tartan: Biological Overview | References
Gene name - tartan
Cytological map position - 70A1-70A1
Function - receptor
Symbol - trn
FlyBase ID: FBgn0010452
Genetic map position - 3L: 13,107,373..13,111,188 [+]
Classification - Leucine-rich repeats (LRRs)
Cellular location - surface transmembrane
The development of the Drosophila eye imaginal disc requires complex epithelial rearrangements. Cells of the morphogenetic furrow are apically constricted and this leads to a physical indentation in the epithelium. Posterior to the furrow, cells start to rearrange into distinct clusters and eventually form a precisely patterned array of ommatidia. These morphogenetic processes include regulated changes of adhesion between cells. Two transmembrane adhesion proteins, Capricious and Tartan, have dynamic and complementary expression patterns in the eye imaginal disc. Novel null mutations in capricious and double null mutations in capricious and tartan are described. They are shown to have redundant functions in regulating the architecture of the morphogenetic furrow and ommatidial spacing. It is concluded that Capricious and Tartan contribute to the adhesive properties of the cells in the morphogenetic furrow and that this regulated adhesion participates in the control of spacing ommatidial clusters (Mao, 2008).
The development of the Drosophila compound eye is a complex process involving the interplay of many signalling pathways. The Drosophila eye is composed of a regular hexagonal lattice of about 800 individual facets known as ommatidia. Each ommatidium consists of a unit of eight photoreceptor neurons (R1-R8) and four cone cells, and is surrounded by pigment cells. The eye develops from a monolayer epithelium known as the eye-antennal imaginal disc. At the start of the third larval instar, the cells in the imaginal disc start to differentiate. This differentiation starts at the posterior of the disc and sweeps anteriorly, preceded by a physical indentation known as the morphogenetic furrow (MF). Developing rows of ommatidia are left in its wake, and this progressive development implies that there is a gradient of developmental stages in a single disc, with the most mature being at the posterior (Mao, 2008).
Most of the cells in the eye disc have a columnar epithelial morphology, but in the morphogenetic furrow they become apically constricted. As a result of this constriction, these cells change from being columnar to bottle-shaped and the consequent change in epithelial packing produces the indentation of the furrow itself. Immediately after the passage of the furrow, and therefore posterior to it, cells begin to rearrange, developing from random packing into first lines of cells, then arcs, and finally morphologically distinct clusters within the epithelium. This process depends on myosin II contractility but presumably also requires precise changes in the adhesive properties of cells as the clusters separate from their neighbours. In fact, adhesive changes can be directly observed-the clusters show increased levels of apical Armadillo/β-catenin, a key component of the adherens junctions, a phenomenon dependent on Atonal and the epidermal growth factor receptor (EGFR) pathway. Beyond this increase in adherens junctions, little is known about the adhesion processes that participate in the clustering process (Mao, 2008).
Capricious (Caps) and Tartan (Trn) are highly similar transmembrane proteins with multiple extracellular leucine rich repeats (LRRs) and shorter intracellular domains (Chang, 1993: Shishido, 1998). They share 67% protein sequence identity in their extracellular domains, which consist of 14 LRR repeats, but only 15% overall identity in their intracellular domains, including a conserved motif of 31 amino acids adjacent to the membrane. Since they lie within 115 kb of each other in the genome, it is likely that they represent a relatively recent gene duplication event. Although their exact molecular function is not well characterised, they can act as homotypic adhesion proteins in cell culture (Shinza-Kameda, 2006) and at least in some contexts their intracellular domains are dispensable (Taniguchi, 2000) supporting the idea that their primary roles are in cell adhesion. Consistent with this, their functions have mostly been associated with their adhesion properties. Caps is required for targeting a subset of embryonic motor neurons to their specific muscles during embryonic development (Taniguchi, 2000) and in targeting R8 photoreceptor axons to the appropriate layers of the optic lobe (Shinza-Kameda, 2006). Caps and Trn have also been implicated in the formation of affinity boundaries between dorsal and ventral compartments in the developing wing imaginal disc (Milán, 2001; Milán, 2002; Milán, 2005). They have halso been shown to have overlapping functions in adhesion of cells in the developing leg imaginal disc (Sakurai, 2007; Mao, 2008 and references therein).
Morphological plasticity in the developing eye involves precisely ordered remodelling of epithelial cell contacts. This study describes the specific and complementary expression patterns of Caps and Trn in the imaginal eye disc and their redundant roles in regulating aspects of epithelial organisation in the morphogenetic furrow and the spacing of developing ommatidia (Mao, 2008).
caps and trn have developmentally regulated expression patterns in the eye. In third instar eye imaginal discs, caps-lacZ is expressed in all cells in the morphogenetic furrow and at a lower level in cells just posterior to the furrow before becoming restricted to single photoreceptor cells. By simultaneous staining with the R8 photoreceptor marker Senseless, it was shown that the single cells eventually expressing caps-lacZ are the R8 cells, the founders of ommatidial development. This result is consistent with the expression pattern reported by Shinza-Kameda (2006), although that study confined description of expression to the later stages when caps is restricted to R8. trn is a close sequence relative of caps and in the wing imaginal disc they are believed to act in partnership as adhesion proteins that regulate cell affinity at compartment borders (Milan, 2001). Therefore the expression pattern in the eye of trn-lacZ was examined. Interestingly, trn-lacZ is also expressed dynamically, initially in all cells in the furrow, then at a lower level in cells just posterior to the furrow, before becoming restricted to a non-overlapping subset of photoreceptor precursors from caps. trn-lacZ colocalised with R1 and R6 markers anti-BarH1. In summary, both caps and trn are widely expressed in the morphogenetic furrow, and each then becomes restricted to non-overlapping subsets of photoreceptors. These complementary expression patterns in the eye suggested that the Caps and Trn proteins might have a previously unrecognised function in eye development (Mao, 2008).
