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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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Molecular and Genetic Characterization of D-mib Mutations(A) Molecular map of the D-mib locus showing the position of the P-element inserted into the 5′ untranslated region (allele D-mib1) and the 13.6 kb deletion that removes the D-mib and the RpS31 genes (allele D-mib2). Transcribed regions are indicated with arrows, and exons are indicated with boxes. Open reading frames are shown in black.(B) Domain composition of D-mib and D. rerio Mib. Both proteins show identical domain organization. D-mib has an N-terminal ZZ zinc finger flanked on either side by a Mib/HERC2 (M-H) domain, followed by two Mib repeats, six ankyrin repeats, two atypical RING domains, and a C-terminal protypical RING that has been associated with catalytic E3 ubiquitin ligase activity. The D-mib3 mutant allele is predicted to produce a truncated protein devoid of E3 ubiquitin ligase activity whereas the D-mib4 protein carries a mutation at a conserved position in the second Mib repeat.(C and C′) Western blot analysis of D-mib (C). The endogenous D-mib protein (predicted size: 130 kDa) was detected in S2 cells (lane 2) and in imaginal discs from wild-type larvae (lane 3) but was not detectable in homozygous D-mib1 (lane 4) and D-mib1/D-mib3 (lane 5) third instar larvae. The D-mib protein produced in transfected S2 cells from the cDNA used in this study (lane 1) runs exactly as endogenous D-mib (lane 2). Panel C′ shows a Red Ponceau staining of the gel with the same protein samples as in panel C.(D–H) Wings from wild-type (D), D-mib1 (E), SerRX82/Serrev6.1 (F), D-mib2/D-mib4 (G), and UAS-D-mib2/+; D-mib1/D-mib2 flies (H). D-mib (E) and Ser (F) mutant flies showed similar wing loss phenotypes. The D-mib mutant phenotype could be almost fully rescued by a leaky UAS-D-mib transgene (H). (D′) and (G′) show high magnification views of (D) and (G), respectively, to show that D-mib2/D-mib4 mutant flies (G′) exhibited ectopic sensilla (arrowheads) along vein L3.(I–N) Nota (I–K) and legs (L–N) from wild-type (I and L), D-mib1 (J and M), and SerRX82/Serrev6.1 (K and N) flies. D-mib mutant flies showed a weak neurogenic phenotype (J) that was not observed in Ser mutant flies (K). Ectopic sensory organs in D-mib mutant flies developed from ectopic sensory organ precursor cells (not shown). D-mib (M) and Ser (N) mutant legs also showed distinct growth and/or elongation defects. Arrows in (J) show ectopic macrochaetes. Arrows in (L–N) indicate the joints. Ti, tibia; t1 to t5, tarsal segments 1 to 5.
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pbio-0030096-g001: Molecular and Genetic Characterization of D-mib Mutations(A) Molecular map of the D-mib locus showing the position of the P-element inserted into the 5′ untranslated region (allele D-mib1) and the 13.6 kb deletion that removes the D-mib and the RpS31 genes (allele D-mib2). Transcribed regions are indicated with arrows, and exons are indicated with boxes. Open reading frames are shown in black.(B) Domain composition of D-mib and D. rerio Mib. Both proteins show identical domain organization. D-mib has an N-terminal ZZ zinc finger flanked on either side by a Mib/HERC2 (M-H) domain, followed by two Mib repeats, six ankyrin repeats, two atypical RING domains, and a C-terminal protypical RING that has been associated with catalytic E3 ubiquitin ligase activity. The D-mib3 mutant allele is predicted to produce a truncated protein devoid of E3 ubiquitin ligase activity whereas the D-mib4 protein carries a mutation at a conserved position in the second Mib repeat.(C and C′) Western blot analysis of D-mib (C). The endogenous D-mib protein (predicted size: 130 kDa) was detected in S2 cells (lane 2) and in imaginal discs from wild-type larvae (lane 3) but was not detectable in homozygous D-mib1 (lane 4) and D-mib1/D-mib3 (lane 5) third instar larvae. The D-mib protein produced in transfected S2 cells from the cDNA used in this study (lane 1) runs exactly as endogenous D-mib (lane 2). Panel C′ shows a Red Ponceau staining of the gel with the same protein samples as in panel C.(D–H) Wings from wild-type (D), D-mib1 (E), SerRX82/Serrev6.1 (F), D-mib2/D-mib4 (G), and UAS-D-mib2/+; D-mib1/D-mib2 flies (H). D-mib (E) and Ser (F) mutant flies showed similar wing loss phenotypes. The D-mib mutant phenotype could be almost fully rescued by a leaky UAS-D-mib transgene (H). (D′) and (G′) show high magnification views of (D) and (G), respectively, to show that D-mib2/D-mib4 mutant flies (G′) exhibited ectopic sensilla (arrowheads) along vein L3.(I–N) Nota (I–K) and legs (L–N) from wild-type (I and L), D-mib1 (J and M), and SerRX82/Serrev6.1 (K and N) flies. D-mib mutant flies showed a weak neurogenic phenotype (J) that was not observed in Ser mutant flies (K). Ectopic sensory organs in D-mib mutant flies developed from ectopic sensory organ precursor cells (not shown). D-mib (M) and Ser (N) mutant legs also showed distinct growth and/or elongation defects. Arrows in (J) show ectopic macrochaetes. Arrows in (L–N) indicate the joints. Ti, tibia; t1 to t5, tarsal segments 1 to 5.

