To investigate the distribution of tensin during development, tensin mRNA distribution was examined in the embryo by in situ hybridization. Expression was first observed in the visceral mesoderm at approximately 5 hr of development; prior to this, expression was not detected. Shortly after its appearance in the visceral mesoderm, tensin mRNA also appears in the developing gut, with particularly strong expression at the foregut/midgut boundary, in the keyhole region of the proventriculus. By stage 16 (14 hr), expression was also detected in the somatic muscles and the epidermal tendon cells, as well as in the central nervous system (Brody, 2002; Torgler, 2004).

To determine the expression stage and transcript complexity of Drosophila tensin, Northern blot analyses were performed at various developmental stages using a probe derived from the Drosophila tensin cDNA. A single transcript of about 3.0 kb was detected throughout all the developmental stages (Lee, 2003).

To examine the subcellular distribution of tensin, green fluorescent protein (GFP) was inserted between the last amino acid of tensin and the stop codon of the 11 kb by genomic rescue construct. This generated GFP-tagged tensin expressed from its normal promoter, which fully rescues by alleles. In the embryo, tensin-GFP was found highly enriched at the major site of integrin adhesion, the muscle attachment sites, both for the somatic muscles and the pharyngeal muscles. Consistent with the high levels of tensin mRNA in the developing proventriculus, tensin-GFP was detected in this tissue. Tensin distribution was confirmed with an antibody raised against the N terminus. Since loss of tensin causes a wing blister, the distribution of tensin-GFP in the pupal wings was examined; it localized to basal adhesive sites between the dorsal and ventral epithelial layers, identical to the distribution of integrins and integrin-linked-kinase (ILK). This demonstrates that the major sites of tensin accumulation in vivo are sites of integrin adhesion (Torgler, 2004).

Effects of Mutation or Deletion

The in vivo function of Drosophila tensin was investigated using genetic approaches. One P-element insertion line, by2, was found that contained a P-element in the 5' flanking sequence of the Drosophila tensin open reading frame. To examine whether the P-element insertion in by2 hampers the transcription of Drosophila tensin, RT-PCR was performed; this showed a highly reduced Drosophila tensin expression level in the whole body. In addition to by2, six revertants and two imprecise excision alleles of by2 were generated. These newly obtained mutants were verified by genomic PCR and subsequent sequencing analyses. The expression levels of Drosophila tensin in these lines were also examined by RT-PCR. The transcript levels of Drosophila tensin were completely normal in the revertant lines compared with wild type. By contrast, Drosophila tensin expression was highly reduced or completely missing in the imprecise excision alleles (Lee, 2003).

Homozygous but not heterozygous by2 mutant flies show blisters in their wings. To see whether this phenotype is related to Drosophila tensin expression, in situ hybridization was performed in wing imaginal discs. Drosophila tensin expression is dramatically reduced in the by2 mutants. Interestingly, the Drosophila tensin transcript is highly enriched in the wing pouch. It was confirmed that the blistered wing phenotype was indeed caused by a deficiency in Drosophila tensin expression, by generating transgenic flies specifically overexpressing Drosophila tensin within a homozygotic by2 genetic background. Ectopic expression of full-length Drosophila tensin in wing imaginal discs dramatically rescues the wing blister phenotype of by2. Moreover, two additional Drosophila tensin loss-of-function alleles, byex10 and byex49 fail to complement the blistered wing phenotype of the by2 mutants. However, byrv8, one of the revertants, fully complemented the by2 mutation. These results unequivocally demonstrate that the blistered wing phenotype in the Drosophila tensin mutants is due to a defect in Drosophila tensin function (Lee, 2003).

All the reported mutant lines with a blistered wing phenotype that mapped around the cytogenetic location of Drosophila tensin, 85D22, were examined and one mutant line was found, blistery1 (by1) --the loss-of-function allele of the blistery (by) gene. The mutation site of by1 has been reported to be located around 85D, but specific details have not yet been determined. The by1 mutants display blisters in the wing subterminal region (Glass, 1934; Prout, 1997; Walsh, 1998), which is identical to the phenotype of the Drosophila tensin mutants such as by2 and byex49. Significantly, the transheterozygotic alleles of by1 and by2 fail to complement each other's blistered wing phenotype, implying that they are alleles of the same gene. Consistently, byex10 and byex49 also failed to complement the blistered wing phenotype of by1 (Lee, 2003).

