InteractiveFly: GeneBrief

unc-5: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The netrins are a conserved family of secreted molecules that guide neuronal growth cones in the developing nervous system. They are bifunctional, capable of attracting some axons while repelling others. Netrins act through receptors belonging to two distinct families: the Deleted in Colorectal Cancer (DCC) and UNC5 families. DCC family receptors include DCC in vertebrates and Frazzled in Drosophila. C. elegans UNC-5 and its three vertebrate homologs UNC5H1, UNC5H2, and UNC5H3/RCM are also single-pass transmembrane receptors. Their extracellular domains consist of two Ig domains and two thrombospondin type I (TSP) domains. The cytoplasmic domains of UNC5 receptors include at least three conserved regions: a ZU5 domain, a DB motif, and a carboxy-terminal death domain (DD). The predicted Drosophila Unc5 protein has the same domain organization as its worm and mammalian orthologs. In Drosophila, Netrins are expressed by midline cells of the CNS and by specific muscles in the periphery. Netrins attract commissural and motor axons expressing the DCC family receptor Frazzled. Drosophila Unc5 receptor is a repulsive Netrin receptor likely to contribute to motor axon guidance. Ectopic expression of Unc5 on CNS axons can elicit either short- or long-range repulsion from the midline. Both short- and long-range repulsion require Netrin function, but only long-range repulsion requires Frazzled (Keleman, 2001).

In C. elegans, UNC-40 (Frazzled homolog) is required primarily for ventral migrations directed toward UNC-6 (Netrin homolog), but also participates in many dorsal migrations away from UNC-6. UNC-5, in contrast, is required only for dorsal migrations away from UNC-6 (Hedgecock, 1990; Hamelin, 1993). Similarly, in vertebrates, DCC is required for the ventral growth of mammalian commissural axons in vivo (Fazeli, 1997), and the attractive Netrin response of isolated Xenopus spinal axons in vitro (Ming, 1997). Expression of UNC-5 family receptors in cultured Xenopus spinal axons switches their response to Netrin from attraction to repulsion (Hong, 1999). Like attraction, repulsion of Xenopus spinal axons also requires DCC function. Together, these data have led to a model in which DCC receptors mediate attraction, and in some cases also repulsion, while UNC5 receptors act only in repulsion (Keleman, 2001 and references therein).

The Xenopus growth cone turning assay has been exploited to investigate the molecular mechanisms of DCC and UNC5 function (Hong, 1999; Stein, 2001). Their work has shown that attraction to Netrin involves the multimerization of DCC receptors, mediated by the association of their cytoplasmic P3 domains (Stein, 2001). Repulsion, in contrast, depends on an interaction between the cytoplasmic domains of DCC and UNC5 receptors. In this case, the association is mediated by the P1 domain of DCC, which binds the DB domain of UNC5 (Hong, 1999). In both attraction and repulsion, binding of Netrin to the extracellular domain of its receptor triggers the interaction between the cytoplasmic domains (Keleman, 2001 and references therein).

A further important conclusion from these studies is that it is the cytoplasmic domain of UNC5 that specifies repulsion. This comes from the finding that a DCC-UNC5H2 chimeric receptor, in which the DCC extracellular domain is fused to the UNC5H2 cytoplasmic domain, can elicit a repulsive Netrin response that is just as strong as that induced by UNC5H2 itself (Hong, 1999). In fact, the extracellular domain of UNC5H2 is not even required: a DCC/UNC5H2 receptor complex can still form and mediate repulsion, even if one of the two proteins lacks its entire extracellular domain. When examining which parts of the UNC5H2 cytoplasmic domain are required for repulsion (Hong, 1999), the DB domain was found to be essential; the death domain was not (Keleman, 2001 and references therein).

Evidence that Drosophila Unc5 is a repulsive Netrin receptor comes from two complementary sets of observations: (1) repulsion (mediated by ectopic NetrinB) of motor axons that exit the CNS ipsilaterally via the segmental nerve root (SNa motor axons) requires Unc5 function, and conversely, (2) midline repulsion of either commissural or intersegmental interneuron-generated Ap axons forced to express Unc5 requires Netrin function (Keleman, 2001).

Within the CNS, Netrins are expressed primarily at the midline, where they act through the DCC family receptor Frazzled to help guide commissural axons toward and across the midline. If Unc5 is indeed a repulsive Netrin receptor, then misexpression of Unc5 on these commissural axons should prevent them from crossing the midline, possibly even redirecting them laterally away from the midline and out of the CNS. To test this, Unc5 was expressed in all postmitotic neurons using a UAS-Unc5 transgene and an elav-GAL4 driver. Commissures are completely lacking in such embryos. The longitudinal connectives are also more widely separated than in wild-type embryos. However, there is no detectable increase in the number of axons that extend laterally away from the midline. Pan-neural expression of Unc5 thus results in a short-range repulsive response in which commissural axons are prevented from crossing the midline, but are not driven directly away from it (Keleman, 2001).

This 'commissureless' phenotype is much stronger than the partial loss or thinning of commissures observed in either Netrin or frazzled mutant embryos, and therefore cannot simply be explained by the ability of Unc5 to sequester Netrin or Frazzled proteins into inactive complexes. Unc5 also appears to be far more potent in preventing midline crossing than the Robo receptors for the midline repellent Slit. Nine independent insertions of the UAS-Unc5 transgene were tested, and all of them give a completely penetrant commissureless phenotype with just single copies of both transgenes. In contrast, robo gain-of-function phenotypes generally require at least two copies of either or both of the UAS-robo and elav-GAL4 transgenes (Keleman, 2001).

The strong commissureless phenotype of these pan-neural Unc5 embryos provides the opportunity to examine the structural requirements for Unc5-mediated repulsion. To this end, a series of UAS constructs encoding mutant Unc5 proteins were generated, each lacking one of the conserved extracellular or intracellular domains, as well as a construct encoding an N-terminal myristoylated form of the Unc5 cytoplasmic domain. None of these mutant Unc5 transgenes, when expressed using the elav-GAL4 driver, resulted in even a weak commissureless phenotype. At least two different insertions of each transgene were tested. Expression and axonal localization of the mutant proteins were confirmed by Western blotting and in situ detection using antibodies against either an N-terminal HA epitope (for the domain deletions) or a C-terminal c-myc epitope (for the myristoylated cytoplasmic domain) (Keleman, 2001).

These results confirm the conclusions from Xenopus in vitro studies (Hong, 1999) that the DB domain is required for Unc5 repulsion, and extend them by showing a requirement also for the ZU5 domain and each of the four extracellular domains. However, the finding of a strict requirement for both the death domain and the extracellular domain of Drosophila Unc5 contrasts with the dispensibility of the corresponding domains of UNC5H2 in the Xenopus assays (Hong, 1999). Deletions were made at analogous positions of the UNC5 proteins in both sets of experiments. It therefore seems unlikely, but cannot be excluded, that the failure of these mutant Drosophila Unc5 proteins to mediate repulsion is just a trivial consequence of protein misfolding or instability. Rather, it suggests that the repulsion of commissural axons by Unc5 in Drosophila may occur through a mechanism distinct from the repulsion of spinal axons by UNC5H2 in the Xenopus assays (Keleman, 2001).

Growth cone repulsion mediated by ectopic UNC5 expression, both in C. elegans and in the Xenopus in vitro assays, has been shown to require not only Netrin function but also that of DCC family receptors (Hamelin, 1993; Colavita, 1998; Hong, 1999). It was therefore anticipated that both Netrin and frazzled function would be required to prevent midline crossing in elav-GAL4/UAS-Unc5 embryos. To test this, embryos carrying both transgenes were generated in the background of either the Netrin deficiency Df(1)NP5 or the frazzled null allelic combination fra³/fra⁴. As expected, midline repulsion by Unc5 does indeed require Netrin function. Surprisingly, however, it is independent of frazzled function. The elav-GAL4/UAS-Unc5 phenotype is just as strong in the frazzled mutant background as in the wild-type background (Keleman, 2001).

Given that Slit can bind directly to Netrin, and can also act via Robo receptors to silence Netrin attraction, might midline repulsion by Unc5 depend in any way on repulsion mediated by Slit and its Robo receptors? To test this, embryos were generated carrying both the elav-GAL4 and UAS-Unc5 transgenes, and that also were homozygous for one or more of the null alleles slit², robo¹, and robo2⁴ (Keleman, 2001).

The commissureless phenotype of pan-neural Unc5 embryos is essentially unaltered in the robo and robo2 single mutant backgrounds. The phenotype is more difficult to interpret when either slit or both robo and robo2 function is eliminated. The CNS phenotype observed in these embryos is intermediate between that of pan-neural Unc5 embryos and either slit or robo robo2 embryos. In some segments, axons are entirely collapsed at the midline as in slit or robo robo2 mutants, but in other segments axons are separated into two bundles, one on each side of the midline. This argues against a direct role for Slit in Unc5-mediated repulsion, since clearly Unc5 misexpression does have an effect in the absence of Slit. It does, however, suggest an indirect role. For example, repulsion by Netrin and Unc5 may only be effective in keeping axons away from the midline when it is added on top of the repulsive signal transduced via Slit and its Robo receptors. Another, not exclusive, possibility is that this intermediate phenotype is due to the ventral displacement of midline cells that occurs in both slit and robo robo2 mutants (Keleman, 2001).

Several recent experiments point to the modular design of axon guidance receptors, in which the extracellular domain determines the ligand specificity while the cytoplasmic domain dictates the response of the growth cone. In particular, Hong (1999) has demonstrated that a DCC-UNC5H2 chimeric receptor consisting of the extracellular domain of DCC and the cytoplasmic domain of UNC5H2 is as effective as wild-type UNC5H2 in repelling Xenopus spinal axons away from a Netrin source in vitro. This finding was tested in vivo. In addition, attempts were made to extend this result by testing the prediction that a reciprocal UNC5-DCC chimera should mediate attraction to Netrin (Keleman, 2001).

