InteractiveFly: GeneBrief

sidestep: Biological Overview | References

BIOLOGICAL OVERVIEW

During development of the Drosophila nervous system, migrating motor axons contact and interact with different cell types before reaching their peripheral muscle fields. The axonal attractant Sidestep (Side) is expressed in most of these intermediate targets. This study shows that motor axons recognize and follow Side-expressing cell surfaces from the ventral nerve cord to their target region. Contact of motor axons with Side-expressing cells induces the down-regulation of Side. In the absence of Side, the interaction with intermediate targets is lost. Misexpression of Side in side mutants strongly attracts motor axons to ectopic sites. Evidence is provided that, on motor axons, Beaten path Ia (Beat) functions as a receptor or part of a receptor complex for Side. In beat mutants, motor axons no longer recognize Side-expressing cell surfaces. Furthermore, Beat interacts with Side both genetically and biochemically. These results suggest that the tracing of Side-labeled cell surfaces by Beat-expressing growth cones is a major principle of motor axon guidance in Drosophila (Siebert, 2009).

Wiring of the nervous system is a precisely controlled process that includes axon outgrowth, axon pathfinding, target recognition, and synapse formation. Outgrowing axons of the same subtype often join and migrate collectively toward their targets, a process called selective fasciculation. Upon arrival in the target region, a subset of growth cones must inevitably defasciculate from the main pathway to select a specific synaptic target. Advancing growth cones are believed to express appropriate receptors that detect and evaluate relevant guidance molecules presented by surrounding cells and tissues. Despite the discovery of several conserved regulators of axonal pathfinding, the spatiotemporal sequence of molecular events that steer growth cones is still unclear. How exactly do growth cones recognize and interpret guidance cues to make pathway decisions? Over the years, several models have been proposed to describe general features of axon navigation across species, including the chemoaffinity hypothesis, the guidepost cell hypothesis, the blueprint hypothesis, and the labeled pathways hypothesis (Siebert, 2009).

The labeled pathways hypothesis postulates that a small number of early differentiating neurons pioneer a stereotypic array of differentially labeled axonal pathways. These primary pathways are selectively recognized by subsequently developing growth cones. While follower axons migrate along a homogenous substrate toward their target region, pioneer axons have to interact with many different substrates, actively search for guidance cues, and interpret relevant guidance information. Pathfinding decisions for pioneer axons are greatly simplified if directional cues would be present in the substrate. However, the experimental evidence for the existence of such substrate pathways is scarce. It has been noted that projections of sensory axons in homeotic mutants of Drosophila involved the specific recognition of 'pre-existing trails.' Similarly, axons emanating from transplanted eyes or Mauthner neurons in Xenopus have been observed to project along defined tracks called 'substrate pathways'. Furthermore, it has been reported that chick optic axons are guided along a preformed adhesive pathway. Despite the appealing simplicity of substrate pathways, the molecular tags that provide directional information have not been identified (Siebert, 2009).

One possible molecule that could serve as a directional cue for Drosophila motor axons is the attractant Sidestep (Side), a transmembrane protein of the immunoglobulin superfamily that is dynamically expressed during embryogenesis but prominently enriched in muscles when motor axons arrive in their target areas (Sink, 2001). In side mutant embryos, motor axons fail to defasciculate at key choice points and hence bypass their muscle targets, suggesting that Side functions as a target-derived attractant (Sink, 2001; de Jong, 2005). Interestingly, mutations in beaten path Ia (beat) lead to similar axon guidance phenotypes, and like Side, Beat has been shown to regulate axon defasciculation at choice points (Fambrough, 1996; Holmes, 1999; Sink, 2001). Based on primary structure predictions, Beat is a secreted protein of the immunoglobulin superfamily that has been shown to function as an anti-adhesive factor on motor axons (Fambrough, 1996; Pipes, 2001). Beat consists of two Ig domains and a Cysteine-rich C-terminal domain that shares similarities with cystine knots (Siebert, 2009).

This study shows that side encodes a candidate directional cue that steers Drosophila motor axons from the place of their birth to their peripheral targets. The spatiotemporal expression pattern of Side thus delineates a linear pathway for motor axons. High levels of Side are consistently found ahead of Beat-expressing growth cones. Contact with motor axons induces the down-regulation of Side and eliminates the pathway, preventing other outgrowing nerves from choosing the same route. Furthermore, biochemical and genetic evidence is provided that Beat functions as a membrane-associated receptor or part of a receptor complex for Side. Taken together, these findings support the concept of pre-existing pathways labeled with molecular markers that are recognized by a specialized subset of axons expressing the appropriate receptors (Siebert, 2009).

This work, provides evidence for a simple guidance mechanism that guides Drosophila motor axons to their target regions. During the period of axonal pathfinding, Side is dynamically expressed on different tissues but its temporal shift forms a spatial pattern as such that high levels are located ahead of motor axonal growth cones. Beat-expressing motor axons recognize and follow Side-labeled surfaces. In order to better visualize the complex spatial and temporal dynamics of axon guidance processes, a FasIIGFP^Mue397-based imaging assay was established that allowed analysis of the activity of growth cones in living wild-type and mutant embryos. These time-lapse observations revealed that in wild-type embryos, the ISN migrates continuously through the lateral body wall until it reaches a choice point near the dorsal trunk. As expected for migration along a substrate pathway, the ISN employs a relatively small growth cone that extends only few filopodia for steering. The observed growth rates are in good agreement with the growth rates of the RP2 axon in filleted, semiviable embryo preparations (Siebert, 2009).

The absence of the attractant Side strongly interferes with axonal growth and results in delayed arrival of the ISN in its dorsal target regions, frequently failing to innervate the dorsal-most muscles. The lack of Side-mediated attraction likely prevents the progression at a normal rate, causing the ISN to develop a complex growth cone that appears to actively search for guidance information. Since Side is a transmembrane protein, it is predicted to function as a contact attractant. The spatiotemporal expression pattern should therefore provide pathway information. The growth cone of the ISN follows Side-positive cell surfaces from its first emergence in the ventral nerve cord to its dorsal target region. Consequently, the tight association of motor axons and their substrates - e.g., sensory axons - is partially lost in side mutants. Since Side is expressed in all sensory clusters (Sink, 2001), it likely also prefigures the SNa and SNc pathways. Based on the positions of the segmental sensory clusters in the body wall, motor axons following Sidestep-labeled sensory axons are guided into the proximity of their target regions. At stage 15, motor axons reach the end of the sensory tracks and begin to defasciculate into the muscle fields that up-regulate Side at this developmental time point. In this respect, it is interesting to note that motor pathways terminate on the cell bodies of sensory neurons when deprived of their target muscles. The opposed migration of efferent motor axons and afferent sensory axons therefore provides a robust mechanism for the establishment of the basic neuromuscular connectivity pattern in Drosophila. In the brain of vertebrates, a similar mechanism controls the wiring of the thalamus and the cortex (Lopez-Bendito, 2003). Corticothalamic and thalamocortical fibers meet at a common intermediate target and continue to grow along each other in opposite directions (Siebert, 2009).

