The caps-LacZ reporter is expressed in four dorsal (1, 2, 9, and 10) and six ventral (12, 14 to 17, and 28) muscles. In muscle 12, caps-LacZ and caps RNA are expressed in a single nucleus of the syncytial muscle, near the contact site of the motorneuronal growth cone. caps-LacZ is also expressed in central nervous system (CNS) motorneurons aCC, RP2, RP5, and the most medial U, all of which innervate caps-LacZ-positive muscles (Shishido, 1998).
Caps protein is expressed on the surface of developing motor axons. In first-instar larvae, Caps protein was detected in the mature synaptic sites of all caps-positive muscles (Shishido, 1998).
Capricious and Tartan, two transmembrane proteins with leucine-rich repeats, contribute to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing development. Engrailed/Invected expression confers posterior (P) identity and Apterous (Ap) confers dorsal (D) identity in the wing disc. P compartment cells lacking engrailed/invected activity do not respect the anterior-posterior boundary. Likewise, dorsal cells lacking ap activity fail to respect the dorsal-ventral (DV) boundary in the wing disc. Modulation of Notch signaling has been implicated in DV boundary formation. Fringe acts as a glycosyltransferase to modify the receptor protein Notch in the dorsal compartment. Fringe activity makes D cells more sensitive to Delta, a ligand expressed by V cells and less sensitive to Serrate, the ligand expressed by D cells. Consequently, signaling by each ligand is limited to nearby cells on the opposite side of the boundary, with the result that high levels of Notch activity are limited to a narrow band of cells along the DV boundary. Although altering the signaling properties of cells by modulation of Fringe activity has been shown to allow cells to cross the boundary, Fringe activity has been shown to be insufficient to support boundary formation. This observation, together with the fact that Notch signaling is activated symmetrically has suggested that other Apterous-dependent cell interactions might be needed for formation of the DV affinity boundary. Evidence suggests that capricious and tartan are targets of Apterous that contribute to DV boundary formation in the wing disc. caps and tartan are expressed in the D compartment during boundary formation. Caps and Tartan confer affinity for D cells, assessed by sorting-out behavior. Caps supports boundary formation without conferring D signaling properties. Fringe, in contrast, confers dorsal signaling properties without affecting DV affinity. Thus, Caps, Tartan, and Fringe have complementary roles in boundary formation (Milán, 2001).
In second and early third instar wing discs, caps-lacZ is expressed in the D compartment. At this stage, expression of Caps protein coincides with that of Apterous, and caps-lacZ expression depends on Apterous activity. Expression of the Apterous inhibitor, dLMO, under control of patched-Gal4 represses caps-lacZ expression. During third instar, dorsal expression of caps-lacZ decreases and new lateral expression domains arise. These domains are initially stronger in the D compartment but become symmetric in D and V compartments in mature third instar discs. tartan expression was monitored using a lacZ reporter gene and antibody to Tartan protein, and is similar to caps expression at all stages. The dynamics of these expression patterns suggested that Caps and Tartan proteins might mediate cell interactions during early DV patterning and subsequently during medial-lateral patterning of the wing (Milán, 2001).
To assess the roles of Caps and Tartan in DV boundary formation, use was made of a rescue assay in which the Gal4-UAS system was used to restore Caps and Tartan expression in D cells of apterous mutant wing discs. apGal4/ap- mutant discs are not able to form a smooth DV boundary and fail to induce Wg expression uniformly along the interface between D and V cell populations. Expression of Caps in D cells under apGal4 control restores a smooth interface between D and V cells in the mutant discs, but does not restore Wg expression along the boundary. Tartan is considerably less effective at producing a smooth interface between D and V cells. Connectin, a GPI-anchored membrane protein that is related to Caps and Tartan in the LRR domains, was tested because Connectin has been shown to mediate homophilic cell adhesion. Connectin was ineffective in the boundary rescue assay. Fasciclin II, an unrelated adhesion protein, was also unable to restore the DV boundary. These observations suggest that Caps expression produces an affinity boundary between D and V cells by a mechanism that is not simply due to increased cohesion among dorsal cells (Milán, 2001).
To test the requirement for Caps and Tartan in boundary formation, clones of cells mutant for caps or tartan were produced. Single mutant clones did not produce observable alterations in the wing disc. Clones simultaneously mutant for caps and tartan do not cause defects at the DV boundary, but do perturb medial-lateral cell interactions. In wild-type discs, loss of caps and tartan activity may be compensated for by other proteins. It was therefore asked whether reduced levels of caps and tartan activity would cause defects when DV boundary formation was compromised by reduction of Ap activity. Two sensitized genetic backgrounds were examined. Bx1 produces a wing scalloping phenotype that is very sensitive to the level of expression of other genes involved in DV patterning. Bx1 is a dominant mutation that overexpresses the dLMO protein. dLMO competes with Ap for binding to its cofactor Chip and thereby reduces Ap activity. The second sensitized genotype was provided by a mutant with reduced expression of the Ap cofactor Chip. Chipe5.5 was selected because it is less sensitive to modification than Bx1 but shows specific genetic interactions with ap, dLMO, Serrate, and fringe. Interactions were scored on the basis of dominant wing scalloping phenotypes in flies heterozygous for Chipe5.5 or Bx1. Deletions in 18 different genes dominantly enhanced both phenotypes. Nine of these uncover genes with known roles in DV patterning, including ap, vestigial, cut, and Serrate. Df(3L)C190, a deletion that removes the caps and tartan genes, enhances the Bx1 and Chipe5.5 phenotypes. The caps tartan double mutant chromosome used for the clonal analysis also enhances both phenotypes. The contributions of caps and tartan were then tested individually. A caps lack-of-function mutant enhances both phenotypes. A tartan mutant lack-of-function mutant enhances Bx1, but does not produce a phenotype in the less sensitive Chipe5.5 background. These observations indicate that reduced caps and tartan activity causes wing defects when the system is sensitized by reduction of Ap activity. These observations suggest that Caps and Tartan contribute to DV boundary formation (Milán, 2001).
Use was made of the flip-out Gal4 system to produce clones of Gal4-expressing cells in the wing disc to examine effects of ectopic Caps and Tartan expression. Of 178 clones examined, 23 contacted the boundary on the D side compared to 25 on the V side. Sixty-four clones were located internally in the D compartment, compared to 66 in the V compartment. Caps-expressing clones differ in two respects from control clones. Fewer Caps-expressing clones were recovered in the V compartment (V/D ratio ~0.7). Nonetheless, twice as many V clones were recovered at the boundary as would be expected if Caps had no effect on their distribution. Comparable results were obtained for clones expressing Tartan or Caps and Tartan together. These observations suggest that clones expressing Caps or Tartan survive poorly in the V compartment. To ask whether poor survival of V clones could be responsible for their accumulation at the DV boundary, Caps and Tartan were co-expressed with the viral apoptosis inhibitor p35. p35 expression suppresses the loss of V clones. Yet, V clones were still overrepresented by ~2-fold at the DV boundary. Control clones expressing GFP and p35 were evenly distributed between D and V compartments. These observations suggest that V clones expressing Caps or Tartan survive poorly if they fail to contact D cells, and that Caps or Tartan expression causes V cells to sort-out toward the D compartment (Milán, 2001).
