226 The Journal of Comparative Neurology K. ROESCH ET AL. (GFAP) in response to neuronal damage (Ridet et al., Dnase I was heat-inactivated, and the samples were la- 1997; Pekny and Pekna, 2004). Gliotic M u¨ller glial cells beled with 25 M biotin N6 ddATP (Enzo Biosciences, also show altered expression of various enzymes, ion chan- Syosset, NY) by using 45 U of TdT (Roche; Tietjen et al., nels, and receptors. The signiﬁcance of this response is 2003). poorly understood. The role of M u¨ller glial cells in disease Mouse genome 430 2.0 Genechip® oligonucleotide ar- has become even more intriguing, because an intimate rays were prehybridized in a Genechip® Hybridization molecular and functional relationship between M u¨ller glia Oven 320 (Affymetrix, Santa Clara, CA) in 1X MES and multipotent progenitors has become appreciated in buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% various systems, leading to the suggestion that M u¨ller Triton X-100) with 0.5 mg/ml acetylated BSA and 0.1 glia might serve as a source of retinal neurons following mg/ml herring sperm DNA. The single cell cDNAs were injury (Fisher and Reh, 2001, 2003; Ooto et al., 2004; heated to 99°C for 5 minutes, cooled to 45°C, added to Blackshaw et al., 2004, Osakada et al., 2007). the microarrays, and incubated in the hybridization Although extensive studies of the physiological and oven at 45°C for 16 hours. The slides were then scanned morphological properties of Mu¨ ller glia have been per- on a Genechip Affymetrix Scanner 7G. Signals from formed (Reichenbach and Robinson, 1995; Newman and microarrays were globally scaled with a target intensity Reichenbach, 1996), much less is known of their molecular set to 500 by using Affymetrix Microarray Suite Soft- biology. In the retina, gene expression in the abundant rod ware (MAS 5.0). Signal levels for all Affymetrix probe photoreceptors is most well characterized (Blackshaw et sets on the mouse 430 2.0 microarray are listed in al., 2001), and advances have been made in understanding Supplemental Table T4 for the ﬁve M u¨ller glial cells the molecular nature of speciﬁc neuronal cell types such proﬁled in this study as well as the 23 single cells (21 as amacrine and ganglion cells (Ivanov et al., 2006; Tri- immature cells, 2 adult rod photoreceptors) used for marchi et al., 2007). Considerably less is understood about the molecular complexity of Mu¨ ller glia. In this study, we comparison. have employed microarray analysis of single cells to ob- Data analysis: hierarchical clustering and tain a detailed proﬁle of the Mu¨ ller glial transcriptome. Fisher’s exact test Expression of Mu¨ ller glia enriched or speciﬁc transcripts was tested by in situ hybridization on retinal sections and Gene Cluster software (Eisen et al., 1998) was used to on dissociated retinal cells. Many novel M u¨ller glial genes determine gene association by hierarchical clustering. The have been found. Clusters of genes with characteristics results were visualized with Treeview software (Eisen et related to Mu¨ ller glial cell function, such as K and water al., 1998). The data were ﬁltered such that any probe set homeostasis, neuronal signaling, and recycling of pho- that failed to reach a signal of 1,000 in any single cell was topigments, were readily identiﬁed. eliminated; 1,000 was found to be a distinct value, where the threshold of a present and absent call on the array was MATERIALS AND METHODS very clear. The data for the remaining probe sets were log Isolation of single M u¨ller glial cells and transformed and normalized according to the software instructions. cDNA ampliﬁcation Additionally, for each probe set that was detected with Single-cell cDNA generation was achieved as described a signal of 1,000 in at least one microarray, the expres- (Brady and Iscove, 1993; Dulac and Axel, 1995; Tietjen et sion values were scaled by dividing each value by the al., 2003; Trimarchi et al., 2007). In short, retinae from maximum value for that probe set. The resulting scaled C57/BL6 adult mice were dissected in phosphate-buffered values were then binned into ﬁve equally sized bins as saline (PBS; pH 7.4) and then dissociated with papain described (Trimarchi et al., 2007). All probe set pairs were (Worthington Biochemical, Freehold, NJ) for 15–20 min- analyzed for association by using the following procedure. utes at 37°C and gentle manual trituration. Cells were First, a contingency table with ﬁve rows and ﬁve columns pelleted and resuspended in PBS containing 0.1% bovine was obtained that recorded the joint distribution over bins serum albumin (BSA). Dissociated cells were visualized for a given probe set pair. A P value for signiﬁcant asso- under an inverted microscope, and M u¨ller glial cells were ciation was then calculated from this table by using Fish- i(tdPraeBnnSts/if0ﬁe.re1rd%edbBawsSeiAtdh) oaannddthrtaehiwrenndigssletaiensdscetdnmeienodrtolpehpoionllotygomyw.erTaashsheeybcwuhfaefreiner ceor’rsreesxpaocntdtteosta. sFionrglmeugeltnipel,ethAeffPymvaelturiexs wideernetigﬁeenresrathteadt reaction (PCR) tubes containing cold lysis buffer. cDNA as follows. First, for each gene, all Affymetrix IDs that was generated from single cells by reverse transcription map to this gene were identiﬁed. Then, for each microar- (Superscript II [Invitrogen, La Jolla, CA]). After poly(A) ray, the maximal expression value for these Affymetrix tailing of the synthesized ﬁrst-strand cDNA product, PCR IDs was identiﬁed. This maximal value was then used as ampliﬁcation of the samples was performed by using the expression value for that gene in that array. This LA Taq polymerase (Takara Mirus Bio, Shiga, Japan; collapsing method is referred to as “each-max-value”. A Trimarchi et al., 2007). The cDNA smears were analyzed new ID was then generated by a comma-separated list of on agarose gels. all Affymetrix IDs that were collapsed for a given gene. For each array and gene, the maximal value among the Affymetrix array hybridization Affymetrix IDs was always used. The resulting expression Ten micrograms of each single-cell cDNA were digested proﬁle of a gene was thus a combination of values mea- for 13 minutes at 37°C with 1 U of RNase free DNase I sured by any of the collapsed Affymetrix probe sets and (Roche, Indianapolis, IN) in One-Phor All buffer (100 mM therefore does not always correspond to a single Af- Tris acetate, 100 mM Mg acetate, 500 mM K acetate). fymetrix probe set proﬁle.