Regulation of Emx2 Expression by Antisense Transcripts in Murine Cortico-Cerebral Precursors

BACKGROUND Emx2 encodes for a transcription factor expressed in the embryonic intermediate mesoderm and central nervous system (CNS). It is implicated in several aspects of cerebral cortex development, including morphogenetic field specification, arealization, precursor proliferation and lamination. Four Emx2-associated antisense transcripts have been found in the urogenital system; one of them, Emx2OS, has been also detected in the adult brain. Until now, however, nothing is known about expression and function of Emx2OS in the developing CNS. METHODOLOGY/PRINCIPAL FINDINGS By quantitative RT-PCR and in situ hybridization, we reconstructed the Emx2OS expression profile in the embryonic CNS, paying special attention to the developing cerebral cortex. Emx2OS was observed in a number of CNS structures expressing also Emx2. Within the cortex, Emx2OS was detectable in periventricular precursors, expressing the sense transcript, and peaked in newly born post-mitotic neurons not expressing such transcript. By integrating lentiviral gene delivery, RNAi, TetON technology, morpholino-mediated gene knock-down, drug-induced perturbation of gene expression, and quantitative RT-PCR, we addressed possible roles of Ex2 antisense RNA in Emx2 regulation, in primary CNS precursor cultures. We found that, in both cortical precursors and their neuronal progenies, Emx2 antisense RNA contributes to post-transcriptional down-regulation of its sense partner, possibly by a Dicer-promoted mechanism. The same RNA, when delivered to rhombo-spinal precursors, stimulates ectopic expression of Emx2, whereas Emx2 knock-out dramatically impairs Emx2OS transcription. This suggests that, within the developing CNS, a reciprocal Emx2/Emx2OS regulatory loop may normally sustain transcription at the Emx2 locus. CONCLUSIONS/SIGNIFICANCE This study shows that antisense transcripts may contribute to developmental regulation of a key transcription factor gene implicated in CNS patterning, possibly by complex and multilevel mechanisms. The activation of Emx2 by a short antisense transcript may be a prototype of a method for overexpressing single specific genes, without introducing additional copies of them into the genome.