Caps- and Tartan-expressing clones of V compartment origin sort-out toward the DV boundary but remain in the V compartment. Although these clones do not cross the boundary, many of them appear to push the Wg stripe toward dorsal. To examine this behavior more closely, clones were produced in early 2nd instar discs, before the DV boundary forms. Some of these clones were bisected by the nascent DV boundary so that they contributed to both compartments (referred to as D+V clones). Control D+V clones expressing GFP or lacZ reporter genes have no effect on the shape of the Wg stripe, and most are of similar size in both compartments. These clones were generally elongated in shape and had irregular borders where they contacted neighboring wild-type cells. In contrast, D+V clones expressing Caps or Tartan are more compact in shape, have smoother borders, and tend to be considerably smaller in the V compartment. Many of these clones distorted the Wg stripe where they crossed the boundary. D+V clones expressing Caps and Tartan together had similar effects (Milán, 2001).
The effects of smaller clones on the shape of the DV boundary were examined, using expression of an ap-lacZ reporter gene to mark dorsal cells. GFP-expressing clones that contact the DV boundary have no effect on the ap-lacZ border or on the Wg stripe. In contrast, ventral Caps- or Tartan-expressing clones often displace both the ap-lacZ border and the Wg stripe toward dorsal. In one case, a ventral Caps-expressing clone was observed that had separated a group of D cells from the rest of the D compartment. The effects of clones expressing Tartan or Caps and Tartan together were similar to those of Caps-expressing clones. D compartment clones had no effect (Milán, 2001).
Although it is not possible to observe how these distortions of the DV boundary arise, it is tempting to speculate that they result from V cells attempting to sort-out into the D compartment. Sorting out could be caused by increased affinity for D compartment cells or by repulsion by V compartment cells. Either mechanism could provide a force to push the clones into the D compartment and displace D cells and the Wg stripe. Since Caps and Tartan are expressed by D cells, it was asked whether V clones are attracted to the D compartment by homophilic cell adhesion mediated by Caps and Tartan. To measure homophilic adhesion, use was made of a cell aggregation assay that involved the use of cultured S2 cells. S2 cells expressing Caps and Tartan do not aggregate more than control cells. Likewise, binding of a secreted Caps-Alkaline Phosphatase fusion protein to cells expressing Caps, Tartan, or both could not be detected. Thus, the sorting-out behavior of Caps- and Tartan-expressing clones is unlikely to depend on homophilic cell adhesion mediated by these proteins. It is proposed that Caps and Tartan interact with other surface proteins expressed in the D compartment (Milán, 2001).
To examine how Caps and Tartan induce sorting behavior, clones that had sorted toward the DV boundary were observed using confocal microscopy. Membrane-bound cellular processes were observed extending from Caps-expressing cells toward cells in the D compartment. Caps protein outlined the cell surface and appeared in bright spots that may be intracellular vesicles. In more apical sections, Caps protein was located on thin structures that extended into the dorsal compartment. In cross-section, these structures can be seen to project from V cells over the apical surface of nearby D cells. Since Caps is a membrane protein, it is inferred that these are membranous cellular processes, perhaps filopodia. Processes were also observed projecting toward the D compartment from V clones that were not in contact with the boundary. Similar structures extending between closely spaced clones within the V compartment were not observed. All projections were oriented toward the D compartment. These observations support the idea that Caps may interact with another cell surface protein in the D compartment (Milán, 2001).
Tartan-expressing clones also sort-out toward the D compartment. Clones of Tartan-expressing cells were examined: cellular projections could not be visualized with anti-Tartan antibody. As an alternative, use was made of a transgene expressing cytoplasmic ß-Gal to mark cellular processes when coexpressed with Tartan. Ventral Tartan-expressing clones also extend cytoplasmic processes toward D cells. Projections produced by cells expressing Caps were similar in appearance when visualized using cytoplasmic ß-Gal. Cellular processes expressing Caps and Tartan may help ventral cells to sort toward the D compartment. These observations suggest that the behavior of Caps- and Tartan-expressing V clones is guided by increased affinity for D cells (Milán, 2001).
Why are V clones expressing Caps and Tartan unable to cross the Wg stripe. Caps and Tartan are not able to restore Notch signaling and Wg expression when expressed in the D compartment of ap mutant discs. Likewise, clones of cells expressing Caps or Tartan do not induce Wg expression in adjacent V cells. Instead, Wg is expressed normally where Tartan or Caps-expressing V cells contacted D cells. Thus, ventral Caps- and Tartan-expressing clones retain ventral signaling properties. In this respect, they differ from ventral Fringe-expressing clones, which acquire the signaling properties of D cells (Milán, 2001).
Fringe acts as a glycosyltransferase enzyme to modify Notch and make it differentially sensitive to its ligands. Consequently, ventral clones of Fringe-expressing cells induce ectopic expression of Wg where they contact other V cells. In cases where ventral Fringe-expressing clones contact the DV boundary, Wg is induced at the interface with other V cells, but not at the interface with D cells. The change in signaling properties of these cells results in relocation of the stripe of Wg expression to the interface between the clone and other V cells. Consequently, Fringe-expressing clones cross the boundary defined by the Wg stripe. Caps- and Tartan-expressing clones retain ventral signaling properties and so cannot reposition the Wg stripe (Milán, 2001).
The behavior of Fringe-expressing clones differs in a second respect. Fringe-expressing clones are not lost from the V compartment and do not accumulate at the DV boundary. Thus, Fringe-expressing clones do not acquire the ability to sort-out toward D cells, an ability that is conferred by Caps or Tartan expression. These properties are reflected in the different abilities of Fringe and Caps to rescue the DV affinity boundary in ap mutant discs. Expression of Fringe restores Notch signaling and induces Wg expression, but is unable to produce a smooth DV affinity boundary. In contrast, Caps produces a smooth boundary but does not restore Notch signaling. Coexpression of Caps and Fringe restores Wg expression and produces a normal DV affinity boundary. Likewise, clones expressing Caps, Tartan, and Fringe sort-out toward the DV boundary and cross into the D compartment. These observations suggest that Fringe and Caps/Tartan play distinct but complementary roles in boundary formation (Milán, 2001).
It is proposed that the activities of Caps and Tartan, as well as those of the Notch ligands and Fringe, are required for DV boundary formation. Apterous controls expression of Serrate and Fringe as well as Caps and Tartan in dorsal cells during boundary formation. The ligands for Notch are transmembrane proteins. Therefore, ligand-receptor binding may contribute to adhesion between D and V cells at the boundary while inducing signaling. By increasing the affinity for Delta, Fringe may promote binding between Notch on D cells and Delta on V cells. Likewise, by reducing the affinity of D cells for Serrate, Fringe may promote binding between Serrate on D cells and Notch on V cells. Increased binding between oppositely specified cells is likely to help to stabilize the interface between the two cell populations, but seems unlikely to help drive the initial segregation of the populations needed to establish a smooth boundary. Indeed, restoring Fringe and Serrate expression in apterous mutant discs is not sufficient to restore a normal DV boundary. It is proposed that the transient expression of Caps and Tartan in D cells initiates the segregation of the two cell populations. Once they are separated, Fringe-dependent cell interactions may stabilize the boundary. Fringe has also been implicated in boundary formation in vertebrate limbs. It will be of interest to learn whether Caps and Tartan homologs play comparable roles in DV boundary formation in vertebrates (Milán, 2001).
Caps and Tartan expression induce the formation of cellular processes that projected from V cells toward D cells. Cytonemes and similar structures have been proposed to mediate long-range cell interactions in imaginal discs. The structures observed appear to differ from cytonemes in that they project across the signaling center into the opposite compartment, rather than projecting toward the signaling center. Filopodia have been implicated in guiding morphogenetic movements in epithelial sheets. Filopodia expressing E-Cadherin have been implicated in the formation of adhesive zippers between epithelial cells, which serve as nucleation centers for reorganization of the cytoskeleton. It is proposed that imaginal disc cells use filopodia that express cell-surface proteins, including Caps and Tartan, to assess the identity of nearby cells and to control cell behavior. Caps and Tartan do not appear to mediate homophilic adhesion. This suggests that dorsal cells express another cell surface protein that is able to bind the LRR domains of Caps and Tartan. Expression screening and systematic searches for membrane proteins expressed on D cells may help to identify the Caps/Tartan binding partner (Milán, 2001).
