BIOLOGICAL OVERVIEW

The gene frazzled was so named because mutants are observed to shake upon revival from ether-induced anesthesia. Once frazzled had been cloned, it was revealed that the protein it encoded was related to the human gene Deleted in Colorectal Cancer (DCC). DCC is somewhat a misnomer, since it was discovered that the gene on chomosome 18q whose loss is involved in generating colorectal cancer is, in fact, Dpc4 (Smad4) the homolog of Drosophila Medea involved in TGFbeta signaling. Thus DCC is no longer a candidated gene for colorectal cancer. Frazzled and DCC share 52% amino acid identity; both belong to the immunoglobulin superfamily. In structure, these particular human and fly proteins are composed of four extracellular immunoglobulin C2 repeats, six fibronectin III repeats and an evolutionarily conserved intracellular domain. fra is expressed on axons in the developing nervous system and on midgut and ectodermal epidermis. Mutation of fra results in partially penetrant defects (defects that are not always apparent) in commissures and axon pathfinding. Similar to two other Drosophila proteins, Fasciclin II and Neuroglian, Frazzled also presents extensive homology to vertebrate neural adhesion molecule L1. Like Frazzled, both Fasciclin II and Neuroglian have extracellular Ig domains and fibronectin III repeats. It has recently been demonstrated that Neuroglian interacts with the membrane cytoskeleton of the cell, acting through Ankyrin (Dubreuil, 1996). It may well be that this is also the case with Frazzled.

What makes Frazzled of particular interest is its homology to DCC and to a C. elegans protein (UNC-40), both of which have been shown to function as netrin receptors. The N-terminal two-thirds of both netrins A/B are homologous to the N-termini of the polypepide chains of Laminin, a large heterotrimeric protein of the extracellular matrix. Does Frazzled likewise act as a receptor for Netrin in Drosophila?

Approximately 40 motor axons in each abdominal hemisegment of the Drosophila embryo extend into the periphery (outside the CNS) where they innervate 30 body wall muscles: all extension and innervation is carried out in a highly stereotyped pattern. A subset of motor axons exit the ventral CNS in the intersegmantal nerve (ISN) and extend dorsally to innervate the Netrin A/B-expressing dorsal muscles 1 and 2 (Mitchell, 1996).

In fra mutant embryos, these ISN axons, which would normally express Frazzled, continue to extend dorsally, but often branch or extend inappropriately once they reach the dorsal muscle region. In a fraction of hemisegments these mutant ISN axons wander into adjacent segements or toward the dorsal midline, and appear to make contact with inappropriate muscles, or branch more extensively over their normal muscle targets.

The ISN motor axon defects in fra mutants strongly resemble those observed for Netrin A/B mutant embryos. In both fra and NetrinA/B mutants, the posterior commissure is more severly affected than the anterior. In Netrin mutants, the ISN axons display a similar frequency of dorsal muscle targeting errors. Innervation of muscles 6 and 7 by the SNb motor axon is similarly affected. In fra and netrin mutants the SNa axons project normally to their lateral muscle targets, however, these are targets which do not normally express netrin. These axons do express frazzled and consequently their trajectory can be altered by ectopic netrin expression on all muscles. In frazzled mutants, ectopic expression of frazzled in all muscles (rather than just in the neurons where it is usually expressed) neither rescues nor enhances frazzled motor axon defects. Paradoxically, ectopic expression of frazzled in all neurons does not appear to cause guidance defects (Kolodziej, 1996).

This work strongly suggests that Frazzled is a receptor or a ligand-binding component of a Drosophila Netrin receptor. This is far from the whole story, however. Both Netrin proteins are expressed in muscles from both the dorsal and ventral muscle groups, and both are strongly expressed by midline cells during the initial period of commissure formation and axonogenesis in the ventral nerve cord. In addition, a pair of large cells located posterior to the posterior commissure also stain strongly for one of the netrins. In the peripheral nervous system motor axons (located above the dorsal and ventral muscle groups) stain for one of the netrins (Mitchell, 1996 and Harris, 1996). With such a complex expression pattern for netrins, it is surprising that the ectopic expression of frazzled does not result in breakdown of axon guidance. Perhaps there exist considerable backup cues that allow proper axon guidance even in an environment where one receptor or just a single component of a receptor is misexpressed.

Frazzled is required in the target for establishment of retinal projections in the Drosophila visual system. Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. The role of the Netrins and their receptor Frazzled have been investigated in the establishment of retinal projections. The Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Frazzled is not required for the differentiation of cells in the target. These data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. The possibility is favored that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor (Gong, 1999).

net A and net B are expressed in identical patterns: both transcripts are expressed in lamina precursors, which in wild type form an arc-shaped ribbon of cells. Thus, the Netrins are expressed in a pattern that would allow them to act as signals for incoming fibers. Fra protein, in contrast, is strongly expressed in photoreceptor axons, suggesting that retinal fibers have the ability to sense Netrin in the target. Interestingly, Fra is also expressed in the target structure, the lamina. fra transcripts are found in an arc-shaped band of cells similar to net transcripts, but double RNA in situ hybridizations reveal that fra and net transcripts do not colocalize to the same cells. Instead, fra transcripts are expressed in more mature lamina precursor cells located posteriorly adjacent to the net-expressing lamina precursor cells. While the transcript is only expressed very transiently, Fra protein expression persists and is thus present throughout the differentiated lamina and in all lamina cells (Gong, 1999).

What is the role of Fra in the target cells? Fra is not required for neuronal or glial differentiation of lamina precursor cells. Non-innervated lamina precursor cells lacking fra can express the early neuronal differentiation marker Dachshund or the glial differentiation marker Repo, as long as the cells are within range of the diffusible differentiation signals emanating from ingrowing retinal fibers. Interestingly, for neuronal differentiation, this range appears to be restricted to a few cell diameters, while for glial differentiation, this range must be much larger, since even very large clones of fra appear to have a normal complement of glial cells. In fact, glial differentiation may be largely independent of retinal innervation, as has been suggested by a previous study which showed that even in uninnervated animals some glial cells are present in the lamina anlage. Together, these findings demonstrate that the presence of differentiated neuronal and glial cells in the target is not sufficient for the attraction of retinal fibers. Moreover, they exclude the possibility that Fra is merely indirectly involved in retinal fiber attraction by mediating target cell differentiation and point instead to a more direct role for Fra in the target for attracting retinal fibers (Gong, 1999).

What is the molecular function of Fra in the target cells? The fact that removal of both Netrins does not affect the retinal projection makes it unlikely that Fra functions as a Netrin receptor in the lamina target. Further, the fact that removal of Fra from the retinal fibers does not affect their projection, makes it unlikely that Fra functions as a homotypic cell adhesion molecule, directly effecting the attractive interaction between retinal fibers and their target cells. Given these findings, a third possibility is favored: Fra in the target cells may interact in a heterotypic fashion with an unidentified component on the surface of retinal fibers. It is possible that this component is another Netrin receptor. This idea is supported by the finding that Netrin misexpression in retinal fibers results in projection defects that phenotypically mimic the removal of Fra from the target, suggesting the presence in retinal fibers of another Netrin receptor in addition to Fra. The existence of additional Netrin receptors in the fly is expected. Apart from an UNC-5 type receptor (see Drosophila unc-5), which has been found in both worms and vertebrates, a second DCC/UNC-40 homolog may also exist in the fly, based on genetic evidence that UNC40 function is partially redundant in the worm: molecular null alleles of unc40 display a less severe phenotype than some truncation alleles, suggesting that the truncated proteins interfere with a second pathway. Of course, alternative models are possible. Whatever the identity of the interacting partner, the presence of Fra on target cells is a prerequisite for any innervation by retinal fibers. Fibers whose designated target area lacks fra avoid the area by rerouting into fra+ regions. It is interesting that, in avoiding fra mutant regions, retinal fibers do not scramble randomly to reach fra+ areas, but rather reroute in an orderly fashion. When foregoing their a-p position, retinal fibers appear to reroute as a cohort and, when misprojecting along the d-v axis, they maintain their relative order. This finding argues that the process of retinotopic map formation relies on two functionally separable mechanisms: one mediating attraction to the target, the other providing positional information. In vertebrates, positional information in the retinotectal system appears to be largely provided by graded repulsive interactions between retinal fibers and target cells mediated by Ephrins and their receptors. Such a repulsive mechanism for defining positional values requires an underlying attraction of innervating fibers to the target. Thus, it will be interesting to learn whether DCC receptors, similar to their role in the Drosophila visual system, serve to attract retinal fibers to their target in the vertebrate visual system as well (Gong, 1999).

