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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 lens marker expression.(A–F) G4 mES cells transfected with either (A–D) Pax6 or (E,F) Six3 expression plasmids exhibit γA-crystallin (A,E) and Prox1 (B,F) expression at day 7. Pax6-transfection also results in expression of (C) αB-crystallin, and (D) Tdrd7. (G) Expression of lens markers in Pax6- and Six3-transfected G4 mESC colonies confirmed by RT-PCR. (H–K) In some cases, γA-crystallin positive mES cells accumulate in aggregates at days 7–14, with further expansion into lentoid bodies at 30 days (J, phase; K, γA-crystallin immunofluorscence). Scale bars: A 75 µm; B–F 50 µm; H–I 25 µm; J–K 50 µm.
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pone-0115106-g003: Pax6 or Six3 expression in G4 mESC cells induces lens marker expression.(A–F) G4 mES cells transfected with either (A–D) Pax6 or (E,F) Six3 expression plasmids exhibit γA-crystallin (A,E) and Prox1 (B,F) expression at day 7. Pax6-transfection also results in expression of (C) αB-crystallin, and (D) Tdrd7. (G) Expression of lens markers in Pax6- and Six3-transfected G4 mESC colonies confirmed by RT-PCR. (H–K) In some cases, γA-crystallin positive mES cells accumulate in aggregates at days 7–14, with further expansion into lentoid bodies at 30 days (J, phase; K, γA-crystallin immunofluorscence). Scale bars: A 75 µm; B–F 50 µm; H–I 25 µm; J–K 50 µm.

Mentions: After 14 days of culture post-transfection, γA-crystallin expression continued to be detected in these ES cells, mainly in the central portions of individual colonies (Fig. 3A,E). We also detected expression of other lens fiber cell differentiation markers, Prox1, αB-crystallin, and Tdrd7 expression by immunostaining in these cultures at this stage whereas control vectors (LvHPV422 and LvHPV570) and vectors encoding either of two other genes, Eya1, Ctnnb (encoding β-catenin), gave negligible staining (Fig. 3B–D,F, and data not shown). These data suggest that Pax6 and Six3 can induce markers of lens fiber cell fate in mES cells. Tdrd7 expression was observed to be partly granular (Fig. 3D), as previously described in lens fiber cells [37]. RT-PCR analyses confirmed the transcript expression of these and other lens expressed genes (Fig. 3G). Thus, expression of either Pax6 or Six3 induces the expression of lens fiber cell markers.


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 lens marker expression.(A–F) G4 mES cells transfected with either (A–D) Pax6 or (E,F) Six3 expression plasmids exhibit γA-crystallin (A,E) and Prox1 (B,F) expression at day 7. Pax6-transfection also results in expression of (C) αB-crystallin, and (D) Tdrd7. (G) Expression of lens markers in Pax6- and Six3-transfected G4 mESC colonies confirmed by RT-PCR. (H–K) In some cases, γA-crystallin positive mES cells accumulate in aggregates at days 7–14, with further expansion into lentoid bodies at 30 days (J, phase; K, γA-crystallin immunofluorscence). Scale bars: A 75 µm; B–F 50 µm; H–I 25 µm; J–K 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0115106-g003: Pax6 or Six3 expression in G4 mESC cells induces lens marker expression.(A–F) G4 mES cells transfected with either (A–D) Pax6 or (E,F) Six3 expression plasmids exhibit γA-crystallin (A,E) and Prox1 (B,F) expression at day 7. Pax6-transfection also results in expression of (C) αB-crystallin, and (D) Tdrd7. (G) Expression of lens markers in Pax6- and Six3-transfected G4 mESC colonies confirmed by RT-PCR. (H–K) In some cases, γA-crystallin positive mES cells accumulate in aggregates at days 7–14, with further expansion into lentoid bodies at 30 days (J, phase; K, γA-crystallin immunofluorscence). Scale bars: A 75 µm; B–F 50 µm; H–I 25 µm; J–K 50 µm.
Mentions: After 14 days of culture post-transfection, γA-crystallin expression continued to be detected in these ES cells, mainly in the central portions of individual colonies (Fig. 3A,E). We also detected expression of other lens fiber cell differentiation markers, Prox1, αB-crystallin, and Tdrd7 expression by immunostaining in these cultures at this stage whereas control vectors (LvHPV422 and LvHPV570) and vectors encoding either of two other genes, Eya1, Ctnnb (encoding β-catenin), gave negligible staining (Fig. 3B–D,F, and data not shown). These data suggest that Pax6 and Six3 can induce markers of lens fiber cell fate in mES cells. Tdrd7 expression was observed to be partly granular (Fig. 3D), as previously described in lens fiber cells [37]. RT-PCR analyses confirmed the transcript expression of these and other lens expressed genes (Fig. 3G). Thus, expression of either Pax6 or Six3 induces the expression of lens fiber cell markers.

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