary phenotype to that of robo; that is, too few axons cross the midline (Seeger, 1993).

Of particular interest to understanding the role of Comm protein in the development of commissural neurons is the fact that neurons crossing the midline are decorated with Comm, but these cells to not in fact express comm (For an update on this observation see the Biological Overview section, Georgiou, and Tear, 2002). During stage 12, Comm protein expression is restricted to a subset of midline cells, mimicking the expression seen for the transcript. The dorsoventral location of these cells suggests that they are the anterior and medial pair of midline glia. Interestingly, the first commissural axons that contact the comm-expressing midline glia also accumulate Comm protein along their axons, despite no COMM transcript being observed in their neuronal cell bodies. Both N- and C-terminal-specific anti-Comm antibodies stain these commissural axons. This axonal presence of Comm protein is restricted to those axons that contact comm-expressing midline cells. It thus appears as if the entire Comm protein may be exported from the midline cells to decorate these commissural axons (Tear, 1996).

Expression of a truncated COMM protein, missing the last two third of the cytoplasmic domain, results in essentially no commissures. Nevertheless, transfer of Comm protein from midline glia to commissural axons still takes place in these mutant embryos, but instead of decorating the midline portions of commissural axons (pathways that do not form in these mutant embryos), accumulation of Comm protein is observed in longitudinal axon pathways. Two important conclusions result from this observation. (1) Commissural growth cones make contact with midline cells in comm mutant embryos, otherwise no Comm protein could accumulate on their axons. This is consistent with a model in which Comm protein is required for axons to cross the CNS midline but not for attracting commissural axons to the CNS midline. (2) These observations suggest that transfer of Comm protein from midline cells to commissural axons is not sufficient for Comm function. Presumably, portions of the Comm cytoplasmic domain, deleted in the truncation mutants, encode essential functions (Tear, 1996).

comm encodes a novel surface protein expressed on midline cells. Comm is thought to be a midline attractant. As commissural growth cones contact and traverse the CNS midline, Comm protein is apparenty transferred from midline cells to commissural axons. The double mutant for robo and comm is indistinguishable from robo. The expression of Robo protein was examined in comm hypomorphic alleles. Normally, Robo is expressed at very low levels on commissural axons and at high levels on longitudinal axons. In mutant embryos, Robo expression in the longitudinal tracts appears as if it might be higher than normal. Interestingly, in comm hypomorphic alleles, the occasional thin commissures express Robo protein at levels that are higher than normally seen in the commissures and closer to what is typically seen in the longitudinal tracts. This result was the first hint that Comm protein might function by suppressing Robo expression on commissural axons, thus allowing axons to cross the midline, an event that occurs as a default state in the absence of both proteins (Kidd, 1998).

To test the hypothesis that increased expression of comm might lead to a robo-like phenotype comm was expressed pan-neurally. A continuous range of robo-like phenotypes is generated under these conditions. The range of phenotypes reveals the comm-gain-of-function phenotype to be dosage-sensitive, since the severity increases in embryos expressing two copies of comm, as compared to those expressing only one. In wild-type embryos, the pattern of Robo protein expression begins in the neuroepithelium as well as in some lateral epidermal stripes, but is conspicuously absent from the midline region. In comm gain-of-function embryos, Robo expression in the neuroepithelium is greatly reduced or absent, while the epidermal expression outside the nervous system is maintained. This same pattern can be observed around the time when the first growth cones are extending. In wild-type embryos during stages 12 and 13, no Robo is seen at the midline, but there is a high level of Robo expression on ipsilaterally projecting growth cones and a significant level throughout the neuroepithelium. In contrast, in comm gain-of-function embryos, such growth cones lack Robo protein, and the neuroepithelium expresses greatly reduced levels of Robo (Kidd, 1998).

The Comm transmembrane protein is required at neuromuscular synaptogenesis. All muscles in the Drosophila embryo express Comm during the period of motoneuron-muscle interaction. It is endocytosed into muscles before synaptogenesis. In comm loss-of-function mutants, motoneuron growth cones fail to initiate synaptogenesis at target muscles. This stall phenotype is rescued by supplying wild-type Comm to the muscles. Cytoplasmically truncated Comm protein fails to internalize. Expressing this mutant protein in muscles phenocopies the synaptogenesis defects of comm mutants. Thus, synaptogenesis initiation is positively correlated with endocytosis of Comm in postsynaptic muscle cells. It is proposed that Comm is an essential part of the dynamic cell surface remodeling needed by postsynaptic cells in coordinating synaptogenesis initiation (Wolf, 1998).