Cytoplasmic domain requirements for Frazzled-mediated attractive axon turning at the Drosophila midline

The conserved DCC ligand-receptor pair Netrin and Frazzled (Fra) has a well-established role in axon guidance. However, the specific sequence motifs required for orchestrating downstream signaling events are not well understood. Evidence from vertebrates suggests that P3 is important for transducing Netrin-mediated turning and outgrowth, whereas in C. elegans it was shown that the P1 and P2 conserved sequence motifs are required for a gain-of-function outgrowth response. This study demonstrates that Drosophila fra mutant embryos exhibit guidance defects in a specific subset of commissural axons and these defects can be rescued cell-autonomously by expressing wild-type Fra exclusively in these neurons. Furthermore, structure-function studies indicate that the conserved P3 motif (but not P1 or P2) is required for growth cone attraction at the Drosophila midline. Surprisingly, in contrast to vertebrate DCC, P3 does not mediate receptor self-association, and self-association is not sufficient to promote Fra-dependent attraction. In contrast to previous findings, the cytoplasmic domain of Fra is not required for axonal localization, and neuronal expression of a truncated Fra receptor lacking the entire cytoplasmic domain (FraDeltaC) results in dose-dependent defects in commissural axon guidance. These findings represent the first systematic dissection of the cytoplasmic domains required for Fra-mediated axon attraction in the context of full-length receptors in an intact organism and provide important insights into attractive axon guidance at the midline (Garbe, 2007).

At first glance, the domain requirements for DCC/Fra/Unc-40 signaling might appear to differ among species. For example, in vertebrates it has been demonstrated that the P3 conserved sequence motif is essential for the outgrowth and turning of cultured Xenopus spinal neurons. Whereas in C. elegans, P3 is not required to generate a gain-of-function phenotype associated with expression of MyrUnc-40; however, P1 and P2 are essential for this response. Can the difference in motif requirements between vertebrates and C. elegans simply be attributed to a divergence in conserved functions for these domains throughout evolution? Given the high degree of sequence similarity of these motifs, this seems unlikely. Here it should be noted that the potential function of the conserved P1-P3 motifs in full-length Unc-40 receptor signaling in the context of an in vivo attractive decision have not been examined. Therefore, perhaps the apparent divergence in function could simply be attributed to the phenotypic readout of each assay in the individual systems (Garbe, 2007).

In the case of axonal attraction, the data are in agreement with previous reports from vertebrates suggesting that P3 is required for a Netrin-mediated turning response whereas they are inconsistent with P1 and P2 playing obligatory roles. These deletion receptors also offer the exciting opportunity to study the signaling and/or domain requirements for additional Netrin-Fra-mediated decisions such as the promotion of axon outgrowth and the steering of motor neurons to their appropriate ventral muscle targets (Garbe, 2007).

In vertebrate systems, P3 has been implicated in mediating two distinct functions. Initially, it was reported that the conserved cytoplasmic P3 sequence motif is necessary and sufficient for ligand-gated receptor multimerization and Netrin-induced attractive turning in cultured Xenopus spinal neurons. Versions of DCC lacking the P3 motif cannot self-associate and neurons expressing this form of DCC are no longer able to respond to Netrin. Replacing the P3 motif with the SAM multimerization domain from Eph tyrosine kinase receptors is sufficient to restore an appropriate Netrin response, suggesting that the major function of the P3 domain is to mediate self-association. Surprisingly, this study found that P3 does not appear to mediate self-association of the Drosophila Fra receptor, as mutants lacking P3 show equivalent biochemical interactions. It seems clear from these data that P3 function in the context of midline attraction is through a mechanism that is independent of mediating receptor multimerization. Although it remains an open question whether the receptor-receptor interactions that were observed in vitro are necessary for attractive guidance, it is clear that in the absence of an intact P3 domain they are not sufficient (Garbe, 2007).

Another set of studies proposed that P3 recruits Fak and this recruitment leads to tyrosine phosphorylation of DCC by Src family non-receptor tyrosine kinases. Both Fak recruitment and tyrosine phosphorylation are important in mediating Netrin-induced outgrowth and attractive turning in cultured neurons in vitro. Specifically, the LD-like motif within P3 was shown to play a critical role in FAK association, although a P3-independent binding site has also been suggested. Interestingly, this study found that mutant Fra receptors in which the LD motif is intact, but the rest of P3 is disrupted are still unable to mediate Fra attraction, suggesting that Fak binding may not be important for Fra function in Drosophila. This is also consistent with the observation that fak mutants do not have a disrupted CNS phenotype. Nevertheless, future studies will test for genetic interactions between mutations in fra and mutations in fak and/or src in the context of the Drosophila midline. It is worth noting that the tyrosine residue in DCC identified as the principal target of Fyn/Src kinases is not conserved in Drosophila Fra or C. elegans UNC-40, suggesting that the precise mechanisms by which Fra/DCC/UNC-40 signaling is regulated by tyrosine kinases may differ between organisms. However, the facts that, (1) tyrosine phosphorylation of UNC-40 has been observed in C. elegans (though the responsible kinase has not been identified) and (2) UNC-40 signaling appears to be negatively regulated by a receptor tyrosine phosphatase is consistent with an evolutionarily conserved role for tyrosine phosphorylation of the DCC/Fra/UNC-40 family of receptors. Furthermore, the Abl kinase has also been implicated in Netrin-Fra signaling in Drosophila suggesting that additional phosphorylation events may be important for DCC/Fra/UNC-40 output (Garbe, 2007).

Previous data determined that the cytoplasmic domain of Fra is necessary and sufficient for normal distribution of the receptor. Therefore, it was surprising to find that the newly generated FraDeltaC-HA localized similarly to the wild-type protein. The original experiments (Hiramoto, 2000) were performed using a truncated Fra receptor that contained the transmembrane domain and 67 juxtamembrane cytoplasmic amino acids of the Robo receptor (FraDeltaC^Robo67-Myc). It is hypothesized that this exongenous protein sequence could be interfering with proper localization of the Fra receptor. Indeed this seems to be the case; whereas the FraDeltaC-HA construct mimics wild-type receptor localization when expressed in all neurons by elavGal4, the original FraDeltaC^Robo67-Myc does not. Therefore, these data suggest that normal Fra localization does not require the cytoplasmic domain. Additional experiments will help determine the specific domains of Fra that are sufficient to control receptor distribution. Finally, all of the above observations are based on overexpression studies and therefore may not completely reflect the localization of endogenous proteins with similar deletions (Garbe, 2007).

Intriguingly, expression of FraDeltaC in a fra mutant background results in a complete commissureless phenotype, suggesting the possibility that it is capable of inhibiting Fra-independent axon attraction. Although this is one of the simplest interpretations, other hypotheses exist. For example, double mutants between fra null alleles and either abl or trio also exhibit a near commissureless phenotype. These data are consistent with Abl and Trio participating in other pathways that are important for guidance toward and across the Drosophila midline. Accordingly, one possibility could be that FraDeltaC is interfering with an independent Abl and/or Trio signaling pathway (Garbe, 2007).

Previous results demonstrated that panneural overexpression of Netrin leads to a phenotype where few axons cross the midline. It was suggested that wild-type Netrin distribution provides a directional cue that attracts axons across the midline and when Netrin is misexpressed in all neurons, axons become confused and are no longer able to decipher the appropriate path. Along these lines, perhaps the extracellular domain of FraDeltaC is binding Netrin and `presenting' it everywhere thereby confusing the axons. Indeed, the Fra receptor has been shown to redistribute Netrin laterally away from its midline source. However, this theory would have to assume that this specific truncation is somehow misregulated upon ligand binding (for example, perhaps it is not efficiently internalized) since other wild-type and deletion receptors - which presumably bind to and relocalize Netrin similarly to FraDeltaC - do not produce this effect when expressed panneurally. Since a commissureless phenotype is seen only when FraDeltaC is overexpressed in a fra background, it would also be necessary to argue that another receptor elicits the response to this un-internalized and redistributed Netrin (Garbe, 2007).

Finally, FraDeltaC expression may cause increased midline repulsion, thereby preventing axons from crossing the midline. Accordingly, the FraDeltaC misexpression phenotype shows a striking similarity to embryos deficient for the gene comm. Comm is a single-pass transmembrane protein that acts to downregulate Robo expression and comm, robo double mutants resemble robo single mutants indicating that comm acts upstream of the Robo receptor. Therefore, if FraDeltaC misregulates Comm, then perhaps Robo levels are increased and axons are repelled away from the midline. This argument would imply that overexpressing FraDeltaC in a robo mutant background should produce robo-like mutants (similar to the comm, robo double mutants). Contrary to this hypothesis, it was observed that FraDeltaC expression can partially suppress a robo loss-of-function phenotype suggesting that FraDeltaC is not simply misregulating Comm. However, since FraDeltaC cannot prevent all axons from approaching the midline in fra, slit double mutants, and given that the FraDeltaC overexpression phenotype in a fra, robo double mutant background is weaker than that seen in fra single mutants, the possibility cannot be ruled out that signaling through Robo is partially required and/or that the upregulation of another Robo family member, for example Robo2, prevents axons from approaching the midline when FraDeltaC is expressed in all neurons. Whatever the mechanism by which FraDeltaC exerts its influence on commissural axon guidance, it may provide an important route to a further dissection of the missing factors that function in addition to Netrin and Fra to guide axons across the midline (Garbe, 2007).

