Fasciclin 2

DEVELOPMENTAL BIOLOGY

Embryonic

In stage 12 embryos Fas2 is expressed in very specific neurons of the CNS. By stage 16, the entire MPI pathway expresses Fas2, as well as several other longitudinal pathways. On the periphery, several motorneuron growth cones in each segment express Fas2. Protein is also found in the ectoderm where spiracles are formed, in Malpighian tubules, along a short length of the midgut, and in patches of ectoderm in the head (Grenningloh, 1991).

Although pioneer neurons are the first to delineate axon pathways, it is uncertain whether they have unique pathfinding abilities. As a first step in defining the role of pioneer neurons in the Drosophila embryonic CNS, the temporal profile and trajectory of the axons of four pioneer neurons (MP1, dMP2, vMP2 and pCC) have been described; it has been shown that they differ from previously published reports. The formation of the longitudinal tracts begins with the extension of the pioneer axons at stage 12. At the onset of axon outgrowth, the axons of vMP2 and pCC fasciculate and extend anteriorly to meet the homologous axons from the next anterior segment. Similarly, the axons MP1 and dMP2 fasciculate and extend posteriorly until they meet the homologous axons from the next posterior segment. At stage 13, the ascending axons of vMP2 and pCC encounter the descending axons of dMP2 and MP1 to form the first joint longitudinal pathway. At stage 14, the vMP2/pCC fascicle separates from the MP1/dMP2 fascicle, to form two pathways that make contact only at the segmental boundary: an outer fascicle, composed of the axons MP1 and dMP2, and an inner one, consisting of pCC and vMP2. From this stage on, it had been thought that all of the pioneer axons run in a combined MP1/MP2 pathway along the most medial tract of the three fascicles that express FASII. However, between stages 14 and 15, the axons of the pioneer neurons have been found to reassort. First, the two axon fascicles move closer together. Next, these two tracts, which were fused at the segment boundary, separate. Finally, the axons change partners: dMP2 defasciculates from MP1 (or is displaced by follower neurons) and fasciculates with pCC, and vMP2 defasciculates from pCc (or is displaced) and runs in a more ventral plane along the same longitudinal pathway. These patterns are maintained for the rest of embryogenesis (Hidalgo, 1997).

Targeted ablation of one, two, three or four pioneer neurons at a time shows that (1) no single pioneer neuron is essential for axon tract formation; (2) the interaction between two pioneers is necessary for the establishment of each fascicle, and (3) pioneer neurons function synergistically to establish the longitudinal axon tracts, to guide the fasciculation of follower neurons along specific fascicles and to prevent axons from crossing the midline (Hidalgo, 1997).

Fasciclin II defines coherent embryonic domains of the brain. One large, coherent domain encompasses part of the tritocerebrum and deuterocerebrum (D/T). Four others are defined (P1, P2, P3 and P4) in the protocerebrum. The formation of the neuropil of the brain is initiated from these centers. Axons extending ventrally from the D/T domain form the cervical connective and the subesophageal (tritocerebral) commissure. In the protocerebrum, ventrally directed axons from P4 and P3 (dorsal to P2), as well as dorsally directed axons from P1 (ventral to P2), converge onto cluster P2. The P2 cluster subsequently elongates medially and links up with its contralateral counterpart, thereby forming a track for the supraesophageal commissure (Younossi-Hartenstein, 1997).

buttonless mutation specifically eliminates the dorsal medial (DM) cells, mesodermal cells just above the central nervous system that express Fas2; this genetic ablation reveals a requirement for DM cells as cellular cues for axonal guidance during transverse nerve outgrowth and bifurcation of the median nerve. Transverse nerves are absent in btn mutants. By stage 16 [Images] of development, abnormally long and thick axon bundles expressing Fasciclin II can be seen in mutant flies, extending along the midline at the dorsal surface of the ventral cord. It is presumed that these bundles result from outgrowth and fasciculation of axons that contribute to the median nerve in multiple segments (Chiang, 1994).

During the formation of the enteric nervous system (ENS) of the moth Manduca sexta, identified populations of neurons and glial cells participate in precisely timed waves of migration. The cell adhesion receptor fasciclin II is expressed in the developing ENS and is required for normal migration. Two isoforms of Manduca fasciclin II (MFas II), a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) and a transmembrane isoform (TM-MFas II), have been identified. Using RNA and antibody probes, it has been found that these two isoforms are expressed in cell type-specific patterns: GPI-MFas II is expressed by glial cells and newly generated neurons, while TM-MFas II is confined to differentiating neurons. The expression of each isoform also corresponds to the motile state of the different cell types: GPI-MFas II is detected on tightly adherent or slowly spreading cells, while TM-MFas II is expressed by actively migrating neurons and is localized to their most motile regions. Manipulations of each isoform in embryo culture shows that they played distinct roles: whereas GPI-MFas II acts strictly as an adhesion molecule, TM-MFas II promotes the motility of the EP cells as well as maintaining fasciculation with their pathways. These results indicate that precisely regulated patterns of isoform expression govern the functions of fasciclin II within the developing nervous system (Wright, 2000).

During embryogenesis, a population of ~300 postmitotic neurons (the EP cells) delaminates from a neurogenic placode in the posterior foregut; concurrently, a second population of glial precursor cells emerges from an adjacent proliferative zone associated with the developing esophageal nerve (formerly named the recurrent nerve). These two cell groups become distributed across the foregut and midgut via a stereotyped sequence of motile behaviors, giving rise to the enteric plexus that spans the foregut- midgut boundary. The EP cells first migrate and then extend axons along specific sets of visceral muscles on both the foregut and the midgut, and then the glial cells spread along these same pathways, ensheathing the postmigratory neurons. Notably, the migrating cells and their pathways remain accessible to manipulation throughout embryogenesis, permitting an in vivo analysis of the mechanisms controlling this migratory sequence (Wright, 2000).

The formation of the ENS in Manduca involves three discrete phases of migration. Neurogenesis in the ENS is complete by 40% of development, at which time the EP cells have formed a condensed packet of postmitotic neurons adjacent to the foregut-midgut boundary. Concurrently, a smaller number of large glial progenitor cells that lie at the anterior margin of this packet have been generated. Over the next 15% of development, the EP cells commence a slow, circumferential phase of migration, during which the packet of neurons spreads bilaterally around the foregut along the foregut-midgut boundary. Throughout this first phase of migration, the leading neurons extend short filopodial processes onto the adjacent epithelial surfaces, but the cells remain tightly adherent within the packet. By 55% of development, the EP cells have encircled the foregut, and groups of neurons align with eight longitudinal muscle bands that have coalesced on the midgut surface. These muscle bands form requisite pathways for the next phase of migration (55%-65% of development), during which subsets of EP cells disperse from the original packet and travel rapidly along the bands onto the midgut. Additional sets of neurons also migrate anteriorly and laterally along radial muscles that form on the foregut (58%-65% of development). In contrast, the glial precursors adjacent to the EP cells continue to divide but undergo only minor changes in position during these first two migratory phases. However, toward the end of EP cell migration, proliferating glial progeny commence a third phase of migration, during which they spread down along the same pathways established by the EP cells to form the glial sheath of the enteric plexus. Unlike the rapid, transient migration of the EP cells, the extension of this glial sheath continues more gradually, so that the postmigratory neurons are not completely enwrapped until 80%-85% of development. Notably, the enteric glia ensheathe the branches of the enteric plexus only to the extent that the neurons have migrated but do not extend along the more distal axons and terminal branches elaborated by the EP cells on the adjacent gut musculature (Wright, 2000).

To investigate whether the two isoforms of MFas II are differentially expressed in the developing ENS, riboprobes were generated against unique sequences within the 3' UTR regions of each isoform. Both the EP cells and the associated glia express MFas II throughout their migratory phases of development. Surprisingly, when the isoform-specific probes were applied to identically staged embryos, GPI-MFas II and TM-MFas II were found to be expressed in complementary patterns within the ENS. At 53% of development, GPI-MFas II transcripts are confined to cells at the anterior margin of the EP cell packet, an area that is populated by glial precursors. In contrast, TM-MFas II expression at 53% was localized to the adjacent population of EP cells that had spread around the foregut- midgut boundary. This distinction persists throughout subsequent periods of development. During the fast phase of migration (58% of development), GPI-MFas II mRNA expression remains confined to the foregut regions (Wright, 2000).

Perturbations of the two isoforms have indicated that they play distinct functional roles during development, suggesting the following model: upon delamination from the foregut epithelium, both the EP cells and the adjacent glial precursors express GPI-MFas II, which serves a strictly adhesive function. Lacking a cytoplasmic anchoring domain, GPI-MFas II becomes uniformly distributed over the entire plasma membrane. While it helps maintain tight apposition between neighboring cells, it does not regulate the slow spreading migration of the EP cells either positively or negatively). However, the presence of GPI-MFas II may contribute to the subsequent orientation of neuronal subsets onto each of the midgut muscle bands, possibly by allowing neighboring cells to cluster with those neurons that first contact the bands as they form (Wright, 2000).

Shortly before fast migration, the EP cells undergo a rapid down-regulation of GPI-MFas II and up-regulation of TM-MFas II, which becomes localized to the leading processes of the neurons. With the onset of MFas II expression by the muscle bands, the dispersive migration of the EP cells along these pathways is facilitated by TM-MFas II. Interference with TM-MFas II expression during this period disrupts the attachment of the EP cells to the muscle bands and neighboring neurons and inhibits their migration and process outgrowth. The localized expression of TM-MFas II in the leading processes and axons suggests that it helps regulate the polarized assembly of actin filaments associated with active neuronal motility, as proposed for NCAM. In contrast to the neurons, the trailing glial precursors continue to express only GPI-MFas II. As with the slow phase of EP cell migration, the subsequent migration of the glial cells along the branches of the enteric plexus involves a slow, spreading movement, during which the glial cells do not disperse but remain tightly adherent to themselves and to the underlying neurons that they ensheathe. Thus, the expression of a particular MFas II isoform is both predictive of cell type (neuronal versus glial) and corresponds with the motile behavior displayed by those cells. A similar relationship also occurs in other regions of the developing nervous system: TM-MFas II is expressed exclusively by identified sets of neurons (primarily motor neurons) and is localized to their growing axons, while GPI-MFas II is primarily confined to glial cell populations that ensheathe the peripheral nerve branches (Wright, 2000).

Precedent for this model can be found in studies of the vertebrate receptor NCAM. Like MFas II, NCAM is expressed as both GPI-linked and transmembrane isoforms, which have been proposed to play distinct roles during axon outgrowth. The predominant transmembrane forms of NCAM (NCAM 140 and 180) are expressed primarily by developing neurons and immature muscle cells, while GPI-linked NCAM (NCAM 120) is expressed by glial cells but not neurons. When tested in vitro, both NCAMs 140 and 120 were found to act as substrates for axon outgrowth, and NCAM 140 could also act as a neuronal receptor (enhancing the outgrowth of cells expressing this isoform). In contrast, the presence of NCAM 120 (the GPI-linked isoform) actually inhibits the elongation of processes by cells grown on substrates expressing NCAM. Similarly, GPI-MFas II is expressed on tightly adherent cells (primarily on glial cells, but also on the EP cells during their slow spreading phase), while the onset of active neuronal motility ia preceded by a rapid switch in isoform expression to TM-MFas II. Whether persistent expression of the GPI-linked isoform would inhibit neuronal motility (as has been suggested for NCAM 120) can now be tested in vivo by expressing ectopic GPI-MFas II in the premigratory EP cells; these experiments are currently in progress (Wright, 2000 and references therein).

An important conclusion from the current studies is that both isoforms of MFas II can be rapidly regulated at the transcriptional and translational levels of expression. For example, the midgut muscle bands exhibit a rapid onset of TM-MFas II shortly before EP cell migration (between 53% and 55% of development) and then subsequently lose the expression of MFas II-specific mRNA and protein once migration is complete (between 63% and 65%). In like manner, GPI-MFas II expression is rapidly eliminated from the EP cells before the onset of fast migration, while TM-MFas II is steadily upregulated. Studies on a variety of other systems have shown that GPI-linked receptors can be released from cell membranes by endogenous phospholipases. Phospholipase activity might similarly contribute to the regulation of GPI-MFas II expression by the EP cells, although it would have to be sufficiently localized to avoid perturbing GPI-MFas II on the adjacent glial cells. Protein turnover may also be affected by the presence of a PEST sequence, a motif that has been identified in one isoform of Fasciclin II in Drosophila. TM-MFas II in Manduca lacks this motif, but both MFas II isoforms contain multiple ATTTA sites in their 3' UTR regions that may affect mRNA stability. A similar cluster of ATTTA sites has been proposed to contribute to the rapid regulation of OCAM, an adhesion receptor related to NCAM that (like Fasciclin II) is transiently expressed in the developing nervous system and undergoes activity-dependent down-regulation at synapses (Wright, 2000).

