BIOLOGICAL OVERVIEW

As a homophilic cell adhesion molecule, Fasciclin 3 aids in cell adhesion, axon pathfinding and fasciculation. Fas3 is also involved in establishing contact between specific nerve growth cones and muscles, ensuring proper muscle innervation. Expression at junctions between tissues of different developmental fate (for example, at segmental grooves in developing epidermis) indicates a role in boundary formation or integrity.

Fas III is a component of septate junctions. Loss of Discs large (DLG), a protein required for septate junction structure, cell polarity, and proliferation control in Drosophila epithelia, affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion. Fasciclin III is highly enriched at the septate junction and is present in lower amounts in the lateral cell membrane, but is excluded from the adherens junction. Neuroglian is enriched at the apical end of the cell, reduced in the septate junction, and again on the rest of the lateral cell membrane. Localization of Fas III and Neuroglian in both salivary glands and imaginal discs is dependent of DLG. When septate junctions are completely eliminated in dlg mutants, both proteins are found apparently unrestricted along the cell membrane. In fact Neuroglian appears to have an elevated level of expression compared with wild type, while FAS III levels are reduced (Woods, 1996).

A variety of cell recognition pathways affect neuronal target recognition. However, whether such pathways can converge at the level of a single growth cone is not well known. The RP3 motoneuron in Drosophila has previously been shown to respond to the muscle cell surface molecules Toll and Fasciclin 3 (Fas3), which are normally encountered during RP3 pathfinding in a sequential manner. Toll and Fas3, putative 'negative' and 'positive' recognition molecules, respectively, affect RP3 antagonistically. Under normal conditions, Toll and Fas3 together improve the accuracy of RP3 target recognition. When presented with concurrent Toll and Fas3 expression, RP3 responds to both, integrating their effects. This was demonstrated most succinctly by single cell visualization methods. When a balance in relative expression levels between the two antagonistic cues is achieved, the RP3 growth cone exhibits a phenotype virtually identical to that seen when neither Toll nor Fas3 is misexpressed. Thus, growth cones are capable of quantitatively evaluating distinct recognition cues and integrating them to attain a net result, in effect responding to the 'balance of power' between positive and negative influences. It is suggested that the ability to integrate multiple recognition pathways in real-time is one important way in which an individual growth cone interprets and navigates complex molecular environments (Rose, 1999).

Toll and Fas3 protein were simultaneously misexpressed in the entire embryonic musculature during the period of motoneuron-muscle interaction. Testing with Toll and Fas3 antibodies indicates that the two proteins are simultaneously expressed at elevated levels in all muscles. As with other muscle cell surface molecules, both Toll and Fas3 appear to accumulate at muscle-muscle contact sites. Misexpression occurs throughout most of the period of motoneuron axon pathfinding and synaptogenesis, i.e., hours 12-20 of embryogenesis. Despite transgenic expression, there is no difference in the number or overall morphology of muscles. Therefore, motoneuron growth cones leaving the CNS will experience both Toll and Fas3 expression on all muscle surfaces that they encounter. This contrasts the wild-type situation in which growth cones first encounter Toll-expressing proximal muscles (15, 16, 17, and 28) before reaching FAS3-expressing proximal-medial muscles a few hours later. Any motoneuron axon defects that result from co-misexpression of Toll and Fas3 may be interpreted as a likely direct result of motoneuron growth cones encountering Toll and Fas3 proteins outside of their normal context. Nevertheless, the nervous system was found to develop normally in Toll/Fas3 co-misexpressor embryos. In each hemisegment of the peripheral nervous system, all five major nerve branches [intersegmental nerve, segmental nerve a (SNa), SNb, SNc, and SNd] extend outside the CNS into characteristic muscle regions. Previous studies using the Toll and Fas3 misexpressing lines have revealed that subsets of motoneuron growth cones are often misguided when encountering either ectopic Toll or Fas3 expression in the musculature. Toll/Fas3 co-misexpressor embryos remain innervated at a high frequency when examined with immunocytochemistry This apparent return to a wild-type innervation pattern could be attributable to one of at least two separate situations. Either RP3 or MN6/7b, another motoneuron that innervates the 6/7 cleft, could once again be attracted to the cleft when the levels of Toll and Fas3 expression there are equalized. Alternatively, the presence of mAb 1D4 staining at the cleft could indicate that a motoneuron other than RP3 or MN6/7b is innervating the cleft ectopically. The latter would imply that co-misexpression of Toll and Fal3 permits ectopic innervation at the 6/7 cleft more readily than misexpression of either cue alone. The former would suggest that the 6/7 cleft becomes 'normalized' when both Toll and Fas3 are co-misexpressed in the musculature. To distinguish between these possibilities, the RP3 growth cone was visualized directly. In all cases, the RP3 axon extends out of the CNS normally. It first crosses the dorsal midline of the CNS and then exits via the intersegmental nerve, one segment posterior to its origin. This supports the notion that co-misexpression of Toll and Fas3 in the embryonic musculature does not affect RP3 axonal pathfinding within the CNS. Once outside the CNS and when encountering concurrent misexpression of Toll and Fas3, the most common decision of the RP3 growth cone is to navigate through the normal peripheral pathway and to innervate the 6/7 cleft, its wild-type site of innervation. Overall, whereas Toll is capable of preventing RP3 innervation of its normal target site, co-misexpression of Fas3 and Toll leads to a phenotype that is similar to wild type. These observations are interpreted as RP3 growth cone integration of two antagonistic signals during its target recognition. Thus the RP3 growth cone is competent to respond to concurrently misexpressed Toll and Fas3, two structurally and physiologically distinct molecules normally expressed by the targets of the growth cone and surrounding cells. Furthermore, in vitro, the growth cone is capable of evaluating the relative contributions of each molecule and responding appropriately. These results support the general idea that signal integration at individual growth cones is an important mechanism by which neural networks are established (Rose, 1999).