The first hint that muscle-supplied Comm protein plays a role during interactions between motoneuron growth cones and muscles came from analysis of neuromuscular development in three comm mutant alleles: comm 5/comm 5 (null), comm 1/comm 1 (loss of function due to truncation of the distal cytoplasmic domain that includes the putative Adaptin recognition site and comm 5 /comm 1 (transheterozygous). These mutant embryos exhibit major axon pathway defects at the CNS midline, presumably due to the loss of the normal Comm expression in the midline glia. The morphological development of muscles proceeds normally in all comm loss-of-function alleles. Muscle cell surface molecules Toll, Fasciclin II, Fasciclin III, and Connectin are all found on the surface of muscles that normally express them. In comm 1/comm 1, the mutant Comm with partial deletion of its cytoplasmic domain fails to internalize. In all three alleles, Comm-independent endocytosis is still observed in muscles. Despite the axon pathway defects at the CNS midline, the nerves containing motoneuron axons are seen to normally extend out of the CNS in all mutant alleles. Subsequent pathway selections by the axons are indistinguishable from wild type until their growth cones reach respective target muscles. Thus, outside the CNS, Comm does not function as an axon guidance molecule. However, on contacting the target muscles, the motoneuron growth cones fail to initiate synaptogenesis. In the hour 18 wild-type embryos, Synaptotagmin, a synaptic vesicle protein, accumulates as punctate bodies at the axon terminals undergoing synaptogenesis. In contrast, the motor axon terminals in the Comm null mutants at the same stage merely show low levels of diffused Synaptotagmin staining. The latter condition is similar to the stage at which the axons are still navigating through the non-target areas. In the majority of the cases, the growth cones stall at or just short of their normal targets. In the remaining cases, they extend beyond the normal stopping points without apparently settling on alternative targets. These phenotypes are evident at frequencies of 14%-63% in all five nerves (ISN, SNa, SNb, SNc, SNd) and persist even past hour 18, the normal period of synaptogenesis. ISN, SNc and the posterior branch of SNa normally contain motoneuron axons that do not cross the CNS midline. In such mutants, no motoneuron axons apparently cross the CNS midline. Yet the growth cones in ISN, SNa and SNc all exhibit the defects similar to those of the other motoneuron groups. The three allelic situations show little variation. Therefore, the neuromuscular synaptogenesis defects are likely to be a direct effect of disrupting muscle-derived Comm protein (Wolf, 1998).

Is Comm internalization necessary for synaptogenesis initiation, as hinted by the situation with the comm 1/comm 1 loss-of-function mutant allele? A complication with this mutant allele is that the modified Comm protein contains a novel amino acid sequence with potentially novel functions. Noting that Comm’s cytoplasmic domain contains a putative Adaptin recognition sequence, it was hypothesized that Comm endocytosis would stop without its cytoplasmic domain. The significance of Comm internalization was tested by misexpressing cytoplasmically truncated Comm in muscles of wild-type embryos. Following the misexpression of truncated Comm, which lacks nearly the entire cytoplasmic domain, high levels of Comm immunoreactivity are seen on the muscle surfaces, while the number of Comm-positive endosomes drops to roughly 25% of the normal level. It therefore appears that not only does this truncated Comm fail to internalize in muscles, but it also acts as a dominant negative form of the protein that prevents internalization of most of the endogenous Comm. Interestingly, the motoneuron growth cones fail to initiate synaptogenesis and often stall just short of the target muscles. Except for SNa, which for an unknown reason exhibits a less severe case, the defects are virtually identical in both rates and appearance to the comm loss-of-function mutants. Thus, the cytoplasmically truncated Comm apparently works as a ‘dominant negative’ factor for synaptogenesis initiation. Furthermore, lack of Comm-mediated endocytosis is once again correlated with a lack of synaptogenesis initiation (Wolf, 1998).

