## 3 Discussion  
The present work examined the genes/transcriptomic alterations that might be involved in TH-induced cone degeneration in mice using scRNAseq and a pathway/network analysis. Our findings reveal that excessive TH signaling has an adverse impact on cone phototransduction, mitochondrial bioenergetics, and cellular homeostasis/viability. Our scRNAseq identified 13 cell clusters in the retina, which were, in turn, classified by cell type using a curated set of known cell identity marker genes. The cluster analysis data are highly consistent with previous reports using immunochemical/morphological approaches [39] and nanoliter droplets [40] (see Supplementary Table S9). The cone transcriptomes demonstrated the most striking changes after the T3 treatment. Treatment with T3 led to the upregulation of 229 DEGs and the down-regulation of 221 DEGs in cones. These data are consistent with the previous findings on the profound/broad roles of TH signaling in cones, ranging from the expression and retinal localization/distribution of cone opsins [29; 41] to cone viability [30; 37; 38], and they support the view that cones are more susceptible to the stimulation of TH signaling among retinal cells. In contrast, the rods responded moderately to the T3 treatment, with 51 DEGs accounting for 4.4% of the total DEGs. Of note, retinal glial cells showed great responses to the T3 treatment, with 179 DEGs in Muller cells, 163 DEGs in astrocytes, and 100 DEGs in microglia, accounting for 15.3%, 14.0%, and 8.6% of the total DEGs, respectively. These data are consistent with the previous findings showing the increased labeling of the glial fibrillary acidic protein, a marker of Muller cell activation, and the increased labeling of Iba1, a marker of microglial activation, in the retinas after the T3 treatment [37; 42]. Interestingly, it was recently reported that TH signaling in Muller cells coordinate retinal intercellular communications during light/dark adaptation [43]. The top 25 down-regulated DEGs in the cone cluster include almost all of the genes involved in phototransduction. Furthermore, both the canonical pathway analysis and the pathway/network analysis revealed that phototransduction was the most significantly inhibited pathway after T3 treatment. It was previously shown that the stimulation of TH signaling induces the expression of _Opn1 mw_ (encoding M-opsin) [28; 29; 32] and inhibits the expression of _Opn1 sw_ (encoding S-opsin). This work shows that excessive TH signaling inhibits the expressions of both _Opn1 mw_ and _Opn1 sw_. The discrepancy on the expression of _Opn1 mw_ might be contributed by the experimental condition variations, including the age of the mice when they received the T3 treatment and the dose/duration of the treatment. Similar to cones, the top 34 down-regulated DEGs in the rods include genes involved in phototransduction. The canonical pathway analysis showed that phototransduction was the most significantly inhibited pathway after T3 treatment. The data showing reduced cone and rod phototransduction are consistent with the findings showing that the stimulation of TH signaling with the T3 treatment or deletion of Dio3 reduces cone and rod light responses [37; 38]. A pathway analysis revealed that the oxidative phosphorylation pathway was profoundly suppressed in the cone cluster after the T3 treatment. The IPA also suggested the dysfunction of the mitochondrion in the cone cluster. The mitochondrion is a known major target of T3 through both a genetic mechanism via TH receptors and a non-genomic mechanism via truncated TH receptors or direct actions [1; 44; 45]. The data from the transcriptomic analysis are consistent with the findings obtained from the mitochondrial respiration activity assays, showing that treatment with T3 reduces the basal respiration, ATP production, and maximal respiration in the retina compared with the untreated controls [42].