To further confirm that the Drosophila tensin mutants are allelic to by, the Drosophila tensin locus was sequenced in the by1 mutants. The by1 allele was found to contain two missense point mutations that convert tyrosine 62 and threonine 163 to asparagine (Y62N) and arginine (T163R) in tensin, respectively. Therefore, it is concluded that Drosophila tensin mutants and by1 are different alleles of by, and Drosophila tensin is henceforth referred to as blistery (Lee, 2003).

by mutant flies exhibit a blistered wing phenotype with varying degrees of severity, which can be classified into three groups; normal wing, class I wing (which contains a small blister < 1/4 size of the total wing area) within a restricted region, and class II wing with a large blister (> 1/4 size of the total wing area) resulting in a crumpled wing phenotype. The severity of the wing blister phenotype was determined by measuring the frequency of each wing class. By comparing the severity in various mutants and their by expression levels, it was found that the severity of wing blisters is closely related to the level of the by transcript. For example, a relatively mild phenotype of the byex10 mutants can be explained by higher expression of by than the other by mutants (Lee, 2003).

Besides the blistered wing phenotype, by mutants exhibit reduced hatching rate of laid eggs. The hatching rate of the mutant eggs is reduced to about 60% of wild type, while the survival rate of mutants after hatching is not significantly affected. These results demonstrate that tensin plays important roles during Drosophila early development as well. The impairment in egg hatching results from defective fertilization; therefore, the by mutants are homozygous viable with decreased fertility (Lee, 2003).

Drosophila wing development after pupariation (AP) consists of two distinct stages: prepupal and pupal wing morphogenesis. Pupal wing morphogenesis is further divided into three stages: separation (11-12 hours AP) of the ventral cell layer from the dorsal layer, re-apposition of the inter-vein cells (21-36 hours AP) and re-separation (60 hours AP) of the two cell layers. Shortly after eclosion, wings expand and unfold by an influx of hemolymph. PS integrins are required for the attachment of the two wing surfaces during pupal wing re-apposition and for the maintenance of the wing bilayer (Lee, 2003).

To determine the detailed roles of tensin during wing morphogenesis, the pupal wings of the by2 flies were examined. No differences were observed in the attachment of two wing surfaces and in the integrin localization between wild type and by2 wings during both prepupal apposition (4-6 hours AP) and pupal reapposition stages (30-36 hours AP) (Lee, 2003).

Because the pupal wing development was not disturbed in the by2 mutants, the expansion and unfolding processes of adult wings in the by2 mutants was investigated. After eclosion, the by2 flies display folded wings similar to the controls. Then, a sudden and rapid influx of hemolymph induces the unfolding of folded wings in the by2 mutants. However, as soon as the wings of by2 flies unfold, fluid-filled blisters began to appear at the distal part of the wings, and the boundary of the blisters expand to a certain extent. After the fluid dries, the wing blisters are fixed in place. Taken together, although the dorsal and ventral layers of a wing can be brought into close association during apposition and re-apposition processes in the by2 flies, the link between them may not be strong enough to resist the hydrostatic pressures during the wing unfolding process (Lee, 2003).

The functional significance of each domain of tensin in normal wing development was examined. UAS lines overexpressing either full-length tensin protein or various deletion mutant forms of tensin were generated. Unlike DeltaN and DeltaC, overexpression of DeltaPTB by MS1096-GAL4 driver completely rescues the blistered wing phenotype of by2. These data suggest that both the N-terminal region and the SH2 domain of tensin are required for proper attachment of two wing surfaces (Lee, 2003).

Since mammalian tensin is known to participate in the integrin signaling, whether tensin genetically interacts with integrin was examined. As expected, the blistered wing phenotype becomes more severe in the if3; by2/+ mutants and extremely severe in the double homozygotic mutants for if3 and by2, compared with if3 homozygotes or by2 heterozygotes. In addition, the rate of flies showing blistered wings in the total population greatly increases in the double mutants (Lee, 2003).

