capricious: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - capricious

Synonyms -

Cytological map position - 70A2--3

Function - cell adhesion

Keywords - axon guidance, boundary formation, wing, CNS, mesoderm

Symbol - caps

FlyBase ID: FBgn0023095

Genetic map position -

Classification - leucine-rich repeats protein

Cellular location - surface

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Kulkarni, A., Ertekin, D., Lee, C. H. and Hummel, T. (2016). Birth order dependent growth cone segregation determines synaptic layer identity in the visual system. Elife 5. PubMed ID: 26987017
The precise recognition of appropriate synaptic partner neurons is a critical step during neural circuit assembly. However, little is known about the developmental context in which recognition specificity is important to establish synaptic contacts. This study shows that in the Drosophila visual system, sequential segregation of photoreceptor afferents, reflecting their birth order, lead to differential positioning of their growth cones in the early target region. By combining loss- and gain-of-function analyses it was demonstrated that relative differences in the expression of the transcription factor Sequoia regulate R cell growth cone segregation. This initial growth cone positioning is consolidated via cell-adhesion molecule Capricious in R8 axons. Further, the initial growth cone positioning was shown to determine synaptic layer selection through proximity-based axon-target interactions. Taken together, this study demonstrates that birth order dependent pre-patterning of afferent growth cones is an essential pre-requisite for the identification of synaptic partner neurons during visual map formation in Drosophila.

Upon reaching the target region, neuronal growth cones transiently search through potential targets. Synaptic connections will only be formed with a subset of these targets. The capricious (caps) gene may regulate these processes in Drosophila. caps encodes a transmembrane protein with leucine-rich repeats (LRRs). During the formation of neuromuscular synapses, caps is expressed in a small number of synaptic partners, including muscle 12 and the motorneurons that innervate it. Loss-of-function and ectopic expression of caps alter the target specificity of muscle 12 motorneurons, indicating a role for caps in selective synapse formation (Shishido, 1998). In addition, Capricious, along with Tartan, another leucine-rich repeat protein, contributes to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing development. caps and tartan are expressed in the dorsal compartment during boundary formation. Caps and Tartan confer affinity for dorsal cells, assessed by sorting-out behavior. Caps supports boundary formation without conferring dorsal compartment signaling properties. Fringe, in contrast, confers dorsal signaling properties without affecting DV affinity. Therefore, Caps, Tartan, and Fringe have complementary roles in boundary formation (Milán, 2001).

The final step in formation of neural connectivity involves the recognition of target cells. Although earlier events of growth cone guidance greatly restrict the target region, neurons still have to choose a specific synaptic partner from among several potential targets. In each abdominal hemisegment of Drosophila larvae, ~40 motorneurons innervate 30 muscle fibers in a specific manner. Once a motor axon enters its target region during late embryogenesis, its growth cone searches over the surface of many muscles but withdraws from most of these contacts, forming stable synapses only with its own target or targets. caps regulates the formation of some of the selective synaptic connections in this system (Shishido, 1998).

A screen was performed for enhancer trap lines that express a reporter gene in specific muscle fibers during the establishment of motorneuron innervation. caps was identified by analysis of one such line, E2-3-27. In E2-3-27 embryos, the reporter (caps-LacZ) is expressed in four dorsal and six ventral muscles. caps-LacZ is also expressed in central nervous system (CNS) motorneurons that innervate caps-LacZ-positive muscles. caps-LacZ is not expressed in motorneurons that have been identified as innervating caps-negative muscles (for example, RP1, RP3, and RP4). Thus, the expression of caps-LacZ is correlated with neuromuscular specificity (Shishido, 1998).

To determine the function of caps in vivo, caps loss-of-function mutant alleles, which lack the first exon and do not express Caps protein, were generated. Most of the caps mutants die late in embryogenesis or soon after hatching, although a few survive to adulthood. Although no gross developmental defects are found in the CNS or musculature of caps mutant embryos and larvae, the target specificity of muscle 12 motorneurons is altered. In wild-type larvae, muscle 12 is innervated by the terminal branch of ISNb, including the RP5 axon, which projects to the boundary between muscles 12 and 13 and forms synaptic endings exclusively on muscle 12. In contrast, in caps mutant larvae, the terminal branch is often accompanied by additional varicosities on muscle 13, a neighboring caps-negative muscle. Thus, caps restricts arborization of the nerve terminal to muscle 12 (Shishido, 1998).

