InteractiveFly: GeneBrief

Amalgam : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - Amalgam

Synonyms -

Cytological map position - 84A5

Function - ligand

Keywords - CNS, PNS, axon guidance

Symbol - Ama

FlyBase ID: FBgn0000071

Genetic map position -

Classification - immunoglobulin superfamily

Cellular location - secreted, transmembrane



NCBI link: Precomputed BLAST | Entrez Gene
Recent literature
Becker, H., Renner, S., Technau, G.M. and Berger, C. (2016). Cell-autonomous and non-cell-autonomous function of Hox genes specify segmental neuroblast identity in the gnathal region of the embryonic CNS in Drosophila. PLoS Genet 12: e1005961. PubMed ID: 27015425
Summary:
In thoracic and abdominal segments of Drosophila, the expression pattern of Bithorax-Complex Hox genes is known to specify the segmental identity of neuroblasts (NB) prior to their delamination from the neuroectoderm. This study identified and characterized a set of serially homologous NB-lineages in the gnathal segments and used one of them (NB6-4 lineage) as a model to investigate the mechanism conferring segment-specific identities to gnathal NBs. It was shown that NB6-4 is primarily determined by the cell-autonomous function of the Hox gene Deformed (Dfd). Interestingly, however, it also requires a non-cell-autonomous function of labial and Antennapedia that are expressed in adjacent anterior or posterior compartments. The secreted molecule Amalgam (Ama) was identified as a downstream target of the Antennapedia-Complex Hox genes labial, Dfd, Sex combs reduced and Antennapedia. In conjunction with its receptor Neurotactin (Nrt) and the effector kinase Abelson tyrosine kinase (Abl), Ama is necessary in parallel to the cell-autonomous Dfd pathway for the correct specification of the maxillary identity of NB6-4. Both pathways repress CyclinE (CycE) and loss of function of either of these pathways leads to a partial transformation (40%), whereas simultaneous mutation of both pathways leads to a complete transformation (100%) of NB6-4 segmental identity. Finally, the study provides genetic evidences, that the Ama-Nrt-Abl-pathway regulates CycE expression by altering the function of the Hippo effector Yorkie in embryonic NBs. The disclosure of a non-cell-autonomous influence of Hox genes on neural stem cells provides new insight into the process of segmental patterning in the developing CNS. 

BIOLOGICAL OVERVIEW

Amalgam (Ama), a secreted member of the immunoglobulin superfamily, has been identified as a ligand for the Neurotactin (Nrt) receptor. Nrt is a member of the serine esterase-like family of membrane proteins. Ama is necessary for Nrt-expressing cells both to aggregate with one another and to associate with embryonic primary culture cells. Aggregation assays performed with truncated Nrt molecules reveal that the integrity of the cholinesterase-like extracellular domain is not required either for Ama binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the Nrt cytoplasmic domain is required for Nrt-mediated adhesion, although not for Ama binding. Using an ama-deficient stock, it has been found that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants. Although the specific roles of Nrt and Ama during Drosophila development remain quite obscure, different aspects of the puzzle are beginning to fall into place. A link has been established between these two enigmatic proteins. One of the critical missing pieces is what lies downstream of the Nrt receptor and how Ama binding to Nrt affects these downstream components. Addressing this issue and identifying orthologs in other organisms are probable key steps in a further understanding of this intriguing receptor-ligand interaction (Fremion, 2000).

Nrt functions as a heterophilic cell adhesion protein in various cell culture assays (Barthalay, 1990), suggesting that its cholinesterase domain functions as a part of a recognition/signaling transmembrane receptor-like protein. Further studies have delineated a specific region of the cholinesterase-like domain located near the membrane that is involved in this recognition process (Darboux, 1996). Ama is able to promote the aggregation of Nrt-expressing S2 cells; Ama interacts specifically with Nrt, and Ama is necessary for the interaction of Nrt-expressing S2 cells with dissociated embryonic primary culture cells, the original assay that established the heterophilic adhesion properties of Nrt. The nearly identical patterns of Ama and Nrt protein accumulation throughout embryogenesis are consistent with a receptor-ligand relationship in vivo. Furthermore, the finding that Ama protein fails to exhibit its normal pattern of accumulation in an nrt mutant is compatible with Nrt acting as the key receptor for Ama during embryogenesis. These are intriguing observations: that during embryogenesis, cells that accumulate Ama on their surface are sometimes different from the cells that are expressing ama transcripts. Certainly these differences can be explained easily for a secreted protein such as Ama. However, the functional significance, if any, of these disparities between spatial patterns of protein expression versus protein accumulation is unknown (Fremion, 2000).

