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Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila.

Le Borgne R, Remaud S, Hamel S, Schweisguth F - PLoS Biol. (2005)

Bottom Line: During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling.Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells.We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, CNRS UMR 8542, Paris, France.

ABSTRACT
Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

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D-mib Co-Localizes with Dl and Ser at the Apical Cell Cortex(A and A′) D-mib (green) is detected in all cells of the wing imaginal disc. In (A), Ser is in red and Discs-large (Dlg) is in blue.(B–D′′′) D-mib (green in B, B′, C, C′, D, and D′) co-localized with Ser (red in [B and B′′]), Dl (red in [C and C′′]), N (red in [D and D′′]), and E-Cadherin (E-Cad; blue in [D and D′′′]) and was found apical to Discs-large (Dlg; blue in [B, B′′′, C, and C′′′]) in notum cells located at the edges of the wing discs.(E–E′′) D-mib (green in [E and E′]) co-localized with Dl (red in [E and E′′]) at the apical cortex of wing pouch cells.(F–F′′) D-mib staining at the apical cortex (blue in [F and F′]) was not detected in D-mib2 mutant clone (marked by loss of nuclear GFP staining; green in [F]). Loss of D-mib activity has no detectable effect on the apical accumulation of Dl (red in [F and F′′]).Bar is 50 μm for (A and A′) and 10 μm for (B–F′′).
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pbio-0030096-g003: D-mib Co-Localizes with Dl and Ser at the Apical Cell Cortex(A and A′) D-mib (green) is detected in all cells of the wing imaginal disc. In (A), Ser is in red and Discs-large (Dlg) is in blue.(B–D′′′) D-mib (green in B, B′, C, C′, D, and D′) co-localized with Ser (red in [B and B′′]), Dl (red in [C and C′′]), N (red in [D and D′′]), and E-Cadherin (E-Cad; blue in [D and D′′′]) and was found apical to Discs-large (Dlg; blue in [B, B′′′, C, and C′′′]) in notum cells located at the edges of the wing discs.(E–E′′) D-mib (green in [E and E′]) co-localized with Dl (red in [E and E′′]) at the apical cortex of wing pouch cells.(F–F′′) D-mib staining at the apical cortex (blue in [F and F′]) was not detected in D-mib2 mutant clone (marked by loss of nuclear GFP staining; green in [F]). Loss of D-mib activity has no detectable effect on the apical accumulation of Dl (red in [F and F′′]).Bar is 50 μm for (A and A′) and 10 μm for (B–F′′).

Mentions: We next studied the subcellular localization of D-mib (Figure 3). Anti-D-mib antibodies were generated that specifically detected D-mib on Western blots (see Figure 1C) and on fixed tissues (Figure 3F–F″). Using these antibodies, we found that D-mib was detected in all imaginal disc cells (Figure 3A and 3B). We then examined D-mib subcellular distribution in epithelial cells located along the edge of the wing discs because cross-sectional imaging affords better resolution along the apical-basal axis. D-mib co-localized with Ser, Dl, and N at the apical cortex (Figure 3B–3D′′′). Dl and Ser were also detected in large intracellular vesicles that probably correspond to multivesicular bodies in that they also stained for hepatocyte growth factor-regulated tyrosine kinase substrate [46] (Figure 3B–3C′′′; data not shown). The intracellular dots seen with the anti-D-mib antibodies were distinct from the Dl- and Ser-positive dots and appeared to result from background staining (data not shown). The reduced cytoplasmic staining seen in D-mib mutant cells (Figure 3F–3F′′) suggests that D-mib is also present in the cytoplasm. A similar localization at the apical cortex and in the cytoplasm was seen for a functional yellow fluorescent protein (YFP)::D-mib fusion protein (see Figure 6 below). These localization data suggest that D-mib may act at the apical cortex to regulate the activity of Dl and/or Ser.


Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila.

