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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 H1 hES cells induces lens marker expression.(A–F) H1 hES cells transduced by Pax6 lentiviral vector express (A–C) Prox1 in partly overlapping fashion (C) by 14 days post transduction. (D–F) By 24 days post-transduction, (D) γA-crystallin and (E) Tdrd7 are expressed, the latter as cytoplasmic granules. Similar results were obtained following Six3 transduction (not shown). (G) RT-PCR confirms induction of lens marker gene expression in Pax6- or Six3-transduced H1 hES cells. Scale bars: A–C 150 µm; D–F 50 µm.
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pone-0115106-g004: Pax6 or Six3 expression in H1 hES cells induces lens marker expression.(A–F) H1 hES cells transduced by Pax6 lentiviral vector express (A–C) Prox1 in partly overlapping fashion (C) by 14 days post transduction. (D–F) By 24 days post-transduction, (D) γA-crystallin and (E) Tdrd7 are expressed, the latter as cytoplasmic granules. Similar results were obtained following Six3 transduction (not shown). (G) RT-PCR confirms induction of lens marker gene expression in Pax6- or Six3-transduced H1 hES cells. Scale bars: A–C 150 µm; D–F 50 µm.

Mentions: To study whether Pax6 or Six3 could induce lens cell fate in human hES cells, we used lentiviral vectors to introduce either mouse Pax6 or Six3 into human H1 ES cells. By 14 and 24 days post-infection, Pax6 lentivirus-infected H1 hES cells exhibited expression of γA-crystallin, Prox1, and Tdrd7 (Fig. 4A–F), similar to expression of the homologous mouse proteins in G4 and Pax6-GFP mES cells. Prox1 expression was detected in Pax6 transduced hES cells (Fig. 4A–C), and so was Tdrd7, which overlapped with a subset of γA-crystallin expressing cells (Fig. 4D–F). Note that depending on the type of lens cells Prox1 staining is observed in both nucleus and cytoplasm [41]. In cells of the lens placode, epithelium and the germinative zone, Prox1 is predominantly cytoplasmic, while in differentiating fiber cells it is predominantly nuclear. It is possible that Pax6 lentivirus-infected H1 hES cells are in the process of differentiation and therefore exhibit staining in both locations. Similar results were obtained with H1 hES cells transduced with a Six3 expressing lentiviral vector (data not shown), and lens marker results were also confirmed by RT-PCR (Fig. 4G).


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 H1 hES cells induces lens marker expression.(A–F) H1 hES cells transduced by Pax6 lentiviral vector express (A–C) Prox1 in partly overlapping fashion (C) by 14 days post transduction. (D–F) By 24 days post-transduction, (D) γA-crystallin and (E) Tdrd7 are expressed, the latter as cytoplasmic granules. Similar results were obtained following Six3 transduction (not shown). (G) RT-PCR confirms induction of lens marker gene expression in Pax6- or Six3-transduced H1 hES cells. Scale bars: A–C 150 µm; D–F 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0115106-g004: Pax6 or Six3 expression in H1 hES cells induces lens marker expression.(A–F) H1 hES cells transduced by Pax6 lentiviral vector express (A–C) Prox1 in partly overlapping fashion (C) by 14 days post transduction. (D–F) By 24 days post-transduction, (D) γA-crystallin and (E) Tdrd7 are expressed, the latter as cytoplasmic granules. Similar results were obtained following Six3 transduction (not shown). (G) RT-PCR confirms induction of lens marker gene expression in Pax6- or Six3-transduced H1 hES cells. Scale bars: A–C 150 µm; D–F 50 µm.
Mentions: To study whether Pax6 or Six3 could induce lens cell fate in human hES cells, we used lentiviral vectors to introduce either mouse Pax6 or Six3 into human H1 ES cells. By 14 and 24 days post-infection, Pax6 lentivirus-infected H1 hES cells exhibited expression of γA-crystallin, Prox1, and Tdrd7 (Fig. 4A–F), similar to expression of the homologous mouse proteins in G4 and Pax6-GFP mES cells. Prox1 expression was detected in Pax6 transduced hES cells (Fig. 4A–C), and so was Tdrd7, which overlapped with a subset of γA-crystallin expressing cells (Fig. 4D–F). Note that depending on the type of lens cells Prox1 staining is observed in both nucleus and cytoplasm [41]. In cells of the lens placode, epithelium and the germinative zone, Prox1 is predominantly cytoplasmic, while in differentiating fiber cells it is predominantly nuclear. It is possible that Pax6 lentivirus-infected H1 hES cells are in the process of differentiation and therefore exhibit staining in both locations. Similar results were obtained with H1 hES cells transduced with a Six3 expressing lentiviral vector (data not shown), and lens marker results were also confirmed by RT-PCR (Fig. 4G).

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