Neurotactin

Gene name - Neurotactin

Synonyms -

Cytological map position - 73C 1-2

Function - cell adhesion

Keywords - neural - axonogenesis

Symbol - Nrt

FlyBase ID:FBgn0004108

Genetic map position - 3-[44]

Classification - transmembrane protein - cholinesterase homolog

Cellular location - surface



NCBI link: Entrez Gene

neurotactin orthologs: Biolitmine
BIOLOGICAL OVERVIEW

Nrt accumulates in the membranes of neuroblasts and their progeny. Between stages 12 and 16 of axogenesis, extensive protein accumulation is observed in the CNS. Analysis of CNS serial sections reveals strong accumulation of Nrt in growing axon tracts and some heterogeneity in Nrt's distribution throughout the cell cortex but not in the motor axons exiting the CNS. It was not possible to resolve the expression of Nrt in growth cones because of its widespread accumulation in cell bodies. After completion of neurogenesis, Nrt is down-regulated during the first larval instar. In the PNS of the embryo, Nrt is detected only in polyinnervated sensory organs. In the developing PNS of the pupae, Nrt is expressed by ocellar pioneer neurons but not by neurons of neighboring bristles, which are monoinnervated sensory organs. Nrt is also expressed by differentiating eye photoreceptors and some sensory neurons in the wing (Speicher, 1998)

Mutations in neurotactin have been isolated and characterized. Analysis of both loss and gain of gene function conditions during embryonic and postembryonic development reveals specific requirements for neurotactin during axon outgrowth, fasciculation, and guidance. Nrt requirements were examined in the adult dorsal head, where different neighbor neurons select alternative substrates for axon projection. During early pupal development, ocellar pioneer axons cover the internal side of the prospective head without contacting the epithelium by extending into the extracellular matrix (ECM). After head eversion, the ocellar pioneer nerve becomes displaced to its final position perpendicular to the epidermal surface and the brain. Mechanoreceptor neurons from ocellar and orbital bristles also start extending their axons before head eversion; however, they seem to follow epithelial cues toward their targets in the brain and remain associated with the epidermis once head eversion has taken place. Recognition of these two different substrates (ECM versus epidermis) by the growth cones and the choice to attach or not to the epidermis is vital for both types of axons to project to their correct targets after head eversion. In order to be displaced perpendicular to the epidermis and the brain after head eversion, ocellar pioneer axons must not adhere to the epidermis. Conversely, mechanoreceptor axons should be associated with the epidermis in order to continue following this surface after head eversion. Therefore, ocellar pioneer axons should recognize and be guided by a different subset of cues than mechanoreceptors (Speicher, 1998).

Nrt is expressed by all ocellar pioneer neurons from the onset of axon extension, but it is not expressed by mechanosensory neurons. Mutations in nrt frequently cause defasciculation of the normally tightly associated ocellar pioneer axons. Before head eversion in mutant pupae, this phenotype is predominant, but the penetrance decreases after head eversion, possibly due to the new association of glial cells with the ocellar pioneer nerve and the subsequent enwrapping of the four fascicles by two sheets of glia. Despite this frequent defasciculation phenotype, ocellar pioneer axons usually reach their brain targets. Occasionally, ocellar pioneer axons associate with the epidermis and remain attached to it, sometimes stalling or connecting with the neighboring mechanoreceptor axons and following them for some distance along the epidermal contour. This latter phenotype suggests a role (direct or indirect) for Nrt in ocellar pioneer growth cone guidance. Bristle axons detaching from the epidermal surface or connecting with the ocellar pioneer nerve are never observed in nrt mutants, conditions that would have revealed that the mechanoreceptor axons had chosen their alternative substrate for extension (Speicher, 1998).

