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Differential requirement for Dab2 in the development of embryonic and extra-embryonic tissues.

Moore R, Cai KQ, Tao W, Smith ER, Xu XX - BMC Dev. Biol. (2013)

Bottom Line: Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels.Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss.However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, 33136, FL, USA. xxu2@med.miami.edu.

ABSTRACT

Background: Disabled-2 (Dab2) is an endocytic adaptor protein involved in clathrin-mediated endocytosis and cargo trafficking. Since its expression is lost in several cancer types, Dab2 has been suggested to be a tumor suppressor. In vitro studies indicate that Dab2 establishes epithelial cell polarity and organization by directing endocytic trafficking of membrane glycoproteins. Dab2 also modulates cellular signaling pathways by mediating the endocytosis and recycling of surface receptors and associated signaling components. Previously, two independent gene knockout studies have been reported, with some discrepancies in the observed embryonic phenotypes. To further clarify the in vivo roles of Dab2 in development and physiology, we designed a new floxed allele to delete dab2 gene.

Results: The constitutive dab2 deleted embryos showed a spectrum in the degree of endoderm disorganization in E5.5 and no mutant embryos persisted at E9.5. However, the mice were grossly normal when dab2 deletion was restricted to the embryo proper and the gene was retained in extraembryonic tissues using Meox2-Cre and Sox2-Cre. Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels.

Conclusion: The study of the new dab2 mutant allele in embryos and embryoid bodies confirms a role for Dab2 in extraembryonic endoderm development and epithelial organization. Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss. Conditional deletion indicates that Dab2 is dispensable for organ development, when the vast majority of the embryonic cells are dab2 . However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.

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Mild defective positioning phenotype in embryoid bodies and compensatory expression of Arh and Numb in Dab2  ES cells and embryos. (A) In some of the dab2 (-/-) ES clones (such as clone dfg15), a monolayer epithelial structure is seen at some of the surface of embryoid bodies (indicated by arrows), as shown by an example of a section stained with Dab2 (green) (no signal in this image), GATA4 (red), and DAPI (blue). (B) Western blot shows that Arh and Numb were variably expressed in embryoid bodies derived from several ES cell clones of different dab2 genotypes. (C) The expression of Dab2, Arh, and Numb adaptors proteins was analyzed by Western blotting of E9.5 embryos from matings between female dab2 (fl/fl) and male dab2 (+/df); Sox2-Cre. The embryos were harvested, dissected free of extraembryonic tissues, lysed and denatured in SDS buffer, and probed for Dab2, Arh, and Numb. The extraembryonic tissues were used for genotyping.
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Figure 7: Mild defective positioning phenotype in embryoid bodies and compensatory expression of Arh and Numb in Dab2 ES cells and embryos. (A) In some of the dab2 (-/-) ES clones (such as clone dfg15), a monolayer epithelial structure is seen at some of the surface of embryoid bodies (indicated by arrows), as shown by an example of a section stained with Dab2 (green) (no signal in this image), GATA4 (red), and DAPI (blue). (B) Western blot shows that Arh and Numb were variably expressed in embryoid bodies derived from several ES cell clones of different dab2 genotypes. (C) The expression of Dab2, Arh, and Numb adaptors proteins was analyzed by Western blotting of E9.5 embryos from matings between female dab2 (fl/fl) and male dab2 (+/df); Sox2-Cre. The embryos were harvested, dissected free of extraembryonic tissues, lysed and denatured in SDS buffer, and probed for Dab2, Arh, and Numb. The extraembryonic tissues were used for genotyping.

Mentions: Nevertheless, phenotypic variations were observed among the five dab2 (-/-) ES clones isolated. A clone of dab2 (-/-) ES cells (dfg15) was observed to have a mild positioning phenotype, in that some monolayer endoderm epithelial structures were observed in a portion of embryoid bodies derived from this clone (Figure 7A, arrows). We suspect that additional endocytic adaptors with activity similar to Dab2, such as Numb and Arh[31], might be able to substitute for Dab2 function and rescue the endoderm disorganization in a subset of Dab2 mutant embryoid bodies. Indeed, we found that Numb and Arh proteins were expressed in differentiated ES cells in embryoid bodies, and the expression varied among clones (Figure 7B). Compared to other ES clones, the cells of the dab2 (-/-) clone dfg15 had higher Arh and Numb expression. Additionally, expression of Arh and Numb appeared to change with passage in culture. Thus, the compensatory expression of Numb and/or Arh may rescue primitive endoderm disorganization in embryoid bodies to some degree when dab2 gene is deleted. Future experiments using combinative deletions or forced overexpression of the adaptors will be needed to test this possibility.


Differential requirement for Dab2 in the development of embryonic and extra-embryonic tissues.