Specific antibodies against Caps and Trn were raised to examine their expression pattern in more detail. Unfortunately the Caps antiserum did not reliably detect the endogenous level of Caps protein in eye discs. In contrast, the Trn antibody successfully recognised Trn protein in wild-type discs. Its specificity was confirmed by the loss of signal in clones of cells mutant for Trn but not in clones of cells mutant for Caps. The antibody staining pattern confirmed the caps-lacZ expression pattern: Trn is expressed broadly in the morphogenetic furrow and in subsets of ommatidial cells after the furrow. Since photoreceptor specific markers are almost all nuclear, and Trn is membrane localised, overlapping staining patterns cannot readily be used to confirm the identity of the specific ommatidial cells stained posterior to the furrow. However, the expression pattern is fully consistent with that of the trn-lacZ line, that is, the staining is localised in the expected location of R1, 6 and 7 but not of R8, 2, 5, 3, 4. Z-sections along the anterior-posterior axis of the disc revealed that it is expressed mostly in the apical membrane of photoreceptor cells but is also visible in some basolateral membranes. Interestingly, Tartan is only expressed in the anterior half of the furrow (Mao, 2008).
Since caps and trn single mutants did not affect eye development, and since Caps and Trn are highly related proteins, it was asked whether they might act redundantly in eye development. To address this, a caps trn double null mutation, capsDel1 trn28.4, was generated by using P-element induced male recombination, to simultaneously delete caps and recombine the new mutation onto the existing trn28.4 null allele. This double capsDel1 trn28.4 null retains the intervening genes CG33262 and CG11281, so represents a clean removal of the two related proteins. Mitotic clones of the capsDel1 trn28.4 mutation (marked by lack of GFP, green) showed subtle but consistent defects. Within the morphogenetic furrow, the mutant cells showed normal levels of apical constriction, and accumulated high levels of Armadillo/β-catenin indistinguishably from the wild type. However, at the clone border between mutant and wild-type cells, there was a consistent reduction in the apical constriction of cells and their Armadillo accumulation in adherens junctions. This phenotype is fully penetrant but appears more pronounced when the clone boundary is perpendicular to the morphogenetic furrow (Mao, 2008).
The apical constriction of morphogenetic furrow cells generates the indentation of the furrow itself. Sagittal sections in the Z-axis of capsDel1 trn28.4 clones along the furrow, showed that the cells at the clone boundary with the enlarged apical profiles were also taller than their neighbours, that is, their apical surfaces were elevated, thereby disrupting the furrow itself (Mao, 2008).
These related phenotypes of relaxation of apical constriction and increase in apical-basal height of the cells were a non-autonomous effect: they were observed in both mutant and wild-type cells at the clone boundary. The range of the phenotype was only 2-3 rows of cells beyond the clone border, and in some cases this non-autonomy was predominantly in the wild-type, and sometimes predominantly in the mutant territory. Given the subtle nature of the effects, the borders of clones of single mutants for caps or trn were reexamined, an it was confirmed that they were never visibly affected (Mao, 2008).
A second phenotype associated with the capsDel1 trn28.4 double null clones (marked by lack of GFP, green) was a perturbation in ommatidial spacing in third instar eye discs. Again, this was apparent only at the boundaries between mutant and wild-type cells. Ommatidia close to these boundaries were often clearly displaced from their normal positions but there was no obvious change to their individual morphology, nor was the total number of ommatidia obviously affected. 25 individual eye discs containing clones were analysed, and the number of ommatidia adjacent to clonal boundaries was counted, along with the number of these ommatidia that were displaced from their normal position. In total, of 846 ommatidia at boundaries, 187 (i.e., 22%) were displaced. This phenotype is also non-autonomous, with both mutant and wild type ommatidia showing mis-positioning. These spacing defects remain later, at pupal stages of eye development, and they are made more apparent by the fusion of neighbouring ommatidia that are abnormally close to each other. These fusions are observed in about 5% of ommatidia adjacent to clone boundaries. Very occasional defects were observed in the normal number of cone cells. As with the third instar eye disc, the pupal phenotypes were not observed in clones mutant for caps or trn alone (Mao, 2008).
Since defects were observed only at the boundaries between capsDel1 trn28.4 mutant and wild type tissue, it was wondered whether the sudden step-like changes in Caps and Trn levels were more important than the overall levels of these adhesion proteins. Therefore clones were made over-expressing Caps and Trn but they did not show any visible furrow or ommatidial spacing defects at the clone boundaries. This implies that the boundary effects seen in clones of capsDel1 trn28.4 cells are caused by the juxtaposition of cells expressing Caps and Trn with cells not expressing them. In summary, it is concluded that there is a redundant function for Caps and Trn in controlling aspects of cell morphology and ommatidial spacing in Drosophila retinal development (Mao, 2008).