Gain-of-function phenotypes produced by ectopic gene expression are another way to reveal function; therefore, the GAL4 system was used to direct altered patterns of Nrt expression in a genetic background devoid of the normal Nrt expression. In pupae of one such gain-of-function mutant, only a small fraction of ocellar pioneer and many mechanoreceptor axons show Nrt accumulation. Under these conditions, the defasciculation phenotype of the ocellar pioneer nerve is not corrected. Nevertheless, in 21 of 42 pupal heads (50%) after head eversion, axons of mechanoreceptors (Ocellar and Postvertical) leave the epidermal surface and associate with the ocellar pioneer nerve. These bristle axons start projection at their normal cell sites but soon after initial extension turn toward the ocellar pioneers and contact them. To improve Nrt expression, nrt expression was driven by the action of the scabrous promoter. Before head eversion, pupae of this gain-of-function mutant show Nrt accumulation in most or all ocellar pioneer axons and in large patches of epithelial cells around the location of ocelli and mechanoreceptors. Three classes of phenotypes are found in these pupae. After head eversion, 12 of 17 pupal heads (70%) are like those just described -- mechanoreceptor axons projecting away from the epidermis in association with the ocellar pioneers. At the same time, 9 of 17 pupal heads (53%) have ocellar pioneer axons projecting into the epidermis that have either stalled or extend for some distance following the mechanoreceptor axons. In 12 of 77 pupal heads (16%) before eversion, there is found an epidermal phenotype in which both sides of the head capsule came closer together due to a fold along the midline of the head. This phenotype correlates with the epithelial expression of Nrt at both sides of the head midline. In this genetic combination, ocellar pioneer axons seem to fasciculate much better than in the nrt mutants; however, the fact that some ocellar pioneer axons attach to the epithelial surface makes inevitable the presence of some splitting in the nerve (Speicher, 1998).

Neurons of the triple row in the anterior margin of the wing, but not sensillum neurons in the third vein, express Nrt in wild type. No obvious phenotype in the axon-extension pattern of wing neurons is found in nrt mutant pupae. However, pupae that ectopically express Nrt in all wing neurons as well as in veins I and III, do show a mutant phenotype. Frequently, vein III sensilla have axons that turn back toward axons of the triple row, creating a loop that prevents both types of axons from leaving the wing in the direction of the CNS. In addition, analysis of the projection patterns of bristle axons of the pupal notum also reveals errors in their extension. For example, axons from Scutellar bristles can erroneously project to the contralateral side or even into the abdomen. These results are consistent with the idea that Nrt has the capacity to provide axons with guidance cues (Speicher, 1998).

Despite its extensive expression in the embryo, Nrt is not essential for fly development. Accordingly, staining of nrt5 embryos with mAb BP102, which marks all CNS axons, does not reveal gross defects. However, a slightly delayed axogenesis, a lack of nerve cord condensation that persists postembryonically, and a mild constriction of the nerve cord at a random location are observed consistently in nrt5 embryos. These defects may be attributed to a reduction in adhesiveness among neural cells. Staining with mAb 1D4 to label Fas II-expressing axons uncovers subtle but distinct defects in Fas II axons, both in homozygous and transheterozygous embryos carrying the alleles nrt1 and nrt5. The defects are observed in 10%-15% of hemisegments, and consist mainly in the stalling or misrouting and subsequent stalling of axons; rarely do misrouted axons cross the midline. These phenotypes are observed in nrt alleles with a different genetic background, indicating a partial requirement for Nrt in axon outgrowth and growth cone guidance in the CNS. They also suggest that nrt1 is a strong hypomorph or even a null allele like nrt5 (Speicher, 1998).