How does Ama facilitate Nrt-mediated adhesion? Observations that an artificially generated membrane-anchored form of Ama can mediate homophilic aggregation in the absence of Nrt indicate that Ama can interact with itself. This may suggest one mechanism whereby Ama could function as a linker between Nrt molecules on opposing cell surfaces. Nrt would bind Ama, which would bind to a second Ama molecule that could interact with Nrt on the surface of a second cell. Thus, Ama would serve as a linker protein between Nrt-expressing cells. Alternatively, Ama could alter the Nrt protein upon binding, such that it could now recognize in a homophilic manner other Nrt molecules (Fremion, 2000).

The binding of Ama to Nrt alone is not sufficient for cell aggregation. The finding that the Nrt cytoplasmic domain is essential for adhesion indicates that important events are taking place in the cytoplasm that are critical for this adhesion process. The amino acid sequence of the Nrt cytoplasmic domain does not reveal any clear motifs such as kinase or phosphatase domains. However, it does encode several putative phosphorylation sites, and Nrt is found to be phosphorylated on serine and threonine residues. Interestingly, Nrt is enriched at sites of cellular contacts in neuronal and epithelial tissues and within Nrt transfectant aggregates (Barthalay, 1990). This recruitment to specific regions of the membrane suggests that Nrt may bind to cytoskeleton components. Nrt-mediated cell adhesion might be considered as a two-step process: (1) binding of the ligand (Ama) to the extracellular domain, a step that can occur without the Nrt cytoplasmic domain; and (2) stabilization and strengthening of the interaction through clustering of Nrt to sites of cell contact and interactions dependent upon the Nrt cytoplasmic domain. This latter step was not achieved with transfectants lacking the Nrt cytoplasmic domain, demonstrating the critical role of the Nrt cytoplasmic domain in this process (Fremion, 2000).

The isolation and characterization of nrt mutants have demonstrated that Nrt is required for outgrowth, fasciculation and guidance of axons (Speicher, 1998). Mutations in nrt produce quite subtle and variable defects in axon guidance within the embryonic CNS. These phenotypes are greatly enhanced when nrt mutations are combined with mutations in other cell adhesion or signaling molecules such as nrg. These results have led to the suggestion that nrt plays a cooperative or partially redundant role in axon guidance. One of the most robust phenotypes of nrt mutants alone is disruption of ocellar nerve axon pathfinding in pupae. In nrt mutants, misrouting, stalling and defasciculation of the ocellar pioneer nerves are observed at high frequencies (Fremion, 2000).

One might expect that if Ama and Nrt indeed interact in vivo as indicated by the co-immunoprecipitation experiment, mutations in these genes would lead to similar phenotypes. Since the ocellar pioneer nerve disruptions are the most consistent phenotype for nrt null mutants, a determination was made whether a loss-of-function ama mutant displays comparable phenotypes. Similar to nrt mutations, a deficiency for ama results in highly penetrant defasciculation phenotypes of the ocellar pioneer nerves in developing pupae. However, there are significant differences in the expressivity of ocellar nerve defects. Stalling or misrouting of these axons as is found in nrt mutants is not observed in ama mutant pupae. This result suggests that rather than having an instructive role in pathfinding, Ama maintains proper cohesion between these axons and contributes to the organization of these fascicles (Fremion, 2000).

The role of Ama during embryonic axon guidance cannot be addressed due to the lack of specific ama mutations. The deficiency that was used in these studies [Df(3R)ama] is also deleted for the embryonic dorsal-ventral patterning gene zerknullt. The lethality associated with zerknullt mutations could not be rescued with a wild-type zerknullt transgene in order to generate an adult viable strain that is deficient for Ama. The difficulty is that partial rescue of zerknullt embryonic functions leads to variable defects in germ band extension and retraction. These disruptions in morphogenetic movements make it impossible to assay reliably for subtle defects in formation of axon pathways in the embryonic CNS. Therefore, it is possible that loss-of-function mutations in ama could result in embryonic axon pathfinding defects that are similar to nrt loss-of-function mutations (Fremion, 2000).

