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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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Lineage analysis of Cldn6+ cells at different times in embryonic development. Tamoxifen injection at E5.5 shows widespread labeling throughout the embryo (A–C). Tamoxifen injection at E7.5 and later shows epithelial-restricted expression of β-galactosidase in definitive endoderm derivatives (D,E, G–J). Epithelia of the kidney is also labeled (G, K). Melanocytes also label (C, F, scattered cells in L), with a decrease in labeling correlating to later times of tamoxifen injection. Embryos administered tamoxifen at E5.5 (A–C) and E7.5 (D–F) were harvested at E12.5 for analysis. Embryos administered tamoxifen at E11.5 (G–L) were harvested at E15.5 for analysis. panc, pancreas; lu, lung; liv, liver; st, stomach; kid, kidney; int, intestine.
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fig04: Lineage analysis of Cldn6+ cells at different times in embryonic development. Tamoxifen injection at E5.5 shows widespread labeling throughout the embryo (A–C). Tamoxifen injection at E7.5 and later shows epithelial-restricted expression of β-galactosidase in definitive endoderm derivatives (D,E, G–J). Epithelia of the kidney is also labeled (G, K). Melanocytes also label (C, F, scattered cells in L), with a decrease in labeling correlating to later times of tamoxifen injection. Embryos administered tamoxifen at E5.5 (A–C) and E7.5 (D–F) were harvested at E12.5 for analysis. Embryos administered tamoxifen at E11.5 (G–L) were harvested at E15.5 for analysis. panc, pancreas; lu, lung; liv, liver; st, stomach; kid, kidney; int, intestine.

Mentions: We experimented with tamoxifen dosage as was done previously (Hayashi and McMahon, 2002) and found that a 4-mg injection of tamoxifen to pregnant females provided maximum labeling of embryos without significant embryonic loss (between 0–2 embryos resorbed per litter). We then performed a time course of tamoxifen injection during embryonic development, which is schematized in Supplemental Figure 4B. Four milligram tamoxifen injections were administered to pregnant females between E5.5 and E12.5, with embryos whose mothers were injected between E5.5 and E10.5, harvested on E12.5, and those whose mothers were injected on E11.5 and E12.5, harvested on E15.5. Selected whole mount images are shown in Figure 4. At all stages examined, neither Cre-independent β-galactosidase expression nor tamoxifen-independent β-galactosidase expression was detected (data not shown). Embryos at each stage of injection were sectioned post-staining to better visualize localization of the signal to the epithelial or mesenchymal compartments (data not shown).


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)

Lineage analysis of Cldn6+ cells at different times in embryonic development. Tamoxifen injection at E5.5 shows widespread labeling throughout the embryo (A–C). Tamoxifen injection at E7.5 and later shows epithelial-restricted expression of β-galactosidase in definitive endoderm derivatives (D,E, G–J). Epithelia of the kidney is also labeled (G, K). Melanocytes also label (C, F, scattered cells in L), with a decrease in labeling correlating to later times of tamoxifen injection. Embryos administered tamoxifen at E5.5 (A–C) and E7.5 (D–F) were harvested at E12.5 for analysis. Embryos administered tamoxifen at E11.5 (G–L) were harvested at E15.5 for analysis. panc, pancreas; lu, lung; liv, liver; st, stomach; kid, kidney; int, intestine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2665265&req=5

fig04: Lineage analysis of Cldn6+ cells at different times in embryonic development. Tamoxifen injection at E5.5 shows widespread labeling throughout the embryo (A–C). Tamoxifen injection at E7.5 and later shows epithelial-restricted expression of β-galactosidase in definitive endoderm derivatives (D,E, G–J). Epithelia of the kidney is also labeled (G, K). Melanocytes also label (C, F, scattered cells in L), with a decrease in labeling correlating to later times of tamoxifen injection. Embryos administered tamoxifen at E5.5 (A–C) and E7.5 (D–F) were harvested at E12.5 for analysis. Embryos administered tamoxifen at E11.5 (G–L) were harvested at E15.5 for analysis. panc, pancreas; lu, lung; liv, liver; st, stomach; kid, kidney; int, intestine.
Mentions: We experimented with tamoxifen dosage as was done previously (Hayashi and McMahon, 2002) and found that a 4-mg injection of tamoxifen to pregnant females provided maximum labeling of embryos without significant embryonic loss (between 0–2 embryos resorbed per litter). We then performed a time course of tamoxifen injection during embryonic development, which is schematized in Supplemental Figure 4B. Four milligram tamoxifen injections were administered to pregnant females between E5.5 and E12.5, with embryos whose mothers were injected between E5.5 and E10.5, harvested on E12.5, and those whose mothers were injected on E11.5 and E12.5, harvested on E15.5. Selected whole mount images are shown in Figure 4. At all stages examined, neither Cre-independent β-galactosidase expression nor tamoxifen-independent β-galactosidase expression was detected (data not shown). Embryos at each stage of injection were sectioned post-staining to better visualize localization of the signal to the epithelial or mesenchymal compartments (data not shown).

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