UAS transgenes were prepared encoding chimeric Fra-Unc5 and Unc5-Fra receptors, in which the cytoplasmic domains of the two Netrin receptors had been swapped immediately proximal to their transmembrane domains. To test the prediction that the cytoplasmic domain of Unc5 specifies repulsion, the CNS of embryos in which one or another of these chimeras was expressed using the elav-GAL4 driver was examined. As expected, pan-neural expression of the Fra-Unc5 chimera results in a commissureless phenotype just as strong as that observed with the full-length Unc5 receptor. Ectopic expression of Unc5-Fra has no obvious effect, as previously found to be the case also for full-length Fra (Keleman, 2001).

Does the Unc5-Fra chimera act as an attractive Netrin receptor? If so, pan-neural expression of this receptor, like that of Fra itself, should at least partially rescue the frazzled mutant phenotype. This is indeed the case. Each of two UAS-Unc5-fra transgene insertions tested almost completely rescue the frazzled null mutant. UAS-Unc5-fra rescues both the commissural and longitudinal axon defects of frazzled mutants just as efficiently as does UAS-fra. It is therefore concluded that Unc5-Fra is an attractive Netrin receptor, formally completing the demonstration that Netrin receptors are modular: the growth cone response (attraction or repulsion) is determined by the cytoplasmic domain (DCC or UNC5, respectively), irrespective of the Netrin binding extracellular domain to which it is attached (Keleman, 2001).

Experiments using the elav-GAL4 driver show that Unc5 is a potent mediator of Netrin repulsion at short range, preventing commissural axons from crossing the midline. In a final set of experiments, these observations were extended by asking how an ipsilateral interneuron -- one that does not normally cross the midline -- would respond to ectopic expression of Unc5. For this, the Ap-GAL4 driver was used. This line expresses GAL4 in three neurons (termed the Ap neurons) in each hemisegment. Their cell bodies are positioned laterally within the nerve cord, several cell diameters from the midline. One is located dorsally, the other two ventrally. All three are intersegmental interneurons. Their axons first grow toward the midline, but they do not cross it, instead turning anteriorly to continue along the medial edge of the ipsilateral longitudinal tract. In the experiments reported here, focus was placed on the behavior of the dorsal Ap neuron (Keleman, 2001).

Expression of Unc5 in this neuron has remarkable consequences. Rather than growing toward the midline, its axon now grows laterally away from the midline to exit the CNS and continue on a motor trajectory into the periphery. This phenotype is highly penetrant: 91% of dorsal Ap axons examined exited the CNS in these embryos. Thus, Unc5 can repel axons away from the midline at long range, forcing them 180° off course. All of the mutant Unc5 proteins tested in the midline crossing assay were also found to be defective in this assay (Keleman, 2001).

This long-range repulsion by Unc5 requires Netrin function, as expected. However, unlike the short-range repulsion of commissural axons at the midline, long-range repulsion of Ap axons is partially dependent on frazzled function. In frazzled mutant embryos, only 59% of Ap axons exited the CNS upon ectopic Unc5 expression. To determine whether this reflects an autonomous requirement for frazzled, its function was restored specifically in the Ap neurons by introducing a UAS-fra transgene into these embryos. The percentage of Ap axons exiting the CNS rose to 97%, demonstrating that potent long-range repulsion of Ap axons requires expression of both Unc5 and Fra in the Ap neurons themselves (Keleman, 2001).

Finally, it was asked whether Slit and its receptor Robo contribute to the long-range repulsion of Ap axons mediated by Unc5. Of the three Drosophila Robo receptors, Ap neurons express only Robo, the founding member of the Robo family. Slit repulsion could therefore be eliminated simply by removing robo function, thus avoiding the problem of the midline collapse that complicates the interpretation of Slit function in the midline crossing assay. In a robo null mutant background, Unc5 expression drives Ap axons out of the CNS just as effectively as it does in a wild-type background. Long-range repulsion mediated by Netrin and Unc5 thus operates independently of repulsion mediated by Slit and Robo (Keleman, 2001).

At this point, it is not clear whether just one or both of the Drosophila Netrins, NetA and NetB, are ligands for Unc5. Clearly, NetB alone is capable of repelling Unc5-expressing SNa motor axons (Mitchell, 1996; Winberg, 1998), while ectopic expression of NetA appears to have no effect on these axons (Winberg, 1998). Thus, while NetA and NetB are both attractants, only NetB may also be a repellent. It will be interesting to determine whether these functional interactions are reflected in the ligand specificity of the Drosophila Netrin receptors, and if so, whether such specificity depends on the unique insert domains found in each of the NetA, NetB, and Unc5 proteins (Keleman, 2001).

The expression patterns of Unc5 and the two Netrins within the developing nervous system are strikingly complementary. SN motor axons that express Unc5 avoid both midline cells and peripheral muscles that express Netrins, while commissural axons that cross the midline and motor axons that innervate Netrin-expressing muscles do not express Unc5. These observations hint at an important role for Netrin repulsion in guiding SN motor axons, first out of the CNS, and then in their choice of specific muscle targets in the periphery. However, in both Netrin-deficient and Unc5 RNAi embryos, SN motor axons still exit the CNS and choose their correct targets. Thus, repulsion by Netrins is likely to be just one of multiple guidance cues that control these pathways (Keleman, 2001).

One obvious candidate for a second repellent that drives motor axons away from the midline is Slit. However, even in embryos lacking both slit and Netrin function, many motor axons still exit the CNS. Evidently, at least one additional midline repellent for Drosophila motor axons remains to be discovered. Multiple cues also appear to repel motor axons in the vertebrate CNS. For example, trochlear motor neurons express Unc5h1 and their axons are repelled by netrin-1 in vitro, but they still project normally in netrin-1-deficient mice (Keleman, 2001).

In the periphery, a host of molecules have been identified that help direct individual motor axons to their specific muscle targets. In addition to NetA and NetB, these include Semaphorins, such as Sema2a, and Ig family cell adhesion molecules, such as FasII. Genetic analysis suggests that muscle target selection depends on a complex set of cooperative and antagonistic interactions between these and other guidance cues. In this context, the loss of just a single guidance cue generally results in only a partially penetrant targeting defect. It is therefore perhaps not so surprising to find that SN motor axons still appear to project normally when either Netrin or Unc5 function is impaired. Detecting a more subtle requirement for Netrin repulsion in muscle targeting will require the generation of loss-of-function mutations in Unc5, and the examination of embryos in which motor axons are defective in their responses to multiple cues, not just Netrins (Keleman, 2001).

A DCC-UNC5H2 chimera has been shown to act as a repulsive Netrin receptor in vitro. Likewise a Fra-Robo receptor also behaves as a repulsive Netrin receptor in vivo, while the reciprocal Robo-Fra receptor functions as an attractive Slit receptor. Together, these two studies have led to the general notion that axon guidance receptors are modular in their design -- the extracellular domain determines the ligand specificity while the cytoplasmic domain specifies the nature of the response (attraction or repulsion) (Keleman, 2001).

This idea has been tested in vivo, specifically for the attractive and repulsive Netrin receptors, Fra and Unc5. As predicted by this model, a Fra-Unc5 chimera acts as a repulsive receptor and Unc5-Fra as an attractive Netrin receptor. Both chimeras prove to be as potent as the wild-type proteins in mediating these responses. These data thus provide a clear in vivo demonstration of the modular design of the two Netrin receptors (Keleman, 2001).

Netrins can act over either a short or a long distance to repel CNS axons forced to express Unc5. Pan-neural expression of Unc5 leads to a short-range response, in which commissural axons are prevented from crossing the midline, but do not grow away from it. Expression of Unc5 in a single lateral interneuron, the dorsal Ap neuron, elicits a long-range response. Rather than growing toward the midline as it normally does, the Ap axon instead grows directly away from the midline and out of the CNS (Keleman, 2001).

Why doesn't pan-neural expression of Unc5 lead to the same long-range response, resulting in a massive exodus of CNS axons? The reason for this is not entirely clear. It may be that only very few neurons are capable of such a dramatic response, and the GAL4 line used to express Unc5 happens to be expressed in one of these. However, the only other GAL4 driver tested that is expressed in a subset of interneurons (eg-GAL4) also produces a long-range response, so it seems that this may indeed be a more general phenomenon. Another possibility is that the pan-neural and subset-specific drivers differ in the timing and/or levels of transgenic expression they provide. While this is certainly the case, it seems unlikely to account for the different responses. Introducing a second copy of the UAS-Unc5 transgene does not result in a detectable increase in the number of axons exiting the CNS with elav-GAL4, nor does the use of another, earlier, pan-neural driver, 1407-GAL4. Similarly, an extra copy of the UAS-Unc5 transgene does not alter the long-range response of Ap axons (Keleman, 2001).

It is suspected that the essential difference in these two sets of experiments is the distribution of free Netrin ligand. Normally, Netrin is likely to diffuse far from the midline, and could thus act as a long-range repellent for SN motor axons that endogenously express Unc5, or Ap axons that are forced to express Unc5. But when Unc5 is ectopically expressed at high levels on all CNS axons, Netrin diffusion may be more limited. In this case, axons might only encounter free Netrin ligand close to the midline, resulting in a short-range response (Keleman, 2001).