During the formation of the insect peripheral nervous system (PNS), the cell adhesion receptor Fasciclin II has been shown to play a prominent role in axonal fasciculation and synapse formation during motor neuron outgrowth. In the moth Manduca, Fasciclin II (MFas II) is expressed both as a transmembrane isoform (TM-MFas II) and a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II). By using RNA and antibody probes, it has been shown that these two isoforms are expressed in nonoverlapping patterns: TM-MFas II is expressed exclusively by neurons and becomes localized to their most motile regions, while GPI-MFas II is expressed primarily by the glial cells that ensheath the peripheral nerves. This cell-type specificity of expression allowed the monitoring of the nature of neuronal-glial interactions during PNS development. The outgrowth of TM-MFas II-positive axons in many regions precedes the arrival of GPI-MFas II-expressing glial processes that enwrap them. In a few key locations, however, GPI-MFas II-positive glial cells differentiate before the arrival of the first axons and prefigured their subsequent trajectories. Prior inhibition of GPI-MFas II expression disrupts the subsequent outgrowth of axons at these locations but not elsewhere in the PNS. These results suggest that the two isoforms of MFas II play distinct roles with respect to cellular motility and nerve formation (Wright, 2001).

Unlike Drosophila, in which motor terminals have been reported to remain incompletely ensheathed, all of the efferent branches and motor terminals in Manduca appear to become wrapped by glial processes during embryogenesis, a relationship that is maintained throughout postembryonic development. In this regard, the neuromuscular junction of Manduca is more similar to that of vertebrates than has been described in flies. However, the behavior of the outgrowing glial cells is notable in that they clearly follow additional guidance cues besides the TM-MFas II-positive motor axons: the leading glial processes are often seen extending in parallel but independent of the motor axons, and only subsequently making contact with the adjacent axons and enwrapping them. Moreover, some GPI-MFas II-positive glial processes grow substantially beyond the TM-MFas II-positive motor terminals on mature muscles. It is possible that a subset of motor neurons (or specific motor terminal branches) do not express TM-MFas II but are nevertheless ensheathed by GPI-MFas II-positive glial cells, although in Drosophila, all motor neurons have been reported to express Fasciclin II during embryogenesis. An examination of Manduca neuromuscular junctions in postembryonic animals has also shown that all motor terminals remain ensheathed by GPI-MFas II-expressing glial cells throughout subsequent life stages. Alternatively, this additional glial elaboration may reflect the ensheathment of unstained sensory afferent fibers en route to the CNS. Simultaneous labeling of sensory neurons and the glial sheath in developing embryos will be needed to address this issue (Wright, 2001).

The results of this paper indicate that at least two distinct types of interaction occur between TM-MFas II-positive axons and GPI-MFas II-positive glial cells. The first type, illustrated by the extension of the DNl branch (branches of the dorsal nerve that grow laterally), occurs when axons expressing TM-MFas II navigate to their targets in advance of glial cells. Axons pioneering the DNl clearly do not rely on GPI-MFas II-expressing glia as a substrate for guidance; rather, the DNl forms in a manner similar to vertebrate peripheral nerves, in which axons extend into target regions prior to the arrival of glial cells. The TM-MFas II-expressing axons subsequently provide one of the substrates followed by the ensheathing glia during their outgrowth, similar to the guidance of Schwann cells by axons during vertebrate development. Based on these results, it is not surprising that manipulations targeting the GPI-linked isoform have no effect on the outgrowth of this nerve, even when the level of GPI-MFas II expression in the glial cells surrounding the DNl are substantially reduced. Although synapse formation in these preparations was not examined in any detail, the overall appearance of the terminal branches of the DNl is normal, consistent with the arrival of the axons at their muscle targets prior to ensheathment by the glial processes. Therefore, in the case of the DNl, the axons conduct their initial pathfinding independent of glial interactions and provide support for subsequent glial differentiation (Wright, 2001).

In contrast, the other class of interaction (illustrated by the formation of the DNa nerve -- the branch of the dorsal nerve that grows anteriorly) suggests that GPI-MFas II-positive glial cells play an essential role during axonal guidance in some specific instances. The glial cells forming the bridge structure are already present and expressing GPI-MFas II prior to the arrival of the DNa pioneer growth cones, and the morphology of this transient structure prefigures the trajectory subsequently taken by the DNa nerve. Moreover, removal of GPI-MFas II (with PI-PLC) or inhibition of its expression (using antisense oligodeoxynucleotides) dramatically alters the formation of this nerve. Both of these manipulations perturbed the trajectory of the DNa axons to some degree in over 90% of the treated preparations, the most dramatic of which involved a complete failure of the DNa to defasciculate from the DNl. More often, the DNa axons initially extended into the area normally occupied by the bridge glia but then either stalled or grew in a variety of inappropriate directions, including back towards the CNS. Treatment with PI-PLC (which selectively cleaves GPI linkages) typically results in a complete loss of GPI-MFas II expression and tends to produce more pronounced errors in DNa formation. While the relatively low concentration of PI-PLC used in these studies did not cause any obvious defects in the DNl branch (which grows in advance of GPI-MFas II-expressing substrates), this enzyme should also cleave other GPI-linked molecules that might be expressed by the bridge glia. The effects of this treatment on DNa branch formation might therefore reflect the function of additional receptors besides GPI-MFas II. However, the successful application of anti-sense oligodeoxyncleotides designed to inhibit GPI-MFas II expression also produce a similar range of defects in DNa formation, most often resulting in a stalled phenotype in which the axons fail to advance normally beyond the malformed glial bridge. These results indicate that in the case of the DNa branch, axonal pathways are prefigured by a defined glial structure. This phenomenon is reminiscent of a 'Blueprint' hypothesis, suggesting that some axon pathways may be pre-specified by glial or other nonneural structures (Wright, 2001).

What is the role of GPI-MFas II with respect to the formation of the DNa branch? Given previous studies demonstrating that Fasciclin II can act as a homophilic binding protein, the initial assumption was that TM-MFas II-positive axons would specifically adhere to GPI-MFas II glial cells either before or after their initial outgrowth. This model would predict that interactions between TM-MFas II receptors on the growth cones and GPI-MFas II on the glial cells normally mediate the guidance of the axons across the glial bridge, and that removal of GPI-MFas II is sufficient to disrupt this process. An alternative possibility is that GPI-MFas II simply acts as an adhesion molecule holding the glial cells in a cohesive group as the bridge structure differentiates, whereupon other molecular components associated with the bridge provide the actual guidance cues for the DNa axons. Contrary to these expectations, instances were observed where TM-MFas II-positive axons grow independently of GPI-MFas II-positive glial cells, and outgrowth of GPI-MFas II-positive glial processes occurs partially independently of TM-MFas II-expressing axons. Although homophilic adhesion between the identical extracellular domains of different MFas II isoforms is formally possible, whether the transmembrane and GPI-linked isoforms directly interact in vivo remains to be determined (Wright, 2001).

An important issue with respect to the immunoglobulin-related superfamily of CAM (IgSF) receptors in general concerns the potential functions of distinct isoforms. While many of these molecules have been shown to be able to promote cell-cell contact via homophilic interactions (including Fasciclin II), ample precedent has been established that some IgSF receptors mediate functions beyond simple adhesion. In the case of transmembrane IgSFs, both NCAM 140 and L1 have been shown to interact with a variety of intracellular signaling pathways in actin assembly and neuronal motility. Similarly, a PDZ-binding domain within the cytoplasmic tail of trans-membrane Fasciclin II has been shown to promote interactions with both the Shaker potassium channel and the membrane-associated guanylate kinase Discs-large, which may in turn mediate synaptic growth and plasticity. The role of the GPI-linked IgSFs is by contrast less well understood. Although a number of GPI-linked cell adhesion receptors have been shown to interact with nonreceptor tyrosine kinases, no such association has been demonstrated for GPI-linked isoforms of the NCAM/apCAM/fasciclin II family (Wright, 2001).

Experiments performed in cell culture have shown that GPI-linked receptors (such NCAM-120) can act as substrate molecules that promote neuronal motility in vitro, although other studies performed in vivo have suggested that GPI-linked isoforms of this family serve a strictly adhesive function (Wright, 2001).

In the case of MFas II, studies using cultured embryos support a model in which the two isoforms serve distinct functions with respect to cellular motility. For example, an identified set of newly generated enteric neurons (the EP cells) initially expressed only GPI-MFas II, and this isoform maintains strong adhesive contact between adjacent neurons but does not contribute to their motility. The EP cells then switched isoforms to express only TM-MFas II shortly before commencing a period of rapid migration and out-growth, events that are inhibited when TM-MFas II expression is perturbed. Subsequent expression of GPI-MFas II in the ENS is entirely confined to the enteric glial cells that, like the glial cells of the PNS, gradually spread along the pathways formed by the neurons and their processes. The membrane distributions of the two isoforms are similar in the ENS as reported in this study: GPI-MFas II protein appears to be uniformly distributed over the entire surface of cells expressing this isoform, while TM-MFas II is localized to the leading processes of migrating neurons or their growth cones. The consistent localization of TM-MFas II to the most motile regions of a neuron suggests that it may help regulate the cytoskeletal dynamics associated with migration and outgrowth, as proposed for other transmembrane isoforms of this receptor family. In contrast, the data argue that GPI-MFas II may act primarily as a simple adhesion molecule, maintaining strong intercellular contacts without directly promoting intracellular signaling events that might affect cellular motility. Whether the two isoforms directly interact (one providing a substrate for cells expressing the other isoform) or whether either isoform may interact heterophilically with other receptor types encountered in vivo remain to be explored (Wright, 2001).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

Using an antibody against the cell membrane glycoprotein Fasciclin 2 (Fas2), it has been found that in the procephalic region Fas2 is first expressed by late stage 10 in an ectodermal patch at the border between the intercalary and antennal segment. Later it also covers the posterodorsal ocular neuroectoderm (including the optic lobe anlage) and part of the labral ectoderm. Fas2 is also detected in brain NBs emerging from the antennal and intercalary neuroectoderm, and at a low level in a few dorsal ocular NBs. It has been found that Fas2 controls proneural gene activity in the eye/antennal imaginal disc, raising the possibility that it functions likewise in the procephalic neuroectoderm. However, Fas2 expression in almost all identified brain NBs is initiated after delamination from Fas2-negative neuroectoderm, suggesting that Fas2 in the procephalic neuroectoderm is not involved in the regulation of proneural genes. It has been shown that Fas2 appears on the surface of neural somata prior to axon outgrowth; these neurons belong to 'fiber tract founder clusters' that pioneer the main axonal tracts in the brain. Considering position and time point of development, it is suggested that the identified Fas2-positive deuto- and trito-cerebral NBs (Tv1, Tv2, Td1, Td2, Td6, Td8; Dv2, Dd9, Dd11) are the precursors of the 'D/T fiber tract founder cluster' (Urbach, 2003).

Using Fas2 to chart the structure of the neuropil

Insect neurons are individually identifiable and have been used successfully to study principles of the formation and function of neuronal circuits. In Drosophila, studies on identifiable neurons can be combined with efficient genetic approaches. However, to capitalize on this potential for studies of circuit formation in the CNS of Drosophila embryos or larvae, it is necessary to identify pre- and postsynaptic elements of such circuits and describe the neuropilar territories they occupy. A strategy for neurite mapping is presented, using a set of evenly distributed landmarks labelled by commercially available anti-Fasciclin2 antibodies that remain comparatively constant between specimens and over developmental time. By applying this procedure to neurites labelled by three Gal4 lines, neuritic territories are shown to be established in the embryo and maintained throughout larval life, although the complexity of neuritic arborizations increases during this period. Using additional immunostainings or dye fills, Gal4-targeted neurites can be targetted to individual neurons and they can be characterized further as a reference for future experiments on circuit formation. Using the Fasciclin2-based mapping procedure as a standard (e.g., in a common database) would facilitate studies on the functional architecture of the neuropile and the identification of candiate circuit elements (Landgraf, 2003).

Working with defined pre- and post-synaptic neurons is a prerequisite for the study of mechanisms that underlie circuit formation. The fact that such neurons establish synaptic contacts with one another requires that some of their neurites project to a common region. Thus, proximity of neurites is a criterion that can be used towards the identification of putative pre- and postsynaptic neurons. In Drosophila (like in other insects), synaptic contacts are restricted to the neuropile, a cell body-free area, which also contains the ascending, descending, and commissural fibers. Unlike the gray matter in the vertebrate spinal cord (where cell bodies and synapses are intermingled), neuronal cell bodies of the Drosophia CNS are restricted to the synapse-free 'cortex' from where they send monopolar projections into the neuropile. These neuropilar accumulations of neurites of CNS neurons (i.e., efferent and interneurons) are joined by projections from peripheral sensory neurons. The functionality of thus established neuronal circuits demands that the spatial arrangements of synapse-bearing neurites in the neuropile are fairly reproducible between different individuals, as has been learned from analyses in larger insects. In order to map these reproducible neurites in the Drosophila neuropile, predominantly anatomical landmarks of the neuropile have been used to date as reference points for the relative positions of neuronal projections. Such landmarks are segmentally repeated nerve roots and commissures, or easily identifiable fiber tracts (so far applied only in the imaginal CNS (Landgraf, 2003).