The mechanisms by which tissue boundaries form are not well understood. Differences in cell adhesion can contribute to tissue boundary formation. Sorting-out of cell populations can be guided by both the amount and types of adhesion proteins that cells express. A different view comes from studies on Ephrins and Eph receptors, which suggest that repulsion or deadhesion can promote segregation of cell populations. Many adhesion proteins form regulated connections with the cytoskeleton and participate in contact-mediated signaling. A useful distinction can be made between initial cell-cell contacts, which may be transient, and formation of stable contacts that may involve substantial reorganization of the cytoskeleton. If signaling promotes reorganization of the cytoskeleton, cell interactions might be destabilized. Repeated cycles of deadhesion and readhesion could lead to sorting out behavior. It is possible therefore that adhesive differences and differences in cell behavior both contribute to forming tissue boundaries. At present, it is not clear which type of explanation best describes formation of the compartment boundaries in imaginal discs. Caps and Tartan are cell surface proteins that mediate cell interactions. These findings suggest that contacts with appropriately specified cells mediated by Caps and Tartan might be stabilized, whereas contacts with inappropriate cells might be destabilized. A deeper understanding of these processes awaits identification of the cell surface proteins with which Caps and Tartan interact (Milán, 2001).
The body wall musculature of the Drosophila larva consists of a stereotyped pattern of 30 muscles per abdominal hemisegment that are innervated by about 40 distinct motoneurons. Proper innervation by motoneurons is established during late embryogenesis. Guidance of motor axons to specific muscles requires appropriate pathfinding decisions as they follow their pathways within the central nervous system and on the surface of muscles. Once the appropriate targets are reached, stable synaptic contacts between motoneurons and muscles are formed. Recent studies have revealed a number of molecular components required for proper motor axon pathfinding and demonstrate specific roles in fasciculation/defasciculation events, a key process in the formation of discrete motoneuron pathways. The gene capricious (caps), which encodes a cell-surface protein, functions as a recognition molecule in motor axon guidance, regulating the formation of the selective connections between the SNb-derived motoneuron RP5 and muscle 12. Krüppel (Kr), best known as a segmentation gene of the gap class, functionally interacts with caps in establishing the proper axonal pathway of SNb including the RP5 axons. The results suggest that the transcription factor Krüppel participates in proper control of cell-surface molecules that are necessary for the SNb neurons to navigate in a caps-dependent manner within the array of the ventral longitudinal target muscles (Abrell, 2001).
Use has been made of a Kr gain-of-function mutation, termed Irregular facets (If), to identify modifiers of Kr activity during eye morphogenesis. One suppressor of ectopic Kr activity in the eye, the P-element insertion l(3)02937, was found to reside within the gene caps. caps encodes a CAM of the 'leucine-rich repeat' family and has been shown to be required for proper pathfinding and synapsing of the RP5 motoneuron with muscle 12. In addition to l(3)02937, a second P-element insertion, l(3)05121, was identified that resides in the first exon of the caps gene. Genetic analyses showed that the two P-element insertions failed to complement each other and the previously identified caps65.2 null-mutation, indicating their allelism to caps. Furthermore, precise excision of the P-element insertion l(3)05121 results in a reversion of the caps mutant phenotype to wildtype, indicating that the P-element insertion is the cause of the mutation. Moreover, the phenotype caused by the newly identified caps alleles is indistinguishable from the caps65.2 loss-of-function phenotype, suggesting that they represent either strong hypomorphic or lack-of-function caps mutations (Abrell, 2001).
While the innervation of muscles occurs during embryogenesis, previous studies on caps function have focussed on mutant defects that were observed in the motoneuronal pattern of third instar larvae, showing that Caps is necessary for proper pathfinding of the RP5 axons. RP5 axons are part of the SNb fascicle. During embryogenesis, the SNb enters the ventral muscle field between muscle 15 and 28 (choice point 'target entry'). Its axons pass the ventral oblique muscle field towards the ventral longitudinal muscle targets in the most internal muscle layer. Close to the second choice point of the SNb nerve, the RP3 axon separates from SNb to target the cleft between muscles 7 and 6. The SNb continues along muscle 14 and enters the ventral longitudinal muscle field. At a position close to muscle 30, the RP1 and RP4 axons separate to target muscle 13. The remaining RP5 bypasses muscle 13 to synapse with muscle 12. This muscle as well as the RP5 axons are characterized by caps expression both in the embryo and in larvae (Abrell, 2001).
The early route of SNb pathfinding and the defasciculation patterns of RP1, RP3 and RP4 are not affected in caps homozygous larvae, whereas the pathway of RP5 is altered. RP5 loses its target specificity and synapses with both muscle 12 and 13 instead of muscle 12 only. Furthermore, overexpression of caps in all muscles causes the same phenotype. These results were taken to indicate that caps plays an important role in selective target recognition and synapse formation by the motoneuron RP5 (Abrell, 2001).
In order to see whether and to what extent caps affects neuromuscular connectivity in the embryo, the phenotypes of the different caps mutants were examined. Of a total of 111 homozygous caps65.2 mutant individuals examined, all embryos developed into normal looking larvae. However, the majority of the caps65.2 mutant larvae (62%) failed to hatch. Out of 81 hatched larvae examined, only two individuals survived to pupal stages and developed into adults. Similar results were obtained with homozygous capsl(3)02937 (64% unhatched larvae; 3% adults) and capsl(3)05121 (73% unhatched larvae; 3% adults) mutants. The two newly identified caps alleles are therefore similar in strength to caps65.2, previously shown to be a lack-of-function mutation. Moreover, these data indicate that caps functions primarily during embryogenesis and that previous studies on caps function in larvae have been performed with the fraction of mutants that develop into larvae (Abrell, 2001).
SNb development was examined in homozygous mutant embryos using monoclonal antibodies directed against the CAM FasII. In all three caps mutants, the SNb enters the ventral muscle field and the RP axons defasciculate normally. Furthermore, RP1, RP3 and RP4 properly synapse with their respective target muscles, whereas the RP5 axons stall, show enlarged growth cone-like structures and fail to contact the target muscle 12. Instead, the RP5 axons are split and found also in direct contact with the transversal nerve (TN), a link never observed in wildtype. These phenotypes suggest that pathfinding of RP5 axons during embryogenesis is retarded and in cases where the RP5 axons defasciculate and elongate, they show erratic targeting and synapsing, as has been described for third instar larvae (Abrell, 2001).
