title: Induction of functional photoreceptor phenotype by exogenous Crx expression in mouse retinal stem cells.
abstract: OBJECTIVE
  This study was undertaken to determine whether exogenous expression of the transcription factor Crx can promote the differentiation of mouse retinal stem cells (RSCs) into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties.
  
  
  METHODS
  RSCs isolated from mouse ciliary epithelium and maintained in serum-free culture were genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6; POU transcription factor Oct3/4; proliferating cell nuclear antigen [PCNA]); of neuronal markers (nestin, neuron-specific class III beta-tubulin [beta III Tub] and neurofilament [NF 200]); and of photoreceptor-specific markers (rhodopsin [Rho], cyclic nucleotide-gated cation channel-3 [CNG3], blue-cone opsin, and cGMP phosphodiesterase [PDE]); were evaluated during differentiation by immunocytochemistry and Western blot analysis. Phototransduction cascade activity was assessed by measuring light-induced hydrolysis of cyclic (c)GMP levels with a cGMP enzyme-linked immunoassay.
  
  
  RESULTS
  Transient Crx transgene expression was observed in 63% of RSCs. Expression of stem cell markers of proliferation and pluripotency Pax6, PCNA, and Oct3/4, was significantly decreased by exogenous Crx expression. Concomitantly, Crx induced expression of the analyzed neuron- and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in RSCs expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels.
  
  
  CONCLUSIONS
  Crx halted proliferation of RSCs and induced them to differentiate into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. This study demonstrates that Crx can successfully induce RSCs to differentiate into cells with functional photoreceptor phenotypes.
authors.affiliation: Retinitis Pigmentosa Research Unit, The Rayne Institute, King's College London School of Biomedical and Health Sciences at Guy's, King's College, and St. Thomas' Hospitals, St. Thomas' Hospital Campus, London, United Kingdom. catherine.jomary@kcl.ac.uk
authors.forename: Catherine
authors.initials: C
authors.lastname: Jomary
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Jones
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D017299:Rod Opsins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*; D014785:Vision, Ocular
chemical_list: D015415:Biomarkers; C518237:Cnga3 protein, mouse; D054815:Cyclic Nucleotide-Gated Cation Channels; D018398:Homeodomain Proteins; D018809:Proliferating Cell Nuclear Antigen; D017299:Rod Opsins; D015534:Trans-Activators; C109288:cone rod homeobox protein; C095853:short-wavelength opsin; D012243:Rhodopsin; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517277:Pde2a protein, mouse