The intracellular domain of the Frazzled/DCC receptor is a transcription factor required for commissural axon guidance

In commissural neurons of Drosophila, the conserved Frazzled (Fra)/Deleted in Colorectal Cancer (DCC) receptor promotes midline axon crossing by signaling locally in response to Netrin and by inducing transcription of commissureless (comm), an antagonist of Slit-Roundabout midline repulsion, through an unknown mechanism. This study shows that Fra is cleaved to release its intracellular domain (ICD), which shuttles between the cytoplasm and the nucleus, where it functions as a transcriptional activator. Rescue and gain-of-function experiments demonstrate that the Fra ICD is sufficient to regulate comm expression and that both γ-secretase proteolysis of Fra and Fra's function as a transcriptional activator are required for its ability to regulate comm in vivo. These data uncover an unexpected role for the Fra ICD as a transcription factor whose activity regulates the responsiveness of commissural axons at the midline and raise the possibility that nuclear signaling may be a common output of axon guidance receptors (Neuhaus-Follini, 2015).

This study has identify the Fra ICD as a transcription factor that regulates the expression of comm, a key modulator of axonal responsiveness at the midline. γ-secretase proteolysis of Fra releases its ICD, which is capable of nuclear translocation and is sufficient to promote midline crossing and regulate comm expression in rescue and gain-of-function assays in vivo. The conserved P3 motif within the Fra ICD functions as a transcriptional activation domain and this activity is required for Fra's regulation of comm expression. Thus, in addition to its canonical role signaling locally to regulate growth cone dynamics, Fra functions as a transcription factor to regulate axonal responsiveness at the midline (Neuhaus-Follini, 2015).

comm is expressed in commissural neurons with exquisite temporal specificity. How might the transcriptional activity of the Fra ICD be regulated to contribute to comm's expression pattern? γ-secretase proteolysis is typically the second cleavage event in a proteolytic cascade, preceded by ectodomain shedding. Indeed, previous pharmacological experiments suggest that DCC's ectodomain is shed as a result of metalloprotease cleavage and that this proteolytic event is required for subsequent γ-secretase-dependent processing. Metalloprotease-dependent ectodomain shedding is often ligand-dependent, while subsequent γ-secretase processing depends on the shape of the membrane-tethered metalloprotease cleavage product. For example, metalloprotease-dependent shedding of the Notch ectodomain is stimulated by the binding of Notch ligands, and the subsequent γ-secretase cleavage of the membrane-tethered ICD is constitutive. As Fra regulates comm independent of Netrins, Fra ectodomain shedding may occur in response to the binding of a different ligand. Alternative ligands for DCC have been identified, including the vertebrate- specific proteins Draxin and Cerebellin 4. In addition, the secreted protein MADD-4 physically associates with the C. elegans ortholog of Fra/DCC, UNC-40, and guides sensory neurons and muscle arms in an UNC-40-dependent manner. The function of the Drosophila ortholog of MADD-4, Nolo, has not been investigated, nor has its ability to bind to Fra (Neuhaus-Follini, 2015).

It seems unlikely that the transcriptional activity of the Fra ICD is controlled at the level of nuclear localization. When Fra ICDDP3 (lacking a NES) was expressed in the commissural EW neurons in vivo, it accumulates in the nucleus at the earliest developmental stages that can be observed, suggesting that the Fra ICD is constitutively imported into the nucleus. Nuclear accumulation of full-length Fra ICD (with a NES) was only observed occasionally, implying that after the Fra ICD translocates to the nucleus, it is rapidly exported. The fact that Fra's NES and activation domain are both encoded by P3 raises the possibility that when Fra is engaged in transcriptional activation, the association of co-activators with P3 might prevent it from associating with nuclear export machinery, coupling Fra's nuclear activity to its nuclear retention (Neuhaus-Follini, 2015).

The finding that Fra's ability to regulate comm expression depends on its function as a transcriptional activator seems to imply that the Fra ICD can associate with chromatin, but the Fra ICD does not contain an obvious DNA-binding domain. A Neo DNA-binding domain has not been identified either, but chromatin immunoprecipitation experiments have demonstrated that the Neo ICD associates with chromatin in vitro. The Fra ICD's DNA-binding activity and specificity probably arise from associations between the Fra ICD and DNA-binding partners, as is the case with Notch. The Notch ICD has no DNA-binding activity of its own and associates with DNA as part of a complex including an obligate CSL (CBF1/ RBPjk, Su(H), Lag-1) DNA-binding partner. If the Fra ICD can associate with multiple DNA-binding proteins, it might allow the Fra ICD to regulate the expression of many different target genes, depending on which of its DNA-binding partners are expressed in particular cell types or developmental contexts (Neuhaus-Follini, 2015).

The observation that a structurally intact P3 is required for Fra-dependent transcription suggests that P3 plays another role in Fra's transcriptional output besides its function as an activation domain. One possibility is that P3 is required for Fra's association with chromatin, perhaps by functioning as a binding interface for Fra's DNA-binding co-factors. This idea is supported by the observation that FraE1354A antagonizes midline crossing in both fra mutants and heterozygotes, while FraDP3 has only a mild effect. Perhaps the ICD of FraE1354A inhibits midline crossing by occupying chromatin sites that are normally targets of both Fra and other transcriptional activators that act in a parallel pathway; the ICD of FraDP3 would not have this effect if P3 is required for Fra's association with chromatin. FraE1354A is not likely to be inhibiting endogenous Fra in rescue experiments, as fra³ is either a strong hypomorphic or null allele. This model predicts that Fra has other transcriptional targets in EW neurons that are relevant for commissural axon guidance. It will be informative to identify additional transcriptional targets of Fra both in embryonic commissural neurons and in other cell types. In the retina, R8 photoreceptor axons have targeting defects that are much milder in Netrin mutants than in fra mutants, raising the possibility that the Netrin-independent output of Fra signaling in this system might be through the transcriptional pathway that this study has identified (Neuhaus-Follini, 2015).

Cleavage of axon guidance receptors has been shown to regulate the activities of these receptors in a number of different ways. Degradation of axon guidance receptors can provide temporal control of axonal sensitivity to guidance cues. In vertebrates, this mode of regulation controls axonal responsiveness to members of the class 3 family of secreted Semaphorins (Sema3s), which signal repulsion through Neuropilin (Nrp)/ Plexin (Plex) co-receptors. Calpain proteolysis of PlexA1 in pre-crossing spinal commissural neurons reduces their sensitivity to Sema3B, which is expressed in the ventral spinal cord as these axons are growing toward the ventral midline. ADAM metalloprotease cleavage of Nrp1 reduces the sensitivity of proprioceptive sensory axons to Sema3A allowing them to terminate in the ventral spinal cord, where Sema3A expression is high. In addition, γ-secretase proteolysis of DCC in vertebrate motor neurons inhibits their responsiveness to midline-derived Netrin, preventing them from ectopically projecting toward the midline (Neuhaus-Follini, 2015).

Proteolytic processing has also been implicated as a requisite step in local repulsive Robo signaling in Drosophila. The Robo ectodomain is cleaved by the ADAM metalloprotease Kuzbanian and this proteolytic event is required for Robo's ability to transduce repulsive signals in vivo and for Slit-dependent recruitment of effectors of local Robo signaling in vitro. As γ-secretase-dependent intramembrane proteolysis is typically constitutive following ectodomain shedding, and occurs subsequent to metalloprotease processing of the human Robo1 receptor, it is likely that Drosophila Robo is cleaved to produce a soluble ICD. The observation that Robo proteolysis is required for local Slit-Robo signaling does not exclude the possibility that the Robo ICD may also have a nuclear function that contributes to axon guidance, but this possibility has not yet been explored (Neuhaus-Follini, 2015).

Proteolysis has also been identified as a regulator of contact- mediated axonal repulsion. Eph receptors signal repulsion in response to their transmembrane ephrin ligands; ephrins can also function as receptors, signaling repulsion in response to Eph binding. Metalloprotease and subsequent γ-secretase cleavage of both Ephs and ephrins have been demonstrated, providing a mechanism through which adhesive interactions can be broken to allow for repulsive signaling. The importance of this mode of regulation for axon targeting has not yet been established in vivo and a recent study using an EphA4 variant that is insensitive to metalloprotease cleavage suggests that EphA4 proteolysis is not required for EphA4-dependent motor axon targeting (Neuhaus-Follini, 2015).

