Differential gene expression profiling of large and small retinal ganglion cells.
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.
Ivanov
Dmitry
D
Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Miami, FL 33136, USA.
Dvoriantchikova
Galina
G
Barakat
David J
DJ
Nathanson
Lubov
L
Shestopalov
Valery I
VI
D000818:Animals; D002469:Cell Separation; D048429:Cell Size; D002470:Cell Survival / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D007150:Immunohistochemistry; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D012165:Retinal Ganglion Cells / Q000145:classification / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014157:Transcription Factors