cDNA clone length - 2465

Bases in 5' UTR -581

Bases in 3' UTR - 369

PROTEIN STRUCTURE

Amino Acids - 508 with signal peptide and 488 as a mature protein

Structural Domains

The extracellular domain of FAS3 contains 326 amino acids; there is a 24 amino acid transmembrane domain and a 138 amino acid intracellular domain. The extracellular domain contains four potential N-linked glycosylation sites. The intracellular domain has a single tyrosine residue, two potential phosphorylation sites and a polyglutamine tract called an opa-repeat sequence (Snow, 1989).

The most N-terminal of the three Fasciclin III immunoglobulin-like domains is expected to be important in mediating cell-cell recognition events during nervous system development. A model structure of this domain was built aligning the protein sequence of the Fasciclin III first domain to the immunoglobulin McPC603 structure. Based on this alignment, a model of the domain was built. The resulting model is compact and has chemical characteristics consistent with related globular protein structures. This model is a de novo test of the sequence-to-structure alignment algorithm and is currently being used as the basis for mutagenesis experiments to discern the parts of the Fasciclin III protein that are necessary for homophilic molecular recognition in the developing Drosophila nervous system (Castonguay, 1995).

Evolutionary Homologies

FAS3 is a novel cell adhesion molecule that does not relate to any of the vertebrate adhesion molecules (Snow, 1989).

REGULATION

Transcriptional Regulation

Epidermal growth factor receptor induces pointed P1 and inactivates Yan protein in the embryonic ventral ectoderm. Two other candidate genes for EGF-R regulation are ventral nervous system defective and Fasciclin III. While early expression of vnd are not affected, no expression of vnd is detected from stage 11 in Egf-r mutants. While fas III expression is unaltered in pointed or yan mutants, hyperactivation of Egf-r expands the Fas III domain (Gabay, 1996).

ebi (the term for 'shrimp' in Japanese) regulates the epidermal growth factor receptor (EGFR) signaling pathway at multiple steps in Drosophila development. Mutations in ebi and Egfr lead to similar phenotypes and show genetic interactions. However, ebi does not show genetic interactions with other RTKs (e.g., torso) or with components of the canonical Ras/MAP kinase pathway. ebi encodes an evolutionarily conserved protein with a unique amino terminus, distantly related to F-box sequences, and six tandemly arranged carboxy-terminal WD40 repeats. The existence of closely related proteins in yeast, plants, and humans suggests that ebi functions in a highly conserved biochemical pathway. Proteins with related structures regulate protein degradation. Similarly, in the developing eye, ebi promotes EGFR-dependent down-regulation of Tramtrack88, an antagonist of neuronal development (Dong, 1999).

