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Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Anchan RM, Lachke SA, Gerami-Naini B, Lindsey J, Ng N, Naber C, Nickerson M, Cavallesco R, Rowan S, Eaton JL, Xi Q, Maas RL - PLoS ONE (2014)

Bottom Line: Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture.In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms.These findings should facilitate investigations of lens development.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.

ABSTRACT
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

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Pax6 or Six3 expression in G4 mESC cells induces γA-crystallin expression.(A–B) Control mESC cultures transfected with vector alone. (C–F) G4 mES cells transfected with (C–D) Pax6 or (E–F) Six3 expression plasmids demonstrate γA-crystallin immunoreactivity by day 7 post-transfection. (G) The number of γA-crystallin immunoreactive colonies following Pax6- or Six3-transfection is>10-fold more than for vector controls. Scale bar: A–F 200 µm.
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pone-0115106-g002: Pax6 or Six3 expression in G4 mESC cells induces γA-crystallin expression.(A–B) Control mESC cultures transfected with vector alone. (C–F) G4 mES cells transfected with (C–D) Pax6 or (E–F) Six3 expression plasmids demonstrate γA-crystallin immunoreactivity by day 7 post-transfection. (G) The number of γA-crystallin immunoreactive colonies following Pax6- or Six3-transfection is>10-fold more than for vector controls. Scale bar: A–F 200 µm.

Mentions: Introduction of Pax6 or Six3 into G4 mES cells via expression plasmid resulted in a>10-fold increase in the percentage of γA-crystallin immunoreactive mES cell colonies by 7 days post-treatment compared to G4 mES cells transfected with control plasmids at similar efficiency (Fig. 2A–G). Similar results were obtained with lentiviral vector transduction (data not shown).


Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Anchan RM, Lachke SA, Gerami-Naini B, Lindsey J, Ng N, Naber C, Nickerson M, Cavallesco R, Rowan S, Eaton JL, Xi Q, Maas RL - PLoS ONE (2014)

Pax6 or Six3 expression in G4 mESC cells induces γA-crystallin expression.(A–B) Control mESC cultures transfected with vector alone. (C–F) G4 mES cells transfected with (C–D) Pax6 or (E–F) Six3 expression plasmids demonstrate γA-crystallin immunoreactivity by day 7 post-transfection. (G) The number of γA-crystallin immunoreactive colonies following Pax6- or Six3-transfection is>10-fold more than for vector controls. Scale bar: A–F 200 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269389&req=5

pone-0115106-g002: Pax6 or Six3 expression in G4 mESC cells induces γA-crystallin expression.(A–B) Control mESC cultures transfected with vector alone. (C–F) G4 mES cells transfected with (C–D) Pax6 or (E–F) Six3 expression plasmids demonstrate γA-crystallin immunoreactivity by day 7 post-transfection. (G) The number of γA-crystallin immunoreactive colonies following Pax6- or Six3-transfection is>10-fold more than for vector controls. Scale bar: A–F 200 µm.
Mentions: Introduction of Pax6 or Six3 into G4 mES cells via expression plasmid resulted in a>10-fold increase in the percentage of γA-crystallin immunoreactive mES cell colonies by 7 days post-treatment compared to G4 mES cells transfected with control plasmids at similar efficiency (Fig. 2A–G). Similar results were obtained with lentiviral vector transduction (data not shown).

Bottom Line: Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture.In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms.These findings should facilitate investigations of lens development.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.

ABSTRACT
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

Show MeSH
Related in: MedlinePlus