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, 2004), producing an additive effect on rod photoreceptor differentiation in insm1a morphants. Finally, we sought to identify genetic pathways that lie upstream of Insm1a activity in the retina. The results of our knockdown experiments provide strong evidence that insm1a is required for proper differentiation of retinal neurons. find more Moreover, the developmental expression pattern of insm1a in the retina matches the pattern of retinal progenitor cell (RPC) exit from the cell cycle and the onset of neurogenesis. This suggests that Notch-Delta signaling may function as an upstream negative regulator of insm1a expression, since one well-known role of this pathway is to preserve a pool of undifferentiated proliferative RPCs during retinal development ( Bernardos et al., 2005, Richard et al., 1995?and?Scheer et al., 2001). This hypothesis is supported by previous work demonstrating that inactivation of Notch-Delta signaling in mouse retinal explants caused an increase in Insm1 expression ( Nelson et al., 2007). Therefore, we examined whether expression of insm1a was similarly affected by reducing Notch activity in vivo in the zebrafish. We blocked all Notch signaling by exposing zebrafish embryos to the ��-secretase inhibitor DAPT from 10.5 to 28?hpf. This period of DAPT treatment allowed specification of the eye field to occur normally, but inhibited Notch activity diglyceride during retinal neurogenesis. We then evaluated insm1a expression in control (DMSO-treated) and DAPT-treated embryos at 28?hpf by WISH. Whereas very little expression of insm1a was observed in the retinas of control embryos at this time ( GDC-0941 cost Fig. 9A), strong expression of insm1a was observed throughout the retina and lens of DAPT-treated embryos ( Fig. 9B). Expression was also increased in other tissues, including the brain. This result indicates that Notch-Delta signaling is an early negative regulator of insm1a expression. To further explore how Notch signaling regulates insm1a expression, we next investigated whether insm1a is a direct target of Notch effector genes. The transcriptional repressor Her4 is a Notch target gene that is expressed throughout the developing nervous system ( Clark et al., 2012?and?Yeo et al., 2007). To determine whether Her4 directly interacts with the insm1a promoter, we carried out in vitro reporter assays using her4 cDNA and a luciferase reporter driven by two different lengths of the insm1a promoter. Co-transfection of HEK293 cells using the her4 expression vector and either a short insm1a regulatory sequence (?282 to +59) or long insm1a regulatory sequence (?2440/+59) demonstrated that Her4 is able to negatively regulate the insm1a promoter in a dose-dependent fashion ( Fig. 9C). In the absence of Her4, the 2.5?kb insm1a promoter drove significantly greater luciferase activity than the 300?bp promoter (t-test p