title: Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.
abstract: BACKGROUND
  Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete.
  
  
  RESULTS
  This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types.
  
  
  CONCLUSIONS
  The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.
authors.affiliation: Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.
authors.forename: Sung Tae
authors.initials: ST
authors.lastname: Doh
authors.forename: Hailing
authors.initials: H
authors.lastname: Hao
authors.forename: Stephanie C
authors.initials: SC
authors.lastname: Loh
authors.forename: Tapan
authors.initials: T
authors.lastname: Patel
authors.forename: Haim Y
authors.initials: HY
authors.lastname: Tawil
authors.forename: David K
authors.initials: DK
authors.lastname: Chen
authors.forename: Anna
authors.initials: A
authors.lastname: Pashkova
authors.forename: Andy
authors.initials: A
authors.lastname: Shen
authors.forename: Huimin
authors.initials: H
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Cai
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002642:Chick Embryo; D018274:Electroporation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000196:embryology*
chemical_list: D049452:Green Fluorescent Proteins