Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors
© Hsieh and Yang. 2009
Received: 24 February 2009
Accepted: 17 August 2009
Published: 17 August 2009
The paired homeobox protein Pax6 is essential for proliferation and pluripotency of retinal progenitors. However, temporal changes in Pax6 protein expression associated with the generation of various retinal neurons have not been characterized with regard to the cell cycle. Here, we examine the dynamic changes of Pax6 expression among chicken retinal progenitors as they progress through the neurogenic cell cycle, and determine the effects of altered Pax6 levels on retinogenesis.
We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates.
These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase re-entry, whereas rapid accumulation or reduction of Pax6 protein during the G2/M phase of the cell cycle may be required for specific neuronal fates. These findings thus provide novel insights on the dynamic regulation of Pax6 protein among neurogenic progenitors and the temporal frame of neuronal fate determination.
green fluorescent protein
Hamburger and Hamilton
proliferating cell nuclear antigen
red fluorescent protein
retinal ganglion cell
small hairpin RNA.
The Pax6 gene encodes an evolutionarily conserved paired homeobox protein critically involved in eye development and retinogenesis [1–5]. In both Drosophila and vertebrates, ectopic expression of Pax6 induces ectopic eyes or lens [2, 6, 7], indicating an instructive role of Pax6 in ocular tissue determination. Genetic studies have demonstrated that the dosages of Pax6 strongly impact the formation and growth of ocular tissues. Homozygous or heterozygous loss of function Pax6 mutants have either no eye or small eye phenotypes [8–11]. Conversely, increasing dosages of the Pax6 gene also lead to microphthalmia and malformed retinas [12, 13]. Furthermore, Pax6 is involved in pattern formation of the vertebrate eye. Aberrant Pax6 expression results in malformation of the anterior segment of the eye [5, 14–16]. Pax6 expression in the optic vesicle is required for establishing the naso-temporal axis and the dorsal characteristics of the retina , whereas Pax6 expressed in the distal optic cup reciprocally suppresses Pax2 in the optic stalk to establish the optic cup/stalk boundary . The complex roles of Pax6 in eye development are reflected by the existence of multiple enhancers that regulate the exquisite spatial and temporal expression of Pax6 transcripts in neural and non-neural ocular tissues [17, 19–21].
Multiple Pax6 isoforms are encoded by the Pax6 gene in vertebrates [22–24]. The canonical Pax6 and the Pax6(5a) isoform, which contains amino acid residues encoded by an alternatively spliced exon inserted within the paired domain, are commonly found among various vertebrates [22, 23, 25]. The mammalian Pax6 and Pax6(5a) isoforms, and their corresponding Drosophila homologs encoded by the eyeless and eyegone genes, exhibit distinct biological activities. Both Pax6 and eyeless can induce ectopic eye formation in the fly wing imaginal disc , whereas Pax6(5a) and eyegone can elicit only cell proliferation but not neuronal differentiation . Similarly, in embryonic mouse cortex, Pax6 influences both cell proliferation and neuronal differentiation, but Pax6(5a) only affects proliferation [27, 28]. In mice missing the transcript Pax6(5a), lens abnormalities and iris hypoplasia are observed, but development of the neural retina remains largely normal , suggesting that Pax6 is able to compensate for the function of Pax6(5a) in the neural retina.
During mammalian central nervous system development, multipotent progenitors undergo a transition from the preneurogenic stage, when symmetrical divisions occur and give rise to proliferative progeny, to the neurogenic stage, during which a subset of progenitors withdraw from the cell cycle and initiate neuronal differentiation. In the cortex, reducing Pax6 expression levels affects the length of the cell cycle and the progression from symmetrical to asymmetrical division among cortical progenitors . Furthermore, decreased Pax6 expression leads to depletion of basal cortical progenitor and precocious neuronal differentiation . In the spinal cord, altering Pax6 levels affects the expression of the proneural gene Ngn2 and the timing of cell cycle exit .
