See the embryonic expression pattern of Ama at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
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).
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).
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date revised: 21 September 2003
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