Transdifferentiation of the retinal pigment epithelia to the neural retina by transfer of the Pax6 transcriptional factor.

The Pax6 gene plays an important role in eye morphogenesis throughout the animal kingdom. The Pax6 gene and its homologue could form ectopic eyes by targeted expression in Drosophila and Xenopus. Thus, this gene is a master gene for the eye morphogenesis at least in these animals. In the early development of the vertebrate eye, Pax6 is required for the instruction of multipotential progenitor cells of the neural retina (NR). Primitive retinal pigment epithelial (RPE) cells are able to switch their phenotype and differentiate into NR under exogenous intervention, including treatment with fibroblast growth factors (FGFs), and surgical removal of endogenous NR. However, the molecular basis of phenotypic switching is still controversial. Here, we show that Pax6 alone is sufficient to induce transdifferentiation of ectopic NR from RPE cells without addition of FGFs or surgical manipulation. Pax6-mediated transdifferentiation can be induced even at later stages of development. Both in vivo and in vitro studies show that the Pax6 lies downstream of FGF signaling, highlighting the central roles of Pax6 in NR transdifferentiation. Our results provide an evidence of retinogenic potential of nearly mature RPE and a cue for new therapeutic approaches to regenerate functional NR in patients with a visual loss.

[1]  R. Sullivan,et al.  Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens , 2002, The Journal of comparative neurology.

[2]  A. Grinberg,et al.  The Rx homeobox gene is essential for vertebrate eye development , 1997, Nature.

[3]  I. Hanson,et al.  Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly , 1994, Nature Genetics.

[4]  G. Schaffner,et al.  DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. , 1993, Genes & development.

[5]  Sanjaya Singh,et al.  Truncation Mutations in the Transactivation Region of PAX6 Result in Dominant-Negative Mutants* , 1998, The Journal of Biological Chemistry.

[6]  I. Gorlov,et al.  PAX6, Paired Domain Influences Sequence Recognition by the Homeodomain* , 2002, The Journal of Biological Chemistry.

[7]  G. Saunders,et al.  Transcriptional Regulation of the Human PAX6 Gene Promoter* , 1997, The Journal of Biological Chemistry.

[8]  J. Wittbrodt,et al.  Six3 overexpression initiates the formation of ectopic retina. , 1999, Genes & development.

[9]  N. Bertrand,et al.  FGF signalling controls the timing of Pax6 activation in the neural tube. , 2000, Development.

[10]  H. Handa,et al.  Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations. , 2003, American journal of human genetics.

[11]  H. M. Petry,et al.  Vision change after sheet transplant of fetal retina with retinal pigment epithelium to a patient with retinitis pigmentosa. , 2004, Archives of ophthalmology.

[12]  K. Yasuda,et al.  Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development , 2000, Mechanisms of Development.

[13]  P. Gruss,et al.  Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6 , 2003, Development.

[14]  C. K. Mitchell,et al.  Calbindin immunoreactivity of horizontal cells in the developing rabbit retina. , 1995, Experimental eye research.

[15]  I. Jackson,et al.  The retinal pigmented epithelium is required for development and maintenance of the mouse neural retina , 1995, Current Biology.

[16]  R. Krumlauf,et al.  ‘Shocking’ developments in chick embryology: electroporation and in ovo gene expression , 1999, Nature Cell Biology.

[17]  R. Lang,et al.  Pax6 induces ectopic eyes in a vertebrate. , 1999, Development.

[18]  T. Reh,et al.  Müller glia are a potential source of neural regeneration in the postnatal chicken retina , 2001, Nature Neuroscience.

[19]  C. Cepko,et al.  Expression of Chx10 and Chx10-1 in the developing chicken retina , 2000, Mechanisms of Development.

[20]  P. Overbeek,et al.  Patterning the optic neuroepithelium by FGF signaling and Ras activation. , 2001, Development.

[21]  P. Emson,et al.  Localization of two calcium binding proteins, calbindin (28 kD) and parvalbumin (12 kD), in the vertebrate retina , 1990, The Journal of comparative neurology.

[22]  R. Kageyama,et al.  Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue , 2001, Nature Neuroscience.

[23]  G. Rubin,et al.  Targeted expression of teashirt induces ectopic eyes in Drosophila. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Reh,et al.  Multipotential stem cells and progenitors in the vertebrate retina. , 1998, Journal of neurobiology.

[25]  S. Inouye,et al.  Chemical nature of the light emitter of the Aequorea green fluorescent protein. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[26]  P. Callaerts,et al.  Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. , 1995, Science.

