Fasciclin 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Fasciclin 2

Synonyms - Fasciclin II - FasII

Cytological map position - 4B1-B2

Function - cell adhesion

Keywords - cell adhesion molecule, neural

Symbol - Fas2

FlyBase ID:FBgn0000635

Genetic map position - 1-[6]

Classification - CAM - Ig superfamily

Cellular location - surface - transmembrane and lipid linked

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Halberg, K.A., Rainey, S.M., Veland, I.R., Neuert, H., Dornan, A.J., Klämbt, C., Davies, S.A. and Dow, J.A. (2016). The cell adhesion molecule Fasciclin2 regulates brush border length and organization in Drosophila renal tubules. Nat Commun 7: 11266. PubMed ID: 27072072
Multicellular organisms rely on cell adhesion molecules to coordinate cell-cell interactions, and to provide navigational cues during tissue formation. In Drosophila, Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. This study shows that Fas2 is essential to brush border maintenance in renal tubules of Drosophila. Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, the study demonstrates a radically different role for this well-known cell adhesion molecule, and proposes that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border.
Ou, M., Wang, S., Sun, M., An, J., Lv, H., Zeng, X., Hou, S. X. and Xie, W. (2018).. The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction. Exp Cell Res. PubMed ID: 30553967
Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. The study analyzed the role of Gef26 in synaptic development and function. Significant decreases were identified in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, the data demonstrate that Gef26-specific small G protein Rap1 works downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a betaPS integrin-mediated signaling pathway. Taken together, these findings define a novel role of Gef26 in regulating NMJ development and function.

Fas2 carries out multiple functions in the Drosophila nervous system. A member of the immunoglobulin superfamily, Fas2 has been implicated in the process of fasciculation, the adherence of growing axons to one another during the process of growth cone guidance. Fas2 has also been implicated in the process of presynaptic functional plasticity, the ability of synapses to undergo long-term changes in structure. Expressed both presynaptically and postsynaptically, Fas2 controls the number and stability of neuromuscular junction synapses. In this function, Fas2 is down regulated by mutations affecting cyclic AMP levels (See the learning pathway). In addition, decreased Fas2 is necessary and sufficient to cause synaptic sprouting. Finally, Fas2 and CREB (the cyclic AMP response element binding protein) act in parallel pathways to cause increases in strength of the neuromuscular synapse.

Fas2 functions in selective fasciculation, that is, adherence of growing axons to the MP1 fascicle. The MP1 fascicle, generated by the anterior extending growth cone of the pCC axon is easily identified because a specific monoclonal antibody will stain only very specific types of growth cones. These include aCC, pCC, MP1, dMP2 and vMP2. The pCC axon pioneers the MP1 fascicle. In the absence of Fas2, growth cones do not extend in the proper rostrocaudal direction and fail to fasciculate (Grenningloh, 1991).

Subsequent studies reveal an even more complex function for Fas2. In addition to selective fasciculation, Fas2 also functions in growth cone guidance. Three major motor nerves were analyzed: the intersegmental nerve (ISN) that innerates dorsal muscles; the segmental nerve a (SNa), innervating lateral muscles, and segmental nerve b (SNb), innervating ventral muscles. High specific defects in axonal guidance occur in over- or under-expression of Fas2. These do not appear to be fasciculation phenotypes because fascicles are not formed. They are instead guidance phenotypes in which axons wander from their normally specified courses. It would appear that guidance is not taking place here solely on the basis of self adhesion, but in the context of other competing attractive and repulsive cues. In this case, cell adhesion per se is not always the sole or the decisive factor (Lin, 1994 a and b).

Muscles 7 and 6 are each innervated by two motoneurons, RP3 and MN6/7b. This innervation occurs at stage 17. The two axons form about 18 synaptic boutons by the time of the initial larval hatch, and the number increases to approximately 180 boutons by the third instar larva. Although Fas2 is initially expressed by all embryonic motor axons and their growth cones during the period of axon outgrowth, the axonic Fas2 decreases upon innervation, and Fas2 is confined to the synaptic terminal. After synapse formation, Fas2 is localized both pre-synaptically and postsynaptically, where it controls synapse stabilization. In Fas2 mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and the resulting lethality is rescued by transgenes that drive Fas2 expression both pre- and postsynaptically; driving Fas2 expression on either side alone is insufficient.

