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Sep 27, 2004 - 1 Department of Ophthalmology, Lied Transplant Center (LTC 11715), ... the early and late stages of histogenesis have distinct .... DIFF call.
Cellular and Molecular Characterization of Early and Late Retinal Stem Cells/Progenitors: Differential Regulation of Proliferation and Context Dependent Role of Notch Signaling Jackson James,1 Ani V. Das,1 Jo¨rg Rahnenfu¨hrer,2 Iqbal Ahmad1 1

Department of Ophthalmology, Lied Transplant Center (LTC 11715), University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6395

2

Max-Plank-Institute for Informatics, Stuhlsatzenhausweg 85, 66123 Saarbru¨cken, Germany

Received 7 January 2004; accepted 1 April 2004

ABSTRACT:

Retinal stem cells/progenitors that define the evolutionarily conserved early and late stages of retinal histogenesis are known to have distinct competence to give rise to stage-specific retinal cell types. However, the information regarding their innate proliferative behavior and phenotypic potential in terms of generating neurons and glia is lacking. Here we demonstrate that, like their counterparts in other central nervous system (CNS) regions during early and late stages of embryonic development, the early and late retinal stem cells/progenitors display different proliferative response to fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) and bias towards generating neurons or glia. Although the former predominantly generate neurons, the latter are partial towards giving rise to glia. Transcription profiling identified classes of genes that are differentially expressed

in early and late retinal stem cells/progenitors in proliferating conditions and suggested that the distinct proliferative response to FGF2 and EGF is likely due to differential expression of FGF receptor 1 (FGFR1) and EGF receptor (EGFR). However, the proliferative maintenance of retinal stem cells/progenitors is likely to include other signaling pathways such as those mediated by insulin-like growth factors (IGFs) and stem cell factor (SCF). Transcription profiling of early and late retinal stem cells/progenitors in proliferating and differentiating conditions suggested a context dependent role for Notch signaling, which may constitute one of the mechanisms underlying the stagedependent phenotypic potential of retinal stem cells/progenitors. © 2004 Wiley Periodicals, Inc. J Neurobiol 61: 359 –376, 2004 Keywords: stem cells; progenitors; retina; Notch; microarray

INTRODUCTION

poral sequence, in which RGCs, cone photoreceptors, horizontal cells, and the majority of amacrine cells are born during early histogenesis while bipolar cells, Mu¨ller glia, and the majority of rod photoreceptors are born during late histogenesis (Kahn, 1974; Young, 1985; LaVail et al., 1991; Prada et al., 1991). The conserved sequence of the generation of retinal cell types suggests that stem cells/progenitors that define the early and late stages of histogenesis have distinct competence to give rise to stage-specific cell types (Morrow et al., 1998; Belliveau and Cepko, 1999; Rapaport et al., 2001). Although emerging evidence suggests important role for factors belonging to bHLH and homeobox classes of transcription factors in the regulation of competence (Hatakeyama et al., 2001),

The vertebrate retina consists of seven major cell types that include rod and cone photoreceptors, retinal ganglion cells (RGCs), horizontal cells, amacrine cells, bipolar cells, and the Mu¨ller glia. Thymidine birth-dating studies have shown that the generation of these cells follows an evolutionarily conserved temCorrespondence to: I. Ahmad ([email protected]). Contract grant sponsors: NIH, and Research to Prevent Blindness. © 2004 Wiley Periodicals, Inc. Published online 27 September 2004 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/neu.20064

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the molecular and cellular properties that define these two progenitor populations remain largely unknown. Here we demonstrate that early and late retinal stem cells/progenitors are distinct in their proliferative responses to different mitogens and possess different potential to generate neurons and glia. Transcriptional profiling of these two subpopulations of cells suggests that the distinct proliferative and differentiation potential of these cells are underpinned by a differential expression of distinct sets of genes that include FGFR1, EGFR, IGFII, and SCF. The transcriptional profiling suggested a context-dependent role for Notch signaling. Analysis of this notion showed that although Notch signaling is utilized by early stem cells/progenitors to maintain themselves in an uncommitted state, it is utilized in late retinal stem cells/ progenitors to generate glia.

METHODS Retinal Cell Culture Timed-pregnant (E14 and E18) Sprague-Dawley rats were obtained from Sasco Laboratories (Wilmington, MA). The gestation day was confirmed by the morphological examination of embryos (Christie, 1964). Embryos were harvested at appropriate gestation periods, and eyes were enucleated. Retinal stem cells/progenitors were isolated from E14 and E18 embryos and cultured as previously described (Ahmad et al., 1999). Briefly, retina were dissected out and dissociated by trypsinization. Optic nerve and remaining mesenchymal tissues were carefully removed before cell dissociation. Retinal neurospheres were generated by culturing the cells for 5 days in culture medium [DMEM/F12, 1⫻N2 supplement (Gibco), 2 mM l-glutamine, 100 U/mL penicillin, 10 mg/mL Streptomycin] supplemented with EGF (20 ng/mL) ⫹ FGF2 (10 ng/mL) ⫹ 0.1% FBS (E14 cells) and EGF (20 ng/mL) (E18 cells). SCF (100 ng/mL)/ IGFII (100 ng/mL) was added to culture medium to analyze the effect on retinal neurosphere generation. For differentiation the clonal neurospheres were plated on poly-D-lysine (250 ␮g/mL)/laminin coated six-well plates and cultured in medium containing 1% FBS for another 6 days.

were captured using cooled CCD-camera (Princeton Instruments) and Openlab software.