This study has identified a new way in which axon guidance receptor proteolysis can influence axon responsiveness to guidance cues. γ-secretase-dependent processing of Fra releases its ICD, which translocates to the nucleus, where it functions as a transcription factor to regulate the guidance of commissural axons. It is proposed that the ability to signal from the nucleus may be a common property of axon guidance receptors and may serve as a general mechanism through which axon guidance receptors regulate their own activities or the activities of other proteins. Human Robo1 is processed by sequential metalloprotease and γ-secretase cleavage and its ICD localizes to the nucleus in vitro. It remains to be seen whether the ICDs of Ephs and ephrins, which are cleaved by γ-secretase, and of Plexins, which are proteolytically processed, but have not yet been identified as γ-secretase substrates, translocate to the nucleus as well. It will also be interesting to determine whether the ICDs of Fra and other axon guidance receptors signal from the nucleus to regulate aspects of neuronal morphogenesis and function besides axon pathfinding. Finally, recent work indicating that the cleaved C terminus of the Drosophila Wnt receptor Frizzled translocates to the nucleus and contributes to the establishment of postsynaptic structures by regulating RNA export serves as a reminder that the trafficking of cell surface receptor fragments to the nucleus may allow these fragments to signal not only by regulating transcription, but in other ways as well (Neuhaus-Follini, 2015).

Islet coordinately regulates motor axon guidance and dendrite targeting through the Frazzled/DCC receptor

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. This study shows that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring (Santiago, 2017).

The RP3 motor neurons innervate the NetrinB-expressing muscles 6 and 7 and are enriched for fra mRNA during the late stages of embryonic development, and it was reported previously that, in the absence of fra or Netrin, there are significant defects in the innervation of muscles 6 and 7. This phenotype is also detected in the absence of hb9/exex or isl/tailup, two transcription factors expressed in RP3 as well as in other ventrally projecting motor neurons, suggesting that hb9 or isl may regulate fra. Interestingly, Hb9, Isl, and the LIM homeodomain factor Lim3 were all recently shown to bind directly to the fra locus in vivo, as determined by a genome-wide DNA adenine methyltransferase identification (DAM-ID) analysis performed in Drosophila embryos. However, DAM-ID results do not provide information about the functional significance of the detected binding events or about the cell types in which they occur. To determine whether Hb9, Isl, or Lim3 regulate the expression of fra in embryonic motor neurons, situ hybridization experiments were performed and fra mRNA expression was analyzed with single-cell resolution in embryos mutant for these factors. Only isl is required for fra expression in the RP3 motor neurons at stage 15, when RP axons have reached the ventral muscle field but their final targets have not been selected. 80% of RP3 neurons in abdominal segments A2-A7 in isl/+ embryos are positive for fra mRNA versus 38% in isl mutant embryos. A significant difference was also observed in fra mRNA levels in RP3 neurons between isl mutants and heterozygotes when quantifying pixel intensity from the fra in situ, whereas no difference was detected in the signal of the isl-H-tau-myc transgene. No change was detected in the number or position of RP3 neurons in isl mutants, consistent with previous data demonstrating that Isl is not required for the generation or survival of Drosophila motor neurons. Importantly, no requirement was found for either hb9 or lim3 in regulating fra mRNA expression in any RP motor neurons, demonstrating that isl's effect on fra is specific and could not have been predicted simply from similarities in loss of function phenotypes or from transcription factor binding data (Santiago, 2017).

Hb9 has been shown to be required for robo2expression in RP3. Interestingly, just as hb9 is not required for fra expression in RP neurons, isl is not required for robo2expression. A previous study reported that isl; hb9 double mutants have a stronger intersegmental nerve b (ISNb) phenotype than either single mutant, but muscle 6/7 innervation defects were not quantified. This study scored motor axon guidance defects in isl; hb9 double mutants and found that the double mutants display significantly more muscle 6/7 innervation defects than either single mutant. Similarly, embryos mutant for both robo2and fra have a stronger motor axon phenotype than either robo2or fra single mutants. Note that, because robo2, fra double mutants have severe defects in midline crossing, motor axon phenotypes should be interpreted with caution . These results show that Hb9 and Isl act in parallel to regulate distinct downstream programs in RP3 neurons, demonstrating how combinations of transcription factors result in specific cell surface receptor profiles and axon trajectories (Santiago, 2017).

To determine whether isl and fra act in the same genetic pathway during RP3 guidance, embryos mutant for both genes were examined. In isl-null mutants, 20% of hemisegments lack muscle 6/7 innervation, whereas fra-null mutants have a significantly stronger phenotype (34% of hemisegments). isl, fra double mutants do not have more muscle 6/7 innervation defects than fra single mutants, consistent with isl and fra acting in the same pathway. If fra acts downstream of Isl during motor axon targeting, then it was reasoned that restoring Fra expression in isl mutant neurons might rescue muscle 6/7 innervation. Indeed, it was found that pan-neural overexpression of Fra in isl mutants partially but significantly rescues these defects. The difference between genotypes was most noticeable when hemisegments were counted in which a growth cone stalls at the 6/7 cleft as well as those in which it fails to reach it. In isl mutants, a growth cone stalls at or fails to reach the 6/7 cleft in 27% of hemisegments compared with 15% of hemisegments in sibling mutants overexpressing Fra. The data was also analyzed by comparing the number of embryos with 6/7 innervation defects. In isl mutants, 0% of embryos have no 6/7 innervation defects in A2-A6, 44% have one defect, and 56% have two or more defects. In contrast, in isl mutants overexpressing Fra, 29% of embryos have no innervation defects, 29% have one defect, and 41% have two or more defects. The incomplete rescue could be due to differences in the timing or levels of GAL4/UAS-mediated expression of Fra compared with its endogenous regulation or could indicate that Isl regulates additional downstream effectors important in this process. Nevertheless, these data strongly suggest that Fra is an essential downstream effector of Isl during the guidance of the RP3 axon to its target muscles (Santiago, 2017).

To further investigate the relationship between isl and fra, whether ectopic expression of isl is sufficient to induce fra expression was tested. These experiments used the apterous (ap) neurons. The axons from this subset of interneurons form a single fascicle on either side of the midline that are labeled by ap-Gal4. The ap neurons express low levels of fra, do not express isl, and do not cross the midline. Fra overexpression causes ectopic midline crossing of ap axons. Overexpression of Isl with ap-Gal4 produced high levels of midline crossing, phenocopying the effect of Fra overexpression. In stage 17 control embryos, ap axons cross the midline in 12% of segments, whereas in embryos overexpressing UAS-Isl with ap-Gal4, ap axons cross the midline in 60% of segments. This phenotype is dose-dependent because embryos with two copies of an UAS-Isl insertion display significantly more ectopic midline crossing than embryos with one insert (Santiago, 2017).

To determine whether Isl overexpression results in fra induction, the expression of fra mRNA in situ was examined in ap neurons. In stage 15 wild-type embryos, a low percentage of ap neurons express fra (25% of ventral ap clusters were fra+). In contrast, in embryos overexpressing isl from two UAS-Isl inserts, 37% of the ventral ap clusters were fra+. To test whether the ectopic crossing phenotype depends on fra function, Isl was overexpressed in embryos homozygous for a null allele of fra. Strikingly, removing fra completely suppresses the crossing phenotype, indicating that fra is required for Isl to produce its gain-of-function effect. Although it cannot be ruled out that Isl affects the expression of other genes in the Fra pathway to cause midline crossing, these results demonstrate that ectopically expressing Isl causes an increase in fra expression and a fra-dependent axon guidance phenotype and suggest that the functional relationship between isl and fra may be used in multiple contexts (Santiago, 2017).

Fra mutants have defects in RP axon midline crossing, as shown by retrograde labeling of single motor neurons. In addition, Netrin-Fra signaling controls the medio-lateral position of dendrites in several groups of motor neurons. Therefore, it was asked whether isl regulates midline crossing or RP3 dendrite development through fra. A genetic strategy was used to label single motor neurons by mosaic expression of a membrane-tethered GFP transgene under the control of lim3b-GAL4, which labels RP motor neurons, sensory neurons, and several other motor and interneuron populations. RP3 neurons were identified by the stereotyped position of the RP3 cell body and by the targeting of its axon to muscles 6 and 7. Because of the axon targeting defects observed in isl and fra mutants, cell body position was used to identify RP3 neurons in mutants. By this approach, significant midline crossing defects were detected in RP3 axons in fra mutants. Surprisingly, however, no defects were observed in RP axon midline crossing in isl mutants (Santiago, 2017).

