As early as six hours, the long alternatively spliced form of Neuroglian is expressed on the surface of specific CNS and PNS neurons, and a few PNS support cells. The longitudinal strip of expression found by seven hours in the CNS prefigures the location where the longitudinal axons will form, and appears even before the longitudinal glia have migrated into this same position (Hortsch, 1990).

Between 11 and 12 hours of development, the short alternatively spliced form of Neuroglian is expressed in glia, and a variety of non-neuronal tissues, including trachea, hindgut, salivary gland and muscle.

The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. The differentiation of the LG has been examined in six mutations in which the longitudinal tracts are absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero have no longitudinal tracts, and glial differentiation remains arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia fail to form an oriented scaffold, but cytological differentiation of the LG is unperturbed. The longitudinal tracts in embryos mutant for slit fuse at the VNC midline and scaffold formation is normal, except that it is medially displaced. Longitudinal tracts have intersegmental interruptions in embryos mutant for hindsight and midline. In hindsight, there are intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracts after initial extension. LG morphogenesis during axonogenesis is abnormal in midline. Commitment to glial identity and glial differentiation also occurs before scaffold formation. In all mutants examined, the early distribution of the glycoprotein Neuroglian is perturbed. This is indicative of early alterations in VNC pattern present before LG scaffold formation begins. Therefore, some changes in scaffold formation may reflect changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation precedes longitudinal axon tract formation (Jacobs, 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 kekkon1, 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).

GPCR signaling is required for blood-brain barrier formation in Drosophila

The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. The G protein-coupled receptor (GPCR) Moody, the G protein subunits Gαi and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).

The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established (Schwabe, 2005).

Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).

The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).

To further gauge the dye-penetration assay, embryos mutant for known septate-junction components were examined: Neurexin IV, which is required for blood-nerve barrier formation in the PNS, Neuroglian, and the sodium-pump component Nervana 2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated. In all three mutants, severe penetration of dye was found, well after the nerve cord is sealed in wild-type (22 hr). These findings provide further evidence that the sealing of the nerve cord is achieved by SJs and suggest that the components of the ectodermal SJs are required for the function of surface glial SJs as well (Schwabe, 2005).

In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171: Trapped in endoderm-1) were identified. Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs. Their expression was examined by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology. In the CNS, moody is expressed in surface glia from embryonic stage 13 onward (10 hr); in addition to cells surrounding the nerve cord (subperineurial glia), this includes cells lining the dorsoventral channels (channel glia). moody is also expressed in the ensheathing glia of the PNS (exit and peripheral glia). Both CNS and Peripheral nervous system expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcmN17), confirming that they are indeed glial. tre1 is expressed in all longitudinal glia and a subset of surface glia, as well as in cells along the midline. As expected, the (lateral) glial expression is lost in gcm mutants, while midline expression is not. Both moody and tre1 are also expressed outside the nervous system in a largely mutually exclusive manner, specifically in the germ cells, the gut, and the heart (Schwabe, 2005).

Several additional G protein signaling components are found in the surface glia. The six extant Gα genes show broad and overlapping expression in embryogenesis, with three of them (Go, Gq, and Gs) expressed throughout the nervous system and Gi expressed more specifically in surface glia. Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis. Finally, the RGS loco is uniformly expressed in early embryos due to a maternal contribution but is then transcriptionally upregulated in surface and longitudinal glia, as well as in other tissues outside the nervous system. The nervous-system expression of loco is lost in gcm mutants. The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry, but at 17 hr of development, when staining is feasible, the protein levels are still quite low (Schwabe, 2005).

In sum, the GPCR Moody, the RGS Loco, and Gi are differentially expressed in surface glia. This expression precedes and accompanies the morphogenesis and sealing of the surface glial sheath (Schwabe, 2005).

To examine protein expression and distribution of the GPCR signaling components in greater detail, third-instar larval nerve cords were examined. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwabe, 2005).

Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP. Loco immunostaining is punctate and more dispersed throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody. To avoid fixation and staining artifacts, fluorescent-protein fusions (Moody-mRFP; Loco-GFP) were generated and expressed using moody-Gal4, which drives weak surface glial expression. In the live nerve-cord preparations, Loco-GFP is much less dispersed and shows strong colocalization with Moody-mRFP at the plasma membrane (Schwabe, 2005).

In the absence of a known ligand, the coupling of G proteins to receptors is difficult to establish, but their binding to RGS proteins is readily determined. Loco physically binds to and negatively regulates Gi, and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go. In S2 tissue-culture assays, it was found that Loco binds to Gi and Go, but not to Gs and Gq. Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Schwabe, 2005).

Thus, Loco physically interacts with Gi and Go and shows subcellular colocalization with Moody, suggesting that the four signaling components are part of a common molecular pathway (Schwabe, 2005).

Using dye penetration as the principal assay, whether the GPCR signaling components that are expressed in surface glia play a role in insulation was examined. moody genomic (Δ17; Bainton, 2005) and RNAi mutants show similar, moderate insulation defects. The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moodyΔ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; (Bainton, 2005) are able to rescue the defect independently, as well as in combination. tre1 genomic (Kunwar, 2003) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi. Thus, despite the close sequence similarity of the two GPCRs and their partially overlapping expression in surface glia, only moody plays a significant role in insulation. Overexpression of moody causes intracellular aggregation of the protein (Schwabe, 2005).

loco is expressed both maternally and zygotically. loco zygotic nulls are paralytic and, on the basis of an ultrastructural analysis, a disruption of the glial seal, has been suggested. In a dye-penetration assay, loco zygotic null mutants show a strong insulation defect, which can be rescued by panglial expression of Loco in its wt or GFP-tagged form. The extant null allele of loco13) did not yield germline clones; therefore loco RNAi was used to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe. Overall, insulation as well as locomotor behavior is affected much more severely in loco than in moody and is close in strength to the SJ mutants. Overexpression of loco is phenotypically normal (Schwabe, 2005).

Thus, positive (moody) and negative (loco) regulators of G protein signaling show qualitatively similar defects in loss of function, suggesting that both loss and gain of signal are disruptive to insulation. Such a phenomenon is not uncommon and is generally observed for pathways that generate a localized or graded signal within the cell (Schwabe, 2005).

Both Gi and Go have a maternal as well as a zygotic component. Gi zygotic null flies survive into adulthood but show strong locomotor defects. In Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody, suggesting redundancy among Gα subunits. To further probe Gi function, the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) were overexpressed in glia using repo-Gal4; such overexpression presumably leads to a masking of any local differential in endogenous protein distribution. Expression of Gi-wt results in very severe dye penetration, while overexpression of Gi-GTP is phenotypically normal. Only Gi-wt but not Gi-GTP can complex with Gβγ; overexpression of Gi-wt thus forces Gβγ into the inactive trimeric state. This result therefore suggests that the phenotypically crucial signal is not primarily transduced by activated Gi but rather by free Gβγ. Similar results have been obtained in the analysis of Gi function in asymmetric cell division (Schwabe, 2005).

Go null germline clones do not form eggs and do not survive in imaginal discs, indicating an essential function for cell viability (Katanaev, 2005). Therefore animals with glial overexpression of constitutively active (Go-GTP), constitutively inactive (Go-GDP), and wt (Go-wt) Go (Katanaev, 2005) were examined. Overexpression of Go-GDP, which cannot signal but binds free Gβγ, leads to severe dye penetration, again pointing to a requirement for Gβγ in insulation. However, Go-GTP and Go-wt show a moderate effect, suggesting that signaling by active Go does contribute significantly to insulation, in contrast to active Gi (Schwabe, 2005).

Overall, it was found that all four GPCR signaling components expressed in surface glia are required for insulation, further supporting the notion that the four components are part of a common pathway. The phenotypic data suggest that this pathway is complex: two Gα proteins, Gi and Go, are involved, but with distinct roles: activated Go and Gβγ appear to mediate most of the signaling to downstream effectors, while activated Gi seems to function primarily as a positive regulator of Gβγ. The loss of moody appears much less detrimental than the loss of free Gβγ (through overexpression of Gi-wt or Go-GDP); this is inconsistent with a simple linear pathway and points to additional input upstream or divergent output downstream of the G proteins. Finally, it was consistently observed that both loss (moody, Gi null, and Go-GDP) and gain (loco and Go-GTP) of signal are disruptive to insulation, suggesting that the G protein signal or signals have to be localized within the cell (Schwabe, 2005).

These complexities of G protein signaling in insulation preclude an unambiguous interpretation of genetic-interaction experiments and thus the linking of moody to Gi/Go/loco by genetic means. Double-mutant combinations between moody and loco were generated using genomic mutants as well as RNAi, with very complex results: in moody loco genomic double mutants, the insulation defect is worse than that of loco alone, while in moody loco RNAi double mutants the insulation defect is similar to that of moody alone. This strong suppression of loco by moody is also observed in the survival and motor behavior of the RNAi-treated animals. Thus the phenotype of the double-mutant combination is dependent on the remaining levels of moody and loco, with moody suppressing the loco phenotype when loco elimination is near complete (Schwabe, 2005).

To understand how the GPCR signaling components effect insulation at the cellular level, the distribution of different markers in the surface glia was examined under moody and loco loss-of-function conditions and under glial overexpression of Gi-wt. To rule out cell fating and migration defects, the presence and position of the surface glia were determined using the panglial nuclear marker Repo. In all three mutant situations, the full complement of surface glia is present at the surface of the nerve cord, with the positioning of nuclei slightly more variable than in wt (Schwabe, 2005).

In the three mutants, the SJ marker Nrg-GFP still localizes to the lateral membrane compartment, but the label is of variable intensity and sometimes absent, indicating that the integrity of the normally continuous circumferential SJ belt is compromised. Notably, the size and shape of the surface glia are also very irregular. While qualitatively similar, the phenotypic defects are more severe in loco and under Gi-wt overexpression than in moody, in line with the results of functional assays. When examining the three mutants with the actin marker GFP-Moesin, it was found that the cortical actin cytoskeleton is disrupted in varying degrees, ranging from a thinning to complete absence of marker, comparable to the effects observed with Nrg-GFP. However, GFP-positive fibrous structures are present within the cells, indicating that the abnormalities are largely restricted to the cell cortex. The microtubule organization, as judged by tau-GFP marker expression, appears normal in the mutants. The light-microscopic evaluation thus demonstrates that, in the GPCR signaling mutants, the surface glia are positioned correctly and capable of forming a contiguous epithelial sheet as well as septate junctions. Instead, the defects occur at a finer scale -- abnormally variable cell shapes and sizes, and irregular distribution of cortical actin and SJ material (Schwabe, 2005).

The changes in cell shape and actin distribution that were observed in the three mutants might simply be a secondary consequence of abnormalities in the SJ belt; to test this possibility, how a loss of the SJ affects the morphology and the actin cytoskeleton of the surface glia was examined. SJ components are interdependent for the formation and localization of the septa, and lack of a single component, such as Nrg, leads to nearly complete loss of the junction and severe insulation defects. In Nrg mutants, the surface glial cell shape and cortical actin distribution show only mild abnormalities. Thus, in contrast to the GPCR signaling mutants, the complete removal of the SJ causes only weak cytoskeletal defects, strongly arguing against an indirect effect. It is concluded that GPCR signaling most likely functions by regulating the cortical actin cytoskeleton of the surface glia, which in turn affects the positioning of SJ material along the lateral membrane (Schwabe, 2005).

More detailed insight into the nature of the defects in GPCR signaling mutants is afforded by electron microscopy. The surface glia in nerve cords of first-instar wild-type and mutant larvae were examined. Initially, dye penetration into the nerve cord was tested using ruthenium red. In wild-type, the dye diffuses only superficially into the surface glial layer, while in moody and loco mutants the dye penetrates deep into the nerve cord, in concordance with light-microscopic data. Tissue organization and SJ morphology were examined under regular fixation in randomly selected transverse sections. It has been reported that the surface glial sheath is discontinuous in loco mutant nerve cords, but this analysis was carried out at 16 hr of development, i.e., at a time when, even in wild-type, SJs are not yet established and the nerve cord is not sealed. In contrast to these findings, in the current study it was observed that, in loco as well as moody mutants, the glial sheath is in fact contiguous at the end of embryonic development. The ultrastructure of individual septa and their spacing also appear normal, indicating that moody and loco do not affect septa formation per se. However, the global organization of the junctions within the glial sheath appears perturbed: in wild-type, the surface glia form deep interdigitations, and the SJs are extended, well-organized structures that retain orientation in the same plane over long distances. In moody and loco mutants, the SJs are much less organized; they are significantly shorter in length and do not form long planar extents as in wild-type (Schwabe, 2005).