cRNA Probe Generation and Microarray Analysis Total RNA was isolated from E14 and E18 neurospheres in proliferating (P) and differentiating (D) conditions using RNeasy mini-RNA isolation kit (Qiagen). First-strand cDNA synthesis was carried out using the Superscript II reverse transcriptase (Gibco) and T7(dT)24 primer at 42°C for 1 h. T4gp32 was added to enhance the first-strand synthesis (Rapley, 1994; Nycz et al., 1998). This was followed by secondstrand synthesis using DNA polymerase I at 16°C for 2 h and incubation with T4 DNA polymerase (10 U) at 16°C for 5 min. The reaction was stopped by adding 0.5 M EDTA and subjected to the phenol:chloroform extraction procedure to remove residual proteins. The final cDNA pellet was suspended in RNAse free water. To prepare cRNA probes, in vitro transcription was carried out in the presence of 10 ␮g of cDNA and biotin-labeled nucleotides using T7 RNA polymerase at 37°C for 5 h. The cRNA probes were quantitated, adjusted to same concentration, and fragmented. Hybridization of biotinylated-cRNA probes to microarrays was carried out by Research Genetics, Inc. using Rat Genome U34A (RGU34A) microarray chips containing sequences corresponding to 8323 known rat neurobiology genes and 417 ESTs. The hybridized arrays were scanned using an Agilent GeneArray scanner, and the raw data was initially normalized against housekeeping genes represented in the array. The analysis of hybridization data, using the Affymetrix MicroArray Suite Software, provided the average difference (AD) values for the expression 8740 genes, for four different conditions (i.e., early retinal stem cells/progenitors in proliferating and differentiating conditions; late retinal stem cells/progenitors in proliferating and differentiating conditions). Thus, for every gene, four AD values were obtained. The AD values were subjected to box plot analysis to normalize any difference between samples. The box plot of all possible values, plotted on log2 scale were similar; therefore, no further normalization was applied. The relative expression of transcripts in four groups was measured using ABS call and DIFF call. The ABS call, based on p-values, obtained using Wilcoxon signed-ranked test, determined whether a particular transcript corresponding to a particular gene is present or absent. Genes thus sorted were ordered by fold change values, that is, the expression ratio, corresponding to any two groups. The microarray results were corroborated by RT-PCR and immunocytochemical analyses.

Immunoflourescence Analysis Immunofluorescence analysis was carried out for the detection of cell-specific markers and BrdU as described previously (Ahmad et al., 1999). Briefly, paraformaldehyde fixed cells were incubated in PBS containing 5% NGS and 0, 0.2, or 0.4% Triton X-100, followed by an overnight incubation in antibodies for Nestin, Map2, GFAP, EGFR, FGFR1, and BrdU at 4°C. Cells were examined for epifluorescence following incubation in IgG conjugated to Cy3/FITC. Images

RT-PCR Total RNA was isolated from cultured cells using RNeasy kit (Qiagen) and RT-PCR carried out with gene-specific primers (Ahmad, 1995; Bhattacharya et al., 2003; James et al., 2003a). Approximately 2 ␮g of RNA was transcribed into cDNA and amplified using gene-specific forward and reverse primers. PCR products were re-

Transcriptional Profiling of Retinal Progenitors solved in 2% agarose gel and visualized with ethidium bromide staining.

Perturbation of Notch Signaling Notch signaling was upregulated by coculturing enriched E14 and E18 stem cells/progenitors with cell line, constitutively expressing the Notch ligand Jagged-1 (Lindsell et al., 1995). Notch signaling was attenuated by culturing E14 and E18 retinal cells in the presence of 5 ␮M of N-[N-(3,5Diflurophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT), a gamma-secretase inhibitor (Calbiochem, CA) as previously described (Ge et al., 2002).

RESULTS Early and Late Retinal Stem Cells/Progenitors Display Different Proliferative and Differentiation Potential The difference in the number and types of cells generated during early and late histogenesis suggests that the proliferative potential of early and late retinal stem cells/ progenitors is different. To test this notion, we examined the relative response of these cells to different mitogens in generating neurospheres. Retinal cells isolated from early (E14) and late (E18) stages of histogenesis were cultured in the presence of EGF/FGF2 and the number of neurospheres generated was determined (Fig. 1). The early and late stem cells/progenitors generated retinal neurospheres in all three conditions. The majority of cells in the retinal neurospheres (⬃90%) were proliferating and expressed progenitor markers, Nestin and Musashi [Fig. 1(A–P)] (Pevny and Rao, 2003). The number of retinal neurospheres generated in different mitogens was remarkably different. In the presence of FGF2, early retinal stem cells/progenitors generated significantly more neurospheres than late retinal stem cells/ progenitors (2917.06 ⫾ 726 vs. 1364.17 ⫾ 225.28; p ⬍ 0.001). In contrast, late retinal stem cells/progenitors generated significantly more neurospheres than early retinal stem cells/progenitors, in the presence of EGF (2260 ⫾ 370 vs. 3412 ⫾ 107.93; p ⬍ 0.01). A synergistic effect on the generation of neurospheres by early retinal stem cells/progenitors was observed when they were cultured in the presence of both EGF and FGF2 (4329.19 ⫾ 811 vs. 1564.77 ⫾ 100.38; p ⬍ 0.001). Such a synergistic effect was not observed in the case of late retinal stem cells/progenitors. On the contrary, the number of neurospheres generated by these cells decreased significantly in the presence of EGF and FGF compared to those cultured in the presence of EGF alone, suggesting that the mitogenic effect of FGF2 that is evident in early retinal stem cells/progenitors has changed into a

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nonmitogenic response for late retinal stem cells/progenitors. The fact that gliogenesis takes place only during late retinal histogenesis suggests that the early and late retinal stem cells/progenitors may be intrinsically different in their ability to generate neurons and glia. To test this premise, neurospheres generated by early and late retinal stem cells/progenitors were cultured in conditions that promote differentiation, and the proportion of cells expressing neuronal (Map2) and glial (GFAP) markers was examined. The proportion of cells expressing Map2 was significantly higher in neurospheres generated by early retinal stem cells/progenitors than those generated by late retinal stem cells/progenitors (17.48% ⫾ 9.33 vs. 8.23 ⫾ 4.00; p ⬍ 0.05). In contrast, the proportion of cells expressing GFAP was significantly higher in neurospheres generated by late retinal stem cells/progenitors compared to those generated by early retinal stem cells/ progenitors (9.33 ⫾ 1.92 vs. 21.47 ⫾ 4.2; p ⬍ 0.01), suggesting the proclivity of early and late retinal stem cells/progenitors to generate neurons and glia, respectively (Fig. 2).