Amalgam (Ama), a secreted member of the immunoglobulin superfamily, has been identified as a ligand for the Neurotactin (Nrt) receptor. Nrt is a member of the serine esterase-like family of membrane proteins. Ama is necessary for Nrt-expressing cells both to aggregate with one another and to associate with embryonic primary culture cells. Aggregation assays performed with truncated Nrt molecules reveal that the integrity of the cholinesterase-like extracellular domain is not required either for Ama binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the Nrt cytoplasmic domain is required for Nrt-mediated adhesion, although not for Ama binding. Using an ama-deficient stock, it has been found that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants. Although the specific roles of Nrt and Ama during Drosophila development remain quite obscure, different aspects of the puzzle are beginning to fall into place. A link has been established between these two enigmatic proteins. One of the critical missing pieces is what lies downstream of the Nrt receptor and how Ama binding to Nrt affects these downstream components. Addressing this issue and identifying orthologs in other organisms are probable key steps in a further understanding of this intriguing receptor-ligand interaction (Fremion, 2000).

Nrt functions as a heterophilic cell adhesion protein in various cell culture assays (Barthalay, 1990), suggesting that its cholinesterase domain functions as a part of a recognition/signaling transmembrane receptor-like protein. Further studies have delineated a specific region of the cholinesterase-like domain located near the membrane that is involved in this recognition process (Darboux, 1996). Ama is able to promote the aggregation of Nrt-expressing S2 cells; Ama interacts specifically with Nrt, and Ama is necessary for the interaction of Nrt-expressing S2 cells with dissociated embryonic primary culture cells, the original assay that established the heterophilic adhesion properties of Nrt. The nearly identical patterns of Ama and Nrt protein accumulation throughout embryogenesis are consistent with a receptor-ligand relationship in vivo. Furthermore, the finding that Ama protein fails to exhibit its normal pattern of accumulation in an nrt mutant is compatible with Nrt acting as the key receptor for Ama during embryogenesis. These are intriguing observations: that during embryogenesis, cells that accumulate Ama on their surface are sometimes different from the cells that are expressing ama transcripts. Certainly these differences can be explained easily for a secreted protein such as Ama. However, the functional significance, if any, of these disparities between spatial patterns of protein expression versus protein accumulation is unknown (Fremion, 2000).

How does Ama facilitate Nrt-mediated adhesion? Observations that an artificially generated membrane-anchored form of Ama can mediate homophilic aggregation in the absence of Nrt indicate that Ama can interact with itself. This may suggest one mechanism whereby Ama could function as a linker between Nrt molecules on opposing cell surfaces. Nrt would bind Ama, which would bind to a second Ama molecule that could interact with Nrt on the surface of a second cell. Thus, Ama would serve as a linker protein between Nrt-expressing cells. Alternatively, Ama could alter the Nrt protein upon binding, such that it could now recognize in a homophilic manner other Nrt molecules (Fremion, 2000).

The binding of Ama to Nrt alone is not sufficient for cell aggregation. The finding that the Nrt cytoplasmic domain is essential for adhesion indicates that important events are taking place in the cytoplasm that are critical for this adhesion process. The amino acid sequence of the Nrt cytoplasmic domain does not reveal any clear motifs such as kinase or phosphatase domains. However, it does encode several putative phosphorylation sites, and Nrt is found to be phosphorylated on serine and threonine residues. Interestingly, Nrt is enriched at sites of cellular contacts in neuronal and epithelial tissues and within Nrt transfectant aggregates (Barthalay, 1990). This recruitment to specific regions of the membrane suggests that Nrt may bind to cytoskeleton components. Nrt-mediated cell adhesion might be considered as a two-step process: (1) binding of the ligand (Ama) to the extracellular domain, a step that can occur without the Nrt cytoplasmic domain; and (2) stabilization and strengthening of the interaction through clustering of Nrt to sites of cell contact and interactions dependent upon the Nrt cytoplasmic domain. This latter step was not achieved with transfectants lacking the Nrt cytoplasmic domain, demonstrating the critical role of the Nrt cytoplasmic domain in this process (Fremion, 2000).