The specific SNb defect of the caps mutants on RP5 pathfinding and its failure to synapse exclusively with muscle 12 correlates with the observation that both RP5 neurons and their target muscle are characterized by caps expression both in embryos and larvae. In addition, overexpression of caps in all muscles causes a phenotype similar to loss-of-function mutations, suggesting that relative levels of Caps are important for pathfinding. Thus, both lack-of-function and gain-of-function studies indicate a specific role for caps in selective target recognition and synapse formation. The results show that this function of caps is required during embryogenesis and that the previously reported phenotype observed in third instar larvae represents only a weak phenotype common to escapers. In all other cases, impairment of caps activity causes the RP5 axons to stall immediately after defasciculation from the SNb fascicle. This observation indicates that caps is a critical component specifically required for RP5 pathfinding after the defasciculation of the RP5 axons from SNb has occurred (Abrell, 2001).
caps is expressed in both motoneuron RP5 and its synaptic target muscle 12 in third instar larvae. In view of the embryonic caps mutant phenotypes, it was asked whether caps is also expressed during early embryogenesis and whether Kr (which is expressed in neuroblasts and in a distinct subset of embryonic muscles) could be responsible for the control of caps expression (Abrell, 2001).
caps is indeed expressed in neuroblasts. However, this aspect of the caps expression pattern was not altered in Kr lack-of-function mutant embryos except in the central region which is distorted due to the earlier segmentation function of Kr. caps expression was monitored in Krres mutant embryos in which the early segmentation function of Kr is specifically rescued. No difference was observed in the caps expression patterns of Krres mutant and wildtype embryos and the Kr neural expression pattern was unchanged in caps mutant embryos, indicating that Kr and caps activities are independently controlled. Since caps acts as a dose-dependent modifier of ectopic Kr activity during eye formation, it was next asked whether later aspects of the expression patterns, caps expression in RP5 neurons and their target muscles and Kr expression in a subset of muscles along the SNb pathway, might reflect the need of the two gene activities for the proper guidance of SNb axons (Abrell, 2001).
Previous work has shown that in the absence of Kr activity, the SNb stalls at the second choice point and RP axons fail to defasciculate. This observation is consistent with the proposal that the two genes act in the same genetic pathway. In order to test this proposal, genetic interaction studies were performed using capsl(3)05121 and Krres mutant combinations, asking whether a reduction of Kr and caps activities causes defects in neuromuscular connectivity (Abrell, 2001).
Heterozygous Krres/+ or capsl(3)05121/+ single mutant embryos developed a normal motoneuron pattern. Each of the RP axons was properly connected to its target muscle as revealed by anti-FasII antibody staining. Thus, a reduction of either Kr or caps activity has no effect on motoneuron development and pathfinding. In contrast, double heterozygous Krres/+;capsl(3)05121/+ embryos, where the dosage of both genes was reduced at the same time, develop a specific SNb nerve phenotype without affecting the ISN and SNa. In about one-third of the cases, the SNb stop along ventral longitudinal muscles, ending with a large growth cone-like structure. In addition, properly defasciculated RP axons fail to continue along their normal paths; a portion of them elongate and stall either in a position very close to the TN or is directly connected to it. Double homozygous Krres;capsl(3)05121 mutants develop an even stronger phenotype; the SNb is absent in most of the double mutants analysed or does not extend beyond its second choice point close to muscle 28. In only few cases, the SNb stalls in the ventral muscle field as had been described for homozygous Krres embryos (Abrell, 2001).
These phenotypes were also obtained with similar frequencies in double homozygous Krres;caps65.2 mutants, indicating that the phenotype is not dependent on a particular caps allele. The results show in addition that the defects are stronger and more pronounced in double mutant embryos than those obtained with single mutant embryos (Abrell, 2001).
The failure to detect the SNb nerve in the majority of homozygous Krres;capsl(3)05121 mutant embryos correlates with a thickening of the ISN. In order to test whether SNb might have lost its identity due to a transformation into ISN identity, the SNb-derived RP neurons were labelled by virtue of the transgenic islH-tau-myc marker gene. The results show that the RP axons were present. However, the SNb fails to separate from the ISN or in the cases separation occurs, it stalls shortly after the defasciculation. These observations indicate that the SNb is not transformed into ISN identity and suggest that the SNb has lost the capability to respond to guidance cues such as CAMs and repellents. Moreover, the results indicate that Kr and caps activities cooperate in a synergistic fashion necessary for proper defasciculation of the SNb axons at the exit junction and for RP axon guidance in the ventral muscle field (Abrell, 2001).
Previous studies have shown that overexpression of caps in all neurons causes a specific misrouting or stalling of RP5 at the second choice point near muscle 30 in about one-third of the embryos. This effect of panneural caps expression is dependent on the extracellular domain of Caps, suggesting that Caps functions as a cell-adhesion component which participates in the guidance of the SNb at the specific choice point near muscle 30. In contrast, panmuscular expression of caps has no effect on SNb guidance and pathfinding, but severely interfers with synapsing of RP5 resulting in connections being formed not only with muscle 12, but also with the neighboring muscle 13 (Abrell, 2001).
In order to investigate whether misexpression of Kr can interfere with SNb formation, Kr was ectopically expressed in all motoneurons or muscles using the Gal4/UAS system. To achieve this, the ftzNG-Gal4 ('panmotoneuronal expressioní) and the 24B-Gal4 driver lines ('panmuscular expression') were used in combination with one and two copies of UAS-Kr transgenes. Panmotoneural Kr expression from one transgene in wildtype embryos results in a minor phenotype of the SNb, in which the distal RP axons fail to reach their target muscles and maintain growth cone-like structures at their ends. Panmotoneural Kr expression from two transgenes causes a stronger phenotype. In 36% of the cases, the most distal RP axons stalls and the RP5 axon does not innervate the target muscle 12, whereas in all other cases, the SNb stalls at the second choice point, a phenotype that is similar to the Krres homozygous mutant phenotype. This observation indicates that the Kr overexpression phenotype is dosage-dependent and that both the lack-of-function and gain-of-function effects of Kr interfere with SNb development. Since Kr is a cell-autonomous transcription factor, it is likely that it is required for and can interfere with the transcription of motoneuronal genes necessary for proper motoneuronalguidance (Abrell, 2001).
In the wildtype embryos, Kr is not only expressed in the nervous system but also in specific subsets of muscle founder cells and muscles. Relevant sites of Kr expression during the formation of neuromuscular connectivity are the ventral oblique muscles 14 and 16, the ventral acute muscle 27 and the ventral longitudinal muscles 6, 7 and 13. Heterozygous Krres mutant embryos develop a normal muscle pattern, whereas in homozygous Krres mutant embryos muscle 27 is transformed into a duplicated muscle 26. The other muscles that normally express Kr either appear to be normal or develop a variably altered morphology. Overexpression of four copies of Kr in all muscles leads to the reverse result, i.e. muscle 26 is transformed into a second muscle 27. Thus, Kr is necessary to determine muscle identity and can alter muscle fate upon ectopic expression in muscles that normally do not express the gene (Abrell, 2001).
Pan muscular expression of Kr in response to one or two copies of the 24B-driven Gal4/UAS-Kr cDNA transgenes does not disturb the muscle pattern. However, it has severe consequences for the formation and defasciculation of SNb. With one copy of Kr, the RP3 axon separates properly from SNb and succeeds in finding the cleft between target muscles 7 and 6, whereas the remaining RP axons fail to defasciculate. In the majority of cases, however, the SNb passes the ventral oblique muscles and enters the ventral longitudinal muscles normally, but it stalls at the second choice point and RP axons fail to defasciculate. Two copies of transgene-derived Kr expression in all cases cause stronger defects that the SNb stalled at the position where RP3 would normally defasciculate. The same Kr-dependent phenotypes are found in response to a different panmuscular Gal4 driver, namely the twi Gal4 line. Thus, in contrast to panmuscular expression of caps, panmuscular expression of Kr appears to interfere with a muscle-specific program that regulates defasciculation of the RP axons and/or the elongation of SNb after the second choice point has been reached. The finding is consistent with the argument that Kr determines the spectrum of molecules expressed in muscles that are used to transmit signals to other cells, namely cell-surface and secreted molecules (Abrell, 2001).