In the vertebrate retina, multipotent progenitor cells give rise to six major types of neurons and one type of glial cell in a conserved temporal order [33–38]. At the onset of retinogenesis, the central retinal progenitors enter the neurogenic stage first, while the peripheral retina remains in a preneural state . When Pax6 is conditionally ablated in the peripheral retina by using the Pax6 α cre mouse, which encodes the Pax6 P0 promoter and the α enhancer driving the expression of cre recombinase, progenitor expansion is affected, resulting in a retina reduced in size . In addition, in the Pax6 α cre-mediated Pax6 conditional knockout mutant, peripheral retinal progenitors are unable to express multiple neurogenic basic helix-loop-helix (bHLH) transcriptional factors and are limited to producing only amacrine interneurons . Recently, it has been reported that Pax6 α cre-mediated Pax6 deletion in the peripheral retina unmasks a suppression of the cone-rod homeobox gene Crx by Pax6, and more centrally localized Pax6 deletion by the Chx10-cre driver also leads to the loss of progenitor multipotency . Thus, Pax6 differentially expressed by the central or peripheral retinal progenitor pools may serve distinct functions during development, but nonetheless is required for generation of multiple retinal neurons.
In the mature retina, various neuronal cell types express disparate levels of Pax6. How this highly differential Pax6 expression is achieved among specific neurons remains unclear. In this study, we have analyzed Pax6 expression at the cellular level among neurogenic progenitors in the embryonic chicken retina. We demonstrate a significant down-regulation of Pax6 protein as progenitors undergo the preneurogenic to neurogenic transition. In addition, we show that while neurogenic progenitors maintain a uniformly low Pax6 level during the S phase, a subset of retinal progenitors rapidly increase or decrease their Pax6 levels as they enter G2/M phase of the cell cycle. The divergence of Pax6 protein levels correlate with the onset of differentiation for certain neurons. Moreover, disruption of Pax6 levels among neurogenic progenitor cells leads to aberrant proliferation and production of neuronal cell types. These results reveal for the first time the dynamic expression of Pax6 during the neurogenic cell cycle and highlight the importance of Pax6 protein regulation as neural progenitors adapt to different cell fates.
Dynamic Pax6 expression during retinogenesis
Together, these results demonstrate that Pax6 expression in the retina decreases as progenitor cells undergo the preneurogenic to neurogenic transition. Moreover, most neurogenic retinal progenitors maintain low levels of Pax6, whereas postmitotic neurons undergo cell type-specific elevation or reduction of Pax6.
Cell cycle-dependent Pax6 protein expression among neurogenic progenitor cells
We next quantified changes of Pax6 protein levels during the transition from the S phase to G2/M phase of the cell cycle. In the E6.5 (HH stage 30) retina when RGC production peaks, following a 20-minute BrdU pulse, an average of 10.7% of the cells were labeled by BrdU (Figure 4B). After a 3 or 6 hour chasing period, BrdU+Pax6Hi cells gradually increased in the G2/M phase population (17% of total cells, with 4n DNA content) compared to the uniform Pax6Lo level of the 0 hour BrdU+ cells (Figure 4B). Quantification of Pax6 levels among 4n BrdU+ cells showed a steady increase of BrdU+Pax6Hi cells (Figure 4C). At 30 minutes after the pulse chase, the BrdU+Pax6Hi cell population already showed a statistically significant increase (Figure 4C). After 6 hours of chase, the percentage of BrdU+Pax6Hi cells had increased from 7.0% to 20.8% (Figure 4C; N = 3). Concomitantly, the Pax6Lo progenitor cells decreased from 87.6% to 72.5% during the 6 hour chase period (Figure 4C; N = 3), reflecting altered Pax6 expression as progenitors progressed through the G2/M phase. However, the Pax6Neg cell populations did not show statistically significant changes during the chase (Figure 4C).
Divergent Pax6 expression in G2/M and neuronal fate specification
We then examined whether G2/M progenitors with no Pax6 corresponded to committed cone photoreceptors, which at maturity contain no Pax6. Confocal imaging revealed that the early cone cell marker Visinin could be detected in PH3+ cells (Figure 6E–H), suggesting that the onset of Visinin expression begins during the M phase. This was further confirmed in dissociated retinal cell cultures, where we detected co-labeling of PH3 with weak Visinin signals in anaphase progenitors (Figure 6I–L). Furthermore, confocal imaging demonstrated that intensely Visinin+ cone photoreceptor precursors expressed no Pax6, whereas cells emerging from the telophase expressed both low levels of Pax6 and Visinin (Figure 6M–P). These data suggested that the emergence of the cone marker Visinin correlated with the down-regulation of Pax6 in a subset of M phase cells. Therefore, the divergence of Pax6 expression among a subset of progenitors in G2/M phase of the cell cycle may reflect neuronal fate specification.