Le Borgne R, Remaud S, Hamel S, Schweisguth F - PLoS Biol. (2005)

D-mib Co-Localizes with Dl and Ser at the Apical Cell Cortex(A and A′) D-mib (green) is detected in all cells of the wing imaginal disc. In (A), Ser is in red and Discs-large (Dlg) is in blue.(B–D′′′) D-mib (green in B, B′, C, C′, D, and D′) co-localized with Ser (red in [B and B′′]), Dl (red in [C and C′′]), N (red in [D and D′′]), and E-Cadherin (E-Cad; blue in [D and D′′′]) and was found apical to Discs-large (Dlg; blue in [B, B′′′, C, and C′′′]) in notum cells located at the edges of the wing discs.(E–E′′) D-mib (green in [E and E′]) co-localized with Dl (red in [E and E′′]) at the apical cortex of wing pouch cells.(F–F′′) D-mib staining at the apical cortex (blue in [F and F′]) was not detected in D-mib2 mutant clone (marked by loss of nuclear GFP staining; green in [F]). Loss of D-mib activity has no detectable effect on the apical accumulation of Dl (red in [F and F′′]).Bar is 50 μm for (A and A′) and 10 μm for (B–F′′).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1064853&req=5

pbio-0030096-g003: D-mib Co-Localizes with Dl and Ser at the Apical Cell Cortex(A and A′) D-mib (green) is detected in all cells of the wing imaginal disc. In (A), Ser is in red and Discs-large (Dlg) is in blue.(B–D′′′) D-mib (green in B, B′, C, C′, D, and D′) co-localized with Ser (red in [B and B′′]), Dl (red in [C and C′′]), N (red in [D and D′′]), and E-Cadherin (E-Cad; blue in [D and D′′′]) and was found apical to Discs-large (Dlg; blue in [B, B′′′, C, and C′′′]) in notum cells located at the edges of the wing discs.(E–E′′) D-mib (green in [E and E′]) co-localized with Dl (red in [E and E′′]) at the apical cortex of wing pouch cells.(F–F′′) D-mib staining at the apical cortex (blue in [F and F′]) was not detected in D-mib2 mutant clone (marked by loss of nuclear GFP staining; green in [F]). Loss of D-mib activity has no detectable effect on the apical accumulation of Dl (red in [F and F′′]).Bar is 50 μm for (A and A′) and 10 μm for (B–F′′).
Mentions: We next studied the subcellular localization of D-mib (Figure 3). Anti-D-mib antibodies were generated that specifically detected D-mib on Western blots (see Figure 1C) and on fixed tissues (Figure 3F–F″). Using these antibodies, we found that D-mib was detected in all imaginal disc cells (Figure 3A and 3B). We then examined D-mib subcellular distribution in epithelial cells located along the edge of the wing discs because cross-sectional imaging affords better resolution along the apical-basal axis. D-mib co-localized with Ser, Dl, and N at the apical cortex (Figure 3B–3D′′′). Dl and Ser were also detected in large intracellular vesicles that probably correspond to multivesicular bodies in that they also stained for hepatocyte growth factor-regulated tyrosine kinase substrate [46] (Figure 3B–3C′′′; data not shown). The intracellular dots seen with the anti-D-mib antibodies were distinct from the Dl- and Ser-positive dots and appeared to result from background staining (data not shown). The reduced cytoplasmic staining seen in D-mib mutant cells (Figure 3F–3F′′) suggests that D-mib is also present in the cytoplasm. A similar localization at the apical cortex and in the cytoplasm was seen for a functional yellow fluorescent protein (YFP)::D-mib fusion protein (see Figure 6 below). These localization data suggest that D-mib may act at the apical cortex to regulate the activity of Dl and/or Ser.

Bottom Line: During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling.Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells.We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, CNRS UMR 8542, Paris, France.

ABSTRACT
Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib) gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

Show MeSH
Related in: MedlinePlus