The isolation and characterization of nrt mutants have demonstrated that Nrt is required for outgrowth, fasciculation and guidance of axons (Speicher, 1998). Mutations in nrt produce quite subtle and variable defects in axon guidance within the embryonic CNS. These phenotypes are greatly enhanced when nrt mutations are combined with mutations in other cell adhesion or signaling molecules such as nrg. These results have led to the suggestion that nrt plays a cooperative or partially redundant role in axon guidance. One of the most robust phenotypes of nrt mutants alone is disruption of ocellar nerve axon pathfinding in pupae. In nrt mutants, misrouting, stalling and defasciculation of the ocellar pioneer nerves are observed at high frequencies (Fremion, 2000).

One might expect that if Ama and Nrt indeed interact in vivo as indicated by the co-immunoprecipitation experiment, mutations in these genes would lead to similar phenotypes. Since the ocellar pioneer nerve disruptions are the most consistent phenotype for nrt null mutants, a determination was made whether a loss-of-function ama mutant displays comparable phenotypes. Similar to nrt mutations, a deficiency for ama results in highly penetrant defasciculation phenotypes of the ocellar pioneer nerves in developing pupae. However, there are significant differences in the expressivity of ocellar nerve defects. Stalling or misrouting of these axons as is found in nrt mutants is not observed in ama mutant pupae. This result suggests that rather than having an instructive role in pathfinding, Ama maintains proper cohesion between these axons and contributes to the organization of these fascicles (Fremion, 2000).

The role of Ama during embryonic axon guidance cannot be addressed due to the lack of specific ama mutations. The deficiency that was used in these studies [Df(3R)ama] is also deleted for the embryonic dorsal-ventral patterning gene zerknullt. The lethality associated with zerknullt mutations could not be rescued with a wild-type zerknullt transgene in order to generate an adult viable strain that is deficient for Ama. The difficulty is that partial rescue of zerknullt embryonic functions leads to variable defects in germ band extension and retraction. These disruptions in morphogenetic movements make it impossible to assay reliably for subtle defects in formation of axon pathways in the embryonic CNS. Therefore, it is possible that loss-of-function mutations in ama could result in embryonic axon pathfinding defects that are similar to nrt loss-of-function mutations (Fremion, 2000).


REGULATION

Transcriptional Regulation

In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).

Protein Interactions

Neurotactin is a transmembrane protein whose extracellular domain is able to bind a ligand(s). Heterotypic binding assays utilizing embryonic cells obtained from gastrula stage embryos or transfected S2 cells expressing Nrt protein has indicated that an Nrt ligand(s) is present on the surface of embryonic cells. This Nrt ligand is also found as a soluble form, since auto-aggregation of Nrt-expressing S2 cells can be induced with a 100,000 g supernatant prepared from embryonic extracts (Fremion, 2000 and references therein).

Fractionation experiments using embryo extracts show that Ama is present in soluble fractions. Western blot analysis with Ama-specific antisera indicates that extracts prepared from embryonic cells contained immunoreactive polypeptides of ~45 kDa within the membrane fraction and in the 100,000g supernatant. Higher molecular weight bands are only observed in the membrane fraction pellet and might be related to Ama molecules trapped in protein complexes. The ama gene encodes a protein with an N-terminal signal sequence and a weakly hydrophobic C-terminal domain. Immunostaining of whole-mount embryos suggests that Ama is a membrane-associated protein, although the weakly hydrophobic C-terminal domain is unlikely to tether Ama directly to the membrane (Fremion, 2000).

In order to confirm further that Ama is a secreted protein, S2 cells were transfected with a plasmid construct encoding the ama cDNA under the control of an inducible metallothionein promoter. After induction with divalent cations, products were immunodetected by Western blot analysis of whole-cell extracts. The culture medium in which Ama transfectants have grown contains soluble Ama protein. Taken together, these data indicate that Ama is a secreted, soluble protein that can associate with the cell surface (Fremion, 2000.

To determine whether Ama plays a role in Nrt-mediated heterophilic adhesion, Nrt transfectants, which are not able to aggregate by themselves, were incubated in culture medium containing secreted Ama protein. Aggregate formation, similar to that in the experiment where the 100 000 g embryonic extract supernatant was used as a soluble fraction containing ligand activity, was observed. Moreover, when a soluble fraction is prepared from embryos deleted for the ama gene, Nrt transfectants do not aggregate. To determine whether Ama interacts specifically with Nrt, an S2 cell pull-down assay was conducted. Untransfected and transfected S2 cells expressing Nrt were incubated with soluble protein fractions prepared from either wild-type or ama-deficient embryos. These S2 cells were then pelleted and total cellular proteins were analyzed by Western blot analysis with Nrt- and Ama-specific antibodies. Nrt-expressing S2 cells are able to pull-down Ama from wild-type embryonic extracts, while control S2 cells do not. Not surprisingly, no Ama-immunoreactive material is found associated with Nrt-expressing S2 cells that are incubated with soluble protein fractions prepared from ama-deficient embryos. These results show that Ama is necessary for Nrt-mediated adhesion. A molecular association between Ama and Nrt has been demonstrated by a co-immunoprecipitation assay (Fremion, 2000).

