Activation of Id genes by BMP signaling influences proliferation and cell fate choices of retinal progenitors

Abstract

Introduction: : Purposes: Bone morphogenetic proteins (BMPs) are multi-functional growth factors, and are well-known for regulating eye development in diverse aspects. It is also known that BMP signaling is essential for embryonic development and cellular functions such as cell growth, fate determination, differentiation and apoptosis. Inhibitors of differentiation (Id) proteins have emerged recently as the critical targets of BMPs. Id proteins are known to be important mediators of BMPs and the regulation of Id protein by BMPs is mainly through a Smad-dependent pathway. Our previous work showed that BMPs and BMPRs were co-expressed with Ids mainly in the ganglion cells and amacrine cells in the inner nuclear layer (INL) of the adult retina, suggesting a possible role for Id in inner retina development and in the terminal differentiation and/or maintenance of INL interneurons and ganglion cells in the adult. In this study, we used retinal progenitor cells (RPCs) from mouse embryos to study (1) the regulation of Id expression by BMPs; and (2) the effects of BMPs on retinal cell differentiation.

Methods: : Expression of Id1-3 gene and protein was examined by Q-PCR and western blot respectively in retinal progenitor cell (RPC) culture prepared from E14.5 mouse embryos in the presence or absence of mouse recombinant BMP-4. Activation of Smad proteins was detected by western blot analysis in RPCs incubated with or without noggin. Id promoter activity was measured by luciferase assay. In addition, the effect of BMP-4 on the differentiation of RPCs was examined by immunohistochemistry.

Results: : Significant increases of Id1-3 mRNA and protein expression levels were observed in the RPCs after treatment with BMP-4, and BMP-4 also activated the Id1 promotor to drive luciferase expression in the RPCs. A decrease in PRC proliferation accompanied these effects. Phosphorylation of Smad proteins was detected 30 min after the addition of recombinant BMP-4. These responses were abolished when cells were co-treated with noggin, a BMP antagonist. Immunoflurorescent staining demonstrated the translocation of phospho-Smad1/5/8 into the nucleus of RPC upon BMP-4 stimulation. Furthermore, BMP-4 promoted RPCs to differentiate into neuronal lineage.

Conclusions: : These results demonstrate for the first time a potentially new pathway for regulating retinogenesis through the interactions between BMP-4, Ids and their downstream signaling molecules.