Ectopic overexpression of caps in all embryonic muscles by G14-GAL4 driver causes formation of more ectopic synapses. In ~70% of the hemisegments, the ISNb terminal formed one or more additional collaterals that formed more robust synaptic endings on muscle 13. The ectopic nerve endings contained type III boutons, which are typical of muscle 12 but not muscle 13 neuromuscular synapses. Since the ectopic synapses were present in the first-instar larvae, caps may function while the connections are being formed. This possibility is further supported by the absence of such ectopic endings when caps expression was induced after completion of synaptogenesis by Mhc82-GAL4 (Shishido, 1998).

It is proposed that caps mediates selective synapse formation. The loss-of-function phenotype may result from improper recognition of the target muscle, whereas the extra synapses on muscle 13 could reflect retention of inappropriate synaptic contacts. In contrast, the gain-of-function phenotype could indicate that the nerve terminal is attracted to muscle 13 and other muscles by ectopic caps. In both cases, however, muscle 12 motorneurons reach their target region normally and extend along muscle 12 before making ectopic synapses on muscle 13. Thus, caps may stabilize specific motorneuronal contacts during a late phase of target selection (Shishido, 1998).

The expression of caps on both sides of the synaptic partners suggests that caps functions homophilically, as has been proposed for the candidate target recognition molecules, Connectin and Fasciclin III. However, expression of caps in S2 cells did not promote cell aggregation. Thus, caps may mediate synaptic target recognition through cell-cell signaling rather than adhesion (Shishido, 1998).

In order to study the role of caps in neural recognition of muscle targets, Caps protein was expressed ectopically in all muscles. Panmuscle Caps alters the target specificity of muscle 12 motorneurons (MNs), indicating that Caps can function in muscles as a target recognition molecule. Panneural Caps alters the pathfinding of muscle 12 MNs. The defect appears to be caused by changes in the steering behavior of muscle 12 MNs at a specific choice point along their pathway to the target muscle. These results revealed a novel function of Caps in axon pathfinding. Deletion analyses of Caps were performed. Caps lacking the intracellular domain was expressed in all neurons or in all muscles, and the ability of altered Caps protein to induce the pathfinding and targeting phenotypes was examined. The function of muscularly expressed Caps in target recognition is intracellular domain dependent, whereas the function of neurally expressed Caps in pathfinding is not, suggesting that Caps may function in neurons and muscles in a different manner. The requirement of the intracellular domain for the function of muscularly expressed Caps suggests the presence of a signaling event within muscle cells that is essential for selective synapse formation (Taniguchi, 2000).

Ectopic and increased expression of Caps was induced in all neurons using the GAL4-UAS system. A GAL4 line, elav-GAL43E1, which expresses GAL4 in all neurons, was crossed with the UAS-caps-Ia+Ib line, which contains two copies of UAS-caps. The progeny (elav-GAL43E1/UAS-caps-Ia+Ib) was raised at 29°C during embryonic and larval development to induce maximal ectopic expression. As expected, Caps was ectopically expressed on all neurons, starting from embryonic stage 12, in elav-GAL43E1/UAS-caps-Ia+Ib individuals. Caps protein was detected in all major axon tracts in the CNS and in the periphery, suggesting that ectopically expressed Caps was properly transported to axons (Taniguchi, 2000).

To analyze the effect of panneural Caps expression on the formation of the nervous system, motor neuron axonal processes were examined in the third-instar larvae by monoclonal antibody 1D4 (anti-Fasciclin II) and monoclonal antibody 22C10 staining. No gross morphological defects were seen in the CNS and musculature, suggesting that their overall development proceeded normally. However, a highly specific change was detected in the trajectory of motoneurons that innervate muscle 12 (muscle 12 MNs). In wild-type larvae, axons of muscle 12 MNs, that fasciculate to form the terminal branch of the intersegmental nerve b (ISNb), project along the internal surface of muscle 13 before reaching their final target, muscle 12. In contrast, in elav-GAL43E1/UAS-caps-Ia+Ib larvae, they pass along the exterior of muscle 13 in 29% of segments. Such a phenotype is not observed in control larvae (elav-GAL43E1/+ raised at 29oC). Despite the abnormality in their trajectory, the axons of muscle 12 MNs reach their normal target by turning interiorly at the cleft between muscles 12 and 13, and establish normal synapses on muscle 12. Thus, ectopic and increased Caps expression on all neurons affects axon pathfinding of muscle 12 MNs, but not their synapse formation. The penetrance of the misrouting phenotype is dependent on the level of expression of ectopic Caps on neurons (Taniguchi, 2000).