Early and Late Retinal Stem Cells/Progenitors Differentially Express Distinct Classes of Genes in Proliferating Conditions To understand the underlying mechanism of distinct proliferation and differentiation potential of early and late retinal stem cells/progenitors, we carried out transcription profiling of these cells in proliferating and differentiating conditions using microarray analysis. Prior to analysis, determination of cellular composition of neurospheres revealed that the majority (⬃90%) of cells in both early and late primary neurospheres in the proliferating condition consists of BrdU- and nestin-positive cells. However, a minor population (⬍10%) of BrdU-negative cells was detected expressing markers characteristic of differentiated cells, suggesting the heterogeneous nature of the neurospheres. Therefore, we expected detection of transcripts corresponding to a few phenotype-specific genes of early or late-born retinal cell types in proliferating condition (see below). Analysis of AD values for the expression of 8740 genes in each of the four conditions revealed similar box plots, suggesting that further normalization for intersample variation is not required [Fig. 3(A)]. Figure 3(B–D) shows scatter plot analysis that identified candidate genes expressed in four different groups that passed the selection on the basis of ABS call and DIFF call and had a fold change above 2 or below 1/2. Using the above criteria, 427 genes were identified that showed a differential expression pattern between early and late retinal stem

Figure 1 .

Figure 1 Early and late retinal stem cells/progenitors display distinct proliferative potential. Retinal cells, isolated from early (E14) and late (E18) stages of histogenesis, when cultured in serum-free medium supplemented with mitogens generate neurospheres containing BrdU-positive cells expressing stem cell markers, nestin (A–D) and Musashi (I– L). The colocalization of BrdU and Nestin (E–H)/Musashi (M–P) is apparent in cells in the retinal neurospheres that have spread out. E14 and E18 stem cells/progenitors display differential ability to generate retinal neurospheres in the presence of EGF, FGF2, and EGF ⫹ FGF2 (E). Arrowhead ⫽ double-labeled cells (200⫻). **p ⬍ 0.01 and ***p ⬍ 0.001 when compared to control. Figure 2 Early and late retinal stem cells/progenitors display distinct differentiation potential. Neurospheres generated by early (E14) and late (E18) retinal progenitors were cultured in differentiating condition and potential of cells in retinal neurospheres to express Map2 and GFAP was examined by double immunocytochemical analysis. E14 retinal neurospheres contained more Map2-positive cells (B, arrows, G) than GFAP-positive cells (C, arrowhead, G), whereas E18 neurospheres contained more GFAP-positive cells (E, arrowheads, G) than MAP2 positive cells (F, arrows; G) (200⫻). *p ⬍ 0.05 and **p ⬍ 0.01 when compared to E14.

Figure 2

Transcriptional Profiling of Retinal Progenitors

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Figure 3 Identification of differentially expressed genes in early and late retinal stem cells/ progenitors by microarray analysis. Retinal neurospheres generated by early (E14) and late (E18) retinal stem cells/progenitors in proliferating (P) and differentiating (D) conditions were subjected to microarray analysis. Box plots analyses the intersample variations (A) and scatter plots of arrays identify outlying genes (red spots) whose expression differs between early and late stem cells/ progenitors in proliferating conditions (B), early progenitors in proliferating and differentiating conditions (C) and late progenitors in proliferating and differentiating conditions (D).

cells/progenitors in proliferating conditions. This represented 4.8% of genes on the array. Out of these genes, 82.45% are expressed in both early and late retinal stem cells/progenitors, 11.4% are expressed predominantly in early retinal stem cells/progenitors, and 5.14% are expressed in late retinal stem cells/ progenitors [Fig. 4(A)]. The result of transcription profiling was corroborated by RT-PCR analysis of some of these genes [Fig. 4(B)]. Northern analysis was not carried out due to the limitation of cells. These genes belong to different functional classes (Table 1). The fidelity of transcription profiling was reflected indirectly by the differential expression of marker genes that identify cells generated by early and late stem cells/progenitors. For example, the transcripts corresponding to Islet-1 [Fig. 4(B); accession # S6329], a marker of early generated neurons, RGC (Brown et al., 2000; Rachel et al., 2002), are predominantly expressed in early retinal stem cells/progenitors, whereas peripherin [Fig. 4(B); accession # X52376], a marker of rod photoreceptors (Molday et al., 1987; Arikawa et al., 1992), which are generated during late retinal histogenesis, is expressed predominantly in late retinal stem cells/progenitors. The expression of both genes is not the characteristic of proliferating retinal stem cells/progenitors, but corresponds to a minor population of differentiated cells in

retinal neurospheres that are likely to be generated due to asymmetrical division of retinal stem cells/ progenitors (Sommer and Rao, 2002; Zhong, 2003). A comparison of transcription profiling of retinal stem cells/progenitors in the proliferating condition has revealed genes that may underlie distinct proliferative potential of early and late retinal stem cells/ progenitors. These include FGFR1, IGF2, and SCF. For example, the levels of FGFR1 expression, which is twofold higher in early retinal stem cells/progenitors compared to those in late retinal stem cells/ progenitors, may explain the preferential mitogenic response of the former to FGF2 (Fig. 1). Similarly, the mitogenic responsiveness of the late retinal stem cells/ progenitors to EGF is likely due relatively high expression of EGFR. Because sequence corresponding to EGFR was not represented in the Affymetrix chip, we tested the premise by immunocytochemical analysis of EGFR immunoreactivity in neurospheres generated by early and late retinal stem cells/progenitors (Fig. 5). We carried out immunocytochemical analysis of FGFR1 to corroborate differential expression FGFR1, as revealed by transcription profiling. The level of FGFR1 immunoreactivity was higher in neurospheres generated by early retinal stem cells/progenitors compared to those generated by late retinal stem cells/progenitors, corroborating transcriptional

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Figure 4 Identification of differentially expressed genes in proliferating early and late retinal stem cells/progenitors. Venn diagram shows overlapping and distinct expression of 427 genes in early and late retinal stem cells/progenitors in proliferating conditions (A). The nonoverlapping expression of some of the genes in early and late retinal progenitors was corroborated by RT-PCR analyses (B). Genes listed by accession number can be identified in Table 1, which represents the functional classification of nonoverlapping genes. 1 ⫽ early stem cells/progenitors; 2 ⫽ late stem ceels/progenitors; M ⫽ markers.