How is the timing of Comm endocytosis regulated? A likely candidate seems to be the arrival of motoneuron growth cones into the muscle fields. To determine if the motoneuron growth cones provide cues, the prospero mutation was used to delay motoneuron growth cone extension. At hour 18, even in the absence of growth cone-muscle contacts, the Comm endocytosis proceeds normally. This suggests that the timing of Comm-mediated endocytosis is independent of motoneuron growth cones. Comm is likely to bind to a factor(s) closely associated with muscles prior to internalization. This process of Comm activation is regulated temporally so that it would, under the normal condition, coincide with synaptogenesis initiation between appropriate pairs of motoneuron growth cones and muscle targets (Wolf, 1998).

The most significant conclusion from the genetic analysis is that there is a positive correlation between Comm endocytosis and synaptogenesis initiation in the embryonic neuromuscular system. Before synaptogenesis initiation, the Comm protein is expressed on the muscle surface. During this time, motoneuron growth cones that contact the muscles extend past them without stopping to innervate. This could be explained by some muscle-associated factors that inhibit synaptogenesis initiation by the growth cones. As the growth cones that are destined to innervate the muscles approach, Comm endocytosis reaches its peak. Two models are presented for Comm’s role that are consistent with all the observations so far. The first model, which is called the ‘janitor’ model, proposes that Comm endocytosis itself serves as a critical element of the membrane remodeling that accommodates synaptogenesis. The second model proposes that Comm’s key role is to mediate a transmembrane signal transduction event. The ‘janitor’ model proposes the following sequence of events: (1) Comm’s extracellular domain binds to Comm activation factors, activating the endocytic pathway, which is likely mediated by the Adaptin/Clathrin complex. The activation factors are independent of motoneuron growth cones, and it is possible that they also serve as synapse inhibition factors. (2) Then Comm acts as an endocytosing molecule. This endocytosis rapidly and specifically removes the synapse inhibition factors from the surface of the muscles. (3) This remodeling of the muscle surface makes it compatible with synaptogenesis initiation. It defines a time frame in which specific synaptic target recognitions take place. The motoneuron growth cones that arrive after Comm endocytosis can initiate synaptogenesis on appropriate muscles. The decision of individual growth cones to select specific target muscles among their neighbors and initiate synaptogenesis is made at most within a few hours after Comm endocytosis. Comm is thought to coordinate the general timing of synaptogenesis by ‘cleaning up’ the target cell surface and thereby facilitating target recognition between specific synaptic partner cells (Wolf, 1998).

The ‘signaling’ model is of a more general nature. One simple scenario proposes the following: (1) Comm binds to Comm activation factors; (2) this leads to the transmembrane signal transduction, whose exact nature remains open to speculation. It could either take place locally within the cytoplasm or involve gene regulation. Presumably, the distal portion of Comm’s cytoplasmic domain contains the critical functional domains, since the truncated Comm cannot promote synaptogenesis. (3) The result of the signal transduction is to resurface the muscle membrane so that it now becomes compatible for synaptogenesis. This could be achieved by either insertion and/or activation of synaptogenesis promotion factors or removal and/or inactivation of synaptogenesis inhibition factors. The two models are not mutually exclusive. Each, however, makes specific predictions. In the first case, the ‘janitor’ model predicts that the Comm-positive endosomes in muscles are enriched with molecules that inhibit synaptogenesis. It will be interesting to determine if any putative inhibitory muscle surface molecules (e.g. Toll, Semaphorin II and Netrins) are co-internalized into the Comm-positive endosomes. The model also predicts that blocking Comm’s endocytic activity during the period of motoneuron-muscle contact should be sufficient to stop its role. One version of the ‘signaling’ model, the alternative case, would predict that inhibiting either transcription or translation in the muscles during the same period prevents synaptogenesis (Wolf, 1998).

Most of the neurons of the ventral nerve cord send out long projecting axons that cross the midline. In the Drosophila CNS, cells of the midline give rise to neuronal and glial lineages with different functions during the establishment of the commissural pattern. The development of midline cells is fairly well understood. In the developing ventral neural cord, 7-8 midline progenitor cells per abdominal segment generate about 26 glial and neuronal cells, i.e. 3-4 midline glial cells, 2 MP1 neurons, 6 VUM neurons, 2 UMI neurons, as well as the median neuroblast and its support cells. The VUM neurons comprise motoneurons as well as interneurons, which project through the anterior and posterior commissures. Genetic studies indicate that the VUM neurons are involved in the initial attraction of commissural growth cones. The MP1 neurons are ipsilateral projecting interneurons, which participate in the formation of specific longitudinal axon pathways. The median neuroblast divides during larval and pupal stages. Contrary to what occurs in the grasshopper CNS, the Drosophila median neuroblast does not generate midline glial cells. In Drosophila, the midline glial cells develop from a set of 2-3 progenitors located in the anterior part of each segment. A function of the midline glial cells during the maturation of the segmental commissures has been found, such that two midline glial cells migrate along cell processes of the VUM-midline neurons to separate anterior and posterior axon commissures. If this migration is blocked, a typical fused commissure phenotype develops. Toward the end of embryogenesis, midline glial cells are required for the formation of individual fascicles within the commissures (Hummel, 1999 and references).

