Otx2 cell-autonomously determines dorsal mesencephalon versus cerebellum fate independently of isthmic organizing activity

During embryonic development, the rostral neuroectoderm is regionalized into broad areas that are subsequently subdivided into progenitor compartments with specialized identity and fate. These events are controlled by signals emitted by organizing centers and interpreted by target progenitors, which activate superimposing waves of intrinsic factors restricting their identity and fate. The transcription factor Otx2 plays a crucial role in mesencephalic development by positioning the midbrain-hindbrain boundary (MHB) and its organizing activity. Here, we investigated whether Otx2 is cellautonomously required to control identity and fate of dorsal mesencephalic progenitors. With this aim, we have inactivated Otx2 in the Pax7⁺ dorsal mesencephalic domain, previously named m1, without affecting MHB integrity. We found that the Pax7⁺ m1 domain can be further subdivided into a dorsal Zic1⁺ m1a and a ventral Zic1^- m1b sub-domain. Loss of Otx2 in the m1a (Pax7⁺ Zic1⁺) sub-domain impairs the identity and fate of progenitors, which undergo a full switch into a coordinated cerebellum differentiation program. By contrast, in the m1b sub-domain (Pax7⁺ Zic1^-) Otx2 is prevalently required for post-mitotic transition of mesencephalic GABAergic precursors. Moreover, genetic cell fate, BrdU cell labeling and Otx2 conditional inactivation experiments indicate that in Otx2 mutants all ectopic cerebellar cell types, including external granule cell layer (EGL) precursors, originate from the m1a progenitor sub-domain and that reprogramming of mesencephalic precursors into EGL or cerebellar GABAergic progenitors depends on temporal sensitivity to Otx2 ablation. Together, these findings indicate that Otx2 intrinsically controls different aspects of dorsal mesencephalic neurogenesis. In this context, Otx2 is cell-autonomously required in the m1a subdomain to suppress cerebellar fate and promote mesencephalic differentiation independently of the MHB organizing activity.