Effects of altering Pax6 levels on proliferation, neuronal fate specification, and cell death
We also examined the influence of Pax6 levels on three major neuronal cell types generated between HH stage 24 and stage 32. Reducing Pax6 by shRNA resulted in a decrease of Brn3a+ retinal ganglion cells from 4.3% to 1.2% (Figure 8C,G). However, Pax6 overexpression did not elevate the production of Brn3a+ RGCs (Figure 8C). Quantification using Visinin showed that reducing Pax6 using shRNA dramatically enhanced cone precursors from 24.6% to 40.5% (Figure 8E,G). Conversely, overexpressing Pax6 caused reduced Visinin+ cone precursor production (Figure 8E). The production of AP2α+ amacrine cells was enhanced when Pax6 was down-regulated and suppressed when Pax6 was elevated (Figure 8D), suggesting that amacrine interneuron specification was also sensitive to Pax6 levels. Together, these results demonstrate that Pax6 expression levels among neurogenic progenitor cells critically affect both cell proliferation and neuronal fate decisions (Figure 8F,G).
In this study, we have analyzed Pax6 protein expression during the preneurogenic to neurogenic transition and among neurogenic chicken retinal progenitor cells. We provide evidence that Pax6 protein levels are highly dynamic in proliferating progenitors and are coupled with cell cycle progression. We also demonstrate that Pax6 levels critically influence cell proliferation and cell fate specification of neurogenic progenitors.
Pax6 is expressed by retinal progenitors throughout development. However, our results show that Pax6 protein levels are significantly down-regulated as retinal progenitors undergo the preneurogenic to neurogenic transition and initiate production of postmitotic neurons. During the remainder of retinogenesis, the peripheral margin progenitor cells retain a higher level of Pax6 compared to the central progenitor cells. How the down-regulation of Pax6 expression is achieved during the preneurogenic to neurogenic transition remains unknown, but it may involve signals that promote cell-intrinsic changes necessary to enable cell cycle exit and neuronal fate specification. The differing levels of Pax6 in the peripheral versus central retina are achieved, in part, at the transcriptional level through the complex arrays of Pax6 enhancers [17, 19]. Our data suggest that translational and/or post-translational regulation may also contribute to the rapid change of Pax6 protein levels during the cell cycle. The cellular distinctions between the central Pax6Lo and peripheral Pax6Hi progenitors are not well understood. Conditional ablations of the Pax6 gene in the peripheral retina by Pax6 α-cre and in the central retina by the Chx10-cre both block expression of proneural genes and limit the pluripotency of retinal progenitors [40, 41]. However, the peripherally expressed Pax6 also plays a role in suppressing the Crx gene , suggesting either a context-dependent or dosage-dependent mechanism. The Pax6 perturbation carried out in our study focused on the central neurogenic retina, where the expression of several proneural genes, including Ath5 and Ngn2, has begun and postmitotic neurons have accumulated. Thus, our experiments address the importance of Pax6 regulation among neurogenic progenitors.
We have established by quantitative flow cytometry analysis that the cycling neurogenic progenitor population expresses a relatively low level of Pax6, which when perturbed interferes with BrdU incorporation. Interestingly, overexpression of Pax6 resulted in a decreased number of cells undergoing DNA synthesis, but did not significantly impact the proportion of PCNA+ cells remaining in the cell cycle. Since PCNA labels proliferating cells in all phases of the cell cycle, this result suggests that high Pax6 levels potentially alter cell cycle distribution and/or lead to cell cycle arrest. Consistent with our observations, studies in the developing cortex have shown that reducing Pax6 perturbs cell cycle length and affects the preneurogenic to neurogenic transition [30, 31]. In addition, activation of Pax6 or Pax6(5a) above endogenous levels in the cortical progenitors disrupts cell cycle progression . Live imaging of a tagged Pax6 isoform has revealed that Pax6p46 is associated with the pericentromeric area of the chromosomes during M phase, and overexpression of this isoform causes abnormal mitosis . Our study shows that low levels of Pax6 are expressed by the majority of neurogenic progenitors, but progenitor cells residing within the S phase appear to maintain a more uniformly low level of Pax6 compared to G1 and G2/M phase cells. This raises the possibility that the G1 to S phase transition of neurogenic progenitors may require a narrow range of Pax6 protein. Further analyses are necessary to determine the precise cell cycle defects of retinal progenitors with aberrant levels of Pax6 expression.