profiling results. In contrast, the level of EGFR immunoreactivity was higher in neurospheres generated by late retinal stem cells/progenitors compared to those generated by early retinal stem cells/progenitors, supporting the notion that EGF-responsiveness of the former correlates with EGFR expression levels. Signaling mediated by IGFs is known to regulate such diverse functions as growth, metabolism, reproduction, and longevity (Oldham and Hafen, 2003). In teleost and chick retina, insulin and IGFs have been observed to promote the proliferative phase of neurogenesis (Mack and Fernald, 1993; Garcia-de Lacoba et al., 1999; Otteson et al., 2002). SCF-mediated signaling is known to play an important role in the maintenance of stem cells during hematopoiesis, gametogenesis, and melanogenesis (Ashman, 1999). We have recently shown that SCF-cKit receptor-mediated signaling regulate proliferation of neural stem cells, isolated from adult ciliary epithelium (Das et al., 2004). It is likely that both IGFs and SCF regulate proliferation of retinal stem cells/progenitors because levels of transcripts corresponding to IGFII and SCF are increased by 14.7- and 5.7-fold (Table 1), respec-

tively, in early retinal stem cells/progenitors compared to those in late retinal stem cells/progenitors. To test this hypothesis, the generation of neurospheres by early and late retinal stem cells/progenitors was analyzed in the presence of SCF or IGFII (Fig. 6). There was a significant difference in the number of retinal neurospheres generated by both early and late retinal stem cells/progenitors, in the presence of either IGF2 (early retinal stem cells/progenitors: 5248.85 ⫾ 217.93 vs. 3552.02 ⫾ 165.85, p ⬍ 0.001; late retinal stem cells/progenitors: 4468.59 ⫾ 195.78 vs. 2608.21 ⫾ 290.93 vs. p ⬍ 0.01) or SCF (early retinal stem cells/progenitors: 6285.2 ⫾ 323.48 vs. 3552.02 ⫾ 165.85, p ⬍ 0.001; late retinal stem cells/progenitors: 6201.42 ⫾ 607.86 vs. 2608.21 ⫾ 290.93 vs. p ⬍ 0.001), compared to controls. However, there was a significant difference in the response of early and late retinal stem cells/progenitors to IGF2 or SCF in the generation of neurospheres; the latter generated more retinal neurospheres than the former (IGFII: 1.47 fold vs. 1.71 fold; SCF: 1.77 fold vs. 2.38 fold).

Early and Late Retinal Stem Cells/Progenitors Differentially Express Distinct Classes of Genes in Differentiating Conditions To identify classes of genes, that may be involved in the differentiation, we carried out K-means analysis to group differentially expressed genes into different clusters based on the similar expression dynamics between early and late retinal stem cells/progenitors in proliferating and differentiating conditions. This was achieved by two filtering steps. First, a log transformation with base 2 was applied to all AD values. To include only those genes that displayed a significant difference in expression, genes with the log ratio less than 5 between any two of the four conditions were omitted. The remaining 1421 genes were grouped into 16 different clusters (Fig. 7). Second, the number of genes belonging to a specific cluster was further refined by using cutoffs for AD values for high and low expression of selected genes. The cutoff for the high expression levels was set to 8 and for low expression to 6 on a log2 scale; therefore, selecting only those among 1421 genes that displayed at least fourfold change in levels of expression. Following this filtering step, 147 genes remained, distributed in 16 different clusters, which were assigned to different functional groups [Fig. 8A–B)]. Cluster 1 is the smallest cluster and contains genes that are highly expressed in all four groups and exemplified by constitutively expressed bHLH transcription factor E12. E12 dimerizes with tissue-spe-

Transcriptional Profiling of Retinal Progenitors Table 1

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Gene-Specific Primers Used for RT-PCR Analyses

Gene Name (Accession No.)

Forward Primer

Reverse Primer

somatostatin (K02248) 5⬘-CAACCAGACAGAGAACGATGC-3⬘ TLP1 (U89282) 5⬘-TTTCGCAACTGCCTGTTCCC-3⬘ ERK5/BMK1 (AJ005424) 5⬘-TGTTCTCAGGCACTCCAAAGGG-3⬘ Isl-1 (S69329) 5⬘-ATTTCCCTATGTGTTGGTTGCG-3⬘ SCF (AF071204) 5⬘-ACTTGGGAAAATAGTGGATGACCTC-3⬘ IGF-BP3 (M31837) 5⬘-AATCATCTGAAGTTCCTCAATGTGC-3⬘ Interferon induced mRNA (X61381) 5⬘-AACCACACTTCTCAAGCCTTC-3⬘ AI169327 (EST215162) 5⬘-AGACCCCAAGGATTGCCAG-3⬘ 33-kDa phototransducing protein (M33528) 5⬘-ACTGCGTGCCTTCCGATTTC-3⬘ Furosemide-sensitive K-Cl cotransporter (U55816) 5⬘-AGGGAAGCAAAGAGCACGAAG-3⬘ NglyR (X57281) 5⬘-AGCCTGCTGATAGTCATTTTGTCC-3⬘ Neural Visinin like Protein (D10666) 5⬘-GGGGAAACAGAATAGCAAACTGG-3⬘ Apo D (X55572) 5⬘-TCCTGTGGAAACTGCCTTCATC-3⬘ c-kit (D12524) 5⬘-AAGGCACAGAAGGAGGCACTTAC-3⬘ Major synaptic vesicel protein p38 (X06655) 5⬘-TGTCACCGTGGCTGTGTTTG-3⬘ Retinal degradation slow (X52376) 5⬘-ATCAAGAGTAATGTGGATGGGAGG-3⬘ Notch1 (NM_008714) 5⬘-TCTGGACAAGATTGATGGCTACG-3⬘ Delta-1 (NM_032063) 5⬘-CGACCTCGCAACAGAAAAC-3⬘ Hes1 (NM_024360) 5⬘-GCTTTCCTCATCCCCAATG-3⬘ ␤-actin (XM037235) 5⬘-GTGGGGCGCCCCAGGCACCA-3⬘

cific bHLH transcription factors such as MyoD (expressed in developing myoblasts) and NeuroD (expressed in developing neuroblasts) to activate tissuespecific gene expression (Bertrand et al., 2002; Maleki et al., 2002). Clusters 12 and 2 represent group of genes whose expression levels are relatively higher in differentiating early and late retinal stem cells/ progenitors, respectively, and therefore, may participate in the process of differentiation or encode differentiation markers. Cluster 12 is exemplified by the preferential expression of c-Met RTK. c-Met RTK, and its ligand hepatocyte stimulating factor (HSF),