The most consistent axonal phenotype in neurotaxin null individuals is defasciculation of the ocellar pioneer nerve. It is clear, however, that Nrt is not the only molecule implicated in ocellar pioneer nerve fasciculation: although the penetrance of ocellar pioneer axon defasciculation is high, expressivity is far from complete and many axons remain fasciculated. In contrast, a similar defasciculation phenotype is not evident in the CNS of nrt null embryos, perhaps because it is difficult to reveal, as has been shown to be the case with FasII null mutants. The apparent lack of defasciculation phenotypes in the CNS might result from redundant adhesion pathways, which may exist in larger numbers in the CNS than in the ocellar pioneer nerve. It is not unreasonable to speculate that additional constraints might be imposed by the high axonal and cell body density in contrast to the unrestrained navigation of ocellar pioneer axons in the ECM. In nrt null mutants, some ocellar pioneer axons can project in the epidermis, but there they eventually stall. This phenotype suggests either that the epidermis inhibits ocellar axon extension (through an active signaling process or a purely mechanical constraint) or that the molecules involved in the interaction of ocellar pioneer growth cones with the epidermal cell surface are not able to produce the signals necessary to maintain ocellar axon extension. Nevertheless, ectopic expression of Nrt in the epidermal cell surface can produce exactly the same phenotype, indicating that ocellar pioneer growth cones recognize and interact with Nrt in the epidermal cells. This last result strongly suggests that the epidermis inhibits ocellar pioneer axon extension. The molecules involved in this inhibition, however, must not operate over mechanoreceptor growth cones (Speicher, 1998).

The stalling phenotype of CNS axons in nrt null embryos could reflect a role for Nrt in promoting axon outgrowth, or it could be a consequence of an altered selection of substrate as in the case of ocellar pioneers. The misrouting phenotypes also suggest that Nrt may function, directly or indirectly, as a guidance cue. A likely requisite for a molecule to carry out a guidance function is that it ought to be unequally distributed within the neural population. Nrt might partially fulfill this requirement since, despite its broad expression in the CNS, it seems to accumulate unevenly in different neural cells. Likewise, the misrouting of sensory axons under ectopic expression of Nrt might indicate that it can indeed function as a guidance molecule. Nevertheless, pathfinding phenotypes in loss-of-function conditions could also reflect a generic requirement for Nrt for fasciculate in order to stabilize growth and recognition along specific pathways. Selective fasciculation is a mechanism of growth cone guidance experimentally distinguishable from directional guidance. In the case of the ocellar pioneer axon guidance, however, fasciculation is revealed as a mechanism that can subserve directional guidance. Thus, fasciculation mediated by Nrt, as in the ocellar pioneer nerve, could serve as a mechanism to integrate the guidance of individual growth cones by CAMs and related molecules of more restricted expression, in order to generate robust and precise pathfinding toward the correct targets. This could be necessary if individual growth cone guidance is not an accurate process. In the course of such cooperative function, the weakening of adhesion between the ocellar pioneer axons would make them prone to errors such as projecting to an alternative substrate (the epidermis). This could explain why an ocellus has so many pioneer neurons (50). Fasciculation of ocellar pioneer axons can then be understood as a mechanism subserving directional guidance. In this regard, it should be noted that the PNS expression of Nrt is prominent in polyinnervated sensory organs, including ocelli and eye ommatidia neurons, the axons of which grow together toward the CNS (Speicher, 1998).


GENE STRUCTURE

cDNA clone length - 3.5 kb and 4.2 kb

Bases in 5' UTR - 508

Bases in 3' UTR - 487 and 1107. There are two polyadenylation signals and both are used.


PROTEIN STRUCTURE

There are three or more transcripts. The larger RNA species appear to have longer 3' UTRs (de la Escalera, 1990 and Hortsch, 1990).

Amino Acids - 846

Structural Domains

There is an extracellular esterase domain which lacks the serine residue which would indicate that the molecule has esterase activity (Hortsch, 1990). There is one predicted transmembrane domain. The cytoplasmic domain consists of 324 amino acids and is highly hydrophilic, with 40% charged residues (de la Escalera, 1990).

