Previously, Sox2 expression was examined in mice in which the coding region of Sox2 was replaced with EGFP
7 ; however, no detailed examination was made of the endogenous Sox2 gene or protein. Thus, we first examined endogenous Sox2 in retinas of wild-type mice with respect to both mRNA transcription and protein expression. Specifically, changes in the mRNA expression of Sox2 over time in the mouse neural retina were examined at various developmental stages using semiquantitative RT-PCR
(Fig. 1A) . Sox2 mRNA was expressed at E16 and after birth; thereafter, its expression gradually decreased, but persisted until adulthood. We next examined the spatiotemporal expression of Sox2 at various developmental stages by immunostaining of frozen retinal sections of wild-type mice. At E17, ganglion cells had started to form the innermost layer, whereas most other areas of the retina were occupied by immature progenitor cells collectively known as the neuroblastic layer (NBL). Sox2 expression was observed in the NBL, except in the outermost region (
Fig. 1B , arrowheads) at E17. At P5, Sox2 expression was dramatically downregulated in the NBL and was observed in the ganglion cell layer (GCL) and on the innermost side of the NBL, corresponding to the inner nuclear layer (INL), which had just begun to form
(Fig. 1B) , suggesting that Sox2 is expressed in differentiating cells. Approximately 2 weeks after birth (
Fig. 1B , P15), Sox2 expression was restricted to the GCL and INL. In the adult retina, Sox2 expression was clearly observed in the INL, which consisted of horizontal, amacrine, bipolar, and Müller glia cells, and was weakly detected in the GCL, which consisted of retinal ganglion cells and displaced amacrine cells. This pattern of expression is quite similar to that reported for the Sox2 promoter-EGFP mouse.
7
We next examined the retinal subtypes of Sox2-expressing cells by examining the expression of various retinal cell-specific markers. Sox2 was expressed in Müller glial and amacrine cells based on the coexpression of GS and HuC/HuD
(Fig. 1C) . While most of the GS-positive cells also expressed Sox2, less than 30% of the HuC/HuD-positive amacrine cells expressed Sox2. These results suggest that not all amacrine cells express Sox2.
Amacrine cells can be classified into several subtypes, and so we examined the expression of amacrine cell subtype markers. In mouse retina, GABAergic neurons compose approximately 40% of amacrine cells.
15 Calretinin was localized in some GABAergic neurons, and nearly all the Calretinin-positive cells coexpressed Sox2
(Fig. 1C) . Many GABAergic amacrine cells in the mouse retina contain other neurotransmitters such as acetylcholine and dopamine in addition to GABA. Islet1 and choline acetyltransferase (ChAT) are markers of cholinergic amacrine cells. Approximately 80% of Islet-1-positive amacrine cells coexpressed Sox2, and all of the ChAT-positive cells also expressed Sox2
(Fig. 1C) . These results suggested that Sox2 could also be a marker for cholinergic amacrine cells. The Sox2 and Islet-1 double-positive cells in the GCL were assumed to be displaced amacrine cells
(Fig. 1C) . Although we could not obtain clear antibody staining against another amacrine cell subset marker that localizes to 30% of all amacrine cells,
15 all the Sox2-positive cells in the row of amacrine cell nuclei of the developing retina expressed GABAergic neuron markers. Therefore, we assume that Sox2 is a marker for this subset of amacrine cells.