Spatiotemporal Control of Pyramidal Neuron Diversity in Cerebral Cortex Development: Intrinsic specification within progenitors and influence of POU-III transcription factors on progeny neuron characteristics

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent generation of upper-layer neurons is genetically instructed in their parental progenitor cells, mechanisms governing cell-intrinsic fate transitions remain obscure. Recent reports have demonstrated that the cortical ventricular zone (VZ), which predominantly houses neural progenitor cells, is composed of heterogeneous parental cells that give rise to distinct classes of progeny neurons and glia. We conducted in vivo manipulations to the Notch and Neurogenin pathways in the developing mouse neocortex that force differentiation of progenitors into neurons. By examining resulting cell identities, we show that the beginning of heterogeneity among VZ precursors coincides in space and time with the appearance of the first differentiating cells committed to a neural fate. This onset of neurogenesis occurs first in the lateral neocortex at E11.5 in the mouse, and spreads medially. Shortly thereafter, upper-layer neuronal classes are specified in an iterative and progressive differentiation process among progenitors. Despite the forced exit from VZ at any time or location, electroporated cells undergo a prespecified program to achieve matched laminar positions and molecular identities. Thus, Notch, Neurogenin and Tbr2 govern differentiation to appropriate neural fates regardless of time of exit, whereas "self-renewal" in the VZ is intimately tied to progressive restriction toward later-born cell fates. Furthermore, by examining regional differences in the onset and progression of neurogenesis, we propose that a combinatorial scheme of time- and location-dependent factors regulates distinct neural outcomes.;Interestingly, the induction of POU-homeodomain transcription factors Pou3f3/Brn1 and Pou3f2/Brn2 follows a spatiotemporal lateral-to-medial gradient in ventricular zone progenitors that accompanies a transition from the production of deep-layer to upper-layer neurons. Brn1/2 protein only accumulates to sufficient levels to be inherited by neural progeny starting at mid-neurogenesis, and labels cells switching from deep-layer Ctip2+ identity to Satb2 + upper-layer fate as they migrate to superficial strata of the thickening cortical plate. Using an Engrailed repressor (EnR) fusion protein to attenuate Pou3f transcription in vivo, we demonstrate that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of the Pou3f factors in ventricular zone progenitors. On the other hand, overexpression of Oct6 (Pou3f1), Brn1, or Brn2 in early neural progenitors is sufficient to cause them to switch to producing Satb2+ upper-layer neurons, many that exhibit robust pia-directed migration. Overexpression of dnMAML, which blocks Notch/RBPJK-mediated transcription, rescues neural commitment lost in Brn-EnR electroporations at E13.5, indicating that the Pou3f class promotes neurogenesis via inhibition of Notch, but generated neurons later display impaired differentiation and migration. This is congruent with our finding that these Pou3fs also regulate transcription factors critical for neurogenic differentiation. We thus identify downstream targets that likely effect these newfound roles of Pou3f factors throughout the entire cascade of late cortical neural differentiation: in neurogenesis, molecular identity, and migratory destination.