This study capitalizes on a set of axon tracts that are labelled by the commercially available antibodies against the intracellular domain of Fasciclin2. These provide a set of standard landmarks that are evenly distributed throughout the neuropile. As shown by double-labellings with presynaptic markers, all Fasciclin2-positive fiber tracts are fully contained within the synaptic neuropile. They can be used in a very easy and efficient way for the charting of neurites in the neuropile. So far, Fasciclin2 fibre tracts have served as one-dimensional (mediolateral) landmarks in younger embryos. This approach has been extended by using the set of Fasciclin2 tracts in three dimensions and at different developmental stages. These analyses were exclusively centered on abdominal neuromeres for two reasons: predominantly, the abdominal motorsystem contributes to larval movement, and abdominal neuromeres face only minor reorganization during larval life (Landgraf, 2003).

Each Fasciclin2 fascicle has been named according to its relative dorsoventral (D, dorsal; C, central; V, ventral) and mediolateral (M, medial; I, intermediate; L, lateral) position. Such a nomenclature is neutral and can therefore be applied to any set of axon fascicles. The pattern of Fasciclin2 tracts remains relatively constant throughout larval development and thereby permits comparisons and extrapolations across different developmental stages. The main change to the embryonic pattern of Fasciclin2 in the neuropile is the addition of further elements, particularly five transverse projections (TP1-5) per neuromere in larval stages, which provide added reference points for the anteroposterior axis. From their association with different motor axons in the larva (TP1 with RP2 and VUM; TP2 with aCC), it is concluded that TP1 represents the pISN and TP2 the aISN nerve root (Landgraf, 2003).

In order to facilitate comparisons with published work, attempts were made to relate the Fasciclin2 pattern of the late embryo and larval stages to existing descriptions. For example, the Fasciclin2 pattern has frequently been used for work on the ventral nerve cord of earlier embryos, usually at 13 h of development. At this stage, three tracts can be resolved in the horizontal plane, of which the intermediate Fasciclin2 tract is formed or at least joined by axons of the MP1-interneurons (targeted by C544-Gal4), the medial tract by MP2-interneurons (targeted by 15J2-Gal4;. A split of the three tracts into vertically distinguishable bundles occurs during the next ca. 3 h. During this interval, it is still possible to trace the MP2/pCC- and MP1-axons via the C544- and 15J2-Gal4 lines when visualizing the Gal4-expressing neurons with the Uas-CD8-GFP reporter gene; later their Gal4-expression patterns change dramatically. Thus, despite the highly dynamic changes in the neuropile during this period (i.e., nerve cord condensation, closer apposition of neuropile at the midline and the fact that the intracellular Fasciclin2 domain vanishes from many cell bodies and axons), it is possible to map the MP2/pCC-axons to the DM (dorsointermediate) axons, and the MP1 interneuron axons to the dorsal CI-fascicles (Landgraf, 2003).

Classical neurobiological work on neuronal circuitry in other insects has been based on mapping strategies that used morphologically distinguishable axonal tracts in the neuropile and relates these to projection patterns of neurons. Similar strategies have been used for the thoracic adult CNS of Drosophila. The DM- and VMd-fascicles serve as reliable landmarks for distinguishing dorsal, intermediate, and ventral commissural tracts. The distinct patterns of sensory projections of different modalities, linked to the classical neurobiological literature, reveal a partitioning of the larval Drosophila neuropile. In an effort to relate the pattern of Fasciclin2 tracts to neuropilar regions, use has been made of three different Gal4 lines that target different subpopulations of sensory neurons (C161-, MJ94-, MzCh-Gal4). Sensory projections are confined to ventral regions, while neurites of motorneurons occupy the very dorsal neuropile. Thus, there is little, if any, physical overlap and contact between afferent sensory projections and central motorneuron neurites during larval stages. However, some overlap might occur lateral to the DM-fascicle, most likely with projections of the dbd and vbd-neurons. Thus, the data suggest that there are few, if any, monosynaptic connections between sensory and motorneurons in the embryonic and abdominal larval ventral nerve cord of Drosophila. However, this is a fairly rough estimation that will have to be tested by more detailed studies in the future (Landgraf, 2003).

Having described some spatial features of the neuropile with the help of the Fasciclin2 pattern, this charting strategy was next applied to three selected Gal4 driver lines. This effort is intended to identify and characterize neurons that are genetically amenable and that could be used for the investigation of neural circuit formation in the embryonic and larval Drosophila CNS. Three neural Gal4 lines were analyzed with precision. Before presenting detailed characteristics of abdominal Gal4-labelled neurons, an overview of the three Gal4 lines is provided: Per abdominal half-neuromere eve-Gal4^RRK expresses Gal4 in two motor-(aCC and RP2) and one interneuron (pCC). DDC-Gal4 displays 9-11 Gal4-neurons, and MzVum-Gal4 12-14 cells plus 3 efferent VUM (Ventral Unpaired Median) neurons located at the ventral midline. In all three lines, Gal4 expression occurs in a defined sequence, and for most cells it is yet unclear to what extent a late onset of expression reflects a late birth and/or differentiation of those cells. Only in the aCC, pCC and VUM neurons is Gal4 expression initiated at the time of their respective births, thus making them amenable to genetic manipulations of axonal pathfinding and differentiation. Next, the relative strengths of Gal4 expression were compared and overall MzVum-Gal4 expresses strongest, followed by DDC-Gal4 and eve-Gal4^RRK. However, Gal4 levels of different neuronal subsets in each Gal4 strain can differ significantly (e.g., in MzVum-Gal4, GABAergic interneurons express low levels while VUM and leucokinin-1-positive neurons express high levels). Because of differences in timing and strength of expression, Gal4-based manipulations would not be expected to affect all cells alike (Landgraf, 2003).

By virtue of the Fasciclin2-positive landmarks, it was possible to work out detailed descriptions of the neuropilar positions of neurites labelled by the three Gal4 lines eve-Gal4^RRK, MzVum-Gal4, and DDC-Gal4. These studies clearly show that the Fasciclin2 framework allows spatial relationships between neurites to be pinpointed even across specimens: for example, neurites of the aCC and RP2 neurons (eve-Gal4^RRK) are concentrated to form an oval in each hemineuromere that is located at the level of the DL-fascicle, medial to the ascending section of transverse projection 2 and anterior to transverse projection 1. At the same level (of the DL-fascicle), MzVum-Gal4-labelled neurites form whirlwind-like arrangements that have oval holes in their centers. These holes map to the region where aCC and RP2 neurites are concentrated, as indicated by the transverse projection 2. Thus, by using Fasciclin2-positive tracts as landmarks, spatial relationships of neurites are reproducibly revealed in three dimensions (Landgraf, 2003).

Since the pattern of Fasciclin2-positive axon tracts remains relatively constant from the late embryo to larva, it can also be used to investigate how neuronal projections change during this developmental period. The larval patterns of neurites described above are prefigured in the late embryo. For example, at late embryonic stages, the central arborisations of aCC and RP2 at the level of the DL-fascicle are also concentrated anterior to pISN (equals TP1 in the late larva), which corresponds to the region that lacks neurites in MzVum-Gal4 embryos. Thus, the principle spatial relationships between these sets of neurites (of aCC and RP2 versus those of MzVum-Gal4) appear to be laid down in the late embryo and maintained to larval stages, though the complexity and the spread of neurites increases over developmental time. This is an important observation because it suggests that: (1) By late embryonic stages neuritic arbors define those territories in the neuropile from which they will elaborate and spread during subsequent larval stages. Thus, principle spatial relationships between neurites are laid down during embryogenesis. (2) Data on the distribution of neurites obtained at one stage of development can be extrapolated and used to interpret other stages (Landgraf, 2003).

As demonstrated so far, using Fasciclin2 stainings significantly improves descriptions of the characteristic patterns of central neurites targeted by different Gal4-lines. However, these patterns of neurites are composites of different neurons. With this in mind, attempts were made to define methods with which to resolve such complex neuritic patterns into their constituent parts (Landgraf, 2003).

The first approach to tackle this problem is to employ antisera, which would reveal the morphologies of particular subsets of neurons. By using antibodies against the neurotransmitter/neuromodulator Serotonin and the neuropeptides Corazonin and Leucokinin-1 on nerve cords displaying Gal4-driven CD8-GFP expression, it is possible to define these neurites among the composite of Gal4-targeted projections that correspond to Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons and the regions of the neuropile that these occupy. Anti-Serotonin stains two and anti-Corazonin one neuron per hemineuromere. These three cells are targeted by the DDC-Gal4 line and appear to give rise to most of the DDC-Gal4-labelled neurites in the abdomen. Anti-Leucokinin-1 labels Gal4-targeted efferent projections forming type-3 terminals on the VL1-muscle (type-3v, DDC-Gal4;) and on the segment border muscle (type-3u, MzVum-Gal4). Of these, only the type-3u neuron is revealed by Leucokinin-1-like immunoreactivity and can thereby be traced back to a ventrolateral cell body in the CNS extending side branches toward the VL-fascicle. There are additional Leucokinin-1-positive projections associated with the DM-fascicle that are not targeted by MzVum-Gal4 but seem to originate from 2-4 (Gal4-negative) neurons at the anterior tip of the nerve cord. This has been confirmed by targeting the cytotoxin Ricin to MzVum::CD8-GFP neurons. This selectively abolishes all MzVum-Gal4-specific CD8-staining and the VL- but not the DM-associated Leucokinin-1-like immunoreactivity (Landgraf, 2003).

In summary, it has been shown that a small range of antisera can readily be used to reveal the projections of particular subsets of neurons. Such specific stainings are well suited to serve as spatial reference points in their own right. Moreover, in this instance, the Serotonin, Corazonin, and Leucokinin-1 immunoreactive neurons were instrumental in revealing some of the constituent parts of the complex projection patterns of the DDC-Gal- and MzVum-Gal4-lines (Landgraf, 2003).

Next, efferent neurons targeted by the three Gal4 lines were characterized and their axonal projections (nerve root and branch), target muscles, and terminal types were described. Based on morphological, molecular, and ultrastructural characteristics of motor terminals, several types of efferent neurons can be distinguished in Drosophila. It should be emphasised that distinctions between terminal types are not only of importance to studies of the Drosophila motor system but also correlate with differences between the central dendritic arbors of particular efferent neuron types. To classify the Gal4-labelled efferent neurons with respect to terminal type, a range of immunohistochemical stainings was employed: Synaptotagmin, Cysteine string protein, and Synapsin all represent presynaptic proteins involved in regulation of synaptic vesicles; Discs large is a predominantly postsynaptic protein, which labels the subsynaptic reticulum; and anti-Leucokinin-1 antisera detect an insect neuropeptide (Landgraf, 2003).

While the visualization of Gal4-labelled neurites via immunostaining is efficient, it is at the same time limited to particular subsets of cells, leaving many neurons unidentified. This limitation can be overcome by using standard neuronal tracers. To reveal the morphologies of those Gal4::CD8-GFP neurons, the neural tracer dye Cascade Blue was iontophoretically applied to individual cells. Thus, it was possible to define the positions of somata and central projections of all efferent neurons and a number of interneurons (Landgraf, 2003).

For the efferent neurons, it was found that their central projections are restricted to the dorsal neuropile (dorsal to the CI-fascicles). The only exception to this was the efferent SBM-neuron (MzVum-Gal4; whose short central arbors reside in the ventral neuropile where they associate with the VL-fascicle (consistent with Leucokinin-1 staining). In addition, it was found that differences in terminal type are reflected by distinctions in the central arbors of efferent neurons: while type-1 motoneurons elaborate extensive dendritic arbors (aCC and RP2; VA), efferent neurons with type-2 and type-3 terminals form comparatively sparse and stunted central arbors (VUM and SBM; VL1). Finally, these analyses suggest that the central projections of the same motoneuron in consecutive neuromeres do not overlap, i.e., they seem to behave in accordance with the tiling principle (Landgraf, 2003).

The interneurons of two of the Gal4-lines have been identified previously: pCC (eve-Gal4^RRK) lies adjacent to the aCC motorneuron; three interneurons of DDC-Gal4 are serotonergic or corazonergic. In addition, two MzVum-Gal4 interneurons were identified via Cascade blue fill. These two intersegmental interneurons seem to contribute to most or all MzVum-Gal4-targeted neurites in the ventral neuropile, ventral to the CI-fascicle (except for intersegmental ascending and descending projections and the leucokinin-1-positive neurites associated with the VL-fascicle). It is possible that additional ventral neurites might be derived from the mVg- and GABAergic neurons of MzVum-Gal4 (Landgraf, 2003).