In order to test whether panmuscular Kr expression interferes with SNb guidance in a Kr- and caps-dependent manner, the phenotype was examined of double homozygous Krres; capsl(3)05121 mutant embryos that receive panmuscular Kr expression. Upon panmuscular Kr expression, the SNb is absent in 70% of the cases. In the other cases, the SNb had formed and separated from the ISN, but stalled in the region of the ventral longitudinal muscles. The observed phenotype is reminiscent of the phenotype obtained with Krres;capsl(3)05121 double homozygous mutants, but a higher proportion of axons defasciculate in the exit junction region. This observation indicates that panmuscular Kr expression can partially rescue the SNb phenotype of the double homozygous mutants. However, defasciculation from the stalled SNb occurs in an erratic manner, implying that axon guidance is still strongly impaired. The lack of a better rescue is likely to be due to the fact that Kr has not been expressed in its wildtype muscular pattern and/or that the correct levels and neuronal Kr activity are not provided through panmuscular expression. Nevertheless, the results are consistent with the proposal that Kr regulates a muscular programme which in turn regulates SNb axon guidance along the muscles. This proposal is supported by the notion that the phenotype is reminiscent of mutants affecting CAMs in motoneurons such as FasII (Abrell, 2001).
The similarity of motoneuronal phenotypes of Kr and CAM mutants, and the interaction between Kr and caps activity suggests that Kr might also interact genetically with additional CAMs which are necessary for proper path finding. This proposal was tested using mutant combinations of Krres and the loss-of-function mutant FasIIeb112. FasII is a more general CAM than Caps. It is expressed on all motoneurons during late embryonic stages and is necessary to maintain adhesion between the axons (Abrell, 2001).
Heterozygous Krres/+ or FasIIeb112/+ embryos develop a normal SNb pattern and all RPs are properly connected to their target muscles. In double heterozygous FasIIeb112/+;Krres/+ mutants, however, the SNb enters the ventral muscle field normally in all cases, but the nerve stops at the second choice point by forming a growth cone-like structure. No individual RP axons could be observed. This phenotype is very similar to the one observed with homozygous Krres mutant embryos, implying that the two gene activities cooperate to allow for proper SNb development (Abrell, 2001).
In addition to position, size and morphology, the innervation of muscles by specific motoneurons represents a diagnostic feature for the determination of muscle identity. Previous results have shown that Kr is expressed in a specific subset of muscle progenitors and is necessary for the acquisition of a specific muscle fate as shown by muscle transformations that occur in response to gain-of-function and lack-of-function Kr mutations. Muscle 27 is transformed into a duplicated muscle 26 in homozygous Kr mutant embryos whereas high level overexpression (four copies of Kr) in all muscles leads to the reverse transformation. The data suggest that Kr contributes not only to identifying characteristics of muscle 27 but also provides adhesion properties to other Kr-expressing muscles along the SNb pathway, i.e. muscles 14 and 16, the ventral acute muscle 27, and the ventral longitudinal muscles 6, 7 and 13. The genetic interactions between Kr and the CAMs FasII and Caps support the hypothesis and imply that the adhesive properties of motoneurons and/or muscles are established in such a way that a concomitant reduction in adhesion of Krres/+ ; caps/+ or Krres/+; FasIIeb112/+ mutants results in a situation which no longer provides sufficient information to allow accurate axonal pathfinding and innervation. This proposal is also consistent with the finding that the presence of one muscle in an otherwise muscle-depleted embryo can be sufficient for the defasciculation of nerve bundles (Abrell, 2001).
In muscles 6, 7 and 13, Kr is known to maintain the expression of a direct target gene, knockout (ko). ko mutant embryos display a Kr-like motoneuron phenotype, suggesting that the gene, which encodes a novel protein with unknown biochemical characteristics, plays a key role in SNb defasciculation and RP pathfinding by acting downstream of Kr. However, in contrast to Kr, ectopic misexpression of ko does not affect SNb branching and synaptic targeting of RP neurons, and no genetic interaction as observed between ko and caps could be found. It is therefore likely that Kr transmits its signal not only via ko, but also through other factors that are still to be identified. It was also found that in contrast to caps, ectopic panmotoneural expression of Kr causes defects similar to the Kr lack-of-function mutation, and a reduction of combined caps or FasII and Kr activities. It is therefore speculated that Kr activity can also directly interfere with the spectrum of CAMs in motoneurons, resulting in non-compatible cell-surface characteristics between axons and muscles, which in turn interfere with neuromuscular connectivity (Abrell, 2001).
During development of multicellular organisms, cells are often eliminated by apoptosis if they fail to receive appropriate signals from their surroundings. Short-range cell interactions support cell survival in the Drosophila wing imaginal disc. Evidence is presented showing that cells incorrectly specified for their position undergo apoptosis because they fail to express specific proteins that are found on surrounding cells, including the LRR transmembrane proteins Capricious and Tartan. Interestingly, only the extracellular domains of Capricious and Tartan are required, suggesting that a bidirectional process of cell communication is involved in triggering apoptosis. Evidence showing that activation of the Notch signal transduction pathway is involved in triggering apoptosis of cells misspecified for their dorsal-ventral position (Milán, 2002).
In second instar wing discs, the LRR proteins Caps and Tartan are expressed in cells of the dorsal compartment. During third instar, dorsal expression of Caps and Tartan decreases and new lateral expression domains arise. The region of low Caps and Tartan expression in the center of the mature third instar wing disc coincides with the domain in which Dpp signaling induces Spalt expression. The reciprocity of Spalt and Caps/Tartan expression in third instar wing discs suggested that Spalt might repress Caps/Tartan at this stage. spalt mutant clones located medially show ectopic expression of Caps protein and a tartan-lacZ reporter gene. Ubiquitous expression of Spalt in the wing pouch reduces the levels of expression of Caps and Tartan in the lateral wing disc. These results indicate that Spalt restricts expression of caps and tartan to lateral cells in third instar wing discs (Milán, 2002).
To determine whether apoptosis might be a general response of cells unable to engage in normal interactions with their neighbors, the effects caused by producing cells with inappropriate dorsal-ventral compartment identity were examined. Clones of cells expressing Apterous (Ap) were produced to examine the survival of D cells in the V compartment. Fewer than 20% of surviving Ap-expressing clones were of V compartment origin. Half of these had sorted out into the D compartment and so were in contact with other Ap-expressing cells. The remaining ~10% of clones were recovered in the V compartment. Ventral Apterous-expressing clones were round in shape and induced Wg expression at their borders. Expression of the Apterous inhibitor dLMO was used to produce cells with V identity in the D compartment. Only 30% of dLMO-expressing clones were of D compartment origin. Most of these had sorted out into the V compartment. Fewer than 5% of dLMO-expressing clones were recovered in the D compartment. These were round in shape and induced Wg expression at their borders. These observations suggested that dLMO-expressing clones are preferentially lost from the D compartment if they are unable to make contact with V cells. Likewise, Ap-expressing clones are preferentially lost from the V compartment if they are unable to make contact with D cells. Loss of the inappropriately specified cells was suppressed by coexpression of p35. Under these conditions 48% of dLMO and p35-expressing clones were of D origin, and 51% of clones expressing Ap and p35 were of V origin. This indicates that inappropriately positioned cells are lost by apoptosis. Apoptosis of these cells occurs when clones were induced in second instar. Clones induced during third instar survive equally in both compartments. Caps and Tartan are expressed in D cells under Ap control in second instar wing discs. Ectopic expression of Caps or Tartan cause clones to sort out toward the D compartment, suggesting that these proteins may confer a preferential affinity for D compartment cells. To test whether loss of Caps or Tartan expression contributes to the poor survival of dorsal dLMO-expressing clones, the recovery was measured of clones coexpressing dLMO with Caps or with Tartan. When coexpressed with Caps, 58% of dLMO-expressing clones were of dorsal origin and were recovered in the D compartment, compared to 30% when dLMO was expressed alone. Coexpression with Tartan yielded 54% dorsal dLMO-expressing clones. Expression of CapsDeltaC and TrnDeltaC is able to support survival of dLMO-expressing clones in the D compartment almost as effectively as the full-length proteins (Milán, 2002).