Neurotactin (Nrt), a Drosophila transmembrane glycoprotein that is expressed in neuronal and epithelial tissues during embryonic and larval stages, exhibits heterophilic adhesive properties. The extracellular domain is composed of a catalytically inactive cholinesterase-like domain. A three-dimensional model deduced from the crystal structure of Torpedo acetylcholinesterase (AChE) has been constructed for Nrt. The model suggests that the Nrt extracellular domain is composed of two sub-domains organized around a gorge: an N-terminal region, whose three-dimensional structure is almost identical to that of Torpedo AChE, and a less conserved C-terminal region. By using truncated Nrt molecules and a homotypic cell aggregation assay that involves a soluble ligand activity, it has been possible to show that the adhesive function is localized in the N-terminal region of the extracellular domain comprised between His347 and His482. The C-terminal region of the protein can be removed without impairing Nrt adhesive properties, suggesting that the two sub-domains are structurally independent. Chimeric molecules in which the Nrt cholinesterase-like domain has been replaced by homologous domains from Drosophila AChE, Torpedo AChE or Drosophila glutactin (Glt), share similar adhesive properties. These properties may require the presence of Nrt cytoplasmic and transmembrane domains since authentic Drosophila AChE does not behave as an adhesive molecule when transfected in S2 cells (Darboux, 1996).

Evolutionary Homologs

The extracellular domain is homologous to cholinesterases, including fly Acetylcholine esterase and glutactin and to rat thyroglobulin. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin (de la Escalera, 1990).


REGULATION

Protein Interactions

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 themselves 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).

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).


DEVELOPMENTAL BIOLOGY

Electron microscopy reveals that NRT is found uniformly along the contacts between neurons or epithelial cells. Non-adhesive cultured cells transfected with NRT cDNA do not become self adhesive, but these cells bind to a subpopulation of embryonic cells (approximately 14%), suggesting that NRT is an heterophilic cell adhesion molecule (Barthalay, 1990). The ligand has not been identified.

Embryonic

Neurotactin is expressed at points of interneuronal cell contact, hence its name. NRT first appears in the presumptive mesoderm, anterior midgut and dorsal transverse furrow prior to gastrulation [Image]. This early Neurotactin is found on the apical surface of cells. By germ band shortening, Neurotactin is found in the proliferating ventral nervous system. Later it is restricted to the surface of neuroblasts and their progeny. In visceral mesoderm, non-neuronal expression diminishes, although it is seen on fat body cells and the dorsal vessel. Peripheral nervous system expression is restricted to sensory cells that send out multiple dendritic projections. Staining is stronger on dendrites than on cell bodies. After the CNS is condensed, Neurotactin is expressed in the CNS, fat body, dorsal vessel and the PNS, including the sensory neurons of the head and tail (Hortsch, 1990 and de la Escalera, 1990).

Accumulation of Amalgam 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 thefat 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).

Larval

Larval imaginal neurons express Neurotactin and this persists through the pupal stage. Neurotactin is found associated with chordotonal neurons of the leg disc, and photoreceptor cells plus their axons posterior to the morphogenetic furrow in the eye disc (de la Escalera, 1990).

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

In contrast to the histolyzing larval muscles, the CNS undergoes dramatic differentiation and restructuring during early metamorphosis. The majority of the CNS is composed of adult-specific neurons that reorganize at this time by extending processes and establishing new connections. Several genes known to be involved in neuronal-specific processes are differentially regulated during the late larval ecdysone pulse (see Developmental control genes induced during metamorphosis.) For example, the Drosophila neurotactin and plexin A genes are induced. These genes are involved in axonal pathfinding and in establishing synaptic connections. The neurotactin (nrt) gene product is involved in growth cone guidance and is localized to the cell surface at points of interneuronal cell contact in the presumptive imaginal neurons within the larval CNS. Nerve cord condensation does not occur normally in the late third instar CNS of nrt mutant animals. In prepupae, nrt is expressed in a tissue- and cell type-specific manner: it is restricted to a small set of ocellar pioneer neurons in the brain, photoreceptors of the eye, and some sensory neurons in the developing wing. It is suggested that nrt, like the control genes induced from >18 hours BPF to PF, is regulated by the late larval ecdysone pulse. The plexin A gene belongs to a family of genes that encode Ca2+-dependent homophilic cell adhesion molecules first identified in the vertebrate CNS and PNS. Drosophila Plexin A also acts as a receptor for class I semaphorins, and both loss of function and overexpression experiments demonstrate that Plexin A is involved in axon guidance and repulsion of adjacent neurons (defasciculation). Many neurons defasciculate in response to ecdysone during nervous system remodeling, and it is suggested that an increase in plexin A expression may be partly responsible for this response. Several more differentially expressed neuronal-specific molecules are shown at The Drosophila Microarray Project. These genes provide several new candidates for factors that are involved in the neuronal outgrowth and morphological remodeling responses to ecdysone (White, 1999).

