Purchase this article with an account.
L. D. Squires, R. L. Brown; DNA Microarray Analysis of Melanopsin-Containing Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5036.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The circadian pacemaker in the mammalian SCN is synchronized with the solar cycle by direct synaptic input from a small subset of retinal ganglion cells (RGCs), which contain the novel photopigment, melanopsin, and are intrinsically photosensitive. Due to the scarcity of these cells (~ 2000/retina), it has proven difficult to elucidate the melanopsin-based phototransduction cascade. The recent production of a mouse strain expressing GFP in the melanopsin-containing (m)RGCs (the gift of S. Hattar, JHU) has enabled us to use florescence-activated cell sorting (FACS) to generate highly-enriched cell populations for differential gene expression profiling.
Transgenic mice were constructed that express GFP in the mRGCs. Retinas from transgenic and control mice were dissociated by treatment with papain, yielding a suspension of single cells. FACS enabled us to obtain > 4, 000 GFP-positive cells from twenty retinas. Control RGCs were specifically labeled with the Thy-1 marker and purified by FACS. Total RNA was isolated from both cell populations, converted to cDNA, and amplified in a linear fashion. This cDNA was applied to an Illumina mouse-6 v1.1 array, containing probes for > 22, 000 transcripts; hybridization results were analyzed using "Genesifter" (VizXLabs).
As expected, the melanopsin transcript (Opn 4) was enriched in mRGCs by 31-fold over whole retina, and 17-fold over the total RGC population. The transcript encoding the pituitary adenylyl cyclase activating peptide (PACAP), another marker for mRGCs, was enriched 38-fold over whole retina and 14-fold over Thy-1+ RGCs. In contrast, the relative abundance of standard photoreceptor markers was reduced by 10- to 25-fold in the mRGCs. Furthermore, TRPC7, previously reported to be enriched in mRGCs, was enriched 9-fold over total retina, and G14 was enriched 11-fold over total retina (10-fold over RGCs).
Elucidation of the melanopsin-based signaling pathway is crucial to the understanding of circadian photoentrainment. Our study has identified several candidate proteins as potential components of the phototranduction cascade; future functional characterization studies will be required to confirm their role.
This PDF is available to Subscribers Only