[27]  A. Maslov,et al.  Neural Stem Cell Detection, Characterization, and Age- Related Changes in the Subventricular Zone of Mice , 2022 .

[28]  S. Saule,et al.  The homeobox-containing Engrailed (En-1) product down-regulates the expression of Pax-6 through a DNA binding-independent mechanism. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[29]  S. Zipursky,et al.  The Eye-Specification Proteins So and Eya Form a Complex and Regulate Multiple Steps in Drosophila Eye Development , 1997, Cell.

[30]  J A Epstein,et al.  Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing. , 1994, Genes & development.

[31]  M. Yamada,et al.  PAX6 missense mutation in isolated foveal hypoplasia , 1996, Nature Genetics.

[32]  R. Riepe,et al.  Müller cell localisation of glutamine synthetase in rat retina , 1977, Nature.

[33]  H. Okano,et al.  Expression of Neural RNA-Binding Proteins in the Postnatal CNS: Implications of Their Roles in Neuronal and Glial Cell Development , 1997, The Journal of Neuroscience.

[34]  C. Cepko,et al.  The Expression and Function of Notch Pathway Genes in the Developing Rat Eye , 1997, The Journal of Neuroscience.

[35]  A. McMahon,et al.  Six3 promotes the formation of ectopic optic vesicle‐like structures in mouse embryos , 2001, Developmental dynamics : an official publication of the American Association of Anatomists.

[36]  T. Reh,et al.  Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos. , 1997, Development.

[37]  G. Mardon,et al.  Ectopic eye development in Drosophila induced by directed dachshund expression. , 1997, Development.

[38]  A. Kawakami,et al.  Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development , 1997, Mechanisms of Development.

[39]  H. Nakamura,et al.  Tbx5 and the retinotectum projection. , 2000, Science.

[40]  G. Mardon,et al.  Dachshund and Eyes Absent Proteins Form a Complex and Function Synergistically to Induce Ectopic Eye Development in Drosophila , 1997, Cell.

[41]  M. Yamada,et al.  Missense mutation in the alternative splice region of the PAX6 gene in eye anomalies. , 1999, American journal of human genetics.

[42]  R. Morgan.  Conservation of sequence and function in the Pax6 regulatory elements. , 2004, Trends in genetics : TIG.

[43]  C. M. Park,et al.  Basic fibroblast growth factor induces retinal regeneration in vivo. , 1989, Developmental biology.

[44]  K. Struhl,et al.  Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience). Host-Range Shuttle System for Gene Insertion into the Chromosomes of Gram-negative Bacteria. , 1988 .

[45]  J. Epstein,et al.  Identification of a Pax paired domain recognition sequence and evidence for DNA-dependent conformational changes. , 1994, The Journal of biological chemistry.

[46]  H. Handa,et al.  Autoregulation of Pax6 transcriptional activation by two distinct DNA‐binding subdomains of the paired domain , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[47]  P. Bovolenta,et al.  Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors , 1998, Mechanisms of Development.

[48]  N. Bonini,et al.  The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between flies and vertebrates. , 1997, Development.

[49]  P. Gruss,et al.  Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos , 2000, Mechanisms of Development.

[50]  W. Gehring.  The master control gene for morphogenesis and evolution of the eye , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[51]  P. Gruss,et al.  Pax-6, a murine paired box gene, is expressed in the developing CNS. , 1991, Development.

[52]  E. de Juan,et al.  Restoration of visual responses following transplantation of intact retinal sheets in rd mice. , 2004, Experimental eye research.

[53]  D. van der Kooy,et al.  Retinal stem cells in the adult mammalian eye. , 2000, Science.

[54]  H. Handa,et al.  The Pax6 isoform bearing an alternative spliced exon promotes the development of the neural retinal structure. , 2005, Human molecular genetics.

[55]  P. Gruss,et al.  Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk , 1999, Mechanisms of Development.

[56]  J. Toy,et al.  The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[57]  W. Halfter.  Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth , 1998, The Journal of comparative neurology.

[58]  Claude Desplan,et al.  Crystal structure of a paired domain-DNA complex at 2.5 å resolution reveals structural basis for pax developmental mutations , 1995, Cell.

[59]  P. Layer,et al.  Histogenesis of the avian retina in reaggregation culture: from dissociated cells to laminar neuronal networks. , 1993, International review of cytology.

[60]  F. Guillemot,et al.  Pax6 Is Required for the Multipotent State of Retinal Progenitor Cells , 2001, Cell.