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).

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.