One of the main tissues in which Caps and Trn have been studied is the developing wing imaginal disc. Despite evidence that caps and trn have an important function in maintaining compartment borders in the wing, previously studied mutants in these genes have not affected the dorsal-ventral boundary. Therefore advantage was taken of having made a previously unavailable double null mutation to look at DV border formation in the wing. Clones of capsDel1trn28.4 double null (marked by lack of GFP) did not perturb or cross the boundary, as marked by staining with an antibody against Senseless. This is consistent with earlier data, where clones of cells simultaneously null for trn and hypomorphic for caps did not cause defects at the DV boundary. The fact that complete loss of both proteins does not affect DV boundary formation or maintenance suggests that Caps and Tartan are not essential for compartmentalising cells in this part of the wing (Mao, 2008).
This study has shown that the related adhesion proteins Capricious and Tartan have redundant functions in the remodelling of epithelial cell contacts that occur during the early stages of Drosophila eye development. Each is expressed in a two phase pattern, first broadly in the furrow and then later in non-overlapping subsets of photoreceptors: Caps in R8 and Trn in R1, 6 and 7. A null mutation was made of caps and also a double null, in which both caps and trn are absent. Analysis of these mutations shows that while removal of either gene alone has no phenotype, the loss of both leads to subtle but reproducible defects in retinal development. The earliest phenotype is a reduction of apical constriction and accumulation of Armadillo in the morphogenetic furrow. Slightly later, displacement of ommatidia from their normal very precise array was seen. Finally, this displacement leads to occasional fusion of neighbouring ommatidia and other minor defects in the pupal retina. Intriguingly, all the defects observed are limited to clone boundaries; cells fully within the mutant clones appear normal (Mao, 2008).
The capsDel1trn28.4 phenotypes in the eye are relatively minor. They are nevertheless reproducible and quite penetrant. Essentially all clones that cross the furrow perpendicularly show a reduction in apical constriction and Armadillo staining at their clone boundaries, and 22% of ommatidia that lie at the clone boundary are detectably misplaced; ommatidial fusion defects in the pupal retina are rarer, at about 5%. It is proposed that these phenotypes are all a consequence of the initial furrow defects, and that these are caused by loss of the furrow expression of Caps and Trn. This implies that the later, photoreceptor-specific expression of Caps and Trn does not participate in the observed phenotypes. This proposal is based on the following logic: (1) the redundant function of the two proteins is difficult to reconcile with non-overlapping expression: if they are in different cells, how can they replace each other's function? Although it would be possible to imagine a scenario where this could occur, a more parsimonious explanation is that the redundant phenotype depends on their function where they are co-expressed, in the furrow. (2) The expression of Caps in R8 is already known to have a quite separate function, in the targeting of the R8 axon growth cones to the appropriate layer of the optic lobe (Shinza-Kameda, 2006). The R8 cell bodies are in the retina, which is why caps-lacZ expression there is seen there, but the protein must be transported to the axon terminals. The discovery of an equally specific but non-overlapping expression of Trn, suggests that it too might have an analogous function in axon targeting, although this prediction has not been tested (Mao, 2008).
The idea that the later, photoreceptor specific expression of Caps and Trn is responsible for axonal guidance defects, but not retinal patterning, appears inconsistent with the protein expression of Trn that is see at the apical surface of the photoreceptors, i.e. in the retina, distant from the axon terminals. Unfortunately, the wild-type protein expression of Caps, which is know to be involved in axonal guidance, could not be detected so it is possible that Caps protein is localised very differently from Trn-only in the axons. Although a better anti-Caps antibody could resolve this fully, on balance it is suspected that the apical expression of Trn, and possibly Caps, either reflects a function distinct from the retinal defects report in this study and also from axonal guidance; or that it is a non-functional consequence of the intracellular trafficking pathways that transport the functional pool to the axon terminal (Mao, 2008).
The third reason (for suggesting that the functions this study has uncovered are dependent on Caps and Trn in the furrow, and that the later defects in spacing are secondary consequences of a primary furrow defect) is that this is consistent with the furrow acting to organise epithelial packing. Detailed inspection of cells in the furrow and immediately after they emerge from it, shows profound rearrangement that starts with straight lines of cells, evolving into arcs and finally into morphologically distinct clusters. Adhesion defects in the furrow may disrupt this process such that ommatidial clusters and their spacing become less ordered. It is not understood why these phenotypes manifest only at clone boundaries, but it is presumed to be a consequence of a discontinuity in adhesive properties. Similarly, the short range non-autonomy of the phenotype is probably due to local cell packing problems caused by adhesion anomalies at the boundaries of wild-type and mutant tissue. Another possible explanation for the non-autonomous effects is that changes in cell shape and epithelial morphology in the furrow could affect the range or efficiency of intercellular signalling molecules, thereby affecting normal retinal development. Little is known about how epithelial characteristics can modulate secreted signals and this will be a fruitful area for future study (Mao, 2008).