In summary, it was found that neurites targeted by MzVum-Gal4 segregate into a dorsal fraction, consisting primarily of motoneuronal side branches, and a ventral fraction derived almost exclusively from interneurons. This pattern simplifies interpretations of experimental results obtained with this Gal4-line (for example, if mutant backgrounds reveal selective impairment of only dorsal or ventral neurites). Having applied a combination of a standardized set of Fasicilin2-positive landmarks, specific antisera, and single cell tracings, it has been possible to (1) assign most neurites of the Gal4-lines to identified neurons, and (2) define the regions of the neuropile that they occupy. Future applications of a standardised mapping strategy to other Gal4 lines will considerably advance the understanding of the functional architecture of the Drosophila neuropile, and it will form a basis with which candidate pre- and postsynaptic circuit elements can be identified (Landgraf, 2003).

An important aspect of this study is that despite its limited scope it reveals an apparent partitioning of the neuropile into (possibly functionally) distinct regions. Facets of a functional architecture of the neuropile have already been documented such as the modality-specific sensory projections that partition the ventral neuropile. Due to the Fasciclin2-based mapping, these areas can now be named and the regions can be related to projection patterns of other neurons. In accordance with work published for other insects, the dorsal neuropile is predominantly occupied by the central arbors of efferent neurons (with the single exception of the ventral type-3u neuron arbors). There is little overlap with sensory areas so that direct connections between sensory and motor neurons will be the exception. In addition, different efferent neurons elaborate their central arbors in distinct anteroposterior regions of the dorsal neuropile. These territories seem to be defined in the embryo and they are maintained through larval stages, although areas of overlap between formally distinct territories increase as central arbors become more elaborate over time. This relative constancy of the topography of the neuropile over time also exists for Serotonin-, Corazonin-, and Leucokinin-1-positive neurons. An important consequence of such constancy for future research work is that neurites can be compared or descriptions extrapolated across different developmental stages (Landgraf, 2003).

Interestingly, neuropeptidergic projections seem to cluster in particular areas. Corazonin and Leucokinin-1 (and also Serotonin) are closely associated with the DM-fascicle. Published data suggest that antibodies against FMRF, molluscan neuropeptide SCP_B, and Substance-P reveal neural structures that might also be localized in this median area. A second neuropeptide 'hot spot' is the VL-fascicles, where staining with anti-Leucokinin-1 antibodies is found. Also antisera against Allatostatin and Insulin appear to stain in this region. The fascicles are innervated by the posterior ascending cells of MzVum-Gal4 and DDC-Gal4 and curiously are detected with antisera against muscle myosin heavy chain. Interestingly, both of these neuropeptidergic 'hot spot' areas bear very prominent Fasciclin2-labelled neurites (Landgraf, 2003).

The influence of pioneer neurons on a growing motor nerve in Drosophila requires the neural cell adhesion molecule homolog FasciclinII

The phenomenon of pioneer neurons has been known for almost a century, but so far few insights have been offered to explain the mechanisms or the identity of the molecules involved. The formation of the Drosophila intersegmental motor nerve (ISN) has been examined. aCC/RP2 and U motor neurons grow together at the leading front of the ISN. Nevertheless, aCC/RP2 neurons are the pioneers, and U neurons are the followers, because only aCC/RP2 neurons effectively influence growth of the ISN. This influence is shown to depend on the neural cell adhesion molecule homolog FasciclinII. (1) Ablation of aCC/RP2 has a stronger impact on ISN growth than U ablation. (2) Strong growth-influencing capabilities of aCC/RP2 are revealed with a stalling approach: when aCC/RP2 motor axons are stalled specifically, the entire ISN (including the U neurons) coarrests, demonstrating that aCC/RP2 neurons influence the behavior of U growth cones. In contrast, stalled U neurons do not have the same influence on other ISN motor neurons. The influence on ISN growth requires FasciclinII: targeted expression of FasciclinII in U neurons increases their influence on the ISN, whereas a FasciclinII loss-of-function background reduces ISN coarrest with stalled aCC/RP2 axons. The qualitative differences of both neuron groups are confirmed through findings that aCC/RP2 growth cones are wider and more complex than those of U neurons. However, U growth cones adopt aCC/RP2-like wider shapes in a FasciclinII loss-of-function background. Therefore, FasciclinII is to a degree required and sufficient for pioneer-follower interactions, but its mode of action cannot be explained merely through an equally bidirectional adhesive interaction (Sanchez-Soriano, 2005 ).

To investigate the role of Drosophila pioneer motor neurons for the establishment of motor neuronal nerves, analyses focused for several reasons on the most dorsal muscle area and its innervation through the ISN. (1) It is the area farthest away from the CNS requiring long-distance navigation of the ISN axons. Such growth is very likely to involve inter-axonal communication as an essential regulatory feature. (2) The muscle and innervation pattern is simpler in the dorsal region than in the ventral muscle field, facilitating analysis of phenotypes. (3) This is the only area in the embryo in which all innervating neurons have been identified and can be genetically manipulated using available Gal4 lines: in more detail, per hemisegment one RP2 and three ventral unpaired median (VUM) neurons have branches on most, if not all, muscles in the dorsal muscle field; one aCC motor neuron innervates muscle DA1, and three different U motor neurons establish contacts on DO1, DO2, and DA3, respectively (a fourth U neuron that terminates on LL1 muscles is not considered here). Several Gal4-driver lines can be used to target UAS-coupled genes to these neurons for their visualization and/or manipulation: RN2-Gal4 directs Gal4 expression to aCC and RP2 neurons, U/CQ-Gal4 to the U neurons, and MzVum-Gal4 to the VUM neurons. Apart from the single axons of the ISN, the entire nerve or all of its terminals can also be labeled to monitor its length. To this end, either neuronal surface markers such as FasciclinII or HRP or synaptic markers such as synapsin or Dlg can be used (Sanchez-Soriano, 2005).

Ultrastructural and ablation studies have suggested that aCC motor neurons might perform pioneer functions during ISN establishment. However, this aspect was never fully resolved, mainly because of technical limitations. Use was made of the refined tools described above to complement former studies and clarify the roles of the different neuron groups during ISN formation (Sanchez-Soriano, 2005).

Previous ultrastructural studies revealed that aCC is the first neuron of the ISN to grow out, thus defining it as the pioneer neuron of this motor nerve. However, these studies were restricted to aCC growth in the CNS (i.e., the very early stage of ISN development). Therefore, these studies were extended and the positions of axonal tips of the three different neuronal subpopulations (labeled with the UAS-mCD8-GFP or UAS-GFP-actin^2-2 reporters) were compared relative to the entire ISN length (visualized with anti-FasciclinII) at early and later stages of development. These studies revealed that the RN2-Gal4-positive neurons (aCC and/or RP2) grow mostly at the leading front of the developing ISN. In contrast, the growth cones of VUM axons keep considerably behind aCC/RP2 at all developmental stages investigated, discarding them as pioneers. The U neurons (visualized via U/CQ-Gal4) are relatively delayed at early stages of ISN formation when they begin to leave the CNS, navigating behind the axonal tips of aCC/RP2. However, once the U neurons join the ISN and navigate in the periphery, axonal tips of this neuron population reach the most distal end of the growing ISN in 70% of cases. Thus, although aCC and/or RP2 neurons are leading initially, U neurons catch up and stay at the nerve front during later ISN development. Therefore, if defining a pioneer neuron merely by its position at the leading edge of a growing nerve, then not only aCC/RP2 but also U neurons would have to be considered pioneers in most ISNs (note that in grasshoppers, U neurons but not aCC seem to be the pioneers) (Sanchez-Soriano, 2005).

To uncover potential functions of leading neurons during the formation of an entire nerve, ablation studies can be used. Ablation studies were performed using RN2-Gal4-driven expression of the cytotoxin ricinA, a tool that was used successfully to test neuronal pioneer functions in the Drosophila CNS. To test for ablation efficiency, the patterns of three independent cell markers were analyzed in control embryos and embryos with RN2-Gal4-induced ricinA expression. Already at the very early stage of axonal growth (early stage 13), marker expression was affected severely but cell specifically in ricinA-expressing aCC/RP2 neurons. This indicates that ricinA induced neuronal degeneration starts at very early stages, strongly suggesting that manipulated aCC/RP2 neurons do not grow motor axons. Effects on ISN morphology resulting from specific aCC/RP2 ablation were analyzed at late stage 17 (i.e., the time of hatch). Although dissections at this stage are difficult, they enable analysis of experimental impacts on matured neuronal connections, whereas analyses at earlier stages may be hampered by transient abnormalities or developmental delays. In the case of ricinA-induced aCC/RP2 ablations, growth of the entire ISN is affected in 26% of cases, as revealed by anti-FasciclinII staining at late stage 17. The defects primarily consist of premature stalling of the nerve mostly at the level of muscles DO2/DA2. This indicates that, at rather low frequency, the nonmanipulated U and VUM neurons fail to reach their dorsal target muscles as a consequence of the absence of aCC/RP2. These data show the same tendency as ablation studies in Drosophila in which another promoter construct and cytotoxin were used (12% of nerves were affected in the absence of aCC at stage 16) (Sanchez-Soriano, 2005).

Might this low impact of aCC/RP2 ablations be attributable to the fact that U neurons share pioneer functions with aCC at the leading ISN front? Because this possibility has never been tested, U neurons were ablated using U/CQ-Gal4-driven ricinA. Although U neurons are as severely affected at early stage 13 as aCC/RP2 in the above experiments, their absence has an impact on the dorsal outgrowth of the ISN in only 2.9% of cases, thus at much lower frequency than in the case of aCC/RP2 ablation (Sanchez-Soriano, 2005).

Together, these data show that ISN growth does not essentially depend on the presence of aCC/RP2 or U neurons. However, although U neurons and aCC/RP2 neurons both grow at the leading edge of the ISN, they are different in their abilities: aCC and/or RP2 neurons play a more important role in ISN development than U neurons (Sanchez-Soriano, 2005).

Effects observed in ablation experiments do not necessarily reflect the full influence that pioneers may have on follower neurons during normal development. If pioneer neurons and the potential cues they display are ablated, pathfinding cues from other sources may gain in importance for the guidance of follower neurons and compensate for the loss of the pioneers. To test this possibility for aCC/RP2, a stalling approach based on targeted expression of the ionotropic GABA receptor Resistant to dieldrin (UAS-Rdl) was used. When Rdl was targeted to aCC/RP2, their axons stalled mostly in the dorsolateral muscle field before reaching their most dorsal target muscles (in 70% of 134 hemisegments). Such stall was not caused by neuronal debility or degeneration, because aCC/RP2 motor neurons expressing Rdl at late larval stages (3 d later using eve-Gal4^RRK) looked relatively normal (axonal and dendritic processes of correct size persisted, and neuromuscular terminals displayed presynaptic markers and boutons). Next the impact that stalling of aCC/RP2 axons might have on the growth behavior of the entire ISN was analyzed. Stalled aCC/RP2 axons cause a coarrest of the complete ISN in 70% of cases, suggesting that aCC/RP2 neurons display influential properties far beyond those revealed by ablation experiments. Rdl usually influences electrical cell properties, but its mode of action in this context is still under investigation. To confirm that defects in other ISN neurons are not a direct non-cell autonomous effect of Rdl but rather a secondary consequence of stalled aCC/RP2 axons, other means to stall aCC/RP2 were used. Thus, targeted expression of DRac1^N17 (dominant-negative form of the Rho-like GTPase) and Notch^ICD (activated form of the transmembrane receptor Notch) equally induced stalls of aCC/RP2 and led to frequent coarrest of the complete ISN. Thus, three very different forms of targeted manipulation used to stall aCC/RP2 axons reveal the same quality of these neurons (i.e., the ability to cause coarrest of other motor axons in the developing ISN). To simplify experimental work, the following experiments were restricted to the use of UAS-Rdl (Sanchez-Soriano, 2005).

To find out whether other neurons of the ISN would differ from aCC/RP2 with respect to their growth-influencing properties, Rdl was targeted by U/CQ-Gal4 or MzVum-Gal4. Like aCC/RP2 axons, axons of U and VUM neurons also stalled after Rdl expression, respectively. However, these stalls had only a mild effect or had no effect on the rest of the ISN neurons (the ISN coarrests with stalling U neurons in 10% of cases and with stalling VUMs in 0% of cases (Sanchez-Soriano, 2005).

