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Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

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Armadillo mutants overexpressed with dppGAL4 induce ectopic bristles and veins in adult wings, with varying degrees of severity.(A, A′) ArmS10 is lethal even at 18°C using dppGAL4, but results in a lawn of ectopic bristles using C5GAL4 (insets). (B, B′) Only a very weak phenotype is caused by overexpression of ΔNArm1–128, inducing ectopic bristles on vein L3 near the wing margin (arrowheads). (C, C′) Although ectopic bristles and veins are induced at 18°C (arrowheads), the phenotype caused by ΔNArm1–155 is predominantly neurogenic, with many ectopic sensillae along the wing veins (white arrows and inset, C5GAL4). (D, D′) In contrast, even at 18°C no pupae eclose when the tethered form is overexpressed with dppGAL4. The phenotype induced by Myr-ΔNArm1–155 is similar to, though more severe than, that of ArmS10, causing a lawn of ectopic bristles to form with C5GAL4 (inset). (E, E′) ArmΔCXM19 has a very weak phenotype, with only few ectopic bristles forming at the wing margin at the tip of vein L3 (arrowheads).
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pone-0002893-g013: Armadillo mutants overexpressed with dppGAL4 induce ectopic bristles and veins in adult wings, with varying degrees of severity.(A, A′) ArmS10 is lethal even at 18°C using dppGAL4, but results in a lawn of ectopic bristles using C5GAL4 (insets). (B, B′) Only a very weak phenotype is caused by overexpression of ΔNArm1–128, inducing ectopic bristles on vein L3 near the wing margin (arrowheads). (C, C′) Although ectopic bristles and veins are induced at 18°C (arrowheads), the phenotype caused by ΔNArm1–155 is predominantly neurogenic, with many ectopic sensillae along the wing veins (white arrows and inset, C5GAL4). (D, D′) In contrast, even at 18°C no pupae eclose when the tethered form is overexpressed with dppGAL4. The phenotype induced by Myr-ΔNArm1–155 is similar to, though more severe than, that of ArmS10, causing a lawn of ectopic bristles to form with C5GAL4 (inset). (E, E′) ArmΔCXM19 has a very weak phenotype, with only few ectopic bristles forming at the wing margin at the tip of vein L3 (arrowheads).

Mentions: As with overexpression of Wingless under dppGAL4, overexpression of ArmS10 or Myr-ΔNArm1–155 was lethal even at 18°C (Figure 13A, A′, D, D′). A few wings, however, were recovered that overexpressed ΔNArm1–155 (Figure 13C, C′), exhibiting expanded sensillae (white arrows), as well as additional bristles associated with ectopic veins near the anterior extent of dpp expression. However, using C5GAL4, which drives late expression throughout the wing pouch with the exception of the D/V boundary, it was possible to compare phenotypes across these constructs. Although the wings were severely folded and blistered, it was evident that while ΔNArm1–155 caused a neurogenic phenotype, the ectopic signalling induced by ArmS10 and Myr-ΔNArm1–155 resulted in a lawn of ectopic bristles (Figure 13A, A′, C, C′, D, D′, insets). In contrast, only weak phenotypes were observed with ΔNArm1–128 and ArmΔCXM19 wings, with few ectopic bristles along and at the tip of the L3 vein, respectively (Figure 13B, B′, E, E′). The presence of ectopic bristles correlated well with the high nuclear levels of endogenous Armadillo induced by overexpression of ArmS10, ΔNArm1–155 and Myr-ΔNArm1–155; with ArmΔCXM19 near the D/V boundary (Figure 12); and with nuclear localisation of αSenseless, which marks sensory organ precursors (not shown). However, the mainly neurogenic and veination defects of ΔNArm1–155, which differ both quantitatively and qualitatively from both the Myr-ΔNArm1–155 and ΔNArm1–128 phenotypes, suggested that the severity of the effects might lie at least partly in disruption of interactions between Armadillo and α-catenin. The ΔNArm1–155 and ΔNArm1–128 mutants differ structurally in the extent of the N-terminal deletion; the absence of part of the 1st Armadillo repeat in ΔNArm1–128 likely reduces its binding to α-catenin (Figure 1).


Wingless signalling alters the levels, subcellular distribution and dynamics of Armadillo and E-cadherin in third instar larval wing imaginal discs.

