232 The Journal of Comparative Neurology K. ROESCH ET AL. Fig. 4. Heatmap of Mu¨ ller glia enriched genes illustrating poten- tial functions of M u¨ller glia. Expression of genes with potentially relevant functions (such as growth factors, cell cycle components, and Notch pathway components) for Mu¨ ller glia physiology was examined. The data are summarized as a heatmap. RGCs, retinal ganglion cells; ACs, amacrine cells; PRs, photoreceptors; MGs, M u¨ller glia cells. binding cassette, subfamily A, member 1)/ced-7 were ob- served to be expressed in a subset of Mu¨ ller glia, whereas others, Gulp1 (GULP: engulfment adaptor PTB domain con- taining 1)/ced-6, Lrp1 (low-density lipoprotein receptor- related protein 1)/ced-1, and Rac1 (RAS-related C3 botuli- num substrate 1)/ced-10, could not be detected in the Mu¨ ller glial cells studied here. Heterogeneity of M u¨ller glia Although the focus of this study was to deﬁne the tran- scriptome of Mu¨ ller glia, it was noted that certain genes were expressed heterogeneously among the M u¨ller glial cells analyzed (Fig. 4, Supplemental Figs. S1, S2), includ- ing common housekeeping genes such as cytoplasmic -actin, -2-microglobulin, heat shock protein 1 , and TATA box binding protein. This might be due to technical or real differences. Indeed, other studies employing Af- fymetrix microarrays to examine housekeeping gene ex- pression among different tissues, as well as our SAGE study of the retina during development, similarly found that housekeeping genes vary considerably in their ex- pression levels (Warrington et al., 2000; Blackshaw et al., 2004; Lee et al., 2007). Little is known about the signiﬁcance and extent of Mu¨ ller glial cell heterogeneity, although it is clear that molecular differences exist because the homeodomain- containing transcription factor Chx10 is expressed in only a subset of all Mu¨ ller glial cells (Rowan and Cepko, 2004). Heterogeneous expression of Chx10 could also be observed in the current study (Fig. 4). Heterogeneity of gene expres- sion among three of the Mu¨ ller glial cells was observed across a large cluster (Supplemental Fig. S1). This was further analyzed for a few of these genes by using in situ hybridization (see below). A recent expression analysis of astrocytes has revealed extensive molecular heterogeneity among that class of glial cells as well (Bachoo et al., 2004). Furthermore, morphological studies have identiﬁed exten- sive anatomical differences between M u¨ller glial cells in the central versus peripheral chick retina (Anezary et al., 2001). Further characterization of the glial genes identi- ﬁed in this study will help clarify the diversity of M u¨ller glial cells and perhaps shed light upon a potential func- tional diversity. Mu¨ ller glia enriched genes: Fisher’s exact test To examine the relationships of gene expression pat- terns by using a method other than hierarchical cluster- ing, the data were subjected to an analysis using the Fisher’s exact test (Trimarchi et al., 2007). This test cal- culates the probability that a particular expression pat- tern for a pair of genes across a number of arrays would occur by chance. Supplementary Table T3A shows the genes that have similar a pattern to that of ApoE, with P values more signiﬁcant than 10 4. This method identiﬁed many of the same genes identiﬁed by using hierarchical clustering around ApoE. The Fisher’s exact test was also applied to look for genes with a similar distribution to that of Aqp4 (Supplementary Table T3B). As expected, very similar genes were obtained as for ApoE, conﬁrming the observation that ApoE and Aqp4 are enriched/speciﬁc in Mu¨ ller glia. Some highly expressed and potentially M u¨ller glia speciﬁc genes shown and discussed in Figure 2 could be identiﬁed through this method as well.