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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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Histological phenotypes of constitutively Dab2  embryos at E4.5 and E5.5. (A) Fixed and paraffin embedded E4.5 embryos in utero from timed matings of dab2 (+/df) mice were sectioned and stained with H&E to identify implanted embryos. Once found, the serial sections were used for staining with Dab2 to identify dab2 deletion mutants, adjacent sections were then stained with GATA4 and GATA6 as markers for primitive endoderm; and with Oct3/4 to identify cells of the inner cell mass. Consecutive sections of a representative Dab2-positive and a Dab2-negative (dab2 (-/-)) E4.5 embryo are shown. (B) Examples of one wildtype (WT) and five Dab2-negative (dab2 (-/-)) E5.5 embryos are shown. Dab2 staining was used to identify mutant embryos. An adjacent section was stained for GATA4 to identify the endoderm cells. The images shown were sections at or near midline (widest area) of the embryos. A spectrum of severity in disorganization of endoderm cells was seen in the dab2  embryos. The arrow indicates a parietal endoderm cell in the wildtype.
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Figure 2: Histological phenotypes of constitutively Dab2 embryos at E4.5 and E5.5. (A) Fixed and paraffin embedded E4.5 embryos in utero from timed matings of dab2 (+/df) mice were sectioned and stained with H&E to identify implanted embryos. Once found, the serial sections were used for staining with Dab2 to identify dab2 deletion mutants, adjacent sections were then stained with GATA4 and GATA6 as markers for primitive endoderm; and with Oct3/4 to identify cells of the inner cell mass. Consecutive sections of a representative Dab2-positive and a Dab2-negative (dab2 (-/-)) E4.5 embryo are shown. (B) Examples of one wildtype (WT) and five Dab2-negative (dab2 (-/-)) E5.5 embryos are shown. Dab2 staining was used to identify mutant embryos. An adjacent section was stained for GATA4 to identify the endoderm cells. The images shown were sections at or near midline (widest area) of the embryos. A spectrum of severity in disorganization of endoderm cells was seen in the dab2 embryos. The arrow indicates a parietal endoderm cell in the wildtype.

Mentions: We initially examined embryos at E4.5 when Dab2 is expressed in the primitive endoderm[17]. At this implantation stage, dab2 (-/-) embryos were identified by immunostaining for the absence of Dab2 protein (Figure 2). In wildtype or heterozygous blastocysts, the Gata4-, Gata6-, and Dab2-positive primitive endoderm cells exclusively located on the surface layer covering the Oct3/4-positive inner cell mass. Among 26 E4.5 embryos found following sectioning of 6 uteri, 4 implanted blastocysts were confirmed as Dab2- based on immunostaining. In the Dab2 knockout embryos, although Gata4- and Gata6-positive primitive endoderm cells were present, the cells were not organized into a monolayer epithelium and some located deep inside the inner cell mass (Figure 2A). Thus the embryonic defect is initiated at E4.5, post differentiation but prior to morphogenesis of the primitive endoderm, although the phenotype is subtle at this stage.


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)

Histological phenotypes of constitutively Dab2  embryos at E4.5 and E5.5. (A) Fixed and paraffin embedded E4.5 embryos in utero from timed matings of dab2 (+/df) mice were sectioned and stained with H&E to identify implanted embryos. Once found, the serial sections were used for staining with Dab2 to identify dab2 deletion mutants, adjacent sections were then stained with GATA4 and GATA6 as markers for primitive endoderm; and with Oct3/4 to identify cells of the inner cell mass. Consecutive sections of a representative Dab2-positive and a Dab2-negative (dab2 (-/-)) E4.5 embryo are shown. (B) Examples of one wildtype (WT) and five Dab2-negative (dab2 (-/-)) E5.5 embryos are shown. Dab2 staining was used to identify mutant embryos. An adjacent section was stained for GATA4 to identify the endoderm cells. The images shown were sections at or near midline (widest area) of the embryos. A spectrum of severity in disorganization of endoderm cells was seen in the dab2  embryos. The arrow indicates a parietal endoderm cell in the wildtype.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Histological phenotypes of constitutively Dab2 embryos at E4.5 and E5.5. (A) Fixed and paraffin embedded E4.5 embryos in utero from timed matings of dab2 (+/df) mice were sectioned and stained with H&E to identify implanted embryos. Once found, the serial sections were used for staining with Dab2 to identify dab2 deletion mutants, adjacent sections were then stained with GATA4 and GATA6 as markers for primitive endoderm; and with Oct3/4 to identify cells of the inner cell mass. Consecutive sections of a representative Dab2-positive and a Dab2-negative (dab2 (-/-)) E4.5 embryo are shown. (B) Examples of one wildtype (WT) and five Dab2-negative (dab2 (-/-)) E5.5 embryos are shown. Dab2 staining was used to identify mutant embryos. An adjacent section was stained for GATA4 to identify the endoderm cells. The images shown were sections at or near midline (widest area) of the embryos. A spectrum of severity in disorganization of endoderm cells was seen in the dab2 embryos. The arrow indicates a parietal endoderm cell in the wildtype.
Mentions: We initially examined embryos at E4.5 when Dab2 is expressed in the primitive endoderm[17]. At this implantation stage, dab2 (-/-) embryos were identified by immunostaining for the absence of Dab2 protein (Figure 2). In wildtype or heterozygous blastocysts, the Gata4-, Gata6-, and Dab2-positive primitive endoderm cells exclusively located on the surface layer covering the Oct3/4-positive inner cell mass. Among 26 E4.5 embryos found following sectioning of 6 uteri, 4 implanted blastocysts were confirmed as Dab2- based on immunostaining. In the Dab2 knockout embryos, although Gata4- and Gata6-positive primitive endoderm cells were present, the cells were not organized into a monolayer epithelium and some located deep inside the inner cell mass (Figure 2A). Thus the embryonic defect is initiated at E4.5, post differentiation but prior to morphogenesis of the primitive endoderm, although the phenotype is subtle at this stage.

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