A novel basic helix-loop-helix (bHLH) transcriptional repressor, NeuroAB,expressed in bipolar and amacrine cells in the retina.

All retinal neurons develop from a common retinal progenitor, cell fate becoming irreversible during the final mitosis. Different cell types are produced in an ordered manner: The most likely order in chick is retinal ganglion cells, cone photoreceptors, amacrine cells and horizontal cells, followed by rod photoreceptors, bipolar cells, and finally Müller glia, with significant overlap in the appearance of these different cell types. This order is largely conserved among the vertebrates, which suggests conserved regulatory mechanisms underlying the onset of specification of each cell type. A variety of extrinsic and intrinsic factors have been demonstrated to regulate retinogenesis. Extrinsic factors including secreted factors have been suggested to influence intrinsic factors. It is known that the basic helix-loop-helix (bHLH) transcription factors play an important role as intrinsic factors in the cell fate determination of retinal cell types. Based on sequence similarity, neuronal bHLH proteins can be largely divided into two families: Achaete-Scute complex (AS-C) related proteins and Atonal-related protein (ARPs). Furthermore, based on phylogenetic tree analysis, four subgroups have been proposed in the ARP family: the Neurogenin, NeuroD, ATO, and BETA3 groups. He identified a novel chick bHLH transcription factor, NeuroAB. A phylogenetic tree prepared from bHLH sequences suggests that NeuroAB is a member of the BETA3 group, which consists of BETA3, BHLHB4, and Olig1-3. In situ hybridization and immunostaining indicated that NeuroAB is expressed predominantly in postmitotic bipolar cells and GABAergic amacrine cells in the retina. These findings suggest that NeuroAB is involved in the maturation and maintenance of bipolar cells and GABAergic amacrine cells. The bHLH transcription factors bind to DNA as dimmers and recognize the consensus sequence CANNTG, called the E-box. A DNA pull down assay indicated that the NeuroAB protein binds to the E-box sequence. bHLH proteins function as a transcriptional activator or repressor in many developmental events. To investigate the transcriptional properties of NeuroAB, he preformed reporter assays using the minimal helpes simplex virus thymidine kinase (TK) promoter with five copies of the E-box sequences and the native GAP-43 promoter containing E-box motifs. The results obtained from the two promoters suggested that NeuroAB functions as a transcriptional repressor. Protein phosphorylation represents a mechanism that is frequently employed by cells to regulate functions of a variety of molecules including transcription factors. Recently, some transcription factors have been demonstrated to be regulated by phosphorylation at a specific serine residue by GSK3β that is a serine/threonine kinase known to play important roles in a variety of developmental events. He found a consensus phosphorylation site for GSK3β in NeuroAB at serine 56. A series of reporter assay showed that the repressive activity of NeuroAB is inhibited by phosphorylation at a specific serine residue in the consensus phosphorylation site for GSK3β. Similar phosphorylation sites are observed in other BETA3 group members: BETA3, BHLHB4, Olig1, and Olig2. This finding suggests that the activities of BETA3 group members are commonly regulated by GSK3β or other kinases sharing the same substrate specificity such as GSK3α. It is conceivable that protein phosphorylation regulates transcription factor activity by modulating cellular localization, protein stability, protein-protein interactions or DNA binding. The results of the DNA binding assays suggested that phosphorylation of NeuroAB by GSK3β leads to reduction of the DNA-binding activity. As the expression profile of GSK3β in the developing retina had yet to be elucidated, he performed in situ hybridization to examine whether the expression of GSK3β is spatially and temporally regulated in the developing retina. Its strong expression was observed in ganglion cells from E8 and a subset of amacrine cells from E12. These findings suggest that NeuroAB is involved in the maturation and maintenance of bipolar cells and GABAergic amacrine cells and regulation by GSK3β plays an important role in retinogenesis