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Genetic targeting of the endoderm with claudin-6CreER.

Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA - Dev. Dyn. (2008)

Bottom Line: The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning.To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette.We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, USA.

ABSTRACT
A full description of the ontogeny of the beta cell would guide efforts to generate beta cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette. Cldn6 mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

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Expression of Cldn6 from commencement of gastrulation to the early stages of gut tube organogenesis. A,D: Expression at E6.5 is quite broad throughout the epiblast, with the exception of the primitive streak and visceral endoderm. Similar expression is seen at E7.5 (B,E). C,F: By E8.5, expression begins to be restricted to the endoderm and by E9.5 (G) and E10.5 (H), expression is restricted to the endoderm, otic vesicle (arrowhead), and mesonephros (arrow). A–C are representative whole mount stainings for the sections in D–F), respectively. Embryos in D–F were sectioned in their deciduas. Plane of section is indicated by a line in the corresponding whole mount embryo. For E, posterior is facing up. dec, decidua; ect, ectoderm; mes, mesoderm; end, endoderm; rm, Reichert's membrane; nf, neural fold; he, heart.
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fig01: Expression of Cldn6 from commencement of gastrulation to the early stages of gut tube organogenesis. A,D: Expression at E6.5 is quite broad throughout the epiblast, with the exception of the primitive streak and visceral endoderm. Similar expression is seen at E7.5 (B,E). C,F: By E8.5, expression begins to be restricted to the endoderm and by E9.5 (G) and E10.5 (H), expression is restricted to the endoderm, otic vesicle (arrowhead), and mesonephros (arrow). A–C are representative whole mount stainings for the sections in D–F), respectively. Embryos in D–F were sectioned in their deciduas. Plane of section is indicated by a line in the corresponding whole mount embryo. For E, posterior is facing up. dec, decidua; ect, ectoderm; mes, mesoderm; end, endoderm; rm, Reichert's membrane; nf, neural fold; he, heart.

Mentions: We examined expression of Cldn6 by in situ hybridization, in sections and whole mounts, from gastrulation to the initial stages of organogenesis (Fig. 1). At E6.5, Cldn6 is broadly expressed throughout the epiblast as well as the hypoblast. It is absent in the primitive streak (Fig. 1A) and in some visceral endoderm surrounding the epiblast (Fig. 1A,D). This lack of expression in the primitive streak is not surprising, as cells undergo an epithelial-to-mesenchymal transition (EMT) as they migrate to form mesoderm and definitive endoderm, thus dissolving adherens and tight junctions (Solursh and Revel, 1978; Tam et al., 1993; Cano et al., 2000; Batlle et al., 2000; Zohn et al., 2006; Ikenouchi et al., 2003; Ohkubo and Ozawa, 2004). At E7.5, expression is still evident throughout most of the epiblast (Fig. 1B,E). The primitive streak and nascent mesoderm are devoid of Cldn6 expression (Fig. 1E). By E8.5, expression begins to become restricted to the definitive endoderm (Fig. 1C,F). This is the stage when initial patterning of the gut tube along the anterior-posterior axis is thought to occur. At E9.5, intense expression is seen in the entire gut, otic vesicles, and a small region of the forebrain (Fig. 1G). This expression pattern at E9.5 is comparable to what is reported by others (Gitton et al., 2002; Sousa-Nunes et al., 2003). At E10.5, expression is similar to E9.5, but staining in the mesonephros and forebrain is now more apparent (Fig. 1H).Fig. 1


Genetic targeting of the endoderm with claudin-6CreER.

Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA - Dev. Dyn. (2008)

Expression of Cldn6 from commencement of gastrulation to the early stages of gut tube organogenesis. A,D: Expression at E6.5 is quite broad throughout the epiblast, with the exception of the primitive streak and visceral endoderm. Similar expression is seen at E7.5 (B,E). C,F: By E8.5, expression begins to be restricted to the endoderm and by E9.5 (G) and E10.5 (H), expression is restricted to the endoderm, otic vesicle (arrowhead), and mesonephros (arrow). A–C are representative whole mount stainings for the sections in D–F), respectively. Embryos in D–F were sectioned in their deciduas. Plane of section is indicated by a line in the corresponding whole mount embryo. For E, posterior is facing up. dec, decidua; ect, ectoderm; mes, mesoderm; end, endoderm; rm, Reichert's membrane; nf, neural fold; he, heart.
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Related In: Results  -  Collection

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fig01: Expression of Cldn6 from commencement of gastrulation to the early stages of gut tube organogenesis. A,D: Expression at E6.5 is quite broad throughout the epiblast, with the exception of the primitive streak and visceral endoderm. Similar expression is seen at E7.5 (B,E). C,F: By E8.5, expression begins to be restricted to the endoderm and by E9.5 (G) and E10.5 (H), expression is restricted to the endoderm, otic vesicle (arrowhead), and mesonephros (arrow). A–C are representative whole mount stainings for the sections in D–F), respectively. Embryos in D–F were sectioned in their deciduas. Plane of section is indicated by a line in the corresponding whole mount embryo. For E, posterior is facing up. dec, decidua; ect, ectoderm; mes, mesoderm; end, endoderm; rm, Reichert's membrane; nf, neural fold; he, heart.
Mentions: We examined expression of Cldn6 by in situ hybridization, in sections and whole mounts, from gastrulation to the initial stages of organogenesis (Fig. 1). At E6.5, Cldn6 is broadly expressed throughout the epiblast as well as the hypoblast. It is absent in the primitive streak (Fig. 1A) and in some visceral endoderm surrounding the epiblast (Fig. 1A,D). This lack of expression in the primitive streak is not surprising, as cells undergo an epithelial-to-mesenchymal transition (EMT) as they migrate to form mesoderm and definitive endoderm, thus dissolving adherens and tight junctions (Solursh and Revel, 1978; Tam et al., 1993; Cano et al., 2000; Batlle et al., 2000; Zohn et al., 2006; Ikenouchi et al., 2003; Ohkubo and Ozawa, 2004). At E7.5, expression is still evident throughout most of the epiblast (Fig. 1B,E). The primitive streak and nascent mesoderm are devoid of Cldn6 expression (Fig. 1E). By E8.5, expression begins to become restricted to the definitive endoderm (Fig. 1C,F). This is the stage when initial patterning of the gut tube along the anterior-posterior axis is thought to occur. At E9.5, intense expression is seen in the entire gut, otic vesicles, and a small region of the forebrain (Fig. 1G). This expression pattern at E9.5 is comparable to what is reported by others (Gitton et al., 2002; Sousa-Nunes et al., 2003). At E10.5, expression is similar to E9.5, but staining in the mesonephros and forebrain is now more apparent (Fig. 1H).Fig. 1

Bottom Line: The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning.To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette.We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts 02138, USA.

ABSTRACT
A full description of the ontogeny of the beta cell would guide efforts to generate beta cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette. Cldn6 mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver.

Show MeSH
Related in: MedlinePlus