TY - JOUR
T1 - Differential gene expression profiling of large and small retinal ganglion cells
AU - Ivanov, Dmitry
AU - Dvoriantchikova, Galina
AU - Barakat, David J.
AU - Nathanson, Lubov
AU - Shestopalov, Valery I.
N1 - Funding Information:
This study was supported an NIH grant EY017991 and a Research to Prevent Blindness (RPB) Career Development Award (V.S.), by the AHA Scientist Development Award 0735014B and Fight For Sight fellowship PD05034 (D.I.), by NIH center grant P30 EY014801 and an unrestricted grant to the University of Miami Department of Ophthalmology from RPB. We thank the staff of the DNA Microarray Core Facility at the University of Miami Miller School of Medicine for expert assistance, Dr. Alexei Sharov for expert advice in the array experiment design and Dr. Dolena Ledee for critical reading of the manuscript.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2008/9/15
Y1 - 2008/9/15
N2 - Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.
AB - Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.
KW - FACS
KW - Gene expression
KW - Microarrays
KW - Networks
KW - Retinal ganglion cells
KW - Soma size
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U2 - 10.1016/j.jneumeth.2008.06.016
DO - 10.1016/j.jneumeth.2008.06.016
M3 - Article
C2 - 18640154
AN - SCOPUS:49549108442
VL - 174
SP - 10
EP - 17
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1
ER -