A very recent paper (Sakurai, 2007) has analysed the functions of Caps and Trn in the developing leg disc. That study also shows a completely redundant function caused by rather subtle adhesion defects. Leg disc development is, however, very different from eye development and the developmental consequences are therefore distinct. In the leg, the sharpening of a progressive border that develops between tarsus 5 and the pretarsus segment was compromised in double mutants. By analysing cell movement within the developing leg disc, Sakurai proposed that Caps and Trn expression allows cell mobility within the epithelium: their downregulation coincides with reduced mobility, while their overexpression leads to cell invasion into inappropriate territories. In the eye, there is no evidence for significant mixing of cells within the epithelium and, the eye model suggests a different use of a rather similar function for these adhesion proteins. In both cases, however, Caps and Trn appear to regulate the ability of cells within an epithelium to reorganise with respect to their neighbours (Mao, 2008).
In summary, the results are interpreted to imply that Caps and Trn expressed in the morphogenetic furrow participate in modulating the adhesivity of epithelial cells. At this stage in development, they are beginning to undergo complex and coordinated rearrangements, with concomitant adhesion changes with their neighbours. Even quite minor disruption of this process leads to alterations in epithelial packing that can have consequent effects on the spacing of ommatidia. The relatively minor retinal phenotype of loss of Caps and Trn implies that other adhesion proteins contribute to the overall regulation of this process. For example, Drosophila E-cadherin, an essential component of adherens junctions, is necessary for epithelial maintenance, and mutant (hypomorph) clones fail to form adherens junctions and lose their epithelial integrity completely. It is suspected that complex regulation of adhesion may require the action of several adhesion systems. The data also leads to the tentative suggestion that Trn may, like Caps, have a later function in photoreceptor neuron development, for example in axon targeting. Finally, and on a separate tack, construction of a double null mutation for caps and trn unambiguously shows that neither are essential for the normal formation of the dorsal-ventral boundary of the wing imaginal disc, a process that overexpressed Caps and Trn can disrupt (Milan, 2001; Mao, 2008 and references therein).
Domain boundary formation in development involves sorting of different types of cells into separate spatial domains. The segment boundary between tarsus 5 (Ta5) and the pretarsus (Pre) of the Drosophila leg initially appears at the center of the leg disc and progressively sharpens and expands to its final position, accompanied by down-regulation of the cell recognition molecule Capricious and Tartan and cell displacement from Ta5 to Pre across the boundary. Capricious and Tartan are controlled by transcription factor Bar and Al, and their loss of function leads to reduction of cell affinity to wild type neighbors and cell displacement activities. In addition, although the mutant cells formed Ta5/Pre boundary, its progression and sharpening were compromised. Cells overexpressing Capricious or Tartan became invasive within Ta5 and Pre, sometimes escaping the compartmental restriction of cell movement. Dynamic spatiotemporal regulation of cell affinity mediated by Capricious and Tartan is a key property of refinement of the Ta5/Pre boundary (Sakurai, 2007).
Segmentation of the distal leg of Drosophila is initiated by the activation of EGFR signaling at the center of the disc, which leads to the activation of Caps and Trn under control of Al and Bar, and the expansion and sharpening of the Ta5/Pre boundary. Since Caps and Trn are dispensable for Ta5/Pre boundary formation marked with the stripe of Fas2, they are subordinate genes determining cell affinity. The rough borders of wild type clones in Ta5/Pre region suggest that those cells frequently exchange their positions with neighbors. Clones of Df(3L)trnΔ17 capsGS1 sorted from neighboring wild type Ta5 cells, suggesting that Caps and Trn are required to keep Caps+ Trn+ cells mixed with neighbors. The results of gain-of-function experiments support this idea, as also described for the analyses in the lateral part of the wing disc. Although it has been suggested that the Caps extracellular domain acts on a putative receptor, recent finding that Caps mediates homophilic cell adhesion in S2 cells (Shinza-Kameda, 2006), and the isolation of a point mutation in the Caps intracellular domain (T501I) that abrogates the invasive activity of Caps suggest that Caps acts as a receptor promoting cell exchange through homophilic cell recognition, an activity termed mixing. The molecular function of Trn is less clear, but the similarity of its structure and activity to Caps suggest that Trn might act redundantly with Caps in homophilic cells recognition and mixing (Sakurai, 2007).
To understand the role of Caps and Trn in Ta5/Pre segment boundary formation, both the cell mixing function of Caps and Trn, and the spatio-temporal regulation of their expression must be considered. At early third instar, Caps and Trn are expressed in the region covering the future Ta5 and Pre, and would permit cells to flexibly exchange their positions with those of their neighbors, thereby maintaining a fluid state of cell mixing. This hypothesis is supported by the slightly increased Circularity index (CI) of caps trn double mutant clones in the future Pre region. Through the mid-to-late third instar, Caps and Trn were down regulated in the central domain. Caps- and Trn-dependent cell mixing persists and could shift cells that have lost Caps/Trn expression into the future Pre region. The imbalance of cell mixing activity in the Caps+ Trn+ and Caps− Trn− domains would allow the observed proximal-to-distal flow of cells across the Bar/Al boundary. Finally, the expression of Fas2 in the row of cells abutting the distal side of the Caps-expressing cells may stabilize the Ta5/Pre boundary (Sakurai, 2007).