These results suggest that a membrane-anchored form of Ama might interact directly with Nrt-expressing cells and facilitate heterophilic aggregation. To test this hypothesis, a transmembrane form of Ama (Ama-TM) was generated. Ama-TM was created by fusing the entire ama open reading frame to the transmembrane and cytoplasmic domain of the Drosophila Neuroglian protein. When plated onto plastic slide flasks, the Ama-TM S2 transfectants were able to bind methylene blue-stained Nrt-expressing S2 cells. This result suggests that the Ama membrane-bound form may also bind the Nrt molecule. Ama-TM-expressing S2 cells form large aggregates in the cell aggregation assay. Thus, it would appear that Ama protein can interact with itself in addition to its interaction with Nrt (Fremion, 2000).

Nrt is a type II transmembrane protein inserted in the lipid bilayer by a single hydrophobic region composed of 22 amino acids that separates the N-terminal cytoplasmic domain (323 amino acids) from the C-terminal extracellular domain (500 amino acids). By expressing truncated Nrt proteins in S2 cells and using a soluble fraction prepared from embryonic extracts that promote cell aggregation, a region within the extracellular domain between His347 and His482 that is essential for the adhesive function of Nrt has been localized (Darboux, 1996). In order to determine if this in vitro recognition process requires only Ama, the same experiments were repeated by replacing the crude extract with culture medium containing secreted Ama protein. Consistent with previous results, Nrt molecules that were truncated downstream of residues Pro452 were found to be inactive, while truncation downstream of His482 generates a molecule that possesses the same adhesive properties as the full-length Nrt. Simultaneously with these assays, aliquots of cells or aggregates were analyzed on SDS-PAGE, and Ama binding to transfectants was evaluated by Western blot analysis. Only Delta EXT3 transfectants are able to form aggregates, and protein analysis demonstrates that these cells bind Ama while Delta EXT1 and Delta EXT2 transfectants stay as single cells and no Ama binding is detected. Among the series of truncated molecules that have been analyzed (Delta EXT1, Delta EXT2 and Delta EXT3), the presence of Ama was found to correlate with the capacity to form aggregates. This suggests that at least in vitro, Ama is the major component involved in this Nrt-mediated cell-cell recognition process (Fremion, 2000).

The role of the Nrt cytoplasmic domain in aggregation was investigated by using a construct designated Delta CYT. The Delta CYT molecule lacks the 293 amino acid cytoplasmic domain, including five putative phosphorylation sites, leaving intact a short terminal sequence for correct initiation of translation as well as sequences near the transmembrane domain for proper membrane insertion and orientation. The Delta CYT construct was used to transfect S2 cells, and protein expression was analyzed by Western blot analysis. The apparent molecular weight of the Delta CYT molecule (60 kDa) is consistent with the predicted size of the glycosylated extracellular domain (522 amino acids). The correct translocation of the Delta CYT was investigated further by mild papain digestion of the transfectants. This treatment releases a polypeptide whose molecular weight (55 kDa) is compatible with the expected accessibility of the extracellular domain to proteases. These observations indicate that the removal of 293 amino acids from the cytoplasmic domain does not impair efficient insertion of Nrt into the membrane or the overall stability of the Nrt protein (Fremion, 2000).

Delta CYT-expressing transfectants does not form aggregates in the presence of the culture medium containing secreted Ama protein, although the controls (full-length Nrt transfectants) aggregate efficiently. This result demonstrates that the Nrt cytoplasmic domain is necessary for cells to aggregate. Interestingly, Ama binding to Delta CYT transfectants is detected. This demonstrates that Ama binding to the Nrt extracellular domain alone is not sufficient to promote aggregation and that the Nrt cytoplasmic domain is also required for Nrt-mediated aggregation (Fremion, 2000).