Taken together, the light- and electron-microscopic evaluations of the GPCR signaling mutants both show defects in the organization of the surface glial epithelium. The reduction in SJ length is consonant with the variability and local disappearance of the Nrg-GFP marker. Since the sealing capacity of the junction is thought to be a function of its length, the reduction in mean SJ length in the mutants provides a compelling explanation for the observed insulation defect (Schwabe, 2005).

Therefore, in addition to a reduction of the insulating SJs, this analysis of the GPCR signaling mutants revealed irregular cell shape and size, as well as weaker and variable accumulation of cortical actin in the surface glia. These data suggest that the primary defect in the mutants lies with a failure to stabilize the cortical actin, whose proper distribution is required for the complex extended morphology of the glia, which then affects SJ formation as a secondary consequence. Several lines of evidence exclude the reverse chain of causality, that is, a primary SJ defect resulting in destabilization of cortical actin and cell-shape change. Surface glia coalesce into a contiguous sheath and show strong accumulation of cortical actin before SJ material accumulates and sealing is completed. In the GPCR signaling mutants, there is misdistribution of SJ material along the cell perimeter, but the junctions do form. Finally, the GPCR signaling mutants show cell-shape and cortical actin defects that are much more severe than those observed in the near complete absence of SJ (Schwabe, 2005).

Compared to the columnar epithelia of the ectoderm and the trachea, the surface glial sheath is very thin. Compensating for their lack in height, surface glia form deep 'tongue-and-groove' interdigitations with their neighbors. This increases the length of the intercellular membrane juxtaposition and thus of the SJ, which ultimately determines the tightness of the seal. It is proposed that the surface glial interdigitations are the principal target of regulation by GPCR signaling. In GPCR signaling mutants, a loss of cortical actin leads to diminished interdigitation and thus to a shortening of the SJ, resulting in greater permeability of the seal. This model integrates all the observations at the light- and electron-microscopic levels (Schwabe, 2005).

Nak regulates localization of clathrin sites in higher-order dendrites to promote local dendrite growth

During development, dendrites arborize in a field several hundred folds of their soma size, a process regulated by intrinsic transcription program and cell adhesion molecule (CAM)-mediated interaction. However, underlying cellular machineries that govern distal higher-order dendrite extension remain largely unknown. This study shows that Numb-associated kinase (Nak), a clathrin adaptor-associated kinase, promotes higher-order dendrite growth through endocytosis. In nak mutants, both the number and length of higher-order dendrites are reduced; these characters phenocopied by disruptions of clathrin-mediated endocytosis. Nak interacts genetically with components of the endocytic pathway, colocalizes with clathrin puncta and is required for dendritic localization of clathrin puncta. More importantly, these Nak-containing clathrin structures preferentially localize to branching points and dendritic tips that are undergoing active growth. Evidence is presented that the Drosophila L1-CAM homolog Neuroglian is a relevant cargo of Nak-dependent internalization, suggesting that localized clathrin-mediated endocytosis of CAMs facilitates the extension of nearby higher-order dendrites (Yang, 2011; see video abstract).

Postmitotic neurons elaborate highly branched, tree-like dendrites that display distinct patterns in accordance with their input reception and integration. Therefore, regulation of dendrite arborization during development is crucial for neuronal function and physiology. Dendrite morphogenesis proceeds in two main phases: lower-order dendrites first pioneer and delineate the receptive field and then higher-order dendrites branch out to fill in gaps between existing ones (Jan, 2010). This process is exemplified by the morphogenesis of Drosophila dendritic arborization (da) neurons, which have a roughly fixed pattern of lower-order dendrites in early larval stages. Higher-order dendrites then branch out to reach the order of more than six, covering the entire epidermal area. These distinct phases of dendrite arborization are manifested by the difference in underlying cytoskeletal composition. While lower-order dendrites are structurally supported by rigid microtubules, higher-order dendrites contain actin and loosely packed microtubules. It is thought that the structural flexibility of higher-order dendrites allows dynamic behaviors like extension, retraction, turning and stalling to explore unfilled areas (Yang, 2011).

The da neurons are classified into four types (I-IV) according to branching pattern and complexity of dendrites. The most complex class IV da neurons have a unique pattern, in which few branches are sent out from proximal dendrites, while dendrites grow extensively in distal regions. Polarized growth of higher-order dendrites requires specialized cellular machineries. For instance, disruption of the ER-to-Golgi transport in class IV ddaC neurons preferentially shreds higher-order dendrites, suggesting that the secretory pathway is needed to sustain membrane addition during dendrite formation. Golgi outposts, hallmark of the satellite secretory pathway in dendrites, move anterogradely and retrogradely during extension and retraction of terminal dendrites, respectively. Arborization in the distal field demands active transport systems mediated by microtubule-based motors, as mutations in dynein light intermediate chain (dlic) or kinesin heavy chain (khc) fail to elaborate branches in the distal region of class IV ddaC neurons. The transport of Rab5-positive endosomes allows branching of distal dendrites, suggesting that the endocytic pathway also has a role in dendrite morphogenesis (Yang, 2011).

The growth of higher-order dendrites seems to require elevated level of endocytosis. Endocytosis is more active in dendrites than in axons in cultured hippocampal neurons. Dynamic assembly and disassembly of clathrin-positive structures, indicative of active endocytosis, are seen at dendritic shafts and tips of young hippocampal neurons. These clathrin-positive structures become stabilized in mature neurons. Endocytosis is known to regulate the polarized distribution of the cell adhesion molecule NgCAM in hippocampal neurons, which is first transported to the somatodendritic membrane and then transcytosed to the axonal surface. Endocytosis is also important for transporting NMDAR to synaptic sites during their formation in dendrites of young cortical neurons. The NMDAR packets transported along microtubules are intermittently exposed to the membrane surface by cycles of exocytosis and endocytosis, at sites coinciding with the clathrin 'hotspots'. Endocytosis can regulate the activities of transmembrane receptors whose signaling activity is important to dendrite growth and maintenance. For instance, the neurotrophin-Trk receptor-mediated signaling that depends on endocytosis could be importantfor dendrite morphogenesis. However, how endocytosis regulates dendrite morphogenesis is not yet clear (Yang, 2011).

Clathrin-mediated endocytosis (CME) is the major route for selectively internalizing extracellular molecules and transmembrane proteins from the plasma membrane. Transmembrane cargos destined for internalization are recruited into clathrin-coated pits through interaction with appropriate clathrin adaptors. One such accessory factor is adaptor protein 2 (AP2), a heterotetrameric complex consisting of a, b, m and s subunits. AP2-dependent cargo recruitment can be regulated by reversible protein phosphorylation by actin-related kinase (Ark) family serine/threonine kinases. In yeast, Ark family genes are known to influence endocytosis by phosphorylating Pan1, an Eps15 homolog, to regulate actin dynamics. Mammals contain two Ark family genes, cyclin G-associated kinase (GAK) and adaptor-associated kinase 1 (AAK1) and both have been implicated in vesicular transport. GAK, best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has multiple functions during clathrin cycle. AAK1 has been shown to bind the a subunit of AP2, phosphorylate the cargo-binding m2 subunit and promote receptor-mediated transferrin uptake. AAK1 also participates in transferrin receptor recycling from the early/sorting endosome in a kinase activity-dependent manner (Yang, 2011).

Numb-associated kinase (Nak), the Drosophila Ark family member, contains the conserved Ark kinase domain and several motifs (DPF, DLL and NPF) mediating interactions with endocytic proteins. To study the function of Nak in development, nak deletion mutants and RNAi lines were generated and it was shown that depletion of nak activity in da neurons disrupts higher-order dendrite development. This function of Nak in dendritic morphogenesis is likely mediated through CME, as Nak exhibits specific genetic interactions with components of CME, colocalizes with clathrin in dendritic puncta and is required for the presence of clathrin puncta in distal higher-order dendrites. More importantly, live-imaging analysis shows that the presence of these clathrin/Nak puncta at basal branching sites correlates with extension of terminal branches. In addition, evidence is presented that the localization of Neuroglian (Nrg) in higher-order dendrites requires Nak, implying that regional internalization of a cell adhesion molecule is crucial for dendrite morphogenesis (Yang, 2011).

This study has shown that disruption of nak during dendrite arborization of da neurons significantly reduces both number and length of dendritic branches. Multiple classes of da neurons were analyzed for the lack of Nak activity, which suggests that its general role in higher-order dendrite morphogenesis. The function of Nak in dendrite arborization is required cell autonomously, as dendritic defects in nak mutants could be rescued by neuron-specific expression of wild-type Nak (Yang, 2011).

Several lines of evidence suggest a functional link between Nak and AP2, the endocytosis-specific clathrin adaptor, in dendrite morphogenesis. First, coimmunoprecipitation results show that Nak predominantly associates with AP2. Second, Nak colocalizea well with GFP-Clc and alpha-adaptin but not with AP1 and AP3 in S2 cells. Third, neuron-specific depletion of AP2 mimics the dendritic defect in nak mutants and reduction of AP2 gene dose enhances nak-induced dendritic defect. These genetic interactions are specific, as mutations in components of AP1 (AP47SAE-10) and AP3 (garnet1) showed no enhancement of nak-associated dendritic phenotypes (Yang, 2011).

Mutations in Nak DPF motifs that are known to interact with alpha-adaptin (DPF-to-AAA), reducing interaction with AP2, render Nak incapable of rescuing the dendritic defects. As AP2 acts to recruit clathrin to endocytic sites, this functional link between Nak and AP2 implies that the dendritic defect in nak mutants is caused by the disruption of Clathrin-mediated endocytosis (CME). Consistent with this notion, mutations in Chc also interact genetically with nak in dendrite morphogenesis and Nak and clathrin are colocalized in dendrites. Thus, it is suggested that Nak functions through CME to promote dendrite development. Being an Ark family kinase implicated in CME, Nak might function similarly to AAK1 that is known to regulate the activities of clathrin adaptor proteins via phosphorylation in cultured mammalian cells). Consistently, it was shown that Nak kinase activity is indispensable for its ability to rescue dendritic defects. Disrupting dynamin activity in shits1-expressing neurons exhibited stronger defects than nak mutants. In addition to endocytosis, dynamin is known to act in the secretory pathway. Given the known role of the secretory pathway in dendrite morphogenesis, it is possible that only endocytic aspect is disrupted in nak mutants, but both secretory and endocytic aspects are affected in shi mutants (Yang, 2011).

Clathrin- and Nak-positive structures in da neurons are preferentially localized to the branching points of higher-order dendrites. Unlike Rab4, Rab5 and Rab11 that are mobile in dendrites, these clathrin/Nak puncta are stationary. Importantly, it was possible to correlate the localization of these stationary clathrin/Nak puncta with motility of local terminal dendrites. The clathrin puncta in higher-order dendrites probably represent sites where populations of clathrin-coated vesicles actively participate in endocytosis. Consistent with this, these clathrin-positive structures are enriched with PI4,5P2, which is known to assemble endocytic factors functioning in the nucleation of clathrin-coated pits. The proximity and tight association between localized endocytic machinery and polarized growth have been described in several systems, including the extension of root hair tips, the budding of yeast cells and the navigation of axonal growth cones. Thus, while the mechanism remains to be determined, the requirement of CME in cellular growth appears conserved (Yang, 2011).

