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S. Mukherjee, A. Giddabasappa, W. Xiao, B. Xu, S. Chaney, D. A. Fox; Gestational Lead Exposure (GLE) Selectively Increases Late-Born Retinal Neurons, but Not Müller Glial Cells, by Differentially Regulating Basic Helix-Loop-Helix (bHLH) Genes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5899.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we showed that GLE increased and prolonged the proliferation of retinal progenitor cells (RPCs) resulting in an increased number of late-born retinal neurons (rods and bipolar cells), but not Müller glial cells [ARVO 2007]. Our present goal was to determine the spatiotemporal mRNA and protein expression profile of bHLH genes and other factors that regulate retinal neuronal and glial differentiation in GLE mice.
C57BL/6 female mice were exposed to water or lead throughout gestation (E) and until postnatal day (PN) 10: a period equivalent to human gestation. Gene expression from E18.5, PN2, PN6 and PN10 retinas was analyzed by Affymetrix array and validated by real-time PCR (qPCR). Retinas from PN2-10 mice were used to determine protein expression by Western blots and spatiotemporal protein expression by immunocytochemical confocal (IHC) studies.
Microarray and qPCR analysis revealed that Mash1 mRNA expression was significantly upregulated at PN6-10 in GLE retinas, but that Otx2, NeuroD1, Hes-1, Notch1, STAT3, CNTF, gp130 and SOCS3 mRNA expression were not significantly different from controls. Immunoblots for GLE retinas showed that Mash1 expression was significantly downregulated at PN2 and upregulated at PN6; pSTAT3 expression was significantly downregulated at PN2; Notch1-ICD and Hes1 expression were significantly downregulated at PN6; and STAT3 expression was not significantly different from controls. In GLE retinas at PN5, IHC revealed that Mash1 was significantly upregulated in postmitotic and differentiating neurons, whereas there was no significant change in the number or expression pattern of Hes1+ve cells.
The downregulation of Mash1 and pSTAT3 protein expression at PN2 is consistent with the observed increase and prolongation of proliferating RPCs in GLE retinas. The increase in mRNA and protein expression of the proneural bHLH factor Mash1 in developing GLE retinas likely underlies the increased differentiation of late-born neurons seen in adult GLE offspring. The decreased protein expression and activation of the Notch1-Hes1 pathway at PN6 did not inactivate pSTAT3, but favored RPC differentiation. These novel findings show that GLE targeted specific bHLH genes at PN6. This resulted in an increased cell-fate specification and differentiation of late-born rods and bipolar cells, but not Müller glial cells.
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