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).

Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells

The class III phosphatidylinositol-3 kinase [PI3K (III)] regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate [PI(3)P]. While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. This study, in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) were examined in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein beta-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of beta-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes (Abe, 2009).

Cell polarity along the apical-basal axis is essential for the function of epithelial cells. This polarity is formed and maintained by distinct localization of membrane spanning and associated proteins, to apical, lateral or basal membrane domains. Membrane proteins localized to the apical or basolateral plasma membrane are endocytosed into early and apical or basolateral endosomes. For example, horseradish peroxidase (HRP) administered to the apical cell surface is incorporated into the apical early endosome. By contrast, HRP or dimeric IgA administered to the basolateral cell surface or transferring receptor (TfR) in the basolateral domain are internalized into the basolateral early endosome, which remain distinct. Sorting of proteins for transcytosis, recycling and degradation takes place in these early endosomes. The proteins, incorporated into apical and basolateral early endosomes, meet in common endosomes, a process that can be observed within 15 min after the onset of internalization in MDCK cells. The significance of keeping the apical and basolateral early endosomes distinct is thought to ensure that proteins from the apical and basolateral plasma membrane remain apart before the sorting processes proceeds. Although it is plausible that the trafficking of proteins in distinct membrane domains is regulated differently, the factors involved in such a differential regulation remain elusive (Abe, 2009).

One of the key molecules regulating membrane trafficking is PI3K (III), a heterodimer of Vps34p and Vps15p/p150, which produces phosphatidylinositol-3-phosphate (PI(3)P). PI(3)P is found to localize with early endosome and internal vesicles of multivesicular bodies (MVBs) in mammalian cells in culture. Genetic and pharmacological analysis, using yeast and mammalian cells in culture, suggests that PI3K (III) is required for five distinct processes. These are: (1) the fusion of clathrin-coated vesicles and early endosomes as well as the fusion between early endosomes; (2) the recycling from early endosomes back to the Golgi complex or other destinations; (3) the entry of proteins into the lysosomal degradation pathway; (4) the formation of internal vesicles of MVBs and (5) autophagy. Moreover, inactivation of PI3K (III) by Vps34 mutation leads to an expansion of the outer nuclear membrane and an abnormal reduction of the LDL receptor at the apical membrane in C. elegans. In Drosophila, dVps34 mutation results in defective endocytosis of the apical membrane protein Notch and a defective onset of autophagy. It has been suggested that PI3K (III) utilizes different effectors at apical and basolateral endosomes. However, the role of PI3K (III) in the regulation of protein localization at different membrane domains has remained unclear (Abe, 2009 and references therein).

To understand the various functions of PI3K (III), it is crucial to clarify which downstream effectors are involved in each of the processes it regulates. PI3K (III) is thought to exert its function through the recruitment of proteins that contain PI(3)P-binding motifs such as FYVE or PX domains. Among such proteins, Rabenosyn-5 (Rbsn-5) has been shown to contribute to endosome fusion and recycling processes in mammalian cells. Genetic studies on C. elegans and Drosophila also show that Rbsn-5 is essential for receptor-mediated endocytosis and endosome fusion, although it is not clear whether or not Rbsn-5 is involved in other PI3K (III)-related phenomena (Abe, 2009).

To determine how the proteins in distinct membrane domains are regulated by PI3K (III) and its effector Rbsn-5 this study analyzed Drosophila wing development. This provides a good model since wing primordial cells have a clear polarity along the apical-basal axis. In addition a number of membrane proteins are known to be transported in an organized manner along the apical-basal axis. For example DE-cadherin, a cell adhesion protein and Fmi, a planar cell polarity (PCP) core protein, are localized in the apical junctions or zonula adherens (ZA), whereas the cell adhesion molecules FasIII and β-integrin are localized in lateral and basal membranes, respectively. This study found that inactivation of PI3K (III) in the wing primordial cells by knockdown of dVps15 affects the localization of these membrane proteins differently. In particular, it was found that dVps15 knockdown results in the accumulation of FasIII at the lateral membrane, whereas it results in intracellular accumulation of DE-cadherin, Fmi and β-integrin. Importantly, inactivation of Rbsn-5 shows accumulation of FasIII and β-integrin at the lateral membrane and intracellular vesicles, respectively, but no effects of DE-cadherin and Fmi localization (see in contrast Mottola, 2010). These results provide evidence for a differential regulation of protein localization by PI3K (III) and Rbsn-5 at distinct membrane domains (Abe, 2009).

This study demonstrated that PI3K (III) differentially regulates the localization of proteins at distinct membrane domains. The intracellular accumulation of Fmi, DE-cadherin and β-integrin induced by the dVps15 knockdown might be due to defects in the degradation pathway, since the maturation of MVBs and the lysosomal trafficking were defective in these cells. However, unlike these proteins, Fas III did not accumulate in the intracellular compartments, but rather accumulated at the surface of the lateral plasma membrane. It is possible that PI3K (III) regulates proteins at the lateral membrane differently from those localized at other membrane domains. It is also possible that PI3K (III) regulates Fas III in a different way, irrespective of the membrane domain to which it is localized. Whichever is the case it will be important to elucidate the mechanism underlying this difference in a future study (Abe, 2009).

