234 Validation of expression in M u¨ller glia To validate the expression of genes expressed in M u¨ller glial cells, in situ hybridization on retinal sections was performed for a subset of genes (Fig. 5). The corresponding Affymetrix signal values are represented in the top panel of Figure 5 as a heatmap. Because the cells used for comparison were mainly immature cells, some of the genes that are indeed speciﬁc to Mu¨ ller glia in the adult retina are also expressed in those immature cells that still contain a certain expression proﬁle of (late) progenitor cells. The similarity of M u¨ller glia gene expression to progenitor cell gene expression has been discussed previ- ously (Fig. 4 and Blackshaw et al., 2004). M u¨ller glia transcripts were localized most prominently in the middle portion of the inner nuclear layer (INL), consistent with the location of the transcript in the cell body. Transcripts with this predicted restriction in expression pattern are Dkk3, ApoE, Spbc25, Apg4B, Pak3, Car2, Rlbp1, deltex 2 homolog (Dtx2), and pleiotrophin (Ptn) (Fig. 5A–F,H,L,M). Certain genes, such as Dbi, GPR37, and Glul (Fig. 5G,N,K, respectively), gave punctuate staining in the mid- dle portion of the INL, as well as diffuse expression in the outer nuclear layer (ONL), consistent with expression in ascending glial processes, and in the ganglion cell layer (GCL), consistent with expression in M u¨ller glial descend- ing processes and endfeet. Expression in the INL was either throughout the INL, as is the case for clu (Fig. 5J), or in a subset of cells in the middle of the INL, as is the case for Dbi (Fig. 5G). The pan INL pattern is suggestive of a gene being expressed in several cell types in the INL, whereas the more speciﬁc middle of the INL pattern is more consistent with expression restricted to M u¨ller glia. Even transcripts expressed in only a subset of M u¨ller glial cells proﬁled, such as Apg4b, Dtx2, and Ptn could be veriﬁed and detected by in situ hybridization (Fig. 5D,M,L, respectively). It was of interest to determine whether some of the genes with moderate to high levels of Affymetrix signals in proﬁled Mu¨ ller glia were speciﬁc to M u¨ller glia. Speciﬁcity might give some insight into function and would indicate that such genes are suitable for use as speciﬁc markers of Mu¨ ller glia. Although analysis of candidate M u¨ller glial genes by section ISH is a fairly rapid and informative assay, it was unclear for some genes whether their expres- sion was restricted to Mu¨ ller glial cells. To determine speciﬁcity of expression more precisely, ISH on dissociated cells was carried out in combination with immunocyto- chemistry for the known glial marker glutamine syn- thetase (Glul). Dissociated cell in situ hybridization (DISH) for Itm2b revealed that it was exclusively ex- pressed in Glul-positive cells. A modulator of the Notch pathway, Apg4b, as well as Dkk3, Aqp4, and ApoE were shown to be fairly speciﬁc to M u¨ller glia as well (Fig. 6). DISH also allowed an assessment of heterogeneity of ex- pression within Muller glial cells. The Rlbp1 transcript, expressed in only three of ﬁve cells in the microarray analysis, was found in only a subset of M u¨ller glia, in 70% of the cells marked with the anti-Glul (Supplemental Fig. S2). In contrast, there was a complete overlap of Itm2b and clusterin transcript expression with anti-Glul stain- ing (Fig. 5, top panel, Supplemental Fig. S2). Additional validation of GPR37 as a M u¨ller glia speciﬁc gene in the murine retina was possible through the use of a transgenic mouse line, GPR37tm1Dgen, available from The Journal of Comparative Neurology K. ROESCH ET AL. the Jackson Laboratory. The GPR37tm1Dgen mice express a transgene consisting of lacZ driven by the endogenous GPR37 promotor. Xgal staining on retinal sections showed lacZ expression in Mu¨ ller glial cells (Fig. 5O). Pax6 expression was further investigated by immuno- cytochemistry by using anti-Pax6 and anti-Glul antibodies (Fig. 5Q,R; see Supplemental Fig. S3 for magenta-green versions of images). Analysis of the sections with confocal microscopy showed that Pax6 is indeed expressed in adult Mu¨ ller glial cells, as well as in some types of retinal neurons, as previously reported for neurons (de Melo et al., 2003), but not previously reported for M u¨ller glial cells. Creation of a mouse line expressing Cre for genetic access to M u¨ller glia To create a line of mice for future experiments in which gene expression speciﬁcally in Mu¨ ller glial cells could be manipulated, a BAC was engineered to express Cre from a promoter that was predicted to be enriched or speciﬁc to glial cells (Kessaris et al., 2006). This BAC was used to create a transgenic mouse. Mice that successfully trans- mitted the BAC were assessed for Cre activity by crossing to mice carrying a nuclear lacZ ﬂoxed indicator allele, RC::PFwe (Farago et al., 2006). Strong nuclear lacZ ex- pression was detected in all or nearly all M u¨ller glial cells in the progeny of this cross (Fig. 5P). This may be a fortuitous ﬁnding, as inspection of the microarray data for Mu¨ ller glia did not show expression of Pdgfra. Some Xgal staining was observed in the ONL and GCL, but it was not distinctly nuclear. Control Xgal staining of littermates did not show this lacZ activity, and thus the origin of the ONL and GCL staining is unclear. DISCUSSION The goal of this study was to deﬁne the transcriptome of mature Mu¨ ller glial cells. A single cell proﬁling approach was used because total tissue preparations of the retina would only contain approximately 5% of cells as Mu¨ ller glia (based on the percentage of cells showing Glul immunoreac- tivity after dissociation into single cells). A PCR-based am- pliﬁcation method was used to generate enough cDNA from a single cell to use as probe on Affymetrix microarrays. The microarray results were then validated by section ISH and DISH. Validation was particularly important because PCR- based protocols may lead to a higher false-positive rate, compared with the higher false-negative rate that is gener- ally associated with linear ampliﬁcation methods (Van Gelder et al., 1990; Iscove et al., 2002; Che and Ginsberg, 2004; Subkhankulova and Livesey, 2006). The microarray data from the single cells conﬁrmed the high expression of many known Mu¨ ller glial genes and also revealed the ex- pression of many genes not previously known to be tran- scribed in Mu¨ ller glia. By characterizing single, freshly iso- lated Mu¨ ller glial cells, this study also circumvented the need to use FACS or in vitro culture, or to manipulate these cells further, and thereby minimized induced gene expres- sion changes. We have found this method to be fairly sensi- tive and reproducible in the current study, as well as a previous study of developing amacrine and ganglion cells (Trimarchi et al., 2007). We also created and characterized a Cre line for recom- bination and expression in Mu¨ ller glial cells. This novel