In larvae that express Caps panneurally, defects were also observed in the formation of the transverse nerve. The nerve was often split and the cell body of a neuron associated with the nerve (lateral bipolar cell) was occasionally mislocated. With the exception of the transverse nerve phenotype, the effect of ectopic Caps on the formation of motor nerves is highly specific to muscle 12 MNs. Notably, the subbranches of ISNb that terminate on ventral muscles other than muscle 12 displayed no abnormalities in their trajectory or targeting. The other motor nerves, the intersegmental nerve (ISN), intersegmental nerve d (ISNd), segmental nerve a (SNa), and segmental nerve c (SNc) also retained their normal morphology, although it remained possible that there were subtle abnormalities that could not be detected with the marker used (Taniguchi, 2000).

To determine when the axon guidance errors of muscle 12 MNs begin to be seen in individuals that panneurally express Caps, the development of the motor nerves in the embryos was examined by mAB 1D4 staining. In wild-type embryos, motor axons exit the CNS through the ISN or segmental nerve (SN) roots. They then divide into five peripheral motor branches (ISN, ISNb, ISNd, SNa, and SNc) that project to different groups of muscle fibers. The axons of muscle 12 MNs follow the ISNb pathway. During embryonic stage 15, the axons of ISNb, including those of muscle 12 MNs, separate from the ISN and enter the ventral muscle field at muscle 28. They then extend between the external surface of muscles 6 and 7 and the internal surface of muscle 14. At a choice point near muscle 30, muscle 12 MNs and motoneurons that innervate muscle 13 shift their trajectory by turning to a more internal muscle layer. Muscle 12 MNs then extend along the internal surface of muscle 13, forming the terminal branch of ISNb. They reach their final target (muscle 12) by late stage 16 and begin to form functional synapses by early stage 17 (Taniguchi, 2000).

In elav-GAL43E1/UAS-caps-Ia+Ib embryos, no abnormality is seen in the development of ISNb until midstage 16; it defasciculates from ISN at the normal branching point, extends along the internal surface of muscle 14, and reaches muscle 30. However, specific defects in the ISNb trajectory are observed at its distal edge during late stage 16 to early stage 17. The terminal branch of ISNb formed by muscle 12 MNs normally extends along the internal surface of muscle 13 and starts to establish synaptic contacts with muscle 12 by this stage. In 18% of hemisegments in elav-GAL43E1/UAS-caps-Ia+Ib embryos, a misrouting phenotype similar to that seen in the larvae is observed. Instead of traveling along the internal surface of muscle 13, the terminal branch of ISNb takes an abnormal path along the external surface of muscle 13 to reach muscle 12. In 13% of hemisegments in elav-GAL43E1/ UAS-caps-Ia+Ib embryos, the ISNb stalled near muscle 30, failing to extend to more interior muscle layers. Occasionally, thin axonal processes were seen to emanate from the stalled nerve terminal, which stopped prematurely and failed to innervate muscle 12. These results suggest that ectopic expression of Caps in all neurons changes the behavior of muscle 12 MNs at their specific choice point near muscle 30 (Taniguchi, 2000).

Capricious is a transmembrane protein with a short intracellular domain that has no known functional motif. To examine the function of Caps in neurons and muscles, additional ectopic expression experiments were performed using modified forms of the Caps protein. Two Caps deletion constructs were generated: CapsID, which lacks the entire intracellular domain, and CapsED, which lacks most of the extracellular domain extending from the second LRR domain to the amino acid just preceding the transmembrane domain (amino acids 72-449). Ectopic expression of these deletion constructs was induced by the GAL4-UAS system. The UAS lines, UAS-CapsID-4 and UAS-capsED-20, were used to ectopically express CapsID and CapsED, respectively. When crossed with GAL4 drivers, these lines induced similar levels of ectopic Caps expression to the UAS-caps-Ia5 line with intact Caps (Taniguchi, 2000).

The UAS lines were first crossed with elav-GAL4 line to study the ability of the deleted forms of Caps to induce the pathfinding defects of muscle 12 MNs. Ectopically expressed CapsID and CapsED were detected on all major nerve tracts, indicating that the modified forms of Caps are processed and transported properly (Taniguchi, 2000).

