Figure 8.

Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies co-precipitate with the promoters of Brn3b, Shh, Brn3a and Isl2. Both antibodies do not precipitate with control Brn3b ORF. Moreover, control IgG precipitation does not show any non-specific binding to these promoters. (B) Functional synergy of ISL1 and BRN3B on Brn3a luciferase reporter gene expression. CV1 cells are co-transfected with Brn3a luciferase reporter construct, different expression plasmids and PRL encoding CMV-renilla luciferase. Cells transfected with reporter, PRL and empty pcDNA expression vector are used as controls. Luciferase activity is determined by normalizing firefly activity with renilla activity. Then fold activation is calculated by dividing the luciferase activity of experimental group with that of control. Each histogram represents the mean + s.d. (n=4) (C) The Math5-Brn3b/Isl1 pathway in the development of RGCs. MATH5 expression endows the post-mitotic precursors with RGC competence by activating BRN3B and ISL1 expression and inhibiting the expression of non-RGC bHLH TFs. In this report, we show that ISL1 cooperates with BRN3B to regulate RGC terminal differentiation by maintaining the expression of Brn3b and activating a RGC-specific gene cascade, including Brn3a, Isl2, Shh, and Irx4. Solid lines indicate the direct regulation identified.