Mentions: The closest Drosophila homolog of the vertebrate mib gene is the predicted gene CG5841, D-mib [18]. The D-mib mutations identified are shown in Figure 1. A P-element inserted into the 5′ untranslated region of the D-mib gene was recently isolated (http://flypush.imgen.bcm.tmc.edu/pscreen/) (Figure 1A). Insertion of this P-element confers late pupal lethality. Lethality was reverted by precise excision of the P-element, suggesting that insertion of this P-element is a D-mib mutation, referred to as D-mib1. A 13.6-kb deletion that removes the entire D-mib coding region was selected by imprecise excision of this P-element. This deletion represents a allele of D-mib and was named D-mib2. This deletion also deletes the 3′ flanking RpS31 gene (Figure 1A). The D-mib1 and D-mib2mutant alleles did not complement the l(3)72CdaJ12 and l(3)72CdaI5 lethal mutations that have been mapped to the same cytological interval as the D-mib gene [35]. This indicates that these two lethal mutations are D-mib mutant alleles, and they were therefore renamed D-mib3 and D-mib4, respectively. The D-mib1 and D-mib3 mutations behave as genetic alleles (see Materials and Methods). In contrast, D-mib4 is a partial loss-of-function allele because flies trans-heterozygous for D-mib4 and any other D-mib alleles are viable.


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)

Molecular and Genetic Characterization of D-mib Mutations(A) Molecular map of the D-mib locus showing the position of the P-element inserted into the 5′ untranslated region (allele D-mib1) and the 13.6 kb deletion that removes the D-mib and the RpS31 genes (allele D-mib2). Transcribed regions are indicated with arrows, and exons are indicated with boxes. Open reading frames are shown in black.(B) Domain composition of D-mib and D. rerio Mib. Both proteins show identical domain organization. D-mib has an N-terminal ZZ zinc finger flanked on either side by a Mib/HERC2 (M-H) domain, followed by two Mib repeats, six ankyrin repeats, two atypical RING domains, and a C-terminal protypical RING that has been associated with catalytic E3 ubiquitin ligase activity. The D-mib3 mutant allele is predicted to produce a truncated protein devoid of E3 ubiquitin ligase activity whereas the D-mib4 protein carries a mutation at a conserved position in the second Mib repeat.(C and C′) Western blot analysis of D-mib (C). The endogenous D-mib protein (predicted size: 130 kDa) was detected in S2 cells (lane 2) and in imaginal discs from wild-type larvae (lane 3) but was not detectable in homozygous D-mib1 (lane 4) and D-mib1/D-mib3 (lane 5) third instar larvae. The D-mib protein produced in transfected S2 cells from the cDNA used in this study (lane 1) runs exactly as endogenous D-mib (lane 2). Panel C′ shows a Red Ponceau staining of the gel with the same protein samples as in panel C.(D–H) Wings from wild-type (D), D-mib1 (E), SerRX82/Serrev6.1 (F), D-mib2/D-mib4 (G), and UAS-D-mib2/+; D-mib1/D-mib2 flies (H). D-mib (E) and Ser (F) mutant flies showed similar wing loss phenotypes. The D-mib mutant phenotype could be almost fully rescued by a leaky UAS-D-mib transgene (H). (D′) and (G′) show high magnification views of (D) and (G), respectively, to show that D-mib2/D-mib4 mutant flies (G′) exhibited ectopic sensilla (arrowheads) along vein L3.(I–N) Nota (I–K) and legs (L–N) from wild-type (I and L), D-mib1 (J and M), and SerRX82/Serrev6.1 (K and N) flies. D-mib mutant flies showed a weak neurogenic phenotype (J) that was not observed in Ser mutant flies (K). Ectopic sensory organs in D-mib mutant flies developed from ectopic sensory organ precursor cells (not shown). D-mib (M) and Ser (N) mutant legs also showed distinct growth and/or elongation defects. Arrows in (J) show ectopic macrochaetes. Arrows in (L–N) indicate the joints. Ti, tibia; t1 to t5, tarsal segments 1 to 5.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064853&req=5