Isl and fra expression both initiate earlier than stage 13, the time at which RP axons cross the midline. Therefore, whether isl is required for fra expression was examined during the early stages of commissural axon guidance. Interestingly, isl was not required for fra expression at stage 13 in any of the ventrally projecting RPs. In contrast, in stage 15 isl mutant embryos from the same collection, a decrease in was observed fra expression in RP1 and RP3. The temporal pattern of fra expression in RP motor neurons is dynamic, so that a larger proportion of RP1 and RP3 neurons express fra mRNA during late embryogenesis than during the stages of midline crossing. A requirement was detected for isl in regulating fra in RP1 and RP3 as early as stage 14, when the RP motor axons have exited the CNS. Taken together, these results suggest that isl is not essential for early fra expression but required for fra expression during the late stages of motor neuron differentiation. The stages at which a requirement was detected for isl in regulating fra correspond to when RP3 axons are exploring their ventral muscle targets, consistent with a model in which Isl instructs the final stages of RP3 axon targeting through Fra (Santiago, 2017).

Another essential feature of Drosophila larval motor neurons that is established late in embryogenesis is the morphogenesis and targeting of their dendrites in the ventral nerve cord. Motor neuron dendrites begin to form as extensions off the primary neurite at stage 15, a stage when a requirement is detected for isl in regulating fra. By early stage 17 (15 hr after egg laying, AEL), RP3 has assumed its stereotyped morphology, consisting of a small ipsilateral projection extending from the soma and a large dendritic arbor forming off the contralateral primary neurite (Santiago, 2017).

The FLP-out genetic labeling strategy was used to visualize individual late-stage RP motor neurons and analyze their dendrites. Focus was placed on the large contralateral arbor of the RP motor neurons that spans one side of the nerve cord in wild-type embryos and forms branches that extend into several medio-lateral zones. Analyses using isl-tau-myc and lim3a-tau-myc transgenes confirmed that the RP cell bodies retain their stereotyped positions in isl mutants and that the relative dorsal-ventral positions of RPs 1/4, 3, and 5 are preserved, allowing identification of distinct classes of RP motor neurons (Santiago, 2017).

Most RP3 neurons in late-stage isl/+ embryos neurons form contralateral dendritic arbors that send projections into the zone between the medial FasII+ axon pathways and the intermediate FasII+ pathways, hereafter referred to as the 'intermediate zone,' consistent with previously published images of RP3 neurons from wild-type embryos. Interestingly, the dendritic morphology of RP3 was distinct from that of a related neuron, RP5, that also expresses Isl and Lim3b-Gal4 and that can be unambiguously identified in both wild-type and mutant embryos because its cell body is found in a more ventral position than the other RP neurons. In wild-type embryos, the RP5 axon targets muscles 12 and 13 (VL1 and VL2) as well as other ventral muscles. Most RP5 neurons in isl/+ embryos exclusively target their dendrites to the lateral zone of the neuropile. Furthermore, the difference observed in the dendritic targeting of RP3 and RP5 neurons correlates with a difference in fra expression. Although fra expression in RP3 and RP5 neurons in control embryos is comparable when RP axons are crossing the midline, by stage 15, significantly fewer RP5 than RP3 neurons express fra. Interestingly, isl is not required for the low levels of fra expression in late-stage RP5 neurons, in contrast to its role in promoting high levels of fra in late-stage RP3 neurons (Santiago, 2017).

Finally, endogenous Netrin expression was monitored in late-stage nerve cords using a Myc-tagged NetB knockin allele, and significant enrichment of Netrin protein was detected in the area between the intermediate and medial FasII+ axon bundles. This area corresponds to the zone where contralateral dendritic projections from RP3 neurons were detected, suggesting that high levels of Fra in RP3 may instruct the formation of dendritic arbors in this region in response to Netrin (Santiago, 2017).

RP motor neuron dendrites were examined in isl mutant embryos to determine whether Isl regulates dendritic position or morphogenesis through Fra or other effectors. No significant difference in the morphology or medio-lateral position of RP5 dendrites was observed between heterozygous and mutant embryos. In striking contrast, many RP3 neurons in isl mutants fail to extend contralateral dendrites into the intermediate zone. Instead, the dendrites of these RP3 neurons remain fasciculated with the intermediate FasII+ axon pathways and do not send medial extensions toward the midline. To more quantitatively measure medio-lateral position and to address the possibility that defects in targeting are secondary to defects in outgrowth, RP3 neurons were traced using Imaris software and total contralateral dendrite lengths and the total number of dendrite tips were measured. Total length of contralateral dendrites was also measured in the intermediate zone of the neuropile, defined as the area between the medial FasII+ and the intermediate FasII+ axon pathways. Although RP3 neurons displayed increased variability in the size of their dendritic arbors in isl mutants, there was no significant difference in the total length or tip number of RP3 dendrites between isl mutants and heterozygotes, suggesting that targeting defects in isl mutants are not likely due to reduced outgrowth. However, the ratio of RP3 dendrites in the intermediate zone over total RP3 dendrite length was significantly reduced in isl mutants, confirming that isl mutant RP3 dendrites are shifted laterally relative to controls (Santiago, 2017).

The dendrites of RP3 neurons were examined in fra/+ and fra mutant embryos. As with isl mutants, cell body position was used to identify RP3 neurons, and neurons with ambiguous positions were excluded. In fra mutant RP3 neurons whose axons fail to cross the midline, a single dendritic arbor forms off the ipsilateral primary neurite, and this arbor was traced. A significant lateral shift was observed in the position of RP3 dendrites in fra mutants both by scoring for the presence of dendrites in the intermediate zone and by quantitative analysis of the dendrites of traced neurons. The lateral shift in fra mutants was more pronounced than in isl mutants, consistent with the observation that some RP3 neurons retain fra expression in the absence of isl. Of note, the lateral shift phenotype did not correlate with whether the RP3 axon had crossed the midline because it was detected at similar frequencies in both contralateral and ipsilateral arbors. Curiously, several RP3 contralateral dendritic arbors appeared reduced in size in fra mutants, whereas this phenotype was not seen in control embryos. However, as in isl mutants, there was no significant change in the total dendrite length or tip number in fra mutants compared with their sibling controls, although there was increased variability in the sizes of dendritic arbors in the mutants. These findings are consistent with previous reports that Netrin-Fra signaling does not play a major role in regulating the outgrowth of motor neuron dendrites in the nerve cord (Santiago, 2017).

A single-cell labeling method allows precise description of the axon targeting defects in isl and fra mutants and determine whether they correlate with defects in dendrite position. Axon and dendrite targeting occur at approximately the same developmental stage, and there is no evidence that one process depends on the other. Importantly, previous studies using retrograde labeling of motor neurons in mutant embryos were not able to address this question because they relied upon motor axons reaching the correct muscles to be visualized (Santiago, 2017).

To determine whether defects in dendrite position correlate with defects in axon targeting, both phenotypes were scored in single labeled RP3 neurons in embryos with muscles fully preserved following dissection. All of the RP3 axons that could be scored in isl heterozygous embryos innervated the muscle 6/7 cleft . In contrast, 18 of 26 isl mutant RP3 axons innervated muscles 6/7, and eight stalled at the 6/7 cleft or earlier along RP3's trajectory or bypassed the choice point. In fra mutant embryos, 10 of 22 RP3 neurons failed to innervate the muscle 6/7 cleft and stalled at or bypassed the choice point. This phenotype is stronger than the frequency at which a complete loss of muscle 6/7 innervation in isl or fra mutants was detected by scoring with anti-FasII. To determine whether this enhancement was due to the heat shock (H.S.) step that is required for genetic labeling, defects were scored using anti-FasII in embryos heat-shocked for either 5 min or 1 hr; it was found that the 1-hr H.S. mildly enhances muscle innervation defects in isl mutants (to 30.4%) whereas a 5-min H.S. does not (to 24.7%, data not shown). Importantly, the two H.S. protocols did not result in any difference in the frequency of dendrite targeting defects observed in isl mutants because 7 of 17 RP3 dendrites in isl mutants are shifted laterally in embryos treated with 1-hr H.S, and 9 of 16 dendrites are shifted after 5-min H.S (Santiago, 2017).

Surprisingly, no correlation was detected between axon and dendrite defects in isl mutants. Although 5 of 26 RP3 neurons displayed defects in both axons and dendrites in isl mutants, 12 of 26 neurons showed defects in one process but not the other). A similar analysis in fra mutants revealed that 8 of 22 RP3 neurons displayed defects in both muscle 6/7 innervation and dendrite position, whereas 8 of 22 displayed normal targeting in one process but not the other. These data suggest that axon and dendrite targeting can occur independently within an individual RP3 neuron and that the central targeting defects observed in isl mutants are not likely to be secondary to defects in muscle innervation (Santiago, 2017).