The Caps/Trn-mediated cell mixing appears to be required for the distal-to-proximal progression of the Ta5/Pre boundary, during which Caps and Trn are gradually repressed in a distal-to-proximal direction. The phenotypes of the Df(3L)trnΔ17 capsC28fs clones suggest that when the prospective Ta5/Pre boundary hit the clones from the distal side, the sudden loss of Caps and Trn activity prevents or delays the boundary progression. It is speculated that this phenotype was caused by the reduction of cell affinity in the mutant clones to mix with both presumptive Ta5 and Pre cells, causing the mutant cells, retaining Ta5 identity, to segregate from both Ta5 and Pre. This change in cell affinity might delay the proximal to distal cell flow by the time Al begins expression and determines the Fas2 expression border. Consistent with these phenotypes, small Pre segment and faulty junction formation were observed in the adult legs with large clones of Df(3L)trnΔ17, capsC28fs. It is suggested that cell mixing promoted by Caps and Trn helps the boundary to sharpen and progress by selectively pulling Caps+ Trn+ cells into the Ta5 region through homophilic cell affinity and by displacing the cells that have turned off Caps and Trn into the future Pre region (Sakurai, 2007).
The classical model of cell affinity boundary formation proposes that differential expression of cell adhesion molecules creates cell affinity boundaries where domain borders forms. Although the coincidence of Caps and Trn expression border at the Ta5/Pre border fits with this model, it was found that the complete removal of those genes still allowed the expression of Fas2, suggesting that cells at the Ta5/Pre border can turn on Fas2 expression and form the unique rectangular shape in the absence of Caps and Trn activities. The Fas2 expression is activated by Al in cells abutting Bar expressing neighbors. Fas2 may stabilize the Ta5/Pre border independently of Caps and Trn functions after the border is placed at the proper position by Caps and Trn. Occasionally in Caps− Trn− cells Fas2 expression expanded to 2–3 cells. This blurred border phenotype might indicate that the border refinement by Caps and Trn is essential for Al+ cells to limit Fas2 expression in a single row of cells. In contrast, Bar mutant clones showed strong cell sorting phenotype in Ta5. It is thus likely that Bar controls additional target genes which, together with Caps, Trn and Fas2, carry out the cell affinity boundary formation, and to promote the Ta5/Pre boundary progression and sharpening (Sakurai, 2007).
In addition to Ta5, caps expression has been observed in proximal cells of pupal tarsal leg segments undergoing joint formation. This study observed that adult legs carrying clones of Df(3L)trnΔ17, capsC28fs are defective in Ta5/Pre and Ta4/Ta5 joint formation. This might reflect that proper boundary formation is prerequisite for leg segmentation, or independent late roles of Caps and Trn in leg joint formation. Further analyses of joint cell differentiation processes will be required to clarify those issues (Sakurai, 2007).
The current finding that Caps and Trn promote cell boundary progression and sharpening is somewhat different from the previously proposed model that these molecules mediate cell sorting at the dorsal–ventral compartment boundary of the wing disc. Previous studies assessed the cell-sorting activity of Caps or Trn by using a rescue assay in which attempts were made to complement a defect in the DV compartment boundary formation in ap mutants. Although apparently sharp DV boundaries were created by the expression of Caps and Trn driven by the ap-Gal4 driver in ap mutants, it is not clear whether the Caps and Trn activities revealed in this assay reflect their endogenous function in cell segregation. This is because Caps and Trn are not normally expressed in D cells at the late third instar, when the wing discs were examined in the assay; the ap-Gal4 expression forced the Caps and Trn expression in D cells at this stage, even in ap mutants. Therefore, the segregation of cells at the DV boundary may simply reflect the cell-sorting effect of ectopic overexpression of Caps and Trn. In addition, caps trn double mutant clones did not alter wing patterning except for cell round up phenotypes that were observed in both D and V compartment. It is therefore suggested that Caps and Trn may act primarily to help cells to locate appropriate place according to their positional identity by maintaining homophilic cell affinity. Their roles in the progression of Ta5/Pre boundary uncovered in this work and in medio-lateral cell positioning in the wing disc can be understood in this context (Sakurai, 2007).
A large number of LRR proteins are encoded in the genome of higher metazoans. It would be interesting to investigate whether cell mixing and cell displacement also play active roles in other cases of cell boundary progression by helping cells to create and maintain sharp boundaries (Sakurai, 2007).
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).
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).
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).
Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities contribute to organizing cells into different tissues. Drosophila limbs and rhombomeres of the vertebrate hindbrain are subdivided into compartments. Cells in adjacent compartments do not mix. The wing primordium is subdivided into dorsal (D) and ventral (V) compartments by the activity of the LIM-homeodomain protein Apterous in D cells. The leucine-rich repeats transmembrane proteins Capricious and Tartan have been shown to contribute to formation of the affinity boundary between dorsal and ventral compartments. This paper describes a structure-function analysis of Capricious and Tartan. Evidence is presented that both the extracellular and intracellular domains are required for the establishment of a DV affinity boundary. The data suggest that the extracellular domains of Capricious and Tartan may work as ligands of an unknown D cell surface protein. Their intracellular domains may be required to transduce a signal necessary for the establishment of the DV boundary (Milán, 2005).