Fas2 can also control synaptic growth: various Fas2 alleles lead to either an increase or decrease in sprouting depending upon the level of Fas2. A 50% decrease in Fas2 can lead to a 50% increase in the number of synaptic boutons (Schuster, 1996a).

Altering the level of neuronal activity or cAMP concentration leads to increased synaptic structure and function at the neuromuscular junction. ether à go-go and Shaker code for potassium channels; mutations in both can result in increased neuronal activity and increase in synaptic structure and branching. Mutation in K+ channels display greatly enhanced nerve activity as a result of reduction in K+ currents. K+ channel mutation is thought to increase motoneuron activity and synaptic transmission by increasing the cAMP second messenger signaling. Mutations in dunce, coding for c-AMP phosphodiesterase, result in increased cAMP levels, heightened neural activation, and also result in increased axonal branching (Schuster, 1996b and references).

Increase in synaptic growth in eag, Shaker and dunce mutants is accompanied by approximately 50% reduction in synaptic levels of Fas2. This decrease in Fas2 is both necessary and sufficient for presynaptic sprouting; Fas2 mutants that decrease Fas2 levels by 50% lead to sprouting similar to eag, Shaker and dunce mutants, while Fas2 transgenetic animals that maintain synaptic Fas 2 levels suppress sprouting in eag, Shaker and dunce mutants. However, Fas2 mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons. Thus these results show a requirement for the presynaptic downreguation of Fas2 in activity and cAMP-induced synaptic sprouting. It is speculated that activity or cAMP may trigger the down-regulation of synaptic Fas2 by actively removing it from the presynaptic terminal (Schuster, 1996b).

Since Fas2 mutants lead to an increase in the number of boutons without affecting synaptic strength, and increased cAMP in dnc mutants increases both synaptic structure and quantal content, there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB, the cyclic AMP response element-binding protein, mediates the transcriptional requirement of cAMP-dependent long-term synaptic change. Thus CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with FAS2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, dCREB2-b (an isoform of CREB), in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, dCREB2-a, increases synaptic strength by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of dCREB2-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity, and cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, 1996).

Fasciclin 2, the Drosophila orthologue of neural cell-adhesion molecule, inhibits EGF receptor signalling

Adhesion proteins not only control the degree to which cells adhere to each other but are increasingly recognised as regulators of intercellular signalling. Using genetic screening in Drosophila, Fasciclin 2 (Fas2), the Drosophila orthologue of neural cell adhesion molecule (NCAM), has been identified as a physiologically significant and specific inhibitor of epidermal growth factor receptor (EGFR) signalling in development. Loss of fas2 genetically interacts with multiple genetic conditions that perturb EGFR signalling. Fas2 is expressed in dynamic patterns during imaginal disc development, and in the eye it was shown that this depends on EGFR activity, implying participation in a negative-feedback loop. Loss of fas2 causes characteristic EGFR hyperactivity phenotypes in the eye, notum and wing, and also leads to downregulation of Yan, a transcriptional repressor targeted for degradation by EGFR activity. No significant genetic interactions were detected with the Notch, Wingless, Hedgehog or Dpp pathways, nor did Fas2 inhibit the FGF receptor or Torso, indicating specificity in the inhibitory role of Fas2 in EGFR signalling. These results introduce a new regulatory interaction between an adhesion protein and a Drosophila signalling pathway and highlight the extent to which the EGFR pathway must be regulated at multiple levels (Mao, 2009).