It was next asked whether isl and fra regulate dendrite development in other classes of motor neurons. RP1 and RP4 also express isl, fra, and lim3b-Gal4. A requirement was detected for isl in regulating fra expression in RP1, but not in RP4, at stage 15. Interestingly, most RP1 neurons, like RP3 neurons, retain high levels of fra at this stage, whereas few RP4 neurons express fra in late-stage control embryos. Previous descriptions of RP1 and RP4 neurons indicate that they form contralateral dendritic arbors of distinct morphologies; RP1's dendritic arbor is taller and found more medially. However, because the axons of RP1 and RP4 target adjacent muscles external to muscles 6 and 13, and their cell bodies are found close to the midline at a similar dorsal-ventral position, they could not nr unambiguously distinguished in single-cell labeling experiments. Nevertheless, when RP1 and RP4 neurons were scored together, a significant lateral shift was observed in the position of RP1 and RP4 dendrites in isl mutants compared with heterozygous siblings: 3 of 22 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in isl heterozygous embryos (14%) compared with 16 of 22 in mutant embryos. A similar phenotype was detected in RP1 and RP4 dendrites in fra mutants. Specifically, 6 of 24 RP1 and RP4 dendritic arbors were excluded from the intermediate zone in heterozygotes (25%) compared with 17/19 in fra mutants. Although additional work will be necessary to determine whether the defects in dendrite position that were detect in RP1 and RP4 neurons in isl mutants correlate with changes in fra expression, these data demonstrate that Isl is required for high levels of fra expression in at least two classes of motor neurons (RP1 and RP3), both of which require isl and fra for dendritic targeting (Santiago, 2017).

To directly test whether isl regulates RP3 dendrite position through its effect on fra expression, a UAS-HA-Fra transgene was overexpressed using lim3b-GAL4 in isl mutants and the hsFLP technique was used to sparsely label RP motor neurons. Strikingly, in isl mutants overexpressing Fra, 0 of 21 RP3 contralateral dendritic arbors were excluded from the intermediate zone compared with 8 of 22 (36%) in sibling mutants lacking the UAS-Fra transgene. To quantitatively measure dendrite position, traces of RP3 dendrites were obtained. A robust rescue of the lateral shift phenotype was detected in isl mutants, as measured by the length of dendrites in the intermediate zone over the total dendrite length. Indeed, the ratio of dendrites in the intermediate zone in rescued mutants was higher than in heterozygous controls, perhaps reflecting a gain-of-function effect caused by artificially high levels of Fra from transgenic overexpression. Importantly, Fra overexpression did not have any effect on total dendritic arbor lengths or tip numbers, strongly arguing that the rescue that was observe is not caused by an increase in the total size of the arbors. Although it cannot be ruled out that Isl regulates dendrite position in part through additional effectors, the observation that cell-type-specific overexpression of Fra in isl mutants rescues dendrite targeting provides compelling support for the model that fra acts downstream of isl to control RP3 dendrite morphogenesis. Together with the demonstration that isl directs RP3 motor axon targeting through the regulation of fra, it is concluded that isl coordinately regulates the targeting of axons in the periphery and of dendrites in the CNS through a common downstream effector (Santiago, 2017).

In the vertebrate spinal cord, the position of motor neuron cell bodies correlates with the targeting of their axons in the periphery (Catela, 2015). This myotopic map may be established through the action of transcription factors that coordinately control cell migration and axon guidance. In particular, Lhx1 and Isl1 are expressed in limb-innervating lateral motor column (LMC) motor neurons and regulate the trajectory of their axons as well as the medio-lateral settling position of their cell bodies . Lhx1 and Isl1 regulate axon guidance through EphA4 and EphB receptors, respectively, and a recent study suggests that Lhx1 regulates cell body position through a distinct effector, the Reelin signaling protein Dab-1 (Santiago, 2017).

In Drosophila, unlike in vertebrates, the position of motor neuron cell bodies does not necessarily correlate with the targeting of their axons in the periphery because neurons that innervate adjacent muscles can be found far apart within a segment. Instead, recent studies have shown that both the larval and the adult Drosophila nervous systems use a myotopic map in which the position of motor neuron dendrites, rather than their cell bodies, correlates with the position of their target muscles (Brierley, 2009, Mauss, 2009). This may be a conserved feature of motor systems across phyla because the dendritic patterning of at least four motor neuron pools in the spinal cord correlates with muscle target identity in the mouse (Santiago, 2017).

Slit-Robo, Netrin-Fra, and Sema-Plexin signaling have been shown to control motor neuron dendrite targeting in Drosophila, and rescue experiments suggest that these guidance receptors act cell-autonomously in this process (Brierley, 2009, Mauss, 2009, Syed, 2016). In addition, the initial targeting of motor neuron dendrites in the embryo is largely unaffected by manipulations that affect the position or the activity of pre-synaptic axons or the presence of muscles, suggesting that this process is likely under the control of cell-autonomous factors, although these remain unidentified (Santiago, 2017).

This study has addressed several key questions about how motor neuron dendrite targeting is specified in Drosophila. First, it was shown that fra expression in two classes of motor neurons (RP3 and RP5) correlates with the medio-lateral position of their dendrites. Previous studies suggested that different classes of motor neurons express different levels of guidance receptors to direct the position of their dendrites, but this has not been demonstrated. Second, Isl, which was previously shown to regulate axon targeting in a subset-specific way, was also shown to regulate dendrite targeting. Third, it was found that Isl regulates both processes through fra. Surprisingly, no correlation was found between axon and dendrite phenotypes in isl mutants. The absence of a correlation suggests that the dendrite positioning defects are not secondary to defects in target selection, consistent with a previous study in which the general patterning of motor neuron dendrites was not disrupted in muscle-less embryos. However, additional experiments that disrupt axon targeting and monitor the medio-lateral position of dendrites will be necessary to confirm that the two occur independently (Santiago, 2017).

Future work will also be necessary to identify additional transcription factors that specify motor neuron dendrite development. A role has been identified for Hb9 in regulating robo2and robo3 expression, but it is not known whether these receptors regulate motor neuron dendrite development. No change was detected in robo1 mRNA levels in RP3 neurons in either hb9 or isl mutants. Robo signaling could be regulated post-transcriptionally. Comm is required for midline crossing of motor neuron dendrites and may endogenously regulate their medio-lateral position. The temporal pattern of comm expression does not support a role in dendrite targeting, however, because comm is not expressed in RP motor neurons at late stages of embryogenesis (Santiago, 2017).

The functional consequences of dendrite targeting defects remain to be explored. It is likely that shifting the position of motor neuron dendrites alters their connectivity, but testing this hypothesis will require identifying the pre-synaptic neurons that impinge on the RPs during locomotive behavior. Forcing a shift in the position of dendrites of dorsally projecting motor neurons does not abolish their connectivity with known pre-synaptic partners but does change the number of contacts established. In mice, the ETS factor Pea3/Etv4 is required for the dendritic patterning of a subset of motor neurons, and electrophysiological recordings reveal changes in connectivity in Pea3 mutant spinal cords. It will be of high interest to investigate whether analogous defects are detected in isl or fra mutant embryos (Santiago, 2017).

Drosophila Isl was initially described as a subset-specific regulator of axon guidance. More recently, Wolfram (2012) demonstrated that Isl also acts instructively to establish the electrophysiological properties of RP motor neurons through repression of the potassium ion channel Shaker. The curreng data show that, in addition to regulating the axonal trajectory and the electrophysiological properties of the RP3 neuron, Isl also establishes its dendritic position. Terminal selectors have been defined as transcription factors that coordinately regulate gene programs conferring multiple aspects of a neuron's identity, including its neurotransmitter phenotype, ion channel profile, and connectivity. Unlike the early-acting factors that function transiently to specify cell fate, terminal selectors are expressed throughout the life of an animal and are required for the maintenance of neural identity. Although there are several examples of transcription factors that act this way, it remains unclear how widespread a phenomenon it is. Does Isl fit the criteria for a terminal selector? Isl is not required for all aspects of RP3 identity because RP neurons retain expression of other motor neuron transcription factors in isl mutants, and their axons exit the nerve cord. Future work will be necessary to determine whether Isl is required throughout larval life for the maintenance of RP3's physiological and morphological features and to what extent Isl coordinately establishes multiple features of RP neuron identity (Santiago, 2017).

Co-expressed transcription factors could act synergistically to regulate specific downstream programs, in parallel through completely distinct effectors, or by some combination of the two mechanisms. Indeed, examples of all of these scenarios have been described. Both in vitro and in vivo studies demonstrate that, in vertebrate spinal motor neurons, Isl1 forms a complex with Lhx3 and that the Isl1-Lhx3 complex binds to and regulates different genes than Lhx3 alone or than a complex composed of Isl1 and Phox2b, a factor expressed in hindbrain motor neurons. In a subset of spinal commissural neurons, Lhx2 and Lhx9 act in parallel to promote midline crossing through upregulation of Rig-1/Robo3. In Drosophila dorsally projecting motor neurons, Eve, Zfh1, and Grain act in parallel to promote the expression of unc5, beat1a, and fas2, although Eve also regulates additional targets important for axon guidance that are not shared by Zfh1 or Grain (Santiago, 2017).