Although Caps and Tartan confer a D-specific affinity, expression of Caps or Tartan in S2 cells is not able to promote cell aggregation and soluble forms of the extracellular domains of Caps and Tartan are not able to bind to full-length forms of Caps and Tartan expressed in S2 cells. In a sorting assay, membrane-bound cellular processes extending from ventral Caps- or Tartan-expressing cells toward cells in the D compartment were observed. Similar cellular extensions were not found between closely spaced clones within the V compartment. These observations support the idea that Caps and Tartan may be interacting with another cell surface protein in the D compartment. To further test this, the ability of Caps and Tartan to rescue the DV affinity boundary in the complete absence of Apterous activity was tested. Caps and Tartan were able to partially rescue the DV affinity boundary, suggesting that Caps and Tartan may confer, independently of any other protein expressed in D cells, an affinity difference. However, the DV boundary was not always smooth, and cells tended to violate it, suggesting that Caps and Tartan may require the binding to another cell surface protein in D cells to generate an affinity boundary. Coexpression of Caps and Tartan gave the same results. Thus, in the strong but not complete loss-of-function background for Apterous activity, the unknown dorsal partner of Caps and Tartan may be expressed at low but sufficient levels to interact with Caps and Tartan and generate a DV affinity boundary. In the complete absence of Apterous activity, their partner may not be expressed at all (Milán, 2005).
The extracellular domain of Caps is required for this interaction to take place. A truncated form of Caps lacking the extracellular domain (Caps-ED) is not able to make V cells sort toward the DV boundary, and its expression in D cells in the partial absence of Apterous activity is not able to rescue the DV affinity boundary. Its subcellular distribution and relative amount of overexpression was comparable to the full-length forms of Caps (Milán, 2005).
It was asked if the intracellular domains of Caps and Tartan are required to induce segregation of D and V cell populations. For this purpose, truncated forms of Caps and Tartan lacking the intracellular domain were tested in the sorting and rescue assays. Of interest, they make V cells sort toward the DV boundary, the same way as full-length forms of Caps and Tartan, suggesting that the lack of intracellular domains is not compromising Caps/Tartan activity in this assay. Clones induced in early second instar discs, before the DV boundary forms, were bisected by the nascent DV boundary so that they contributed to both compartments. Control D+V clones had no effect on the shape of the Wg stripe and were of similar size in both compartments. In contrast, D+V clones expressing truncated forms of Caps or Tartan tended to be considerably smaller in the V compartment, the same way as full-length forms of Caps and Tartan. Many of these clones distorted the Wg stripe where they crossed the boundary. These results suggest the extracellular domains of Caps and Tartan are not only required but also are sufficient to make V cells sort toward the DV boundary probably through interaction to an unknown D partner. Expression in D cells of truncated forms of Caps and Tartan lacking the intracellular domain in the partial absence of Apterous activity is able to partially rescue the DV affinity boundary. However, the DV boundary is not always smooth, and cells violate it. Are the rescue and sorting assays qualitatively or just quantitatively different tests of function? That the truncated forms behave quantitatively as full-length forms in the sorting assay and the subcellular distribution of both types of proteins were comparable suggests the inability of the truncated forms to rescue the DV affinity boundary and reflects a qualitative requirement of the intracellular domain in this process (Milán, 2005).
Fusion proteins consisting of the extracellular and transmembrane domains of Caps and Tartan and the β-galactosidase protein, a protein well-known to oligomerize, in the intracellular domain were not able to completely rescue the DV affinity boundary in the partial absence of Apterous activity, suggesting the intracellular domains of Caps and Tartan are not require for oligomerization. Their capability to partially rescue the DV affinity boundary was comparable to Caps-ID and Trn-ID, suggesting the β-galactosidase protein is not interfering with the activity of Caps and Tartan. Because β-galactosidase protein makes a tetramer, the possibility cannot be ruled out that Caps and Tartan may need to be arranged in a higher order multimer to be functional. Even though the intracellular domains of Caps and Tartan are quite different, they are completely interchangeable. Protein swaps consisting of the extracellular and transmembrane domains of Caps or Tartan and the intracellular domains of Tartan or Caps, respectively, were able to completely rescue the DV affinity boundary in the rescue assay (Milán, 2005).
With all these results, it is proposed that Caps and Tartan may work both as ligands and receptors to generate a stable DV affinity boundary in the wing primordium. In the sorting assay, Caps and Tartan may work as ligands of an unknown D cell surface protein. The extracellular domains of Caps and Tartan are necessary and sufficient to make V cells sort toward the DV boundary, probably being attracted by a D receptor. The extracellular domains of Caps and Tartan contain 14 LRRs and are 67% identical in terms of amino acid sequence, suggesting that they may bind to a common protein. In the rescue assay, both the extracellular and intracellular domains of Caps and Tartan are required. The extracellular domains of Caps and Tartan partially rescue the DV affinity boundary, suggesting Caps and Tartan are behaving as ligands in this assay. In the presence of the intracellular domains, the rescue is complete. Their intracellular domains would then either transduce a signal or interact with the cytoskeleton, thus generating a strong DV affinity difference (Milán, 2005).