The glial cells present repulsive signals to the Roundabout receptor in addition to a permissive contact-dependent signal helping commissural growth cones across the midline. A novel repulsive component is encoded by the karussell gene. In stage 12 karussell mutant embryos, several short FasII-positive cell processes project toward the CNS midline. In older embryos, FasII expression is found on axons crossing the midline in more than 50% of the neuromeres. The distribution of the 22C10 antigen in karussell embryos uncovers only a few abnormalities. Thus a small subset of normally ipsilateral axons project contralateral in karussell mutants. The majority of axons found in the circles around the RP1 neurons are likely to be commissural axons that cross the midline more than once. This suggests that karussell encodes a novel component of the repulsive signaling pathway. karussell is shown to act in parallel to commissureless and roundabout gene functions, or it may act downstream in a common regulatory hierarchy (Hummel, 1999).

To determine which CNS midline cells present the repulsive signal recognized by Robo, double mutant embryos were analyzed. In commissureless mutant embryos no commissures are formed. The gene pointed is specifically expressed in the midline glial cells and controls differentiation of this cell type. In commissureless pointed mutant embryos few commissures are formed. Similarly in commissureless/slit mutant embryos commissures do form. Since pointed as well as slit affect differentiation of the midline glial cells it is suggested that these cells present the repulsive ligand to Robo. In the absence of differentiated midline glial cells no repulsive ligand can be present and growth cones can cross the midline. This finding implies that disruption in glial differentiation at the midline should lead to a robo-like phenotype as well (Hummel, 1999).

What is the function of midline glia in commissure formation? Genetic data suggest that, in addition to being a permissive substrate for commissural growth, the midline glial cells present the repulsive signal to axons that should not cross the midline. Indeed, many examples of FasciclinII-positive axons crossing the midline are found in mutants in which the development of the midline glial cells is affected. The same defect can be observed when differentiation of the midline glia is impaired by directed overexpression of argos, which is a negative regulator of the EGF-receptor pathway. This finding was not unexpected since the Commissureless protein, which regulates Robo expression, is found in high levels in these cells. It is proposed that one important function of the midline glial cells is to act as a control post dictating who can and cannot cross. They prevent commissural axons from crossing the midline more than once and ensure that ipsilateral projecting axons never cross the midline. These two processes might be regulated by different processes (karussell/roundabout) (Hummel, 1999).

The following model is proposed for commissure formation. The initial growth of commissural growth cones towards the midline in stage 12 embryos is guided by an attractive signal expressed by the midline neurons. Presumably, this attraction is mediated by early Netrin expression in the midline neurons or alternatively by the action of a Schizo/Weniger attractive system. At this early developmental stage the midline glial cells are elongated in shape, contacting the epidermis with their basal side and are assumed to send out cellular processes contacting the VUM-midline neurons at the dorsal side of the nervous system. The midline glial cells express a repulsive signal that is conveyed to lateral axons via the Robo receptor and/or the karussell gene product. This repulsive function restricts the first axons to cross the midline just anterior of the VUM neurons. The midline glial cells also express a contact dependent permissive guidance cue helping the axons to cross the midline. Subsequently, neuron-glia interaction at the midline results in the migration of the midline glial cells along processes of the VUM neurons (Hummel, 1999).

Roundabout (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 comm, and 13 alleles of slit. At the time, because slit had such a similar axon phenotype to sim, 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. Indeed, Slit was found to be the midline repellent for the Robo receptor (Kidd, 1999 and references).