How does regionalized endocytosis contribute to dendrite branching? It is proposed that region-specific internalization and recycling of the cell adhesion molecule Nrg is a mechanism for generating local Nrg concentration optimized for higher-order dendrite morphogenesis. In the advance of mammalian axonal growth cones, adherent L1 can provide the tracking force for growth cone extension. As the growth cone advances, L1 is endocytosed in the central region to release unnecessary adhesion and recycled back to the peripheral region. Similarly, continuously recycling of Nrg along the dendritic membrane may help its delivery to growing dendrites that potentially function in promoting dendrite extension or stabilizing newly formed dendrites. Excessive Nrg in higher-order dendrites as in da neurons overexpressing Nrg may inhibit dendrite arborization by generating superfluous adhesion. Thus, Nak-mediated endocytosis could alleviate this inhibition by internalizing Nrg from the cell surface, allowing dendrite elongation (Yang, 2011).

Arborization of higher-order dendrites in Drosophila da neurons requires branching out new dendrites and elongation of existing ones, which requires two other cellular machineries. First, transporting the branch-promoting Rab5-positive organelles to distal dendrites by the microtubule-based dynein transport system is essential for branching activity. In the absence of Rab5 activity, dendritic branching is largely eliminated and lacking the dynein transport activity limits branching activity to proximal dendrites. Second, the satellite secretory pathway contributes to dendrite growth by mobilizing Golgi outposts to protruding dendrites. Similar to Rab proteins, the Golgi outposts labeled by ManII-GFP were only partially colocalized with YFP-Nak and their dendritic distribution is independent of Nak activity. Also, in lva-RNAi larvae in which the transport of Golgi outposts is disrupted, YFP-Nak puncta were localized normally to distal dendrites. These findings suggest that localization of Golgi outposts in dendrites is not dependent on Nak activity and localization of YFP-Nak is not dependent on transport of Golgi outposts. It is envisioned that arborization of dendrites is achieved by transporting the branch-promoting factors like Rab5 distally via the dynein transport system. Following the initiation of new branches, dendrite extension requires growth-promoting activity provided by the anterograde Golgi outposts and localized clathrin puncta to promote local growth. To actively distribute clathrin puncta in distal dendrites that are far away from the soma, Nak can participate in the condensation of efficient endocytosis into the punctate structures in higher-order dendrites. It is possible that both stationary Nak/clathrin puncta and secretory Golgi outposts are spatially and temporally coupled to promote extension of dendrites, thus coordinating several events like adhesion to the extracellular matrix, membrane addition/extraction, cargo transport, and cytoskeletal reorganization, eventually building up the sensory tree in the target field (Yang, 2011).

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. This assumption was tested in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. These findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions (Bergstralh, 2015).

Previous work demonstrated that metaphase spindles in the cuboidal follicle epithelium are oriented between 0° and 35° relative to the plane of the layer, roughly perpendicular to the apical-basal axis of the cell. Metaphase spindle orientation in this tissue relies on the canonical factors Mud and Pins, and mutants in either gene randomize spindle orientation. Unexpectedly, this study found that the organization of the epithelium is maintained in mud and pins mutants. This is not due to post-metaphase correction of division angles, as vertically oriented spindles persist into telophase in mud mutants (Bergstralh, 2015).

To disrupt spindle orientation more severely, Inscuteable was ectopically expressed in follicle cells. In neuroblasts, this protein recruits Pins and Mud to the apical cortex of neuroblasts so that mitotic spindles are oriented along the apical-basal axis. It has a similar effect on spindle orientation when ectopically expressed in follicle cells. Rather than randomizing spindle orientation as in pins and mud mutants, Inscuteable orients almost all spindles perpendicular to the epithelial plane. Divisions are thus horizontal and produce an apical and a basal daughter. Like spindle randomization, this has no effect on tissue organization. In the neuroblast, spindle orientation controls cell fate by ensuring the asymmetric segregation of fate determinants to one daughter cell. Inscuteable expression in the follicle epithelium does not confer neural cell fate, because it does not cause expression of the transcription factor Deadpan. It was also observed that female flies expressing UAS-Inscuteable under the control of the strong follicle cell driver Traffic Jam-Gal4 are fertile, indicating that reorienting most divisions in the follicular epithelium does not disrupt egg chamber development (Bergstralh, 2015).

In the imaginal wing disc, misoriented cell division is associated with basal cell extrusion and apoptosis. The possibility was therefore considered that the apically misplaced cells produced by horizontal divisions in the follicle cell layer are also eliminated by programmed cell death. However, misplaced cells show neither cleaved caspase-3 immunoreactivity nor pyknosis. Furthermore, expression of the apoptotic inhibitor p35 has no effect on follicular epithelia expressing Inscuteable or containing pinsp62 mutant clones. Live imaging reveals that rather than dying, misplaced daughter cells simply reintegrate back into the epithelial monolayer (Bergstralh, 2015).

The findings prompted a closer examination of mitosis in wild-type follicle cells. These cells divide only during the early stages of egg chamber maturation, switching from mitosis to endocycling at stage 6. Live imaging reveals that the monolayer has an uneven, 'bubbly' appearance in early stages. This is because mitotic cells round up, exhibiting a concomitant increase in cortical phospho-myosin, and often move apically, pulling away from the basement membrane. Daughter cells are frequently born detached from the basement membrane. These cells then reinsert into the monolayer. These results are consistent with the earlier observation that metaphase spindle angles, which determine the angle of division, are not strictly parallel to the plane of the tissue. They also show that in the follicle epithelium reintegration is not only a backup mechanism, but occurs as a normal feature of division. It is speculated that apical movement and angled cell divisions may help to relieve local tension caused by cell expansion and division, which crowds the tightly packed neighbouring cells (Bergstralh, 2015).

Reintegration of newly born epithelial cells has previously been observed in two specific developmental contexts. In mammalian ureteric buds, cells move apically into the lumen to divide and one daughter cell then re-inserts into the epithelium at a distant site. This may contribute to branching. Second, neuroepithelial cells of the zebrafish neural keel normally orient their spindles vertically, and the apical daughter then intercalates into the opposite side of the neural tube in a process that depends on planar cell polarity signalling. In both of these cases, reintegration occurs at a distant site. In contrast, reintegration in the follicle epithelium is always local, and therefore acts to maintain, rather than to alter, epithelial architecture (Bergstralh, 2015).

As local reintegration can be detected only by live imaging, it is possible that it is a general feature of epithelial tissues that has been largely overlooked. To test this possibility, two other types of Drosophila epithelium were examined: the columnar epithelium of the early embryonic ectoderm and the neuroepithelium of the developing optic lobe. It has previously been shown that ectopic expression of Inscuteable reorients spindles in these tissues without affecting tissue integrity. The neuroepithelium is pseudostratified and undergoes interkinetic nuclear migration before division. Expression of Inscuteable in this tissue efficiently reorients divisions, producing one daughter cell that protrudes apically from the layer, as in the follicular epithelium. Live imaging reveals that these apical cells then reintegrate into the epithelium over the next 30min. Inscuteable expression also causes misoriented divisions in the columnar cells of the early embryonic ectoderm, resulting in misplaced daughter cells that lie below, rather than above, the monolayer. Three-dimensional tracking over time shows that these basally misplaced daughter cells can move apically to reintegrate (Bergstralh, 2015).

Reintegration seems to be an active process, because cells undergo a series of shape changes as they reinsert into the monolayer. One possibility is that this is a cell migration process driven by actomyosin constriction at the rear (the apical surface), which squeezes the basal side of the cell back into the epithelium. However, no obvious enrichment of the Myosin Regulatory Light Chain (Spaghetti Squash) or Heavy Chain (Zipper) was observed at the apical surface of reintegrating cells. Myosin is most obviously enriched at the adherens junctions. This correlates with a planar constriction of the reintegrating cell at this level, which would be predicted to hinder rather than help reintegration. Furthermore, reintegrating cells often show a large, transient expansion of their apical free surface, which suggests that the apical membrane is pushed out to accommodate the compression of the basal side of the cell as it squeezes between its neighbours. This behaviour is incompatible with a reintegration mechanism initiated by a contractile force at the rear of the cell, although myosin may play a role in retracting the apical projection during the final stages of reintegration (Bergstralh, 2015).

These observations raise the question of how cells born above or below the monolayer are induced to move in the correct direction to reintegrate. The apical polarity factors aPKC, Bazooka and Crumbs have been observed to disappear from the apical cortex of the follicle cells during mitosis, so it is unlikely that they act as polarity cues for reintegration. Similarly, misplaced cells have no obvious attachment to the basement membrane, and there is no evidence that they form basal stalks, which in any case would be inherited only by the basal daughter of a horizontal division. In Drosophila, cadherin-based adherens junctions localize to the apical side of the lateral membrane, in contrast to mammals where they lie more basally. Cells born apical to the epithelium remain attached to the monolayer by these apical adherens junctions, as revealed by Armadillo (Drosophila β-catenin) staining. In wild-type tissues, both daughter cells inherit part of the apical belt of adherens junctions from the mother cell, whereas the more apical daughter inherits all of the adherens junctions following a horizontal division. Live imaging reveals that the basal cell generates a new junction with its sister and a transient junction that extends along its lateral cortex. Thus, adherens junctions link both apical and basal daughters to cells within the epithelium (Bergstralh, 2015).

To test for a role for adherens junctions in reintegration, the strong hypomorphic allele armadillo3 (previously called armXP33) was used, that encodes a truncated protein and causes intermittent gaps in the epithelium. No misplaced cells or multilayering was observed in armadillo3 clones expressing Inscuteable and no cell death was observed. Reintegration of an armadillo3 mutant cell expressing Inscuteable was also observed directly. These results argue against a major role for adherens junctions in this process (Bergstralh, 2015).

In addition to their apicolateral adherens junctions, follicle cells adhere laterally through functionally redundant homophilic adhesion molecules, such as the IgCAM Neuroglian167 (Nrg167) and the N-Cam-like protein Fasciclin II (Fas2). Both Nrg167 and Fas2 are highly expressed along the length of follicle cell lateral membranes during the first half of oogenesis, when follicle cells are dividing, but their expression is downregulated in post-mitotic stages. This pattern of expression suggests that these proteins are important during division. They are also expressed along lateral membranes in the embryonic epithelium and neuroepithelium. Furthermore, Nrg is localized along the cortex throughout the course of reintegration. In agreement with earlier work, short hairpin RNA (shRNA)-mediated depletion of Nrg167 causes the appearance of occasional follicle cells lying apical to the epithelial monolayer, which is otherwise unperturbed. Apical cells are also observed in mutant clones of Fas2G0336, a P-element allele that behaves as a protein null. Similar phenotypes have been previously attributed to the loss of apical-basal polarity, but the Nrg shRNA and Fas2 mutant cells within the monolayer seem to have normal polarity, as shown by the wild-type distributions of aPKC, Par-6, Bazooka, DE-cadherin, Arm and Dlg. It was therefore reasoned that the apically extruded cells represent failed reintegrations. To test this possibility, the number of cells born above the layer was increased by overexpressing Inscuteable in Nrg knockdown or Fas2 mutant cells. Inscuteable expression increased the mean number of apically positioned cells more than twofold when combined with Nrg shRNA and more than tenfold in Fas2G0336 mutant egg chambers. Live imaging confirmed that cells born apically remain above the epithelium and never reintegrate. Cumulatively, these results show that normal levels of lateral adhesion are required for reintegration (Bergstralh, 2015).

On the basis of these results, it is proposed that tissue surface tension drives reintegration by acting to maximize cell-cell adhesion. As this process is driven by lateral adhesion, it should be able to pull cells back into the monolayer from either side of the epithelium, and this may explain how misplaced cells in the embryonic ectoderm reintegrate from the basal side, whereas follicle and optic lobe cells reintegrate from the apical side. Although these three epithelia reintegrate misplaced cells, this does not seem to be the case in the wing disc epithelium. This difference may arise because lateral adhesion molecules such as Neuroglian are concentrated in apical septate junctions in the wing disc, rather than along the entire lateral membrane as seen in most other mitotic epithelia. These lateral adhesion proteins will therefore segregate into only the apical daughter of a horizontal division in the wing disc, thereby preventing the basal daughter from integrating by maximizing lateral adhesion (Bergstralh, 2015).