The quaternary structure of amalgam, a Drosophila neuronal adhesion protein, explains its dual adhesion properties

Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. This study reports its biophysical characterization; small-angle x-ray scattering was used to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each 'arm' of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane (Zeev-Ben-Mordehai, 2009).


DEVELOPMENTAL BIOLOGY

Embryonic

Accumulation of Ama is first observed during early stage 8 of embryogenesis, shortly after the formation of the three germ layers during gastrulation. At this stage, Nrt is already expressed throughout the ectoderm and mesoderm. During germ band extension, Ama begins to be expressed within a row of midline cells that appear to be a subset of mesectodermal cells (Seeger, 1988); Nrt is expressed by midline cells and also more generally by the ectoderm layer. During embryonic stages 11 and 12, from the fully extended germ band through germ band shortening, neuroblasts undergo a series of asymmetric cell divisions to produce ganglion mother cells that in turn divide symmetrically, generating two neurons. Nrt is expressed ubiquitously in the ectoderm layer, outlining the epithelial cells and the developing neuroblasts and their progeny (Piovant, 1998). The Ama expression pattern is more restricted: Ama is not found on the neuroblasts; however, high levels of protein are found on their neural but not GMC progeny (Seeger, 1988). Both proteins are expressed in a subset of mesodermal derivatives including the fat body and the dorsal vessel. By stage 13 of embryogenesis, mature neurons are extending axons along stereotyped pathways forming the segmentally repeated arrays of commissural and longitudinal axon bundles or fascicles. During these stages, Ama and Nrt accumulation is seen within the fat body and throughout the central nervous system (CNS) on both neuronal cell bodies and their axons (Piovant, 1998; Seeger, 1988; de la Escalera, 1990; Hortsch, 1990). During early stages of peripheral nervous system (PNS) development, Nrt is expressed weakly on axon pathways that connect the ventral, lateral and dorsal clusters, while Ama expression is not detectable. However, in later stage embryos, both proteins concentrate on external sensory organ precursors. Overall, the patterns of Ama and Nrt accumulation during embryogenesis are strikingly similar (Fremion, 2000 and references therein).

Since the data from S2 cell transfection experiments show that Ama is secreted into the culture medium, one might expect to find differences between AMA RNA and protein expression within tissues during development. For nrt, there is a precise correspondence between patterns of nrt RNA and protein localization (de la Escalera, 1990; Hortsch, 1990). In the case of AMA, there are interesting differences between RNA and protein accumulation profiles. During early stages of embryogenesis, there is a strong correlation between patterns of AMA RNA and protein expression. For example, high levels of both AMA RNA and protein are observed in the mesoderm invagination during early stages of germ band extension. During later stages of embryogenesis, differences between the patterns of RNA and protein accumulation become apparent. By stage 14, Ama protein is found predominantly within the CNS, while in situ hybridizations show no RNA expression within this tissue. The same observation was made for the fat body, which accumulates large amounts of Ama protein. Once again, low levels of AMA RNA are detected in this tissue. By stage 14, weak AMA RNA signals are found around the gut, but never accumulated in discrete areas such as the external sensory organs in the PNS where Ama protein is concentrated. Clearly, there are regions where Ama protein accumulation does not correspond to high levels of AMA transcripts, suggesting that Ama protein turnover is low (Fremion, 2000).

The overall patterns of Ama protein accumulation during embryogenesis are interrelated with patterns of NRT RNA and protein expression (Seeger, 1988; de la Escalera, 1990; Hortsch, 1990). Differences in the patterns of AMA RNA and Nrt expression are observed. For instance, in a stage 10 embryo double stained for AMA RNA and with anti-Nrt antibody, differences are apparent. AMA transcripts, which identify cells producing the ligand, are located more apically than Nrt-expressing cells. Clearly, many cells that express high levels of AMA transcript do not express Nrt protein. The normal pattern of Ama protein accumulation is also dependent upon the presence of Nrt. In nrt mutant embryos, the pattern of Ama protein expression is clearly aberrant (Fremion, 2000).