Although Caps and Tartan are able to support the survival of dLMO-expressing clones in the D compartment, the reverse is not true. Caps and Tartan expression are induced by Apterous but obviously cannot support the survival of Ap-expressing clones in the V compartment. Moreover, ectopic expression of Caps or Tartan cause loss of clones by apoptosis in the V compartment. In these experiments ventral Caps- or Tartan-expressing clones that made contact with cells in the D compartment either by sorting out or by sending long cytoplasmic extensions are able to survive. The remaining clones are lost by apoptosis but can be rescued by coexpression of p35. Thus, Caps and Tartan do not appear to function as general survival factors. Their ability to support cell survival depends on the developmental context. Caps and Tartan can mediate cell interactions that prevent apoptosis of misspecified cells in areas where these proteins are expressed by the surrounding cells (Milán, 2002).
One of the most striking observations is that the extracellular domains of Caps and Tartan are sufficient to convey these cues in two developmental contexts. By analogy to the dual roles of EphB and EphrinB proteins as both ligands and receptors, it is suggested that Caps and Tartan proteins serve as ligands to identify cells to their neighbors, perhaps by engaging a cell surface receptor. Failure to receive this signal may cause neighboring cells to elicit a signal that triggers apoptosis of the mispositioned cell. In this context it is worth noting that Caps and Tartan can perform this function in situations where the surrounding cells also express these proteins but not where the surrounding cells don't express them, for example, in the early V compartment. Thus, their ability to mediate cell interactions that support the survival of misspecified cells is context specific. They do not appear to act as general survival factors (Milán, 2002).
Cells in the wing disc often die in small groups, raising the possibility that death signals may not be targeted precisely at the defective cell. Although the nature of the proposed death signal is not known, the results have implicated activation of the Notch signaling pathway in elimination of cells mispositioned with respect to DV identity. Blocking Notch activation in these cells using the dominant-negative NotchECD receptor or using a dominant-negative form of the Notch effector Mastermind is sufficient to prevent removal of these cells by apoptosis. This indicates that loss of cells is due to activation of the conventional Notch signaling pathway. There is a similar requirement for Notch activation in programmed cell death in the eye imaginal disc. It is clear that Notch signaling is not dedicated to elimination of cells. On the contrary, wing disc cells unable to transduce the Notch signal are lost. Thus, it is evident that Notch signaling is used to cause apoptosis in a specific context, in conjunction with other signals. Cells may die when they receive a combination of signals that indicate incorrect position. Dorsal cells expressing dLMO lack Caps and Tartan, which mediate dorsal cell interactions, as well as Serrate and Fringe, which influence Notch signaling. Restoring either category of cell interaction is sufficient to suppress apoptosis of these cells (Milán, 2002).
In the second larval instar, cells in the wing disc assess their DV position. Cells that are misspecified with respect to DV compartment identity tend to sort out into the appropriate compartment. dLMO-expressing cells sort-out into the V compartment. Ap-expressing cells sort-out into the D compartment. If mispositioned cells are able to contact similarly specified cells, they can survive. Although Caps and Tartan are able to trigger cell interactions that provide dLMO-expressing cells with the information that they need to survive in the D compartment, Caps and Tartan are not essential for survival of dorsal cells. Clones simultaneously mutant for both genes survive equally well in D and V compartments. Caps and Tartan are also used to generate a difference in medial-lateral cell affinity during the third instar. Lateral Spalt-expressing cells are eliminated by apoptosis. Caps or Tartan can suppress loss of these cells, but they are not required for survival of lateral cells. These observations indicate that there must be additional cell surface proteins that are capable of mediating the cell interactions that are needed in the dorsal compartment and in the lateral region of the wing disc to support cell survival. In this context it is worth noting that medial spalt mutant cells are not eliminated by apoptosis. Survival of medial cells may depend on the activity of a second Dpp target gene, optomoter blind (omb). Cells lacking omb in the center of the disc are lost, and large mutant clones produce extensive loss of wing tissue. This type of strong wing-scalloping phenotype is typically associated with massive cell death and activation of the JNK pathway (Milán, 2002).
The findings indicate that apoptosis of misspecified cells is associated with loss of expression of specific cell surface proteins. This correlates with alterations in cell affinity. However, alteration in cell affinity per se does not appear to be sufficient to drive cells into apoptosis. Removal of Caps and Tartan laterally caused affinity differences without compromising clone survival. Likewise, spalt mutant clones caused ectopic expression of Caps and Tartan and became rounded due to differences in cell affinity in the medial part of the wing disc. Caps- and Tartan-expressing clones survived normally and remained well integrated in the disc epithelium, despite these differences in affinity. Loss of spalt mutant clones during pupal stages is due to sorting out of the clones from the epithelial sheet to form vesicles of mutant tissue, and not due to apoptosis. Taken together, these observations suggest that alterations in cell affinity are not the cause of apoptosis of mispositioned Spalt-expressing cells. Apoptosis of these clones appears to be due to the absence of specific cell surface cell interactions that can be mediated by Caps and Tartan proteins (Milán, 2002).
Layer-specific innervation is a major form of synaptic targeting in the central nervous system. In the Drosophila visual system, photoreceptors R7 and R8 connect to targets in distinct layers of the medulla, a ganglion of the optic lobe. Capricious (CAPS), a transmembrane protein with leucine-rich repeats (LRRs), is a layer-specific cell adhesion molecule that regulates photoreceptor targeting in the medulla. During the period of photoreceptor targeting, caps is specifically expressed in R8 and its target layer but not in R7 or its recipient layer. caps loss-of-function mutations cause local targeting errors by R8 axons, including layer change. Conversely, ectopic expression of caps in R7 redirects R7 axons to terminate in the CAPS-positive R8 recipient layer. CAPS promotes homophilic cell adhesion in transfected S2 cells. These results suggest that CAPS regulates layer-specific targeting by mediating specific axon-target interaction (Shinza-Kameda, 2006).
Different classes of R-cell axons are targeted to distinct layers of the optic lobe. The present study demonstrates that CAPS is expressed in R8 and in the medulla layer M3 where R8 axons terminate. In caps mutant animals, R8 does not precisely target to this region in the medulla. Ectopic expression of CAPS in R7 redirects R7 terminals to the CAPS-positive M3 layer. In vitro, CAPS mediates homophilic cell adhesion. These observations are consistent with the idea that CAPS mediates homophilic interaction between pre- and post-synaptic cells during the formation of layer-specific neural connections (Shinza-Kameda, 2005).
Its expression in specific synaptic partners provided the first clue to the function of CAPS in the visual system. Previous studies showed that during the development of the neuromuscular system, CAPS expression is closely correlated with synaptic connectivity: caps-positive motoneurons innervate caps-positive muscles, whereas CAPS-negative motoneurons connect with caps-negative muscles. Genetic analysis suggested that caps functions as an attractive target cue that guides motoneurons to the correct muscles. Thus, in the two Drosophila systems in which neural connectivity has been well characterized, caps is expressed in both partners of specific synaptic pairings and appears to mediate the interaction between innervating axons and their targets. This idea is further supported by the in vitro cell aggregation assay, which showed that CAPS binds homophilically. However, it should be noted that caps may regulate target specificity through signal transduction rather than by simple adhesion. During the formation of neuromuscular connectivity, motor axons choose among individual muscle cells present in the target region. In contrast, during R-cell targeting in the medulla, afferent axons establish specific synaptic connections by selecting the correct target layer. CAPS thus can mediate cell-to-cell specificity in two different types of targeting system (Shinza-Kameda, 2006).