During its journey through the lateral body wall, the ISN completely ignores the nearby ventral and lateral muscle fields. At this stage, muscle fibers are not yet differentiated, and hence do not express endogenous Side. If these muscle precursors, however, are forced to express Side prematurely under control of Mef2-Gal4, the ISN diverts from its normal path and grows straight into the muscle field. The premature attraction drastically slows down the migration toward dorsal targets, leading to a permanent lack of NMJs on dorsal muscles. Thus, both gain and loss of Side cause migratory delays that result in the lack of NMJs on dorsal muscles but for different reasons. Ectopic expression of Side leads to excess attraction in wrong directions, whereas lack of Side leads to reduced attraction along the predestined path (Siebert, 2009).

Since mutations in both beat and side cause similar axon guidance phenotypes in embryos and innervation defects in larvae, it is assumed that the products of both genes might interact functionally. Several pieces of genetic evidence suggest that beat and side function in a common pathway: (1) the double mutant phenotype is similar to the respective single mutant phenotypes; (2) overexpression of Side in muscles of wild-type embryos leads to premature attraction of the ISN into ventral and lateral muscle fields. This gain-of-function phenotype is completely suppressed by coexpression of Beat. (3) Overexpression of Side in muscles of beat mutants renders motor axons unresponsive to ectopic Side; (4) endogenous Side is not down-regulated in beat mutants. In addition, Beat and Side interact in vitro. S2 cells expressing Beat and Side form large cell clusters in aggregation assays when individually transfected cell populations are mixed. Moreover, Beat coimmunoprecipitates with Side, supporting the idea that Beat interacts with Side or a Side-containing complex. The formation of cell aggregates further argues that Beat-Side interactions lead to the formation of heterophilic adhesion complexes (Siebert, 2009).

The spatiotemporal expression of Side appears to be strictly regulated. The levels of Side expression are highest in front of motor axonal growth cones. Side disappears from cell surfaces once these cells have been contacted by motor axons, indicating that motor nerves neutralize attractive surfaces and thereby disguise the path they are following. Motor nerve bundles that exit the CNS at a later time point are thus prevented from choosing the same route. In beat mutants, this regulatory mechanism appears to be nonfunctional. Side is constitutively expressed in peripheral nerves. Expression of exogenous Beat in post-mitotic neurons but not in muscles rescued the regulatory defects, suggesting that Beat induces the down-regulation of Side cell-autonomously. If Beat would be a secreted protein, one would expect that expression in muscles down-regulates Side on sensory axons. The secreted metalloprotease tolloid-related as well as the secreted TGF-β ligand Dawdle have been shown to rescue axonal guidance defects tissue independently. Biochemical data from transiently transfected S2 cells further support a cell-autonomous function for Beat. Beat was not secreted into the medium, and a fraction of it was associated with membranes. However, since Beat is not normally expressed in S2 cells the lack of a coreceptor or chaperone might prevent its secretion. Candidate coreceptors are the remaining members of the beat multigene family (Pipes, 2001). Several family members are expressed in the ventral nerve cord, and might function together with Beat in the regulation of its subcellular localization and/or function (Pipes, 2001). Further experiments will be necessary to determine the composition of Beat-containing complexes, and how they transduce guidance signals into the growth cone (Siebert, 2009).

If Side is an instructive signal for Beat-expressing motor axons it should be possible to redirect their paths in a side mutant background; i.e., in the absence of endogenous Side but in the presence of exogenous Side on a defined tissue. Regardless of whether Side is expressed in trachea, muscles, or hemocytes in a side mutant background, motor axons head toward the ectopic source of Side. The growth cones find and recognize Side-expressing cell surfaces and adopt their route accordingly. In the most extreme case, motor axons strongly interacted with hemocytes, which are highly motile cells. Side therefore potently controls the path of motor axons. Although Side-mediated attraction is likely not the only mechanism to reach the muscle targets, these results, together with the high penetrance of sidestep mutant phenotypes, suggest that it is one of the major mechanisms. In this respect, it is important to note that Side is not required for motor axon outgrowth per se, but rather for the specification of the growth direction. Based on the experimental evidence, a model is proposed for the navigation of motor axons from the ventral nerve cord to their target area in Drosophila. Beat-expressing motor axon fascicles recognize, extend on, and subsequently mask a pre-existing, Side-labeled substrate pathway that determines their growth direction. In the absence of the labeled pathway, in side mutants, axonal migration is delayed or growth cones head into aberrant directions. In either case they will miss their targets. In beat mutants, the pathway is constitutively labeled but cannot be recognized, leading to similar phenotypes. Since Beat and Side are conserved in insects, similar guidance principles might occur in all organisms, in which the peripheral nervous system develops from sensory organ precursors (Siebert, 2009).

Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development

An 'interactome' screen of all Drosophila cell-surface and secreted proteins containing immunoglobulin superfamily (IgSF) domains discovered a network formed by paralogs of Beaten Path (Beat) and Sidestep (Side), a ligand-receptor pair that is central to motor axon guidance. This study describes a new method for interactome screening, the Bio-Plex Interactome Assay (BPIA), which allows identification of many interactions in a single sample. Using the BPIA, four more members of the Beat-Side network were 'deorphanized'. Interactions were confirmed using surface plasmon resonance. The expression patterns of beat and side genes suggest that Beats are neuronal receptors for Sides expressed on peripheral tissues. side-VI is expressed in muscle fibers targeted by the ISNb nerve, as well as at growth cone choice points and synaptic targets for the ISN and TN nerves. beat-V genes, encoding Side-VI receptors, are expressed in ISNb and ISN motor neurons (Li, 2017).