This study shows that Isl and Hb9 act in parallel through at least two distinct effectors and proposes that they regulate their targets by different mechanisms. Hb9 likely indirectly promotes robo2 expression by repressing one or multiple intermediate targets because its conserved Engrailed homology repressor domain is required for its function in motor axon guidance and for robo2 regulation. In vertebrate motor neurons, Isl1 forms a complex with Lhx3 to directly activate several of its known targets. A recent genome-wide DAM-ID analysis found that Isl binds to multiple regions within and near the fra locus in Drosophila embryos, suggesting that it may directly activate fra. The finding that lim3 is not required for fra expression in RP motor neurons, together with evidence that Isl can alter the electrical properties of muscle cells independently of Lim3, suggest that Drosophila Isl does not need to form a complex with Lim3 for all of its functions. Future research will be necessary to detect Isl binding events in embryonic motor neurons, although these experiments are challenging when binding occurs transiently or in a small number of cells. Interestingly, overexpression of Isl using ap-Gal4 or hb9-Gal4 induces fra only in certain subsets of these neurons, consistent with a model in which Isl binds to the fra locus in a cell-type-specific manner. The generation of many large-scale datasets for transcription factor binding sites presents the field with the task of reconciling these data with clearly defined genetic relationships during specific biological processes. This study and others have initiated this effort, but it will be important to investigate the functional significance of other putative transcription factor-effector relationships to achieve a better understanding of how transcriptional regulators control cell fate (Santiago, 2017).

Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs

Frazzled (Fra) and deleted in colorectal cancer (Dcc) are homologous receptors that promote axon attraction in response to netrin. In Drosophila, Fra also acts independently of netrin by releasing an intracellular domain (ICD) that activates gene transcription. How neurons coordinate these pathways to make accurate guidance decisions is unclear. This study shows that the ADAM metalloprotease Tace cleaves Fra, and this instructs the switch between the two pathways. Genetic manipulations that either increase or decrease Tace levels disrupt midline crossing of commissural axons. These conflicting phenotypes reflect Tace's function as a bi-directional regulator of axon guidance, a function conserved in its vertebrate homolog ADAM17: while Tace induces the formation of the Fra ICD to activate transcription, excessive Tace cleavage of Fra and Dcc suppresses the response to netrin. It is proposed that Tace and ADAM17 are key regulators of midline axon guidance by establishing the balance between netrin-dependent and netrin-independent signaling (Zang, 2022).

This study reports a conserved role for Tace and ADAM17 in coordinating different receptor signaling outputs. In the context of commissural axon guidance, strong biochemical and genetic evidence is provided demonstrating that Tace functions in both the canonical netrin-dependent Fra pathway and the non-canonical netrin-independent Fra pathway. Specifically, this study has shown that in the non-canonical pathway, Tace cleaves Fra to induce the formation of a transcriptionally active ICD fragment that regulates the expression of Fra's target gene comm. In the canonical pathway, overexpression of Tace results in excessive cleavage of Fra that inhibits the receptor's ability to respond to netrin. Because both Fra pathways are essential for proper midline attraction, such bi-directional regulation by Tace could assist the neurons in orchestrating appropriate signaling outputs. Thus, a model is proposed in which the levels and/or activity of Tace must be correctly regulated to ensure that Fra signals effectively. When the levels or activities of Tace are low, the commissural neuron favors the netrin-dependent canonical pathway to promote midline crossing. In contrast, when Tace is activated, the commissural neuron decreases its responsiveness to netrin and switches to the non-canonical Fra pathway to regulate gene transcription. Together, these two pathways are maintained in a tightly controlled balance and cooperate to facilitate commissural axon midline crossing. Importantly, this study also demonstrated a clear functional similarity between Tace and ADAM17, arguing that ADAM17-dependent cleavage of Dcc likely allows for a similar segregation of receptor signaling outputs in vertebrate systems (Zang, 2022).

Previous studies have revealed that ADAM-dependent proteolysis can impinge on downstream signaling pathways of guidance receptors in many ways. First, ADAM cleavage can terminate receptor signaling by reducing receptor surface levels. Second, ADAM cleavage can facilitate association between the receptor and its downstream effector proteins.Third, ADAM cleavage can physically separate receptor-ligand complexes to switch adhesion to repulsion. Previous studies have demonstrated that both of Fra's vertebrate homologs, Dcc and neogenin, are also ADAM substrates, yet the physiological significance and the precise mechanisms of how proteolysis affects downstream signaling of these receptors remain unclear. This study has shown that in Drosophila, Tace induces the formation of both Fra ECDs and ICDs and is required for the expression of Fra's transcriptional target gene comm. Unlike the mechanisms described above, the current data establish a distinct pathway in which an ADAM can activate an axon guidance receptor by initiating subsequent γ-secretase cleavage to induce transcriptional activity of the receptor. It remains to be seen whether the same non-canonical mechanism is conserved for ADAM17 and Fra's vertebrate homologs Dcc and neogenin. In Drosophila commissural neurons, γ-secretase cleaves Fra to generate an ICD fragment, allowing it to translocate to the nucleus to regulate the transcription of comm (Zang, 2022).

Previous studies have demonstrated that the neogenin ICD is present in the nucleus in both chick retinal ganglion cells and zebrafish embryos. In addition, in vitro evidence from cell lines suggests that both neogenin and Dcc ICDs can localize to the nucleus to modulate gene transcription, suggesting that similar non-canonical pathways exist for Dcc and neogenin as well. This study has shown that conditional removal of Adam17 disrupts midline crossing in vivo. If the only function of ADAM17 at the vertebrate midline is to downregulate netrin and Dcc signaling, it would be expected to see increased crossing when ADAM17 levels are reduced. Thus, in vertebrates, ADAM17 behaves similarly to Tace in Drosophila to positively regulate midline crossing. This observation supports a potential role for ADAM17 in activating the transcriptional activities of Dcc at the vertebrate midline (Zang, 2022).

While Dcc is arguably the most likely substrate, it should be noted that analysis in embryonic mouse spinal cords does not exclude the possibility that ADAM17 functions by regulating other substrates. One possibility is neogenin, which, in addition to netrin, also binds to its canonical ligand repulsive guidance molecule (RGM) with much higher affinity. Cleavage of neogenin by ADAM17 desensitizes axons to RGM, which abolishes its repulsive effects on neurite outgrowth. Importantly, neogenin contributes to commissure formation in the mouse spinal cord by binding and facilitating Dcc and netrin signaling through its ECDs (Zang, 2022).

Thus, it is unlikely that neogenin is the primary ADAM17 substrate, as shedding of its ECDs should have deleterious effects on midline guidance, which is contrary to what wsd observed in Adam17 cKO embryos. Another possible substrate is the repulsive receptor Robo1. While it has not been determined whether Robo1 is a substrate of ADAM17, it is possible that cleavage of Robo1 inhibits its repulsive function, which could explain the reduced commissure formation observed in Adam17 cKO embryos. One would predict that, as a result, overexpression of Tace or ADAM17 should inhibit Robo1-mediated repulsion, which is contrary to what was observed in Drosophila. This again argues against Robo1 as the primary substrate mediating the phenotypes observed in Adam17 cKO embryos (Zang, 2022).

The data suggest that Tace and ADAM17 are strictly regulated to achieve a precise balance of Fra and Dcc signaling outputs. What are the mechanisms involved in commissural neurons to modulate Tace and ADAM17 expression and/or activity? In principle, this regulation could occur either at the metalloprotease level or at the substrate level. First, the surface expression or the activity of Tace and ADAM17 could be regulated. This study has demonstrated that tace and Adam17 transcripts are highly expressed in the embryonic CNS of both invertebrates and vertebrates. In Drosophila, the expression of Tace mRNA and protein does not appear to be temporally controlled, suggesting that Tace activity is likely to be regulated post-translationally. Indeed, a number of molecules have been identified as regulators of ADAM17 activity, including tissue inhibitor of metalloproteinases-3 (TIMP-3) which suppresses ADAM17 catalytic activity, and the adapter proteins iRhom1 and iRhom2, which are involved in ADAM17 maturation and stability. Thus, it would be interesting to explore the potential roles of TIMP-3 and the iRhoms and their Drosophila orthologs during axon guidance (Zang, 2022).

Alternatively, regulation of Tace and ADAM17 function could occur at the substrate level. Ligand binding could induce conformational changes in the substrate to facilitate its association with the metalloprotease, or an interacting protein could bind to the substrate to block association with the metalloprotease. Previous work has shown that netrin does not activate the transcriptional activity of Fra,sugge sting that an alternative ligand may exist for the non-canonical pathway. In mouse cortical neurons, leucine-rich repeats and immunoglobulin-like domains 2 (Lrig2) bind to neogenin to inhibit premature ADAM17 cleavage (Zang, 2022).