Contrary to expectation, spindle misorientation does not disrupt the organization of typical cuboidal, columnar or pseudostratified epithelia in Drosophila. Instead, misplaced cells reintegrate, providing a robust mechanism to protect epithelial monolayers from the consequences of misoriented divisions. Indeed, this mechanism may act more generally to safeguard epithelia against any processes that might disrupt their organization. It will therefore be interesting to investigate whether reintegration also occurs in vertebrate epithelia, where the main lateral adhesion molecule is E-cadherin, and whether a role in reintegration contributes to E-cadherin’s function as a tumour suppressor (Bergstralh, 2015).

Effects of Mutation

Drosophila Neuroglian (Nrg) and its vertebrate homolog L1-CAM are cell-adhesion molecules (CAM) that have been well studied in early developmental processes. Mutations in the human gene result in a broad spectrum of phenotypes (the CRASH-syndrome) that include devastating neurological disorders such as spasticity and mental retardation. Although the role of L1-CAMs in neurite extension and axon pathfinding has been extensively studied, much less is known about their role in synapse formation. A single extracellular missense mutation in nrg849 mutants disrupts the physiological function of a central synapse in Drosophila. The identified giant neuron in nrg849 mutants make a synaptic terminal on the appropriate target, but ultrastructural analysis reveals in the synaptic terminal a dramatic microtubule reduction, which is likely to be the cause for disrupted active zones. The results reveal that tyrosine phosphorylation of the intracellular ankyrin binding motif is reduced in mutants, and cell-autonomous rescue experiments demonstrate the indispensability of this tyrosine in giant-synapse formation. This function in giant-synapse formation is conserved in human L1-CAM but not in either human L1-CAM with a pathological missense mutation or in two isoforms of the paralogs NrCAM and Neurofascin. It is concluded that Nrg has a function in synapse formation by organizing microtubules in the synaptic terminal. This novel synaptic function is conserved in human L1-CAM but is not common to all L1-type proteins. Finally, the findings suggest that some aspects of L1-CAM-related neurological disorders in humans may result from a disruption in synapse formation rather than in axon pathfinding (Godenschwege, 2006).

The function of L1-type proteins in early neuronal development has been intensively studied, but less is known about their contribution to synapse formation. Recently, it has been shown that the L1-type protein Neurofascin (but not L1-CAM) is important in directing GABAergic innervation of the Purkinje axon initial segment and that this involves ankyrin. This study provides evidence that the ankyrin-independent form of Drosophila L1-type protein Nrg has a function in synapse formation and this novel function in synapse formation is conserved in the human L1-CAM protein but is not common to all L1-type isoforms (Godenschwege, 2006).

L1-type proteins are multifunctional proteins with many interaction partners, and the complete loss of function of Nrg protein results in defects in neurite outgrowth and axonal guidance as well as in lethality in Drosophila. In contrast, nrg849 flies carrying a single missense mutation in the second Ig domain are viable, but synapse formation was disrupted in virtually all animals and pathfinding was rarely affected. This suggests that the nrg849 missense mutation does not affect the overall function of Nrg but rather disrupts a subset of functions that is important for giant-synapse formation, but nonessential for the outgrowth or guidance of the GF axon. This implies that the functions of Nrg during axon pathfinding and synapse formation are distinct and separable (Godenschwege, 2006).

Earlier studies revealed that the extracellular domain of Nrg and L1-CAM functioning as a ligand is sufficient for many developmental processes including the stimulation of neurite outgrowth, sensory axon pathfinding, and eye development. For example, expression of NrgGPI is able to rescue certain aspects of pathfinding in a nrg loss-of-function background and is able to activate Echinoid, epidermal growth factor, (EGF), and fibroblast growth factor (FGF) receptor signaling. In addition, NrgGPI has normal homophilic binding properties and can induce ankyrin recruitment when binding to full-length Nrg. More recently, it has been shown that Nrg180, human L1-CAM, rat-NrCAM, human-NCAM, and Drosophila FasII are able to rescue axon pathfinding defects of specific sensory neurons under nrg loss-of-function conditions, suggesting a functional overlap of these proteins in axonal guidance. However, although neuronal Nrg180 and its human homolog L1-CAM were able to rescue the synaptic dysfunction in the nrg849 missense mutants, no functional overlap was found with NrgGPI, rat-NrCAM, NCAM, or FasII. This supports the hypothesis that Nrg function during synapse formation is distinct from its well-known function during neurite extension and axonal guidance and shows that the intracellular domain of Nrg is indispensable for synapse formation (Godenschwege, 2006).

The highly conserved tyrosine of the ankyrin binding site (FIGQY) in the intracellular domain is essential for Nrg function in giant-synapse formation, and the ultrastructural phenotypes suggest that one of its functions may be to stabilize microtubules in the synaptic terminal. L1-type proteins have been shown to generate different microdomains that are either ankyrin free or ankyrin containing, as determined by the phosphorylation status of the tyrosine in the ankyrin binding motif. The homophilic interaction of L1-type proteins in trans or heterophilic interaction in cis induces the recruitment of ankyrin to the unphosphorylated FIGQY, which in turn interacts with the spectrin cytoskeleton. Recently, it has been shown that the spectrin cytoskeleton is important for stabilization of the neuromuscular junction and that the loss of spectrin can induce synapse disassembly and retraction. Interestingly, synaptic boutons in the NMJ lacking spectrin exhibit ultrastructural phenotypes similar to the giant synapse of nrg849 mutants: they are devoid of microtubules, and the synaptic vesicle density is severely reduced. Hence, FIGQY-unphosphorylated Nrg may have an effect on the microtubule cytoskeleton by its connection to the spectrin cytoskeleton via ankyrin (Godenschwege, 2006).

Although a function cannot generally be excluded for ankyrin binding Nrg in giant-synapse formation, a disruption in this pathway seems not to be the primary cause for the phenotypes in nrg849 mutants. Strong evidence is provided that in nrg849 mutants, signaling via ankyrin-independent Nrg is disrupted and this affects giant-synapse formation. In nrg849 flies, tyrosine phosphorylation of the FIGQY motif is reduced. Furthermore, NrgY1234F, which still has residual binding affinity for ankyrin, has no capacity to rescue mutant synapses but rather has a dominant-negative effect on synapse formation when expressed in a wild-type background. Interestingly, Nrg/L1 with a phosphorylated FIGQY motif is found at cell-cell contact sites in the nervous system and does not recruit ankyrin but has been proposed to bind to phospho-FIGQY-specific proteins. In vertebrates, one of these phospho-FIGQY-specific proteins has been identified as doublecortin, a neurogenesis-specific protein that stabilizes microtubules. Hence, it is proposed that the phosphorylated FIGQY Nrg has a function in giant-synapse formation possibly by anchoring of microtubules in the synaptic terminal via a protein similar to doublecortin (Godenschwege, 2006).

Nrg links the plasma membrane to the cytoskeleton, where it organizes and stabilizes the synaptic terminal, and when this function is disrupted, it may lead to the synaptic phenotypes seen in nrg849 animals. Hence, Nrg may be important for the stability of the active zones by providing a scaffolding function at the synapse that affects local signaling. Alternatively, nrg849 synaptic terminals may represent nascent synapses that have never matured because the dramatic cytoskeletal defects in large synaptic terminals may affect retrograde signaling. Finally, it should be noted that Nrg may not only have a scaffolding function that affects signaling important for synapse formation but may also signal itself. For example, vertebrate L1-CAM can activate mitogen-activated protein kinases (MAPKs) and extracellular-signal-regulated kinases 1 and 2, which are known to play a role in synaptic plasticity and memory formation (Godenschwege, 2006).

Unfortunately, the results do not allow unequivocal distinguishing of which interaction on the extracellular side is affected by the mutation in nrg849 flies. Missense mutations of a surface residue in the second Ig domain in L1-type proteins have been shown to affect homophilic interactions, heterophilic interactions, or both simultaneously. The finding that expression of Nrg180 on both sides of the synapse has rescue capacity could be simply due to the fact that heterophilic interaction on both sides of the synapse may contribute to synapse formation. However, because NrgS213L protein has some residual function, it is also possible that homophilic interaction between wild-type and mutant Nrg protein could be the reason for a rescue capacity of Nrg180 on either side of the synapse. Hence, it is suggested that the missense mutation in nrg849 flies probably disrupts both homophilic and a heterophilic interaction during giant-synapse formation and that the combination of these interactions results in signaling via the phosphorylated, ankyrin binding motif that is important for synapse assembly (Godenschwege, 2006).

The finding that vertebrate L1-CAM, but not the tested paralog isoforms of NrCAM and Neurofascin, rescue the synaptic defects in the Drosophila nrg849 mutants suggests that the synaptic function is conserved but is not a feature of all L1-type proteins and therefore is highly specific. A few of the over 140 identified pathological mutations in L1 have been studied in cell culture, and the results suggest that some pathological defects may be the result of disrupted neurite outgrowth, extension, and branching. However, until now, none of the pathological mutations have been characterized with respect to their affect on synapse formation. It is shown that human L1 with a corresponding pathological mutation (L1H210Q) in the second Ig domain is not able not rescue the synaptic defects in the Drosophila nrg849 allele. Hence, it is possible that some of the clinical L1 phenotypes in humans may also be attributed to synaptic defects rather than axonal growth and pathfinding errors. Therefore, future studies of human L1 mutations in the GFS may not only give new insights into the endogenous role of Nrg and L1 in synapse formation but may also help to understand the pathology of L1-related neurological disorders (Godenschwege, 2006).

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. The functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), have been studied during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, these findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM (Kristiansen, 2005).

This study presents an analysis of the requirements and the functional specificity of Drosophila L1- and NCAM-type proteins during the postembryonic development of the Drosophila peripheral sensory nervous system. The partially penetrant phenotypes, which have been reported for L1- and NCAM-LOF mutants in Drosophila and different vertebrate model systems, suggest that the requirement for these neural CAMs is not absolute and that the lack of either L1- or NCAM-type proteins during nervous system development can be partially compensated for by other gene products. Moreover, considering the unique specificity of L1's and NCAM's homo- and heterophilic adhesive interactions, a molecular redundancy between these protein families may be unexpected. The specificities of the homophilic adhesive interactions within the L1 and the NCAM protein families have undergone considerable evolutionary changes. Drosophila Nrg and FasII exhibit a very low cross-reactivity with their vertebrate homologs, L1-CAM and NCAM. Although only the neuronal isoforms of human (L1-CAMRSLE+) and of Drosophila Neuroglian (Nrg180) have been directly tested for their ability to interact with each other, these results indicate that the ability of vertebrate CAMs to rescue the Nrg LOF phenotype most likely relies on homotypic adhesion, rather than on an interaction with endogenous Drosophila CAMs. This conclusion is also supported by the observation that the GOF phenotype in the wing sensory nervous system is only observed when the vertebrate transgene is expressed in both the wing epithelium and the sensory neurons. In addition, endogenous Nrg expression is not required for the production of the GOF axonal misguidance phenotype in the Drosophila wing (Kristiansen, 2005).

Although axonal growth and guidance involve a large array of different neuronal adhesion molecules, there appears to be a limited number of signaling pathways that are shared among structurally different CAM families. The two major signaling pathways, which are triggered by Ig-CAMs, involve nonreceptor tyrosine kinases or receptor tyrosine kinases, such as FGFR and EGFR. Both of these signaling pathways may act synergistically or in a redundant manner. L1-CAM-, NCAM-, as well as N-cadherin-mediated neuronal cell adhesion all activate neuronal FGF receptors and thereby induce neurite outgrowth in vitro. This suggests that structurally different neural CAMs are capable of feeding into the same signaling pathway and that multiple adhesive specificities coordinately influence axonal growth and guidance (Kristiansen, 2005).

