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).

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: 28 MAY 97 

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.