Here we describe mechanisms regulating area patterning of developing mammalian neocortex, referred to as arealization. Current findings indicate an interplay between intrinsic genetic mechanisms and extrinsic information relayed to cortex by thalamocortical input. Intrinsic mechanisms are based on morphogens and signaling molecules secreted by patterning centers, positioned at the perimeter of dorsal telencephalon, that generate across nascent cortex the graded expression of transcription factors in cortical progenitors. Two major patterning centers are the commissural plate, which expresses Fgf8 and Fgf17, and the cortical hem, which expresses Bmps and Wnts. Four transcription factors, COUP-TFI, Emx2, Pax6, and Sp8, with graded expression across the embryonic cortical axes, are shown to determine sizes and positions of cortical areas by specifying or repressing area identities within cortical progenitors. They also interact to modify their expression, as well as expression of Fgf8. We review these mechanisms of arealization and discuss models and concepts of cortical area patterning.

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of a family of cysteine proteases called caspases, and caspase-activated DNase (CAD/DFF40) and lamin protease (caspase-6) have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensation. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.

Arealization of the neocortex is controlled by a regulatory hierarchy beginning with morphogens secreted from patterning centers positioned at the perimeter of the dorsal telencephalon. These morphogens act in part to establish within cortical progenitors the differential expression of transcription factors that specify their area identity, which is inherited by their neuronal progeny, providing the genetic framework for area patterning. The two patterning centers most directly implicated in arealization are the commissural plate, which expresses fibroblast growth factors, and the cortical hem, which expresses bone morphogenetic proteins and vertebrate orthologs of Drosophila wingless, the Wnts. A third, albeit putative, patterning center is the antihem, identified by its expression of multiple signaling molecules. We describe recent findings on roles for these patterning centers in arealization. We also present the most recent evidence on functions of the four transcription factors, Emx2, COUP-TFI, Pax6, and Sp8, thus far implicated in arealization. We also describe screens for candidate target genes of these transcription factors, or other genes potentially involved in arealization. We conclude with an assessment of a forward genetics approach for identifying genes involved in determining area size based in part on quantitative trait locus mapping, and the implications for significant differences between individuals in area size on behavioral performance.

Summary Radial glia (RG), the progenitors of cortical neurons and basal progenitors (BPs), differentiate from neuroepithelial cells (NCs) with stem cell properties. We show that the morphogen, Fgf10, is transiently expressed by NCs coincident with the transition period of NC differentiation into RG. Targeted deletion of Fgf10 delays RG differentiation, whereas overexpression has opposing effects. Delayed RG differentiation in Fgf10 mutants occurs selectively in rostral cortex, paralleled by an extended period of symmetric NC divisions increasing progenitor number, coupled with delayed and initially diminished production of neurons and BPs. RG eventually differentiate in excess number and overproduce neurons and BPs rostrally resulting in tangential expansion of frontal areas and increased laminar thickness. Thus, transient Fgf10 expression regulates timely differentiation of RG, and through this function, determines both length of the early progenitor expansion phase and onset of neurogenesis, and ultimately the number of progenitors and neurons fated to specific cortical areas.

Telencephalic patterning centers, defined by the discrete expression domains of distinct morphogens, Fgfs in the commissural plate (CoP), Wnts and Bmps in the cortical hem, and a ventral domain of Sonic hedgehog (Shh), are postulated to establish during development the initial patterning of the telencepahlon, including the neocortex. We show that the expression patterns of Sp5, Sp8, and Sp9, members of the Sp8-like family that are homologues of Drosophila buttonhead, correlate during early embryonic development with these three telencephalic patterning centers. To study potential functional relationships, we focused on Sp8, because it is transiently expressed in the CoP coincident with the expression of Fgf8, a morphogen implicated in area patterning of the neocortex. We also show that Sp8 is expressed in cortical progenitors in a high to low anterior-medial to posterior-lateral gradient across the ventricular zone. We used in utero electroporation of full-length and chimeric expression constructs to perform gain-of-function and loss-of-function studies of interactions between Sp8 and Fgf8 and their roles in cortical area patterning. We show that Fgf8 and Sp8 exhibit reciprocal induction in vivo in the embryonic telencephalon. Sp8 also induces downstream targets of Fgf8, including ETS transcription factors. In vitro assays show that Sp8 binds Fgf8 regulatory elements and is a direct transcriptional activator of Fgf8. We also show that Sp8 induction of Fgf8 is repressed by Emx2 in vitro, suggesting a mechanism to limit Fgf8 expression to the CoP. In vivo expression of a dominant negative Sp8 in the CoP indicates that Sp8 maintains expression of Fgf8 and also its effect on area patterning. Ectopic expression of Sp8 in anterior or posterior cortical poles induces significant anterior or posterior shifts in area patterning, respectively, paralleled by changes in expression of gene markers of positional identity. These effects of Sp8 on area patterning oppose those induced by ectopic expression of Fgf8, suggesting that in parallel to regulating Fgf8 expression, Sp8 also activates a distinct signaling pathway for cortical area patterning. In summary, Sp8 and Fgf8 robustly induce one another, and may act to balance the anterior-posterior area patterning of the cortex.