Protein-protein interactions (PPIs) control a vast array of processes in metazoans, ranging from signal transduction and gene regulation within cells to signaling between cells via cell surface and secreted proteins (CSSPs). The strength of PPIs varies widely, from high-affinity interactions that create stable protein complexes to weak transient interactions. Defining global PPI patterns ('interactomes') has been the focus of much recent research. Progress has been made in generating high-throughput protein interaction data for a variety of organisms, including S. cerevisiae, C. elegans and D. melanogaster. Methods used to create interactomes include affinity purification/mass spectrometry (AP-MS) and the yeast two-hybrid assay (Y2H) (Li, 2017).

It is estimated that up to one sixth of human genes encode CSSPs. CSSPs control signaling from the extracellular milieu to cells and the flow of information between cells. Due to their importance and accessibility, CSSPs are often the targets for therapeutic agents, including humanized monoclonal antibody drugs such as checkpoint inhibitors, the non-Hodgkin's lymphoma drug Rituxan, and the breast cancer drug Herceptin. However, the biochemical properties of many CSSP interactions prevent them from being detected by commonly used techniques employed in high-throughput PPI screens, and CSSPs are underrepresented in global interactomes. There are several reasons for this. First, these proteins are usually glycosylated and have disulfide bonds, so they need to be expressed in the extracellular compartment. CSSP interactions between monomers are also often weak, with KDs in the μM range, making them difficult to capture due to their short half-lives. Lastly, insoluble transmembrane domains on cell surface proteins preclude their purification with standard biochemical techniques, which makes them difficult to study using methods such as AP-MS (Li, 2017).

Despite these difficulties, recent advances have been made in the study of global CSSP interaction patterns. Interactions among cell-surface proteins (CSPs) often occur between clusters of proteins on cell surfaces, and avidity effects (stronger binding due to clustering) can make these cell-cell interactions stable even when monomers bind only weakly. To facilitate detection of interactions among CSSP extracellular domains (ECDs) in vitro, several groups have taken advantage of avidity effects by attaching ECDs to protein multimerization domains and expressing ECD fusions as soluble secreted proteins. These methods have been shown to be effective, allowing detection of interactions that otherwise would not have been observable (Li, 2017).

Özkan (2013) scaled up the avidity-based approach, developing a high-throughput ELISA-like screening method, the Extracellular Interactome Assay (ECIA). The ECIA was used to define interactions among 202 Drosophila CSSPs, comprising all CSSPs within three ECD families. These were the immunoglobulin superfamily (IgSF), fibronectin type III (FNIII) and leucine-rich repeat (LRR) families. The ECIA utilized dimers as 'bait' and pentamers as 'prey'. It detected 106 interactions, 83 of which were previously unknown (Li, 2017).

The most striking finding from the ECIA interactome was that a subfamily of 21 2-IgSF domain CSPs, the Dprs, selectively interacts with a subfamily of 9 3-IgSF domain CSPs, the DIPs, forming a network called the 'Dpr-ome' (Özkan, 2013). Each Dpr and DIP that has been examined is expressed by a small and unique subset of neurons at each stage of development. One Dpr-DIP pair is required for normal synaptogenesis and influences neuronal cell fate. In the pupal optic lobe, neurons expressing a Dpr are often presynaptic to neurons expressing a DIP to which that Dpr binds in vitro. The Dpr-ome initially defined by the global interactome contained several 'orphans', proteins with no binding partner (Özkan, 2013). By expressing new versions of Dprs and DIPs, including chimeras, and using these to conduct a 'mini-interactome' analysis of the Dpr-ome, it was possible to find partners for all but one orphan. That protein, Dpr18, has changes to conserved binding interface residues and may lack the capacity to bind to any DIPs (Li, 2017).

The ECIA also identified a second IgSF network, formed among members of the Beaten Path (Beat) and Sidestep (Side) protein subfamilies. Beat-Ia and Side were identified by genetic screens for motor axon defects, and were later shown to have a ligand-receptor relationship. They control the projection of motor axons to muscle targets. Beat-Ia is expressed on motor axons, where it binds to Side, which is expressed on muscles. This binding causes motor axons to decrease their adhesion to each other, allowing them to leave their bundles and turn onto the muscle fibers. beat-Ia and side have strong motor axon phenotypes. In the absence of either protein, motor axons often remain in their fascicles and never leave to arborize on their target muscles (Li, 2017).

The ECIA detected the known Beat-Ia::Side interaction, and also uncovered other interactions between members of the Beat and Side subfamilies. Seven of the 14 Beats were found to bind to four of the eight Sides. The remaining Beats and Sides were still orphans with no binding partners in the other subfamily. The functions of the newly defined interactions between Beats and Sides were unknown. Most beat genes are expressed in embryonic neurons. Some Beats were genetically characterized using deletion mutations and RNAi, but loss of these Beats did not cause strong motor axon phenotypes. None of the other Side subfamily members had been examined (Li, 2017).

This paper describes the development of a new method for interactome screening, which is called the BPIA (Bio-Plex Interactome Assay). This method uses the 'Bio-Plex' system, which employs Luminex xMAP technology. This method detects binding of a prey protein to many bait proteins, each conjugated to a bead of a different color, in each assay well. For the ECIA, the number of assays required for the interactome screen was the square of the number of proteins examined, while with the Bio-Plex the number of assays could be equal to the number of proteins. In principle, then, the Bio-Plex might greatly speed up interactome screening, and might also be more sensitive, since the available signal-to-background ratio is much greater for the Bio-Plex than for the ECIA. As a test of the method, a Bio-Plex 200 was used to do a mini-interactome screen of the Beat-Side network. Based on the the fact that the Dprs and DIPs that were initially orphans were later shown to have binding partners, it was hypothesized that some of the orphan Beats should have Side partners, and vice versa. Consistent with this hypothesis, it was possible to deorphanize two more Beats and two Sides using the BPIA (Li, 2017).

To further understanding of Beat and Side function during embryonic development, this study characterized expression of several Beats and Sides, focusing primarily on Side-VI and the three Beat-Vs, which were the strongest interactors in both the ECIA and BPIA screens. The three beat-Vs exhibit differential expression in identified motor neurons, while side-VI is expressed at motor axon choice points and in a subset of target muscle fibers (Li, 2017).