These results demonstrate that the NCAM orthologue Fasciclin 2 specifically inhibits EGFR signalling activity during the normal development of the Drosophila eye, notum and wing. Interestingly, like other Drosophila EGFR inhibitors, Fas2 participates in a potential negative-feedback loop to regulate signalling, although the developmental significance of this remains to be established. The evidence for the interaction between Fas2 and EGFR relies on genetic interactions, diagnostic phenotypes of loss of function fas2 mutants, and a direct readout in fas2 clones of reduction of Yan, a transcriptional repressor targeted for degradation by EGFR activity. Furthermore, the results in the eye are supported by similar genetic logic in the developing notum and wing. Despite this, fas2 phenotypes are not identical to those of other known EGFR inhibitors. This is less surprising than it first appears, as the phenotypes of none of the known EGFR inhibitors in Drosophila (which currently include Argos, Kekkon-1, Echinoid, Sprouty, as well as some less specific proteins such as Gap-1) are as strong as constitutive activation of the receptor, and all are distinct. The explanation for the variation in strength and detail of phenotype is that each of the inhibitors has a different molecular mechanism and site of action in the pathway, as well as different sites of expression. For example, Argos is specific to the EGFR and is a diffusible molecule that sequesters ligand. By contrast, Sprouty, a cytoplasmic protein, inhibits a range of receptor tyrosine kinases, whereas Echinoid and Kekkon-1 are cell surface proteins that bind directly to the EGFR. It is evident that EGFR regulation depends on a patchwork of overlapping effects of multiple different types of modulators, each of which has greater or less importance in different developmental contexts. Presumably, this network of regulators underlies the observed precision and robustness of signalling (Mao, 2009)

Loss of Fas2 in the eye triggers at least two distinct types of extra photoreceptor recruitment. The ectopic mini-clusters appear at the same time that the normal outer photoreceptors are recruited and, by analogy with argos mutations, it is believed that they are caused by transformation of the 'mystery cells'. In normal development these form part of the precluster, but are ejected prior to the onset of photoreceptor differentiation. It is also possible that some of the mini-clusters are derived from de novo photoreceptor determination occurring in undifferentiated interommatidial cells, which is known to be triggered by excess EGFR activity. The second recognisable type of extra photoreceptors are the R7-like, Prospero-positive cells. These are presumably the product of abnormal recruitment of cone cell precursors as R7s, a switch of fates within the R7 equivalence group, which is sensitive to altered levels of receptor tyrosine kinase signalling (Mao, 2009).

The genetic data do not reveal a molecular mechanism for the inhibition of EGFR by Fas2 - that will require future biochemical analysis - but its location at the plasma membrane and the non-autonomy that was detected at the border of mutant clones point to three classes of models. (1) Fas2 reduces EGFR ligand production, presumably the TGFα homologues Spitz or Keren, for example by direct sequestration of the mature ligand. (2) Fas2 inhibits EGFR signalling, either by direct interaction with the receptor, or by indirectly downregulating its level or activity; in this case the observed non-autonomy would be indirect and caused by the well established positive feedback loop, whereby EGFR signalling activates expression of Rhomboid 1, which itself generates processed ligands. (3) Perhaps slightly less plausibly, the extracellular domain of Fas2 might be able to span the intercellular gap, thereby interacting with and inhibiting EGFR molecules on adjacent cells (Mao, 2009).

Precedence leads to a favouring of the second model. Two other adhesion proteins, Kekkon-1 and Echinoid, interact directly with the EGFR. Similarly, mammalian E-cadherin can inhibit the EGFR by direct binding. Of particular relevance to this work, it has recently been reported that mammalian EGFR can be inhibited by NCAM, the Fas2 orthologue (Povlsen, 2008). In these experiments using explanted mouse neurons combined with transfected mammalian cell lines, NCAM stimulates neurite outgrowth by blocking EGFR function. Preliminary results lead the authors to favour a mechanism of NCAM-induced downregulation of EGFR levels, although direct parallels with the current work are difficult to draw because the cytoplasmic domains of NCAM and Fas2 are not similar (Mao, 2009)