Effects of Mutation or Deletion

Mutants of Neurotactin have embryonic defects in axon patterning within the embryonic CNS (Jimenez, 1993).

The lack of widespread axonal defects in the CNS of neurotactin mutants suggests that the function of Nrt in CNS morphogenesis might be largely replaced by functionally related molecules. If so, embryos lacking Nrt as well as one of these other molecules may display synergistic mutant phenotypes. To test this possibility, embryos lacking function of both nrt and one of several genes encoding neural CAMs were examined. Embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon-1, display phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes (Speicher, 1998).

Neuroglian (Nrg) is a Drosophila neural CAM related to several vertebrate CAMs, though most closely to mouse L1. Two forms of Nrg that differ in their cytoplasmic domains and patterns of expression are known . The long Nrg isoform is neural-specific; it is initially (early stage 12) found in a fraction of CNS neurons, but during stage 13 it can be detected in most (and probably all) differentiating neurons. The short Nrg isoform is expressed by glia, is widely expressed in other tissues, and is probably expressed throughout the entire CNS. nrg1, a loss-of-function mutation for both Nrg forms, is lethal and causes motor neuron pathfinding defects, but the overall CNS structure of mutant embryos looks normal. Furthermore, unlike nrt5 embryos, no defects are detected with mAb 1D4 in nrg1 embryos. In contrast, nrg1; nrt5 double mutant embryos have a severe CNS phenotype. With mAb BP102, thinning or complete interruption of longitudinal connectives, as well as fusion of commissures are observed. Fas II fascicles exhibit similar abnormalities as those observed in nrt5 embryos, albeit with a much higher expressivity and penetrance. Most notably, interruptions of the longitudinal axon bundles are frequent, as are misguidance phenotypes. Double mutant embryos, like single nrt- embryos, also show a local constriction of the ventral nerve cord with a variable expressivity. This defect may be a consequence of the impaired axogenesis and condensation of the nerve cord. No defects outside the CNS are evident in the double mutants (Speicher, 1998).

Using mAb 22C10 (see Futsch), which recognizes a subset of neurons, and mAb 1D4, the behavior of several identified pioneer axons were examined during early stages of axogenesis in nrg1;nrt5 embryos.The pioneer axon of the intersegmental nerve, aCC, as well as the pioneer axon of the segmental nerve, establish their correct pathways. Likewise, the axons of the U neurons follow the aCC pathway correctly. In contrast, in 37 of 128 cases (29%), the axons of the dMP2 and MP1 neurons, pioneers of the MP1 pathway, do not normally defasciculate from the aCC axon and turn to the posterior; instead, they either become and remain stalled or they delay their extension for a considerable time. Other axons showing misguidance phenotypes are those of the six ventral unpaired medial (VUM) neurons. In the wild type, the VUM axons initially fasciculate together before splitting into two fascicles that grow laterally on either side of the midline, passing the RP2 neuron and fasciculating with the corresponding anterior aCC axon. In 19 of 128 (15%) double mutant segments, the fascicle of VUM axons either does not split or splits into more than two fascicles, each joining a different aCC axon, including that of the same hemisegment. The first two axons of the vMP2 pathway, pCC (the pioneer) and vMP2, grow correctly in most hemisegments; only in 4 of 128 cases was a misrouted vMP2 axon observed. Anomalies in the trajectory of the SP1 axon are also observed, though rarely. nrg1; nrt5 embryos also display, due to slight mispositioning of cells, a somewhat irregular appearance of what is normally a highly stereotyped pattern of neurons. However, the relative positions of neurons are maintained (Speicher, 1998).