In principle, the Bio-Plex system can allow 500 unique protein-protein (bait-prey) interaction pairs to be analyzed in a single well. In this method, capture of proteins from media with streptavidin-coupled beads bypasses the purification step for bait proteins. The assay is also compatible with the use of unpurified prey proteins, thereby reducing the workload for multiplexed screenings. The small size of the beads, the ability to probe multiple interactions simultaneously, and the small volume of the binding reactions all help reduce the amount of protein and reagents needed for the assay. It was possible to produce enough bait and prey proteins for the mini-interactome described in this study (a 23 x 23 matrix) with a single 10 cm dish transfection per protein (Li, 2017).

As a test of the system, the BPIA was used to examine interactions between the Drosophila Beat and Side IgSF protein subfamilies. Beat-Ia is a receptor on motor growth cones that recognizes Side expressed on muscles, and in the absence of Beat-Ia or Side motor axons fail to leave their bundles and arborize on their muscle targets. There are 14 Beat subfamily members and 8 Side subfamily members, but all of these proteins except Beat-Ia and Side itself were orphans until the global IgSF interactome revealed interactions between six other Beats and three Sides (Li, 2017).

In the Dpr-ome, the other IgSF network uncovered by the interactome, every Dpr protein likely to be capable of binding has an interaction partner in the DIP subfamily. Based on this, it is predicted that there should be additional interactions to be discovered within the Beat-Side subfamily network. Using the BPIA, three new interactions were found: Beat-VI::Side-II, Beat-Ic::Side-III, and Beat-Ic::Side. These results suggest that the BPIA is more sensitive than the ECIA. Like the ECIA, the BPIA should be able to find new receptor-ligand interactions even if proteins not previously known to have any interactions were tested. Of course, for both assays any candidate receptor-ligand pairs need to be confirmed as genuine using other methods. For the Beat-Side network, all three new interactions found by the BPIA, as well as the interactions between the three Beat-Vs and Side-VI found by the ECIA, were verified by SPR. This study also demonstrated that Beat-Vs and Side-VI interact using cell-based binding assays and binding to live-dissected embryos (Li, 2017).

There are still five Beats and two Sides that remain orphans. Since the structure of Beat-Side complexes is unknown, it cannot be determined whether these Beats and Sides are likely to be able to bind, but it is speculated that at least the three Beat-IIIs are likely to have Side partners. The Beat-II and Beat-V clusters each interact with a single Side partner, and perhaps the Beat-IIIs interact with one of the two orphan Sides. It is possible that changes in methodology, such as using more highly multimerized preys and/or baits, could increase sensitivity and allow detection of additional interactions (Li, 2017).

The expression patterns of side and beat genes were examined in order to obtain insights into their possible functions. Most sides are expressed in cells in the periphery as well as in the CNS, while most beats are expressed only by CNS neurons, including motor neurons. Beat-Ia::Side interactions are required for normal motor axon guidance, and highly penetrant motor axon defects in which muscles remain uninnervated are observed in mutants lacking either protein. By contrast, partial loss of function of beat-Ib, beat-Ic, both beat-IIs, or beat-VI causes motor axon defects with less than 20% penetrance. Genetic redundancy is a common theme in motor axon guidance , so it is not surprising that only low-penetrance defects are observed when Beat paralogs are not expressed. Given that Beat-Ia and Side both interact with other partners, it is perhaps remarkable that beat-1a and side have such strong phenotypes as single mutants (Li, 2017).

Beat-V and Side-VI also have redundant functions in motor axon guidance. side-VI is expressed in motor axon targets, including muscles 12 and 13 and interacts with the three Beat-Vs, at least two of which are expressed in subsets of motor neurons. Beat-V::Side-VI interactions produced the strongest signals in both the ECIA and BPIA. Sow-penetrance (~1/5 of stage 17 embryonic hemisegments affected) muscle 12 innervation defects were observed in side-VI insertion mutants or in deletion mutants lacking all three beat-V genes. There were also low-penetrance ISN guidance defects in both mutants. The fact that most muscle 12 s are innervated normally in beat-V or side-VI mutants indicates that, while Beat-V::Side-VI interactions may contribute to correct targeting of the RP5 axon to muscle 12, other cues must also be involved. Muscles 12 and/or 13 also express Wnt-4 (a repulsive ligand) and the LRR protein Capricious (Caps; probably an adhesion molecule), and low-penetrance RP5 targeting defects are observed in Wnt-4 and caps mutants. Perhaps muscle 12 is distinguished from other nearby muscles by a set of partially redundant cues, so that strong targeting phenotypes are not observed in any single mutant (Li, 2017).

Although Beat and Side paralogs may not be central to motor axon guidance, their expression patterns suggest that they could be important for determining synaptic connections within the CNS. side-VIII, encoding an orphan Side, is expressed in a small subset of embryonic CNS neurons. In the optic lobe of the pupal brain, an RNAseq analysis of two photoreceptors (R7 and R8) and five types of lamina neurons (L1-L5) revealed that beats and sides have highly specific expression patterns. For example, beat-VII is specific to L2, beat-VI to L5, beat-IIa to L3 (with lower levels in L4), and beat-IIIc to R8, being expressed at much higher levels in those cells relative to all other cells. side and side-III are specific to L3, side-II is specific to L1, side-IV is specific to L2, and side-V is specific to L5. Each of the L neuron types as well as R7 and R8 synapse with different sets of neurons in the medulla, a ten-layered structure that processes visual information from the retina and lamina. It has been observed that R and L neurons expressing specific Dprs often form synapses on medulla neurons expressing DIPs to which those Dprs bind in vitro. In a similar manner, perhaps some of the medulla neurons that are postsynaptic to L or R neurons expressing specific Sides or Beats express their in vitro binding partners, and these Beat-Side interactions might be important for synapse formation or maintenance (Li, 2017).

Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles

Despite several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, evidence is provided that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, the expression of the guidance molecule-encoding genes sidestep and side IV in AMPs is reported. Altogether, these data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs (Lavergne, 2020).

