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First published online 17 July 2007
doi: 10.1242/jcs.008417

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Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling

Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE 68198-5840, USA

View larger version (21K): [in this window] [in a new window]   Fig. 1. Temporal and spatial patterns of Abcg2 expression in the developing retina. (A,B) RT-PCR analysis carried out on cDNA obtained from different stages of late retinal histogenesis and adult retina revealed that levels of transcripts corresponding to ABCG2 decreased as differentiation progressed. (C,D) ABCG2 immunoreactivity in a PN1 retinal section is predominantly confined to the outer neuroblastic layer. (E,F) In dissociated PN1 retinal cells pre-exposed to BrdU, ABCG2 is expressed in cells that also incorporated BrdU (arrows). ONbL, outer neuroblastic layer. IR, inner retina. Magnification, 200x.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Temporal decrease in SP cells in the developing retina. (A-E) Hoechst dye efflux assay, carried out on cell dissociates from different stages of late retinal histogenesis, revealed a temporal decrease in the proportion of SP cells (within triangle). (F) RT-PCR analysis carried out on SP and non-SP (NSP) cells from E18 retina revealed that the former predominantly expressed transcripts corresponding to Abcg2, Nestin, Notch1, Pax6 and Sox2 whereas the latter expressed those corresponding to differentiated cell markers, i.e. rhodopsin kinase (rods), mGluR6 (bipolar cells) and Brn3b (retinal ganglion cells).

View larger version (47K): [in this window] [in a new window]   Fig. 3. Perturbation of Abcg2 expression affects SP cell phenotype of late retinal progenitors. (A-C) E18 retinal progenitors, enriched as neurospheres, were transduced with ABCG2 retrovirus (G1-ABCG2) or empty retrovirus (G1-Control), followed by Hoechst dye efflux assay. There was a significant increase in the proportion of SP cells in G1-ABCG2 retrovirus-transduced neurospheres, compared with controls. (D-F) Neurospheres were transduced with retrovirus to express ABCG2 siRNA (pSuper-ABCG2siRNA) or missense siRNA (pSuper-ABCG2missense) as controls, followed by Hoechst dye efflux assay. There was a significant decrease in the proportion of SP cells in pSuper-ABCG2siRNA retrovirus-transduced neurospheres, compared with controls. (G-L) The specificity of siRNA-mediated attenuation of Abcg2 expression is demonstrated by a decrease in levels of Abcg2 transcript (G) by RT-PCR analysis and ABCG2 protein by western blot analysis (H) and by immunocytochemical analysis (I-L) in pSuper-ABCG2siRNA-transduced neurospheres, compared with controls. Data are expressed as the mean ± s.e.m. from triplicate cultures of two different experiments. ***P<0.005 compared with levels in the control. Magnification, 200x.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Ectopic Abcg2 expression promotes pan neural and retinal progenitor properties in late retinal progenitors. (A-I) E18 neurospheres, transduced with G1-ABCG2 retrovirus to overexpress ABCG2 or empty retrovirus as controls. There was a significant increase in the proportion of BrdU+ cells expressing pan neural progenitor marker, Nestin (A-D,I) and retinal marker, Pax6 (E-H,I) in G1-ABCG2 retrovirus-transduced neurospheres, compared with controls. (J) RT-PCR analysis revealed an increase in levels of transcripts corresponding to Pax6, Nestin and Sox2 in G1-ABCG2-transduced cells, compared with controls, corroborating the immunocytochemical analysis. Data are expressed as the mean ± s.e.m. of triplicate cultures from two different experiments. ***P<0.005 compared with levels in the G1-control. Magnification, 200x.