Whatever the reason for this difference, it has enabled the establishment of in vivo assays that clearly distinguish between short- and long-range repulsion by Netrins. This is important, as it allows the resolution of a long-standing uncertainty concerning the role of DCC proteins in UNC5-mediated repulsion. The origin of this debate goes back to the initial report of Hedgecock (1990), showing that both C. elegans unc-5 and unc-40 are required for dorsal migrations, but unc-40 to a lesser extent than unc-5. Subsequently, expression of unc-5 in lateral mechanosensory neurons was found to redirect their axons dorsally (Hamelin, 1993), and this response shows a strong requirement for unc-40 function (Colavita, 1998). unc-40 has been shown to act autonomously to mediate ventral migrations, but an autonomous role for unc-40 in dorsal migrations has not yet been demonstrated. This is a critical issue, as experiments in Drosophila have demonstrated nonautonomous guidance functions for the unc-40 homolog frazzled. It thus remains unclear what role UNC-40 actually plays in Netrin/UNC-6 repulsion in C. elegans. Further experiments in Drosophila and vertebrates have not yet clarified the situation. In Drosophila, repulsion of SNa motor axons by ectopic NetB, which has been shown to be mediated by Unc5, does not require Frazzled (Winberg, 1998). In contrast, UNC5 receptors (Hong, 1999) do require DCC function to repel Xenopus spinal axons away from a Netrin source in vitro (Keleman, 2001).

In attempting to draw a conclusion from this disparate set of results, several authors have speculated that UNC5 receptors might be able to act alone at high Netrin concentrations, but require DCC coreceptors at lower concentrations. Until now, this idea has not been directly tested. This study offers strong evidence in support of such a model: short-range repulsion of commissural axons by Netrin and Unc5 does not require Frazzled. In contrast, Frazzled is needed for potent long-range repulsion, and this is an autonomous requirement (Keleman, 2001).

Does UNC5 act alone at short range, or might it require yet another coreceptor? This remains to be addressed in future studies. It is, however, interesting to note that short-range repulsion requires the DB (DCC binding) domain of Unc5, but does not require Frazzled. The requirement for the DB domain of Unc5 thus cannot be due to its role in binding the P1 domain of Frazzled, but may instead indicate a role in binding other proteins, perhaps even another coreceptor. Another intriguing difference between UNC5-mediated repulsion in Drosophila assays and in the Xenopus assays of Hong (1999) is that the Drosophila assays show a strict requirement for the Unc5 death domain. This domain was required in both the long- and short-range assays used in this study, but not for the repulsive Netrin response of Xenopus spinal axons in vitro. Perhaps at very low Netrin concentrations, as encountered by Xenopus axons in the in vitro turning assays, UNC5 repulsion does not require the death domain and is mediated entirely through its interaction with DCC, whereas at the high Netrin concentrations likely to be encounted by Drosophila commissural axons in vivo, UNC5 repulsion requires the death domain but not DCC. At intermediate Netrin concentrations, which may operate in long-range assays, UNC5 may require both the death domain and, partially, DCC receptors. There may thus be an inverse relationship between the requirement for either DCC as a coreceptor or for the death domain of UNC5, with the relative requirements depending on the concentration of ligand. In light of this, it is interesting to note that, in several other receptors, death domains mediate self-association. The hypothesis that this may also be the case for UNC5 receptors is currently being tested. This is an attractive idea, as it would imply an appealing symmetry in the signaling modes of Netrin receptors: attraction involves the formation of DCC homodimers (mediated by their C-terminal P3 domains); short-range repulsion would involve the formation of UNC5 homodimers (mediated by their C-terminal death domains), and long-range repulsion the formation of DCC/UNC5 heterodimers (mediated by their P1 and DB domains, respectively) (Keleman, 2001).

Notch-dependent binary fate choice regulates the Netrin pathway to control axon guidance of Drosophila visual projection neurons

Notch-dependent binary fate choice between sister neurons is one of the mechanisms to generate neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well understood. This study reports that Notch-dependent binary fate choice in Drosophila medulla neurons is required to regulate the Netrin axon guidance pathway, which controls targeting of transmedullary (Tm) neurons to lobula. In medulla neurons of Notch-on hemilineage composed of mostly lobula-targeting neurons, Notch signaling is required to activate the expression of Netrin-B and repress the expression of its repulsive receptor Unc-5. Turning off Unc-5 is necessary for Tm neurons to target lobula. Furthermore, Netrin-B provided by Notch-on medulla neurons is required for correct targeting of Tm axons from later-generated medulla columns. Thus, the coordinate regulation of Netrin pathway components by Notch signaling ensures correct targeting of Tm axons and contributes to the neuropil assembly (Zhang, 2023).

During nervous system development, neural diversity is generated through integration of temporal patterning, spatial patterning, and Notch-dependent binary fate choice between sister neurons. How these upstream neural specification programs are translated into highly organized neuropils is not well understood. In this work, using the Drosophila visual system as an example, it was demonstrated that Notch signaling regulates the expression of Netrin pathway components in Notch-on hemilineage neurons, and that the Netrin pathway controls targeting of visual projection neurons (Tm) to the higher order neuropil (lobula). Notch signaling is required to repress Unc-5 expression in Tm neurons. The absence of Unc-5 is essential for Tm neurons to target lobula, and also required for Tm neurons to further enrich NetB in lobula using the Fra receptor. Furthermore, Notch signaling is required to activate NetB expression in Tm neurons, and medulla-derived NetB has an essential role in promoting targeting of Tm axons from later-born medulla columns to lobula through the IOC. Yhe regulation of NetB distribution and the regulation of Netrin pathway components by Notch signaling and possibly other factors is discussed (Zhang, 2023).

Previous studies found that, in other neuropils, Fra captures NetB to enrich Netrin pathway components locally. This study also found that Fra is colocalized with NetB in lobula neuropil layers. Without Fra expressed in medulla projection neurons, NetB enrichment at lobula is greatly diminished. However, since Fra also guides the targeting of Tm neurons to lobula and Tm neurons are also part of the Netrin source, it is possible that both mechanisms contribute to the diminished NetB in the lobula (Zhang, 2023).

Although Fra is required for NetB enrichment, not all Fra is colocalized with NetB in the optic lobe. Furthermore, Unc-5 may be a negative regulator of NetB enrichment. In unc-5 mutants, NetB spreads to lobula plate and medulla. Moreover, overexpression of Unc-5 results in disrupted NetB distribution. However, how Unc-5 regulates NetB distribution requires future investigation. Unc-5 may affect NetB distribution directly or indirectly by affecting the guidance of specific neurons, which affect NetB distribution (Zhang, 2023).

Since mis-expressing Unc-5 in Tm neurons results in their repulsion to the lobula plate, Unc-5 expression should be strictly inhibited in Tm neurons during axon targeting. Unc-5 expression was found to be activated ectopically without Notch signaling, likely in most Tm neurons. However, the regulation of Unc-5 expression in medulla neurons is far more complex and may involve multiple factors/mechanisms. First, besides Tm neurons, Notch-on hemilineage also includes Mi1 neurons, which terminate at the proximal medulla neuropil, and they do keep expressing Unc-5. Therefore, Notch signaling does not inhibit Unc-5 expression in all Notch-on neurons, and in these exceptions, like Mi1 neurons, other factors, such as transcriptional/translational feedback (TTF) or neuronal-specific transcription factors, might override the inhibition of Unc-5 expression by Notch signaling. Consistent with this hypothesis, loss of Hth or Bsh transforms Mi1 into a Tm-type neuron. Second, there should be other factors or mechanisms that repress Unc-5 expression in medulla neurons. A middle layer of neurons (likely born in and around the Ey temporal stage) were observed that turn off Unc-5 regardless of their Notch status. Although most Tm and TmY neurons belong to the Notch-on hemilineage, there are a few exceptions; for example, scRNA-seq data showed that Tm5c, Tm29, and a few TmY neuron types do not express Ap. Among them, Tm29, TmY5a, and TmY14 are shown to express Knot (Kn), and are thus generated from the Notch-off hemilineage in the Ey stage (corresponding to the middle-layer gap in unc-5 expression). Thus, these neurons likely turn off Unc-5 by a different mechanism independent of Notch signaling. It will be interesting to examine whether the TTF(s) or neuronal transcription factors such as Kn are involved. Third, while Unc-5 is expressed in most young immature neurons, it is turned off later in Notch-on neurons, suggesting that a later-acting factor that is specifically activated in the Notch-on hemilineage is required to turn off Unc-5 expression (Zhang, 2023).

In addition to repression of Unc-5, this study also showed that Notch signaling is required to activate NetB expression in Notch-on medulla neurons. Thus, the results suggest that Notch-dependent binary fate choice ensures that lobula-targeting neurons will turn on NetB expression and provide more NetB to the lobula NetB pool. Tm axons from later-born posterior medulla columns may require a higher concentration of NetB to target lobula, because of the longer distance they need to migrate. Consistently, when medulla-derived NetB was knocked down using SoxN-Gal4, Tm axons from posterior columns showed disrupted IOC formation (Zhang, 2023).

Taken together, these results suggest that, in the Notch-on hemilineage neurons, Notch signaling is required to repress the repulsive receptor Unc-5 and also to activate the expression of NetB, which ensures that projection neurons targeting lobula can further enrich NetB with their Fra receptors as well as secrete more NetB. This positive feedback mechanism is required for the correct formation of interommatidial cells (IOC) through which visual project neurons connect to their target neuropil. Thus, Notch-dependent binary fate choice in medulla neurons coordinately regulates the expression of an axon guidance cue and its receptors, which is required for the correct assembly of neuropils (Zhang, 2023).

This study demonstrated that Notch signaling is required to regulate Netrin pathway components. However, it is not known whether this regulation is direct or indirect. In addition, the regulation of Netrin pathway components is complex, and should involve integration of Notch signaling with other factors such as temporal factors and spatial factors (Zhang, 2023).