During Drosophila embryogenesis, a stereotypical pattern of AMPs per abdominal hemisegment in ventral (V-AMP), lateral (L-AMPs), dorsolateral (DL-AMPs) and dorsal (D-AMPs) positions can be distinguished. This study has investigated the relationship between AMPs and motor axons, and their dynamics, during development using embryos carrying the M6-gapGFP transgene, which allows visualization of the membrane of AMP cells. The intersegmental nerve (ISN) established contacts with the DL-AMPs during the embryonic stage 13 and then navigated toward the D-AMP to contact it at stage 15. Within the lateral field, the segmental nerve a (SNa) is sub-divided into two branches, dorsal (D-SNa), which innervates the lateral transverse muscles (LTs 1-4), and lateral (L-SNa), which targets the segmental border muscle (SBM). The SNa sub-division takes place during stage 15 and it was observed that the L-SNa branch migrated towards the L-AMPs before innervating the SBM. In parallel, the anterior L-AMP underwent shape changes and directional migration towards the L-SNa. In a similar way, one of the DL-AMPs moves dorsally following ISN migration and the D-AMPs appear to extend toward the ISN. However, AMPs survival and behavior are not affected in the absence of motor axons, as shown in the prospero mutant, where motor axons fail to exit the CNS. To better characterize dynamics of AMP-motor axons interactions, the M6-GAL4; UAS-Life-actin GFP reporter line was used that allows in vivo visualization of both the motor axons and the AMPs. The M6-Gal4 and M6-gapGFP lines are both driven by the same regulatory elements; however, the expression in motor axons, which is low and difficult to distinguish in M6-gapGFP embryos, is enhanced by the GAL4/UAS system and is clearly present in the M6>lifeActGFP context. Live imaging revealed that, among the numerous oriented cytoplasmic projections sent out by the AMPs, those contacting the growth cones of motor axons became stabilized. In particular, stabilization of filopodial connections between L-AMPs and SNa coincided with the setting of the SNa branching point and specification of its lateral branch. Oriented filopodial dynamics were found in the dorsal region with the contact between D-AMP projections and ISN growth cone prior to ISN migration toward the D-AMP. As previously demonstrated, muscle founders are needed for terminal defasciculation of the main motor axon branches. In this context, AMP positioning and the fact that they actively engage with the navigating motor axons might also participate in this process by acting as spatial check-points that either induce and or attract targeted defasciculation of ISN and SNa (Lavergne, 2020).

To investigate the impact of L-AMPs on the SNa pathway and branching, the effect of a genetic ablation of the AMP cells was assessed using the M6-GAL4-driven expression of the pro-apoptotic gene reaper. This enabled targeted induction of apoptosis in AMPs, leading to AMP cell loss without strong defects in the ISN and SNa trajectory, despite the expression of M6-GAL4 in the motor neurons. This differential effect could be due to a lower expression level of M6-GAL4 in motor neurons than in AMPs, and/or a stronger resistance of neural cells to the Reaper-induced apoptosis. Importantly, in 86% of hemisegments, complete loss of L-AMPs was associated with absence of the lateral branch of SNa (L-SNa), strongly suggesting that L-AMPs play an instructive role in L-SNa formation and/or stabilization. In contrast, loss of L-SNa in hemisegments where the L-AMPs were still present occurred in 5.6% of analyzed hemisegments. The L-SNa loss in this context was thus higher than the one observed in the M6-GAL4 line with only 1.8% of hemisegments without L-SNa. To explain this difference, an effect of Reaper expression in the motor system cannot be excluded, but this could also be a consequence of early stages of apoptosis in L-AMPs. Thus, M6-GAL4-induced apoptosis created a context in which loss of the L-SNa branch was observed in L-AMP-devoid segments where the L-SNa target muscle (SBM) was still present. This suggests that L-SNa branch formation might not be dependent on its muscle target, and so prompted a test of whether the L-SNa would form or persist in an SBM-devoid context. reaper expression was targeted to the developing SBM using the SBM(lbl)-GAL4 driver. The SBM(lbl)-GAL4-driven apoptosis resulted in a systematic loss of the SBM and only sporadic loss of the L-AMPs (12% hemisegments). In the SBM-devoid context but with L-AMP cells correctly located, the L-SNa branch was still present (73% of hemisegments). Additionally, in a subset of SBM-deficient embryos, L-AMPs shifted toward the navigating SNa, leading to a shortened L-SNa (13% of the hemisegments). These observations thus suggest an instructive role for AMPs in L-SNa establishment, and reveal that SBM might not be needed for this process and is at least dispensable for its stabilization. To further test the role of L-AMPs in lateral defasciculation of the SNa, different genetic contexts were analyzed in which AMP specification is affected. First a perturbation of asymmetric cell divisions was induced. To adversely affect divisions of progenitor cells that give rise to AMPs, the asymmetry determinant Numb was ectopically expressed using the pan-mesodermal driver Twist-GAL4. In the lateral region, this led predominantly to the loss of one of the L-AMPs and a duplication of the SBM with no major impact on L-SNa formation compared with the control Twist-GAL4 line. However, in a small subset of hemisegments, loss of both L-AMPs but not SBM (often duplicated) was observed. In this rare context, the L-SNa was absent in 88% of hemisegments, supporting the view that L-AMPs are required for L-SNa branching. These findings are also consistent with the effects of generalized mesodermal expression of the identity gene Pox meso (Poxm), which can lead to a loss of L-AMPs without affecting SBM. In such a context, the L-SNa formation is impaired in 89% of L-AMP-devoid segments against 37% in random Twi>Poxm hemisegments. Interestingly, pan-mesodermal expression of Pox meso can also induce misplacement of L-AMPs along the SBM, leading to aberrant L-SNa trajectory. Hence, L-AMPs and their spatial positioning appear crucial to achieve the formation and correct pathfinding of the L-SNa (Lavergne, 2020).

The findings described above suggest that L-AMPs are a source of attractive signals that promote lateral sub-branching of the SNa, making it competent to innervate the SBM. Interestingly, the SBM is the only lateral muscle innervated by the Connectin-positive SNa, which does not express this homophilic target recognition molecule. In such a context, L-AMP-mediated lateral sub-branching of SNa offers a way to drive L-SNa to its specific muscle target. As L-AMPs seem not to express Connectin either, in contrast to previous suggestions, their role in attracting SNa and inducing the L-SNa sub-branching might rely on other guidance molecules (Lavergne, 2020).

