Abstract
Purpose: :
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.
Methods: :
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).
Results: :
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).
Conclusions: :
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.
Keywords: ganglion cells • gene microarray • circadian rhythms