Loss of ebi affects Egfr-dependent expression of genes in the embryo. The EGFR ligand Spitz is expressed along the ventral midline and induces expression of different target genes, including fasciclin III (fasIII) and orthodenticle (otd), in cells located in more lateral positions. In zygotic null Egfr mutants both otd and FasIII expression are lost. In wild-type stage 11/12 embryos, FasIII protein is broadly distributed in the visceral mesoderm and in a bilaterally symmetric cluster of cells flanking the midline of the ventral ectoderm. In ebi mutant embryos lacking both maternal and zygotic contribution, FasIII expression is largely abolished, although some residual patches of staining remain. Egfr-independent expression of FasIII in the anterior-most region of the embryo is unaffected in ebi mutants. In wild-type stage 10/11 embryos, otd mRNA is expressed in the preantennal head region and in the ventral-most ectoderm. In ebi mutant embryos, otd expression is markedly reduced. These data suggested that ebi may be a component in the EGFR signal transduction pathway (Dong, 1999).

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, which encodes a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans, the t(2;5) translocation, which involves the ALK locus, produces an active form of ALK, which is the causative agent in non-Hodgkin's lymphoma. The physiological function of the Alk RTK, however, is unknown. Loss-of-function mutants in the Drosophila Alk gene are described that cause a complete failure of the development of the gut. It is proposed that the main function of Drosophila Alk during early embryogenesis is in visceral mesoderm development (Lorén, 2003).

The function of Alk in visceral mesoderm development was analysed using the immunoglobulin domain adhesion molecule Fasciclin III (FasIII), which is a marker for differentiated visceral mesoderm in the Drosophila embryo. In wild-type embryos, Alk and FasIII expression patterns overlap perfectly in the visceral mesoderm as the midgut forms. Further analysis of Alk mutant embryos shows that there is a complete loss of Alk-positive and FasIII-positive cells, whereas FasIII staining in the epidermis was normal. Similar results were obtained when antibodies to Drosophila Myocyte enhancer factor 2 (Mef2), which is produced in all muscle lineages of the Drosophila embryo, were used. Furthermore, anti-myosin-heavy-chain (MHC) staining, which showed the thin layer of gut mesoderm in wild-type embryos, was absent from Alk mutant animals (Lorén, 2003).

To test whether Alk is sufficient for FasIII activation, UAS-Alk (an Alk transgene under the control of the yeast Gal4 upstream activating sequence) was expressed using the mesodermal twist-Gal4 driver. Indeed, Alk induces ectopic expression of FasIII, supporting the idea that Alk controls FasIII expression. This is an exciting possibility, since the forkhead-domain transcription factor, Drosophila FoxF/Biniou, has been reported to drive expression of visceral mesoderm markers, including FasIII, and Drosophila FoxF/Biniou could potentially be activated by an Alk RTK signalling pathway, since Alk is a member of the Insulin receptor RTK superfamily (Lorén, 2001). Since induction of FasIII expression was seen upon Alk expression, Alk mutant embryos were re-examined. Using confocal microscopy, it was possible to locate scattered Alk-positive cells in Alk1 mutant animals. This is possible because the anti-Alk antibodies are raised against the extreme amino-terminal end of Alk and therefore the Alk1 truncated protein could be detected. On closer inspection, these cells are also seen to be weakly FasIII-positive. Thus, although FasIII expression seems to be significantly reduced in visceral mesoderm cells in Alk mutants, it is not absent. Nevertheless, full FasIII expression may still require Alk signalling through a FoxF/Biniou-mediated pathway, especially since it has been reported that there may be a positive-feedback pathway that reinforces FasIII expression (Lorén, 2003).