5⬘-ATGGGATTTGGAGGAGAGGG-3⬘ 5⬘-TCCTTTCCTCTGAGACGCACC-3⬘ 5⬘-AGACTCAATGTCAGCGGGGTTC-3⬘ 5⬘-TTCTTGCTGAAGCCTATGCTGC-3⬘ 5⬘-CAGGACCTAATGTTGAAGAGAGCAC-3⬘ 5⬘-TTTCCCCTTGGTGTCATAGCC-3⬘ 5⬘-TATCACCTGAGCCCATCTCTGC-3⬘ 5⬘-TGTTTCCCTGTTCAGCCATC-3⬘ 5⬘-TCTGCTGAGCACCTTCTGTAGTCTG-3⬘ 5⬘-GGAAATGGCTGTGAGCATCG-3⬘ 5⬘-TTCCTTATTCTGCCTCTTCTGTCG -3⬘ 5⬘-CACAGATGAACTCTCGGAAGTCG-3⬘ 5⬘-GCTTCACCCTCAACTTGGTTCAG-3⬘ 5⬘-TCCAAATGGTGACACAGACGC-3⬘ 5⬘-TCCCTTGATAATGTTCTCTGGGTCC-3⬘ 5⬘-AGAGCCAGATGAGAAGCGTGAC-3⬘ 5⬘-CGTTGACACAAGGGTTGGACTC-3⬘ 5⬘-ATGGAGACAGCCTGGGTATC-3⬘ 5⬘-CGTATTTAGTGTCCGTCAGAAGAG-3⬘ 5⬘-CTCCTTAATGTCACGCACGATTTC-3⬘

also known as the scatter factor, are expressed in developing central and peripheral nervous system, and signaling mediated by them is thought to regulate cell-survival, differentiation, axonal growth, guidance, and migration (Maina et al., 1998; Thewke and Seeds, 1999; Powell et al., 2001; Giacobini et al., 2002; Sun et al., 2002). The high expression of c-Met RTK in differentiating early retinal stem cells/progenitors, besides promoting survival of neuroblasts and their differentiation (Maina et al., 1998), may mediate chemoattraction for axonal guidance of early born neurons, such as RGCs. Cluster 2 is exemplified by

366 Table 2

James et al. Genes Expressed in Early and Late Retinal Progenitors

Accession No.

Fold Change

A09811 AJ005424 AF016296 AF071204 M31837 M33528 X57281 AF084576 M96601 O12524 K02248 M91595 L21192 J04486 X17012 L34049 J03624 U55816 X06655 X61381 D10666 U89282 D12516 AF000942 D82074 S69329 M87634 AF001417 L16995 L26268 D38492 D00913 M60921 U23146 M72422 X12459 D90048 M19257 U02553 U89529 X07729 U18729 AF001898 M24324 M32062 J02962 M61875 M25638 Z12152 X86789 U88958 X81449 Y17048 X55572 L00191

74.8 16.2 9.4 7.5 13.8 ⫺7.5 27.6 ⫺4.7 ⫺4.1 ⫺2.4 10.9 7.3 10.7 4.8 14.7 8.6 5.8 ⫺55.2 ⫺4.3 5.7 ⫺7.5 19.2 ⫺41.2 15.6 ⫺15.5 20.4 6.4 20.5 ⫺11.8 ⫺2.6 ⫺5.1 5.8 ⫺3.7 15.9 ⫺4.2 ⫺13.4 ⫺9.8 25 20.8 ⫺27.6 ⫺4.4 40.6 ⫺4.4 7.6 7.0 3.8 ⫺19.9 34.6 87.1 165.8 38.6 ⫺6 ⫺6.3 ⫺3.9 6.9

Descriptions BRL-3A binding protein BMK1/ERK5 protein Neuropilin Stem cell factor (KL) IGF-BP3 33-kDa phototransducing protein Neonatal glycine receptor Delta 3 Taurine transporter c-Kit receptor tyrosine kinase Somatostatin-14 IGF-BP2 GAP-43 IGF-BP IGF II Megalin/gp330 Galanin Furosemide-sensitive K-Cl cotransporter [KCC2] Major synaptic vesicel protein p38 Interferon induced mRNA Neural Visinin like protein Telomerase protein component 1 (TLP1) Hes5 Id3a BHF-1 Isl-1 BF-1, a member of HNF-3/fork head gene family Zf9, a Kruppel-like transcription factor ADD1, a novel HLH transcription factor Antiproliferative factor (BTG-1) Neural adhesion molecule Intercellular adhesion molecule-1 NGF-inducible anti-proliferative secreted protein Mitogenic regulation SSeCKS (322) GAD-65 Arginosuccinate synthetase Na⫹K⫹ ATPase Cytosolic retinol-binding protein (CRBP) Protein tyrosine phosphatase Fatty acid transport protein Neuron-specific enolase Cytochrome b558 Aldehyde dehydrogenase MHC class I RT1 (RTS) Fc-gamma receptor IgE binding protein CD44 Neurofilament (NF-L) Neurofilament (NF-M) Sensory neuron synuclein Neuritin Keratin Caldendrin ApoD Fibronectin

Biological Function Signal transduction

Transcritpion/DNA binding

Cell cycle

Metabolism/mitochondrial

Immune related

Cytoskeleton

Transcriptional Profiling of Retinal Progenitors Table 2

(Continued)

Accession No.

Fold Change

U27767 U40999 X52376 M60737 AF031878 AA800882 AA875531 AA875659 AA891596 AA892506 AA894087 AA894092 AI171962 AI172064 AI228669

⫺5.3 ⫺157.3 ⫺23.0 ⫺27.4 25.6 ⫺27.3 24.2 17.4 ⫺11.2 13.3 ⫺15.5 16.7 22.6 10.8 ⫺48.1

AI169327

367

16.9

Descriptions

Biological Function

RGP4 RRG4 Retinal degeneration slow (rds) Retinal S-antigen Peripherin EST190379 rat cDNA clone RLUAM60 3⬘ end 48.44% homologous to rat cytochrome b 96% homologous to mouse clone: 6230400G14 83.29% homotogous to mouse clone AC116713 99% homologous to rat actin binding protein 1A NM_130411 38% homologous to mouse clone AL596331 54.4% homologous to mouse clone BC031449 99% homologous to rat annexin 1 98.23% homologous to rat lectin (BC058476) 51.18% homologous to rat GABA transporter protein (NM_024371) 100% homologous to rat tissue inhibitor of metalloproteinase-1

Cell-specific

the preferential expression of GFAP, RRG4, and CB90. The relatively high expression of GFAP in differentiating late stem cells/progenitors corroborates our observation that these cells are preferential towards generating glia. The potential of late retinal stem cells/progenitors to generate late-born neurons is reflected in the high expression of photoreceptorspecific gene RRG4, a homolog of Caenorhabditis elegans gene Unc-119 (Higashide et al., 1998). CBP90 binds to brain-specific protein, cortactin, which is known to regulate the actin cytoskeleton in the brain (Ohoka and Takai, 1998; Cheng et al.,

Ests

2000). It is likely that CBP90 may participate in morphogenesis of differentiating late stem cells/progenitors.