Axons in the bilateral CNS of Drosophila decide whether or not to cross the midline before following their specific subsequent pathways. In commissureless mutants, the RP3 and V motoneuron axons often fail to cross the midline but subsequently follow the mirror-image pathways and innervate corresponding muscle targets on the ipsilateral side. Conversely, in roundabout mutants, the RP2 and aCC motoneuron axons sometimes cross the midline abnormally but their subsequent pathways and synaptic targeting are the perfect mirror images of those seen in wild type. Furthermore, within a single segment of these mutants, bilateral pairs of motoneuron axons can make their midline decisions independent of one another. Thus, neither the particular molecular experience of the growth cones nor the decision at the midline caused by these mutations affects growth cone ability to respond normally to subsequently presented cues (Wolf, 2000).

The logic motivating these experiments is that growth cones will retain their ability to respond normally to all subsequent cues if they rely on an experience-independent preprogramming (Preprogram model). But, if their normal mode of operation is one of continual reprogramming, and the final axon pathways are a result of a specific sequence of interactive reprogramming, missing the normal cues at the midline should lead to a subsequent deviation from the normal pathways at one point or another (Reprogram model). It was reasoned that by following the axon pathways of individual neurons affected by midline mutations, these scenarios could be examined experimentally. The first set of experiments with comm mutants provided cases in which growth cones were prevented from crossing the midline. However, due to the bilateral symmetry of the molecular and cellular organizations across the midline, the RP3 and V motoneuron growth cones, when they failed to cross the midline, still found themselves surrounded by the same microenvironment that they would normally have experienced after crossing the midline. The net effect is essentially equivalent to a cellular transplant across the midline. Without experiencing the Comm protein on the midline glia, and despite the abnormal decision to not cross the midline, these growth cones are nevertheless perfectly capable of responding normally to all subsequent cues, allowing them to follow the mirror-image peripheral pathways all the way to their respective target muscles and to initiate synaptogenesis there (Wolf, 2000).

The second set of experiments with robo mutants provided complementary cases in which growth cones were made to abnormally cross the midline, presumably due to either full or partial loss of the ability of the growth cones to respond to midline repulsion signals. The Robo protein is a widely expressed neuronal growth cone receptor, and its deletion offers a means to disrupt the midline decisions of growth cones independent of comm mutations. In all cases, when they cross the midline abnormally, the RP2 and aCC motoneuron growth cones retain normal responsiveness to all subsequent cues, selecting the mirror-image pathways and muscles on the other side of the midline (Wolf, 2000).

These results clearly demonstrate that neither disrupted midline decisions nor a lack of midline signaling molecules affect the ability of the motoneuron growth cones to respond normally to cues encountered beyond the midline. It is concluded that, under the situations examined, the growth cones rely on an experience-independent preprogramming for their navigation through complex in vivo environments (Wolf, 2000).

Roundabout (Robo) in Drosophila is a repulsive axon guidance receptor that binds to Slit, a repellent secreted by midline glia. In robo mutants, growth cones cross and recross the midline, while, in slit mutants, growth cones enter the midline but fail to leave it. This difference suggests that Slit must have more than one receptor controlling midline guidance. In the absence of Robo, some other Slit receptor ensures that growth cones do not stay at the midline, even though they cross and recross it. The Drosophila genome is shown to encode three Robo receptors and Robo and Robo2 have distinct functions, which together control repulsive axon guidance at the midline. The robo,robo2 double mutant is largely identical to slit (Simpson, 2000).

Commissureless protein can downregulate Robo2 as well as Robo. comm overexpression in midline glia and early neurons using Scabrous-GAL4 can reduce the level of Robo2 protein in CNS axons just as it reduces the levels of Robo. In comm gain-of-function embryos, the phenotype is robo like, but there is more disorganization of the outer (i.e., intermediate and lateral) pathways, presumably because Comm is downregulating Robo2 as well as Robo. In comm null mutants, Robo2 is still localized to the lateral pathways of the CNS scaffold (and Robo3 to the intermediate and lateral pathways), indicating that Comm is not required for the lateral restriction of Robo2 and Robo3. This restriction of Robo2 and Robo3 to specific subsets of neurons appears to be largely transcriptional as revealed by in situ hybridization (Simpson, 2000).

In contrast, the dramatic increase of Robo protein levels as growth cones cross the midline is, at least in part, regulated by Comm. The distinction is as follows: which neurons express any particular Robo family member (or combination of Robos) appears to be largely transcriptionally controlled, whereas when a given neuron displays on its axons any particular Robo family member (after the onset of transcription) appears to be controlled by other mechanisms, including Comm. Moreover, where a neuron expresses any particular Robo family member (i.e., the commissural versus longitudinal axon segment) also appears to be controlled by other mechanisms (Simpson, 2000).