Thus, only axons of aCC/RP2 but not axons of U neurons and VUMs have a strong impact on ISN growth (shown by stalling), although aCC/RP2 neurons are not absolutely required for ISN formation (shown by ablation). Therefore, aCC/RP2 are referred to as the pioneers and to U neurons and VUMs as follower neurons of the ISN. This definition refers to their influential properties rather than to their positions in the growing ISN. Consistent with this assignment is the finding that aCC/RP2 and U neurons display different growth cone shapes typical of pioneer and follower neurons (see below) (Sanchez-Soriano, 2005).

Next, potential mechanisms were studied involved in the influence of aCC/RP2 axons on ISN growth. One molecule that could play a role in this process is the homophilic neural cell adhesion molecule (N-CAM) FasciclinII, which is expressed on the surface of all ISN motor neurons. One way to analyze FasciclinII function is through overexpression studies. This approach was used in combination with the stalling strategy to determine whether FasciclinII has the potential to influence pioneer-follower interactions. U neurons frequently grow at the leading front of the ISN. Nevertheless, they behave as followers. However, when FasciclinII is coexpressed together with Rdl using the U/CQ-Gal4 driver, 54% of ISNs coarrest, whereas in control experiments, ISNs coarrested with Rdl-stalled U neurons in only 10% of cases. Thus, targeted expression of FasciclinII in U motor neurons is sufficient to increase their influence on the growth of other ISN motor neurons. This result also confirms previous findings that U neurons are physically placed in a location at the tip of the growing ISN from which they can potentially influence nerve growth. Thus, it is not the position of U growth cones but rather other properties that make U neurons different from aCC/RP2 neurons. Similarly to U neurons, stalled aCC/RP2 axons also have a larger influence on follower neurons when FasciclinII is coexpressed with Rdl using RN2-Gal4 (Sanchez-Soriano, 2005).

Does endogenous FasciclinII also play a role in pioneer-follower interaction? Absence of endogenous protein in fasciclinII^eb112 mutant embryos causes very subtle motor neuronal growth phenotypes that do not allow any functional statement. However, it is speculated that if endogenous FasciclinII is involved in pioneer-follower interactions, lack of FasciclinII should lead to a reduction of ISN coarrest with stalled aCC/RP2 axons. It was found that ISN neurons are coarrested in fasciclinII^eb112 mutant background in only 36% of cases compared with 70% in wild-type background. Thus, removal of FasciclinII causes a severe suppression of the aCC/RP2-induced phenotype and significantly restores the ability of follower neurons to grow to their natural length and targets. This is taken as a strong indication that endogenous FasciclinII is required for the influence of pioneers on ISN growth (Sanchez-Soriano, 2005).

So far, in the context of motor neuronal outgrowth, endogenous FasciclinII has been seen merely as a homophilic cell adhesion molecule that mediates fasciculation. The aCC/RP2 and U neurons all express FasciclinII endogenously and mostly grow together at the leading front of the ISN. Nevertheless, they show asymmetric influential properties (i.e., aCC/RP2 neurons behave as pioneers and U neurons behave as followers). This asymmetry is unlikely to represent the mere result of homophilic adhesion of endogenous FasciclinII between the surfaces of pioneer and follower neurons. To compare the influential capabilities of aCC/RP2 and U neurons, the behavior of the third group of neurons, the later-growing VUMs that are likewise FasciclinII-positive were analyzed in greater detail. If U neurons are stalled, aCC/RP2 axons escape in 90% of cases. Thus, VUM neurons can choose to coarrest with U terminals or grow on with aCC/RP2. Only an estimated 4.4% of VUM neurons coarrests. If aCC/RP2 neurons are stalled, all ISN neurons including VUMs coarrest in at least 70% of cases. More interesting are those 30% of cases in which aCC/RP2 axons stall but U axons escape, providing VUM neurons again with a choice. Twenty-two such cases were identified, of which 27% showed coarrest of VUM neurons with aCC/RP2 and 73% showed escape. Thus, although aCC/RP2 axons are not influential enough to coarrest U axons in these cases, they still managed to coarrest VUM neurons at a much higher percentage than stalled U neurons in the experiment above. Thus, VUM neurons are influenced more through aCC/RP2 than through U axons. A possible explanation would be that aCC/RP2 neurons express FasciclinII at higher levels than U neurons. However, surprisingly, the levels of FasciclinII immunoreactivity (antibody against the intracellular Fasciclin2 domain) in many instances appear rather low on freely visible surfaces of aCC/RP2 axons and growth cones, whereas U neurons always show strong immunoreactivity. Therefore, the molecular modes of FasciclinII function during motor nerve growth may involve regulations beyond mere homophilic adhesive interactions. In support of this view, a signaling function of Fasciclin2 has been demonstrated recently in Drosophila cell culture, as is likewise the case for its homologous proteins in other species (Sanchez-Soriano, 2005).

Having studied the pioneer and follower interactions mostly at the level of nerve growth behavior, studies were extended to the growth cone level. To this end, the growth cones of U and aCC/RP2 motor neurons were visualized during ISN formation using targeted expression of GFP-actin. This way the growth cones of the targeted neurons are selectively labeled, also revealing their filopodial processes. The pioneer growth cones of aCC/RP2 were found to take on a wide shape with multiple scattered filopodia, whereas growth cones of U neurons are narrow and appear simpler. To measure these differences, the area and perimeter of growth cones was determined and the complexity index P²/A (square of the perimeter divided by area) was calculated. Indeed, the P²/A value of aCC/RP2 neurons is significantly higher than of U neurons, indicating that aCC/RP2 growth cones are more complex (i.e., display more individually distinguishable filopodia). The narrow shape of U growth cones most likely reflects the fact that their filopodia are predominantly attracted by cues on the pioneer neurons. Such cues are likely to involve those that help to influence the U neurons to coarrest with aCC/RP2 (for example, FasciclinII) (Sanchez-Soriano, 2005).

Does FasciclinII (which is required for the influence of pioneers on ISN growth) mediate the simple and narrow appearance of U growth cones? To address this question, U growth cones were analyzed in a fasciclinII^eb112 mutant background. Under these conditions, U growth cones take on a wide shape that is reminiscent of aCC/RP2 pioneer growth cones. Measurements of the P²/A complexity index clearly support this observation. The fact that U growth cones acquire pioneer-like shapes provides a good explanation for the finding that follower neurons more frequently escape stalled aCC/RP2 axons in a fasciclinII^eb112 mutant background, and also explains their ability to grow out to full length after aCC/RP2 ablation (Sanchez-Soriano, 2005).

Together, the observations on growth cones are consistent with the findings that aCC/RP2 neurons represent pioneers of the ISN that influence their follower neurons via molecular pathways involving FasciclinII. The fact that aCC/RP2 growth cones behave differently from U growth cones strongly supports the view that their pathfinding and growth-influencing properties are not the same (Sanchez-Soriano, 2005).

Larval

Fas2 is expressed in the antennal imaginal disc where it regulates both mechanosensory neuron fate and ocellar photoreceptor precursor fate (Garcia-Alonso, 1995).

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, the effects of a loss of function of neuronal Synaptobrevin, a protein required for synaptic vesicle release, was studied. Expression of tetanus toxin light chain (which cleaves neuronal Synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. Targeted toxin expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and Chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal Synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal Synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link may exist to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS (Hiesinger, 1999).

The finding of increased Fasciclin II immunoreactivity under conditions of blocked neurotransmitter release corresponds to earlier studies that have shown the opposite effect with an opposite approach: apCAM, the Aplysia cell adhesion protein, is downregulated after application of serotonin, and Drosophila synaptic Fasciclin II is reduced in mutants with abnormally high neuronal activity. Although it was demonstrated for apCAM that it is downregulated via endocytosis, the mechanism of activity-dependent Fasciclin II downregulation at the Drosophila neuromuscular junction remains unknown. Possible downregulation mechanisms to be considered include endocytosis, extracellular cleavage, and reduced transcription or translation in combination with a continuous turnover of the protein. The upregulation of two different types of CAMs (Fasciclin II and Chaoptin) in the same cell type under conditions of blocked neurotransmitter release poses the question of the specificity of the mechanism. In the absence of functional Synaptobrevin increased numbers of docked synaptic vesicles accumulate presynaptically. Assuming that this would result in a significant sequestration of membrane material and that the synaptic vesicle cycle is continuously replenished from cell surfaces carrying adhesion molecules, deactivation of Synaptobrevin could result in decreased intake of CAMs and thus increased CAM immunoreactivity. However, current understanding of the recycling mechanism in the synaptic vesicle cycle and different localization of CAM isoforms does not support this hypothesis. Alternatively, and specifically, CAMs on active terminals and fibers could be downregulated to serve as markers for the competence of the synapses for sprouting (Hiesinger, 1999 and references therein).

In wild-type third instar larvae Fasciclin II is found on R7 and R8 retinal axons. During certain phases of pupation, Fasciclin II is detectable at low levels on photoreceptor cell bodies. It is possible that Fasciclin II is never completely downregulated from R7 and R8 terminals but is expressed mostly below threshold for visualization with confocal microscopy. Upregulation of Fasciclin II levels in photoreceptors lacking functional Synaptobrevin after P + 75% may thus be attributable to an accumulation of the protein, when its downregulation would normally occur via a Synaptobrevin-dependent mechanism as part of a continuous protein turnover. The finding that IrreC-rst immunoreactivity remains unaltered in photoreceptors without functional Synaptobrevin, but is increased in proximal neuropils after widespread TeTxLC expression, can be interpreted in two different ways: either IrreC-rst protein is not present on photoreceptor terminals at the addressed time of pupation, or the Synaptobrevin-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding IrreC-rst is expressed on photoreceptors. In pupal stages IrreC-rst has been shown to be localized on rhabdomeres but not on axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to be a preferred localization for IrreC-rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation mechanism for photoreceptors (Hiesinger, 1999 and references therein).

Taken together, these results clearly show a requirement of Synaptobrevin for optic lobe development. Either Synaptobrevin has a previously unknown activity-independent function, or synaptic transmission is involved in optic lobe development, or both. Investigations of the underlying regulatory processes now have to be undertaken: immunoelectron microscopic analysis of CAM localization at neuron-neuron synapses and a possible causal link to neuronal activity during CNS development (Hiesinger, 1999).

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs have been performed. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (Fas II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that Fas II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, ectopic expression of Fas II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain (Kurusu, 2002).

Studies of MB development with mosaic clones have shown that MB neurons in the adult brain are classified into three groups that project dorsally to the alpha and alpha' lobes and medially to the ß, ß' and gamma lobes. Based on this classification, all the Kenyon cells born before the mid-third larval instar belong to the gamma group. Only in the late third instar, the second group of neurons projecting into the alpha' and ß' lobes is produced. In this study, using various MB markers, it has been demonstrated that the larval Kenyon cells can be further subdivided into distoproximal concentric groups surrounding each of the neuroblasts. Furthermore, the axonal projections of the Kenyon cells are also organized into concentric layers in the peduncle and lobes. Axons of newly born Kenyon cells project into the core that is constituted of densely packed thin fibers rich in actin filaments (Kurusu, 2002).

Distoproximal expression patterns of nuclear regulatory genes in the larval MB cell clusters have been described. In particular, whereas ey is expressed in all the MB cells, including the neuroblasts and ganglion mother cells (GMCs), dac is expressed in differentiated Kenyon cells but not in the centrally located proliferating cells. GAL4 MB markers, such as 201Y and c739, are expressed in an outer group of the differentiated Kenyon cells that is located several cell diameters away from the proliferating neuroblasts (Kurusu, 2002).

While the four MB neuroblasts continue dividing up to the late pupal stage supplying increasing numbers of Kenyon cells, the newly formed larval MB axons follow the medial and the dorsal lobe projections that were pioneered at the embryonic stage with a concomitant increase in the sizes of the lobes. By contrast, a set of genes is turned on in the Kenyon cells after the hatching of the first instar larvae in slightly different patterns in both the cell bodies and their projections. As development proceeds, these differential gene expression patterns became more evident in the second instar larval stage. While the Dac protein is expressed in most of the Kenyon cells, dnc-lacZ is expressed in a small subset of cells peripherally positioned in each of the Kenyon cell clusters originated by the four MB neuroblasts. Expression of 201Y is detected in another subset of cells located more centrally in each of the Kenyon cell clusters, whereas c739 is widely expressed in most of the Kenyon cells (Kurusu, 2002).

Remarkably, these differential expression patterns observed in the Kenyon cells were topologically reflected in their axonal projections in the peduncle and lobes: dnc-lacZ is detected in the outermost surface layer of the peduncle and lobes; 201Y is detected in both the surface and middle layers; and c739 is detected in most axons, a pattern similar to that of FAS II (Kurusu, 2002).