Beyond the evidence for inhibition of the EGFR described in this study and in the recent paper discussed above, Fas2/NCAM has now been implicated in several other signalling systems. The best characterised of these is an interaction with FGFR signalling, where, both in Drosophila and mammals, FGFR activity is required for Fas2/NCAM induced neurite outgrowth and direct binding of NCAM activates FGFR (Kiselyov, 2003; Christensen, 2006). By contrast, and an illustration of the context dependence of such interactions, it has also recently been reported that NCAM can inhibit FGFR activation by its ligand FGF (Francavilla, 2007). Less well studied links between NCAM and growth factors include the observation that NCAM can act as a signalling receptor for GDNF, and that it participates in the response of oligodendrocyte precursors to PDGF. The work reported in this study is the first genetic evidence to imply a role for Fas2 in the physiological inhibition of EGFR activity. It is important to set this discussion in the context of the well established role of Fas2/NCAM as a neural cell-adhesion molecule, with roles in axonal growth and pathfinding, as well as in synaptic maturation (Mao, 2009)

Overall, it is becoming clear that the EGFR pathway is regulated by multiple partially overlapping mechanisms, presumably because of the importance of regulatory precision and robustness of such a central and pleiotropic pathway. Notably, negative-feedback control is a recurring theme. Much less is known about physiologically significant regulators of EGFR signalling in mammals, and it will be interesting to determine whether feedback control is a conserved strategy. As there are many other signalling pathways and adhesion proteins that contribute to normal development, the total potential number of regulatory interactions between these key cell surface proteins is enormous and, indeed, many have been observed in vivo and in vitro. Of course, some of these might not occur in normal biological contexts, emphasising the value of a genetic approach to revealing which relationships between adhesion proteins and signalling pathways are physiologically relevant (Mao, 2009).

Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII

Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, the first invertebrate Glial cell line-derived neurotrophic family receptor-like cDNA (DmGfrl) was cloned and characterized from Drosophila melanogaster, and a DmGfrl mutant allele was generated. It was found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediates neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. a DmGfrl null allele was generated by genomic FLP deletion, and it was found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. These results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that were describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes (Kallijarvi, 2012).

There is ample suggestive evidence that neurons in invertebrates require trophic support similarly to vertebrate neurons, although the identification of neurotrophic ligands in e.g., Drosophila has progressed only recently. The first Drosophila homologs of vertebrate neurotrophin family proteins, Drosophila neurotrophin 1 (DNT1), DNT2 and SpƤtzle, were identified in silico several years ago and recently characterized in detail and shown to possess neurotrophic activity in vivo. Additionally, DmManf, the Drosophila homolog of the novel mammalian CDNF/MANF family of neurotrophic factors, is required for the development of the Drosophila embryonic nervous system (Kallijarvi, 2012 and references therein).

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are secreted growth factors distantly related to the TGF-β superfamily. GFLs are crucial for the development and maintenance of distinct populations of central and peripheral neurons, as well as for the organogenesis of the kidney, and spermatogenesis. In mammals, four different GFL-coreceptor pairs exist. They all signal intracellularly through the RET receptor tyrosine kinase. Neural cell adhesion molecule (NCAM) is an alternative signaling receptor for GDNF in mammals. NCAM binds GFRα1 and GDNF and downregulates NCAM-mediated cell adhesion, which activates cytoplasmic protein tyrosine kinase signaling in the absence of RET. Through NCAM, GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons in mouse brain (Kallijarvi, 2012).

Mammalian GDNF family alpha receptors (GFRα) contain a conserved arrangement of extracellular cysteine-rich GFRα domains and a C-terminal GPI anchor. Homologs of GFLs, RET and the four mammalian GFRα receptors exist in all vertebrates. RET homologs seem to be present in insects but not in echinoderms. The Drosophila melanogaster RET homolog is expressed in many tissues analogous to the tissues where the gene is expressed in vertebrates, suggesting similar functions in development. GFR-like proteins have been identified in silico in sea urchin, insects and worms, including D. melanogaster and C. elegans. In Drosophila, two partial mRNA sequences encoding fragments of GFR-like proteins have been identified. However, GDNF family ligand genes have not been found in invertebrates by in silico methods. To shed light on the evolutionary origin and function of invertebrate GFR-like proteins, this study set out to characterize the Drosophila melanogaster Gfr-like gene (DmGfrl) gene and protein, to investigate its interaction with the mammalian GDNF receptors and to generate a DmGfrl null allele to investigate the in vivo functions of the receptor (Kallijarvi, 2012).