Previous findings show that heterozygous Pax6 loss of function mutations lead to microphthalmia in mice [8–12], which is primarily due to defects in early eye morphogenesis. In transgenic mice carrying multiple copies of Pax6, the eye morphology and retinogenesis are initially normal, but RGC axons show dose-dependent projection defects accompanied by postnatal cell loss [12, 13]. This suggests that Pax6 levels affect RGC differentiation and survival. Since Pax6 positively regulates transcription of the proneural gene Math5 required for RGC development , high levels of Pax6 may cause inappropriate Math5 expression, leading to RGC death. Consistent with the finding that aberrant Pax6 levels cause increased apoptosis during cortical development , we have detected increased cell death due to reduced Pax6 or increased Pax6(5a) levels in the neurogenic retina. These findings indicate that stringent regulation of Pax6 levels is critical for cell survival at different stages of eye/retinal development.
We have demonstrated that various postmitotic retinal neurons exhibit distinct Pax6 levels, suggesting that Pax6 expression is readjusted when neurogenic progenitors become postmitotic, and that Pax6 may, therefore, function in cell fate determination of progenitors and/or subsequent neuronal differentiation. Our data show that perturbing Pax6 during chicken retinogenesis impacts cell fate specification. In close correlation with the contrasting levels of Pax6 in mature RGCs and cone photoreceptors, decreasing Pax6 led to reduced RGC and enhanced photoreceptor genesis. Conversely, forced expression of Pax6 suppressed photoreceptor production. However, we did not detect increased RGC production due to forced Pax6 expression as previously observed in a low density monolayer chicken retinal culture . This might be due to the progenitors in our retinal explants already expressing sufficient amounts of Pax6 to facilitate RGC fate specification. Alternatively, Pax6 overexpression enhances early neuronal markers but not the more mature RGC marker Brn3a. The majority of postmitotic amacrine cells express high levels of Pax6 in the chicken retina. Our results show that both reducing and elevating Pax6 altered amacrine production, supporting a requirement for Pax6 in either specification or differentiation of amacrine interneurons. This result is consistent with phenotypes of Pax6 conditional knockout in the more centrally localized progenitors, which also lose their pluripotency . The fact that both gain and loss of Pax6 function impede amacrine cell development in the chicken retina suggests that Pax6 is likely required for amacrine cell fate specification rather than differentiation, but direct perturbation of Pax6 in postmitotic amacrine cells is necessary to distinguish these possibilities.
In this study, we present the first analysis of Pax6 protein dynamics with regard to the neurogenic cell cycle. We provide evidence that a subset of neurogenic progenitor cells begins to display divergent Pax6 protein levels upon entering into the G2 phase of the cell cycle. This cell cycle-correlated Pax6 protein level change is more readily detectable in the later neurogenic retina (Figures 4, 5, and 6). The dramatic changes of Pax6 levels among the subset of progenitors, coupled with the relatively short G2 phase, indicate that Pax6 protein in neurogenic progenitors may be subjected to rapid proteolytic degradation. In addition, the correlation between decreased Pax6 expression and the appearance of the neuronal marker Visinin in late M phase cells suggests that the subset of progenitor cells showing divergent Pax6 expression in G2/M are likely those that have committed to certain cell fates. Therefore, neuronal fate determination is likely to have occurred prior to the G2 phase of the neurogenic cell cycle, which produces at least one postmitotic neuron.
During the preneurogenic to neurogenic transition, retinal progenitor cells undergo dramatic down-regulation of Pax6 protein. A low level of Pax6 protein is essential for S phase entry and normal proliferation of neurogenic progenitors. A subset of neurogenic progenitors rapidly increase or decrease Pax6 levels in the G2/M phase of the cell cycle, forecasting cell fate selections prior to G2. The dynamic regulation of Pax6 protein within the cell cycle is critical for the expansion and fate specification of neurogenic progenitors.
Materials and methods
Fertilized White Leghorn chicken eggs were purchased from Charles River (Wilmington, MA, USA) and were incubated at 38°C in a rotating humidified incubator. Embryos were staged (HH stage) according to Hamburger and Hamilton .