As development proceeds further, further subdivisions emerge in the third instar larval stage with increasing numbers of Kenyon cells and their axons. Moreover the expression patterns of the 201Y and c739 markers change in both cell bodies and their projections; 201Y is then detected in many of the Kenyon cells and their projections, obscuring the 201Y peripheral pattern in the previous larval instar; c739 is then detected in a group of cells located near each of the neuroblasts. The axons of the c739-expressing cells project into an inner layers of the peduncle and lobes. By contrast, dnc-lacZ is maintained in the peripheral subdivisions both in the Kenyon cells and their projections. Double staining with anti-Fas II antibody confirms discrete internal organization of the peduncle and lobes, which are concentrically subdivided into at least three layers surrounding a core that is not labeled with the MB markers, including Fas II (Kurusu, 2002).

Interestingly, the reporter molecule for dnc-lacZ exhibits a characteristic patchy appearance in the calyx, peduncle and lobes, suggesting uneven distribution of the dnc-lacZ fibers. Indeed, higher magnification of the calyces double labeled with anti-ß-gal and anti-synaptotagmin antibodies reveals extensive arborization of the dnc-lacZ expressing neurons around the synaptic terminals, which are likely to represent the afferent terminals of axonal collaterals of the antennocerebral neurons (Kurusu, 2002).

Based on these expression profiles of nuclear regulatory genes and GAL4 markers in the cell bodies, it is suggested that the Kenyon cells that are labeled with both Dac and 201Y project their axons into the concentric layers that also are labeled with Fas II. However, the proximally located Kenyon cells that are labeled with DAC but not 201Y may correspond to the newly differentiated MB neurons that project thin fibers into the core of the peduncle and lobes. Recently described (using a DsRed variant) has been a similar concentric generation of Kenyon cell fibers in the surrounding layers of the peduncle and lobes, in which younger axons extend into the inner layer to shift older fibers into the outer layers. Clonal studies on the larval projection patterns support this temporal order of layer generation and further show that axons of the newly produced Kenyon cells first project into the core as actin-rich thin fibers to shift to the surrounding layers as they undergo further differentiation (Kurusu, 2002).

A bundle of thin fibers centrally located in the peduncle and lobes has been described, and it has been postulated that the central fibers might be a set of larval fibers that remain throughout metamorphosis as a guide for the ingrowing imaginal fibers. The current results are consistent with this hypothesis and further demonstrate that the core fibers are derived from newly produced Kenyon cells to undergo a dynamic translocation to the surrounding layers as they differentiate. As the adult alpha' and ß' neurons are generated in the late larval stage and may project into the larval MB core, it is presumed that their axons might remain as premature core fibers during the early stages of metamorphosis (Kurusu, 2002).

Layer subdivisions of MBs have been described in other insects. The MB lobes of the honey bee Apis mellifera are subdivided into discrete layers, which correspond to the stratified arrangements of dendritic trees of efferent neurons. In the cockroach Periplaneta americana, afferent terminals segment the calyces into four discrete zones, which receive afferents from distinct sets of olfactory glomeruli. In the Drosophila larval MBs, it has yet to be seen how the layers of the peduncle and lobes are represented in the calyces and whether the layer subdivisions correspond to functional compartments that are wired to different sets of afferent interneurons of the larval antennal lobes. The dendritic arborization in the Drosophila larval calyces is highly condensed and hinders direct anatomical examination of its internal structure. It is speculated that micro subdivisions that are beyond the anatomical resolution achieved in this work could exist in the calyces as well (Kurusu, 2002).

The Fas II protein, a member of the Ig superfamily, mediates axon fasciculation through homophilic adhesion. In the Drosophila ventral nerve cord, Fas II is expressed on a subset of embryonic axons, many of which selectively fasciculate in three distinct longitudinal axon pathways. In fas II loss-of-function mutants, the axons that normally fasciculate together in the three Fas II pathways fail to do so and these axon fascicles do not form. However, overexpression of the Fas II protein results in a gain-of-function phenotype in which pairs of pathways that should normally remain separate instead become abnormally joined together, indicating that Fas II controls specific patterns of selective fasciculation and axon sorting in the central nervous system. Notably, Fas II is not required for several aspects of growth cone guidance: despite the severe defects in fasciculation, the follower growth cones find their way normally in the ventral nervous system (Kurusu, 2002).

These in vivo functions of Fas II in the ventral nerve cord correspond with the loss- and gain-of-function phenotypes in the developing MBs. Although the axons of the embryonic MBs initially grow along the Fas II-expressing cells, they can find their pathways in the developing brain in the absence of the Fas II protein. Later in the larval stages, decreases in Fas II protein level result in abnormal development of the lobes and the internal layers. Particularly noteworthy is that Fas II is intrinsically required for the clonal integrity of the axonal fascicles and hence generation of correct organization of the internal layers (Kurusu, 2002).

In the course of MB development, Fas II becomes detectable at late stage 17 in the embryonic peduncle and lobes. Later in the larval stages, Fas II is expressed in the MB layers but not in the core fibers. The robust gain-of-function phenotypes caused by the ectopic overexpression of Fas II argue for the functional importance of this temporal and spatial regulation of Fas II expression in MB development. Ectopic Fas II expression with OK107 in MB axons including the newly generated core fibers results in major disruption of the branching patterns of the lobes whereas overexpression with 201Y only in the surrounding layers results in no abnormality. These results on the Fas II functions emphasize the importance of cell adhesion properties for the correct branching of the MB lobes and the development of the internal axonal layers (Kurusu, 2002).

It has long been appreciated that the mammalian cerebral cortex is organized into layers, which are connected to different functional neural circuits. Neurons in different layers are generated at different stages during development and migrate away from the ventricular zone, where they are generated. As a result, the deepest layers are formed by neurons born at early stages and the more superficial layers are formed by neurons that are born later and migrate past the deep layers. Similarly, during vertebrate retinal development, six types of neurons and one type of glia are generated in an orderly fashion and form discrete layers (Kurusu, 2002).

In this study, it has been shown that the Drosophila MB cell bodies and their axonal projections are organized into layers. Furthermore, there is a temporal sequence in layer formation, in which younger neurons project first into the core to shift to the surrounding layers as they differentiate. Unlike the mammalian cortex, neuronal migration has not been demonstrated so far in MB development. However, MB neurons originating from the quadruple Kenyon cell clusters initially form four axon bundles, yet eventually converge into a single tract in the peduncle: a process that calls for dynamic sorting and fasciculation of the growing axons during the formation of the ordered internal layers of the peduncle and lobes. The identification of cell adhesion molecules that underlie this process is a subject for intriguing future investigation. Determining how different type neurons are sequentially generated and allocated to different topographical subdivisions is enormously important for developmental neuroscience. It is anticipated that studies of the MB development in the Drosophila brain will lead to important insights into the molecular mechanisms that control the sequential generation of neurons and their positioning into layers during the development of both vertebrate and invertebrate brains (Kurusu, 2002).

Ras-dependent signaling cascade regulates Fasciclin II-mediated cell adhesion at synaptic terminals during larval synaptic growth

Ras proteins are small GTPases with well known functions in cell proliferation and differentiation. In these processes, they play key roles as molecular switches that can trigger distinct signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide-3 kinase pathway, and the Ral-guanine nucleotide dissociation stimulator pathway. Several studies have implicated Ras proteins in the development and function of synapses, but the molecular mechanisms for this regulation are poorly understood. The Ras-MAPK pathway is involved in synaptic plasticity at the Drosophila larval neuromuscular junction. Both Ras1 and MAPK are expressed at the neuromuscular junction, and modification of their activity levels results in an altered number of synaptic boutons. Gain- or loss-of-function mutations in Ras1 and MAPK reveal that regulation of synapse structure by this signal transduction pathway is dependent on Fasciclin II localization at synaptic boutons. These results provide evidence for a Ras-dependent signaling cascade that regulates Fasciclin II-mediated cell adhesion at synaptic terminals during synapse growth (Koh, 2002).

Synapse stability and synapse expansion during muscle growth are regulated by changes in FasII expression at presynaptic and postsynaptic membranes and FasII expression is in part controlled by electrical activity. One mechanism through which electrical activity alters FasII levels is by regulating its synaptic clustering via CaMKII-dependent phosphorylation of Discs large. An additional mechanism by which the levels of FasII at the presynaptic terminal are modified has been documented in this study: the activation of the Ras-MAPK pathway. This redundant mechanism may serve the differential regulation of FasII localization at the presynaptic and postsynaptic site or may represent FasII regulation in response to different signals. Whereas activation of CaMKII is elicited by an increase in electrical activity, activation of the MAPK pathway may be triggered by activity or by an as yet unknown but different signaling mechanism (Koh, 2002).

Studies in Aplysia indicate that activity-dependent endocytosis of ApCAM results in an increase in the number of synaptic contacts during long-term facilitation. ApMAPK is likely to induce ApCAM internalization in a process that depends on ApMAPK activity in dissociated neurons. However, its involvement in the intact organism has not been tested (Koh, 2002 and references therein).

In this study, Drosophila larval neuromuscular synapses have been used to determine the involvement of the Ras-MAPK pathway in the regulation of synaptic FasII levels and in morphological synaptic plasticity. Both Ras and MAPK are expressed at the NMJ, where they regulate presynaptic expansion. This regulation is accomplished by altering FasII levels at synaptic boutons. A ras hypomorph mutant and anti-Ras antibodies have been used to determine that Ras1 is specifically expressed at the larval NMJ. Although Ras1 immunoreactivity at synapses and muscles is severely reduced in ras1 hypomorphic mutants, nuclear staining persists (Koh, 2002).

Two antibodies were used to demonstrate the synaptic localization of MAPK at the NMJ, an antibody that recognizes all forms of the MAPK Rolled (DmERK-A) and an antibody that exclusively labels active, double-phosphorylated MAPK (DpMAPK). Interestingly, although both antibodies labeled synaptic boutons, their distribution was not identical. In particular, the antibody against active MAPK-labeled hot spots was more restricted in its localization than general MAPK staining. This suggests that active MAPK is recruited to specific domains within the synaptic bouton or that MAPK activation occurs at discrete regions within the boutons. Interestingly, the same domain that is occupied by active MAPK has lower levels of FasII, consistent with the idea that MAPK activation might be involved in the downregulation of FasII. It has been suggested that the regions of low FasII concentration correspond to the active zone, suggesting that active MAPK is localized to the active zone. The localization pattern of Ras1 and MAPK at synapses is also consistent with the localization protein 14-3-3, another protein that has been involved in the Ras1-Drosophila Raf-MAPK signal transduction pathway (Koh, 2002).

Expression of constitutively active Ras (Ras1V12) drastically increases the number of synaptic boutons. This change is indistinguishable from the increase in boutons observed in the Ras1V12S35 variant and the constitutively activated Raf^F179, suggesting that these changes are induced by activation of the MAPK pathway. Consistent with these results is the observation that a hypomorphic mutation in ras1, ras1⁵⁷⁰³, has the opposite phenotype, a decrease in bouton number, and that a gain-of-function mutation in rl leads to an increase in bouton number. The finding that Ras1V12 and Ras1V12S35 elicit identical phenotypes at the NMJ is consistent with findings in other tissues, such as in the retina, in which the epidermal growth factor receptor-Ras1 pathway is involved in photoreceptor survival, or in the wing discs, where the Ras pathway is involved in hyperplastic growth (Koh, 2002).

Notably, expression of Ras variants that activate the PI3-K and Ral signal transduction pathways and a constitutively active RalA also induce an increase in bouton number that is similar in extent to RasWT and considerably lower than Ras1V12. These results raise the possibility that Ras1V12G37 and Ras1V12C40 may still retain some degree of affinity for Raf or, alternatively, that other Ras-mediated pathways might also influence NMJ development. All known ras genes encode a protein region, the effector loop, that is highly conserved in all species. Mutations in this loop interfere with the ability of Ras to bind to specific effectors without altering its catalytic activity. A series of mutations in the effector loop that allow almost exclusive activation of a single effector havs been isolated in mammals. The specificity of these mutants has been tested by in vitro binding assays as well as by genetic and biochemical approaches in cell culture. In Drosophila, a genetic approach has been used to demonstrate specificity. These studies suggest that Ras1V12 and RasV12S35 phenotypes are emulated by a hyperactivated form of Raf and suppressed by Raf, MEK, and MAPK mutants (Koh, 2002).

Studies in vertebrate cells and in Drosophila suggest that although Ras activation by receptor tyrosine kinases is blocked by the putative dominant-negative RasN17, Ras activation by PKC and the Ras1V12C40/PI3-K effect on cytoskeletal reorganization in fibroblasts are not. At the NMJ, Ras1N17 does not behave as a dominant negative. Thus, taken together, this analysis of NMJ structure in the different Ras strains suggests that Ras1 regulates the number of type I glutamatergic synapses in Drosophila and this regulation depends to a considerable extent on the activation of the MAPK pathway. Although activation of PI3-K and Ral-GDS-Ral by presumably PKC activation also points to a role for these pathways, their effect on NMJ growth is less prominent than the MAPK pathway (Koh, 2002).