The effects of panneuronal expression of CapsID and CapsED were studied in third-instar larvae. Misexpression of CapsED causes no defects compared to controls in the trajectory of muscle 12 MNs, indicating that the extracellular domain of Caps is essential for the induction of the misrouting phenotype. However, when CapsID was expressed, the misrouting phenotype of muscle 12 MNs was observed to the same extent as when the intact Caps was expressed (19% in elav-GAL43E1/UAS-CapsID-4 compared to 23% in elav-GAL43E1/UAS-caps-Ia5). Thus, the intracellular domain of Caps is not required for the ability of neurally expressed Caps to cause pathfinding defects of muscle 12 MNs (Taniguchi, 2000).

The effects of panmuscle expression of the Caps deletion constructs were examined. When intact Caps is ectopically expressed on all muscles, muscle 12 MNs extend their axons to and establish aberrant synapses with the neighboring muscle 13, indicating that Caps can function on muscles as a target recognition molecule. The requirement of the extracellular and intracellular domains of Caps in this process was examined by crossing the UAS lines for CapsID and CapsED with 24B-GAL4 drivers. Panmuscle expression of CapsID and CapsED was confirmed by staining with antibody against Caps. When intact Caps is ectopically expressed in all muscles, the protein is not uniformly distributed on the muscle surface but is strongly concentrated at neuromuscular synaptic sites. CapsID and CapsED are similarly localized at synaptic boutons when expressed in muscles, indicating that the deleted regions play no role in the synaptic localization of the protein (Taniguchi, 2000).

Whether the deletion constructs can cause the mistargeting phenotype of muscle 12 MNs was examined by staining the third-instar larvae of 24B-GAL4/UAS-capsID-4 and 24B-GAL4/UAS-CapsED-20. In the control larvae that misexpressed intact Caps at a similar level, terminals of muscle 12 MNs formed ectopic synapses on muscle 13 in 40% of segments. In contrast, in 24B-GAL4/UAS-CapsID-4 and 24B-GAL4/UAS-CapsED-20 larvae, the extent of targeting errors by muscle 12 MNs was dramatically decreased to the background level. Thus, both extracellular and intracellular domains are required for muscle Caps to mediate target recognition. This is in contrast with the observations in relation to panneurally expressed Caps, where the intracellular domain is not essential for inducing the misrouting phenotype. These results suggest that neurally expressed Caps and muscularly expressed Caps may function in different manners (Taniguchi, 2000).

By what mechanism does panneurally expressed Caps alter the behavior of muscle 12 MNs at this specific choice point? Axons have to complete two steps -- defasciculation from the main nerve tract and steering into the specific target region -- to change their trajectory successively at discrete choice points along the motor pathway. The defasciculation event would mainly relate to changes in axon-axon interactions, whereas the steering event relates to changes in axon-target region interactions. Although Caps does not promote cell adhesion when expressed on S2 cells, it is conceivable that overexpressed Caps on motor axons changes their behavior by increasing axon-axon interactions. However, this is considered to be unlikely. If pan-neurally expressed Caps increases the axon-axon interactions in a general manner, one would expect to see defasciculation defects at many other choice points along the motor pathway. Instead, the phenotype specificity in individuals that express Caps panneurally is highly restricted to the most distal branch of the ISNb. Furthermore, although the stall phenotype was seen in some of the ISNb terminals in the embryos, these axons appear eventually to separate from the main ISNb. These observations suggest that panneurally expressed Caps affects the pathway choice of muscle 12 MNs by influencing their proper steering events rather than defasciculation (Taniguchi, 2000).

In this model, ectopically expressed Caps on muscle 12 MNs affects their behavior by changing their affinity for the neighboring cells on their pathway. Since Caps is normally expressed on muscle 12 MNs, this effect is likely to be due to an increase in the quantity of Caps protein on the motoneurons. This notion is supported by the dose dependency of the phenotype. During normal development, Caps on muscle 12 MNs could function as a receptor that interacts with molecular cues expressed on their pathway along the external muscle layer (e.g., muscles 28, 14, and 30). Overexpression of Caps on the motoneurons may increase their affinity for the external muscle layer so as to prevent the growth cone from navigating to more interior muscle layers (e.g., muscles 12 and 13). Since Caps is normally expressed on muscles 14 and 28, this may involve a homophilic interaction between the Caps on motoneurons and on muscles. Alternatively, Caps on the motoneurons may interact with an unknown ligand expressed on the muscles. In any event, the results indicate that the steering of muscle 12 MNs at the choice point near muscle 30 is highly sensitive to the amount of Caps protein on the motoneurons (Taniguchi, 2000).