REGULATION

Enhancer loops appear stable during development and are associated with paused polymerase

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. This study generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. These results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase (Ghavi-Helm, 2014).

Drosophila embryogenesis proceeds very rapidly, taking 18 h from egg lay to completion. Underlying this dynamic developmental program are marked changes in transcription, which are in turn regulated by characterized changes in enhancer activity. However, the role and extent of dynamic enhancer looping during this process remains unknown. To address this, 4C-seq (chromosome conformation capture sequencing) experiments were performed, anchored on 103 distal or promoter-proximal developmental enhancers (referred to as 'viewpoints'), and absolute and differential interaction maps were constructed for each, varying two important parameters: (1) developmental time, using embryos at two different stages, early in development when cells are multipotent (3-4 h after egg lay; stages 6-7), and mid-embryogenesis during cell-fate specification (6-8 h; stages 10-11); and (2) tissue context, comparing enhancer interactions in mesodermal cells versus whole embryo. To perform cell-type-specific 4C-seq in embryos, a modified version of BiTS-ChIP (batch isolation of tissue-specific chromatin for immunoprecipitation) was established. Nuclei from covalently crosslinked transgenic embryos, expressing a nuclear-tagged protein only in mesodermal cells, were isolated by fluorescence-activated cell sorting (FACS; (>98% purity) and used for 4C-seq on 92 enhancers at 6-8 h and a subset of 14 enhancers at 3-4 h. The same 92 enhancers, and 11 additional regions, were also used as viewpoints in whole embryos at both time points. The enhancers were selected based on dynamic changes in mesodermal transcription factor occupancy between these developmental stages and the expression of the closest gene. This study was thereby primed to detect dynamic three-dimensional (3D) interactions, focusing on developmental stages during which the embryo undergoes marked morphological and transcriptional changes (Ghavi-Helm, 2014).

All 4C-seq experiments had the expected signal distribution, with high concordance between replicates. To assess data quality further, ten known enhancer-promoter pairs (of the ap, Abd-b, E2f, pdm2, Con, eya, stumps, Mef2, sli and slp1 genes) were compared, and in all cases the expected interactions were recovered. For example, using an enhancer of the apterous (ap) gene, the expected interaction was detected with the ap promoter, 17 kilobases (kb) away, illustrating the high quality and resolution of the data (Ghavi-Helm, 2014).

In chromosome conformation capture assays, interaction frequencies decrease with genomic distance between regions. To adjust for this, the 4C signal decay was modelled as a function of distance using a monotonously decreasing smooth function. Subtracting this trend, the residual interaction signal was converted to z-scores and interacting regions defined by merging neighbouring high-scoring fragments within 1 kb. Using this stringent approach, 4,247 high-confidence interactions were identified across all viewpoints and conditions, representing 1,036 unique interacting regions (Ghavi-Helm, 2014).

Each enhancer (viewpoint) interacted with, on average, ten distinct genomic regions, less than half (41%) of which were annotated enhancers or promoters. Distal enhancers had a higher than expected interaction frequency with other enhancers. Similarly, promoter-proximal elements had extensive interactions with distal active promoters, 98% of which are >10 kb away. Enhancer-promoter interactions, although not significantly enriched, involve active promoters, with high enrichment for H3K27ac and H3K4me3, and active enhancers, defined by H3K27ac, RNA Pol II and H3K79me3. These results are similar to recent findings in human cells and the mouse β-globin locus, indicating similarities in 3D regulatory principles from flies to human (Ghavi-Helm, 2014).

The extent of 3D connectivity is surprising given the relative simplicity of the Drosophila genome. On average, each promoter-proximal element interacted with four distal promoters and two annotated enhancers, whereas each distal enhancer interacted with two promoters and three other enhancers. These numbers are probably underestimates, as 60% of interactions involved intragenic or intergenic fragments containing no annotated cis-regulatory elements. Despite this, the level of connectivity is similar to that recently observed in humans, where active promoters contacted on average 4.75 enhancers and 25% of enhancers interacted with two or more promoters. The multi-component contacts that were observed for Drosophila enhancers indicate topologically complex structures and suggest that, despite its non-coding genome being an order of magnitude smaller than humans, Drosophila may require a similar 3D spatial organization to ensure functionality (Ghavi-Helm, 2014).

Insulators, and associated proteins, are thought to have a major role in shaping nuclear architecture by anchoring enhancer-promoter interactions or by acting as boundary elements between topologically associated domains (TADs). Occupancy data from 0 to 12 h Drosophila embryos revealed a 50% overlap of interacting regions with occupancy of one or more insulator protein. Insulator-bound interactions are enriched in enhancer elements, suggesting that insulators may have a role in promoting enhancer-enhancer interactions. In contrast to mammalian cells, this study observed no association between insulator occupancy and the genomic distance spanned by chromatin loops, although there was a modest increase in average interaction strength. Conversely, 50% of interacting regions are not bound by any of the six Drosophila insulator proteins, suggesting that these 3D contacts are formed in an insulator-independent manner, or are being facilitated by neighbouring interacting regions (Ghavi-Helm, 2014).

If enhancer 3D contacts are involved in transcriptional regulation, then genes linked by interactions with a common enhancer should share spatio-temporal expression. For the four loci examined-pdm2, engrailed, unc-5 and charybde-this is indeed the case. For example, the pdm2 CE8012 enhancer interacts with both the pdm2 and nubbin (nub, also known as pdm1) promoters, located 2.5 and 47 kb away, respectively. Both genes, producing structurally related proteins, are co-expressed in the ectoderm, overlapping the activity of the pdm2 enhancer. Although there are examples of long-range interactions in Drosophila, often involving Polycomb response elements (PREs) and insulator elements, the vast majority of characterized enhancers are within 10 kb of their target gene, with few known to act over 50 kb. However, as investigators historically tested regions close to the gene of interest, characterized Drosophila enhancers are generally close to the gene they regulate. In contrast, although 4C cannot assess the full extent of short-range interactions, it provides an unbiased systematic measurement of the distance of enhancer interactions, far beyond 10 kb (Ghavi-Helm, 2014).

The distance distribution of all significant interactions reveals extensive long-range interactions within the ~180 megabase (Mb) Drosophila genome; 73% span >50 kb, with the median interaction-viewpoint distance being 110 kb. Two striking examples of long-range interactions are the unc-5 and charybde loci. The unc-5 promoter interacts with multiple regions, including a weak but significant interaction with the promoter of slit (sli), at a distance of >500 kb. These genes produce structurally unrelated proteins that are co-expressed in the heart, and are essential for heart formation (Ghavi-Helm, 2014).

A promoter-proximal element near the charybde (chrb) promoter has a strong interaction with the promoter of the scylla (scyl) gene, almost 250 kb away. Both genes are closely related in sequence and co-expressed throughout embryogenesis. These long-range interactions were confirmed by reciprocal 4C, using either the promoter of chrb or scyl, or an interacting putative enhancer as viewpoint. This interaction was further verified using DNA fluorescence in situ hybridization (FISH) in embryos. As a control, the distance was assessed between the chrb promoter (probe A) and an overlapping probe A' or a region on another chromosome (probe D), to determine the distances between regions very close or far away, respectively. Comparing the distance between the chrb and scyl promoters (probes A and B) showed a high, statistically significant co-localization, in contrast to the distance between the chrb promoter and a non-interacting region with equal genomic distance (probes A and C) (Ghavi-Helm, 2014).

The reciprocal 4C revealed several intervening interactions that are consistently associated with loops to both the scyl and chrb promoter. The activity was examined of two of these in transgenic embryos. Both interacting regions can function as enhancers in vivo, recapitulating chrb expression in the visceral mesoderm and nervous system (Ghavi-Helm, 2014).

When considering a 1-Mb scale around this region, the 4C interaction signal drops to almost zero just after the promoters of both genes. This 'contained block' of interactions is reminiscent of TADs, although the boundaries don't exactly match TADs defined at late stages of embryogenesis, which may reflect differences in the developmental stages used. However, the boundaries do overlap a block of conserved microsynteny between drosophilids spanning ~50 million years of evolution, suggesting a functional explanation underlying the maintained synteny. Expanding this analysis across all viewpoints, ~60% of interactions are located within the same TAD and the same microsyntenic domain as the viewpoint. In the case of the chrb and scyl genes, this constraint may act to maintain a regulatory association between a large array of enhancers, facilitating their interaction with both genes' promoters (Ghavi-Helm, 2014).

These examples, and the other 555 unique interactions >100 kb, provide strong evidence that long-range interactions are widely used within the Drosophila genome, potentially markedly increasing the regulatory repertoire of each gene. As enhancer-promoter looping can trigger gene expression, it follows that enhancer contacts should reflect the dynamics of transcriptional changes during development and therefore be temporally associated with gene expression. To assess this, looping interactions were directly compared between the two different time points and tissue contexts. Given the non-discrete nature of chromatin contacts, the quantitative 4C-seq signal was used to identify differential interactions based on a Gamma-Poisson model, and they were defined as having >2-fold change and false discovery rate <10% (Ghavi-Helm, 2014).

Despite the marked differences in development and enhancer activity between these conditions, surprisingly few changes were found in chromatin interaction frequencies, with ~6% of interacting fragments showing significant changes between conditions. Of these, 87 interactions were significantly reduced during mid-embryogenesis (6-8 h) compared to the early time point (3-4 h), and 90 interactions significantly increased. Similarly, 105 interactions had a higher frequency in mesodermal cells, compared to the whole embryo, and For example, a promoter-proximal viewpoint in the vicinity of the Antp promoter identified many interactions, two of which are significantly decreased at 6-8 h, although the expression of the Antp gene itself increases. For one region, the reduction in 4C interaction at 6-8 h corresponds to a loss in a H3K4me3 peak from 3-4 h to 6-8 h, suggesting that this 3D contact is associated with the transient expression of an unannotated transcript. The activity of the other interacting peak was examined in transgenic embryos, and it was shown to act as an enhancer, driving specific expression in the nervous system overlapping the Antp gene at 6-8 h. Along with the two enhancers discovered at the chrb locus, this demonstrates the value of 3D interactions to identify new enhancer elements, even for well-characterized loci like Antp (Ghavi-Helm, 2014).