Overexpression constructs were generated by cloning the human Pax6 and Pax6(5a) cDNAs downstream of the chicken beta actin enhanced cytomegalovirus promoter (CAG) followed by an internal ribosomal entry site upstream of the human placental alkaline phosphatase. For shRNA-mediated knockdown, hairpin sequences specifically targeting Pax6 or the control RFP message (HcRed N1; Clonetech, Mountain View, CA, USA) were cloned downstream of the mouse U6 promoter in the vector pBS/U6 (U6) . In addition, a cassette containing the CAG promoter and a nuclear GFP was cloned downstream of the of U6 shRNA cassette in the same orientation. The set of specific primers used to generate the shRNAs targeting Pax6 were: XJY276 (5'GGTCTGTACCAACGATAACAA); XJY277 (5'AGCTTTGTTATCGTTGGTACAGACC); XJY278 (5'AGCTTTGTTATCGTTGGTACAGACCCTTTTTG); XJY279 (5'AATTCAAAAAGGGTCTGTACCAACGATAACAA). The two sets of specific primers used to generate the shRNAs Redi1 and Redi2 targeting HcRed N1 (Clonetech) were:
Redi1, XJY694 (5'GGCACCGTGAACGGCCACTACTTA), XJY695 (5'AGCTTAAGTAGTGGCCGTTCACGGTGCC), XJY696 (5'AGCTTAAGTAGTGGCCGTTCACGGTGCCCTTTTTG), XJY697 (5'AATTCAAAAAGGGCACCGTGAACGGCCACTACTTA); Redi2, XJY698 (5'GGAGAGAACCACCACCTACGAA), XJY699 (5'AGCTTTCGTAGGTGGTGGTTCTCTCC), XJY700 (5'AGCTTTCGTAGGTGGTGGTTCTCTCCCTTTTTG), XJY701 (5'AATTCAAAAAGGGAGAGAACCACCACCTACGAA).
Retinal cultures and transfection assays
For transfection of monolayer cells, dissociated HH stage 27 retinal cells were plated on poly-D-lysine coated 8-well LabTek (Nalge Nunc, Rochester NY, USA) slides at 20,000 cells/mm2, 3 hours prior to Lipofectamine PLUS (Invitrogen, Carlsbad, CA, USA) transfection using Pax6 shRNA knockdown vectors or Pax6 overexpression vectors with a CAG-nGFP plasmid at 2:1 ratio . For transfection of retinal tissues, DNA at 2 μg/ml in phosphate-buffered saline (PBS) was injected into the subretinal space of the eye at HH stage 24/25. The injected eyes were placed between paddle electrodes and subjected to five pulses at 10 V/mm for 25 ms with 975-ms intervals using ECM 830 Square Wave Electroporation System (BTX, Holliston, MA, USA). Central retinas (posterior halves) were dissected from electroporated eyes and cultured on Nucleopore membrane discs (Millipore, Billerica, MA, USA) floating on a retinal culture medium containing an equal ratio of F12:DMEM (JRH Biosciences, Lenexa, KS, USA), 2% fetal calf serum, 0.2% chicken serum, 10 mM HEPES, and penicillin/streptomycin at 37°C in 5% CO2 for 72 hours.
Transfection of 293T cells was carried out using Lipofectamine PLUS (Invitrogen) and 0.65 μg of U6-Pax6i, U6-Redi1, U6-Redi2, or U6-GFP with 0.5 μg of either RCAS(B)-hPax6 DNA or CMV-HcRedN1 (RFP) as well as 0.35 μg of CAG-nGFP as transfection control. At 48 hours post-transfection, cells were lysed in RIPA buffer without deoxycholate, protein extracts were resolved on a 10% BisTris SDS-PAGE gel using MOPS buffer (Invitrogen), and transferred onto nitrocellulose membrane. Blots were probed with antibodies against GFP (1:500; Invitrogen), HcRedN1 (1:500; Becton Dickenson, Laguna Hills, CA, USA), Pax6 (1:1,000; Chemicon/Millipore, Billerica, MA, USA), and followed by secondary antibodies conjugated with horseradish peroxidase and detected by enhanced chemiluminescence (ECL plus; Amersham, Piscataway, NJ, USA).
Immunohistochemistry and imaging
Heads of chicken embryos or retinal explants were fixed in 4% paraformaldehyde in PBS overnight at 4°C, followed by cryoprotection with 30% sucrose in PBS and embedding in OCT. Coronal sections 20 μm thick were collected and used for immunolabeling. For BrdU pulse labeling, the posterior half of HH stage 30 or stage 33 to 34 retinas were dissected and incubated in the retinal culture medium supplemented with 20 μm BrdU for 20 minutes, followed by extensive washes in fresh culture medium without BrdU, and then further cultured as free floating explants for designated time periods. Retinal cryosections labeled with BrdU were treated with 95% formamide in 1× standard sodium citrate (SSC) pH7.4 for 10 minutes at 70°C prior to antibody incubation.