EFFECTS OF MUTATION

Given the requirement for Ama for Nrt-mediated adhesion, additional in vivo approaches were undertaken in order to question whether Ama and Nrt are involved in the same aspects of neural development. Like the nrt gene, ama is not essential for development. Adults that are deficient for either ama or nrt can be generated. In these studies, ama requirements in ama-deficient pupae were analyzed, since the most reliable phenotype for mutations in nrt was found to be defasciculation of the ocellar pioneer nerves (Speicher, 1998). During early pupal development, ocellar pioneer axons extend in the extracellular matrix (ECM) that covers the internal side of the prospective head without contacting the epithelium. This choice of ECM versus epidermis is crucial for normal pathfinding of these axons. Lack of ama results in defasciculation of the normally tightly associated ocellar pioneer axons. These defects occur frequently: the penetrance is 87% exhibiting defasciculation compared with the wild-type background where defects were observed in only 8% of the individuals. The presence of any single split within the fascicles was considered to be a defasciculation defect. These data are similar to those published by Speicher (1998), for a null mutation in nrt. As in the case of nrt mutants, this phenotype is predominant in those pupae that do not go through the head eversion process. Despite these frequent defasciculation abnormalities, ocellar pioneer axons usually reach their brain targets. In contrast to nrt mutants, no association is observed of the epidermis or connections with the neighboring mechano-receptor axons in ama-deficient pupae (Fremion, 2000).

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. An unusual missense ama allele, amaM109, has been identified that dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the AmaM109 mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone (Liebl, 2003).

Genetic screens for second-site modifiers are useful tools for identifying components of signaling networks. Over the past decade, work in Drosophila has identified multiple modifiers of the Abl mutant phenotype. With the exception of the transcription factor prospero, all of the dominant modifiers identified have been cytoplasmic and co-expressed with Abl in axons. The biochemical characterization of some of the proteins encoded by these dominant enhancers has lead to an emerging model whereby the Abl tyrosine kinase supplies multiple inputs into actin cytoskeleton dynamics in the growth cone (Liebl, 2003).

The dosage-sensitive genetic interactions of ama and nrt with Abl provide unique information regarding Abl signaling networks. Five independent nrt alleles have been identified that remove Nrt function. Three are null alleles (nrtM2, nrtM29, nrtM54), while two (nrtM100 and nrtM221) are missense alleles that behave as protein nulls. Thus, simply reducing wild-type Nrt activity in an Abl-null background impairs viability, suggesting Abl and Nrt lie within one or more common signaling networks. The fact that these genetic combinations have clear effects on axon pathfinding, strongly suggests that at least one of these common signaling networks has its in vivo output in the growth cone. This is confirmed by the severe axon guidance phenotype produced by disruption of Abl and Nrt function through RNAi or homozygous zygotic mutation. Disruption of Abl and Nrt by zygotic mutation results in strong, but less severe CNS phenotypes than RNAi, probably as a result of elimination of maternally loaded Abl mRNA (Liebl, 2003).

Ama and Nrt have been shown to functionally interact to mediate cell:cell adhesion. Heterozygous null alleles of ama have no detectable dominant effects on axon pathfinding in an Abl-mutant background, presumably because the biochemical activity of secreted Ama is not directly associated with the cytoplasmic tyrosine kinase activity of Abl. However, disruption of Abl and Ama by homozygous zygotic mutation or by RNAi techniques does show clear synergistic disruptions of the CNS architecture. As with Abl and Nrt, the RNAi-induced phenotype is the more severe of the two, presumably because of the elimination of maternally supplied Abl mRNA (Liebl, 2003).

The identification of the unusual missense ama allele amaM109 as a strong dominant enhancer of the Abl mutant phenotype, affecting both viability and axon pathfinding, strengthens the conclusion that Ama, Nrt and Abl are functionally intertwined in the growth cone. AmaM109, which alters a cysteine residue needed to stabilize the first Ig domain of Ama, eliminates Ama homophilic adhesion but not the ability of AmaM109 to bind Nrt, and this is probably responsible for its unique character. The biochemical activity of this protein is clearly not wild type, since its ability to support aggregation of Nrt-expressing S2 cells is impaired (Liebl, 2003).

Genetically, the amaM109 allele phenocopies heterozygosity for nrt in the Abl1/Abl4 mutant background. Both genotypes result in 100% pre-pupal lethality, and both result in approximately one-third of embryo segments having defective commissures. Thus, it seems likely that, whatever its biochemical mode of action, the AmaM109 protein disables Nrt activity in a way that simply reducing the dose of wild-type Ama (by heterozygous null mutation) does not (Liebl, 2003).