Notch Signaling Subserve Different Function in Early and Late Stem Cells/ Progenitors Cluster 13 (Fig. 7) consists of genes that encode components of canonical Notch signaling pathway, i.e., Notch1, Delta1, and Hes1. The expression of these components in early and late stem cells/progen-

Figure 5 Early and late retinal progenitors display differential expression of FGFR1 and EGFR. Retinal neurospheres generated by early (E14) and late (E18) retinal progenitors were analyzed for the expression of FGFR1 and EGFR by immunocytochemical analysis. The levels of FGFR1 immunoreactivity were higher in early (A,B) than in late (C,D) retinal progenitors, whereas levels of EGFR were higher in late (G,H) than in early (E,F) retinal stem cells/progenitors (200⫻).

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Figure 6 IGFII and SCF influence the generation of neurospheres by retinal stem cells/progenitors. Retinal cells isolated from early (E14) and late (E18) stage of histogenesis were cultured in the presence of IGFII and SCF and the generation of neurospheres was examined. Controls included E14 and E18 cells cultured in EGF ⫹ FGF2 and EGF, respectively. Both SCF and IGFII significantly influenced the number of retinal neurospheres generated by early and late retinal stem cells/progenitors *p ⬍ 0.05.

itors in proliferating and differentiating conditions was overlapping. However, the trend of their expression, which was verified by RT-PCR analysis (Fig. 9), was different between early and late stem cells/progenitors in differentiating conditions. The expression levels of Notch1, Delta1, and Hes1 transcripts were higher in early retinal stem cells/progenitors in differentiating condition compared to those in proliferating condition, supporting the notion that Notch signaling is activated during early neurogenesis to maintain a pool of stem cells/progenitors by keeping them uncommitted and undifferentiated (Artavanis-Tsakonas et al., 1999). The apparent difference in levels of expression of Hes1 in K-mean (Fig. 7) and RT-PCR (Fig. 9) analyses is due to the fact that the red line in the former represents the average expression patterns of 120 genes in cluster 13. Assessment of the expression of individual genes, such as Hes1, showed a pattern similar to that obtained by the RT-PCR analysis, and therefore, suggested that Notch signaling is activated in early retinal stem cells/progenitors in differentiating conditions. In contrast, the expression levels of Notch signaling components decreased in differentiating late retinal stem cells/progenitors, suggesting a decreased utilization of Notch signaling for the maintenance of stem cell population during late neurogenesis. However, the persistence of their expression, albeit at low levels, was indicative of Notch function other than regulation of cell commitment. To test this notion we examined the neural differentiation of early and late retinal stem cells/progenitors in response to perturbation in Notch signaling. Accentua-

tion of Notch signaling was achieved by coculturing retinal stem cells/progenitors with fibroblast expressing Jagged1 (JT) (Lindsell et al., 1995). Controls included coculturing with untransformed fibroblasts (LTK⫺). Inhibition of Notch signaling was achieved by culturing retinal stem cells/progenitors in the presence of DAPT, a gamma secretase inhibitor (Ge et al., 2002). There was no significant difference in the proportion of cells expressing Map2 or GFAP (Map2: 8.29 ⫾ 1.24% vs. 8.97 ⫾ 1.11%; GFAP: 6.29 ⫾ 1.52% vs. 8.95 ⫾ 1.01%) when early retinal stem cells/progenitors were cocultured with either JT or LTK⫺ cells [Fig. 10(A–D,Q)]. However, when early stem cells/progenitors were cultured in the presence of DAPT, the proportion of both Map2- and GFAPpositive cells increased significantly (Map2: 7.33 ⫾ 0.70% vs. 18.17 ⫾ 3.22%, p ⬍ 0.05; GFAP: 5.23 ⫾ 0.89% vs. 21.23 ⫾ 0.18%, p ⬍ 0.001) compared to those cultured in controlled conditions [Fig. 10(I– L,R)], suggesting that a decrease in Notch signaling leads to the generation of both neurons and glia. When late retinal stem cells/progenitors were cocultured with Jagged1 expressing JT cells, a significant decrease and increase in Map2- and GFAP-positive cells were observed (Map2: 5.59 ⫾ 0.83% vs. 3.12 ⫾ 0.27%, p ⬍ 0.05; GFAP: 8.89 ⫾ 1.46% vs. 19.22 ⫾ 2.83%, p ⬍ 0.001), respectively, compared to controls [Fig. 10(E–H,S)], suggesting that an increase in Notch signaling, unlike its effect on early retinal stem cells/progenitors, promotes glial differentiation in late retinal stem cells/progenitors. This notion was confirmed by exposing late retinal stem cells/progenitors to DAPT; while the proportion of Map2-positive cells increased, those expressing GFAP decreased significantly (Map2: 3.15 vs. 0.32 vs. 7.57 ⫾ 0.38, p ⬍ 0.001; GFAP: 16.11 ⫾ 1.91 vs. 4.93 ⫾ 0.32, p ⬍ 0.001) compared to controls [Fig. 10(M–P,T)].

