The Journal of Comparative Neurology SINGLE CELL TRANSCRIPTIONAL PROFILE OF MGCS shaw et al., 2004) validates the general performance of the assay. Biological processes and molecular functions were systematically assigned to genes with average expres- sion values 10,000 by using IPA (Ingenuity Systems, Redwood City, CA). Genes with the highest signiﬁcance can be categorized into protein synthesis, cell-to-cell signaling, cellular assembly and organization, cellular function and maintenance, carbohydrate metabolism, cell morphology, and lipid metabolism (Supplemental Table T6). Comparison of expression of M u¨ller glia enriched transcripts to expression in other retinal cells The Affymetrix signal values from the M u¨ller glial cells were compared with those of several different types of single retinal single cells in order to gain an appreciation of how Mu¨ ller glial cells differ from neurons and also to identify markers for M u¨ller glial cells. This comparison included 2 immature rod photoreceptors, 2 adult rod pho- toreceptors, and 19 immature amacrine and ganglion cells (Trimarchi et al., 2007). The data were sorted according to decreasing average values from the ﬁve M u¨ller glia sam- ples (Supplemental Table 1) and are represented in Figure 2 as a heat map. The 100 transcripts expressed with the highest averaged values show an almost uniform general expression pattern across all M u¨ller glial samples. Many genes that had high expression values in M u¨ller glia were also highly expressed in rod photoreceptors and immature amacrine and ganglion cells. Some of these genes are involved in generic cellular functions, e.g., ribosomal sub- units, eukaryotic translation elongation factor 1 1 (Eef1a1), ubiquitins (Ubb and Ubc), and glyceraldehyde- 3-phosphate dehydrogenase (Gapd). This initial comparison revealed a dozen genes that appear to be preferentially expressed by M u¨ller glia, in- cluding the tRNA ligase BING4 and the transcription factor Sox2. They also include the lipid transporter ApoE, the major apolipoprotein in the central nervous system (Pitas et al., 1987). Interestingly, ApoE has also been implicated in cell proliferation (Mahley, 1988) and in mod- ulation of the innate and acquired immune response (Las- kowitz et al., 2000). Another gene involved in lipid metab- olism that was found enriched in Mu¨ ller glia is the diazepam binding inhibitor (Dbi), which is also known to be expressed in glial cells of both the central and periph- eral nervous systems (Yanase et al., 2002). Other M u¨ller glia enriched genes are the water channel, Aqp4, and Abca8a, a transporter with ATPase activity, the isopre- noid binding protein Rlbp1, and the receptors Gnai2 and Gpr37. Gnai2 (also called Gi2) mediates signaling from the endothelin-B receptor to maintain mitogenic activity of neural progenitors in the brain (Sinohara et al., 2004). Expression of the Gpr37 and Dkk3 transcripts in Mu¨ ller glia was shown in a previous study using serial analysis of gene expression (SAGE; Blackshaw et al., 2004). Dkk3 is a gene involved in cell differentiation and negative regula- tion of Wnt signaling. Further, caveolin1 (Cav), which is important in nitric oxide metabolism and vesicle-mediated transport, Spbc25, a component of the Ndc80 kinetochore complex (McCleland et al., 2004), and genes with un- known biological function such as Itm2b, Jagn1, and 2310032F03Rik can be classiﬁed as enriched in M u¨ller 229 glia. The data suggest that these genes are speciﬁc for Mu¨ ller glia in the adult murine retina. However, some genes are also expressed in retinal progenitor cells, as has been shown for Sox2 and Dkk3 (Blackshaw et al., 2004), and for Spbc25 (data not shown; Trimarchi and Cepko, in preparation). Mu¨ ller glia enriched transcripts for synap- toporin (Synpr) and type IX procollagen alpha1 (Col9a1) might also be Mu¨ ller glia speciﬁc, although both of these are also detectable in one immature ganglion cell (P0 A6). Some of the potentially Mu¨ ller glia speciﬁc transcripts, such as BING4, Prss2, Ube1c, and Gnb1l could only be detected in three of ﬁve Mu¨ ller glial cells. This might be an indication of heterogeneity within M u¨ller glial cells, or it might be due to a technical issue. The Mu¨ ller glia marker clu, expected to be speciﬁc to Mu¨ ller glia in this comparison, is also detected in one of the rod photoreceptors. Because the retinal pigmented epithelium (RPE) expresses clu as well, it is possible that this expression occurred through RPE contamination of the rod single-cell preparation (Trimarchi et al., 2007). A summary of these data can be found in Table 1. Veriﬁca- tion of the expression pattern of these genes by another method is required, such as in situ hybridization on reti- nal sections, as was done for a subset in this study (below). Identiﬁcation of genes characteristic for Mu¨ ller glial cells To identify Mu¨ ller glial enriched or speciﬁc genes, the analytical tool Cluster 3.0 (Eisen et al., 1998) and Tree- view software (Eisen et al., 1998) were used for clustering the single cell proﬁles, as well as to create a visual map of the data. The ﬁve Mu¨ ller glial cells were once again com- pared with immature amacrine and ganglion cells and with rod photoreceptors. However, instead of considering only the most highly expressed transcripts, as was done in the previous analysis (Fig. 2), the complete data set ob- tained from the microarrays was used for comparison. The data were ﬁltered such that any probe set that failed to reach a signal of 1,000 in any single cell was eliminated. The data for the remaining probe sets was log transformed and normalized according to Eisen et al. (1998). A representative part of the resulting expression level matrix is shown in Supplemental Figure S1 and a portion of that same cluster around Aqp4 is shown enlarged in Figure 3A. Interestingly, tight clustering of genes in a given cell type can be observed, with Figure 3A showing a portion of a Mu¨ ller glial cluster, and Figure 3B showing a rod photoreceptor cluster. All Mu¨ ller glial cells shared a similar gene expression proﬁle over a wide range of genes (Supplemental Fig. S1). Other cell types showed expres- sion values that were considerably distinct from those of the Mu¨ ller glia. The data in Figure 3A of genes enriched in Mu¨ ller glia suggest a Mu¨ ller glia core transcriptome, which likely comprises genes encoding some of the key functions of Mu¨ ller glia. One of the most prominent exam- ples is the inwardly rectifying potassium channel (Kcnj10). Mu¨ ller glia have been previously shown to play an active role in regulating retinal functions by allowing transport of small molecules through speciﬁc ion chan- nels. Other potassium channels expressed in M u¨ller glial cells include Kir4.1 and Kir2.1 (Newman and Reichen- bach, 1996; Bringmann et al., 2000). Genes included in this cluster are also ApoE and Abca8a, which have been identiﬁed to be among the most highly expressed genes in the previous analysis (Fig. 2). Genes not previously iden-