Analysis of the effects of caps loss-of-function and misexpression in R cells supports the notion that CAPS functions as an attractive target recognition molecule in the visual system. In caps mutants, R8 axons reach the medulla and are initially guided to the correct layer. However, R8 axons then fail to stabilize in the target region and instead extend abnormally to nearby nontarget regions. The onset of the phenotype corresponds to the developmental stage when CAPS expression in specific layers becomes eminent in the medulla. These observations suggest that caps is required for the stabilization of the contact between R8 and the medulla layer. The same phenotype was observed in the mosaic animals, in which caps mutant R cells innervated largely wild-type medulla and in MARCM mutant clones. Thus, CAPS is required in the R cells where it is specifically expressed in R8 for proper targeting. Because CAPS is also expressed in the R8 recipient layer in the medulla, it is likely that CAPS also functions in the medulla to mediate afferent-target interaction. However, because of technical limitations, it was not possible to make large caps mutant clones that include all or most of the medulla target neurons. Thus, it remains to be determined if caps is also required in the medulla for correct targeting of R8 cells (Shinza-Kameda, 2005).
Misexpression experiments further support the notion that CAPS mediates specific interaction between R cells and medulla layers. CAPS expression on R7 axons is sufficient to redirect them to terminate in a CAPS-positive M3 layer. Although previous loss-of-function analyses have implicated several other cell surface receptor and adhesion molecules in layer-specific targeting in this system, this is the first molecule whose misexpression was shown to switch the target specificity. Two lines of evidence suggest that the misexpression phenotype is caused by the alteration in axon-target recognition rather than that in axon-axon interaction, such as defasciculation defects. (1) In GMR-GAL4/UAS-caps animals, R7 axons initially do defasciculate from R8 to reach their target region before retracting back to the R8 recipient layer. (2) CAPS misexpression only in R7 is sufficient to induce the mistargeting phenotype. Taken together, loss-of-function and gain-of-function analyses support the notion that CAPS mediates interaction between specific R-cell growth cones and specific layers in the medulla neuropil, both of which express the molecule (Shinza-Kameda, 2006).
Layer-specific targeting is widely observed in vertebrate and invertebrate brain and appears to provide a major means to segregate synaptic connections between specific pre- and post-synaptic cells. Although several axon guidance molecules have been shown to be expressed in particular layers, their roles in layer-specific targeting in vivo remain largely unknown. The best-characterized cues expressed on specific layers are Sidekicks, cell adhesion molecules belonging to the immunoglobulin superfamily, which are expressed both pre- and post-synaptically in specific synaptic partners that project to the same layers in the retina (Yamagata, 2002). When misexpressed on the presynaptic cells, Sidekicks can change their target-layer specificity. The loss-of-function and gain-of-function analyses presented in this study show that CAPS functions in a similar manner to promote layer-specific connectivity in the Drosophila visual system. These examples provide evidence for the role of local cues in the formation of layer-specific connectivity. In contrast, recent studies using the Drosophila visual system have revealed the roles of several receptors and cell adhesion molecules, such as N-cadherin, LAR, and Flamingo, in target layer selection. Although their expression is not layer specific, they contribute to specific aspects of R-cell targeting. Interestingly, the gain-of-function phenotype of CAPS is similar to the of loss of function for N-cadherin or DLAR, suggesting that they may participate in the same molecular mechanism that determines the target specificity in this system. Understanding how such general and cell-specific molecules cooperate to establish layer-specific connectivity will be an important subject in future studies (Shinza-Kameda, 2006).
A key step in organogenesis of the Drosophila tracheal system is the integration of isolated tracheal metameres into a connected tubular network. The interaction of tracheal cells with surrounding mesodermal cells is crucial in this process. In particular, single mesodermal cells called bridge-cells are essential for the guided outgrowth of dorsal trunk branches to direct formation of the main airway, the dorsal trunk (Wolf, 2000). This study presents evidence that the two leucine-rich repeat transmembrane proteins Capricious and Tartan contribute differently to the formation of branch interconnections during tracheal development. Capricious is specifically localized on the surface of bridge-cells and facilitates the outgrowing dorsal trunk cells of adjacent metameres toward each other. Capricious requires both extracellular and intracellular domains during tracheal branch outgrowth. In contrast, Tartan is expressed broadly in mesodermal cells and exerts its role in tracheal branch outgrowth through its extracellular domain. It is proposed that Capricious contributes to the instructive role of bridge-cells whereas Tartan provides permissive substrate for the migrating tracheal cells during the network formation (Krause, 2006).
The initial outgrowth of tracheal cells from ectodermal clusters is triggered by branchless (bnl), a gene encoding a Drosophila FGF homolog (dFGF/Bnl). Bnl is expressed dynamically in small groups of cells surrounding the tracheal primordia and acts as a chemoattractant that guides outgrowth of primary tracheal branches. In addition to the Bnl-signaling, a cellular guidance mechanism is essential for normal outgrowth of dorsal trunk branches. This guidance requires single mesodermal cells called bridge-cells, which are positioned posteriorly next to the tracheal cell clusters and serve as guidance posts for the outgrowing dorsal trunk branches. Initially, filopodia-like extensions from anterior and posterior dorsal trunk cells get in touch with bridge-cells, slide along the bridge-cell surface and contact the opposite extensions. The dorsal trunk fusion process then starts with deposition of the epidermal adhesion protein DEcadherin (DEcad) at the contact point between two fusion cells. Localized at the adherens junctions, DEcad interacts with a and β-catenin (Armadillo) that subsequently bind the actin cytoskeleton. A cytoskeleton-associated plakin Short Stop (Shot), which interacts with both actin and microtubules, is required for DEcad accumulation at the fusion site. Finally, each of fusion cells forms an intracellular tube and the two lumens fuse and become continuous (Krause, 2006).
The molecular mechanisms underlying the initial cell-to-cell contacts between the extending dorsal trunk cells and the guiding bridge-cells are not yet known. The transcription factor Hunchback (Hb) was shown to play a key role in bridge-cell differentiation, which in turn is necessary for dorsal trunk fusion. In addition, the transcription factors Extradenticle (Exd) and Homothorax (Hth) are also expressed in the bridge-cells. However, neither of these transcription factors nor Bnl may be directly involved in the recognition of bridge-cells by tracheal extensions. Thus, it has been speculated that this process is mediated by extracellular matrix and adhesion molecules that are expressed on the surfaces of tracheal and bridge cells (Krause, 2006).
Caps is specifically expressed in the bridge-cells. When caps is lacking, the bridge-cells may not bind the dorsal trunk cells effectively and thus fail to provide sufficient guidance. Consequently, discontinuous dorsal trunks are formed. Since only dorsal trunk cells require bridge-cell's guidance, other tracheal branches are not affected by lack of caps activity (Krause, 2006).
What is the function of Caps in the bridge-cells? Caps might be involved in directly mediating communication between the bridge-cells and the tracheal cells, since it represents a transmembrane protein with 14 leucine-rich repeats (LRR) in the extracellular domain. LRRs are arranged consecutively and parallel to a common axis so that the conformation resembles a horseshoe, which is ideal for mediating cell-to-cell interactions. Possibly, the extracellular domain of Caps assumes the horseshoe-like conformation as well and binds molecules localized on the surface of dorsal trunk cells. Thus, Caps may provide an important link between the guiding bridge-cells and the outgrowing tracheal cells (Krause, 2006).