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 indicate that a signaling cascade downstream of both ligands and receptors is required to induce repulsion or de-adhesion, thus promoting segregation of cell populations. Many adhesion proteins form regulated connections with the cytoskeleton and participate in contact-mediated signaling. The data suggest that the establishment of the DV affinity boundary in the Drosophila wing relies not only on the types of cell adhesion proteins the cells express but also on cell communication downstream of these proteins (Milán, 2005).
In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, a screen was performed of 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, it was shown that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron (Kurusu, 2008).
A database was assembled of 976 Drosophila cell-surface and secreted (CSS) proteins that are likely to be involved in cell recognition events during development. EP lines were found that allowed expression of 410 of these genes in muscles. All the genes were defined that altered presynaptic NMJ terminal patterning and structure without visibly affecting the muscles themselves. The screen identified 30 genes that cause mistargeting of axons within the VLM field with a penetrance of ~30% and 55 genes that produce major alterations in synaptic boutons or the structures of NMJ arbors with ~60% penetrance (Kurusu, 2008).
LRR genes represented 5 of the 12 mistargeting genes of interest. To evaluate the roles of the LRR genes in synaptic targeting, the genes of LOF mutants (and also knocked down or overexpressed mutants) were examined using both panmuscle drivers and drivers expressed only in specific muscle fibers. For mechanistic analysis, focus was placed primarily on results obtained by driving targeting genes or RNAi constructs in muscle 12 only and examining the consequences for innervation of muscle 12 by RP5 and the 1s neuron. These results are interpretable because the axons are known and only the last stage of their targeting should be affected. By contrast, mistargeting phenotypes observed with panmuscle expression could result from errors at any point along the axonal trajectory, and the axons that mistargeted cannot be identified in many cases. There may also be targeting errors that cannot be detected using mAb 1D4, because it labels all motor axons. Only NMJs that display morphological abnormalities can be seen and it was possible to miss phenotypes in which an axon from one motor neuron is replaced by another one, if it forms a similar NMJ. Ideally, these experiments should be performed using reagents that label single identified motor axons, but these are not yet available (Kurusu, 2008).
LRRs are ~24 aa protein domains that can be found outside the cell or in cytoplasmic proteins. A chain of LRRs forms a concave binding surface that is used for interactions with other proteins. The LRR domains of Trn and Caps are interchangeable, suggesting that they can interact with a common receptor (Milán, 2005). Trn and Caps are involved in cell-cell interactions in tracheae and imaginal discs, and Caps regulates layer-specific targeting in the optic lobe (Kurusu, 2008).
Within the VLM field, Caps is expressed on muscle 12, while Trn appears to be expressed on all VLMs, but with higher levels on muscles 6 and 7. trn caps double mutant embryos have stronger motor axon phenotypes than trn single mutants, and they exhibit ISNb terminal loop phenotypes that are suggestive of RP5 mistargeting (Kurusu, 2008).
Because muscle 12 NMJs send loopback branches onto muscle 13 when Caps is expressed in all muscles, it was proposed to be an attractive cue which facilitates targeting of RP5 to muscle 12. However, the actual situation may be more complex, because selective overexpression of either Caps or Trn on muscle 12 produces phenotypes in which muscle 12-destined axons either stall on muscle 13, so that muscle 12 remains uninnervated, or grow under muscle 13 rather than over it to reach muscle 12. These apparently nonautonomous effects (alteration of axonal extension over an adjacent muscle) might be explained by Trn- or Caps-induced alterations in the pattern of myopodia, projections from the muscle that reach out to contact innervating axons and direct their growth. Myopodia can extend over distances similar to the width of a muscle fiber. Perhaps when Trn or Caps is expressed on muscle 12, the myopodia extend under muscle 13 rather than over it. If RP5 axons contact these aberrant myopodia, they may grow under 13 to reach 12; if they fail to contact them, they may stall on muscle 13 (Kurusu, 2008).
CG14351, which is denoted in this study as Haf, is a large protein (1316 aa), and LRRs occupy only aa ~100-350 of the XC domain. Haf has a signal sequence, a single transmembrane region, and a large cytoplasmic domain (~500 aa). It appears to be expressed by all VLMs (Kurusu, 2008).
The embryonic motor axon phenotypes observed in the haf insertion mutant and in haf RNAi x pan-muscle-GAL4 embryos indicate that ISNb cannot innervate any of the VLMs in a normal manner if Haf is not expressed in muscles. Only 40%-50% of ISNbs have a normal morphology. The remainder bypass onto the ISN or SNa or follow abnormal trajectories within the VLM field, sometimes contacting inappropriate targets. The phenotypes are highly variable, suggesting that many different kinds of errors are produced by loss of Haf (Kurusu, 2008).
It is suggested that in the embryo Haf is a permissive muscle factor that is required for target selection by all muscles within the VLM field but does not define the identities of specific fibers. This model is also consistent with the larval phenotypes that result from knocking down Haf expression on muscle 12 only. In this case, the RP5 and 1 s axons seem to reach muscle 12 and form NMJs in a normal manner, but they also extend further and form ectopic synapses on lateral muscles 5 or 8. This phenotype suggests that a stable NMJ on muscle 12 sometimes cannot form when Haf is knocked down, and in these cases the axons (or NMJ branches) continue to grow until they reach the lateral muscles (Kurusu, 2008).