Immunohistochemistry was performed as previously described . Cryosections or monolayer cells were incubated with the following primary antibodies: Neurofilament 68 (mouse, 1:200; Sigma Aldrich, St. Louis, MO, USA), Neurofilament 145 (rabbit, 1:500; Chemicon), Phospho-histone H3 ser28 (rabbit, 1:500; Millipore), Phospho-histone H3 ser10 (rat monoclonal, 1:250; Sigma), Pax6 (mouse, 1:10; Developmental Study Hybridoma Bank, Iowa City, IA, USA), Pax6 (rabbit, 1:500; Chemicon and Covance, Princeton, NJ, USA), PCNA (mouse, 1:200; Sigma), Brn3a (mouse, 1:200; Chemicon), AP2α (mouse, 1:5; DSHB), Islet 1/2 (mouse clone 39.4D5, 1:10; DSHB), Visinin (mouse, 1:50; DSHB), activated caspase 3 (rabbit, 1:250; Upstate/Millipore), BrdU (mouse, 1:5; Amersham), and GFP (mouse monoclonal and rabbit polyclonal; Invitrogen). Sections were then incubated with either biotinylated secondary antibodies followed by horseradish peroxidase detection (Vector Laboratories, Burlingame, CA, USA) using 3,3'-diaminobenzidine (Sigma), or appropriate secondary antibodies conjugated to Alexa 488, 568, 594, or 647 (Invitrogen). For nuclear staining, 1 mg/ml of 4',6-diamidino-2-phenylindole (DAPI; Roche, Palo Alto, CA, USA) was used. Epifluorescent and DIC images were captured using a Nikon E800 microscope equipped with a SPOTII digital camera. Confocal optic sections 1 μm thick were obtained using a Zeiss LSM 410 confocal laser-scanning system with a Zeiss Axiovert 135M microscope, or a Leica TCS SP MP confocal microscope.
The anterior (st25 analysis) and posterior halves of retinas were dissected and collected in Hank's buffer saline solution (HBSS+; Invitrogen). Retinas were washed in HBSS without Mg+ and Ca+ (HBSS-; Invitrogen) and incubated with 0.1% trypsin (Sigma) at 37°C for 10 minutes. Soybean trypsin inhibitor (Sigma) at a final concentration of 0.1 mg/ml or chicken serum (Sigma) to 10% of total volume was added to stop trypsin, followed by addition of DNaseI (Sigma) to 50 mg/ml and trituration to obtain single cell suspensions. Single cell suspensions were fixed in 2% paraformaldehyde in PBS for 30 minutes, washed with HBSS+, followed by 0.1% Triton-X in HBSS+ incubation prior to staining with primary and secondary antibodies as described above with washes in between. Anti-PCNA and anti-BrdU antibody labeled cells were also treated with 2 N and 1 N HCl, respectively, prior to antibody labeling. Cells were filtered and collected using a tube with a 35 mm nylon mesh cap (BD Biosciences/Falcon, San Jose, CA, USA) before flow cytometry analyses using an LSR flow cytometer (BD Biosciences).
Quantification of cell markers and statistical analysis
Quantification of various cell markers for dissociated retinal cells was performed by counting non-overlapping fields through the microscope directly or by analyzing acquired digital images using ImagePro PLUS software (Media Cybernetics, Bethesda, MD, USA). For each sample, a minimum of 200 marker-positive cells or the entire 7-mm diameter well was counted. Quantification of dissociated retinal cells by FACS was performed using FlowJo v5.7.2 according to the manufacturer's guidelines (Ashland, OR, USA). For each sample, a minimum of 30,000 cells was analyzed. Student t-test was applied to data sets where P < 0.05 is considered statistically significant. Data were presented as mean ± SEM.
We thank Dr Joseph Horwitz for advice and support, and Drs Mathew Schibler and Ingrid Schmidt for assistance on confocal imaging and flow cytometry, respectively. This work was supported in part by NEI T32 training grant to Y-WH, and grants from the Research to Prevent Blindness Foundation, the Karl Kirchgessner Foundation, the Foundation Fighting Blindness and the National Eye Institute to X-JY.
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