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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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Injection of tamoxifen post-E7.5 results in epithelial-specific expression of β-galactosidase. In all images, epithelia is outlined in black dashed lines. A–D: In the E15.5 lung, injection at E5.5 (A) or E6.5 (B) labels mesenchymal cells, yet injections at E7.5 and later, shown here at E11.5 (C) or E12.5 (D), only label epithelia. Stage of harvesting: (A,B), E12.5; (C,D), E15.5. E,F: In the E12.5 pancreas, injection at E6.5 (E) leads to strong staining in the epithelia as well as in some scattered mesenchyme. Injection at E7.5 (F) is restricted to the epithelia. No mesenchymal staining was seen in any section examined. G,H: In the E15.5 kidney, injection at E11.5 (G) and E12.5 (H) are shown. All magnification at 10× except F at 20×. inj., injection.
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fig05: Injection of tamoxifen post-E7.5 results in epithelial-specific expression of β-galactosidase. In all images, epithelia is outlined in black dashed lines. A–D: In the E15.5 lung, injection at E5.5 (A) or E6.5 (B) labels mesenchymal cells, yet injections at E7.5 and later, shown here at E11.5 (C) or E12.5 (D), only label epithelia. Stage of harvesting: (A,B), E12.5; (C,D), E15.5. E,F: In the E12.5 pancreas, injection at E6.5 (E) leads to strong staining in the epithelia as well as in some scattered mesenchyme. Injection at E7.5 (F) is restricted to the epithelia. No mesenchymal staining was seen in any section examined. G,H: In the E15.5 kidney, injection at E11.5 (G) and E12.5 (H) are shown. All magnification at 10× except F at 20×. inj., injection.

Mentions: Tamoxifen injection to pregnant females at E5.5 labels cells throughout the embryo, both in the epithelia and mesenchyme (Figs. 4A–C, 5A). This is consistent with the ubiquitous expression of Cldn6 at the time Cre recombinase would be most active (roughly E6.0–7.0). Based on the composite data, it appears E7.5 is the best time point for tamoxifen administration (Fig. 4D–F) if one aims to mostly label definitive endoderm and its derivatives without ubiquitous labeling of the embryo. From this stage forward, Cre-mediated recombination is detected in epithelia including all endoderm-derived organs (Figs. 4D,E,G–J, 5C,F), the kidney (Figs. 4G,K, 5G,H), olfactory epithelia, lens epithelia, and semicircular canal (data not shown). Cells that appear to be melanocytes also label (Fig. 4C,F,L), although Cldn6 expression in these cells is too low to visualize by in situ hybridization. Based on the kinetics of tamoxifen, E7.5 would be the optimal stage for misexpression in definitive endoderm as Cre recombinase would be active around the time the endoderm is patterned and before organogenesis begins. Of note, given that the degree of Cre-mediated recombination is dependent upon the dose of tamoxifen administered, not all Cldn6-expressing cells undergo recombination at the dose used here.Fig. 4


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)

Injection of tamoxifen post-E7.5 results in epithelial-specific expression of β-galactosidase. In all images, epithelia is outlined in black dashed lines. A–D: In the E15.5 lung, injection at E5.5 (A) or E6.5 (B) labels mesenchymal cells, yet injections at E7.5 and later, shown here at E11.5 (C) or E12.5 (D), only label epithelia. Stage of harvesting: (A,B), E12.5; (C,D), E15.5. E,F: In the E12.5 pancreas, injection at E6.5 (E) leads to strong staining in the epithelia as well as in some scattered mesenchyme. Injection at E7.5 (F) is restricted to the epithelia. No mesenchymal staining was seen in any section examined. G,H: In the E15.5 kidney, injection at E11.5 (G) and E12.5 (H) are shown. All magnification at 10× except F at 20×. inj., injection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2665265&req=5

fig05: Injection of tamoxifen post-E7.5 results in epithelial-specific expression of β-galactosidase. In all images, epithelia is outlined in black dashed lines. A–D: In the E15.5 lung, injection at E5.5 (A) or E6.5 (B) labels mesenchymal cells, yet injections at E7.5 and later, shown here at E11.5 (C) or E12.5 (D), only label epithelia. Stage of harvesting: (A,B), E12.5; (C,D), E15.5. E,F: In the E12.5 pancreas, injection at E6.5 (E) leads to strong staining in the epithelia as well as in some scattered mesenchyme. Injection at E7.5 (F) is restricted to the epithelia. No mesenchymal staining was seen in any section examined. G,H: In the E15.5 kidney, injection at E11.5 (G) and E12.5 (H) are shown. All magnification at 10× except F at 20×. inj., injection.
Mentions: Tamoxifen injection to pregnant females at E5.5 labels cells throughout the embryo, both in the epithelia and mesenchyme (Figs. 4A–C, 5A). This is consistent with the ubiquitous expression of Cldn6 at the time Cre recombinase would be most active (roughly E6.0–7.0). Based on the composite data, it appears E7.5 is the best time point for tamoxifen administration (Fig. 4D–F) if one aims to mostly label definitive endoderm and its derivatives without ubiquitous labeling of the embryo. From this stage forward, Cre-mediated recombination is detected in epithelia including all endoderm-derived organs (Figs. 4D,E,G–J, 5C,F), the kidney (Figs. 4G,K, 5G,H), olfactory epithelia, lens epithelia, and semicircular canal (data not shown). Cells that appear to be melanocytes also label (Fig. 4C,F,L), although Cldn6 expression in these cells is too low to visualize by in situ hybridization. Based on the kinetics of tamoxifen, E7.5 would be the optimal stage for misexpression in definitive endoderm as Cre recombinase would be active around the time the endoderm is patterned and before organogenesis begins. Of note, given that the degree of Cre-mediated recombination is dependent upon the dose of tamoxifen administered, not all Cldn6-expressing cells undergo recombination at the dose used here.Fig. 4

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