The homeodomain protein Nkx6 is a key member of the genetic network of transcription factors that specifies neuronal fates in Drosophila. Nkx6 collaborates with the homeodomain protein Hb9/ExEx to specify ventrally projecting motoneuron fate and to repress dorsally projecting motoneuron fate. While Nkx6 acts in parallel with hb9 to regulate motoneuron fate, Nkx6 plays a distinct role to promote axonogenesis; axon growth of Nkx6-positive motoneurons is severely compromised in Nkx6 mutant embryos. Furthermore, Nkx6 is necessary for the expression of the neural adhesion molecule Fasciclin III in Nkx6-positive motoneurons. Thus, this work demonstrates that Nkx6 acts in a specific neuronal population to link neuronal subtype identity to neuronal morphology and connectivity (Broihier, 2004).

Protein Interactions

Drosophila Neurexin is required for septate junction and blood-nerve barrier formation and function. NRX is localized apicolaterally, adjacent to Crumbs, which delimits the zonula adherens. These two proteins are not coexpressed, placing NRX apicolaterally. Both Fasciclin3 and NRX colocalize at salivary gland synaptic junctions. NRX precisely colocalized with D4.1/Coracle except in the PNS and CNS where D4.1/Coracle is only expressed in a few cells.

No defects in the localization of Discs large protein is detected in nrx mutants. However, D4.1/Coracle is not restricted to septate junctions in nrx mutants. These results suggest that the short cytoplasmic portion of NRX that shows homology to glycophorin C is required to localize D4.1/Coracle to septate junctions, creating a parallel with red blood cell cytoskeletal anchoring proteins (Baumgartner, 1996).

The cell adhesion molecule Fasciclin III (FAS3) mediates synaptic target recognition through homophilic interaction. FAS3 is expressed by the RP3 motoneuron and its target muscles during synaptic target recognition. The RP3 growth cone can form synapses on muscles that ectopically express FAS3. This mistargeting is dependent on FAS3 expression in the motoneurons. When the FAS3-negative aCC and SNa motoneuron growth cones ectopically express FAS3, they gain the ability to recognize FAS3-expressing muscles as alternative targets. It is proposed that homophilic synaptic target recognition serves as a basic mechanism for neural network formation (Kose, 1997).

DEVELOPMENTAL BIOLOGY

Embryonic

Fas3 is expressed during neurogenesis in a small subset of neural cells, and possibly also in glial cells. Interestingly, the molecule is not present throughout the axon of single neurons, but only on specific portions within the commissural fascicles (Patel, 1987).

After germ-band extention [Image], Fas3 is expressed transiently on segmentally repeated patches of neuroepithelial cells, and on specific, more mature neuronal lineages.

FAS3 is also found on segmentally repeated stripes of cells at the anterior margin of the segmental grooves. It is also expressed on patches of epithelial cells near the stomodeal and proctodeal invaginations, on visceral but not somatic mesoderm, and on the luminal surface of the salivary gland epithelium.

By the end of germ band retraction Fas3 is expressed in repeated stripes across all body segments (Patel, 1987). Fas3 is expressed by the growth cones of several specific motoneurons, and is expressed as well on their peripheral muscle targets during embryogenesis at the period when the first neuromuscular contacts are made (Halpern, 1991).

Larval

The distribution of proteins has been analyzed in the apico-lateral cell junctions in Drosophila imaginal discs. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous actin, label the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III (FasIII) and Coracle (Cor) label the more basal septate junction. The junctional proteins labeled by these antibodies undergo specific changes in distribution during the cell cycle. Previous work has shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions. This study was extended to examine the effects of mutation in other genes that also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat (coding for a novel cadherin) , discs overgrown, giant discs and warts (coding for a homolog of human myotonic dystrophy kinase). However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).