Immunocytochemical studies of FasII immunoreactivity at synaptic terminals of MAPK gain- and loss-of-function mutants suggest that MAPK regulates levels of synaptic FasII, a cell-adhesion molecule that plays a key role in the maintenance and expansion of NMJs in Drosophila. This model was supported by experiments in which only type I synaptic FasII was immunoprecipitated. This was accomplished by using anti-DLG antibodies, because DLG binds directly to FasII at type I boutons but not at other bouton types. The immunoprecipitation experiments demonstrate that enhancing the levels of MAPK activity at synaptic terminals results in a reduction of type I synaptic FasII. Conversely, decreasing levels of MAPK activity results in an increase in type I synaptic FasII levels. These results are in agreement with the studies in Aplysia dissociated neurons, which show that ApMAPK is involved in the internalization of ApCAM (Koh, 2002).

Additional support for the idea that the changes in bouton number elicited by alterations in Ras1 and MAPK activity are mediated by alterations in FasII levels was demonstrated by examining the overall expression of FasII in MAPK gain- or loss-of-function alleles, examining the distribution of FasII within single synaptic boutons in relation to active MAPK, and using hypomorphic fasII mutants. The studies with rl mutants demonstrate that there is an inverse relationship between levels of synaptic FasII and MAPK activity. Furthermore, active MAPK localization coincides with regions of the bouton that have no or low FasII levels (Koh, 2002).

Two main functions of FasII in the regulation of synapse number have been demonstrated. (1) FasII is critically required for synapse maintenance: below threshold FasII levels, synaptic boutons are not maintained. (2) FasII operates by constraining synaptic growth, similar to the Aplysia system. Therefore, a decrease in FasII to a level still sufficient for maintenance results in an increase in synaptic arbor size. On the basis of this model, the following interpretation of the results is proposed. The dramatic decrease in FasII levels in the homozygous fasII mutant does not allow any influence of MAPK activity changes on NMJ structure. Similarly, when FasII levels are decreased to approximately one-half the wild-type levels (fasII^e76/+), an increase in MAPK activity does not induce an additional increase in bouton number, probably because an additional decrease in FasII compromises synaptic maintenance, thus preventing NMJ growth. However, the increase in FasII levels induced by a reduction of MAPK activity (rl^10a/+) in a fasII^e76/+ background suppresses the increase in boutons observed in fasII^e76/+ alone. This result suggests that MAPK regulates FasII levels and exists upstream of FasII at signal transduction pathways that regulate the number of type I synaptic boutons (Koh, 2002).

Notably, the hypomorph rl^10a/+ has no significant decrease in bouton number, although these mutants have a striking increase in FasII levels compared with wild-type controls. An explanation for this result is that FasII is a homophilic cell-adhesion molecule that is required both in the presynaptic and in the postsynaptic cell for function. If the Ras-MAPK pathway functions to regulate FasII at the presynaptic cell, as suggested by studies with cell-specific Gal4 drivers, then an asymmetric increase in FasII levels in the presynaptic cell alone may not have much of an effect. Previous studies also show that although the NMJ is very sensitive to a decrease in FasII levels, an increase in FasII over wild-type levels does not have much of an effect (Koh, 2002).

Although the results are consistent with a regulation of FasII-mediated synapse growth by the Ras-MAPK pathway, it is important to note that several other molecules in addition to FasII are involved in the regulation of synapse growth. Moreover, several studies suggest that many changes at the fly NMJ are compensated by yet unknown homeostatic mechanisms. Therefore, further understanding of these regulatory and compensatory signals will be necessary to fully explain these observations (Koh, 2002).

In conclusion, a signaling pathway intimately involved in the regulation of synaptic growth at the NMJ has been identified. Identification of the mechanisms involved in the activation of this pathway may provide valuable clues toward understanding the plasticity of this synapse (Koh, 2002).

Control of axon-axon attraction by Semaphorin reverse signaling

Semaphorin family proteins are well-known axon guidance ligands. Recent studies indicate that certain transmembrane Semaphorins can also function as guidance receptors to mediate axon-axon attraction or repulsion. The mechanisms by which Semaphorin reverse signaling modulates axon-surface affinity, however, remain unknown. This study reveals a novel mechanism underlying upregulation of axon-axon attraction by Semaphorin-1a (Sema1a) reverse signaling in the developing Drosophila visual system. Sema1a promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. It is proposed that Sema1a reverse signaling activates Moe, which in turn upregulates Fas2-mediated axon-axon attraction by inhibiting Rho1 (Hsieh, 2014).

Oogenesis

Little is known about how intercellular communication is regulated in epithelial cell clusters to control delamination and migration. This problem has been investigated using Drosophila border cells as a model. Just preceding cell cluster delamination, expression of transmembrane immunoglobulin superfamily member, Fasciclin 2, is lost in outer border cells, but not in inner polar cells of the cluster. Loss of Fasciclin 2 expression in outer border cells permits a switch in Fasciclin 2 polarity in the inner polar cells. This polarity switch, which is organized in collaboration with neoplastic tumor suppressors Discs large and Lethal-giant-larvae, directs cluster asymmetry essential for timing delamination from the epithelium. Fas2-mediated communication between polar and border cells maintains localization of Discs large and Lethal-giant-larvae in border cells to inhibit the rate of cluster migration. These findings are the first to show how a switch in cell adhesion molecule polarity regulates asymmetry and delamination of an epithelial cell cluster. The finding that Discs large and Lethal-giant-larvae inhibit the rate of normal cell cluster movement suggests that their loss in metastatic tumors may directly contribute to tumor motility. Furthermore, these results provide novel insight into the intimate link between epithelial polarity and acquisition of motile polarity that has important implications for development of invasive carcinomas (Szafranski, 2004).

Drosophila border cells (BCs) provide a simple in vivo model for deciphering the mechanisms of cell cluster movement. BCs are a cluster of six to eight somatic follicle cells that differentiate within the anterior follicular epithelium during mid oogenesis, maintaining some aspects of epithelial polarity, while losing others. The BCs then delaminate from the follicular epithelium, a process that requires the polarized cluster to coordinately break contact from adjacent epithelial cells, while simultaneously directing invasion between the germ cells. Once the BCs exit the epithelium, they take ~6 hours to migrate roughly 150 µm between the nurse germ cells to the oocyte (Szafranski, 2004).

The BC cluster includes two polar cells (PCs) that reside at the center of the cluster and that do not contact the migration substrate. Preceding BC differentiation, PCs secrete Unpaired, which determines how many adjacent epithelial cells will activate the Jak-Stat pathway, and thus become BCs. Stat is sufficient for expression of the C/EBP transcription factor, Slow Border Cells (Slbo). Slbo directs BC differentiation and upregulation of E-Cadherin (Shotgun), a cell-adhesion molecule essential for movement. Two tyrosine kinase receptors that appear to function redundantly, Pvr and Egfr, guide the BCs to the oocyte. In contrast to these genes, Discs large (Dlg) prevents BCs from reaching the oocyte prematurely (Szafranski, 2004).

Several functions are thought to be essential for movement as a cluster, as opposed to single cells: (1) intercellular interactions that modulate movement; (2) directional mass motion between cells individually capable of motion in any direction; (3) determination of locomotive-active regions of individual cells, and (4) integration of these processes through organization of cluster polarity. In vertebrates, expression of cell-adhesion molecules in a subset of cells within a migrating cluster has suggested that they may have the requisite properties to execute these functions. Genetic and cell biological analysis provides the first direct evidence that a cell adhesion molecule, Fas2, organizes the activities outlined above. A model is presented that explains how Fas2 organizes epithelial clusters to control delamination and migration. Just preceding cluster migration, Fas2 is polarized in PCs with an orientation that predicts the direction of BC movement. Polarization precedes delamination and migration by 4-6 hours. Furthermore, Fas2 polarity is not perturbed in clusters that fail to migrate. It is concluded that Fas2 polarization is not a consequence of delamination or migration. The timing of Fas2 polarization is determined by the temporal specificity of developmentally programmed loss of Fas2 in surrounding BCs at stage 8. Since loss of Fas2 in anterior follicle cells is controlled transcriptionally, and includes stretch cells, not just BCs, it is unlikely that BC specific transcription factors such as Slbo, Jing or Stat control developmentally programmed loss of Fas2 expression. Other factors, such as Eyeless, are expressed in precisely the cells in which Fas2 is lost, while being lost in PCs. Eyeless thus may be part of a transcriptional regulatory network that developmentally programs loss of Fas2 expression, as well as programs expression of other genes specifically involved in morphogenesis of anterior follicle cell motility (Szafranski, 2004).

How does developmentally programmed loss of Fas2 expression in BCs permit Fas2 polarization in PCs? The data indicate that this is a multistep process. Initially, Fas2 homophilic interactions between BCs and PCs are lost, and several experiments indicate that they are replaced by Fas2 heterophilic interactions with a putative BC receptor. These interactions are essential for maintaining Fas2 in PC membranes contacting BCs. Next, loss of Fas2 from BCs causes relocation of the majority of PC Fas2 to the interface between PCs, where it is maintained because of homophilic interactions with Fas2 from the adjacent PC. In support of this interpretation, misexpression of Fas2 in PCs appears to oversaturate Fas2 between PCs, causing its circumferential accumulation at the contact sites with BCs. It is concluded that the accumulation of Fas2 between PCs ensures that Fas2 is kept at sufficiently low levels at the sites of contact with BCs to allow its polarization to the leading half of PCs. Fas2 polarization is then directed by PC Dlg and Lgl, as evidenced by the observation that loss of function of either protein causes loss of Fas2 polarity. However, Fas2 can also polarize Dlg and Lgl; loss of Fas2 causes loss of Dlg and Lgl polarity, while ectopic Fas2 redirects Dlg and Lgl localization. Thus, Fas2 is in a positive feedback loop with Dlg and Lgl that ensures the build up of a PC signaling and adhesion complex at the leading half of the PCs. These data indicate that Fas2 is involved in intercellular interactions crucial for organizing polarity, an important criterion for a function specifically involved in regulation of motility in multicellular clusters (Szafranski, 2004).

Significantly, the results indicate that molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. The timing of the reorganization of epithelial polarity is crucial for timing delamination. Fas2 therefore plays a direct role in mediating intercellular interactions that modulate movement, a second property proposed for a function specifically involved in regulating cluster motility as opposed to single cells. It is concluded that the Fas2 morphogentic switch facilitates development of motile polarity essential for timely BC delamination. A similar switch mechanism may be important in other processes that crucially depend on timing of Fas2 activity, such as axon pathfinding, and learning and memory (Szafranski, 2004).

Fas2 polarity appears to compartmentalize PCs into distinct functional domains in order to control functionally distinct intercellular communication with leading versus trailing BCs. Leading BCs play a functionally distinct role by pioneering invasion between germ cells while simultaneously detaching from the epithelium. Trailing BCs are likely to play a less active role in invasion, but must mediate precisely timed detachment from the epithelium. Fas2 polarization is thus likely to be crucial for facilitating coordination of the distinct functional requirements of leading versus trailing BCs, by establishing distinct sets of intercellular contact and communication between the PCs and leading versus trailing BCs. In support of this hypothesis, previous studies have suggested that leading and trailing BCs are functionally distinct. In BC clusters comprising a mixture of wild type and slbo, jing, taiman or DE-cadherin mutant cells, wild-type BCs always lead invasion. Furthermore, additional structural evidence has been documented for cluster asymmetry. Amphiphysin, a vesicle trafficking protein that regulates Dlg and Lgl localization, is expressed at higher level in trailing BCs compared to leading BCs. Amph, Dlg and Lgl, are thus good candidates for proteins that differentially regulate cortical and cell surface activities needed to mediate distinct interactions of leading and trailing BCs with adjacent epithelial cells and germ cells during the delamination process (Szafranski, 2004).

Since only Dlg and Lgl are mislocalized in Fas2 clusters, but not Fas3, alpha-Spec or Crb, the data suggest that Fas2 directs localization of specific molecules within distinct regions of different cells of the cluster to control motility. A putative Fas2-binding BC receptor may be another molecule whose polarity is controlled by Fas2. Interaction with this putative receptor appears to facilitate organization of the global polarity of the cluster, since the orientation of delamination, mediated by the BCs, directly correlates with Fas2 polarity in PCs. These data thus suggest that Fas2 coordinates directional mass motion between cells that are potentially capable of motion in any direction, and that it helps to determine the locomotive-active regions of these cells, additional criteria for a function specifically involved in regulating cluster motility. Thus, because Fas2 is required for regulation of several activities that distinguish how single cells versus clusters move, the data provide the first molecular model for understanding the organization of epithelial cluster polarity during delamination and movement. One argument against this proposal might be that the PCs appear to be highly specialized. However, it is thought that this is likely to be of less significance, since PCs express epithelial polarity proteins in a pattern similar to adjacent follicle epithelial cells (Szafranski, 2004).