It remains to be seen whether similar mechanisms regulate Fra and Dcc cleavage. Tace and ADAM17 coordinate the canonical and non-canonical pathways ADAMs can facilitate a switch in responses to guidance cues. For example, ADAM10 and ADAM17 cleave Neuropilin-1 to regulate proprioceptive axon responsiveness to Sema3A. In addition, ADAM10 cleaves ephrinA5 to convert EphA3-ephrinA5-mediated adhesion to repulsion. It is proposed that Tace and ADAM17 can instruct neurons to coordinate the canonical and non-canonical pathways. It is important to point out that, instead of producing opposite signaling outcomes, the canonical and non-canonical pathways both promote midline crossing of commissural axons, which suggests that the two pathways cooperate instead of competing. This distinguishes the current model from existing mechanisms, but at the same time inevitably poses the question: how does the neuron decide between the two pathways? It is speculated that both pathways could be engaged in the same cell but are separated spatially and/or temporally. The canonical pathway is activated at the tip of the axon, where Fra responds to netrin to locally regulate the cytoskeleton (Zang, 2022).

It is possible that the non-canonical Fra pathway is activated in the soma instead, where the cleaved Fra ICDs are within close proximity to the nucleus. This is supported by the observation that both endogenous Tace and overexpressed Tace are almost exclusively detected in the cell soma but not on the axons. In addition, the two pathways could be activated by distinct ligands, so that they are controlled at different developmental time points. Given the diverse roles of ADAM family metalloproteases in development and disease, continued investigation into their regulation and mechanism of action will undoubtedly offer important biological insights (Zang, 2022).

While this study demonstrated that Tace/ADAM17 is a key regulator of Fra/Dcc signaling and controls midline axon guidance, this study has its limitations. First, tace zygotic mutants in Drosophila show midline crossing defects with low penetrance, which is likely the result of a maternal effect. Yet it was not possible to definitively test this by generating maternal zygotic tace mutants, due to developmental arrest potentially linked to its function in the Notch pathway. Second, it remains to be seen if the same non-canonical signaling mechanism is conserved for Dcc in vertebrate systems. Future transcriptomic studies comparing gene expression levels in controls and Dcc cKO embryos or cell lines, or cell lines overexpressing Dcc ICD, could help resolve its role in transcriptional regulation. Third, it is unclear if Tace/ADAM17 is required cell autonomously in commissural neurons or cell non-autonomously in neighboring cells to cleave Fra/Dcc. Generating commissural neuron-specific cKO animals in the future could provide definitive answers. Fourth, the commissure formation deficits observed in Adam17 mutants are not causatively linked to a lack of Dcc cleavage. Finally, it remains to be determined what the biological stimulus that activates the non-canonical pathway is (Zang, 2022).

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex (de Torres-Jurado, 2022).

A precise regulation of ASCD is critical in development, tissue homeostasis, and tumorigenesis. Extrinsic signals from specialized microenvironments, the niches, importantly contribute to that regulation by promoting the stem cell fate in the daughter cell that receives those signals, whereas the other daughter cell enters a differentiation program. Intriguingly, the ASCD of some stem cells, including the Drosophila CNS stem cells called NBs, the subject of this study, seems to depend exclusively on intrinsic regulatory mechanisms. Then, are those stem cells completely independent of their surrounding environment? Also, how general is the requirement of niches and the signals secreted from them for maintaining the stem cell fate in an ASCD (de Torres-Jurado, 2022)?

This study shows that the ASCD of Drosophila NBs, a traditional paradigm for studying intrinsic ASCD regulatory mechanisms, do also depend on extrinsic cues secreted by a glial niche, which are in close contact with those neural stem cell lineages in the larval brain. However, these extrinsic signals are not required to maintain the stem cell fate. They ultimately impact on the regulation of intrinsic factors to induce differentiation in one daughter cell, repressing the self-renewal 'basal state' in this cell. Different studies have shown the possibility of growing in culture isolated larval NBs, which are able to form crescents and divide asymmetrically without any additional extrinsic signal. However, most of these experiments were performed using central brain type I NB lineage (NBI) NBs, which do not require Fra and Robo1 signaling for their correct development, or included type II NB lineage (NBII,) but only particular markers were analyzed (i.e., Baz and Pon). This is relevant as, for example, it was observed that the localization of some ASCD regulators were not affected without extrinsic signals (i.e., Insc, Mira, and Brat). In the experiments in culture, some of the latter regulators are frequently used. It is also key to properly quantify the cases of crescent formation at metaphase, as the phenotypes are never fully penetrant and can be even totally rescued at telophase. For example, in the system used in this study, aPKC showed about 50% localization failures at metaphase, implying that there are NBIIs that show aPKC crescents. Nevertheless, finding that glia-secreted cues are specifically required in NBII lineages was indeed very intriguing. NBII lineages are larger than NBI, as they undergo an additional proliferation phase through INPs and hence are more prone to induce tumor-like overgrowth when ASCD fails. Thus, additional levels of regulation might have evolved in these lineages to ensure the correct division of the NB and INPs to avoid overgrowths. This issue will be further examined in the future (de Torres-Jurado, 2022).

This work has unveiled a novel function for the axon guidance cues Netrins and their Fra/DCC-like receptor in regulating self-renewal versus differentiation in neural stem and progenitor cells of larval NBII lineages. The cortex glial niche that surrounds those lineages secretes Netrins, which modulate Robo1 signaling threshold levels through Fra and Abl kinase in stem and progenitor cells. Whereas Robo1 signaling is activated by its ligand Slit, also secreted by the glial niche, Abl kinase represses this signaling, and these balanced Robo1 signaling levels appear to be critical for the cell-fate commitment of the daughter cell prone to differentiate. The cortex glia dynamically undergoes remodeling through larval stages; only at late third instar larval stages (L3) the glia chamber enwrapping each NB lineage forms completely. This study already observed the presence of the secreted ligands Slit and NetA in the cortex glia at L2, suggesting that these cues are being secreted from the glia since the reactivation of dormant NBs at early L2 (de Torres-Jurado, 2022).

Slit-Robo signaling regulates progenitor cell proliferation in the mammalian CNS and promotes the terminal asymmetric division of a differentiation-committed cell in the Drosophila CNS. Likewise, in mammary stem cells, Robo1 favors their asymmetric mode of cell division. Slit-Robo signaling is also required in other stem or progenitor cells to regulate their lineage specification, identity, or their adhesion/anchoring to the niche. No role for Netrin-Fra/DCC signaling has been previously described in all those contexts (de Torres-Jurado, 2022).

Ultimately, a transcriptional control has been pointed out as the most common way of action by which Robo signaling regulate the above-mentioned cellular processes. This study shows a novel, transcription-independent mechanism by which Robo1 signaling regulates ASCD. Robo1 signaling would be required to activate the small GTPases Rac1 and Cdc42 by repressing its inhibitor RhoGAP93B as well as by recruiting the Dock-Pak complex, which, through Pak, can also bind activated forms of both Rac1 and Cdc42 Rac1 and Cdc42 downregulation directly impacted on the intrinsic machinery that modulate ASCD in neural stem and progenitor cells. Specifically, compromising those small GTPases led to defects in the localization of the ASCD regulators, Par-6, aPKC, Cno, and Numb, and the concomitant formation of ectopic NBs (eNBs) within brain neural lineages, a phenotype that recapitulates that of Slit-Robo1 signaling impairment. The overexpression of robo1 caused similar defects than the loss of robo1. It was also a similar phenotype than for the loss of fra, the downregulation of Abl, or the expression of a kinase dead form of Abl (unable to repress Robo1). Hence, based on all those experiments, a working model proposes that Netrin-Fra signaling would be modulating, through Abl kinase, the Robo1 signaling threshold levels necessary to regulate in turn the correct activity of the small GTPases Rac1 and Cdc42. In fact, and according to this, the expression of Rac1V12 within NBII lineages caused the formation of eNBs and led to defects in the localization of aPKC in NB and progenitor cells, a similar phenotype than that observed after overexpressing robo1 in these NBII lineages. It would be interesting to determine whether this novel function of Netrin-Fra/DCC signaling regulating ASCD is also conserved in vertebrates, and whether Robo/Rac1-Cdc42 signaling threshold levels in the above-mentioned contexts are also critical for and dependent on Netrin-DCC signaling, as was have found in Drosophila (de Torres-Jurado, 2022).

GENE STRUCTURE

Exons - 10

PROTEIN STRUCTURE

Amino Acids - 1355 and 1506

Structural Domains

frazzled encodes two isoforms exhibiting 43% overall sequence identity to Deleted in Colorectal Cancer (DCC) and Neogenin, immunoglobulin (Ig) superfamily members. The extracellular domains of the two predicted Frazzled isoforms contian four Ig C2 type repeats followed by six fibronectin repeats, as do DCC and Neogenin. The two isoforms differ by an insertion of 151 amino acids between the fourth immunoglobulin repeat and the first fibronectin repeat. They share a membrane-spanning domain and a cytoplasmic domain that is 278 amino acids in length (Kolodziej, 1996).

date revised: 22 August 2023

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.