In mammalian cells, tensin has been implicated in signaling related to cell adhesion, such as signal transduction through Src, JNK and PI3K. To examine the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by2 mutants with heterozygotic mutations of the JNK signaling components bsk1 or hep1 (the loss-of-function mutants for Drosophila JNK and MKK7, respectively) display a highly severe blistered wing phenotype, compared with either homozygous by2, heterozygous bsk1 or heterozygous hep1 mutants. Notably, the rate of flies, which show Class II blistered wings, increases from 46.5% to 70% for these double mutants compared with homozygous by2 mutants, and about 15% of these flies had multiple blisters in their wings. Furthermore, the double homozygotic mutants for by2 and hep1 die at pharate adult stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).

Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by the MS1096-GAL4 driver does not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 2003).

To further confirm the genetic interaction between tensin and the JNK pathway, the effect of tensin on JNK activity in vivo was tested. The extent of JNK phosphorylation was tested using anti-phosphospecific JNK antibody in the by overexpression line and the by2 mutants. As expected, JNK phosphorylation is dramatically increased in the wing imaginal discs overexpressing by; this directly demonstrates increased JNK activity by by. On the contrary, JNK phosphorylation in the imaginal discs of the by2 mutants is reduced compared with the control (Lee, 2003).

Moreover, the reduced size and the convex wing phenotype observed in the wings overexpressing by can be most easily explained by apoptosis in the wings. Since the induction of apoptosis by the JNK signaling is well established, it was expected that the wing phenotype induced by by overexpression might be due to apoptosis. To confirm the by-induced apoptosis in vivo, Acridine Orange staining of the relevant wing imaginal discs was carried out. As expected, the overexpression of by dramatically increases apoptotic cell death compared with the control (Lee, 2003).

Thus, tensin genetically interacts with the components of the JNK signaling pathway, and regulates JNK activity during wing development. The supporting evidence for the engagement of tensin in the JNK signaling pathway comes from a recent report that transfected mammalian tensin activates JNK signaling in HEK 293T cells (Katz, 2000). Interestingly, in mammalian cells, JNK is also activated via adaptor proteins p130 CAS and Crk, which both receive a signal from the FAK/Src tyrosine kinase complex in the cell adhesion sites when cells attach to the ECM. Since tensin is a possible substrate for FAK, and p130 CAS is able to interact with the C terminus of tensin, it is highly possible that tensin is involved in this signaling cascade and mediates signals from integrin and FAK to the JNK signaling pathway (Lee, 2003).

The first allele of blistery was isolated as a spontaneous mutation with a recessive wing blister phenotype (Glass, 1934). The blister is found at the distal tip of the wing and extends between 1/3 and 1/2 of the length of the wing. A new by allele was isolated through a P element insertion that gave a wing blister phenotype. Inverse PCR revealed that the P element is inserted 57 nucleotides upstream of the start codon of the gene encoding the Drosophila ortholog of tensin (CG9379). Two further experiments confirmed that by corresponds to the tensin gene: (1) excision of the P element inserted upstream of tensin resulted in a reversion of the by allele to wild-type, demonstrating that the insertion causes the by mutation; (2) a P element rescue construct, containing an 11 kb genomic DNA fragment that included the tensin transcription unit and the flanking regions up to edge of the adjacent genes, fully rescued the by phenotype (Torgler, 2004).

The two by alleles described above are both viable, with a fully penetrant wing blister phenotype. However, tensin is found at integrin junctions in the embryo as well as the developing wing, suggesting a role for tensin at earlier stages of development. Therefore it was possible that these mutations only partly inactivate tensin and a complete loss of function allele would cause lethality, as seen with integrin gene mutations (Torgler, 2004).

In order to generate a mutation that completely eliminated tensin, the P element was excised to generate a small deficiency that removed just the by locus. All excisions were tested for lethality over a larger deficiency (Df(3R)by416). In all cases, the lethal excisions deleted one or more genes in addition to the by locus, suggesting that the adjacent genes are essential for viability but by is not. This was confirmed by the recovery of a new viable allele, called by33c, that contains a deletion removing the start of transcription and the majority of the coding region, and is therefore a null mutation by molecular criteria. The homozygous by33c adult flies are also viable with a fully penetrant wing blister phenotype. In addition, by mutant flies derived from by mutant mothers have the same phenotype, demonstrating that maternally provided tensin is not required for earlier development. As there is only one tensin encoded by the fly genome, it is concluded that flies can survive without tensin (Torgler, 2004).