It has been previously shown that the mutants of sidestep and beat-1a, which encode interacting membrane proteins of the immunoglobulin superfamily, displayed loss of L-SNa, a phenotype similar to the one observed when L-AMPs are missing. However, the mechanisms leading to the loss of the L-SNa in sidestep and beat-1a mutants have not been elucidated. In addition, the embryonic expression pattern of sidestep has been only partially characterized. By using in situ hybridization, this study found that sidestep mRNA is strongly enriched in all the AMPs, suggesting its potential involvement in the dialogue between AMPs and motor axons. It was therefore decided to test Sidestep protein distribution at the time when L-SNa sub-branching is taking place. By examining stage 14 to 15 embryos, a previously unreported faint and transient expression of Sidestep was found specifically in L-AMPs. To confirm this observation, the expression of Sidestep was analyzed in a mutant for beat-1a. It has been reportedthat the contact of Beat-1a-expressing motor axons with Sidestep-expressing cells leads to a negative regulation of the expression of sidestep. If this contact is missing, cells normally expressing sidestep transiently and at a low level will continue to do so, leading to continuous and higher Sidestep level in these cells. Analyses of beat-1a mutants confirmed that the L-AMPs are Sidestep-expressing cells and that Sidestep expression onset coincides with L-SNa sub-branching. The high Sidestep expression resulting from the lack of beat-1a was still detected in late-stage embryos in which it became gradually restricted to the most anterior L-AMPs. This late differential Sidestep expression may point to a leading role for the anterior L-AMPs in the process of interaction with SNa and in its lateral sub-branching. Additionally, an increased Sidestep expression in L-AMPs was also observed in SNa-devoid pros mutants and in the Duf-GAL4; UAS-NetrinB (NetB) context. Importantly, the SBM does not express Sidestep, making the L-AMPs the only Sidestep-expressing cells in the L-SNa pathway. Thus, this newly reported expression pattern suggests that L-AMPs could attract the L-SNa through the temporally and spatially restricted expression of sidestep (Lavergne, 2020).

Interestingly, the sidestep mutants also display a stall phenotype of the ISN suggestive of a potential role of the D-AMPs. Indeed, this study observed that the aberrantly located D-AMPs, after the mesodermal overexpression of the activated form of the Notch receptor (NICD), are able to attract the ISN, suggesting that they are a source of guiding signals. However, because only faint sidestep transcript expression was observed in D-AMPs and Sidestep protein was not detected, it is expected that other guiding cues may be in play. It is important to notice that to visualize the attractive potential of mis-positioned D-AMPs induced pan-mesodermal expression of NICD was observed via a GAL4/UAS system known to be thermo-sensitive. High mesodermal expression of NICD induced at 29°C leads to the loss of majority of muscles but, as is shown in this study, several muscles persist in Twi-GAL4;UAS-NICD embryos incubated at 25°C, thus allowing uncouple effects of delocalized D-AMPs from potential influence of muscles loss on ISN trajectories. However, loss of D-AMPs, observed in this study in a Poxm gain-of-function context, appears to have only a minor effect on the capacity of ISN motor axons to target dorsal muscles, which are correctly innervated by the ISN in 65% of hemisegments without D-AMP. These results highlight differential requirements of AMPs for motor axons defasciculation and navigation with L-AMPs being mandatory for L-SNa branching and D-AMPs acting as guiding cells for the ISN. However, the functional significance underlying the guidance of motor nerves by muscle stem cells remains to be determined (Lavergne, 2020).

It is also important to state that the loss of L-SNa in the sidestep mutants is not fully penetrant (observed in less than 10% of hemisegments), suggesting that sidestep is not the only player in L-SNa sub-branching. This could be due to functional redundancy between several members of Side and Beat families comprising 8 and 14 members, respectively. Expression and function of Side and Beat family members remain largely unexplored, but the fact that Sidestep labels L-AMPs and its paralog, side VI, is expressed in the DL-AMPs suggests there might be a 'Side expression code' that operates in AMPs and makes them competent to interact with navigating motor axons. In support of this hypothesis, this study found that side IV, another member of the Side family, is also expressed in AMPs with a higher transcript levels detected in L-AMPs, suggesting it could contribute to the interactions between L-AMPs and the SNa. To gain insight into AMP functions of sidestep and side IV in setting interactions with motor neurons, the selective AMP-targeting tools need to be developed to generate AMP-specific mutant rescue (Lavergne, 2020).

It has previously been shown that the nervous system is required for the establishment of the adult muscle pattern and that motor axons serve as a support for migration of AMPs during larval and pupal development. More recently, it has also been suggested that the nervous system could be involved in the selection of founder cells from the pool of AMPs. This study took advantage of a previously described genetic context (pan-muscular expression of NetB) to affect the SNa and tested impact of SNa loss on L-AMPs. In stage 16 DUF>NetB embryos, loss of the SNa observed in 84% of the hemisegments does not affect the number of L-AMPs. However, in surviving 3rd instar larvae in hemisegments lacking the SNa, number of L-AMPs is dramatically reduced. Interestingly, a specific depletion was observed of normally associated with SNa anterior L-AMPs (complete loss in 10 out of 26 hemisegments analyzed), while the posterior L-AMPs associated with the transverse nerve (TN) remained unaffected. Thus, this data provides evidence for a cross-talk between AMPs and motor axons, with the AMPs attracting navigating motor axons, which in turn are required for AMP maintenance during larval stages. The loss of anterior L-AMPs in SNa-devoid larva suggests that SNa-derived signals promote survival of associated L-AMPs, but precise underlying mechanisms remain to be elucidated (Lavergne, 2020).

Thus, in Drosophila, the dynamic interactions and close association between AMPs and the motor axon network contribute to setting ISN trajectory and are required for SNa sub-branching and proper innervation of lateral muscles, which is itself needed in larvae for the maintenance of anterior L-AMPs. In vertebrates, it has previously been described that muscle pioneers can impact motor axon pathfinding in zebrafish and more recently in mice that muscle stem cells activate and contribute to neuromuscular junction regeneration in response to denervation, and that depletion of muscle stem cells induced neuromuscular junction degeneration. This study, conducted in Drosophila, represents the first demonstration that, during development of neuromuscular system, muscle stem cells interact with motor neurons and contribute to proper muscle innervation (Lavergne, 2020).

Target recognition and synaptogenesis by motor axons: responses to the Sidestep protein

Sidestep is a pivotal molecular player in embryonic motor axon pathfinding. But questions about its functional repertoire remain: (1) can Side permanently overturn targeting preferences? (2) does it promote synaptogenesis, and (3) can Side facilitate synaptic stabilization? To address these questions, Side was temporally and spatially misexpressed and the visible consequences for neuromuscular junction morphology were assessed. When Side was misexpressed either broadly or selectively in muscles during targeting in a wildtype background motor axon targeting preferences were permanently overturned. However the misexpression of Side in all muscles post-targeting neither changed synapse morphology, nor compensated for a lack of the synapse-stabilizing protein Fasciclin II (FasII). Rather Side appears to be dependent on FasII, instead of on intrinsic ability, for sustaining targeting changes. It is proposed that Side helps to bring motor axons to their correct muscle targets and promotes synaptogenesis, then FasII serves to stabilize the synaptic contacts (de Jong, 2005).