The data from the deletion analysis shows that panneural expression of CapsID is as potent as that of the intact Caps in inducing the pathfinding defects, indicating that the intracellular domain is not required for this function of the molecule. Thus, if Caps functions as a receptor in muscle 12 MNs as proposed above, it must transduce the signal by interacting with other molecules on the membrane. When Caps is expressed in all muscles, muscle 12 MNs form ectopic synapses on muscle 13 (Shishido, 1998). Since Caps is normally expressed on muscle 12 but not on muscle 13, it has been proposed that Caps functions as a specific molecular label on muscle 12 to be recognized by muscle 12 MNs. Three other candidate target recognition molecules -- Connectin, Fasciclin III, and Toll -- are similarly expressed on the surface of a subset of muscles. How do these molecules mediate selective synapse formation? Are they simply presented by muscles as ligands that are passively recognized by motoneurons? Or do they play more active roles in the initiation of synapse formation? To obtain insights on Caps function in selective synapse formation, the effect of deletion of its intracellular domain was studied. The ability of panmuscularly expressed Caps to induce targeting errors by muscle 12 MNs is completely abolished when the intracellular domain is deleted. This is not due to nonspecific conformational change of the molecule, because the CapsID retains its activity when ectopically expressed on neurons. Furthermore, panmuscularly expressed CapsID is found to be present at synaptic sites as is intact Caps, suggesting that the deletion does not affect the transportation or localization of the protein. Taken together, the results suggest that some molecular event(s) in muscles mediated by the intracellular domain of Caps are required for the ectopic synapse formation (Taniguchi, 2000).

In what way might the intracellular domain of Caps be critical for its function? One possible role played by the intracellular domain could be its interaction with cytoplasmic components necessary for synaptic localization. Such a role was shown for the intracellular domain of Fasciclin II, a homophilic cell adhesion molecule essential for the growth and maintenance of neuromuscular junctions. Indeed, there is evidence for the existence of synaptic targeting mechanisms for Caps. The Caps protein is normally localized on newly formed synaptic boutons in subsets of muscles in the first-instar larvae. Ectopically expressed Caps is concentrated at neuromuscular synaptic sites of third-instar larvae. However, the intracellular domain appears to be unnecessary for this process, since CapsID is normally concentrated at synaptic sites. Thus, the essential role played by the intracellular domain in selective synapse formation is not in the synaptic targeting of the molecule. Interestingly, CapsED, which lacks most of the extracellular domain, is also normally localized at synapses. Thus, the functional domain necessary for synaptic targeting may be in the transmembrane domain. Alternatively, there may be multiple targeting systems that act both through the intracellular and extracellular domains of Caps (Taniguchi, 2000).

Cell biological evidence is accumulating to support the notion that postsynaptic cells play active roles in synapse formation. During axodendritic synaptogenesis in vertebrates, dendritic filopodia and their protrusive motility are proposed to be essential for the initiation of synaptic contacts. Similarly, during neuromuscular synaptogenesis in Drosophila, muscles extend fine processes (called myopodia) that actively interact with motoneuronal growth cones. It is an interesting possibility that Caps on muscles participates in such processes by interacting with receptors on specific motoneurons via its extracellular domain and by transmitting the signal into muscles via its intracellular domain. Although the exact nature of the signal mediated by the intracellular domain of Caps is unknown, the present study supports the notion that molecular events occurring in postsynaptic cells are essential for the formation of specific synapses (Taniguchi, 2000).


cDNA clone length - 2951

Bases in 5' UTR - 895

Exons - 1

Bases in 3' UTR - 467


Amino Acids - 532

Structural Domains

caps encodes a transmembrane protein with 14 Leu-rich repeats (LRRs) in its extracellular domain. LRR, a ~24 amino acid motif found in various proteins from sources as diverse as yeast to human, may mediate protein-protein interactions. Among the proteins with LRRs, Caps protein is most closely related to the product of the tartan gene from Drosophila (Chang, 1993), with amino acid similarity extending beyond the LRR region into the cytoplasmic region. The predicted Caps and Tartan proteins are 65% identical in the extracellular domain, which consists of 14 LRR domains. They also share a conserved domain adjacent to the membrane in the cytoplasmic tail, but differ at their C termini. Proteins with LRRs expressed on the cell surface may function in cell adhesion or recognition (Shishido, 1998).

capricious: Developmental Biology | Regulation | Effects of Mutation | References

date revised: 6 October 2001

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