The comm gain of function shows that Comm can downregulate both Robo and Robo2. But does it normally regulate more than just Robo? In the original midline mutant screen paper, the robo;comm double mutant was described as looking just like robo when stained with mAb BP102 (which labels all CNS axons). If the double mutant was indeed indistinguishable from robo alone, then this would suggest that Comm normally only regulates Robo. But this is not the case; distinct differences are observed when the double (robo;comm) mutant is compared with robo alone, using mAb 1D4 to stain the three major Fas II pathways. In a robo mutant, the axons in the medial Fas II pathway cross and recross the midline, while the axons in the intermediate and lateral Fas II pathways do not cross the midline. In contrast, in a robo;comm double mutant, the intermediate Fas II pathway is also perturbed and can be seen crossing the midline. At the very least, this result shows that, in the absence of Robo, Comm still has some additional function that is revealed by removing them both together. Since this additional function affects midline guidance, it is speculated that this additional function involves its regulation of Robo2 and/or Robo3. There are several alternative ways in which one might interpret the additional phenotypes seen in the robo;comm double mutant. Distinguishing between these models requires having probes for the different subsets of Fas II axons (medial versus intermediate versus lateral); such probes are not yet available, although work is underway to generate these tools (Simpson, 2000).

Can Comm also downregulate Robo3? It is very difficult to do the same experiment as with Robo and Robo2. Both Robo and Robo2 proteins are expressed early in both the CNS and surrounding tissues. Comm can be overexpressed early only in the CNS, and differential reduction of Robo or Robo2 protein in the CNS compared to the surrounding tissue can be assessed. However, Robo3 is neither expressed early enough nor in tissues outside the nervous system for a similar comparison. The fact that the robo,robo2;comm triple mutant looks like the robo,robo2 double mutant (in which no axons leave the midline) suggests that if loss of Comm increases the level of Robo3, it does not do so sufficiently to allow any axon to escape the midline. But Robo3 may simply be too weak on its own, even when released from putative Comm downregulation, to repel axons away from the midline. All of these results and interpretations are further complicated by the existence in the Drosophila genome of a gene encoding a second Comm-like protein. Both Comms are all capable when overexpressed of downregulating Robo and Robo2. How they function to regulate the different Robos is under investigation (Simpson, 2000).

Trophic mechanisms in which neighboring cells mutually control their survival by secreting extracellular factors play an important role in determining cell number. However, how trophic signaling suppresses cell death is still poorly understood. The survival of a subset of midline glia cells in Drosophila depends upon direct suppression of the proapoptotic protein Hid via the Egf receptor/RAS/MAPK pathway. The TGF alpha-like ligand Spitz is activated in the neurons, and glial cells compete for limited amounts of secreted Spitz to survive. In midline glia that fail to activate the Egfr pathway, Hid induces apoptosis by blocking a caspase inhibitor, Diap1. Therefore, a direct pathway linking a specific extracellular survival factor with a caspase-based death program has been established (Bergmann, 2002).

The genetic requirement of mapk for MG survival and of hid for MG apoptosis prompted the assumption that MAPK promotes survival of the MG by inhibition of HID activity. According to this model, the MG would be unprotected from HID-induced apoptosis in mapk-deficient embryos, and die. Consistent with this idea, HID protein is detectable in the MG of late stage wild-type embryos. To test this further, embryos that were mutant for both mapk and hid were examined. In early stage mapk;hid double mutant embryos, the initial generation of the MG appears to be normal. However, in contrast to mapk mutants alone, the MG is rescued in mapk;hid double mutant embryos although the survival function of MAPK is missing in these embryos. Dissection revealed that the MG are located directly at the cuticle of the embryos. Because segmental fusions occur in these embryos, some of the MG cluster in groups of up to 20 cells. In individual segments, five to six MG are visible. This number is larger compared to wild-type (three MG per segment), and is remarkably similar to the number of surviving MG in hid mutant embryos alone, indicating that MAPK promotes MG survival largely through inhibition of HID (Bergmann, 2002).