A viewpoint in the vicinity of the Abd-B promoter interacted with a number of regions spanning the bithorax locus, three of which correspond to previously characterized Abd-B enhancers; iab-5, iab-7 and iab-8. The iab-7 and iab-8 enhancers are active in early embryogenesis, and have much reduced or no activity at the later time point. Notably, although the loop to those two enhancers is strong at the early time point, it becomes significantly reduced later in development, when both enhancers' activities are reduced. Conversely, the iab-5 enhancer contacts the promoter at a much higher frequency later in development, at the stage when the enhancer is most active. This locus therefore exhibits dynamic 3D promoter-enhancer contacts that reflect the transient activity of three developmental enhancers. It is interesting to note that in all loci examined, the dynamic contacts of specific elements are neighboured by stable contacts, as seen in the Antp and Abd-B loci. Dynamic changes, therefore, appear to operate in the context of larger, more-stable 3D landscapes (Ghavi-Helm, 2014).

Ninety-four per cent of enhancer interactions showed no evidence of dynamic changes across time and tissue context, which is remarkable given the marked developmental transitions during these stages. To investigate this further, enhancer-promoter interactions were examined of genes switching their expression state between time points or tissue contexts. The ap gene, for example, is not expressed at 2-4 h but is highly expressed during mid-embryogenesis (6-8 h). Despite the absence of expression, the interaction between the apME680 enhancer and the ap promoter is already present at 3-4 h, several hours before the gene's activation. To examine this more globally, differentially expressed genes, going either from on-to-off or off-to-on, were selected. Even for these dynamically expressed genes, there was no correlation with changes in their promoter-enhancer contacts. Similar 'stable' interactions were observed between tissue contexts. Genes predominantly expressed in the neuroectoderm at 6-8 h, for example, have interactions at the same locations in whole embryos and purified mesodermal nuclei at 6-8 h, despite the fact that they are not expressed in the mesoderm at this stage (Ghavi-Helm, 2014).

Pre-existing loops were recently observed in human and mouse cells, and suggested to prime a locus for transcriptional activation. However, why they are formed and how transcription is eventually triggered remains unclear. To investigate this, this study focused on the subset of genes that have both off-to-on expression and no evidence for differential interactions (20 genes; differentially expressed with stable loops (DS) genes). Despite changes in their overall expression, DS genes have similar levels of RNA polymerase II (Pol II) promoter occupancy at both time points. The presence of promoter-bound Pol II in the absence of full-length transcription is indicative of Pol II pausing. Using global run-on sequencing (GRO-seq) data to define a stringent set of paused genes, it was observed that most (75%) DS genes are paused (15 of 20 DS genes), and have a significantly higher pausing index. This percentage is significantly higher than expected by chance when sampling over all off-to-on genes, and is robust to using a strict or more relaxed) definition of Pol II pausing. This association is very evident when examining specific loci, showing Pol II occupancy, short abortive transcripts, and loop formation before the gene's expression. Taken together, these results indicate that 'stable' chromatin loops are associated with the presence of paused Pol II at the promoter (Ghavi-Helm, 2014).

To understand how transcription is ultimately activated, changes were examined in DNase I hypersensitivity at the promoter of DS genes. DNase I hypersensitivity is significantly increased at interacting promoters at the stages when the gene is expressed, suggesting that the recruitment of additional transcription factor(s) later in development might act as the trigger for transcriptional activation (Ghavi-Helm, 2014).

In summary, these data reveal extensive long-range interactions in an organism with a relatively compact genome, including pairs of co-regulated genes contacting common enhancers often at distances greater than 200 kb. Comparing enhancer contacts in different contexts revealed that chromatin interactions are very similar across developmental time points and tissue contexts. Enhancers therefore do not appear to undergo long-range looping de novo at the time of gene expression, but are rather already in close proximity to the promoter they will regulate. Within this 3D topology, highly dynamic and transient contacts would not be visible when averaging over millions of nuclei. As transcription factor binding is sufficient to force loop formation, these results suggest a model where through transcription factor-enhancer occupancy, an enhancer loops towards the promoter and polymerase is recruited, but paused in the majority of cases. The subsequent recruitment of transcription factor(s) or additional enhancers at preformed 3D hubs most likely triggers activation by releasing Pol II pausing. Such preformed topologies could thereby promote rapid activation of transcription. At the same time, as paused promoters can exert enhancer-blocking activity, the presence of paused polymerase within these 3D landscapes could safeguard against premature transcriptional activation, but yet keep the system poised for activation (Ghavi-Helm, 2014).

Transcriptional Regulation

A GATA/homeodomain transcriptional code regulates axon guidance through the Unc-5 receptor

Transcription factor codes play an essential role in neuronal specification and axonal guidance in both vertebrate and invertebrate organisms. However, how transcription codes regulate axon pathfinding remains poorly understood. One such code defined by the homeodomain transcription factor Even-skipped (Eve) and by the GATA 2/3 homologue Grain (Grn) is specifically required for motor axon projection towards dorsal muscles in Drosophila. Using different mutant combinations, genetic evidence is presented that both Grn and Eve are in the same pathway as Unc-5 in dorsal motoneurons (dMNs). In grn mutants, in which dMNs fail to reach their muscle targets, dMNs show significantly reduced levels of unc-5 mRNA expression and this phenotype can be partially rescued by the reintroduction of unc-5. It was also shown that both eve and grn are required independently to induce expression of unc-5 in dMNs. Reconstitution of the eve-grn transcriptional code of a dMN in dMP2 neurons, which do not project to lateral muscles in Drosophila, is able to reprogramme those cells accordingly; they robustly express unc-5 and project towards the muscle field as dMNs. Each transcription factor can independently induce unc-5 expression but unc-5 expression is more robust when both factors are expressed together. Furthermore, dMP2 exit is dependent on the level of unc-5 induced by eve and grn. Taken together, these data strongly suggests that the eve-grn transcriptional code controls axon guidance, in part, by regulating the level of unc-5 expression (Zarin, 2012).

Different transcriptional codes regulate axon guidance but the guidance systems they regulate are still unknown. The GATA factor, Grn, is a major determinant of guidance within dorsal MNs. A strong guidance phenotype occurs in grn mutants, in which ISN axons fail to reach their targets in >85% of the segments. However, none of the downstream molecules required for guidance downstream of grn has been identified to date. Several lines of evidence indicate that the Unc-5 receptor mediates guidance downstream of the GATA transcription factor Grn. First, both genes interact genetically in trans and this type of genetic interaction is often seen between two genes the gene products of which directly interact, such as Slit and Robo. Second, there is also a partial requirement of grn for unc-5 expression in dMNs as unc-5 mRNA levels are reduced in aCC and RP2 in grn mutants. Further support for the role of unc-5 downstream of grn comes from the partial rescue of the grn phenotype obtained by exogenously providing unc-5 specifically in aCC and RP2 (Zarin, 2012).

Among transcriptional codes that regulate motor-axon pathfinding, specific Lim-HD codes are required for the proper guidance of vertebrate motoneurons, in part, through the regulation of the EphA. In Drosophila, Nkx6 (HGTX -- FlyBase) is important for vMN specification and has been proposed to promote guidance through the expression of fasciclinIII (Fasciclin 3 -- FlyBase). Similarly, eve regulates guidance of dMNs through unc-5. In at least some of these situations the regulation is unlikely to be direct because the regulators are thought to mediate their function through a repressive activity. Indeed, eve repressive activity has been shown to be responsible for its guidance function in aCC and RP2, suggesting that its function might be to repress the expression of other transcription factors, such as HB9 (Exex -- FlyBase), which would confer those motoneurons with a ventral fate. Analysis of the unc-5 neuronal enhancer regulated by eve failed to identify any conserved Eve consensus homeodomain binding site, suggesting that eve regulation of unc-5 is not mediated through a direct binding to this element (Zarin, 2012).

Combinatorial codes of transcription factors play an instructive role in the generation of subclass diversity within the vertebrate spinal cord. In invertebrates, in which it is possible to analyse individual motoneurons within a subclass, a further level of complexity is revealed. Within the subclass of motoneurons that project to dorsal muscles, aCC and RP2, subclass determinants (eve, grn and zfh1) can work in a sequential order in aCC specification or independently, in parallel pathways, within RP2. Whereas Zfh1 is a general factor required in all motoneurons, eve and grn are specific to dMNs. It is plausible that each one of them might be important for specific aspects of specification within the same subclass of neurons but together might be responsible for the regulation of common targets such as unc-5. Although grn is required in both aCC and RP2 for unc-5 expression, it is not the only factor required because unc-5 mRNA is not completely absent from those cells and it also requires the presence of eve. In fact, grn or eve can independently induce unc-5 expression in dMP2 neurons but only both factors expressed in combination are able to induce axon exit towards the muscle field. This combinatorial expression of eve and grn might bring unc-5 above the threshold required for exit. unc-5 levels are definitely important for dMP2 exit because removing 50% of the gene dosage significantly suppresses the exit phenotype and this suppression is almost complete in an unc-5 null background. Transheterozygous interactions identified between unc-5 and eve, or unc-5 and grn also suggest that their levels are tightly controlled. As both eve and grn can regulate unc-5 it is likely that their combined activity in aCC and RP2 is essential to express the required levels of unc-5 in both neurons. A model is presented of unc-5 regulation by grn and eve in each individual neuron (Zarin, 2012).