Axonal guidance in the Drosophila ocellar sensory system (OSS) and the wing sensory nervous system involves the Nrg-mediated activation of FGF and EGF receptors. Constitutive activation of FGFR or EGFR can rescue the nrg3 LOF phenotype in the OSS, and Nrg GOF axonal misguidance in the developing wing is reversed by a hypomorphic allele of the Drosophila EGF receptor. The two types of neurons in the Drosophila OSS, ocellar pioneer (OP) and bristle mechanosensory (BM) neurons, differ in their expression of Nrg and FasII protein and in their requirement for both proteins during axonal growth and guidance. Whereas the neuron-specific isoforms Nrg180 and FasIIPEST+ are coexpressed in OP axons, BM axons only express Nrg180, but not FasII. The surrounding epidermis, which interacts with BM but not with OP axons, expresses the nonneuronal Nrg167 isoform (Kristiansen, 2005).

The nrg LOF rescue experiments reveal strikingly different requirements for Nrg and FasII protein in the two neuronal cell populations. The requirement for Nrg in OP axons can be sustained by either the neural Nrg180 or FasIIPEST+, but not by the nonneuronal Nrg167 isoform. The two Nrg protein isoforms have identical extracellular domains and only differ in the size of their respective cytoplasmic domain. The capacity of FasII to fulfil the Nrg180 requirement in OP axon guidance suggests that these structurally different proteins share a redundant function in these axons. This conclusion is further supported by the observation that the partially penetrant nrg LOF OP axonal misguidance phenotype is significantly amplified by a reduction of the fasII gene dosage. Remarkably, ectopic FasIIPEST+ expression in BM neurons enhances the deleterious effect of the Nrg loss, a situation that fits within the concept of antiredundancy or opposing functional capacities (Kristiansen, 2005).

The scenario of cell-specific redundant functions of Nrg180 and FasIIPEST+ is maintained by their vertebrate homologs L1-CAM/Nr-CAM and NCAM140, respectively. This indicates that the redundant specificities of L1 and NCAM proteins in neuronal subsets and the corresponding molecular interactions have been conserved in both CAM families over a long evolutionary time period. However, in contrast to the nonneuronal Nrg167 isoform, which exhibits an antiredundant capacity compared with Nrg180, the nonneuronal (RSLE−) vertebrate L1-CAM isoform is able to rescue the Nrg deficiency in OP axons. In contrast to Drosophila Nrg, the two vertebrate L1-CAM isoforms differ by the inclusion or exclusion of two small exons. The insertion of the five additional amino acid residues, which are encoded by exon2, into the L1-CAM extracellular domain modifies the homo- and heterophilic functions of vertebrate L1-CAMs. The inability of the human L1-CAMRSLE+ isoform to efficiently interact with Drosophila Neuroglian suggests that the L1-CAMRSLE+ GOF phenotype is the result of homotypic molecular interactions. Moreover, the rescue of nrg3 OP axonal phenotype by L1-CAMRSLE− occurs in an Nrg deficient background, suggesting that vertebrate L1-CAMRSLE− proteins are able to engage in homotypic molecular interactions in Drosophila. Interestingly, the nonneuronal human L1-CAMRSLE− protein, for which a lower homophilic interaction capacity has been postulated, causes a much weaker GOF phenotype than the neuronal mouse L1-CAMRSLE+ isoform. Nevertheless, the results indicate that this lower homophilic binding activity of the RSLE− isoform is sufficient to support the functional replacement of Nrg180 in OP axons in Drosophila (Kristiansen, 2005).

Inclusion of the cytoplasmic miniexon generates a tyrosine-based endocytosis signal (RSLEY) in the neuronal vetebrate L1-CAM isoform. The AP-2-mediated endocytosis of the neuronal L1-CAMRSLE+ isoform appears to be an important step in the activation of the MAPK signaling cascade by L1-CAM. Since neither Drosophila Nrg isoform contains an equivalent endocytosis signal in their cytoplasmic domain, Drosophila Nrg function either does not involve endocytosis or uses a different type of sorting signal than vertebrate L1 proteins (Kristiansen, 2005).

Although the analysis of the two Nrg isoforms indicates a specific requirement for Nrg180 in OSS neurons, analysis of GOF conditions in the wing peripheral nervous system reveals an underlying common ability to activate RTK signaling. Since the Nrg-mediated activation of EGFR kinase only requires the extracellular Nrg domain for its interaction with the EGFR, both Nrg isoforms are able to exhibit an identical RTK-dependent axonal misguidance GOF phenotype. The ability of homologous vertebrate L1- and NCAM proteins to elicit the same response in Drosophila sensory neurons indicates a common, conserved specificity to influence RTK activity and thereby to regulate axonal growth and guidance. However, the different ability of the neuronal versus the nonneuronal Nrg isoform to rescue the nrg LOF phenotype in the OSS indicates that Nrg-mediated axonal guidance is also regulated by cytoplasmic interactions (Kristiansen, 2005).

Since the separation of arthropods and chordates, there has been an enormous diversification in the size and organization of metazoan nervous systems. At the same time, there has also been an increase in the number of L1- and NCAM-type paralogous genes in vertebrates (but not in Drosophila), as well as structural divergence and acquisition of new specific functions within each protein family. Both types of proteins have conserved an average of 25%–30% amino acid identity between their vertebrate and Drosophila homologues. The two groups of genes are of roughly similar size, and both have undergone independent events that resulted in the generation of different tissue-specific isoforms in Drosophila and vertebrates. Although both the vertebrate and invertebrate proteins are normally coexpressed in specific axonal tracts, their respective realms of expression have shifted in insect versus vertebrate nervous systems. As a result, NCAM expression is more widespread than L1-CAM or Nr-CAM in vertebrates, while FasII is more restricted than Nrg in insects. Therefore, all these genes are evidently highly accessible to mutation and genetic drift, and the current situation most probably reflects a selective pressure to maintain NCAM- and L1-type protein coexpression in specific axonal tracts of the nervous system. Nevertheless, although both L1 and NCAM proteins have acquired many new functions in both arthropod and chordate species, it appears that they initially had at least partially overlapping roles in growth cone signaling during axon guidance. Both CAM families have apparently maintained some of these shared functions and a common specificity, including a basic function as activators of RTK signaling, over a long time period (Kristiansen, 2005).

Therefore, it seems that the functional redundancy between L1- and NCAM-type proteins could constitute an important evolutionary constraint. It prevents the drift of these molecules into completely different functional entities, while at the same time, it allows their structures to further diverge and acquire separate and additional specificities. It has been proposed that functional redundancy is one mechanism for the canalization (stability after developmental perturbation and during evolution) of developmental processes. The requirement for a shared specificity between L1- and NCAM-type proteins in the control of RTK signaling during axon guidance might therefore reflect a requisite for redundancy that is found in any complex communication process. Redundancy is an essential component in any communication process for ensuring reliability by compensating the naturally occurring perturbations. Neuronal wiring is a cell communication-driven process where a highly complex set of signaling systems operates in parallel. As the number of different signals involved in axon guidance enlarged concomitant with an increase in complexity during evolution, the system noise affecting growth cone signal integration during development also increased. Unspecific adhesive interactions may also constitute a major source of noise for navigating growth cones. Therefore, cooperative redundancy might contribute to establishing a “buffered” physiological context required for ensuring process fidelity. It is postulated that this is the reason why the ancestral functional redundancy between L1- and NCAM-type molecules has been conserved over the last 600 million years of evolution (Kristiansen, 2005).

Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila

Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. This study reports the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allow visualization the glial ensheathment of axons. Individual or a groups of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, these data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species (Banerjee, 2006a).

The localization of Nrx IV, Cont, and Nrg in the embryonic nervous system was studied. Nrx IV, Nrg, and Cont show colocalization at the nerve glial membranes. Nrx IV, Cont, and Nrg are interdependent for their epithelial SJ localization. Having established that Nrx IV, Cont, and Nrg colocalize in the peripheral nerves, whether these proteins are interdependent for their localization in the peripheral nerves was studied. The effect of the absence of each of these three proteins on the localization of the other two was studied in the embryonic peripheral nerves using nrx IV, cont, and nrg null mutants. nrx IV mutant embryos were stained with anti-Nrx IV, anti-Cont, and anti-Nrg. Cont and Nrg proteins showed a rather diffused localization when compared with their wild-type localization. In addition, Cont is present as puncta in the cytoplasm of the glial cells because of its failure to get targeted properly to the membrane in the absence of Nrx IV. Similarly, cont null mutant embryos also showed less defined distribution and reduction of Nrx IV and Nrg in the glial membrane. nrg null mutant embryos display significant reduction in Nrx IV and Cont localization in the glial membranes. These results demonstrate that Nrx IV, Cont, and Nrg are interdependent for their proper localization in the embryonic peripheral nerves (Banerjee, 2006a).

Ultrastructural analyses of the embryonic epithelia in nrx IV, cont, and nrg mutants showed that these genes are required for the formation and/or organization of epithelial SJs. Nrx IV, Cont, and Nrg are interdependent for their proper localization both in the epithelia and in the peripheral nerves. These phenotypic similarities raised an interesting possibility that these proteins are part of a macromolecular protein complex that exists in the nervous system. To determine whether these three proteins form a biochemical complex in the nervous system, Drosophila heads, which are a rich source of both neurons and glial cells, were used. Coimmunoprecipitation experiments using Nrx IV, Cont, and Nrg antibodies efficiently coprecipitated Nrx IV, Cont, and Nrg. Interestingly, both isoforms of Nrg (180 kDa neuronal and 167 kDa epithelial) were immunoprecipitated by Nrx IV and Cont antibodies, suggesting that both isoforms are part of a protein complex that includes Nrx IV and Cont. However, at this point, it is not possible to differentiate whether isoform-specific complexes are formed or whether both isoforms are in the same complex. In addition, sucrose density gradient analysis of the fly head lysates was performed to determine how Nrx IV, Cont, and Nrg distribute in buoyant density gradients. Nrx IV and Cont cosediment in overlapping fractions. Nrg shows distribution in the lighter sucrose density fractions that do not overlap with Nrx IV and Cont but partially overlaps with that of Nrx IV and Cont, indicating that these proteins are associated with subcellular structures of the same density and may associate into a biochemical complex that is partially maintained during subcellular fractionation (Banerjee, 2006a).

The immunofluorescence analysis of nrx IV, cont, and nrg null mutants showed that in each of these mutants, the other two proteins show qualitatively reduced fluorescence intensities under identical confocal settings. The possible explanation for reduced fluorescence intensity could be that loss of any of these proteins affects the localization or stability of the other proteins. Whether the levels of the other two proteins had changed in nrx IV, cont, and nrg null mutants was examined using immunoblot analysis. Nrx IV protein levels did not seem to be affected in cont and nrg mutants when compared with wild type. Cont protein levels were severely affected in nrx IV mutants compared with wild-type and nrg mutant embryos. Nrg protein levels were also affected in nrx IV mutants but showed no change in cont mutants. At this stage, it cannot be rule out whether the change in Cont and Nrg protein levels in nrx IV mutants are attributable to reduced stability or less synthesis of these proteins. Together, the biochemical data indicate that Nrx IV, Cont, and Nrg form a protein complex in the nervous system and that, in the absence of Nrx IV, the stability of Cont and Nrg is severely affected (Banerjee, 2006a).

Inner glial membrane processes are involved in the ensheathment of either an individual axon or a group of axons. This glial ensheathment not only provides insulation of axons but also generates unique junctions between either glial membranes or between axons and glial membranes. Nrx IV, Cont, and Nrg are involved in the establishment of glial–glial SJs. Although additional components involved in these interactions need to be identified, these findings provide a basis for additional analysis of neuronal SJs, which would be relevant to the understanding of their vertebrate counterparts: the paranodal axo-glial SJs (Bhat, 2003; Hortsch, 2003; Salzer, 2003; Banerjee, 2006a and references therein).