DISCUSSION The early and late retinal stem cells/progenitors underpin the generation of distinct cell types in two stages of retinal histogenesis. Their competence for stage-dependent cell fate specification is reflected by differential responsiveness to growth factors and potential to generate neurons and glia. Like their counterparts in other regions of the CNS during early and late embryonic development (Tropepe et al., 1999; Temple, 2001), early and late retinal stem cells/progenitors show preference for FGF2 and EGF, respectively, for proliferation. The responsiveness of early retinal stem cells/progenitors to FGF2 is likely due to relatively high expression of FGFR1, the receptor that

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Figure 7 Differential expression of genes in proliferating and differentiating early and late stem cells/progenitors. K-mean analysis was used to group 1421 genes in 16 clusters based on similar expression dynamics between early (E14) and late (E18) retinal progenitors in proliferating (P) and differentiating (D) conditions. The representative gene in each cluster is identified in the box and referenced in Figure 8.

is thought to primarily mediate the effects of FGF2 (Johnson and Williams, 1993). The preferential proliferative responsiveness to FGF2 is also characteristic of ciliary epithelial neural stem cells/progenitors (Ahmad et al., 2000; Fischer and Reh, 2003), which is likely to be an antecedent to early retinal stem cells/ progenitors because of shared properties (Ahmad et al., 2004). There is a significant decrease in levels of both FGFR1 transcripts and immunoreactivity in late retinal stem cells/progenitors that may explain their lack of responsiveness to FGF2 for proliferation. Their proliferative responsiveness to EGF is correlated with high levels of EGF-R immunoreactivity in retinal neurospheres. This observation is consistent with the in vivo finding that progenitors during late retinal histogenesis (E18) express higher levels of EGF-R than those in early retinal histogenesis (E14) (Lillien and Wancio, 1998). The mechanism underlying the temporal changes in the proliferative behavior

of early and late retinal stem cells/progenitors is not well understood. Because early and late retinal stem cells/progenitors represent two temporally segregated subpopulations of retinal stem cells/progenitors, it could be suggested that that the former may influence the proliferative property of the latter, as it has been for progenitors elsewhere in the CNS (Burrows et al., 1997; Tropepe et al., 1999; Lillien and Raphael, 2000). The importance of the maintenance of a pool of early retinal stem cells/progenitors for sustaining both early and late retinal histogenesis, suggests that multiple signaling mechanisms, besides those mediated by FGF2 and EGF, may regulate their proliferation. Evidence is emerging that stem cells/progenitors recruit disparate signaling pathways to maintain their proliferation (Ahmad et al., 2004; Reya et al., 2003). Transcription profiling of early and late retinal stem cells/progenitors in proliferating condition identified

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Figure 8 Relative gene expression in proliferating and differentiating stem cells/progenitors. Genes (147) distributed in different clusters by K-mean analysis are represented by their relative expression levels using red (highest) and green (lowest) color intensities. Their assignments to functional classes are provided. P ⫽ proliferating condition; D ⫽ differentiating condition; R ⫽ color range corresponding to fold change in expression.

two additional signaling pathways for the regulation of cell proliferation. Signaling mediated by IGFs is known to promote cell proliferation (Heyner et al., 1989; Ferry et al., 1999). The relatively high expression levels of IGFII and IGF binding proteins and positive influence of IGFII on the generation of neurospheres by early and late retinal stem cells/progen-

itors suggests that IGFII-mediated signaling may play an important role in proliferation and represent an evolutionarily conserved mechanism for the maintenance of retinal stem cells/progenitors. This notion is supported by evidence of insulin and IGFs-mediated signaling in the developing fish and chick retina and the recent observation that the IGFs-mediated signal-

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Figure 8 (Continued)

ing sustains the proliferative phase of persistent retinal neurogenesis in the adult fish (Mack and Fernald, 1993; Garcia-de Lacoba et al., 1999; Otteson et al., 2002). Signaling mediated by SCF is known to regulate proliferation of stem cells during hematopoiesis, gametogenesis, and melanogenesis (Ashman, 1999). It has recently been observed that SCF plays an important role in proliferation and differentiation of neural stem cells, isolated from the ciliary epithelium of

adult rat (Das et al., 2004). The expression of SCF and its receptor c-Kit in retinal stem cells/progenitors, and SCF-dependent increase in the generation of neurospheres suggest that SCF-mediated signaling may represent a mechanism utilized by stem cells in the optic neuro-epithelium, in which both retina and ciliary epithelium have their common origin. The increased proliferative responsiveness of late retinal stem cells/ progenitors to IGF/SCF compared to their early

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Figure 9 Components of canonical Notch pathway (cluster 13) are differentially expressed in early and late retinal stem cells/progenitors. Expression of transcripts corresponding to Notch1, Delta1, Hes1, and Hes5 was analyzed in early (E14) and late (E18) retinal stem cells/progenitors in proliferating (P) and differentiating (D) conditions by RT-PCR. The expression of Notch pathway components transcripts increased in differentiating early retinal stem cells/progenitors compared to those in proliferating early retinal stem cells/progenitors. In contrast, their expression decreased in differentiating late retinal stem cells/progenitors compared to those in proliferating late retinal stem cells/progenitors.

counter parts is likely due to relatively higher levels of expression of respective receptors in the former. This notion is supported by the observation that the level of expression of c-Kit is higher in late retinal progenitors than in the early progenitors. The phenotypic potential of early and late retinal stem cells/progenitors in terms of generating neurons and glia is similar to that of neural stem cells/progenitors elsewhere in the brain during the specific stages of development. Evidence obtained from a variety of approaches suggests that during the early stage of brain development, stem cells/progenitors are partial towards generating neurons, whereas during the late stage when gliogenesis prevails, stem cells/progenitors acquire proclivity towards generating glia (Zhu et al., 1999; Lillien and Raphael, 2000; Zhu et al., 2000; Temple, 2001). Although the development of distinct mitotic responsiveness of early and late stem cells/ progenitors to FGF2 and EGF correlates with phenotypic potential, it is unlikely to be causally related to neuronal-glial biases of these two cell population. For

