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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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Epithelial-restricted expression of Cldn6 in the developing lung and pancreas. A–D: Whole mount in situ hybridization shows Cldn6 expression throughout the entire epithelia of the lung from E11.5 to E14.5 [(A) E11.5, (B) E12.5, (C) E13.5, (D) E14.5)]. E,F: Expression of Cldn6 in the E14.5 lung is restricted to the epithelia with no mesenchymal expression. E: Expression of Cldn6 can be seen in the tracheal epithelia, as well as both the proximal and distal airways. F: A higher magnification view of staining from a different lung. Epithelia is outlined by a black dashed line. G: Expression of Cldn6 in the E14.5 pancreas. As with the lung, expression is restricted to the epithelia. Staining in the epithelial lining of the stomach can be seen faintly. H: Cldn6 expression in the E13.5 pancreas. The pancreas is still attached to the stomach. Strong epithelial staining can be seen in the branching epithelia. panc, pancreas; st, stomach.
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fig02: Epithelial-restricted expression of Cldn6 in the developing lung and pancreas. A–D: Whole mount in situ hybridization shows Cldn6 expression throughout the entire epithelia of the lung from E11.5 to E14.5 [(A) E11.5, (B) E12.5, (C) E13.5, (D) E14.5)]. E,F: Expression of Cldn6 in the E14.5 lung is restricted to the epithelia with no mesenchymal expression. E: Expression of Cldn6 can be seen in the tracheal epithelia, as well as both the proximal and distal airways. F: A higher magnification view of staining from a different lung. Epithelia is outlined by a black dashed line. G: Expression of Cldn6 in the E14.5 pancreas. As with the lung, expression is restricted to the epithelia. Staining in the epithelial lining of the stomach can be seen faintly. H: Cldn6 expression in the E13.5 pancreas. The pancreas is still attached to the stomach. Strong epithelial staining can be seen in the branching epithelia. panc, pancreas; st, stomach.

Mentions: To examine whether Cldn6 maintains epithelial expression in endoderm-derived organs, we performed whole mount and section in situ hybridization on embryonic lung and pancreas. Cldn6 is strongly expressed in the epithelia of both organs (Fig. 2A–F, 2G–H, pancreas). On sections, no staining in the mesenchyme is evident (Fig. 2E,F,H).Fig. 2


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)

Epithelial-restricted expression of Cldn6 in the developing lung and pancreas. A–D: Whole mount in situ hybridization shows Cldn6 expression throughout the entire epithelia of the lung from E11.5 to E14.5 [(A) E11.5, (B) E12.5, (C) E13.5, (D) E14.5)]. E,F: Expression of Cldn6 in the E14.5 lung is restricted to the epithelia with no mesenchymal expression. E: Expression of Cldn6 can be seen in the tracheal epithelia, as well as both the proximal and distal airways. F: A higher magnification view of staining from a different lung. Epithelia is outlined by a black dashed line. G: Expression of Cldn6 in the E14.5 pancreas. As with the lung, expression is restricted to the epithelia. Staining in the epithelial lining of the stomach can be seen faintly. H: Cldn6 expression in the E13.5 pancreas. The pancreas is still attached to the stomach. Strong epithelial staining can be seen in the branching epithelia. panc, pancreas; st, stomach.
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Related In: Results  -  Collection

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fig02: Epithelial-restricted expression of Cldn6 in the developing lung and pancreas. A–D: Whole mount in situ hybridization shows Cldn6 expression throughout the entire epithelia of the lung from E11.5 to E14.5 [(A) E11.5, (B) E12.5, (C) E13.5, (D) E14.5)]. E,F: Expression of Cldn6 in the E14.5 lung is restricted to the epithelia with no mesenchymal expression. E: Expression of Cldn6 can be seen in the tracheal epithelia, as well as both the proximal and distal airways. F: A higher magnification view of staining from a different lung. Epithelia is outlined by a black dashed line. G: Expression of Cldn6 in the E14.5 pancreas. As with the lung, expression is restricted to the epithelia. Staining in the epithelial lining of the stomach can be seen faintly. H: Cldn6 expression in the E13.5 pancreas. The pancreas is still attached to the stomach. Strong epithelial staining can be seen in the branching epithelia. panc, pancreas; st, stomach.
Mentions: To examine whether Cldn6 maintains epithelial expression in endoderm-derived organs, we performed whole mount and section in situ hybridization on embryonic lung and pancreas. Cldn6 is strongly expressed in the epithelia of both organs (Fig. 2A–F, 2G–H, pancreas). On sections, no staining in the mesenchyme is evident (Fig. 2E,F,H).Fig. 2

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