The fundamental basis of axonal ensheathment in any species is to faithfully transmit neuronal signals along the nerve fibers and optimize desired cellular responses. To maximize the speed of conduction and/or to minimize the loss of nerve signals, many species evolved mechanisms in which axonal lengths remained short (as seen in insects) by increasing the diameter of the axons or by clustering voltage-gated Na+ channels to discrete unmyelinated regions of the axon, the node of Ranvier, as seen in myelinated nerve fibers of vertebrates. Most invertebrate species use some type of glial cells to ensheath their axons without generating a myelin sheath. The insulation is contiguous without any breaks, suggesting that primitive nodal structures or clustering of voltage-gated Na+ channels may not exist in invertebrates. However, recent reports have challenged some of these notions. In copepod crustaceans, ultrastructural analysis of the first antenna and the CNS has revealed extensive myelination of sensory and motor axons. These studies have raised some fundamental questions about axonal insulation and origins of myelination (Banerjee, 2006a and references therein).

In invertebrates like Drosophila, two types of glial cells are involved in insulation. The inner peripheral glial cells are involved in axonal ensheathment, and the outer (perineurial) glial cells wrap around the inner glial cells to provide another level of ensheathment. This two-cell ensheathment in Drosophila peripheral nerves may be advantageous to ensure that high K+ containing hemolymph does not interfere with action potential propagation. Although the cellular aspects of axonal insulation are being unraveled, the molecular mechanisms underlying the axonal ensheathment remain to be investigated. Most importantly, what are the protein constituents of the insulating membranes, and whether some of the vertebrate myelin proteins have their homologs in invertebrates? A detailed molecular analysis of the nature of the glial cells that ensheath axons as in Drosophila or produce myelin-like structures and the type of myelin in copepods may provide insights into whether myelinating glial cells arose from a common ancestor. However, a genetic dissection of the axonal ensheathment in Drosophila will uncover some of the basic aspects of the neuron-glial interactions that lead to ensheathment of nerve fibers across species (Banerjee, 2006a).

Cell adhesion molecules play a pivotal role in establishing intercellular junctions [e.g., cadherins and associated catenins form a protein scaffold that establish adhesion contacts at the adherens junctions and link them to the actin cytoskeleton. Similarly, claudins and associated cellular scaffolding proteins are required for establishing TJs. In both of these examples, transmembrane proteins bring two opposing membranes together to establish junctions through homophilic and/or heterophilic interactions (Banerjee, 2006a and references therein).

The finding that nrg null mutant nerves display increased spacing between the outer and inner glial membranes suggest that Nrg may be involved in cell–cell interactions and cell–cell adhesion between glial membranes. In contrast, the observation that loss of nrx IV and cont does not affect the membrane spacing between the inner and outer glial membranes suggests that Nrx IV and Cont are not involved in membrane adhesion or bringing the glial membranes in close apposition. Together, these data suggest that Nrg is critical for both the adhesion and SJ formation, whereas Nrx IV and Cont are critical for the formation of the septa at SJs. In addition, the missing axonal fascicles in nrg mutant nerves could result from axon fasciculation defects or axonal degeneration as a secondary consequence resulting from the loss of glial support. Axonal fasciculation defects have been observed in nrg mutants. Alternatively, axonal loss in nrg mutants might result from a disruption in axonal cytoskeleton, because Nrg possesses domains that could potentially interact with and stabilize the axonal cytoskeleton. Based on Nrg expression in S2 cells, Nrg is predicted to recruit membrane skeleton assembly within specialized domains of neurons in response to cell adhesion. Both Nrg protein forms Nrg167 and Nrg180 contain a short cytoplasmic domain as a binding site for ankyrin. Ankyrins are linker proteins that connect various membrane proteins with the actin-spectrin network in the cell. Loss of axons observed in nrg mutants is clearly suggestive of axon-glial interdependence that may alter axonal survival. In vertebrates, axolemmal-myelin interactions are critical for the formation of the paranodal axo-glial SJs (see Garcia-Fresco, 2006). This raises an interesting possibility that axon-glial interactions in Drosophila may use similar interactive mechanism for establishing axo-glial SJs. Thus, the current studies on Nrx IV, Cont, and Nrg suggest that these proteins are critical for the formation and/or organization of the SJs between either glial cells and possibly between axons and glial cells, which remains to be further investigated (Banerjee, 2006a).

Invertebrate axons are insulated from their salt-rich environment through a glial-dependent BBB, which plays a crucial role in electrical and chemical insulation. Ultrastructural studies have demonstrated SJs between perineurial glial cells and inner glial cell membrane to form the structural basis of BBB of some insects. Absence of SJs in nrx IV, cont, and nrg mutants, and a compromised BNB in the PNS as evidenced by dye exclusion analyses, provide additional confirmation in support of SJs as a prerequisite for blood-nerve barrier (BNB) formation. Not just in the PNS, recent reports on the BBB formation and function also in the CNS of Drosophila underscore the importance of SJs in proper sealing and insulation of the nerve cord (Schwabe, 2005), thereby supporting that insulation and establishment of functional SJs in both PNS and CNS go hand in hand. Although, G-protein-coupled receptor signaling pathway members have been identified to establish the BBB in Drosophila CNS, the signaling mechanisms that operate in the PNS still remain to be established (Banerjee, 2006a).

The axo-glial SJs in the myelinated axons share many anatomical features similar to those of invertebrate SJs, especially the electron-dense ladder-like transverse septa (Bhat, 2003; Banerjee, 2006a). Ensheathment of Drosophila axons by perineurial glial cells in the absence of myelin-producing glial cells would have predicted that the molecular components of the invertebrate SJs would be different from those of the vertebrate axo-glial SJs. Surprisingly, the fly SJ molecular components are present at the vertebrate axo-glial SJs and not at the TJs, which serve similar functions. Thus, the molecular similarities reflect an evolutionarily conserved function of creating an ionic barrier both at Drosophila SJs and at axo-glial SJs in the paranodal region. More importantly, Drosophila nerves contain a large number of axons, which are collectively held together by glial membranes not only to maintain nerve fasciculation but also to maintain the insulation of individual axons. It would be of significant interest to establish whether axo-glial SJs are present in Drosophila nerves and to identify downstream components of these junctions in Drosophila that link these junctions to the glial or axonal cytoskeleton. Identification of such molecules will provide insights into whether these junctions play a much broader role in axon-glial signal transduction. In summary, the molecular and functional similarities between the Drosophila SJs and vertebrate axo-glial SJs should allow a genetic and molecular dissection to be undertaken of the formation and function of these junctions in Drosophila (Banerjee, 2006a).

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo

Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. The sensory system of the Drosophila embryo was used to shed light on the mechanism by which the L1-type cell adhesion molecule, Neuroglian regulates axon growth. A highly penetrant sensory axon stalling phenotype was found in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild type embryos when advancing along nerves. Driving expression of the wild type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. It is concluded that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensible for this function. It is therefore unlikely that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, it is suggested that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. The finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion (Martin, 2008).

nrg LOF mutants show a highly penetrant sensory axon stalling phenotype. This defect is displayed by all neurons examined, except for the dorsal cluster neuron, dbd, which apparently leads other dorsal cluster axons as it grows along the ISN into the CNS. The fact that this pioneering neuron is unaffected by loss of nrg function may suggest that Nrg is necessary for axon advance only along axonal substrates. This idea is supported by the observation that lateral cluster axons in nrg mutants almost always advance successfully along their initial non-neuronal growth substrate, the spiracular branch of the trachea, and stall only after they have subsequently joined the ISN. As sensory axons in nrg mutants generally advance for some distance along nerves before stalling, Nrg is apparently required to maintain axon growth along axonal substrates, rather than to initiate it. Most axon stalls occur in the periphery, but their locations are quite variable and do not obviously coincide with particular structures, such as nerve branches, pointing to a stochastic component in the stalling process (Martin, 2008).

Stalled sensory axons in nrg mutants are always found in their correct nerve pathways. The absence of axon misprojection phenotypes shows that Nrg is not necessary for recognition of correct growth substrates by sensory axons. It was also observed that sensory axons in nrg mutants fasciculate normally with other axons. The simple growth cone morphology of the stalled axons -- club-shaped with few filopodia -- is similar to that seen in wild-type sensory axons when they are growing along nerves. These observations show that Nrg is not necessary for axon-axon fasciculation in this system. The Ig-CAM Fasciclin2 (Fas2) is a key regulator of motor axon fasciculation in the Drosophila embryo and also acts redundantly to Nrg in the regulation of OP axon growth in the adult OSS. However, Fas2 is unlikely to be acting redundantly with Nrg in regulating sensory axon fasciculation in the embryo, since embryonic sensory axons do not express Fas2. In addition, the pattern of 22C10 sensory nerve staining is normal in fas2 LOF mutants and in embryos over/misexpressing Fas2 in sensory neurons and their growth substrates. Whether Nrg acts redundantly with some other CAM to mediate sensory axon fasciculation remains to be determined (Martin, 2008).

The sensory axon defects seen in nrg mutant embryos are similar to those reported for motor axons, although motor axons stall at a later stage of growth, close to their synaptic targets, the body wall muscles, and unlike sensory axons, some motor axons misproject in nrg mutants. The nrg LOF phenotype in the embryonic sensory system is much more stereotypic than in the adult OSS. This difference may indicate that Nrg performs more diverse functions in the adult OSS, including regulation of axon fasciculation and guidance in addition to axon advance. While Nrg is expressed in both sensory axons and several of their growth substrates, including trachea and motor axons, mutant rescue experiments demonstrate that expression in sensory axons alone is sufficient to mediate their advance. It is unlikely, therefore, that the sensory axon defects are secondary to other known morphological defects in nrg mutants, such as motor axon stalling or failure of glial cell ensheathment of peripheral nerves (Martin, 2008).

An important, unresolved question is whether the effects of Nrg on axon growth are mediated by homophilic interactions between Nrg on the growth cone and its substrate or whether heterophilic interactions are involved. Substrate-bound Nrg has been shown to promote neurite extension from Nrg-expressing neurons in vitro, but it is unclear whether the same holds true in vivo. Expression of Nrg in sensory neurons completely rescues the axon stalling phenotype of lch5-1 seen in nrg mutants. In wild-type embryos and in nrg mutants, lch5-1 is the pioneer for the lateral cluster fascicle and associates with motor axons, rather than dorsal cluster sensory axons as it grows towards the CNS. Lch5-1 mutant rescue findings might suggest, therefore, that Nrg is acting in a heterophilic fashion to promote advance of this axon. This conclusion rests upon the assumption that, in nrg mutant embryos in which the lch5-1 stall phenotype has been rescued, the lch5-1 growth cone does not employ the dorsal sensory axons as a growth substrate as it advances along the ISN. Observations of the dynamics of lch5-1 growth cone activity, and its association with sensory and motor axons in the ISN as it advances along the nerve in these rescued embryos, would help to resolve this question (Martin, 2008).

Mutant rescue experiments show that the intracellular region of Nrg is largely dispensable for its role in promoting sensory axon advance. It has long been known that Nrg-mediated cell-cell adhesion does not require the intracellular region of the molecule. However, mutation of the intracellular ankyrin-binding domain of L1- type CAMs does have clear effects on their membrane mobility and their coupling to retrograde actin flow in growth cones. Given the linkage between ankyrins and the actin-spectrin cytoskeleton, this has led to suggestions that L1-type CAMs may transmit traction force generated by actin flow to the extracellular substrate, as posited by the 'molecular clutch' hypothesis. The recent finding that MAO kinase phosphorylation of the ankyrin-binding site regulates neurite growth from cerebellar granule neurons on a Ng-CAM coated substrate highlights the potential importance of the intracellular region of L1-type CAMs in mediating their effects on axon growth. However, this view is difficult to reconcile with the current results. One possible explanation is that the in vitro models used in most vertebrate studies to date do not accurately reflect the in vivo functions of L1- type CAMs. Alternatively, Nrg, the Drosophila L1 homologue, may function differently to vertebrate L1-type CAMs in promoting axon growth (Martin, 2008).

The finding that the intracellular region of Nrg is not essential for its ability to promote sensory axon advance raises the question of how Nrg-mediated adhesion of the growth cone to its substrate is coupled to retrograde F-actin flow. Current models assume that such a coupling provides the motive force for growth cone advance. One possibility is that Nrg interacts in cis with another receptor or CAM, which in turn is coupled to the cytoskeleton of the growth cone. A genetic screen, using the sensory axon stalling phenotype described in this study as an assay, would be one way of identifying such a Nrg-interacting molecule (Martin, 2008).

