228 The Journal of Comparative Neurology K. ROESCH ET AL. Fig. 1. Prospective identiﬁcation of single Mu¨ ller glial cells by morphology. Micrograph of cells dissociated from an adult mouse retina. A: Immunostaining for glutamine synthetase (Glul). B: Disso- ciated cell in situ hybridization for clusterin. C: Brightﬁeld image. D: Merged image. Scale bar 10 m in A (applies to A–D). carried out by using modiﬁed oligo dT primers. cDNA preparations from ﬁve individual M u¨ller glial cells were hybridized to Affymetrix Genechip® Mouse genome 430 2.0 arrays. These arrays provide almost complete cov- erage of the mouse genome with 45,000 probe sets. The identity of cells as M u¨ller glial cells was conﬁrmed by expression of known marker genes for M u¨ller glia. Glu- tamine synthetase (Glul), clusterin (Clu), dickkopf ho- molog 3 (Dkk3) (Blackshaw et al., 2004), and S100 cal- cium binding protein A16 (Seigel et al., 1996) were successfully detected (Fig. 2 and Supplemental Fig. S1). All of the cells share expression of these key marker transcripts. The values for the Affymetrix signals from the ﬁve M u¨l- ler glial cells were averaged (Supplemental Table T2). There were 7,377 Affymetrix IDs with averages 1,000, of which 871 had values 10,000. Many known M u¨ller glial speciﬁc genes, such as Glul, aquaporin 4 (Aqp4), and Clu/ApoJ were some of the most highly expressed genes (Fig. 2), perhaps facilitating their previous discovery as Mu¨ ller glial markers (Jenne and Tschopp, 1992). Addi- tionally, this comparison of highly expressed genes with the data in the literature (Nagelhus et al., 1998; Black- Fig. 2. Comparison of highly expressed transcripts in M u¨ller glia with other retinal cell types. The 100 most highly expressed tran- scripts, averaged over the ﬁve single Mu¨ ller glial cells (MGs) and sorted in decreasing order, are illustrated in a heat map and com- pared with their expression in other cell types. Values above 10,000 are represented in dark purple. Rows correspond to different genes (with abbreviated name and Affymetrix ID), and columns represent the single cell probes. Shown are 19 immature amacrine (ACs) and ganglion cells (RGCs), 2 immature rod photoreceptors (PRs) from postnatal day 0 (P0), and 2 mature rod photoreceptors (PRs) (Trima- rchi et al., 2007).