Sidestep (Side) profoundly influences embryonic Drosophila motor axon pathfinding (Sink, 2001). The extreme pathfinding defects observed in the side loss-of-function mutant confounded analysis of post-axon pathfinding responses to the protein. To overcome this technical hurdle Side was misexpressed in the present study in suitable spatio-temporal paradigms, allowing assessment of motor axon responses during targeting, synaptogenesis, and synaptic stabilization. It was found that Side can permanently influence motor axon targeting and synaptogenesis events, but requires FasII for synaptic stabilization (de Jong, 2005).

In addition to expression in muscles, Side protein is observed at low levels in the motor axons and CNS longitudinal connectives. This expression spans embryonic motor axon targeting and early post-targeting periods, and continues across larval stages (Sink, 2001). Strong Side misexpression in all post-mitotic neurons causes motor axon over-fasciculation in the embryo (Sink, 2001). These over-fasciculation defects endure in the larva, with innervation frequently absent on the muscles examined in the present study. By using a weaker neuronal GAL4 driver, the early over-fasciculation defects were avoided. This driver also subsequently failed to significantly alter the morphology of the larval neuromuscular synapse. Hence the role of ongoing Side expression in axons remains undefined, although the robust neuronal misexpression phenotype (Sink, 2001) indicates it could serve to encourage and maintain axon-axon contact and hence aid fascicle organization (de Jong, 2005).

Post-synaptic Side misexpression in the larva did not visibly alter neuromuscular synapse morphology. This suggests that Side is unable to encourage growth of that larval synapse, unlike the BMP signaling pathways. In contrast Side misexpression commencing in muscles during embryogenesis produces targeting changes that remain to the end of larval development. These changes included the attraction to and retention of innervation at unconventional locations on muscles; the incorrect targeting by motor axons native to the muscle region, and the attraction of non-native innervation to Side-misexpressing muscles (de Jong, 2005).

In both vertebrates and Drosophila neuromuscular synapses form at stereotypic locations on somatic muscle fibers. Furthermore, the stereotypic locations arise in response to muscle-derived cue(s) rather than through imposition upon the muscle by the innervating axon (de Jong, 2005).

This study found that misexpression of Side on muscles can overturn stereotypic synapse formation. In both global and select Side misexpression paradigms innervation is observed at novel locations on the muscle fibers. These novel sites include: close to the segmental boundary; diametrically opposite the normal innervation site, and on the external rather than internal surface of the muscle. This shows that the presence of excess Side signal across muscle fiber surfaces can overwhelm the potentially restrictive cues residing at the muscle clefts (normal innervation region), and axonal explorations and contacts are encouraged beyond these sites. As a consequence innervation develops, and becomes stabilized, at non-stereotypic locations. Hence, the current observations lend additional support for the model that intrinsically pre-patterned cues in the muscle fiber determine the innervation site (de Jong, 2005).

Altered expression of several putative targeting molecules readily perturbs the embryonic motor projection arborization pattern. Yet few studies examined whether the changes observed in the embryo are maintained in the larva. In the case of the leucine-rich repeat family protein Capricious the changes are permanent, whereas in the case of the tetraspanin family protein Late bloomer the changes are transient. Evidently, observations in the embryo do not necessarily translate to larval phenotypes. It has now been found that changes induced by Side in the embryo are sustained in the larva (de Jong, 2005).

The global misexpression of Side in muscles with the C142-GAL4 driver permanently altered targeting choices on the muscles of interest. In some larvae this manifested as an excess of innervation, while in others it appeared as a reduction. This suggests that the relative level of Side on the muscle is a deciding factor as to whether innervation was increased or decreased (de Jong, 2005).

The H94-GAL4 line drives expression in two adjacent muscles in the ventral musculature, muscles 6 and 13. When H94-GAL4 drives Side expression in these muscles, at the temporal juncture between pathfinding and targeting, the motor axon growth cones are robustly attracted to the adjoining edges of the two muscles (Sink, 2001). Examination of the subsequent effects on final innervation at muscles 6/7 and 13 showed that excess Side permanently overturned innervation preferences in favor of muscle 13 - often at the expense of muscle 6, and occasionally of muscle 12. This preference for muscle 13 correlates with H94-GAL4 driving more strongly in muscle 13 than in muscle 6 (de Jong, 2005).

A novel feature of this reorganization in targeting was that the loss of native innervation on the Side-expressing 6 muscle was often not significantly compensated for by either 'reach-back' innervation (e.g., Capricious) or ectopic innervation from the transverse nerve (e.g., Late bloomer). Rather, it appears that once the motor axons normally innervating muscle 6 are drawn away to muscle 13, other motor axons in the region do not provide compensatory innervation to muscle 6. Innervation on muscles 6, 7, 13 and 12 derives predominantly from the RP motoneurons. The RP motoneurons have their somata closely grouped at the CNS midline, extend their axons out of the CNS along a shared pathway, and are tightly fasciculated up until the time of targeting. They also express several proteins in common. The Side-sensitiveness of the RP motor axons in misexpression experiments suggests they also share the molecular machinery required to respond to an elevated, muscle-derived Side signal during targeting. This could explain the common attraction to the highest Side-misexpressing muscle (13) on which excess arborizations are evident, and the failure of (RP) motor axons to be drawn to the other lower, Side-misexpressing muscle (6) and its non-misexpressing partner (7) (de Jong, 2005).

Type II innervation is the most common type of novel innervation observed in response to post-synaptic Side misexpression. Type II innervation derives from the VUM neurons; is octopaminergic, and is thought to be have neuromodulatory functions. In wildtype larvae it is not restricted to stereotypic locations on the muscles, nor does it have structurally elaborate postsynaptic partners like the type Ib and Is glutamatergic motor axons. This could, in part, explain its promiscuous innervating capabilities. Ectopic type II innervation can arise in response to decreases in neuronal activity, following the loss of native innervation at a muscle, and in response to elevated FasII levels (de Jong, 2005).

