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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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Endoderm disorganization of Dab2 knockout embryoid bodies. Clones of ES cells isolated from blastocysts derived from matings between dab2 (+/df) parents were genotyped by PCR and allowed to form embryoid bodies in suspension cultures for 5 days. Representative examples of heterozygous dab2 (+/-) and homozygous dab2 (-/-) embryoid bodies were analyzed by immunofluorescence microscopy. (A) Sections were stained for the presence of Dab2 (green), endoderm marker GATA4 (red), and counterstained with DAPI (blue). Merged images at low (top panels) and higher magnification (lower panels) are shown. (B) Sections were stained for the pluripotent marker Oct3/4 (green), laminin (red) to indicate primitive endoderm epithelia, and counter-stained with DAPI (blue). Merged images are shown at the top (low magnification) and middle (higher magnification) panels. Laminin staining (red) alone from the corresponding middle panels is presented at the bottom, and the presence of a thin basement membrane underlying the endoderm epithelium in the wildtype embryoid bodies is indicated by an arrow, and no such basement membrane layer was observed in the dab2  embryoid bodies.
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Figure 6: Endoderm disorganization of Dab2 knockout embryoid bodies. Clones of ES cells isolated from blastocysts derived from matings between dab2 (+/df) parents were genotyped by PCR and allowed to form embryoid bodies in suspension cultures for 5 days. Representative examples of heterozygous dab2 (+/-) and homozygous dab2 (-/-) embryoid bodies were analyzed by immunofluorescence microscopy. (A) Sections were stained for the presence of Dab2 (green), endoderm marker GATA4 (red), and counterstained with DAPI (blue). Merged images at low (top panels) and higher magnification (lower panels) are shown. (B) Sections were stained for the pluripotent marker Oct3/4 (green), laminin (red) to indicate primitive endoderm epithelia, and counter-stained with DAPI (blue). Merged images are shown at the top (low magnification) and middle (higher magnification) panels. Laminin staining (red) alone from the corresponding middle panels is presented at the bottom, and the presence of a thin basement membrane underlying the endoderm epithelium in the wildtype embryoid bodies is indicated by an arrow, and no such basement membrane layer was observed in the dab2 embryoid bodies.

Mentions: We generated several lines of both dab2 heterozygous (+/-) and homozygous (-/-) mutant ES cells from blastocysts for further study. Previously, embryoid bodies derived from ES cells in which Dab2 expression was suppressed by shRNA were defective in the organization of primitive endoderm layer[18]. The ability of dab2- ES cells to form embryoid bodies was tested. In several Dab2 mutant clones analyzed, primitive endoderm differentiation occurred, but the Gata4-positive endoderm cells failed to form an epithelial layer on the surface of the embryoid bodies (Figure 6A). In heterozygous and wildtype controls, a layer of laminin-positive basement membrane was observed (indicated by arrows in Figure 6B), indicating the formation of an epithelium. In Dab2-negative embryoid bodies, the differentiated primitive endoderm cells were positive for laminin; however, neither an endoderm epithelium nor a basement membrane layer was evident (Figure 6B).


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)

Endoderm disorganization of Dab2 knockout embryoid bodies. Clones of ES cells isolated from blastocysts derived from matings between dab2 (+/df) parents were genotyped by PCR and allowed to form embryoid bodies in suspension cultures for 5 days. Representative examples of heterozygous dab2 (+/-) and homozygous dab2 (-/-) embryoid bodies were analyzed by immunofluorescence microscopy. (A) Sections were stained for the presence of Dab2 (green), endoderm marker GATA4 (red), and counterstained with DAPI (blue). Merged images at low (top panels) and higher magnification (lower panels) are shown. (B) Sections were stained for the pluripotent marker Oct3/4 (green), laminin (red) to indicate primitive endoderm epithelia, and counter-stained with DAPI (blue). Merged images are shown at the top (low magnification) and middle (higher magnification) panels. Laminin staining (red) alone from the corresponding middle panels is presented at the bottom, and the presence of a thin basement membrane underlying the endoderm epithelium in the wildtype embryoid bodies is indicated by an arrow, and no such basement membrane layer was observed in the dab2  embryoid bodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Endoderm disorganization of Dab2 knockout embryoid bodies. Clones of ES cells isolated from blastocysts derived from matings between dab2 (+/df) parents were genotyped by PCR and allowed to form embryoid bodies in suspension cultures for 5 days. Representative examples of heterozygous dab2 (+/-) and homozygous dab2 (-/-) embryoid bodies were analyzed by immunofluorescence microscopy. (A) Sections were stained for the presence of Dab2 (green), endoderm marker GATA4 (red), and counterstained with DAPI (blue). Merged images at low (top panels) and higher magnification (lower panels) are shown. (B) Sections were stained for the pluripotent marker Oct3/4 (green), laminin (red) to indicate primitive endoderm epithelia, and counter-stained with DAPI (blue). Merged images are shown at the top (low magnification) and middle (higher magnification) panels. Laminin staining (red) alone from the corresponding middle panels is presented at the bottom, and the presence of a thin basement membrane underlying the endoderm epithelium in the wildtype embryoid bodies is indicated by an arrow, and no such basement membrane layer was observed in the dab2 embryoid bodies.
Mentions: We generated several lines of both dab2 heterozygous (+/-) and homozygous (-/-) mutant ES cells from blastocysts for further study. Previously, embryoid bodies derived from ES cells in which Dab2 expression was suppressed by shRNA were defective in the organization of primitive endoderm layer[18]. The ability of dab2- ES cells to form embryoid bodies was tested. In several Dab2 mutant clones analyzed, primitive endoderm differentiation occurred, but the Gata4-positive endoderm cells failed to form an epithelial layer on the surface of the embryoid bodies (Figure 6A). In heterozygous and wildtype controls, a layer of laminin-positive basement membrane was observed (indicated by arrows in Figure 6B), indicating the formation of an epithelium. In Dab2-negative embryoid bodies, the differentiated primitive endoderm cells were positive for laminin; however, neither an endoderm epithelium nor a basement membrane layer was evident (Figure 6B).

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