Oogenesis

The Drosophila egg chamber provides an excellent model for studying the link between patterning and morphogenesis. Late in oogenesis, a portion of the flat follicular epithelium remodels to form two tubes; secretion of eggshell proteins into the tube lumens creates the dorsal appendages. Two distinct cell types contribute to dorsal appendage formation: cells expressing the rhomboid-lacZ (rho-lacZ) marker form the ventral floor of the tube and cells expressing high levels of the transcription factor Broad form a roof over the rho-lacZ cells. In mutants that produce defective dorsal appendages (K10, Ras and ectopic decapentaplegic) both cell types are specified and reorganize to occupy their stereotypical locations within the otherwise defective tubes. Although the rho-lacZ and Broad cells rearrange to form a tube in wild type and mutant egg chambers, they never intermingle, suggesting that a boundary exists that prevents mixing between these two cell types. Consistent with this hypothesis, the Broad and rho-lacZ cells express different levels of the homophilic adhesion molecule Fasciclin 3. Furthermore, in the anterior of the egg, ectopic rhomboid is sufficient to induce both cell types, which reorganize appropriately to form an ectopic tube. It is proposed that signaling across a boundary separating the rho-lacZ and Broad cells choreographs the cell shape-changes and rearrangements necessary to transform an initially flat epithelium into a tube (Ward, 2005).

Effects of Mutation or Deletion

Homozygotes are viable and have no gross morphological abnormalities. Expression of Fas3 in transfected cultured cells promotes their adhesion to each other but not to non-expressing cells, indicating that Fas3 is a homophilic adhesion molecule (Patel, 1987).

Fasciclin III is expressed by motor neuron RP3 and its synaptic targets (muscle cells 6 and 7) during embryonic neuromuscular development. RP3 often incorrectly innervates neighbouring non-target muscle cells when these cells misexpress fasciclin III, but still innervates normal targets in the fasciclin III null mutant. Fasciclin III manipulations do not influence target selections by other motor neurons, including fasciclin III-expressing RP1. It has been proposed that Fasciclin III acts as a synaptic target recognition molecule for motor neuron RP3, and also that its absence can be compensated for by other molecule(s) (Chiba, 1995).

REFERENCES

Baumgartner, S., et al. (1996). A Drosophila Neurexin is required for septate junction and blood-nerve barrier formation and function. Cell 87: 1059-68

Broihier, H. T., Kuzin, A., Zhu, Y., Odenwald, W. and Skeath, J. B. (2004). Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis. Development 131: 5233-5242. 15456721

Castonguay, L. A., et al. (1995). A proposed structural model of domain 1 of fasciclin III neural cell adhesion protein based on an inverse folding algorithm. Protein Sci 4: 472-483

Chiba, A., et al. (1995). Fasciclin III as a synaptic target recognition molecule in Drosophila. Nature 374: 166-168

Dong, X., et al. (1999). ebi regulates epidermal growth factor receptor signaling pathways in Drosophila. Genes Dev. 13(8): 954-65.

Gabay, L., et al. (1996). EGF receptor signaling induces pointed P1 and inactivates Yan protein in the Drosophila embryonic ventral ectoderm. Development 122: 3355-3362

Halpern, M.E., Chiba, A., Johansen, J. and Keshishian, H. (1991). Growth cone behavior underlying the development of stereotypic synaptic connections in Drosophila embryos. J. Neurosci. 11(10): 3227-38

Kose, H., et al. (1997). Homophilic synaptic target recognition mediated by immunoglobulin-like cell adhesion molecule Fasciclin III. Development 124(20): 4143-4152.

Lorén, C. E., Englund, C., Grabbe, C., Hallberg, B., Hunter, T. and Palmer, R. H. (2003). A crucial role for the Anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila melanogaster. EMBO Rep. 4: 781-786. 12855999

Patel, N,H., Snow, P.M. and Goodman, C.S. (1987). Characterization and cloning of Fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophia. Cell 48: 975-988

Rose, D. and Chiba, A. (1999). A single growth cone is capable of integrating simultaneously presented and functionally distinct molecular cues during target recognition. J. Neurosci. 19(12): 4899-4906.

Snow, P.M., Bieber, A.J. and Goodman, C.S. (1989). Fasciclin III: a novel homophilic adhesion molecule in Drosophila. Cell 59: 313-323

Ward, E. J. and Berg, C. A. (2005). Juxtaposition between two cell types is necessary for dorsal appendage tube formation Mech. Dev. 122: 241-255. 15652711

Woods, D. F., et al. (1996). Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila Epithelia. J. Cell Biol. 134: 1469-1582.

Woods, D. F., Wu, J. W. and Bryant, P. J. (1997). Localization of proteins to the apico-lateral junctions of Drosophila epithelia. Dev. Genet. 20(2): 111-118.

date revised: 10 July 2005  

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