In embryonic post-synaptic Side misexpression experiments it was observed that type II innervation can occur when there is a reduction in type Ib and type Is innervation at muscles 6 and 7. Yet in the same paradigms it was seen the opposite scenario occur, where type II innervation is drawn along with type Ib and Is motor axons onto a Side misexpressing muscle to give an excess of innervation. This would indicate that the presence of type II innervation arises directly in response to the Side signal rather than as a result of altered native innervation quality or quantity (de Jong, 2005).

FasII regulates the pattern of neuromuscular synapse formation, and is also required for synaptic stabilization. To elucidate the potential interplay between Side and FasII in sculpting the neuromuscular synapse, Side was misexpressed in (a) a muscle subset during targeting and (b) all muscles post-targeting in the fasII^e76 mutant background. The fasII^e76 allele is hypomorphic and expresses only 5%-10% of wildtype FasII protein levels (de Jong, 2005).

A previous study shows that elevated FasII expression in muscles 6 and 13 during targeting increases the presence of ectopic innervation on these muscles. This effect is further enhanced if the FasII misexpression is done in a fasII^e76 background. In contrast, this study found that misexpression of Side in muscles 6 and 13 in a wildtype background leads to increased ectopic innervation on muscle 13 (highest expression), and reduced innervation on muscle 6 (lower expression). When the same Side misexpression paradigm was employed in a fasII^e76 background, the attraction of innervation to muscle 13 was still enhanced while muscle 6 again showed a decrease in innervation levels (de Jong, 2005).

These findings suggest that endogenous FasII levels do not impact the responsiveness of motor axon targeting to increased Side levels. There was, however, an approximately 30% increase in the number of bouton on muscle 13 of fasII^e76; UAS-side24; H94-GAL4 larvae compared to muscle 13 in fasII^e76 larvae. This raised the question of whether increased bouton number on muscle 13 in the Side misexpression paradigm was due solely to changes in axonal targeting, or also due to later synaptic stabilization by Side (de Jong, 2005).

It has been found that fasII^e76 bouton number is reduced by ~35% in larval stages whereas early first instar larva bouton numbers are normal. This demonstrates a role for FasII in synapse stabilization. Accordingly bouton number was restored by returning FasII to both the muscle and axon (de Jong, 2005).

In order to assess if Side also has synapse stabilizing ability, it was misexpressed in the fasII^e76 mutant in all muscles from the end of targeting to the end of larval development with MHC⁸²-GAL4. It was found that in spite of the elevated post-synaptic Side levels, the number of boutons was not increased. The fasII^e76 mutant still had 25% fewer boutons than wildtype, irrespective of Side levels. In addition Side misexpression with the H94-GAL4 driver in a wildtype background results in 58 boutons on muscle 13, whereas misexpressing in the fasII^e76 background produced only 43. Again, this represents 25% fewer boutons in the fasII^e76 mutant compared to wildtype. This points to the remaining FasII present in the fasII^e76 allele being able to stabilize some, but not all of the new synaptic contacts attracted by the presence of excess Side. It also suggests that Side has no (or negligible) ability to stabilize synapses (de Jong, 2005).

In summary, this study shows that increasing Side levels on muscle fibers encourages motor axon targeting changes with concomitant synaptogenesis. The sustained retention of these novel contacts, however, in dependent on FasII levels (de Jong, 2005).

sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila

At specific choice points in the periphery, subsets of motor axons defasciculate from other axons in the motor nerves and steer into their muscle target regions. Using a large-scale genetic screen in Drosophila, the sidestep (side) gene was identified as essential for motor axons to leave the motor nerves and enter their muscle targets. side encodes a target-derived transmembrane protein (Side) that is a novel member of the immunoglobulin superfamily (IgSF). Side is expressed on embryonic muscles during the period when motor axons leave their nerves and extend onto these muscles. In side mutant embryos, motor axons fail to extend onto muscles and instead continue to extend along their motor nerves. Ectopic expression of Side results in extensive and prolonged motor axon contact with inappropriate tissues expressing Side (Sink, 2001. Full text of article).

Search PubMed for articles about Drosophila Sidestep

de Jong, S., et al. (2005). Target recognition and synaptogenesis by motor axons: Responses to the sidestep protein. Int. J. Dev. Neurosci. 23: 397-410. PubMed ID: 15927764

Fambrough, D. and Goodman, C.S. (1996) The Drosophila beaten path gene encodes a novel secreted protein that regulates defasciculation at motor axon choice points. Cell 87: 1049-1058. PubMed ID: 8978609

Holmes, A. L. and Heilig, J. S. (1999). Fasciclin II and Beaten path modulate intercellular adhesion in Drosophila larval visual organ development. Development 126: 261-272. PubMed ID: 9847240

Lavergne, G., Zmojdzian, M., Da Ponte, J. P., Junion, G. and Jagla, K. (2020). Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles. Development 147(4). PubMed ID: 32001438

Li, H., Watson, A., Olechwier, A., Anaya, M., Sorooshyari, S. K., Harnett, D. P., Lee, H. P., Vielmetter, J., Fares, M. A., Garcia, K. C., Ozkan, E., Labrador, J. P. and Zinn, K. (2017). Deconstruction of the beaten Path-Sidestep interaction network provides insights into neuromuscular system development. Elife 6. PubMed ID: 28829740

Lopez-Bendito, G. and Molnar, Z. (2003). Thalamocortical development: How are we going to get there? Nat. Rev. Neurosci. 4: 276-289. PubMed ID: 12671644

Ozkan, A. and Berberoglu, H. (2013). Cell to substratum and cell to cell interactions of microalgae. Colloids Surf B Biointerfaces 112: 302-309. PubMed ID: 24004676

Pipes, G. C., Lin, Q., Riley, S. E. and Goodman, C. S. (2001). The Beat generation: A multigene family encoding IgSF proteins related to the Beat axon guidance molecule in Drosophila. Development 128: 4545-4552. PubMed ID: 11714679

Siebert, M., Banovic, D., Goellner, B. and Aberle, H. (2009). Drosophila motor axons recognize and follow a Sidestep-labeled substrate pathway to reach their target fields. Genes Dev. 23(9): 1052-62. PubMed ID: 19369411

Sink, H., Rehm, E. J., Richstone, L., Bulls, Y. M. and Goodman, C. S. (2001). sidestep encodes a target-derived attractant essential for motor axon guidance in Drosophila. Cell 105: 57-67. PubMed ID: 11301002

date revised: 15 April 2020

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