The combinatorial nature of substrate recognition during motoneuron guidance and targeting is well established. Mutants for guidance receptors or ligands often show partially penetrant phenotypes and this includes the unc-5 phenotype in dMNs. By contrast, phenotypes observed for transcription factor mutants that affect dMN guidance are far more severe. For example, Lim1 regulates EphA4 in vertebrate motoneurons but the EphA4 mutant phenotype is less severe than Lim1 mutant phenotype. Similarly, 100% of ISNs in eve mutants or 85% in grn mutants are affected, suggesting that they regulate several guidance systems. It will be interesting to investigate the full array of guidance systems regulated by the eve-grn code. One potential candidate is FasII (Fas2 -- FlyBase) as it is an essential molecule for the pioneer function of aCC and RP2 (Zarin, 2012).

In summary, this study has identified the guidance receptor Unc-5 as a novel target of the GATA transcription factor Grn. unc-5 is key common target that mediates guidance downstream of both eve and grn. Furthermore, both transcription factors can promote transcription of unc-5 independently, suggesting that their combined action is essential to attain the proper expression levels of the Unc-5 receptor in dMNs. Future investigations in cell type-specific expression profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis will hopefully unravel other guidance systems regulated by the eve-grn code in dMNs. This knowledge will bring us closer to understanding the cell-specific transcriptional regulation of guidance (Zarin, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

The expression pattern of Unc5 during Drosophila embryogenesis was determined using in situ hybridization with antisense Unc5 probes and immunohistochemistry using antisera against Unc5 protein. Prior to gastrulation, Unc5 mRNA is strongly expressed in the presumptive mesoderm. Mesodermal expression begins to fade during stages 13-14, persisting only in the cells that form the dorsal vessel. Expression within the CNS begins during late stage 13, shortly after the first axons have extended. Unc5 mRNA can be detected in several dispersed clusters of cells within the CNS, increasing in number as development proceeds. Prominent Unc5 staining is also seen in the peripheral and exit glia, which migrate laterally out of the CNS between stages 14 and 17 (Keleman, 2001).

Anti-Unc5 antisera were generated using a peptide corresponding to the Unc5 amino terminus. Labeling with these antisera reveals accumulation of Unc5 protein on motor axons that exit the CNS ipsilaterally via the segmental nerve root (SN). No staining could be detected on either commissural or longitudinal axons within the CNS, nor on motor axons that exit via the intersegmental nerve (ISN). In the periphery, Unc5 protein can be detected on all branches of the SN. Exit and peripheral glia along both the SN and ISN also express high levels of Unc5 protein. By staining glial cells missing mutants, which lack these glia, it was confirmed that Unc5 protein is expressed on motor axons, not just glia, of the SN, while being undetectable on motor axons of the ISN and its branches (Keleman, 2001).

There is thus a striking complementarity between the expression patterns of Unc5 and the Netrins. Unc5-expressing motor axons avoid midline cells in the CNS and muscles in the periphery that express Netrins. Conversely, commissural axons that are attracted by Netrins do not express Unc5, nor do those motor axons that innervate Netrin-expressing muscles. The finding that Unc5 is expressed on SNa motor axons was particularly satisfying since these axons can be repelled by ectopic expression of NetB, either on all muscles (Mitchell, 1996) or on specific target muscles (Winberg, 1998). These data therefore strongly suggest that repulsion mediated by Netrins and Unc5 helps to guide SNa motor axons out of the CNS and on to their specific muscle targets (Keleman, 2001).

EVOLUTIONARY HOMOLOGS

C. elegans UNC5

Three known genes guide circumferential migrations of pioneer axons and mesodermal cells on the nematode body wall. unc-5 affects dorsal migrations, unc-40 primarily affects ventral migrations, and unc-6 affects migrations in both directions. Circumferential movements still occur, but are misdirected, whereas longitudinal movements are normal in these mutants. Pioneer growth cones migrating directly on the epidermis are affected; growth cones migrating along established axon fascicles are normal. Thus these genes affect cell guidance and not cell motility per se. It is proposed that two opposite, adhesive gradients guide circumferential migrations on the epidermis. unc-5, unc-6, and unc-40 may encode these adhesion molecules or their cellular receptors. Neurons have access to the basal lamina and the basolateral surfaces of the epidermis, but mesodermal cells contact only the basal lamina. These genes probably identify molecular cues on the basal lamina that guide mesodermal migrations. The same basal lamina cues, or perhaps related molecules on the epidermal cell surfaces, guide pioneer neurons (Hedgecock, 1990).

Growth cones in developing nervous systems encounter a sequence of extracellular cues during migration. In theory, a growth cone can navigate by selectively expressing or activating surface receptor(s) that recognize extracellular cues appropriate to each migratory phase. Using the simple C. elegans nervous system, attempts were made to demonstrate that path selection by migrating growth cones can be predictably altered by ectopic expression of a single receptor. The unc-5 gene of C. elegans encodes a unique receptor of the immunoglobulin superfamily (UNC-5), required cell-autonomously to guide growth cone and mesodermal cell migrations in a dorsal direction on the epidermis. The UNC-5 receptor induces dorsally oriented axon trajectories when ectopically expressed in the touch receptor neurons, which normally extend pioneer axons longitudinally or ventrally on the epidermis. These errant trajectories depend on unc-6, which encodes a putative epidermal path cue, just as do normal dorsally oriented axon trajectories (such as those of certain motor neurons), suggesting that UNC-5 acts to reorient the touch cell growth cones by using its normal guidance mechanisms. These results support previous evidence that UNC-5 and UNC-6 play instructive rules in guiding growth cone migrations on the epidermis in C. elegans, and indicate that pioneering growth cones, which normally migrate in different directions, may use equivalent intracellular signaling mechanisms for guidance (Hamelin, 1993).

The UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis of Caenorhabditis elegans. When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, like unc-6 mutations, suppress unc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance by unc-5. This paper describes eight genes that are required for ectopic unc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genes [unc-6 (the ligand for UNC-5), unc-40 (ankyrin repeat proteins serving as a putative link between UNC-5 and the cytoskeleton), unc-34, and unc-44]; mutations in four others identify the novel genes unc-129, seu-1, seu-2, and seu-3. Several of these mutations cause axon guidance defects similar to those found in unc-5 mutants. It is proposed that some or all of these genes may function in a developmentally important unc-5 signaling pathway (Colavita, 1998).

Cell migrations play a critical role in animal development and organogenesis. Here, a mechanism is described by which the migration behaviour of a particular cell type is regulated temporally and coordinated with over-all development of the organism. The hermaphrodite distal tip cells (DTCs) of C. elegans migrate along the body wall in three sequential phases that can be distinguished by the orientation of their movements, which alternate between the anteroposterior and dorsoventral axes. The ventral-to-dorsal second migration phase requires the UNC-6 netrin guidance cue and its receptors UNC-5 and UNC-40, as well as additional UNC-6-independent guidance systems. Evidence is provided that the transcriptional upregulation of unc-5 in the DTCs is coincident with the initiation of the second migration phase, and that premature UNC-5 expression in these cells induces precocious turning in an UNC-6-dependent manner. The DAF-12 steroid hormone receptor, which regulates developmental stage transitions in C. elegans, is required for initiating the first DTC turn and for coincident unc-5 upregulation. Evidence is also presented for the existence of a mechanism that opposes or inhibits UNC-5 function during the longitudinal first migration phase and for a mechanism that facilitates UNC-5 function during turning. The facilitating mechanism presumably does not involve transcriptional regulation of unc-5 but may represent an inhibition of the phase 1 mechanism that opposes or inhibits UNC-5. These results, therefore, reveal the existence of two mechanisms that regulate the UNC-5 receptor and are critical for responsiveness to the UNC-6 netrin guidance cue and for linking the directional guidance of migrating distal tip cells to developmental stage advancements (Su, 2000).

Cell and growth cone migrations along the dorsoventral axis of C. elegans are mediated by the UNC-5 and UNC-40 receptor subtypes for the secreted UNC-6 guidance cue. To characterize UNC-6 receptor function in vivo, genetic interactions were examined between unc-5 and unc-40 in the migrations of the hermaphrodite distal tip cells. Cell migration defects as severe as those associated with a null mutation in unc-6 are produced only by null mutations in both unc-5 and unc-40, indicating that either receptor retains some partial function in the absence of the other. Hypomorphic unc-5 alleles exhibit two distinct types of interallelic genetic interactions. In an unc-40 wild-type genetic background, some pairs of hypomorphic unc-5 alleles exhibit a partial allelic complementation. In an unc-40 null background, however, unc-5 hypomorphs exhibit dominant negative effects. It is proposed that the UNC-5 and UNC-40 netrin receptors can function to mediate chemorepulsion in DTC migrations, either independently or together, and the observed genetic interactions suggest that this flexibility in modes of signaling results from the formation of a variety of oligomeric receptor complexes (Merz, 2001).

Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, mutations were sequenced in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. The data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. These data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5 (Killeen, 2002).

Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored. This study shows that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the C. elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this anti-synaptogenic activity is interchangeable with that of LIN-44/Wnt despite being transduced through different receptors, suggesting that extracellular cues such as netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion (Poon, 2009).

