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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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Cfl1 gene expression is not regulated by GDNF/Ret signaling in developing kidney.(A–A'), Cfl1 in situ hybridization in E11.5 wild-type kidney cultured with control, BSA-soaked bead. A' shows a higher magnification of the boxed area in A. mRNA is detected both in metanephric mesenchyme (m) and ureteric epithelium (u). (B–B'), Similar expression levels of Cfl1 in kidneys cultured with GDNF-soaked beads, which induce enlargement of ureteric tips and extra budding from Wolffian duct (arrows). B' shows a higher magnification of the boxed area in B. (C), Cofilin1 antibody staining of Ret+/− (control) E10.5 embryo, where Wolffian duct shows epithelial thickening as a hallmark of early ureteric bud outgrowth. (D), Normal expression of Cofilin1 protein (red) in Ret−/− Wolffian duct epithelium at E10.5. (E,F) F-actin localization in control and Ret−/− kidneys at E10.5. Scale bars 100 µm for A–B, 50 µm for C–F.
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pgen-1001176-g008: Cfl1 gene expression is not regulated by GDNF/Ret signaling in developing kidney.(A–A'), Cfl1 in situ hybridization in E11.5 wild-type kidney cultured with control, BSA-soaked bead. A' shows a higher magnification of the boxed area in A. mRNA is detected both in metanephric mesenchyme (m) and ureteric epithelium (u). (B–B'), Similar expression levels of Cfl1 in kidneys cultured with GDNF-soaked beads, which induce enlargement of ureteric tips and extra budding from Wolffian duct (arrows). B' shows a higher magnification of the boxed area in B. (C), Cofilin1 antibody staining of Ret+/− (control) E10.5 embryo, where Wolffian duct shows epithelial thickening as a hallmark of early ureteric bud outgrowth. (D), Normal expression of Cofilin1 protein (red) in Ret−/− Wolffian duct epithelium at E10.5. (E,F) F-actin localization in control and Ret−/− kidneys at E10.5. Scale bars 100 µm for A–B, 50 µm for C–F.

Mentions: Cfl1 was identified as one of the potential Ret-induced genes in Ret-expressing NIH3T3 cells [14] and we were therefore interested in determining if Cfl1 could be induced by Ret signaling in the ureteric epithelium. Both gain- and loss-of-function strategies were used to analyze Cfl1 mRNA and protein regulation by GDNF/Ret signaling. Cfl1 is expressed in ureteric epithelium and metanephric mesenchyme of E11.5 kidneys cultured for 24 h (Figure 8A–8A'). While GDNF-soaked beads induced ectopic ureteric budding and local swelling of the UB tip, confirming the functionality of the protein, no change in Cfl1 mRNA expression was observed (Figure 8B–8B'). Two different Ret mutant mouse lines were used to study the effect of either reduced Ret signaling (Ret-hypomorphic mice) or lack of Ret signaling (Ret−/− mice) on cofilin1 protein levels. The Ret-hypomorphic mutant Rettm2(RET)Vpa has reduced UB branching [27] while UB formation fails in most of the Ret−/− kidneys [28]. Cofilin1 was present at normal levels in the early UB of E10.5 Ret−/− mutants (Figure 8C, 8D) as well as in E15 Ret-hypomorphic kidneys (data not shown). Furthermore, the distribution of F-actin appeared normal in both types of Ret-mutant kidneys (Figure 8E, 8F and data not shown). Therefore, although the failure of UB outgrowth in Ret−/− kidneys is phenotypically similar to the phenotype of Cfl1;Dstn double mutant UBs, this is apparently not due to reduced expression of Cfl1 in the Ret mutant.


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)

Cfl1 gene expression is not regulated by GDNF/Ret signaling in developing kidney.(A–A'), Cfl1 in situ hybridization in E11.5 wild-type kidney cultured with control, BSA-soaked bead. A' shows a higher magnification of the boxed area in A. mRNA is detected both in metanephric mesenchyme (m) and ureteric epithelium (u). (B–B'), Similar expression levels of Cfl1 in kidneys cultured with GDNF-soaked beads, which induce enlargement of ureteric tips and extra budding from Wolffian duct (arrows). B' shows a higher magnification of the boxed area in B. (C), Cofilin1 antibody staining of Ret+/− (control) E10.5 embryo, where Wolffian duct shows epithelial thickening as a hallmark of early ureteric bud outgrowth. (D), Normal expression of Cofilin1 protein (red) in Ret−/− Wolffian duct epithelium at E10.5. (E,F) F-actin localization in control and Ret−/− kidneys at E10.5. Scale bars 100 µm for A–B, 50 µm for C–F.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965756&req=5

pgen-1001176-g008: Cfl1 gene expression is not regulated by GDNF/Ret signaling in developing kidney.(A–A'), Cfl1 in situ hybridization in E11.5 wild-type kidney cultured with control, BSA-soaked bead. A' shows a higher magnification of the boxed area in A. mRNA is detected both in metanephric mesenchyme (m) and ureteric epithelium (u). (B–B'), Similar expression levels of Cfl1 in kidneys cultured with GDNF-soaked beads, which induce enlargement of ureteric tips and extra budding from Wolffian duct (arrows). B' shows a higher magnification of the boxed area in B. (C), Cofilin1 antibody staining of Ret+/− (control) E10.5 embryo, where Wolffian duct shows epithelial thickening as a hallmark of early ureteric bud outgrowth. (D), Normal expression of Cofilin1 protein (red) in Ret−/− Wolffian duct epithelium at E10.5. (E,F) F-actin localization in control and Ret−/− kidneys at E10.5. Scale bars 100 µm for A–B, 50 µm for C–F.
Mentions: Cfl1 was identified as one of the potential Ret-induced genes in Ret-expressing NIH3T3 cells [14] and we were therefore interested in determining if Cfl1 could be induced by Ret signaling in the ureteric epithelium. Both gain- and loss-of-function strategies were used to analyze Cfl1 mRNA and protein regulation by GDNF/Ret signaling. Cfl1 is expressed in ureteric epithelium and metanephric mesenchyme of E11.5 kidneys cultured for 24 h (Figure 8A–8A'). While GDNF-soaked beads induced ectopic ureteric budding and local swelling of the UB tip, confirming the functionality of the protein, no change in Cfl1 mRNA expression was observed (Figure 8B–8B'). Two different Ret mutant mouse lines were used to study the effect of either reduced Ret signaling (Ret-hypomorphic mice) or lack of Ret signaling (Ret−/− mice) on cofilin1 protein levels. The Ret-hypomorphic mutant Rettm2(RET)Vpa has reduced UB branching [27] while UB formation fails in most of the Ret−/− kidneys [28]. Cofilin1 was present at normal levels in the early UB of E10.5 Ret−/− mutants (Figure 8C, 8D) as well as in E15 Ret-hypomorphic kidneys (data not shown). Furthermore, the distribution of F-actin appeared normal in both types of Ret-mutant kidneys (Figure 8E, 8F and data not shown). Therefore, although the failure of UB outgrowth in Ret−/− kidneys is phenotypically similar to the phenotype of Cfl1;Dstn double mutant UBs, this is apparently not due to reduced expression of Cfl1 in the Ret mutant.

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