At the start of this project, two Drosophila melanogaster cDNA fragments predicting amino acid sequence with similarity to the GFRα domains of mammalian GDNF receptor proteins had been annotated in Genbank. Starting from these cDNA fragments, RACE, RT-PCR and in silico sequence analysis was used to assemble what was presumed to be the full genomic structure of the gene, and altogether six transcripts produced from this locus were identified. Based on previously suggested nomenclature, this gene was named Drosophila melanogaster Glial cell line-derived neurotrophic factor family receptor-like, or DmGfrl. The two major DmGfrl transcripts (A and B) detectable on Northern blots were found to differ only in their 5' untranslated regions and the 5' coding sequence preceding the first GFRα-like domain, including the translation initiation site and a predicted signal sequence. The exons harboring the translation start sites for transcript A and B are separated in the genome by ~27 kb, which indicates that the two main transcripts are very likely to have separate promoter regions. Such differential promoter usage may serve to allow regulation of the same gene product by separate sets of transcription factors in different developmental and/or physiological contexts. Indeed, DmGfrl transcript A is predominant in embryos. Both major DmGfrl transcripts encode a protein with four cysteine-rich GFRα-like domains, which is in line with previous in silico predictions. Similarity to the mammalian GFRα receptors is restricted to these domains, which have a characteristic arrangement of 10 cysteine residues in each domain. Interestingly, a Gfr-like gene in C. elegans predicts a similarly large protein of >1000 amino acids with four GFRα-like domains. Based on gene structures a common origin has been proposed for the exons encoding D1 to D3 in insect and sea urchin Gfr-like proteins and vertebrate GFRα genes, which suggests that a protoGFRα receptor evolved before the protostome-deuterostome divergence (Kallijarvi, 2012).

Insects lack GDNF family ligands, but having cloned the Drosophila receptor homologs it was asked whether they might respond to mammalian GDNF and whether DmGfrl could mediate mammalian RET phosphorylation. Both experiments suggested that DmRet and DmGfrl are not structurally sufficiently conserved to bind to mammalian GDNF or interact with the mammalian receptor homologs. It is interesting to speculate that one of the seven Drosophila TGF-β ligands could function as a soluble ligand ('protoGDNF') for DmRet and/or DmGfrl (Kallijarvi, 2012).

During Drosophila embryogenesis, DmRet is expressed in many tissues that are functionally analogous to those in which mammalian RET is expressed, including foregut neurons, the excretory system, peripheral ganglia and the central nervous system. DmGfrl and DmRet expression in the embryonic nervous system and in the larval and adult brain was compared using in situ hybridization. The expression pattern of DmGfrl was generally concordant with the neuronal cell expression of GFRα1 and GFRα2 in mice, in which expression at both the mRNAs and proteins has been reported in several brain areas, the spinal cord and various peripheral ganglia. Interestingly, however, DmGfrl expression was detected in the Malpighian tubules, the Drosophila analog of mammalian kidney. In line with previously published in situ hybridization data, DmRet was found to be first expressed in the yolk sac and subsequently in the ventral neuroectoderm starting from embryonic stage 13. DmRet and DmGfrl expression coincided temporally but not spatially during embryogenesis. In the larval and adult brain, DmGfrl and DmRet expression patterns were also completely non-overlapping. Thus, it is concluded that DmRet and DmGfrl likely do not function as an in cis receptor-co-receptor pair as do mammalian RET and GFRα receptors. However, the data do not rule out the possibility that DmRet and DmGfrl could interact via an alternative mode, for example in trans (cell-to-cell) or by cleavage and diffusion of soluble DmGfrl. In the absence of a DmRet null allele or a suitable hypomorphic allele, a genetic interaction between DmRet and DmGfrl was sought in misexpression experiments. No evidence was found that DmGfrl coexpression could modify ectopic DmRet-induced phenotype in the eye. The ectopic expression experiment is, however, inconclusive, and progress in this direction will require the generation of a DmRet allele suitable for genetic interaction experiments (Kallijarvi, 2012).