When caps is ectopically expressed in mesodermal cells, discontinuous dorsal as well as lateral trunks are formed. Moreover, some dorsal trunk cells grow dorsally or ventrally rather than anteriorly towards their adjacent targets. The outgrowing tracheal cells may adhere to ectopic Caps on the surface of mesodermal cells, which normally do not express Caps, rather than binding endogenous Caps on the bridge-cells. As a result, these tracheal cells may become disoriented and extend in abnormal directions. This ability of ectopic Caps to stall normal development of dorsal trunk branches suggests that selective expression of Caps in the bridge-cells is important for the specific local guidance for the outgrowing dorsal trunk cells. However, more severe dorsal trunk defects were observed in hb mutant embryos, which lack the bridge-cells (Wolf, 2000). This observation suggests that additional components besides Caps are involved in the bridge-cell's guidance. Such components may include the chemoattractant Bnl, which is also expressed in the bridge-cells (Krause, 2006).
Previous results demonstrated that Caps plays an essential role during pathfinding of motorneurons, layer-specific targeting in the visual system and boundary formation in wing imaginal discs. Normally, Caps is expressed in a subset of CNS neurons including aCC, RP2, RP5 and U motorneurons. When Caps is overexpressed in all neurons, the axons of muscle 12 motorneurons (MNs) become misrouted. Similar results are obtained when CapsEd lacking the intracellular domain is misexpressed in neurons. However, when CapsId lacking the extracellular domain is misexpressed, no defects can be observed in pathfinding of muscle 12 MNs. These results clearly indicate that a neural expression of CapsEd is sufficient to misroute muscle 12 MNs. During boundary formation of the wing discs, cells incorrectly specified for their position undergo apoptosis because they fail to express Caps. Again, exclusive expression of CapsEd is sufficient to prevent apoptosis of misspecified cells. During these developmental events, Caps is thought to function as a cell adhesion molecule providing specific affinity between different cells and thus, may require only its membrane-anchored extracellular domain. In contrast, muscularly expressed Caps requires extracellular and intracellular domain to establish aberrant synapses of muscle 12 MNs. Similarly, only embryos misexpressing complete Caps reveal interruptions in dorsal and lateral trunks. These results suggest that Caps requires its intracellular domain both for its function during the establishment of motorneuron synapses and during tracheal morphogenesis. This hypothesis was further strengthened by investigations using Caps and Trn hybrid proteins in ectopic expression assays. Misexpression of TrnEdCapsId in mesodermal cells results in discontinuous tracheal branches. Moreover, misexpression in trn mutants aggravates the tracheal defects. These observations suggest that the TrnEdCapsId hybrid protein functions similar to Caps even though it contains only the intracellular domain of Caps. Thus, it is the intracellular domain that determines specificity of Caps function during tracheal morphogenesis (Krause, 2006).
It has been postulated that Caps might interact with receptors on specific motorneurons via its extracellular domain and transmit the signal into muscles via its intracellular domain. Likewise, Caps may function as a signal transmitter between the tracheal cells and the bridge-cells. Interestingly, proteins containing LRRs are predicted to undergo conformational changes upon binding the ligand or other proteins. These changes do not involve the usual movement of separate domains relative to each other, but rather an elastic alteration of the entire structure. Such conformational changes may subsequently induce Caps to interact with other intracellular proteins involved in signaling pathways or regulation of cytoskeletal structures. It appears that depending on the cell type, Caps may act as a cell adhesion molecule or as a receptor that relays signals from the outside to the inside of cells (Krause, 2006).
Sequence alignment of Caps from Drosophila and Anopheles reveals evolutionary conservation of three putative motifs within the intracellular domain: a putative tyrosine phosphorylation site, a predicted PDZ binding motif and a conserved RHR motif. Site-directed mutagenesis of these putative functional motifs and in vivo analysis shows that only the RHR motif is essential for Caps function during tracheal formation. This RHR motif is not yet recognized as a functional motif by protein databases. Therefore, no information regarding other proteins containing such a motif or prediction about its putative function is known. Located immediately after the transmembrane domain, the RHR motif may contribute to conformational changes, which enable Caps to transmit extracellular signals to small membrane-associated proteins that bind components of signaling pathways or cytoskeleton (Krause, 2006).
During the Drosophila wing development, Caps and Trn share redundant function and contribute evenly to the formation of affinity boundary between the dorsal and ventral compartments. This result is rather expected since only the extracellular domain of Caps or Trn is required during the establishment of boundary in wing discs and the extracellular domains of Caps and Trn are 65% identical. Similarly, Trn requires only its extracellular domain to mediate its function during the establishment of tracheal network. The current results demonstrate that the extracellular domain of either Trn or Caps can rescue the trn tracheal phenotype. Thus, the extracellular domains of both Caps and Trn provide the mesodermal cells with a substrate that mediates a normal tracheal branch progression in trn mutants (Krause, 2006).
In contrast, Trn and Caps are expressed in divergent patterns and contribute differently to the formation of continuous tracheal branches. Whereas caps is expressed selectively in the bridge-cells, trn is detected in broad subsets of mesodermal cells excluding the bridge-cells. Furthermore, ectopic expression of Caps in mesodermal cells disrupts formation of normal tracheal interconnections while ectopic expression of Trn in mesodermal cells does not affect tracheal development. Rather, it rescues the tracheal defects of trn mutant embryos. Finally, although Caps requires both its extracellular and intracellular domain for proper function during the formation of tracheal branches, Trn needs only its extracellular domain (Krause, 2006).
Based on these observations, the following model is proposed for Caps and Trn functions during tracheal development: Caps is important for the bridge-cell, which provides instructive cues for the extending dorsal trunk cells, while Trn contributes to permissive matrix function of mesodermal cells for normal tracheal branch outgrowth. Localized at the surface of bridge-cells, Caps may bind to cell surface molecules on tracheal cells and allow the tracheal cells to extend along the bridge-cells so that they can find the correct targets. In the absence of caps, the bridge-cells cannot mediate their local guidance as effectively and consequently, the dorsal trunk cells fail to interconnect to their targets. In contrast, when caps is expressed in additional mesodermal cells besides the bridge-cells, then the nearby tracheal cells can also adhere to these mesodermal cells through Caps and extend in unspecified directions. Thus, disconnected tracheal branches are formed. Previous studies indicate that specific interactions between cell surface proteins on the tracheal cells and the surrounding mesodermal cells are crucial for migration of tracheal cells. The mesodermal cells may serve as a matrix enabling (and facilitating) the tracheal cells to recognize the correct path and to migrate efficiently. These cell-to-cell interactions might involve transmembrane proteins such as Trn. Localized at the surface of mesodermal cells, Trn may interact directly with other molecules on the tracheal cells and thereby support the tracheal cells to extend across the mesodermal sheet. When trn is absent, the navigation of outgrowing tracheal cells is partially hampered and they cannot migrate along their paths as efficiently. Consequently, they fail to connect to their targets. Overexpression of Trn in mesodermal cells mimics the wild-type expression of Trn and does not affect the progression of tracheal cells (Krause, 2006).
These results suggest that migrating tracheal branches require an adhesive substrate provided by the broad expression of Trn in the immediate proximity. The major airway, the dorsal trunk, relies additionally on instructive guidance by the extending bridge-cells expressing Caps, which may bind to surface molecule(s) on the progressing tracheal cells. The intracellular domain of Caps might induce signaling leading to cytoskeletal changes that generate a 'pulling' force of the bridge-cells on the migrating tracheal cells. Identification of extracellular binding partners on tracheal cells and intracellular interaction partners of Caps may elucidate molecular mechanisms underlying transmission of external cues in the bridge-cells that induce intracellular events leading to cellular guidance (Krause, 2006).
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date revised: 26 November 2001
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