Moore R, Cai KQ, Tao W, Smith ER, Xu XX - BMC Dev. Biol. (2013)

Mild defective positioning phenotype in embryoid bodies and compensatory expression of Arh and Numb in Dab2  ES cells and embryos. (A) In some of the dab2 (-/-) ES clones (such as clone dfg15), a monolayer epithelial structure is seen at some of the surface of embryoid bodies (indicated by arrows), as shown by an example of a section stained with Dab2 (green) (no signal in this image), GATA4 (red), and DAPI (blue). (B) Western blot shows that Arh and Numb were variably expressed in embryoid bodies derived from several ES cell clones of different dab2 genotypes. (C) The expression of Dab2, Arh, and Numb adaptors proteins was analyzed by Western blotting of E9.5 embryos from matings between female dab2 (fl/fl) and male dab2 (+/df); Sox2-Cre. The embryos were harvested, dissected free of extraembryonic tissues, lysed and denatured in SDS buffer, and probed for Dab2, Arh, and Numb. The extraembryonic tissues were used for genotyping.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924344&req=5

Figure 7: Mild defective positioning phenotype in embryoid bodies and compensatory expression of Arh and Numb in Dab2 ES cells and embryos. (A) In some of the dab2 (-/-) ES clones (such as clone dfg15), a monolayer epithelial structure is seen at some of the surface of embryoid bodies (indicated by arrows), as shown by an example of a section stained with Dab2 (green) (no signal in this image), GATA4 (red), and DAPI (blue). (B) Western blot shows that Arh and Numb were variably expressed in embryoid bodies derived from several ES cell clones of different dab2 genotypes. (C) The expression of Dab2, Arh, and Numb adaptors proteins was analyzed by Western blotting of E9.5 embryos from matings between female dab2 (fl/fl) and male dab2 (+/df); Sox2-Cre. The embryos were harvested, dissected free of extraembryonic tissues, lysed and denatured in SDS buffer, and probed for Dab2, Arh, and Numb. The extraembryonic tissues were used for genotyping.
Mentions: Nevertheless, phenotypic variations were observed among the five dab2 (-/-) ES clones isolated. A clone of dab2 (-/-) ES cells (dfg15) was observed to have a mild positioning phenotype, in that some monolayer endoderm epithelial structures were observed in a portion of embryoid bodies derived from this clone (Figure 7A, arrows). We suspect that additional endocytic adaptors with activity similar to Dab2, such as Numb and Arh[31], might be able to substitute for Dab2 function and rescue the endoderm disorganization in a subset of Dab2 mutant embryoid bodies. Indeed, we found that Numb and Arh proteins were expressed in differentiated ES cells in embryoid bodies, and the expression varied among clones (Figure 7B). Compared to other ES clones, the cells of the dab2 (-/-) clone dfg15 had higher Arh and Numb expression. Additionally, expression of Arh and Numb appeared to change with passage in culture. Thus, the compensatory expression of Numb and/or Arh may rescue primitive endoderm disorganization in embryoid bodies to some degree when dab2 gene is deleted. Future experiments using combinative deletions or forced overexpression of the adaptors will be needed to test this possibility.

Bottom Line: Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels.Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss.However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, University of Miami Miller School of Medicine, Miami, 33136, FL, USA. xxu2@med.miami.edu.

ABSTRACT

Background: Disabled-2 (Dab2) is an endocytic adaptor protein involved in clathrin-mediated endocytosis and cargo trafficking. Since its expression is lost in several cancer types, Dab2 has been suggested to be a tumor suppressor. In vitro studies indicate that Dab2 establishes epithelial cell polarity and organization by directing endocytic trafficking of membrane glycoproteins. Dab2 also modulates cellular signaling pathways by mediating the endocytosis and recycling of surface receptors and associated signaling components. Previously, two independent gene knockout studies have been reported, with some discrepancies in the observed embryonic phenotypes. To further clarify the in vivo roles of Dab2 in development and physiology, we designed a new floxed allele to delete dab2 gene.

Results: The constitutive dab2 deleted embryos showed a spectrum in the degree of endoderm disorganization in E5.5 and no mutant embryos persisted at E9.5. However, the mice were grossly normal when dab2 deletion was restricted to the embryo proper and the gene was retained in extraembryonic tissues using Meox2-Cre and Sox2-Cre. Adult Dab2-deficient mice had a small but statistically significant increase in serum cholesterol levels.

Conclusion: The study of the new dab2 mutant allele in embryos and embryoid bodies confirms a role for Dab2 in extraembryonic endoderm development and epithelial organization. Experimental results with embryoid bodies suggest that additional endocytic adaptors such as Arh and Numb could partially compensate for Dab2 loss. Conditional deletion indicates that Dab2 is dispensable for organ development, when the vast majority of the embryonic cells are dab2 . However, Dab2 has a physiological role in the endocytosis of lipoproteins and cholesterol metabolism.

Show MeSH
Related in: MedlinePlus