pbio-0030096-g001: Molecular and Genetic Characterization of D-mib Mutations(A) Molecular map of the D-mib locus showing the position of the P-element inserted into the 5′ untranslated region (allele D-mib1) and the 13.6 kb deletion that removes the D-mib and the RpS31 genes (allele D-mib2). Transcribed regions are indicated with arrows, and exons are indicated with boxes. Open reading frames are shown in black.(B) Domain composition of D-mib and D. rerio Mib. Both proteins show identical domain organization. D-mib has an N-terminal ZZ zinc finger flanked on either side by a Mib/HERC2 (M-H) domain, followed by two Mib repeats, six ankyrin repeats, two atypical RING domains, and a C-terminal protypical RING that has been associated with catalytic E3 ubiquitin ligase activity. The D-mib3 mutant allele is predicted to produce a truncated protein devoid of E3 ubiquitin ligase activity whereas the D-mib4 protein carries a mutation at a conserved position in the second Mib repeat.(C and C′) Western blot analysis of D-mib (C). The endogenous D-mib protein (predicted size: 130 kDa) was detected in S2 cells (lane 2) and in imaginal discs from wild-type larvae (lane 3) but was not detectable in homozygous D-mib1 (lane 4) and D-mib1/D-mib3 (lane 5) third instar larvae. The D-mib protein produced in transfected S2 cells from the cDNA used in this study (lane 1) runs exactly as endogenous D-mib (lane 2). Panel C′ shows a Red Ponceau staining of the gel with the same protein samples as in panel C.(D–H) Wings from wild-type (D), D-mib1 (E), SerRX82/Serrev6.1 (F), D-mib2/D-mib4 (G), and UAS-D-mib2/+; D-mib1/D-mib2 flies (H). D-mib (E) and Ser (F) mutant flies showed similar wing loss phenotypes. The D-mib mutant phenotype could be almost fully rescued by a leaky UAS-D-mib transgene (H). (D′) and (G′) show high magnification views of (D) and (G), respectively, to show that D-mib2/D-mib4 mutant flies (G′) exhibited ectopic sensilla (arrowheads) along vein L3.(I–N) Nota (I–K) and legs (L–N) from wild-type (I and L), D-mib1 (J and M), and SerRX82/Serrev6.1 (K and N) flies. D-mib mutant flies showed a weak neurogenic phenotype (J) that was not observed in Ser mutant flies (K). Ectopic sensory organs in D-mib mutant flies developed from ectopic sensory organ precursor cells (not shown). D-mib (M) and Ser (N) mutant legs also showed distinct growth and/or elongation defects. Arrows in (J) show ectopic macrochaetes. Arrows in (L–N) indicate the joints. Ti, tibia; t1 to t5, tarsal segments 1 to 5.
Mentions: The closest Drosophila homolog of the vertebrate mib gene is the predicted gene CG5841, D-mib [18]. The D-mib mutations identified are shown in Figure 1. A P-element inserted into the 5′ untranslated region of the D-mib gene was recently isolated (http://flypush.imgen.bcm.tmc.edu/pscreen/) (Figure 1A). Insertion of this P-element confers late pupal lethality. Lethality was reverted by precise excision of the P-element, suggesting that insertion of this P-element is a D-mib mutation, referred to as D-mib1. A 13.6-kb deletion that removes the entire D-mib coding region was selected by imprecise excision of this P-element. This deletion represents a allele of D-mib and was named D-mib2. This deletion also deletes the 3′ flanking RpS31 gene (Figure 1A). The D-mib1 and D-mib2mutant alleles did not complement the l(3)72CdaJ12 and l(3)72CdaI5 lethal mutations that have been mapped to the same cytological interval as the D-mib gene [35]. This indicates that these two lethal mutations are D-mib mutant alleles, and they were therefore renamed D-mib3 and D-mib4, respectively. The D-mib1 and D-mib3 mutations behave as genetic alleles (see Materials and Methods). In contrast, D-mib4 is a partial loss-of-function allele because flies trans-heterozygous for D-mib4 and any other D-mib alleles are viable.

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