236 retinal progenitor cells (Blackshaw et al., 2004). Similarly, a microarray analysis of genes enriched in 4N retinal progenitor cells showed enrichment for genes expressed in Mu¨ ller glia, relative to retinal neurons (Livesey et al., 2004). In addition, some genes expressed at a relatively high level in mature Mu¨ ller glia, such as Glul and Car2, are also expressed in late retinal progenitor cells. To this list of progenitor genes expressed in M u¨ller glia, we can now add Pax6, a gene required for formation of the retina and other ocular structures (Hill et al., 1991; Glaser et al., 1992). This extensive overlap in gene expression between Mu¨ ller glial cells and progenitors, coupled with the obser- vation that Mu¨ ller glia can divide in response to some stimuli, might indicate that Mu¨ ller glia are a form of retinal progenitor cell. Late retinal progenitor cells might become Mu¨ ller glia without an irreversible cell fate determination event, such as occurs when a neuron is generated. Progenitor cells becom- ing Mu¨ ller glia might retain much of the progenitor gene expression program, including those genes that allow a cell to divide (Fig. 3C), whereas upregulating and/or initiating expression of genes required for specialized Mu¨ ller glial functions, such as ApoE, Glul, and GFAP. Candidate genes for regulation of this transition from retinal progenitor cell to Mu¨ ller glia are the genes Notch1, Hes1, Hes5, and Rax, as overexpression of each of these genes in retinal progenitor cells leads to production of cells with at least some properties of Mu¨ ller glia (Bao and Cepko, 1997; Furukawa et al., 2000; Hojo et al., 2000; Jadhav et al., 2006a,b). In contrast, over- expression of positive bHLH genes, such as Math 3, NeuroD, and many others, leads to a reduction in Mu¨ ller glial pro- duction (Morrow et al., 1999; Cai et al., 2000; Inoue et al., 2002). The transition of a late progenitor cell to a Mu¨ ller glia cell might thus depend on the balance of positive and nega- tive bHLH genes, which might normally be regulated by Notch signaling. It appears that Notch activation generally favors formation of cells with glial characteristics not only in the retina, but also elsewhere in the nervous system (Mor- rison et al., 2000; Gaiano and Fishell, 2002). The potential of Mu¨ ller glial cells to produce neurons in the adult retina might suggest that a discrete set of genes limits this ability. In light of this notion, it is interesting to consider the Notch pathway genes further, not only as regulators of the formation of Mu¨ ller glia as discussed above, but also as regulators of the re-entry into the cell cycle and/or as negative regulators of the production of neurons by M u¨ller glia. The single-cell proﬁles for mature M u¨ller glia include Notch1, Notch2, Hes1, Nrarp, and Cntn1, as well as reg- ulators of Notch signaling, such as (Fig. 4), which bear examination in this context. Hes1, a downstream target of NICD, is also highly expressed by neural stem cells in the forebrain (Ohtsuka et al., 2001; Chojnacki et al., 2003), perhaps reﬂecting a similar role. Similarity to other glial cell types A comparison of the Mu¨ ller glia transcriptome with those of other glial cell types of the central nervous system (Bachoo et al., 2004) failed to reveal common clusters of gene expression. This might be due to technical differ- ences or might suggest a different genetic constitution among them. However, there were some transcripts with shared expression among M u¨ller glia and other glial cell types. Mu¨ ller glia enriched transcripts that are also en- riched in astrocytes are peroxiredoxin 6, Basigin (also called CD147), caveolin, CD9 antigen, carboxypeptidase The Journal of Comparative Neurology K. ROESCH ET AL. E, cystatin C, phospho protein enriched in astrocytes (Pea15), Ptn, Ret4, Slc2a1, and Slc3a2 (Bachoo et al., 2004; Vincent et al., 2005). Preliminary comparisons also imply that Mu¨ ller glial cells are less related to Schwann cells (peripheral glia), because many transcripts highly expressed in Mu¨ ller glia are depleted in Schwann cells, i.e., ApoE, clu, cystatin C (Cst3), enolase1 (Eno1), Gapd, Glul, malate dehydrogenase (Mdhl), and myotrophin (Mtpn) (Vincent et al., 2005). Lastly, on the basis of the investigation presented here, we hope to continue our efforts to understand better the role(s) of Mu¨ ller glia in retinal degeneration. Several dis- eases of the retina, such as retinitis pigmentosa and mac- ular degeneration, lead to a severe loss of photoreceptor neurons and involve “activation” of M u¨ller glia. The iden- tiﬁcation of the cellular events and changes that take place within Mu¨ ller glia during the progressive neuronal cell death might be applicable to a therapeutic strategy. ACKNOWLEDGMENTS The authors thank Susan Dymecki for RC::PFwe mice and members of the Cepko and Tabin labs for helpful discussions and advice. LITERATURE CITED Amaratunga A, Abraham CR, Edwards RB, Sandell JH, Schreiber BM, Fine RE. 1996. Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted to the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells. J Biol Chem 271:5628–5632. Anezary L, Medina JI, Sanchez-Nogueiro J, Lopez-Gallardo M, Prada C. 2001. Shape diversity among chick retina Mu¨ ller cells and their post- natal differentiation. J Comp Neurol 438:32–49. Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K. 2006 Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron 50:855–867. Bao ZZ, Cepko CL. 1997. The expression and function of Notch pathway genes in the developing rat eye. J Neurosci 15:1425–1434. Bachoo RM, Kim RS, Ligon KL, Maher EA, Brennan C, Billings N, Chan S, Li C, Rowitch DH, Wong WH, DePinho RA. 2004. Molecular diversity of astrocytes with implications for neurological disorders. Proc Natl Acad Sci U S A 101:8384–8389. Blackshaw S, Fraioli RE, Furukawa T, Cepko CL. 2001. Comprehensive analysis of photoreceptor gene expression and the identiﬁcation of candidate retinal disease genes. Cell 107:579–589. Blackshaw S, Harpavat S, Trimarchi J, Cai, L, Huang H, Kuo WP, Weber G, Lee K, Fraioli RE, Cho S, Yung R, Asch E, Wong W, Ohno-Machado L, Weber G, Cepko CL. 2004. Genomic analysis of mouse retinal devel- opment. PLoS Biol 2:E247. Brady G, Iscove NN. 1993. Construction of cDNA libraries from single cells. Methods Enzymol 225:611–623. Bringmann A, Skatchkov SN, Pannicke T, Biedermann B, Wolburg H, Orkand RK, Reichenbach A. 2000. Mu¨ ller glial cells in anuran retina. Microsc Res Tech 50:384–393. Cai L, Morrow EM, Cepko CL. 2000. Misexpression of basic helix-loop-helix genes in the murine cerebral cortex affects cell fate choices and neuro- nal survival. Development 127:3021–3030. Chang ML, Wu CH, Jiang-Shieh YF, Shieh JY, Wen CY. 2007. Reactive changes of retinal astrocytes and Muller glial cells in kainate-induced neuroexcitotoxicity. J Anat 210:54–65. Chan-ling T. 1994. Glial, neuronal and vascular interactions in the mam- malian retina. Prog Ret Eye Res 13:357–389. Che S, Ginsberg SD. 2004. Ampliﬁcation of RNA transcripts using termi- nal continuation. Lab Invest 84:131–137. Chen CM, Cepko CL. 2000. Expression of Chx10 and Chx10–1 in the developing chicken retina. Mech Dev 90:293–297. Chojnacki A, Shimazaki T, Gregg C, Weinmaster G, Weiss S. 2003. Glyco- protein 130 regulates Notch1 expression and activation in the self-