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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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Proximity of lens marker and Pax6-GFP or Six3-GFP expressing mES cells.(A–F) mES cell cultures transduced with Pax6-GFP under the constitutive E1a promoter show close proximity but generally non-overlapping expression of GFP with γA-crystallin (A–C) or Tdrd7 (D–F) at 21 days. (G–I) Similar results were obtained for E1a driven Six3-GFP transduction and γA-crystallin expression. These results suggest recruitment of undifferentiated mES cells to a lens fate by Pax6- or Six3-expressing cells. Scale bar: A–I 30 µm.
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pone-0115106-g005: Proximity of lens marker and Pax6-GFP or Six3-GFP expressing mES cells.(A–F) mES cell cultures transduced with Pax6-GFP under the constitutive E1a promoter show close proximity but generally non-overlapping expression of GFP with γA-crystallin (A–C) or Tdrd7 (D–F) at 21 days. (G–I) Similar results were obtained for E1a driven Six3-GFP transduction and γA-crystallin expression. These results suggest recruitment of undifferentiated mES cells to a lens fate by Pax6- or Six3-expressing cells. Scale bar: A–I 30 µm.

Mentions: Immunohistochemical analyses of G4 mES cell cultures transduced with Pax6- or Six3-GFP expressing lentivirus suggested that cells expressing either Pax6 or Six3 appeared to induce their neighbors to enter the lens differentiation pathway, but not necessarily themselves. For example, cells that expressed the Pax6-GFP vector localized alongside of but were distinct from cells that expressed γA-crystallin and Tdrd7 (Fig. 5A–F). Similarly, when Six3-GFP was expressed in G4 mES cells at 21 days post-infection, Six3 GFP expressing cells were often found close to γA-crystallin positive cells, but the two markers only rarely co-localized to the same cell (Fig. 5G–I).


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)

Proximity of lens marker and Pax6-GFP or Six3-GFP expressing mES cells.(A–F) mES cell cultures transduced with Pax6-GFP under the constitutive E1a promoter show close proximity but generally non-overlapping expression of GFP with γA-crystallin (A–C) or Tdrd7 (D–F) at 21 days. (G–I) Similar results were obtained for E1a driven Six3-GFP transduction and γA-crystallin expression. These results suggest recruitment of undifferentiated mES cells to a lens fate by Pax6- or Six3-expressing cells. Scale bar: A–I 30 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0115106-g005: Proximity of lens marker and Pax6-GFP or Six3-GFP expressing mES cells.(A–F) mES cell cultures transduced with Pax6-GFP under the constitutive E1a promoter show close proximity but generally non-overlapping expression of GFP with γA-crystallin (A–C) or Tdrd7 (D–F) at 21 days. (G–I) Similar results were obtained for E1a driven Six3-GFP transduction and γA-crystallin expression. These results suggest recruitment of undifferentiated mES cells to a lens fate by Pax6- or Six3-expressing cells. Scale bar: A–I 30 µm.
Mentions: Immunohistochemical analyses of G4 mES cell cultures transduced with Pax6- or Six3-GFP expressing lentivirus suggested that cells expressing either Pax6 or Six3 appeared to induce their neighbors to enter the lens differentiation pathway, but not necessarily themselves. For example, cells that expressed the Pax6-GFP vector localized alongside of but were distinct from cells that expressed γA-crystallin and Tdrd7 (Fig. 5A–F). Similarly, when Six3-GFP was expressed in G4 mES cells at 21 days post-infection, Six3 GFP expressing cells were often found close to γA-crystallin positive cells, but the two markers only rarely co-localized to the same cell (Fig. 5G–I).

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