To gain insight into the in vivo function of the DmGfrl receptor a DmGfrl null allele by was generated by FLP-mediated genomic deletion. DmGfrl null flies were grossly normal and viable. However, they displayed a severe defect in both male and female fertility. The reduced female fertility results from an oogenesis defect as the mutant females laid fewer eggs than normally and a large fraction of those were small and had abnormal dorsal appendages. The egg morphology defect was efficiently rescued by transgene expression under the widely active daughterless and actin drivers, indicating that the phenotype is specific to loss of DmGfrl expression, and likely dependent on the somatic tissue of the ovary. However, the transgene did not rescue the reduced viability of the eggs or the reduced fecundity of the females. This suggests that the reduced egg viability is either dependent on germline cells, in which this transgene should not be expressed, or is not rescued by the DmGfrlA isoform used in these experiments. Similarly to females, in DmGfrl null males a fertility defect was observed that was not fully penetrant. Because the fecundity of DmGfrl null males was much more reduced than their absolute fertility, it was reasoned that a defect in spermatogenesis is a likely cause. Dissection of the testis histology and function in the mutant flies, as well as further rescue experiments will likely clarify the mechanism of the fertility defect in DmGfrl null males. Interestingly, on the basis of a proteomics study DmRet protein is present in adult spermatozoa, which warrants studies of the putative conserved function of DmRet in spermatogenesis (Kallijarvi, 2012).

Finally, on the basis of molecular evidence from mammals, it was of interest to look if DmGfrl might interact with the Drosophila NCAM homolog FasII. In mammals NCAM binds GDNF and GFRα1 and functions as an alternative signaling receptor for GDNF, mediating neuronal migration and axonal growth. FasII is widely expressed in the embryonic VNC, making it likely that it is also expressed in the DmGfrl-expressing neurons. WA hypomorphic FasII allele was combined with the delDmGfrl allele and whether the former could modify the male fertility phenotype of DmGfrl null flies was investigated. Strikingly, the double homozygous males were completely infertile, indicating a strong genetic interaction between DmGfrl and the FasII allele. There is currently little data linking NCAM/FasII function to reproduction. Nevertheless, on the basis of in silo data both DmGfrl and FasII are expressed at low levels in the testis and ovary. There is evidence for a role of FasII in the hormonal control of the development of Drosophila male genitalia, as a FasIIspin allele has been shown to disrupt the looping of the male genitalia and spermiduct. Interestingly, in FasIIspin flies, the innervation of corpora allata in the ring gland is disrupted, which the authors suggest may lead to elevated level of juvenile hormone and eventually to the looping defect. In preliminary inspection, no rotation defect was observed in the external male genitalia in FasIIe76;;del/Df males, but this lead will be worth further study. No gross embryonic or adult neuronal phenotypes was observed in the DmGfrl null flies. As subtle developmental or behavioral phenotypes may be present this question will require careful further studies (Kallijarvi, 2012).

The strong genetic interaction between DmGfrl and FasII that was described is corroborated by data showing biochemical interaction between the ectopically expressed receptors. These data are the first to suggest that the GFRα1-NCAM interaction described in mammalian systems is evolutionarily conserved. Together with the results suggesting that DmRet and DmGfrl do not function in cis in Drosophila, which lacks GDNF ligands, these data imply that DmGfrl may be an evolutionarily ancient binding partner for NCAM/FasII. Whether or not a soluble ligand exists in Drosophila and is needed to activate the putative FasII-DmGfrl signaling complex needs to be tackled in future studies (Kallijarvi, 2012).


Amino Acids

There is a phosphatidylinositol-linked form with 811 amino acids and a transmembrane form with 873 amino acids. They differ only in their C terminal amino acids (Grenningloh, 1991).

Structural Domains

FAS2 is a member of the immunoglobulin superfamily containing five Ig-like domains. It also contains two fibronectin-type III repeats (Grenningloh, 1991). The protein contains a signal sequence serving for protein secretion.

Fasciclin 2: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 28 MAY 97 

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