Taken together, these results suggest that Nrg may mediate sensory axon advance along nerves in the Drosophila embryo by promoting adhesion between the surface of the growth cone and its axonal substrates. This adhesive interaction is likely to involve a heterophilic interaction between Nrg on the growth cone and some other, as yet unidentified molecule, on the substrate. Intracellular signaling by the Nrg molecule does not appear to be essential for its function in this context. These findings for Nrg are mirrored by the recent discovery that the adhesive, rather than the signaling activity, of another CAM, Drosophila N-cadherin, is essential for its role in target selection of photoreceptor afferents (Martin, 2008).

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. This study describes the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. A novel role was uncovered for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1 (Goossens, 2011).

This study demonstrates a requirement for Neuroglian signaling in different steps of mushroom body (MB) axonogenesis, namely (1) axonal projection into the peduncle, and (2) branching, outgrowth and guidance of axonal sister branches. The two steps in mushroom body axonogenesis are genetically separable and seem to involve distinct NRG signaling complexes (Goossens, 2011).

In peduncle formation, NRG signaling does not rely on the NRG-180-specific intracellular domain, but on the extracellular domain and the part of the cytoplasmic domain common to both NRG isoforms. The extracellular domain contributes intercellular adhesive properties, necessary for axon fasciculation into a peduncle. This conclusion is supported by the defective adhesive properties of the NRG849 mutant protein in cell aggregation assays, and by the fact that Nrg849 hemizygotes frequently lack the peduncle. Interaxonal fasciculation in the peduncle probably involves binding to and stabilization by the actin cytoskeleton network via the ankyrin-binding domain shared by the two NRG isoforms. This conclusion is supported by previous aggregation experiments in Drosophila S2 cells in which it was shown that homophilic binding of NRG leads to recruitment of ankyrin to the contact sites and by the observation that RNAi-mediated knockdown of neuron-specific ank2-RNA results in MB phenotypes similar to those seen in Nrg mutants (Goossens, 2011).

MB lobe development, on the other hand, requires the NRG-180-specific intracellular fragment. This study showed that PYD acts downstream of NRG-180 during the formation of α and γ lobes. Consistent with this, axon stalling defects (i.e. lack of peduncle formation) were never observed in pyd mutants, whereas defects were observed in lobe outgrowth, branching and guidance. Furthermore, the neuron-specific NRG-180 isoform can bind directly to the first PDZ-domain of this MAGUK protein. The observation that NRG and PYD interact to mediate proper sister neurite projections defines a novel role for the ZO-1 homolog PYD in axonogenesis. Thus far, the best-known role of MAGUKs in the nervous system has been in synapse development and function, as is the case for one of the prototypic MAGUKs, Drosophila Discs Large 1 (Dlg1), whereas PYD is known as a component of adherens junctions (Goossens, 2011).

Sema-1a, trio and Rac1 were also found to be a part of the genetic network that interacts with Nrg. The observation that heterozygosity for mutations in Sema-1a and trio both suppress NRG-180 overexpression induced MB phenotypes indicates that Sema-1a and trio are genetically downstream of Nrg and possibly in the same pathway. By contrast, the introduction of a Rac1 mutation in a Nrg gain- or loss-of-function background results in both cases in enhancement of MB phenotypes. This argues against a one-to-one signaling model between NRG and RAC1, in which RAC1 acts only downstream of NRG-180. Consistent with the genetic data, no direct physical interaction could be detected between NRG-180 and RAC1, but preliminary co-immunoprecipitation data suggest that NRG-180 can bind to TRIO. Further experiments will be necessary to assess whether this binding also occurs in vivo, and whether it is instrumental for NRG-180-dependent modulation of RAC1 signaling (Goossens, 2011).

Contrary to the observed genetic interaction between Nrg and Sema-1a, this study found no evidence for interaction between Nrg and two genes that code for well-characterized Semaphorin receptors, plexin A and plexin B. This is an unexpected observation in light of the fact that Sema-1a and plexin A and plexin B interact during mushroom body development. Therefore, this suggests that during mushroom body development Sema-1a acts both in a plexin-dependent and a plexin-independent way. A plexin-independent role in axon outgrowth has previously been described for vertebrate Sema7a (Goossens, 2011).

The distinct requirement for NRG in peduncle and lobe formation is reminiscent of what has been shown for DSCAM. This protein has an early and essential role for selective fasciculation of young axons in the peduncle and is subsequently required for bifurcation and branch segregation. In light of this, it is interesting to note that the different cell-adhesion molecules that have been implicated in MB development have a different MB expression pattern or temporal requirement for MB development. NRG is expressed in the MBs throughout its entire development, but no essential function was found for Neuroglian in larval MB development. By contrast, DSCAM expression disappears with fiber maturation and mutants have larval MB phenotypes. Likewise, Fas2 mutants display larval lobe defects, whereas no lobe defects were found in adult mutants. Taken together, these observations suggest that different steps in MB axonogenesis depend on combinations not only of isoforms of the same cell-adhesion molecule (e.g. DSCAM) but also of different cell surface molecules (e.g. NRG, FAS2 and SEMA-1A). Jointly, the cell-surface molecule complement of any given axon combined with guidance signals will then control the signaling required for proper neural circuit formation in the MBs (Goossens, 2011).

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. Previous work has shown that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche that are directly attached to GSCs through E-cadherin) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. This study has further dissected the molecular and cellular mechanisms underlying these processes. Insulin and Notch ligands were shown to directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. It is also reported that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, it was also found that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function (Hsu, 2011).

The Notch pathway plays a central role in many stem cell systems, and how systemic signals impact Notch signaling in stem cell niches is a question of wide relevance to stem cell biology. Notch controls cap cell number in the Drosophila female GSC niche, and recent studies showed that insulin-like peptides control Notch signaling in the niche (Hsu, 2009), although the underlying cellular mechanisms remained unclear. This study dissected the specific cellular requirements for Notch pathway components and the insulin receptor and reveals that insulin signaling controls cell–cell communication via Notch signaling within the niche (Hsu, 2011).

To summarize, from this study in combination with previous work, a fairly complex model emerges of how insulin-like peptides -- systemic signals influenced by diet -- impact the function of GSCs and their niche through multiple mechanisms. In adult females under favorable nutritional conditions, insulin-like peptides signal directly to GSCs via PI3K to inhibit FOXO and thereby increase their division rates by promoting progression through G2. In parallel to this direct effect on GSC proliferation, insulin-like peptides also act directly on cap cells (a major cellular component of the GSC niche) to control two separate processes. Stimulation of the insulin pathway, also via PI3K inhibition of FOXO, within cap cells intrinsically increase their responsiveness to the Notch ligand Delta (likely at a step upstream of nuclear translocation of the intracellular domain of Notch), which is likely produced by neighboring cap cells. (A similar process likely occurs during niche formation in larval/pupal stages, although in this case, Delta produced in basal terminal filament cells clearly contributes to the specification of cap cells.) Notch signaling within cap cells leads to their maintenance and, indirectly, to GSC maintenance. Independently of its effect on Notch signaling, insulin/PI3K/FOXO pathway activation in cap cells intrinsically promotes stronger cap cell-GSC adhesion (presumably via E-cadherin; Hsu, 2009), which also promotes GSC maintenance. Further, aging also appears to influence insulin signaling levels in Drosophila females (Hsu, 2009), suggesting that physiological changes caused by diverse factors can impinge on this GSC regulatory network. Together, these studies underscore the importance of investigating how whole organismal physiology impacts stem cell function via effects on stem cells and on their niche, potentially via changes in local signaling (Hsu, 2011).

Notch signaling requires direct cell-cell contact because Notch ligands are membrane-bound proteins that induce Notch activation in neighboring cells. In addition to transactivating Notch in adjacent cells, the Notch ligand Delta also inhibits Notch in cis, thus creating a potent switch between high Delta expression/low Notch activity and high Notch activity/low Delta expression (Sprinzak, 2010). Differential Notch activation often underlies binary cell fate decisions. For example, during Drosophila sensory organ development, cells with high levels of Delta and low Notch activity become neurons, while those with elevated Notch activity and low Delta become epidermal cells (Hsu, 2011).

In the Drosophila GSC niche, Notch activity is detected in all cap cells, and Dl-lacZ is expressed in all terminal filament cells. A subset of cap cells also expresses Dl-lacZ, suggesting that some cap cells may express Delta and have high Notch activity simultaneously. The basal terminal filament cell, in which Dl is required for cap cell formation, does not contact all cap cells directly, and it was also found that Dl and Ser are not required within GSCs for cap cell formation or maintenance. It is therefore proposed that cap cells may signal to each other via Delta to activate Notch signaling, and that, in cap cells, Delta might not consistently act in cis to inhibit Notch activation (Hsu, 2011).

The observation that a subset of cap cells can express Dl-lacZ and Notch activity simultaneously is consistent with recent findings. Human eosinophils express both Notch and its ligands, and autocrine Notch signaling controls their migration and survival (Radke, 2009). Similarly, Notch is co-expressed with its ligands in rat hepatocytes following partial hepatectomy and also in normal human breast cells, although it is unclear if autocrine signaling occurs. It is therefore conceivable that Delta expressed in cap cells may stimulate Notch signaling via both paracrine and autocrine manners (Hsu, 2011).

Alternatively, Notch ligands might be secreted from terminal filament cells to stimulate Notch signaling in all cap cells and thereby promote their maintenance. In fact, a soluble form of Delta capable of stimulating Notch has been identified in Drosophila S2 cell cultures, and the ADAM disintegrin metalloprotease Kusbanian is required for the production of soluble Delta in culture. Further, Dl and kuzbanian genetically interact, raising the possibility that soluble forms of ligands might modulate Notch signaling in vivo (Hsu, 2011).

neur encodes an E3 ubiquitin ligase that mediates the endocytosis of Notch ligands in signal-sending cells, thereby enhancing their signaling strength. Contrary to a previous report, this study found no evidence that Notch ligands produced from GSCs are required for self-renewal. In contrast, neur is intrinsically required for GSC maintenance. Similarly, in the Drosophila testis, neur, but not Dl and Ser, is required for GSC maintenance, further indicating that Neuralized maintains GSCs via a Notch-independent pathway (Hsu, 2011).

neur mutant cysts exhibit large and highly branched fusomes, another Notch-independent phenotype. In principle, this aberrant fusome morphology might result from a defect in fusome growth and/or partitioning, or be secondary to an excessive number of cyst division rounds. Nevertheless, the close association of some of these abnormal fusomes with the cap cell interface suggests that fusome defects might lead to GSC loss. Ubiquitination regulates many processes, including protein degradation and vesicular trafficking. It is therefore possible that Neuralized ubiquitinates specific substrates that regulate fusome-related vesicular trafficking during cyst division. Future studies should test whether E3 ligase activity is indeed required for the role of neur in early germline cysts, identify key ubiquitination targets, and elucidate the molecular mechanisms they regulate (Hsu, 2011).

Under low insulin signaling, the FOXO transcriptional factor is required for extended longevity, reduced rates of proliferation, and stress resistance, among other processes. FOXOs are conserved from yeast to humans, and they control many target genes, different subsets of which modulate distinct processes. Drosophila FOXO negatively controls GSC division when insulin signaling is low (Hsu, 2008). It was also shown that insulin signaling modulates niche-stem cell interactions and Notch signaling in the niche (to control cap cell number), and that insulin signaling declines as females become older, leading to stem cell loss (Hsu, 2009). This study has shown that FOXO is required to negatively regulate Notch signaling within cap cells under low insulin activity and that FOXO also modulates the physical interaction between cap cells and GSCs. The multiplicity of FOXO roles in stem cell regulation is further underscored by studies in other stem cell systems. For example, FOXOs regulate several processes, including cell cycle progression, oxidative stress, and apoptosis, in the hematopoietic stem cell compartment, thereby influencing stem cell number and activity. It will be important to investigate how the specificity of FOXO is controlled and also whether or not FOXO regulates other stem cell niches, perhaps acting as a mediator of changes in niche size and/or activity during aging or cancer development (Hsu, 2011).

