Identification and characterization of BRN3A/BRN3B as DLX homeobox gene transcriptional targets in retinal development
Introduction: The Dlx1/Dlx2 double knockout mouse has reduced numbers (33% fewer) of retinal ganglion cells (RGC), due to enhanced apoptosis. Brn3a and Brn3b are closely related members of the Class IV POU-domain gene family, and play functionally interchangeable roles in retinal development. We hypothesized that Brn3a and/or Brn3b are direct DLX transcriptional targets during retinal development. Methods: Chromatin immunoprecipitation (ChIP) assays were performed on E16.5 retinas to identify DLX proteins bound to the Brn3a or Brn3b promoters. Electrophoretic mobility shift assays (EMSA) were used to confirm the specificity of this binding. Luciferase reporter gene assays were performed to confirm the functional significance of DLX binding to the Brn3 or Brn3ba promoters in vitro. In utero retinal electroporation was used to study the effect of DLX gain-of-function on Brn3a/Brn3b expression in vivo. Compound Dlx1/Dlx2/Brn3a and Dlx1/Dlx2/Brn3b knockout mice were generated for analysis of retinal phenotypes. Results: Both DLX1 and DLX2 proteins bound to the Brn3b promoter, but only DLX2 bound to the Brn3a promoter in vivo. Using EMSA, recombinant DLX1 and DLX2 bound to Brn3b and specific supershifted bands resulted from the addition of specific DLX1 or DLX2 antibodies; only recombinant DLX2 bound to Brn3a and the supershifted band resulted from the addition of DLX2 antibody. Both DLX1 and DLX2 binding to the Brn3b promoter activated transcription of a luciferase reporter gene in vitro. Only Dlx2, but not Dlx1, co-transfection with Brn3a activated luciferase reporter gene expression. In utero retinal electroporation showed that ectopic DLX2 expression promoted both Brn3b and Brn3a expression in vivo. Loss of Dlx1/Dlx2 and Brn3b resulted in loss of 90% of RGC and increased cholinergic amacrine cell differentiation. Conclusion: Brn3b is transcriptionally regulated by both DLX1 and DLX2, whereas Brn3a is only regulated by DLX2 in vitro and in vivo. Dlx1/Dlx2 and Brn3b play combinatorial roles in retinogenesis.