Regulation of Emx2 Expression by Antisense Transcripts in Murine Cortico-Cerebral Precursors (original) (raw)
Figure 7
Ectopic activation of _Emx2_-mRNA and _Emx2OS_-ncRNA in rhombo-spinal neurospheres, upon OS1-179(+) delivery.
(A) Ectopic activation of the endogenous Emx2 locus by OS1-179(+). The two graphs show _Emx2_-mRNA and _Emx2OS_-ncRNA expression levels in primary neural precursors from distinctive portions of the E10.5 CNS, rhombo-spinal tract (rh/sc) and telencephalon (te), acutely infected with “driver” and “expressor” lentivectors (Fig. 1C), kept under doxycycline and added or not with the Li+/cyclopamine mix. Samples are RNA-profiled 72 hours after infection. Data are normalized on telencephalic precursors exposed to Li+/cyclopamine, infected by control lentivirus and kept under doxycycline. Basal Emx2 expression by rhombo-spinal cells, very low as compared to telencephalic precursors, rises about 2 (p<0.01), 4 (p<0.001) and 30 times (p<0.001), upon treatment of these cells with the OS1-179(+) virus, the drug mix, or both, respectively. Two-ways ANOVA indicates that a specific interaction between OS1-179(+) and the drug mix takes place, with p<0.001. A similar course is shown by _Emx2OS_-ncRNA. (B) Reversibility of OS1-179(+)-dependent activation of the endogenous Emx2 transcription unit. The four panels to the top show time course analysis of eGFP fluorescence in neural precursors, infected by driver and OS1-179(+) lentiviruses and kept under doxycycline for 72 or 120 hours. The absence of fluorescence in the bottom-right panel means that withdrawal of doxycycline at 72 hours is sufficient to reset levels of the eGFP/OS1-179(+) chimaeric transcript to zero by 120 hours. The two graphs to the bottom show _Emx2_-mRNA and _Emx2OS_-ncRNA expression levels in primary neural precursors from the E10.5 rhombo-spinal (rh/sc) tract, acutely infected with “driver” and “expressor” lentivectors (Fig. 1C), kept under doxycycline for 72 or 120 hours and chronically exposed to Li+/cyclopamine throughout the experiment. Samples are RNA-profiled 120 hours after infection. Data are normalized on rhombo-spinal cells exposed to Li+/cyclopamine, infected by OS1-179(+) lentivirus and kept under doxycycline throughout the experiment. Removal of doxycycline at 72 hours abolishes (p<0.01) the 4-fold up-regulation of _Emx2_-mRNA, detectable in Li/cyclopamine-treated rhombo-spinal precursors, upon their further infection by lentivirus OS1-179(+) (p<0.01). Similar consequences are elicited by doxycycline removal on levels of _Emx2OS_-ncRNA.