Changes in axon outgrowth patterns are often associated with synaptogenesis. Members of the conserved Pam/Highwire/RPM-1 protein family have essential functions in presynaptic differentiation. This study shows that C. elegans RPM-1 negatively regulates axon outgrowth mediated by the guidance receptors SAX-3/robo and UNC-5/UNC5. Loss-of-function rpm-1 mutations cause a failure to terminate axon outgrowth, resulting in an overextension of the longitudinal PLM axon. PLM overextension observed in rpm-1 mutants is suppressed by sax-3 and unc-5 loss-of-function mutations. PLM axon overextension is also induced by SAX-3 overexpression, and the length of extension is enhanced by loss of rpm-1 function or suppressed by loss of unc-5 function. Loss of rpm-1 function in genetic backgrounds sensitized for guidance defects disrupts ventral AVM axon guidance in a SAX-3-dependent manner and enhances dorsal guidance of DA and DB motor axons in an UNC-5-dependent manner. Loss of rpm-1 function alters expression of the green fluorescent protein (GFP)-tagged proteins, SAX-3::GFP and UNC-5::GFP. RPM-1 is known to regulate axon termination through two parallel genetic pathways; one involves the Rab GEF (guanine nucleotide exchange factor) GLO-4, which regulates vesicular trafficking, and another that involves the F-box protein FSN-1, which mediates RPM-1 ubiquitin ligase activity. glo-4 but not fsn-1 mutations affect axon guidance in a manner similar to loss of rpm-1 function. Together, the results suggest that RPM-1 regulates axon outgrowth affecting axon guidance and termination by controlling the trafficking of the UNC-5 and SAX-3 receptors to cell membranes (Li, 2008).

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. This study shows that UNC-6/netrin, UNC-5 and UNC-40/DCC modulate the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants display increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants causes decreased protrusion. In contrast to previous studies, this work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controls polarity of growth cone protrusion and F-actin accumulation that correlates with attraction versus repulsion. However, filopodial dynamics are affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, this study showa that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion (Norris, 2011).

Mammalian UNC-5 homologs

Two vertebrate homologs of UNC-5 have been identified that along with UNC-5 and the product of the mouse rostral cerebellar malformation gene (rcm) define a new subfamily of the immunoglobulin superfamily. Their messenger RNAs show prominent expression in various classes of differentiating neurons. UNC5H1 and UNC5H2 are more similar to one another (52% identity) than to UNC-5 (28% identity in each case). Both have two predicted immunoglobulin-like domains and two predicted thrombospondin type-1 repeats in their extracellular domains, a predicted membrane-spanning region, and a large intracellular domain. The cytoplasmic domains do not contain obvious motifs, but do possess a small region of homology to Zona Occludens-1, a protein that localizes to adherens junctions and is implicated in junction formation. ZO-1 contains PDZ domains, structures implicated in protein clustering. Unc5h1 transcripts are detected at the early stages of neural tube development in the ventral spinal cord. At embryonic day 11, when motor neurons begin to differentiate in that region, transcripts are present throughout the ventral spinal cord, excluding the midline floor region, but are most intense in the ventricular zone and at the lateral edges. Unc5h2 transcripts are not detected at significant levels in the spinal cord until E14, when they are found in the roof plate region. These genes are also expressed in non-neural structures. Netrin-1 can bind cells expressing these proteins (Leonardo, 1997).

Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, the attractive responses, mediated by DCC, of Xenopus spinal axons to netrin-1 were studied. Attraction is converted to repulsion by expression of UNC5 proteins in these cells. This repulsion requires DCC function; the UNC5 cytoplasmic domain is sufficient to effect the conversion, and repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. Evidence is presented that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion (Hong, 1999).

To test whether the ectodomain of UNC5 proteins is required for repulsion, an examination was made of the effect of expressing a chimeric receptor in which the transmembrane and cytoplasmic domains of UNC5H2 (a human UNC5 homolog) were fused to the extracellular domain of DCC. Neurons expressing this DCC/UNC5H2 chimera show the same repulsive response to netrin-1 as do neurons expressing UNC5H2. To determine whether the transmembrane and cytoplasmic domains of UNC5H2 need to be fused to a netrin-binding ectodomain (as is the case for DCC), a chimeric receptor was examined in which the transmembrane and cytoplasmic domains of UNC5H2 were fused to the ectodomain of the NGF receptor TrkA, which does not bind netrin-1. Xenopus spinal neurons do not express TrkA endogenously and do not respond to an NGF gradient with either attraction or repulsion. Neurons expressing the TrkA/UNC5H2 chimera are repelled by netrin-1, a response that is blocked by the anti-DCC antibody; NGF has no effect on these neurons. These results suggested that the cytoplasmic domain of UNC5H2 might be sufficient for repulsion. This possibility was tested by generating a cDNA coding for the cytoplasmic domain of UNC5H2 preceded by a myristoylation sequence that targets cytoplasmic proteins to the inner leaflet of the plasma membrane. Neurons expressing this myristoylated UNC5H2 cytoplasmic domain construct exhibit marked repulsive responses to netrin-1. Thus, expression of the cytoplasmic domain of UNC5H2 is sufficient to convert netrin-mediated attraction to repulsion. It was then shown that netrin-1 triggers the formation of a heterodimeric or heteromultimeric complex involving DCC and UNC5H2 (Hong, 1999).

To further dissect the interaction between DCC and UNC5H2, attempts were made to identify regions in the DCC cytoplasmic domain required for the interaction. The first 46 amino acids are both necessary and sufficient for the interaction. Deletion of the juxtamembrane (JM) region (aas 1120-1148) does not abolish the interaction when performed in the context of the full-length cytoplasmic domain, and conversely, a construct comprising the JM domain alone does not suffice for the interaction. This shows that the JM domain is neither necessary nor sufficient for the interaction and identifies amino acids 1149-1166 as a key stretch required for the interaction. These 18 amino acids comprise the P1 domain, previously identified as a conserved domain among members of the DCC family. However, a construct comprising the P1 domain alone (aas 1149-1466) is not sufficient for the interaction. It is possible that the P1 domain does not fold properly in the absence of some adjacent sequences on either its amino- or carboxy-terminal ends; alternatively, the juxtamembrane region may be redundant with some other region of the cytoplasmic domain, with either one being sufficient but at least one being necessary (Hong, 1999).

Attempts were then made to identify the regions of UNC5 cytoplasmic domains required for DCC binding. Whereas a construct comprising UNC5H2 residues 707-946 is functional, a construct comprising residues 724-946 is not functional. Thus, residues 707-724 are required for binding the DCC cytoplasmic domain. These 18 residues are highly conserved among all previously described UNC5 proteins, and this domain has been termed the DB domain (since it is required for DCC binding). The DB domain is not the only domain required for repulsion, however. Deleting both the C-teminal Death Domain and 113 amino acids between the DB and the DD domains, but leaving the rest intact, including the DB domain, also results in a dominant-negative construct. Thus, sequences between the DD and DB domains are also important for repulsion, as could arise if these sequences are important for binding adaptor proteins. Deletion of the DB domain and all sequences carboxy terminal to it or deletion of all cytoplasmic domain sequences also results in the generation of dominant-negative constructs (Hong, 1999).

A paradox was raised by the finding that the isolated cytoplasmic domains of DCC and UNC5 proteins can interact, yet the full-length proteins do not coprecipitate in the absence of netrin-1. This raises the possibility that the interaction between cytoplasmic domains might be repressed in the context of the full-length proteins. To explore this possibility, a myristoylated cytoplasmic domain of one of the receptors (DCC or UNC5H2) was coexpressed with the full-length version of the other to see if they would coprecipitate. Full-length DCC coprecipitates with the myristoylated UNC5H2 cytoplasmic domain, but only in the presence of netrin-1. Similarly, only a low level of interaction of full-length UNC5H2 with the myristoylated DCC cytoplasmic domain is observed constitutively, and addition of netrin-1 dramatically increases the interaction. These results imply that in the absence of ligand, the UNC5H2 and DCC cytoplasmic domains are largely inaccessible to one another and that addition of netrin-1 causes some change in UNC5H2 and DCC that enables association of their cytoplasmic domains (Hong, 1999).

Why have a mechanism that switches from attraction to repulsion? The answer presumably lies in the fact that growth cones, as they navigate to their targets, change their responsiveness to guidance cues as they progress. Once a growth cone has reached a particular intermediate target, it must change its priorities in order to be able to move on to the next target. For example, commissural axons are initially attracted to the floor plate using netrin-1, but upon crossing the midline, they lose responsiveness to netrin-1. Since the axons continue to express DCC, the switching off must involve some other change. Another switch in growth cone sensitivity at the midline is the acquisition of Slit responsiveness by upregulation of expression of the Robo receptor in Drosophila. Although not yet demonstrated in vivo, it seems likely that there are circumstances where it is desirable not just to switch on or off responsiveness to a particular cue, but rather to convert the responsiveness from attraction to repulsion, to help move the growth cone along. The ability of one receptor to switch responses mediated by another receptor provides an economical means to achieve this end and avoid confusing the growth cone with simultaneous conflicting signals for attraction and repulsion (Hong, 1999 and references).

A recently described recessive mouse mutant, rostral cerebellar malformation (rcm/rcm), demonstrates a swaying gait at approximately 12 days of age. The mutant cerebellar (Cb) phenotype consists of cerebellar tissue that extends rostrally, beyond the usual distinct anterior cerebellar boundary, into the midbrain. Interestingly, the cerebellar ectopia occurs in the absence of any significant alterations in the distribution of nuclear groups within the brainstem. The ectopic Cb tissue is (1) adherent to the posterior and lateral aspects of the inferior colliculus and to the lateral aspect of the rostral brainstem and (2) contains acellular regions within the inner granular layer (igl) and ectopic, calbindin-immunoreactive Purkinje cells (PCs) deep relative to the igl. Within the Cb proper, PC organization is generally normal, as revealed by zebrin II immunoreactivity. In the ectopic Cb tissue PCs also exhibit a banded zebrin distribution. Analysis of the spinocerebellar projection in the mutant suggests a lobular distribution similar to that seen in the normal mouse. Within the anterior region, however, the normal parasagittal banding pattern is somewhat obscured. Spinocerebellar innervation of the ectopic Cb tissue exists, but it is almost exclusively confined to the region adjacent to the caudal inferior colliculus. In conjunction with the recent finding that the mutation appears to affect a UNC-5-like receptor protein for netrin-1 (a molecule that may be involved in axonal guidance and cell migration), these results suggest that this mutant is an important model for the analysis of cerebellar development and regionalization (Eisenman, 1998).

