title: Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.
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.
authors.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.
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Anchan
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America; Department of Biological Sciences, Center for Bioinformatics and Computational Biology, University of Delaware, Newark, Delaware, 9716, United States of America.
authors.forename: Salil A
authors.initials: SA
authors.lastname: Lachke
authors.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.
authors.forename: Behzad
authors.initials: B
authors.lastname: Gerami-Naini
authors.affiliation: 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.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lindsey
authors.affiliation: 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.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ng
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Catherine
authors.initials: C
authors.lastname: Naber
authors.affiliation: 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.
authors.forename: Michael
authors.initials: M
authors.lastname: Nickerson
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Resy
authors.initials: R
authors.lastname: Cavallesco
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Sheldon
authors.initials: S
authors.lastname: Rowan
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Eaton
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Qiongchao
authors.initials: Q
authors.lastname: Xi
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Maas
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000502:physiology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097607:Homeobox Protein SIX3
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012333:RNA, Messenger; D012097:Repressor Proteins; D000097607:Homeobox Protein SIX3; C000605730:PAX6 protein, human; C000605732:Pax6 protein, mouse