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Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

Kuure S, Cebrian C, Machingo Q, Lu BC, Chi X, Hyink D, D'Agati V, Gurniak C, Witke W, Costantini F - PLoS Genet. (2010)

Bottom Line: The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis.Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration.The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT
The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

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Efficiency of Hoxb7/CreGFP-mediated deletion of Cfl1 as evaluated by Cofilin1 antibody staining.Calbindin (green) was used to visualize Wolffian duct epithelium in (A–D). (A–B'), cofilin1 (red) and calbindin (green) in E10.5 Wolffian duct (sections through the expanded region that will give rise to the UB). Cofilin1 is expressed in every cell in both ureteric bud and metanephric mesenchyme, and seems slightly enriched in UB epithelial cells. At E10.5, there is no detectable difference in cofilin1 protein amount or localization between control (Dstn+/−) and Hoxb7/CreGFP; Cfl1F/F; Dstn−/− kidneys. (C–D'), At E12.5, cofilin1 localization in the Dstn+/− control is similar to that at the earlier stages. However, Hoxb7/CreGFP has efficiently deleted the gene in the UB, as the protein is absent in ureteric epithelium of Cfl1F/F kidneys. Arrows point to ureteric buds, scale bar 50 µm.
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pgen-1001176-g003: Efficiency of Hoxb7/CreGFP-mediated deletion of Cfl1 as evaluated by Cofilin1 antibody staining.Calbindin (green) was used to visualize Wolffian duct epithelium in (A–D). (A–B'), cofilin1 (red) and calbindin (green) in E10.5 Wolffian duct (sections through the expanded region that will give rise to the UB). Cofilin1 is expressed in every cell in both ureteric bud and metanephric mesenchyme, and seems slightly enriched in UB epithelial cells. At E10.5, there is no detectable difference in cofilin1 protein amount or localization between control (Dstn+/−) and Hoxb7/CreGFP; Cfl1F/F; Dstn−/− kidneys. (C–D'), At E12.5, cofilin1 localization in the Dstn+/− control is similar to that at the earlier stages. However, Hoxb7/CreGFP has efficiently deleted the gene in the UB, as the protein is absent in ureteric epithelium of Cfl1F/F kidneys. Arrows point to ureteric buds, scale bar 50 µm.

Mentions: The finding that the UB always formed in Cfl1;Dstn double mutant kidneys, but failed to branch, raised the possibility that the activities of cofilin1 and destrin are required for UB branching, but not for Wolffian duct growth or initial UB formation. Alternatively, there might be a delay in the elimination of cofilin1 by Hoxb7/CreGFP, such that there is still sufficient cofilin1 at the time of UB outgrowth (E10.5) but not when branching initiates (E11.5). To study the timing and efficiency of cofilin1 elimination we stained mutant kidneys with anti-cofilin1 antibody. While the UB epithelium was clearly devoid of cofilin1 at E11.5 (data not shown) and E12.5 (Figure 3C–3D'), confirming the activity of Hoxb7/CreGFP, we found that cofilin1 protein levels at E10.5 were normal in the forming ureteric bud (Figure 3A–3B'). Thus, the ability of the UB to grow out in double mutants, but not to branch subsequently, is most likely due to residual cofilin1 expression at E10.5, which is eliminated by E11.5. A similar delay in cofilin1 protein elimination was observed when the floxed Cfl1 allele was deleted in the brain using nestinCre, a result that was attributed to the long half-life of the protein [5].


Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis.

Kuure S, Cebrian C, Machingo Q, Lu BC, Chi X, Hyink D, D'Agati V, Gurniak C, Witke W, Costantini F - PLoS Genet. (2010)

Efficiency of Hoxb7/CreGFP-mediated deletion of Cfl1 as evaluated by Cofilin1 antibody staining.Calbindin (green) was used to visualize Wolffian duct epithelium in (A–D). (A–B'), cofilin1 (red) and calbindin (green) in E10.5 Wolffian duct (sections through the expanded region that will give rise to the UB). Cofilin1 is expressed in every cell in both ureteric bud and metanephric mesenchyme, and seems slightly enriched in UB epithelial cells. At E10.5, there is no detectable difference in cofilin1 protein amount or localization between control (Dstn+/−) and Hoxb7/CreGFP; Cfl1F/F; Dstn−/− kidneys. (C–D'), At E12.5, cofilin1 localization in the Dstn+/− control is similar to that at the earlier stages. However, Hoxb7/CreGFP has efficiently deleted the gene in the UB, as the protein is absent in ureteric epithelium of Cfl1F/F kidneys. Arrows point to ureteric buds, scale bar 50 µm.
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Related In: Results  -  Collection

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pgen-1001176-g003: Efficiency of Hoxb7/CreGFP-mediated deletion of Cfl1 as evaluated by Cofilin1 antibody staining.Calbindin (green) was used to visualize Wolffian duct epithelium in (A–D). (A–B'), cofilin1 (red) and calbindin (green) in E10.5 Wolffian duct (sections through the expanded region that will give rise to the UB). Cofilin1 is expressed in every cell in both ureteric bud and metanephric mesenchyme, and seems slightly enriched in UB epithelial cells. At E10.5, there is no detectable difference in cofilin1 protein amount or localization between control (Dstn+/−) and Hoxb7/CreGFP; Cfl1F/F; Dstn−/− kidneys. (C–D'), At E12.5, cofilin1 localization in the Dstn+/− control is similar to that at the earlier stages. However, Hoxb7/CreGFP has efficiently deleted the gene in the UB, as the protein is absent in ureteric epithelium of Cfl1F/F kidneys. Arrows point to ureteric buds, scale bar 50 µm.
Mentions: The finding that the UB always formed in Cfl1;Dstn double mutant kidneys, but failed to branch, raised the possibility that the activities of cofilin1 and destrin are required for UB branching, but not for Wolffian duct growth or initial UB formation. Alternatively, there might be a delay in the elimination of cofilin1 by Hoxb7/CreGFP, such that there is still sufficient cofilin1 at the time of UB outgrowth (E10.5) but not when branching initiates (E11.5). To study the timing and efficiency of cofilin1 elimination we stained mutant kidneys with anti-cofilin1 antibody. While the UB epithelium was clearly devoid of cofilin1 at E11.5 (data not shown) and E12.5 (Figure 3C–3D'), confirming the activity of Hoxb7/CreGFP, we found that cofilin1 protein levels at E10.5 were normal in the forming ureteric bud (Figure 3A–3B'). Thus, the ability of the UB to grow out in double mutants, but not to branch subsequently, is most likely due to residual cofilin1 expression at E10.5, which is eliminated by E11.5. A similar delay in cofilin1 protein elimination was observed when the floxed Cfl1 allele was deleted in the brain using nestinCre, a result that was attributed to the long half-life of the protein [5].

Bottom Line: The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis.Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration.The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Development, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT
The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.

Show MeSH
Related in: MedlinePlus