example, treatments of stem cells/progenitors with EGF or blocking EGFR signaling does not influence the bias of stem cells/progenitors to generate neurons or glia (Lillien and Raphael, 2000; Zhu et al., 2000). Given the notion that the developmental change in phenotypic potential is influenced by epigenetic cues, a differential response to Notch signaling by early and late retinal stem cells/progenitors may explain their relative preference to give rise to neurons or glia. Recent observations that Notch signaling could promote gliogenesis in retina and other brain regions suggested a new function for Notch signaling other than its classical role in maintaining cells in uncommitted state (Furukawa et al., 2000; Wang and Barres, 2000). The expression patterns of components of the canonical Notch pathway suggested that the role of Notch signaling as the regulator of cell commitment and glial differentiation is context dependent. During early histogenesis, Notch signaling is upregulated both in proliferating and differentiating conditions to maintain a pool of stem cells/progenitors in an uncommitted state by suppressing differentiation. Therefore, the accentuation of Notch signaling in early retinal stem cells/progenitors did not lead to a significant change in neuronal or glial differentiation. However, when Notch signaling was attenuated in these cells by DAPT, there was a significant increase in cells expressing neuronal and glial markers. Given the classic inhibitory influence of Notch signaling on cell differentiation, the generalized neural differentiation in response to decrease in Notch signaling was expected. In contrast to the response of early retinal stem cells/progenitors to the perturbation of Notch signaling, accentuation and inhibition of Notch signaling in late retinal stem cells/progenitors led to a significant increase and decrease in glial differentiation, respectively, suggesting a gliogenic effect of Notch signaling during late histogenesis. The gliogenic ffect of Notch signaling in late retinal stem cells/progenitors is consistent with Notch-mediated instructive gliogenesis in the CNS and PNS (Wang and Barres, 2000). However, the mechanism related to the stage-dependent switch in Notch function from a regulator of cell commitment to the promoter of gliogenesis is not well understood. It is likely that the developmental changes confer the ability on glialspecific genes such as GFAP to be positively influenced by Notch signaling in late stem cells/progenitors. This notion is suggested by observations that the constitutive activation of Notch signaling by over expression of Notch intracellular domain (NICD) induces astrocytic differentiation and activates transcription from GFAP promoter in neural stem cells/ progenitors (Tanigaki et al., 2001; Ge et al., 2002).

Transcriptional Profiling of Retinal Progenitors

Figure 10 Context-dependent role of Notch signaling in early and late retinal stem cells/progenitors. BrdU-tagged (green epifluorescence) early (E14) and late (E18) retinal progenitors were cocultured with JT cells expressing Jagged1 to accentuate Notch signaling. Controls included coculturing of BrdU-tagged progenitors with untransformed parental LTK- cells. BrdU-tagged progenitors were cultured in the presence of DAPT to attenuate Notch signaling. Effects of accentuation/attenuation of Notch signaling on differentiation were examined by the expression of Map2 and GFAP immunoreactivities represented by red epifluorescence. There was no significant difference in the proportion of Map2-positive and GFAP-positive cells when early retinal progenitors were cocultured with JT cells or LTK- cells (A–D, Q). However, there was significant increase in Map2-positive and GFAP-positive cells when early retinal progenitors were exposed to DAPT compared to controls (E–H, S). When late progenitors were cocultured with JT cells, there was a significant decrease and increase in Map2-positive and GFAP-positive cells, respectively, compared to controls (I–L, R). In contrast, the proportion of Map2-positive and GFAP-positive cells increased and decreased, respectively, when late retinal progenitors were exposed to DAPT compared to controls (M–P, T). Inset shows the levels of Hes5 in response to exposure to JT cells (Q and R, insets; lane 2) or DAPT (S and T, insets; lane 2) as a measure of perturbation of Notch signaling. a ⫽ Hes5 (199 bp); b ⫽ ␤-actin (548 bp); 1 ⫽ control; M ⫽ marker (200⫻). *p ⬍ 0.05, **p ⬍ 0.01, ***p ⬍ 0.001.

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However, NICD expression fails to activate GFAP promoter in early stem cells/progenitors, suggesting a context-dependent gliogenic function of Notch signaling (Ge et al., 2002). The question remains as to why Notch signaling is ineffective in the activating glialspecific gene in early stem cells/progenitors. A likely explanation could be the relative inaccessibility of Notch-dependent transcription factors to promoters of glial-specific genes in early stem cells/progenitors. The acquisition of a competence to generate Mu¨ller glia by late retinal stem cells/progenitors may be accompanied by chromatin remodeling that may provide accessibility to the NICD-CSL complex to promoters of glial-specific genes that were not available before. This premise is supported by identification of an EST by transcription profiling [Fig. 7, cluster 2; Fig. 8(b); accession number AA875609], which shows homology to human ATPase chromatin remodeling enzyme, Brahma (Brm). Brm represents a subunit of SWI/SNF chromatin remodeling complexes (Narlikar et al., 2002), whose expression level is tightly correlated with the late retinal histogenesis (James et al., 2003b). In addition, Brm has been shown to interact with both CSL (Kadam and Emerson, 2003) and NICD (James et al., 2003b). Therefore, it is tempting to speculate that in late retinal stem cells/progenitors, Brm binds to the CSL–NICD complex and recruits it to promoters of glial-specific genes and activate their transcription. The specification of a particular cell type is accompanied or followed by the process of morphogenesis. Transcription profiling has identified two classes of genes whose involvement in cellular differentiation and morphogenesis in retina is not known, to the best of our knowledge. These include those that encode c-Met RTK and regulators of F-actin biding protein, cortactin. c-Met RTK is expressed in the developing CNS and PNS, and c-Met RTK-mediated signaling is thought to regulate cell survival, differentiation, axonal growth, axonal guidance, and migration (Maina et al., 1998; Thewke and Seeds, 1999; Powell et al., 2001; Giacobini et al., 2002; Sun et al., 2002). It is likely that c-Met RTK signaling, besides promoting cell survival and differentiation, is utilized by newly born RGCs to mediate chemoattraction for axonal guidance. Cortactin, a linker protein that binds both F-actin and actin polymerization complex Arp2/3, is involved in the stabilization and branching of actin filaments (Wu and Parsons, 1993; Weed et al., 2000; Weaver et al., 2001). Recently, cortactin has been shown to play an important role in dendritic spine morphogenesis (Hering and Sheng, 2003) and neuritogenesis (Martinez et al., 2003). Although sequence corresponding to cortactin is not represented in the

Affymetrix array, its involvement in retinal progenitor differentiation and morphogenesis is apparent from the expression of genes encoding two of its regulator, CBP90 and delta catenin. CBP90 is a brain-specific protein that binds cortactin and regulates its functions (Ohoka and Takai, 1998). Delta-catenin, a brain-specific protein, belongs to p120ctn family of protein that are characterized by 10 armadillo motifs (Ho et al., 2000). It has the ability to bind both cadherins and cortactin and modulate their effects on neural morphogenesis (Kim et al., 2002; Martinez et al., 2003). It is likely that both CBP90 and delta-catenin play an important role in neuronal morphogenesis in the developing retina by regulating the effect of cortactin on actin polymerization and branching. Further insight into their roles in differentiation and maturation of retinal stem cells/progenitors can be obtained by examination of their cell-specific expression and functional involvement. We are thankful to Dr. Gerry Weinmaster for the gift Jagged1-expressing cell line and Frank Soto Leon for his excellent technical assistance.

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