TY - JOUR
T1 - Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques
AU - Doh, Sung Tae
AU - Hao, Hailing
AU - Loh, Stephanie C.
AU - Patel, Tapan
AU - Tawil, Haim Y.
AU - Chen, David K.
AU - Pashkova, Anna
AU - Shen, Andy
AU - Wang, Huimin
AU - Cai, Li
N1 - Funding Information:
We would like to thank Dr. Connie Cepko for a reporter construct of plasmid DNA pCAG-GFP, Dr. Robert S. Molday for Rho-4D2 antibody, Dr. Richard Nowakowski for the use of a confocal microscope (Nikon Eclipse 80i), and Evangeline Tzatzalos and Shannon M. Smith for proof-reading the manuscript. Antibodies: Lim1+2 (4F2), Pax6, Vimentin (H5), Visinin (7G4), Xap-1 (clone 3D2) and Xap-2 (clone 5B9), were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported in part by the grant EY018738 from the National Institute of Health; the New Jersey Commission on Spinal Cord Research grant (08-3074-SCR-E-0); and the Charles and Johanna Busch Award.
PY - 2010
Y1 - 2010
N2 - 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, Pkc, 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. Conclusion. 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.
AB - 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, Pkc, 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. Conclusion. 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.
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U2 - 10.1186/1471-213X-10-8
DO - 10.1186/1471-213X-10-8
M3 - Article
C2 - 20089190
AN - SCOPUS:76949095664
SN - 1471-213X
VL - 10
JO - BMC Developmental Biology
JF - BMC Developmental Biology
M1 - 8
ER -