Mutation of the Unc5h3 (formally known as rcm) gene has important consequences on neuronal migration during cerebellar development. Unc5h3 transcripts are expressed early (embryonic day 8.5) in the hindbrain region and later in the cerebellar primordia. In Unc5h3 mutant embryos, both the development and initial migration of Purkinje cell progenitors occur as in wild-type controls. The rhombic lip, from which granule cell precursors arise, also appears to form normally in mutants. However, at E13.5, an abnormal subpopulation of granule cell and Purkinje cell precursors becomes detectable in rostral areas of the Unc5h3 mutant brain stem. These ectopic cerebellar cells increase in number and continue moving in a rostral direction throughout the remainder of embryogenesis and early stages of postnatal development invading the lateral regions of the pontine area and eventually the inferior colliculus. Cell proliferation markers demonstrate the mitotic nature of these subpial ectopic granule neurons, indicating the displacement of the rostral external germinal layer in mutant animals. These data suggest that establishment of the rostral cerebellar boundary may rely on chemorepulsive signaling events that require UNC5H3 expressed by cerebellar neurons and extracellular ligands that are functionally related to the UNC5H3-binding and guidance molecule, netrin1. Although the phenotype resulting from the Unc5h3 mutation is apparently limited to the formation of the cerebellum, additional sites of Unc5h3 expression are also found during development suggesting the compensatory function of other genes (Przyborski, 1998).

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and its Caenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5, is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. The Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, corticospinal tract fibers respond differently to loss of UNC5H3. In addition, corticospinal tract defects are observed in mice homozygous for a spontaneous mutation that truncates the Dcc transcript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrate unc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates (Finger, 2002).

The crystal structure of netrin-1 in complex with DCC reveals the bifunctionality of netrin-1 as a guidance cue

Netrin-1 (see Drosophila Netrins) is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. This study presents the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC; see Drosophila Frazzled) receptor.One netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, this study demonstrates that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. It is proposed that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses (Finci, 2014).

Phosphorylation of mammalian UNC-5 homologs

C. elegans UNC-5 and its mammalian homologs such as RCM are receptors for the secreted axon guidance cue UNC-6/netrin and are required to mediate the repulsive effects of UNC-6/netrin on growth cones. C. elegans UNC-5 and mouse RCM are phosphorylated on tyrosine in vivo. C. elegans UNC-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK-293 cells, demonstrating that phosphorylation of UNC-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activated Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmic tyrosine residues creating potential binding sites for cytoplasmic signaling proteins. Indeed, the NH(2)-terminal SH2 domain of the Shp2 tyrosine phosphatase binds specifically to a Tyr(568) RCM phosphopeptide. Furthermore, Shp2 associates with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitates with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphorylated on tyrosine than the wild type receptor. These results suggest that netrin-stimulated phosphorylation of RCM Tyr(568) recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function (Tong, 2001).

Mammalian UNC-5 homologs and cell survival

Acting as receptors for netrin-1, the membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Dependence receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand, but inhibiting apoptosis in the presence of ligand. The netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Evidence is presented that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H (Llambi, 2001).

Since UNC5H proteins are cleaved by protease and more specifically by caspase, an interesting model suggests that this cleavage allows the release or the exposure of a fragment that induces cell death. However, while expression of cleavage fragments issued from DCC, RET and AR allow cell death induction, expression of the UNC5H2 C-terminal fragment lying after Asp412 has no pro-apoptotic activity unless a myristoylation signal peptide is added. This observation then suggests the requirement of a sub-membrane localization of this fragment for cell death induction. Interestingly, both DCC and UNC5H proteins show oligomeric properties, which may explain heterodimeric binding of full-length UNC5H molecules with caspase-cleaved C-terminal fragments. One hypothesis then is that a heterodimeric complex allows, within membrane proximity, the exposure of the pro-apoptotic fragment lying downstream of the caspase cleavage site (Llambi, 2001).

It is of interest that this pro-apoptotic fragment contains a death domain. Such death domains have been found in various receptors including death receptors Fas and tumor necrosis factor receptor (TNFR) and the dependence receptor p75NTR. They are considered as 'adaptor' domains, allowing interaction of these receptors with 'adaptor' proteins. Death domains can be divided into two types (i.e. I or II) depending on their ability to homodimerize. Sequence alignment reveals that the UNC5H2 death domain is more related to the type II death domain of p75NTR than to the type I death domain of Fas, suggesting that the death domain of UNC5H probably displays no ability to homodimerize. In any case, both Fas and p75NTR death domains have been reported to be crucial for cell death induction. Remarkably, the deletion of the UNC5H2 death domain totally abrogates UNC5H2 pro-apoptotic activity. Taken together these results suggest that in the absence of netrin-1, UNC5H proteins induce cell death via the requirement of their death domain, which is probably exposed via the caspase cleavage. The role of this death domain is, however, completely unknown. The death domain of Fas allows, via the recruitment of the 'adaptor' molecule FADD, the formation of a caspase-activating complex that drives caspase-8 activation. It is also interesting to note that DCC, while without a death domain, recruits a caspase-activating complex allowing caspase-3 activation via the interaction of DCC with caspase-9. Whether the UNC5H death domain is involved in another caspase-activating complex through the recruitment of 'adaptor' molecules via its death domain needs now to be analysed further (Llambi, 2001).

The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP

UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. Previous studies have discovered that attractive UNC-40/DCC receptor signaling (see Drosophila Frazzled) stimulates growth cone filopodial protrusion and that repulsive UNC-40-UNC-5 heterodimers inhibit filopodial protrusion in C. elegans. This study identified cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. The Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition (Norris, 2014).

Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target (Larrivée, 2007).

These results clearly show that UNC5B functions as a receptor for Netrin-1 in vivo, confirming and extending previous studies. It remains to be determined if Netrin-1 represents the (only) relevant in vivo ligand for UNC5B in mice. In zebrafish embryos, MO-mediated knockdown of unc5b or netrin-1a led to increased filopodial extensions and aberrant vessel branching of intersegmental vessels (ISV). The data in zebrafish are consistent with netrin-1a as a negative regulator of vessel branching. However, the results reported here do not exclude a possible proangiogenic role of Netrin-1. Nonendothelial cells in the ischemic area expressing unc5b (and perhaps other Netrin receptors) could respond to Netrin-1 and perhaps contribute to ischemic revascularization. In addition, no endothelial unc5b expression was observed following femoral artery ligation, and stimulation of UNC5B-negative endothelial cells by Netrin-1 could elicit proangiogenic responses. The present study provides multiple lines of evidence indicating that repulsive responses following Netrin-1 stimulation are consistently observed during neovascularization processes where unc5b is expressed, including tumor angiogenic sprouting. Development of UNC5B-selective agonists may be considered as potential therapeutic tools in anti-angiogenic strategies (Larrivée, 2007).

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3alpha/beta and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. This study shows that the axon guidance cue Netrin-1 (see Drosophila Netrin-A and Netrin-B) promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, this study demonstrates that Netrin-1 can substitute for blockade of Gsk3alpha/beta and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, this study reveals how Netrin-1 and the balance of its receptors Neo1 (a DCC-related protein; see Drosophila Frazzled) and Unc5B (see Drosophila Unc-5) co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase (see Drosophila Fak) to inactivate Gsk3alpha/beta (see Drosophila Shaggy) and stabilize beta-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment (Huyghe, 2020).

REFERENCES

Search PubMed for articles about Drosophila unc-5

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Finger, J. H., et al. (2002). The Netrin 1 receptors Unc5h3 and Dcc are necessary at multiple choice points for the guidance of corticospinal tract axons. J. Neurosci. 22(23): 10346-10356. 12451134

Ghavi-Helm, Y., Klein, F. A., Pakozdi, T., Ciglar, L., Noordermeer, D., Huber, W., Furlong, E. E. (2014) Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512(7512): 96-100. PubMed ID: 25043061

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Hong, K., Hinck, L., Nishiyama, M., Poo, M. M., Tessier-Lavigne, M. and Stein, E. (1999). A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97: 927-941. 10399920

Huyghe, A., Furlan, G., Ozmadenci, D., Galonska, C., Charlton, J., Gaume, X., Combemorel, N., Riemenschneider, C., Allegre, N., Zhang, J., Wajda, P., Rama, N., Vieugue, P., Durand, I., Brevet, M., Gadot, N., Imhof, T., Merrill, B. J., Koch, M., Mehlen, P., Chazaud, C., Meissner, A. and Lavial, F. (2020). Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling. Nat Cell Biol 22(4): 389-400. PubMed ID: 32231305

Keleman, K. and Dickson, B. J. (2001). Short- and long-range repulsion by the Drosophila Unc5 Netrin receptor. Neuron 32: 605-617. 11719202

Killeen, M., et al. (2002). UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but Its UNC-40-independent functions also require a region between the ZU-5 and death domains. Dev. Bio. 251: 348-366. 12435363

Larrivée, B., et al. (2007). Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis. Genes Dev. 21(19): 2433-47. PubMed citation: 17908930

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date revised: 20 December 2024

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