Rbsn-5, a FYVE domain-containing protein, shares a part of the functions of PI3K (III), in that it is necessary for the regulation of Fas III and β-integrin localization, but not that of DE-cadherin and Fmi localization. Although the Rbsn-5^C241 null mutant clones may not completely lack Rbsn-5 activity, the requirement of Rbsn-5, or at least the requirement of an appropriate amount, differs between these proteins with respect to normal trafficking. It appears that Rbsn-5 preferentially controls the events at the basolateral regions, given that Rbsn-5 is necessary for the formation of large endosomes at the basal region, whereas it is indispensable for the formation of actin bundles at the apical surface (Abe, 2009).

PI3K (III) has been implicated in the differential regulation of vesicle trafficking at apical and basolateral regions. For instance, a reduction of PI(3)P dissociates EEA1, a FYVE-domain containing protein essential for early endosome fusion, selectively from basolateral endosomes. However, which proteins, including EEA1, regulate the different trafficking pathways downstream of PI3K (III) has remained unknown. Rbsn-5 has been proposed to be a PI3K (III) effector, since Rbsn-5 harbors a FYVE domain. The current results provide further evidence supporting a possible functional interaction between these two molecules, based on their genetic interaction on the wing morphogenesis and the PI3K (III)-dependent Rbsn-5 immunostaining. Importantly, the different requirement of Rbsn-5 for trafficking at apical junction and basolateral membrane domains suggests that Rbsn-5 may a selective regulator under the control of PI3K (III) (Abe, 2009).

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular junction synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. The localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, this study found that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage it was found that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins (Lu, 2014).

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).

Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps

Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. This study used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, the Flp/FRT system was used to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle (Hudson, 2008).

Two protein trap lines showed a striped pattern in ovarian muscle. The GFP transposon in these lines was inserted into the genes for two uncharacterized proteins, CG30084 and CG6416, both of which contain a PDZ domain and a ZASP motif (ZM). CG30084 also contains four LIM domains in its C-terminus. Both CG30084 and CG6416 proteins show homology to the human Z-disc Alternatively Spliced Protein (ZASP) that localizes to the Z-disc in skeletal muscle. Like the ZASP gene, both CG30084 and CG6416 encode a number of alternative splice forms, almost all of which could be tagged based on the locations of the protein trap insertions . An additional isoform of CG30084 has been annotated that encodes only LIM domains and cannot be tagged. Due to the homology of these genes to ZASP, CG30084 was designated as Zasp52, and CG6416 as Zasp66 based on the cytological location of the genes (Hudson, 2008).

The availability of protein trap lines was especially useful for describing intercellular contacts (e.g., Fas3::GFP, Ilk::GFP inserted into Integrin linked kinase) and for showing the location of 'new' sarcomere components (e.g., Zasp52::GFP, Zasp66::GFP). The muscle cells of the oviducts, which are derived from genital disks, have highly organized sarcomeres and a myotube organization typical of somatic muscles in insects, and likely arise by myoblast fusion during development. In contrast, the visceral muscle layers surrounding ovarioles and ovaries have a mesh-like shape similar to the visceral muscles of the gut. Surprisingly, it was found that the epithelial sheath muscles contain one nucleus rather than two as in gut visceral muscles, making them the only known adult somatic or visceral muscle that does not arise by myoblast fusion. It remains to be determined whether the peritoneal sheath cells are mononuclear as well (Hudson, 2008).

The nature of the epithelial sheath muscle makes it ideally suited for analysis of muscle function. The morphology of the cells can be viewed in great detail with an array of available sarcomere and cellular markers. Importantly, the ability to produce cells with a defined genotype by mitotic clone induction provides a new opportunity for studying the phenotypes in adult muscles caused by homozygous mutations in muscle genes, even if the mutations are lethal during early development. Finally, it is straightforward to do short-term in vitro culturing of ovarioles with the epithelial sheath intact and contracting; thus, the behavior of cells bearing mutations can be studied in live tissue under controlled conditions (Hudson, 2008).

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

Abe, M., et al. (2009). Membrane protein location-dependent regulation by PI3K (III) and rabenosyn-5 in Drosophila wing cells. PLoS ONE 4: e7306. PubMed Citation: 19798413

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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. PubMed Citation: 7795530

Chiba, A., et al. (1995). Fasciclin III as a synaptic target recognition molecule in Drosophila. Nature 374: 166-168. PubMed Citation: 7877688

Dong, X., et al. (1999). ebi regulates epidermal growth factor receptor signaling pathways in Drosophila. Genes Dev. 13(8): 954-65. PubMed Citation: 10215623

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date revised: 25 October 2014  

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