This study suggests a potentially novel mechanism by which the Notch and insulin pathways interact. In the Drosophila female GSC niche, insulin signaling does not control ligand transcription, and it is not required for ligand function (i.e., Dl is required in basal terminal filament cells during cap cell formation, but InR is not). Instead, both InR and N are cell autonomously required for cap cell maintenance, and insulin receptor function (via repression of FOXO) is required for proper Notch signaling. Expression of the intracellular domain of Notch rescues the low cap cell and GSC numbers of InR mutants (Hsu, 2009), and ovarian Notch expression does not appear altered in InR mutants. Therefore, it is speculated that FOXO inhibits the ability of cap cells to respond to Notch ligands by regulating a target that negatively regulates the series of proteolytic events responsible for the release of the intracellular domain of Notch. It cannot, however, be rulef out that Notch and FOXO normally interact at the level of target gene regulation but that overexpression of the intracellular domain of Notch overrides the normal inhibition by FOXO (Hsu, 2011).

These findings contrast with other types of interactions between FOXO and Notch that have been reported. During muscle differentiation in myoblast cultures, FOXO promotes (instead of antagonizing) Notch activity via a physical interaction that leads to activation of Notch target genes. Positive interactions between Notch and PI3K signaling have also been reported. Specifically, activation of the PI3K pathway potentiates Notch-dependent responses in CHO cells, T-cells, and hippocampal neurons. The suggested mechanism, however, involves the inactivation of GSK3 by Akt phosphorylation upstream of FOXO, which is distinct from the involvement of FOXO in the insulin-Notch signaling interaction within the GSC niche. These examples illustrate the diversity of modes of interaction between Notch and insulin signaling. It is conceivable that the positive interaction that is describe between insulin and Notch signaling pathways in the GSC niche may occur in other stem cell niches (Hsu, 2011).

Deregulated Notch signaling is associated with many types of cancers and, in some cases, it is thought that altered Notch signaling promotes cancer development by overstimulating the self-renewal of normal stem cells (Wang, 2009). Hyperactivation of insulin/IGF pathway is also linked to increased cancer risk and poor cancer prognosis. The Notch and insulin/IGF pathways have been reported to interact in cancerous cells via yet another mechanism. Specifically, upregulation of the Notch ligand Jagged 1 leads to PI3K activation in human papillomavirus-induced cancer lines. It is speculated that additional types of interactions between Notch and insulin/IGF signaling, such as the positive regulation of Notch activity by the insulin/PI3K/FOXO pathway that occurs in the Drosophila GSC niche, may also contribute to cancer progression (Hsu, 2011).

Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. Previous studies have shown that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. This study has used the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM) binding site to investigate the signaling requirements for neuronal development. Different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis (Kudumala, 2013).

In vertebrates, L1CAM is proteolytically cleaved by various enzymes, such as neuropsin, plasmin, ADAM10/17 and a yet unidentified serine protease. This study found that expression of L1CAM in the Drosophila nervous system resulted in proteolytic cleavage to a 65 kDa fragment detected by antibodies directed against the intracellular L1CAM domain. It is important to note that this proteolytic cleavage was not seen in larvae but in adults, suggesting that this cleavage is dependent on the differentiation status of the nervous syste. The overall ratio of cleaved L1CAM to full-length L1CAM increased from pupa to adult and the cleavage rate remained constant when comparing adults of different ages. However, it remains to be determined if the cleavage in Drosophila occurs at a site homologous to those that have been described in vertebrates and if this particular cleavage is of functional relevance. Although, proteolytic cleavage of Nrg has not yet been described, this study finds evidence that it does get cleaved. Transgenically expressed L1CAM proteins with L120V, Y1070C, H210Q, E309K and R184Q mutations were proteolytically cleaved and had similar or higher expression levels as the wild-type L1CAM-construct. This allowed for a functional analysis of the pathogenic L1CAM mutant proteins in terms of their ability to support axonal guidance and synapse formation in Drosophila mutants (Kudumala, 2013).

The overall L1-C264Y expression was reduced comparatively but in Western blots no cleavage was observed for L1-C264Y protein even with ten heads. This is similar to L1-C264Y expression in the vertebrates. In vitro the C264Y mutation leads to reduced cell surface expression of the mutant protein, while in vivo L1-C264Y protein seems to be absent or below detection threshold at the cell surface. Therefore, the absence of L1-C264Y cleavage in the vertebrate nervous system was associated with its lack of cell surface expression. However, expression of L1-C264Y in Drosophila was able to fully rescue the synaptic defects of half of the GF-TTMn connections in nrg14;P[nrg180ΔFIGQY] animals suggesting that L1-C264Y protein was expressed at the cell surface. Correlating with the electrophysiological data, it was found that transgenically expressed L1-C264Y protein was present in numerous though not all synaptic terminals at the cell surface as well as in vesicular clusters inside the terminal. Therefore, the results suggest that in the Drosophila GF the pathogenic C264Y mutation not only affects proteolytic processing indirectly by reducing cell surface expression but may also affect it directly in L1-C264Y protein that is expressed at the cell surface (Kudumala, 2013).

Homophilic L1-L1 interactions mediated by the extracellular Ig-domains are involved in various L1-dependent signaling processes required for axonal growth, guidance and synapse stability. The S213L mutation in the nrg849 allele is at a site analogous to the position of the H210Q mutation in humans and both mutations affect homophilic L1 binding. However, although L1CAM-H210Q proteins are unable to interact homophilically with each other, they can efficiently bind to wild-type L1CAM. This study has shown that Neuroglian is required pre- and postsynaptically for GF synapse formation and that expression of Nrg180 on either side of nrg849 mutant synapses can partially rescue the synaptic phenotypes. This suggests that Nrg-S213L similar to L1-H210Q is able to bind to a wild-type extracellular domain (Kudumala, 2013).

This study tested whether the expression of different pathological mutations can compensate for the loss of homophilic interaction in nrg849 mutants. Expression of wild-type human L1CAM, as well as L1-L120V and L1-E309K, both of which mediate normal homophilic binding, was able to efficiently rescue the guidance defects of nrg849 mutants. However, previously it was shown that expression of L1-E309K in a null mutant background did not rescue the guidance defects of bristle mechanosensory (BM) neurons. This suggests that either the functional Nrg/L1CAM requirements for guidance in BM and GF neurons are different or that homophilic interaction of L1-E309K protein with Nrg849 protein induces signaling that is sufficient for GF guidance (Kudumala, 2013).

In contrast, several L1-CAM constructs failed to rescue the guidance phenotypes. This may be due to one or a combination of three most likely causes. First, L1CAM missense mutations that severely reduce or prevent cell surface expression of the mutant protein are likely to affect the rescue capacity, e.g., the C264Y mutation. Secondly, L1CAM missense mutations that affect homophilic binding are unable to fully complement for the lack of homophilic binding of the Nrg-S213L protein. This appears to be the case for the H210Q, C264Y and R184Q mutations. Finally, it is conceivable that some pathological missense mutations with normal homophilic binding have impaired outside-in signaling or heterophilic interactions that are required for axon guidance. These mutant proteins are also unlikely to rescue the nrg849 guidance phenotype. Although the L1-Y1070C construct showed a strong improvement in restoring nrg849 guidance defect, despite its normal homophilic binding capacity, it did not reach statistical significance. However, this mutation has been shown to reduce Epidermal Growth Factor Receptor (EGFR) signaling critical for axon growth in Drosophila. In addition, it was found that expression of L1-4A failed to rescue the nrg849 guidance phenotypes. The ERM-binding site has been shown to be critical for axon growth and branching in vertebrates, suggesting it is also required for axon guidance in Drosophila. (Kudumala, 2013).

While many pathological L1CAM mutations have been studied for their effects on neurite outgrowth, axon guidance and branching, virtually no information is available with respect to their impact on synapse development. Previously studies have demonstrated that intracellular signaling of the Nrg180 FIGQY motif is essential for synapse formation of the GF, but not for neurite outgrowth or axon guidance (Enneking, 2013). These published results demonstrate that expression of wild-type Nrg180 on either side of NrgΔFIGQY synaptic terminals is able to rescue the morphological and functional defects. This finding suggests that transcellular Nrg180 signaling can superimpose the molecular information to the other synaptic side, which lacks Nrg-FIGQY signaling. However, it remains to be determined if this transcellular interaction is homophilic, heterophilic or both (Kudumala, 2013).

The finding that human L1CAM mutations like L1-L120V, L1-E309K, L1-4A and partially L1-C264Y were able to rescue the synaptic defects of Drosophila nrg14;P[nrg180ΔFIGQY] mutant animals suggests that they do not affect outside-in signaling via the FIGQY motif. However, the results do not exclude a synaptic function via other signaling mechanisms that are independent of the FIGQY motif and are unaffected in the Nrg180ΔFIGQY protein. Several L1CAM mutations were identified that had expression levels comparable to L1CAM wild-type control flies, but did not rescue the synaptic phenotype. Although L1-H210Q protein is able to bind to wild-type L1CAM, both human L1CAM mutations with disrupted homophilic binding (L1-H210Q and L1-R184Q) were unable to rescue the synaptic defects when expressed pre- and postsynaptically in the GF circuit of nrg14;P[nrg180ΔFIGQY] mutant flies. This suggests that L1-H210Q and L1-R184Q may not interact transcellularly with NrgΔFIGQY and homophilic interactions are also essential for synapse formation. Alternatively, L1-H210Q and L1-R184Q may be able to interact transcellularly with NrgΔFIGQY, which has a wild-type extracellular domain. However, in this scenario the H210Q and R184Q mutations disrupt outside-in signaling processes via the FIGQY motif. The latter hypothesis is supported by the finding that the nrg849 mutants with the analogous L1-H210Q mutation have reduced tyrosine phosphorylation of the FIGQY motif and synaptic phenotypes similar to nrg14;P[nrg180ΔFIGQY] mutants in addition to guidance defects. Finally, it was found that L1-Y1070C mutant protein failed to rescue the synaptic defects of nrg14;P[nrg180ΔFIGQY] mutant flies although its homophilic interactions are not affected by the mutation. Both, Y1070C and E309K mutations have been shown to reduce EGFR signaling to a similar extent. However, because the L1-Y1070C transgenic expression level was higher than the expression of L1-E309K, the lack of rescue capacity of the L1-Y1070C protein is unlikely to be due to reduced EGFR signaling. This suggests that the Y1070C mutation either affects FIGQY phosphorylation or the localization of phosphorylated and non-phosphorylated L1-type protein at the cell surface. Phosphorylated and non-phosphorylated L1-type proteins have been shown to localize to distinct areas and interestingly, heterophilic binding to Tag-1/Axonin-1 and contactin/F11 are increased for the L1-Y1070C mutant protein (Kudumala, 2013).

In summary, this study has found that extracellular human pathogenic L1CAM missense mutations not only affect adhesive properties but also intracellular signaling pathways distinctly, which are required for axon guidance, synapse formation or both. Mutations that affect homophilic binding are most detrimental because they affect adhesive properties but often also heterophilic interactions and intracellular signaling. In contrast, intracellular mutations as well as extracellular mutations that only affect heterophilic interactions are more likely to only result in a partial loss of L1CAM biological function, which is also reflected by the fact that pathogenic intracellular missense mutations are known to result in less detrimental pathological phenotypes. Therefore, these types of mutations may affect different biological processes such as guidance and synapse formation distinctively and their characterization in vivo is essential in order to gain a complete understanding of L1CAM function. This study found that outside-in signaling via the ERM-motif and via the FIGQY motif are required for GF guidance and synapse formation in Drosophila, respectively. Novel evidence was provided that the H210Q, R184Q and Y1070C but not the L120V and E309K L1CAM mutations affect outside-in signaling via the Ankyrin binding domain, which is essential for synapse formation but not for axon guidance. Thus, the broad variability of pathological phenotypes observed between humans with L1CAM mutations is based on the differential effects on distinct signaling pathways required for developmental biological processes (Kudumala, 2013).


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Neuroglian: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology

date revised: 30 September 2016

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