Somorjai IM, Martinez-Arias A - PLoS ONE (2008)

Armadillo mutants overexpressed with dppGAL4 induce ectopic bristles and veins in adult wings, with varying degrees of severity.(A, A′) ArmS10 is lethal even at 18°C using dppGAL4, but results in a lawn of ectopic bristles using C5GAL4 (insets). (B, B′) Only a very weak phenotype is caused by overexpression of ΔNArm1–128, inducing ectopic bristles on vein L3 near the wing margin (arrowheads). (C, C′) Although ectopic bristles and veins are induced at 18°C (arrowheads), the phenotype caused by ΔNArm1–155 is predominantly neurogenic, with many ectopic sensillae along the wing veins (white arrows and inset, C5GAL4). (D, D′) In contrast, even at 18°C no pupae eclose when the tethered form is overexpressed with dppGAL4. The phenotype induced by Myr-ΔNArm1–155 is similar to, though more severe than, that of ArmS10, causing a lawn of ectopic bristles to form with C5GAL4 (inset). (E, E′) ArmΔCXM19 has a very weak phenotype, with only few ectopic bristles forming at the wing margin at the tip of vein L3 (arrowheads).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2483348&req=5

pone-0002893-g013: Armadillo mutants overexpressed with dppGAL4 induce ectopic bristles and veins in adult wings, with varying degrees of severity.(A, A′) ArmS10 is lethal even at 18°C using dppGAL4, but results in a lawn of ectopic bristles using C5GAL4 (insets). (B, B′) Only a very weak phenotype is caused by overexpression of ΔNArm1–128, inducing ectopic bristles on vein L3 near the wing margin (arrowheads). (C, C′) Although ectopic bristles and veins are induced at 18°C (arrowheads), the phenotype caused by ΔNArm1–155 is predominantly neurogenic, with many ectopic sensillae along the wing veins (white arrows and inset, C5GAL4). (D, D′) In contrast, even at 18°C no pupae eclose when the tethered form is overexpressed with dppGAL4. The phenotype induced by Myr-ΔNArm1–155 is similar to, though more severe than, that of ArmS10, causing a lawn of ectopic bristles to form with C5GAL4 (inset). (E, E′) ArmΔCXM19 has a very weak phenotype, with only few ectopic bristles forming at the wing margin at the tip of vein L3 (arrowheads).
Mentions: As with overexpression of Wingless under dppGAL4, overexpression of ArmS10 or Myr-ΔNArm1–155 was lethal even at 18°C (Figure 13A, A′, D, D′). A few wings, however, were recovered that overexpressed ΔNArm1–155 (Figure 13C, C′), exhibiting expanded sensillae (white arrows), as well as additional bristles associated with ectopic veins near the anterior extent of dpp expression. However, using C5GAL4, which drives late expression throughout the wing pouch with the exception of the D/V boundary, it was possible to compare phenotypes across these constructs. Although the wings were severely folded and blistered, it was evident that while ΔNArm1–155 caused a neurogenic phenotype, the ectopic signalling induced by ArmS10 and Myr-ΔNArm1–155 resulted in a lawn of ectopic bristles (Figure 13A, A′, C, C′, D, D′, insets). In contrast, only weak phenotypes were observed with ΔNArm1–128 and ArmΔCXM19 wings, with few ectopic bristles along and at the tip of the L3 vein, respectively (Figure 13B, B′, E, E′). The presence of ectopic bristles correlated well with the high nuclear levels of endogenous Armadillo induced by overexpression of ArmS10, ΔNArm1–155 and Myr-ΔNArm1–155; with ArmΔCXM19 near the D/V boundary (Figure 12); and with nuclear localisation of αSenseless, which marks sensory organ precursors (not shown). However, the mainly neurogenic and veination defects of ΔNArm1–155, which differ both quantitatively and qualitatively from both the Myr-ΔNArm1–155 and ΔNArm1–128 phenotypes, suggested that the severity of the effects might lie at least partly in disruption of interactions between Armadillo and α-catenin. The ΔNArm1–155 and ΔNArm1–128 mutants differ structurally in the extent of the N-terminal deletion; the absence of part of the 1st Armadillo repeat in ΔNArm1–128 likely reduces its binding to α-catenin (Figure 1).

Bottom Line: Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification.Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling.Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Cambridge, United Kingdom. ildiko.somorjai@obs-banyuls.fr

ABSTRACT

Background: Armadillo, the Drosophila orthologue of vertebrate ss-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex" is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets.

Methodology/principal findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional" and "adhesive" pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, DeltaNArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-DeltaNArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin.

Conclusions/significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-Cadherin.

Show MeSH
Related in: MedlinePlus