View larger version (67K): [in this window] [in a new window]   Fig. 5. Attenuation of Abcg2 expression adversely affects pan neural and retinal progenitor properties in late retinal progenitors. (A-I) There was a significant decrease in the proportion of BrdU+ cells expressing the neural progenitor marker, Nestin (A-D,I) and retinal marker, Pax6 (E-H,I) in pSuper-ABCG2siRNA retrovirus-transduced neurospheres, compared with controls. (J) RT-PCR analysis revealed a decrease in levels of transcripts corresponding to Pax6, Nestin and Sox2 in pSuper-ABCG2siRNA-transduced neurospheres compared with controls, corroborating the immunocytochemical analysis. Data are expressed as the mean ± s.e.m. of triplicate cultures from two different experiments. Magnification, 200x.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Ectopic Abcg2 expression adversely affects differentiation of late retinal progenitors. (A-M). There was a significant decrease and increase in the proportion of Map2+ (A-D,M)/GFAP+ (E-H,M) and Nestin+ (I-L,M) cells, respectively, in G1-ABCG2 retrovirus-transduced neurospheres, compared with controls. (N) RT-PCR analysis revealed a decrease and increase in levels of transcripts corresponding to Map2/GFAP and Nestin, respectively, in G1-ABCG2-transduced neurospheres, compared with controls, corroborating the immunocytochemical analysis. (O) RT-PCR analysis revealed a decrease in the expression of transcripts encoding the specific retinal cell markers, rhodopsin kinase (RK, rods), mGluR6 (bipolar cells), Syntaxin1 (amacrine cells) and glutamine synthetase (GS, Müller cells) in G1-ABCG2 transduced neurospheres compared with controls, demonstrating the effects of ABCG2 on retinal-cell-specific differentiation. Data are expressed as the mean ± s.e.m. of triplicate cultures from two different experiments. ***P<0.005 compared with levels in the control. Magnification, 200x.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Attenuation of ABCG2 promotes differentiation of late retinal progenitors. (A-M) There was a significant increase and decrease in the proportion of Map2+ (A-D,M)/GFAP+ (E-H,M) and Nestin+ (I-L,M) cells, respectively, in pSuper-ABCG2siRNA retrovirus, compared with controls. RT-PCR analysis revealed an increase and decrease in levels of transcripts corresponding to Map2/GFAP and Nestin, respectively, in pSuper-ABCG2siRNA retrovirus-transduced neurospheres, compared with controls, corroborating the immunocytochemical analysis (N). RT-PCR analysis revealed an increase in the expression of transcripts corresponding to specific retinal cell markers, rhodopsin kinase (RK, rods), mGluR6 (bipolar cells), Syntaxin1 (amacrine cells) and glutamine synthetase (GS, Müller cells) in pSuper-ABCG2siRNA retrovirus-transduced neurospheres as compared to controls, demonstrating the effects of ABCG2 on retinal cell specific differentiation (O). Data are expressed as the mean ± s.e.m. of triplicate cultures of two different experiments. Magnification, 200x.

View larger version (76K): [in this window] [in a new window]   Fig. 8. Ectopic Abcg2 expression influences Mash1 and cell cycle regulators expression. SP and NSP cells were enriched by Hoechst dye efflux assay from neurospheres, transduced with G1-ABCG2 retrovirus or empty retrovirus, followed by examination of expression of transcripts corresponding to a proneural gene (Mash1), cell cycle regulators (Ccnd1, Ki67, p27Kip1) and pan neural marker (Map2). Levels of Mash1, CyclinD and Ki67 transcripts were increased in SP cells obtained from ABCG2 retrovirus-transduced neurospheres compared with those from control neurosheres. Whereas levels of p27Kip1 and Map2 transcripts were detected only in NSP cells and their levels decreased in those transduced with ABCG2 retrovirus, compared with those in controls.

View larger version (63K): [in this window] [in a new window]   Fig. 9. Perturbation of Notch signaling affects ABCG2 expression in late retinal progenitors. (A-G). There was a significant increase and decrease in BrdU+ cells, expressing ABCG2 immunoreactivities in neurospheres transduced with NICD and those cultured in DAPT, respectively. (H) RT-PCR analysis revealed that levels of transcripts encoding ABCG2, and those to progenitor markers, i.e. Pax6, Sox2 and Nestin, increased and decreased in neurospheres transduced with NICD and those cultured in DAPT, respectively, compared with controls. The specificity of the perturbation of Notch signaling was displayed by changes in the expression of Notch target gene Hes1. Data are expressed as the mean ± s.e.m. from triplicate cultures of two different experiments. Magnification, 200x.

View larger version (28K): [in this window] [in a new window]   Fig. 10. Perturbation of Notch signaling affects SP cell phenotype of late retinal progenitors. To determine whether or not Notch signaling influenced SP cell phenotype of late retinal progenitors, E18 neurospheres were transduced with NICD retrovirus or cultured in the presence of DAPT to accentuate or attenuate Notch signaling, respectively. There was an increase and decrease in the proportion of SP cells in neurospheres, transduced with NICD and those cultured in DAPT, respectively, compared with levels in the controls (A-D).

View larger version (38K): [in this window] [in a new window]   Fig. 11. Notch signaling influences ABCG2 expression directly. (A-D) Immunocytochemical analysis of E18 neurospheres showed that immunoreactivities corresponding to ABCG2 (arrow) and Notch1 (arrowhead) were co-localized in retinal progenitors. Examination of ABCG2 promoters revealed the presence four CSL binding sites (TGGGA) between –1285 and –657 (E). To determine the CSL-mediated effect on the activities of ABCG2 promoter, ABCG2 promoter deletion constructs were transfected into 293T cells, transduced with NICD retrovirus. (F) There was a significant increase in reporter activities with constructs containing CSL sites (b), compared to those without (a), which displayed base level reporter activities. (G) Mobility shift assay carried out with labeled wild-type ABCG2 promoter sequences containing one of the CSL sites (sequence) using HEK293T cell nuclear extracts revealed the formation of complexes, which were similar to those formed with in vitro translated suppressor of hairless (SuH) protein. These complexes could be competed with an excess of unlabeled sequences and were not formed with sequences in which the CSL site was mutated. (H) Nucleosomal DNA immunoprecipitated with CSL antibody in a ChIP assay on E18 retinal progenitors contained sequences corresponding to the Abcg2 promoter, suggesting the presence of CSL on Abcg2 regulatory complexes in vivo.

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