The wing blisters in tensin mutant flies appeared shortly after eclosion and were localized at the distal end of the wings. This differs from flies carrying an integrin mutation inflated3: these flies have blisters in the center of the wing that are visible immediately after eclosion. By observing the process of wing expansion in the by mutant flies, it was noted that the blister gradually appears as the flies stroke their wings with their back legs. This normal behavior presumably aids the expansion and flattening of the wings. Since in by33c the wing blister appears concomitantly with stroking, it was thought that mechanical shear stress from the legs might cause the wing blister. To test this, the legs of the mutant flies were fixed to a glass slide with glue, just as the wings started to unfold after eclosion: this treatment suppressed the wing blister phenotype. Since the intervein cells apoptose and the cuticle hardens during wing expansion, this rescue is permanent. Examination of actin distribution in the pupal wing showed no detectable difference from wild-type. Thus, tensin is required to stabilize adhesion in the wing so that it can resist the normal mechanical abrasion associated with wing flattening after eclosion. This is distinct from mutations in integrins or talin, where the blister forms during pupal development and therefore could not be 'rescued' by glue (Torgler, 2004).

Tensin is the fourth cytoplasmic protein found to be tightly colocalized with integrins at the ends of the muscles, along with ILK, talin, and PINCH. The effect of the absence of either integrins or the other cytoskeletal adaptors on the localization of tensin was examined. Mutations in the genes encoding integrins, talin, and ILK all disrupt the tight localization of tensin at the muscle ends and at the basal surface of the tendon cells. In all three mutants, tensin was diffusely distributed throughout the cytoplasm, with elevated expression detectable in the tendon cells. Conversely, in mutants lacking PINCH, tensin is still localized, even at late stages when muscles start to detach. These data show that tensin recruitment is dependent on talin and ILK, suggesting it modifies the function of the complexes assembled by these proteins, rather than playing a role in their initial recruitment or stable assembly. This was confirmed by the normal distribution of talin and ILK in tensin mutant embryos. Actin distribution was examined in the absence of tensin, in embryos and pupal wings, but no gross changes were detected . Thus, tensin appears to be a late addition to this integrin-associated adhesion complex (Torgler, 2004).


Reference names in red indicate recommended papers.

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Brody, T., Stivers, C., Nagle, J. and Odenwald, W.F. (2002). Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen. Mech. Dev. 113: 41-59. 11900973

Calderwood, D. A., et al. (2003). Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling. Proc. Natl. Acad. Sci. 100: 2272-2277. 12606711

Chen, H., Ishii, A., Wong, W. K., Chen, L. B. and Lo, S. H. (2000). Molecular characterization of human tensin. Biochem. J. 351: 403-411. 11023826

Chen, H., Duncan, I. C., Bozorgchami, H. and Lo, S. H. (2002). Tensin and a previously undocumented family member, tensin2, positively regulate cell migration. Proc. Natl. Acad. Sci. 99: 733-738. 11792844

Chen, H. and Lo, S.H. (2003). Regulation of tensin-promoted cell migration by its focal adhesion binding and Src homology domain 2. Biochem. J. 370: 1039-1045. 12495434

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Cui, Y., Liao, Y. C. and Lo, S. H. (2004). Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3. Mol. Cancer Res. 2(4): 225-32. 15140944

Davis, S., Lu, M. L., Lo, S. H., Lin, S., Butler, J. A., Druker, B. J., Roberts, T. M., An, Q. and Chen, L. B. (1991). Presence of an SH2 domain in the actin-binding protein tensin. Science 252: 712-715. 1708917

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Katz, B. Z., Zohar, M., Teramoto, H., Matsumoto, K., Gutkind, J. S., Lin, D. C., Lin, S. and Yamada, K. M. (2000). Tensin can induce JNK and p38 activation. Biochem. Biophys. Res. Commun. 272: 717-720. 10860821

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blistery: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 June 2004

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