It seems most likely that the phenotypes of nrg1; nrt5 embryos result from a direct requirement for these two CAMs during axogenesis, and not as a secondary consequence of a previous requirement during neurogenesis. Thus, expression of the nuclear proteins Eve, Ftz, and En, markers of the specification of subsets of neurons that are arranged in characteristic patterns, is found to be normal in nrg1; nrt5 embryos between stages 12 and 16. This suggests that a failure of proper cell fate determination does not cause the axonal mutant phenotype. Likewise, glial cells expressing Repo, a specific marker for most of the CNS glia, form at the correct time and place and in normal numbers in nrg1; nrt5 embryos. The longitudinal glia (LG), which could provide a matrix for longitudinal axon extension, migrate and arrange normally in the double mutant, prefiguring the longitudinal connectives. It is from stage 14 onward, when the LG normally stretch in the anterior-posterior direction and enwrap the connectives, that gaps in the LG begin to appear, overlapping with gaps in the connectives. It is most likely, therefore, that this LG phenotype in late mutant embryos is a consequence, rather than the origin, of the interruptions observed along the axonal connectives (Speicher, 1998).

The drl gene encodes a receptor tyrosine kinase required by a subset of interneurons to make correct axonal pathway choices. Axons expressing Drl project contralaterally across the anterior commissure and then turn to the anterior to form two fascicles: DD and DV. Within the CNS, homozygous drl mutants display partial defasciculation of Drl axon bundles, but their projection patterns are essentially normal. drl;nrt5 double mutant embryos show strong misguidance and stalling phenotypes of Drl axons in many segments. Interestingly, mAbs BP102 and 1D4 in both double mutant combinations show that many axons that do not normally express Drl display defects similar to those exhibited by Drl axons. This reveals a nonautonomous requirement for Drl in these axons. Although at stage 16 Drl and Fas II axon bundles do not appear to contact each other, at mid-stage 13 the extending axon of dMP2, a Fas II-expressing neuron, appears to contact Drl neurons. Nrt expression in differentiating CNS neurons of double drl- nrt- embryos is able to restore the normal projection patterns of both Drl and Fas II fascicles. This result confirms that the double mutant phenotype is produced by the specific lack of Nrt in the CNS during axogenesis (Speicher, 1998).

kek1 is one of two closely related genes that encode transmembrane proteins with structural homology to CAMs and signaling molecules. The gene is expressed in many CNS neurons and midline cells during axogenesis. No major abnormalities are observed in the CNS of kek1 mutant embryos, although partial defasciculation of Fas II axon bundles can be sometimes detected in kek mutant embryos. In kek1; nrt5 double mutants stained with mAb BP102, extension of longitudinal axons through the intercommissural region is frequently affected. A complex phenotype is also detected with mAb 1D4: Fas II axons display defasciculation, stalling, and guidance defects in all double mutant embryos examined. Most notably, Fas II axons frequently cross the midline, a phenomenon rarely observed in nrt5 embryos. These defects are indeed due to the absence of Nrt in differentiating neural cells (Speicher, 1998).

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

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 2120048

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. 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. 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 2100266

Jimenez, F., de la Escalera, S., Martin-Bermudo, M.D. and Gomez, J.M. (1993). Genetic interactions between Neurotactin and other cell adhesion molecules. J. Neurogenet. 8: 234-235. PubMed Citation:

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. 89064560

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. 89028670

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

White, K., et al. (1999). Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179-2184. 10591654

date revised: 10 September 2003 
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.