To better understand the function of Nrt in the CNS, Speicher (1998) carried out an extensive genetic analysis, looking for cell adhesion molecules (CAMs) that are functionally redundant to Nrt. This was achieved by generating animals null for nrt and null for a variety of other CAM-encoding genes in pair-wise combinations. Removal of Nrt does not result in a strong CNS phenotype, Three different genetic combinations showed synergistic interactions in the CNS: nrt and neuroglian (nrg), nrt and derailed (drl), and nrt and kekkon1 (kek1), with the nrt, nrg combination showing the most profound synergy. This work suggests the role of Nrt in CNS cell adhesion is at least partially redundant to Nrg, Drl and Kek1. Interestingly, it has been reported that nrg and Abl have no genetic interaction when the morphology of the CNS is assayed by mAb BP102 staining (Liebl, 2003).

Whether Nrt-mediated adhesion provides novel inputs into Abl-mediated signaling networks in the growth cone or whether Nrt-mediated adhesion represents a novel output of the role of Abl in cytoskeleton dynamics can be determined by the genetic experiments that have been carried out. Intriguingly, deletion of the cytoplasmic region of Nrt eliminates its ability to promote cell:cell adhesion. Since many transmembrane cell adhesion molecules require functional interactions with the actin-based cytoskeleton, it is plausible that Ama:Nrt-mediated adhesion requires interaction of the cytoplasmic region of Nrt with actin-based cytoskeleton components. To clarify this issue molecular genetic screens are currently being conducted to identify protein:protein interactions involving the cytoplasmic domain of Nrt (Liebl, 2003).

Molecular and genetic characterization of nrt as a dominant enhancer of the Abl mutant phenotype has shown that all five mutations previously attributed to dab are nrt alleles. How were these mutations initially attributed to dab? The answer lies in incomplete characterization of proximal and distal breakpoints of Abl deletions, and mistaking the effects of dab near the proximal breakpoint with the effects of fax near the distal breakpoint. In retrospect, the difference in genetic activity between different deletions can be accounted for by the difference in the distal breakpoints of these chromosomes. Null mutations in fax dominantly enhance the Abl mutant phenotype (Liebl, 2003 and references therein).


REFERENCES

Search PubMed for articles about Drosophila Amalgam

Barthalay, Y., Hipeau-Jacquotte, R., de la Escalera, S., Jimenez, F. and Piovant, M. (1990). Drosophila Neurotactin mediates heterophilic cell adhesion. EMBO J. 9(11): 3603-3609. PubMed Citation: 2120048

Beltran, S., et al. (2003). Transcriptional network controlled by the trithorax-group gene ash2 in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100(6): 3293-8. 12626737

Darboux, I., et al. (1996). The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties. EMBO J. 15(18):4835-43. PubMed Citation: 8890157

de la Escalera, S., Bockamp, E. O., Moya, F., Piovant, M. and Jimenez, F. (1990). Characterization and gene cloning of Neurotactin, a Drosophila transmembrane protein related to cholinesterases. EMBO J. 9(11): 3593-3601. PubMed Citation: 2120047

Fremion, F., et al. (2000). Amalgam is a ligand for the transmembrane receptor neurotactin and is required for neurotactin-mediated cell adhesion and axon fasciculation in drosophila. EMBO J. 19(17): 4463-72. PubMed Citation: 10970840

Hortsch, M., Patel, N.H., Bieber, A. J., Tranquina, Z. R. and Goodman, C. S. (1990). Drosophila Neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis. Development 110(4): 1327-40. PubMed Citation: 2100266

Liebl, E. C., et al. (2003). Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding. Development 130: 3217-3226. 12783792

Piovant, M. and Lena, P. (1988) Membrane glycoproteins immunologically related to the human insulin receptor are associated with presumptive neuronal territories and developing neurones in Drosophila melanogaster. Development 103: 145-156. PubMed Citation: 3143540

Seeger, M. A., Haffley, L. and Kaufman, T. C. (1988). Characterization of amalgam: a member of the immunoglobulin superfamily from Drosophila. Cell 55: 589-600. PubMed Citation: 3141062

Speicher, S., et al. (1998). Neurotactin functions in concert with other identified CAMs in growth cone guidance in Drosophila. Neuron 20(2): 221-33. PubMed Citation: 9491984

Zeev-Ben-Mordehai, T., et al. (2009). The quaternary structure of amalgam, a Drosophila neuronal adhesion protein, explains its dual adhesion properties. Biophys J. 97(8): 2316-26. PubMed Citation: 19843464


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date revised: 21 October 2000

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