As has been shown for BC clusters, several vertebrate studies have shown that transmembrane proteins are differently expressed within different cell subpopulations in migrating clusters. Furthermore, the structure and functions of Fas2, Dlg and Lgl homologs are conserved across phylogeny. Thus, the involvement of Fas2, Dlg and Lgl in organizing cell cluster motility also may be conserved. It is concluded that although the precise mechanism of cluster movement may not be conserved in vertebrates, the information gleaned about how BCs regulate epithelial polarity to dynamically organize cluster polarity and movement will be generally useful for understanding how cell cluster motility is organized across phylogeny (Szafranski, 2004).

The role of Fas2 in regulating migration is discussed. Loss- and gain-of-function experiments demonstrate that PC Fas2 acts as a signal to inhibit the rate of BC migration. This work builds on previous studies demonstrating the importance of PCs in determining BC fate. However, this work is the first example of an intercellular signal that specifically organizes cluster movement, rather than determining cell fate. Fas2 clearly has a signaling function, since PCs do not contact the migration substrate. Thus, these data demonstrate for the first time the existence of intercellular communication between cells of a migratory cluster, that is specifically required to modulate migration (Szafranski, 2004).

PC Fas2 signaling inhibits the rate of cluster movement by maintaining Dlg and Lgl localization in BCs. The putative BC receptor with which Fas2 interacts may control Dlg and Lgl localization in BCs. Since Dlg is localized to the cortex of BCs, Dlg must inhibit the rate of migration through cortical activities in BCs. One cortical activity controlled by Dlg is the recruitment of Lgl to the membrane. Since lgl clusters have very similar migration phenotypes to dlg clusters, the data indicate that Lgl and Dlg cooperate to inhibit BC movement. The importance of Dlg and Lgl in regulating cell movement probably derives from the same scaffolding activities they use to organize and control membrane, cytoskeletal and signaling specialization during the polarization of epithelial and neuronal cells. It is proposed that Dlg and Lgl scaffolding organizes and integrates transmembrane signaling and adhesion proteins with signaling, trafficking and cytoskeletal effectors in the cortex of BCs to mediate contact-inhibition of cluster movement (Szafranski, 2004).

BCs resemble mutant dlg invasive tumor cells in that they lose epithelial polarity by accumulating Dlg and Lgl around their circumference, but in contrast to BCs, mutant dlg tumor cells migrate between germ cells without temporal or spatial control. The data demonstrate that Dlg and Lgl not only control polarity and delamination of epithelial clusters, but also actively inhibit movement. Thus, mutant dlg tumor invasion is likely to be caused by a combination of loss of epithelial polarity and over-activation of motility pathways. In this context the results appear to be paradoxical in that loss of epithelial polarity is generally considered to be crucial for facilitating acquisition of motility, but it is seen that loss of polarity in normal migrating clusters delays initiation of movement. The data resolve this paradox in that during normal development, molecules used for polarizing epithelial cells are reorganized to polarize a motile cell cluster. It therefore seems likely that in carcinomas, inappropriate loss of epithelial polarity simultaneously disrupts acquisition of motile polarity, but this phenomenon is not appreciated because ultimately the tumor cells migrate. Thus, it is postulated that overactivation of motility pathways, as is seen with loss of Dlg and Lgl in BCs, may be especially crucial for achieving carcinoma invasion. Consistent with this hypothesis, some dlg mutations that cause loss of epithelial polarity do not lead to tumor invasion, suggesting that acquisition of motility is a separate Dlg function (Szafranski, 2004).

Gene expression data for human cancers suggests that mutations that promote tumor formation, through loss of epithelial polarity and increased proliferation, may be the same mutations that subsequently cause tumor cell invasion. Based on the observation that Dlg is required to maintain polarity, inhibit proliferation and inhibit movement, it is proposed that tumor suppressors such as Dlg that regulate signaling and adhesion at epithelial junctions may unify human gene expression data by providing an ultrastructural target that controls contact inhibition of both proliferation and movement. Progressive deterioration of epithelial junctions may thus provide a common mechanism through which multiple tumor suppressor pathways impact the cascade from cell proliferation to tumor invasion, either through mutation or mislocalization of critical junctional proteins (Szafranski, 2004).

Lin-28 regulates oogenesis and muscle formation in Drosophila melanogaster

Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation (Stratoulias, 2014).

The Cold-Shock Domain (CSD) protein Lin28 was initially identified in Caenorhabditis elegans (C. elegans) as a component of the heterochronic pathway that regulates the timing of cell fate specification (Ambros, 1984). Subsequent discovery of gene expression regulation through small non-coding RNAs clarified the role of Lin28 in this pathway. The lin-28 mRNA is a conserved target of the let-7 micro-RNA (miRNA) family both in C. elegans and vertebrates. On the other hand, Lin28 inhibits let-7 processing. At the molecular level, Lin28 protein interacts with the let-7 precursor (pre-let-7), resulting in inhibition of let-7 maturation. The let-7 inhibition occurs through the physical interaction of the pre-let-7 loop and Lin28 protein, preventing further processing of pre-let-7 towards the mature form of let-7. Together, these interactions create a feedback loop between Lin28 and let-7, leading to a strict regulation of let-7 maturation (Stratoulias, 2014 and references therein).

Lin28 raised further interest when it was used, along with Nanog, to replace the factors c-Myc and Klf4 in somatic cell reprogramming. These experiments, together with data from human embryonic stem cells, underscored the important role of lin-28 in pluripotency regulation and maintenance. Besides acting as a negative regulator of let-7 maturation, Lin28 has also been shown to have a direct effect on translation through the recruitment of the RNA Helicase A. This mode of function, independent of let-7 maturation, has been demonstrated in the case of Insulin-like Growth Factor 2 during mouse myogenesis. Lin28 binding on IGF-2 mRNA increases its translation efficiency and therefore facilitates skeletal myogenesis in mice (Stratoulias, 2014 and references therein).

The Lin28 protein is composed of four domains: a positively charged linker that binds two Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs to the CSD. In mammalian genomes, two paralogs of lin-28 are found, Lin28A and Lin28B. While Lin28B represses let-7 processing in the nucleus to prevent the formation of the precursor form from the primary let-7, Lin28A also blocks cytoplasmic processing of let-7 (Piskounova, 2011). It has recently been shown in mouse that deletion of the Lin28 linker domain alters the protein’s three-dimensional structure and is sufficient to disrupt sequestration of the precursor form of let-7 (pre-let-7) (Stratoulias, 2014).

The miRNA let-7 family is conserved across diverse animals, functioning to control late temporal transitions during development. During the last decade, the involvement of let-7 in regulating cell differentiation has been analyzed in various contexts, including neural cell specification, stem cell maintenance and hematopoietic progenitor differentiation. While eight different let-7 miRNA genes are annotated in the human genome, only one is found in Drosophila melanogaster. Like in C. elegans, in Drosophila the loss of let-7 expression leads to the modification of temporal regulation of the metamorphosis process. During fly metamorphosis, the expression of let-7 complex (let-7C), a polycistronic locus encoding the let-7, miR-100 and miR-125 miRNAs, is under direct control by the steroid hormone ecdysone. Ecdysone is the central regulator of insect developmental transitions. Therefore, let-7 has been proposed to be part of a conserved, ecdysone regulated pathway that controls the timing of the larva to adult transition (Stratoulias, 2014).

In addition to affecting the metamorphosis clock, Sokol and colleagues have shown that the let-7 deletion also affects the neuromuscular remodeling that takes place during the larva to adult transition. During neuromuscular remodeling, and under normal conditions, the dorsal internal oblique muscles (DIOMs) disappear 12 hours after emergence of the adult fly from the pupa. However, the adult let-7 mutants retain the DIOMs through adulthood. Deletion of the let-7 gene is sufficient to induce this phenotype, while deletion of either miR-100 or miR-125 genes is not enough to recapitulate the DIOM phenotype. Furthermore, let-7 has been shown to govern the maturation of neuromuscular junction of adult abdominal muscles, through regulation of Abrupt expression (Stratoulias, 2014 and references therein).

While previous studies have demonstrated that the let-7 target Abrupt and ecdysone signaling are required for oogenesis in fruit fly ovaries, and that the let-7 miRNA family is abundantly expressed both in newborn mouse ovaries and in fly ovaries, no study has been conducted on the role of Lin-28/let-7 network in Drosophila ovaries. Therefore, a study was undertaken of the effects of lin-28 during Drosophila melanogaster development from the egg to the adult, and more particularly during oogenesis (Stratoulias, 2014).

A lin-28 mutant was generated, and the consequent increase of let-7 maturation was validated. lin-28 knockout resulted in reduced muscular performance and defects in DIOM morphogenesis. These results were in line with the let-7 knock out muscular phenotype described earlier. Moreover, this study identified multiple defects during oogenesis due to abnormal follicle and germline stem cell (FSCs and GSCs respectively) differentiation. A link is proposed between ovarian defects and ectopic expression of Fasciclin2 (Fas2), a known downstream target of the Ecdysone pathway, and a predicted let-7 target (Stratoulias, 2014).

Because of their role during stem cell differentiation, members of the let-7 miRNA family have been extensively studied. However, the role of lin-28 is still poorly documented. Deletion of let-7 in Drosophila impairs the musculature remodeling during the larva to adult metamorphosis. For instance the DIOMs, muscles which are required for eclosion and which are lost within 12 hours after eclosion, they are maintained during adulthood upon let-7 deletion. By generating the first lin-28 deletion in flies, this study has successfully confirmed the involvement of Lin-28/let-7 regulatory network in DIOM remodeling. This study has shown that deletion of lin-28 leads to over maturation of let-7, which negatively affects, and sometimes prevents DIOM formation. This drastic phenotype leads to a suboptimal muscular phenotype. However, due to a variable penetrance of the lin-28 deletion phenotype, a proportion of the flies could eclose and live as fertile animals (Stratoulias, 2014).

In addition, a link was discovered between Lin-28 function and oogenesis. The data indicates a role of let-7 during GSC differentiation and egg chamber formation. Because of the importance of these processes, let-7 maturation has to be strictly regulated by Lin-28 activity. It is suggested that a potential network involving Lin-28/let-7/Ecdysone signaling/Abrupt/Fas2 is needed during GSC differentiation and BC migration. The role of Abrupt in downregulating the steroid hormone Ecdysone has previously been demonstrated. Indeed, the loss of Taiman, a target of the transcription factor Abrupt and co-activator of Ecdysone receptor, leads to an increase of undifferentiated GSCs in the germarium due to disruption of Ecdysone signaling. Therefore, by regulating the expression pattern of Abrupt, Lin28/let-7 may adjust the domain of Ecdysone activity, providing a control over the GSCs differentiation and egg chamber maturation during the oogenesis. Indeed, it has been shown that the Ecdysone titre rises during oogenesis at stage 9. While the precise Ecdysone expression pattern is not known, it is suggested that the uniform EcR expression pattern in follicle cells in lin-28 mutants may break the Ecdysone signaling asymmetry needed during proper oogenesis (Stratoulias, 2014).

Furthermore, a previous study demonstrated the activation of let-7 expression via Ecdysone activity. This study showed that lin-28 deletion, resulted in the alleviation of Lin28's inhibitory role on let-7 maturation. This led to loss of Abrupt, which in turn inhibited Ecdysone activity and maintained Fas2 expression, resulting in BC migration impairment. To test whether the increase of Ecdysone signaling amplifies let-7 expression through a positive feedback loop, a system was generated in which there is no control of either let-7 expression nor of Ecdysone activity. This situation leads to an early cyst fusion, a loss of proper GSC differentiation and a mitotic defect, as was observed in the homozygous lin-28^dF30 ovaries. The accumulation of these defects may be enough to trigger apoptosis at mid-oogenesis, a well-known checkpoint previously described (Stratoulias, 2014).

Interestingly, the variable penetrance of the phenotype allows proper oogenesis and appearance of subfertile adult flies. This suggests a robust molecular network where feedback loops can rescue the system if one component disturbs the balance (Stratoulias, 2014).

By combining these results with previously published studies, a conserved link is suggested between hormonal signaling and germline stem cell differentiation, involving the let-7 miRNA family. This suggestion is reinforced in the last couple of years by the discovery of dormant ovarian follicles and mitotically active germ cells in adult mammalian ovaries, which are responsive to gonadotropin hormone. Moreover, it has been demonstrated that Lin-28 is involved in germline stem cell regulation in human ovary and in the ovarian surface epithelium of severe ovarian infertility patients axonal projection is critical for assembly of a functional sensory circuit (Stratoulias, 2014).

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 pins^p62 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 30 min. 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 arm^XP33) 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 Fas2^G0336, 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 Fas2^G0336 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).

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