CG8561 is a 1092 aa protein that has a signal sequence but lacks a transmembrane region. Its C-terminal sequence is characteristic of proteins that are attached to membranes by glycosyl-phosphatidylinositol anchors. It appears to be expressed by all muscles (Kurusu, 2008).
CG8561 muscle RNAi and muscle overexpression produce mistargeting and NMJ arbor phenotypes in larvae. When CG8561 RNAi is expressed in muscle 12 only, the muscle 12 NMJ sends loopback branches to muscle 13 in 70% of affected hemisegments. This implies that CG8561 confers a preference for the RP5 and 1 s axons to choose muscle 12, and in its absence these axons do not strongly prefer muscle 12 to the adjacent muscle 13 (Kurusu, 2008).
CG8561 has a vertebrate ortholog, the acid-labile subunit (Als) of the IGF-1 binding complex. CG8561 mRNA is expressed in the larval fat body (FB) and in a group of neurosecretory cells (NSCs) that express insulin-like peptides. Starvation causes downregulation of the mRNA in the FB and NSCs. These data suggest that CG8561 (dAls) may be involved in insulin/IGF-1 signaling. Interestingly, the single fly insulin/IGF-1 receptor, InR, is expressed in neurons and is required for guidance of photoreceptor axons into the optic lobe (Kurusu, 2008).
Identification of 30 mistargeting genes among the 410 CSS genes that were screened suggests that there may be ~70 mistargeting genes in the entire cell-recognition database, and perhaps twice that many if genes with lower mistargeting percentages are included. If it is assumed that the screen is capable of identifying all CSS proteins involved in targeting, the results seem inconsistent with a simple version of a lock-and-key model, because mRNAs or proteins within the mistargeting set were not found that were expressed in small subsets of muscles. Also, the four LRR proteins examined in this paper have complex effects on targeting that are not explainable by a simple model (Kurusu, 2008).
Fifty-three LRR proteins were screened, and 16 of these produced either mistargeting, NMJ phenotypes, or both. If the failure of an LRR protein to produce a phenotype when overexpressed in muscles in the course of the screen indicates that it is not involved in targeting or synapse development, then there are 48 signal sequence-containing LRR proteins that remain to be examined for expression patterns, GOF phenotypes, and LOF phenotypes. A comprehensive analysis of this large family may help to define mechanisms involved in target selection and synaptic growth (Kurusu, 2008).
Search PubMed for articles about Drosophila Tartan
Chang, Z., Price, B. D., Bockheim, S., Boedigheimer, M. J., Smith, R. and Laughon, A. (1993). Molecular and genetic characterization of the Drosophila tartan gene. Dev. Biol. 160: 315-332. PubMed ID: 8253267
Krause, C., Wolf, C., Hemphälä J., Samakovlis, C. and Schuh, R. (2006). Distinct functions of the leucine-rich repeat transmembrane proteins capricious and tartan in the Drosophila tracheal morphogenesis. Dev. Biol. 296(1): 253-64. PubMed ID: 16764850
Kurusu, M., Cording, A., Taniguchi, M., Menon, K., Suzuki, E. and Zinn, K. (2008). A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection. Neuron 59(6): 972-85. PubMed ID: 18817735
Mao, Y., Kerr, M. and Freeman, M. (2007). Modulation of Drosophila retinal epithelial integrity by the adhesion proteins Capricious and Tartan. PLoS ONE 3(3): e1827. PubMed ID: 18350163
Milán, M., et al. (2001). The LRR proteins Capricious and Tartan mediate cell interactions during DV boundary formation in the Drosophila wing. Cell 106: 785-794. PubMed ID: 11572783
Milán, M., Pérez, L. and Cohen, S. M. (2002). Short-range cell interactions and cell survival in the Drosophila wing. Dev. Cell 2: 797-805. PubMed ID: 12062091
Milán, M., Pérez, L. and Cohen, S. M. (2005). Boundary formation in the Drosophila wing: functional dissection of Capricious and Tartan. Dev. Dyn. 233(3): 804-10. PubMed ID: 15830355
Sakurai, K. T., Kojima, T., Aigaki, T. and Hayashi, S. (2007). Differential control of cell affinity required for progression and refinement of cell boundary during Drosophila leg segmentation. Dev. Biol. 309: 126-136. PubMed ID: 17655839
Shinza-Kameda, M., et al. (2006). Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, Capricious. Neuron 49: 205-213. PubMed ID: 16423695
Shishido, E., Takeichi, M. and Nose, A. (1998). Drosophila synapse formation: Regulation by transmembrane protein with leu-rich repeats, CAPRICIOUS. Science 280: 2118-2121. PubMed ID: 9641918
Taniguchi, H., et al. (2000). Functional dissection of Drosophila capricious: its novel roles in neuronal pathfinding and selective synapse formation. J. Neurobiol. 42: 104-116. PubMed ID: 10623905
date revised: 7 March 2007
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