The surviving MG in late stage embryos are in close contact to commissural axons. In embryos lacking the commissureless (comm) gene, the commissural axons are absent. In comm embryos the MG die prematurely, and some survivors become misplaced laterally along the longitudinal axon tracts. The location of the MG along the longitudinal axons in comm mutant embryos as well as their close contact to commissural axons in wild-type embryos has prompted the suggestion that axon contact is required for MG survival. Axon contact appears to permit the MG to respond to trophic signaling, which is necessary for its survival. Consistent with this notion trophic signaling provided by sSPI/Egfr is present only in MG associated with longitudinal axons in comm;hid double mutants. Thus, it was asked whether axon contact-mediated Egfr signaling in the MG is required to activate MAPK, which in turn suppresses the cell death-inducing ability of HID (Bergmann, 2002).

To address this question, the fate of the MG was examined in comm mutant embryos which are at the same time mutant for hid (comm;hid double mutants) or carry the dominant active mapk allele, mapkSem (mapkSem;comm double mutants). Strikingly, a substantial number of the MG survive even in the absence of axonal contact if hid function is removed or if MAPK is activated. This strongly suggests that axon contact is necessary to suppress HID via MAPK. Only MG in proximity to neurons undergo Egfr signaling. The additional MG that survive along the midline in comm;hid mutants do not express spry, that is, do not receive an Egfr signal, and survive only because hid is absent in this experimental condition (Bergmann, 2002).

pCC/MP2 neurons pioneer the longitudinal connectives by extending axons adjacent to the midline without crossing it. These axons are drawn toward the midline by chemoattractive Netrins, which are detected by their receptor Frazzled (Fra). However, these axons are prevented from crossing by Slit, an extracellular matrix ligand expressed by glial cells and recognized by Roundabout (Robo), a receptor on the axons of most neurons. Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons (Kim, 2002).

Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues (Kim, 2002).

The general importance of regulating myosin II activity during axon guidance decisions is confirmed by observation that pan-neural expression of ctMLCK, but not wtMLCK, in Drosophila embryos causes axons within the pCC/MP2 pathway to project across the midline incorrectly. In crossing the midline, axons in the pCC/MP2 pathway either over-respond to midline attractive cues leading them across the midline or fail to respond to repulsive signals preventing them from crossing. Indeed, it is likely that both processes are operating. Axons within the pCC/MP2 pathway move toward the midline as Fra receptors detect chemoattractive Netrins. However, they are prevented from crossing by the repulsive ligand Slit, detected by Robo, the cell surface receptor present on most growth cones. Expression of ctMLCK does not alter the onset of axon extension nor the initial pioneering events of pCC/MP2 neurons, but is sufficient to allow these axons to overcome the repellent Slit barrier and cross the midline. If midline repulsive signals are reduced by using heterozygous mutations of either slit or robo, ctMLCK expression induces many more pCC/MP2 axons to cross the midline, and decreasing myosin II activity using sqh mutations that lower the activity of the regulatory light chains suppresses some of the crossovers observed in heterozygous robo mutants. Thus, it seems that myosin II activity must be maintained below a certain threshold in order for Robo to prevent axons from crossing the midline. When myosin II activity exceeds that threshold, as in embryos expressing ctMLCK, the growth cone is unable to respond appropriately to activation of Robo (Kim, 2002).

The importance of attractive systems at the midline is further supported by the ability of ctMLCK expression to rescue comm mutant phenotypes. In comm mutants, a failure to remove Robo from the membrane increases midline repulsive activity and thus commissures do not form because axons are prevented from crossing the midline. But attractive cues are also present in comm mutants and, at least in early stages, axons orient toward and explore the midline as if they are trying to respond to midline attractive cues. With ctMLCK expression, these tentative explorations appear to be converted into positive movement across the midline. This suggests that, when myosin II activity is increased by ctMLCK expression, even transient activation of midline adhesive systems, and consequent coupling to actin filaments, will provide sufficient traction to move the growth cone partially over the midline. Once over, the continued presence of Slit at the midline would actually help propel the growth cone all the way across to the contralateral connective, thus forming part of the commissure. The thickness of many of the rescued commissures suggests that fasciculation with early axons may aid later axons in continuing the formation of a commissure. Together, the data indicate that a growth cone’s response to midline attractive cues is sensitive to the overall level of myosin II activity (Kim, 2002).


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date revised: 30 June 2004

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