We used conditional inactivation to study the role of Sp8, the ortholog of the Drosophila transcription factor buttonhead , during murine forebrain development. We report that the absence of Sp8 provokes a morphological dysplasia of the rostromedial forebrain, perturbs A/P patterning and enhances apoptosis of neuronal progenitors. A marker analysis further revealed that although the layering of the mutant cerebral cortex seems normal, Sp8 function is required for the specification of neuronal subpopulations.
Sp8 has an essential role in the formation of the telencephalic midline
One morphologically apparent defect was the dysgenesis of the septum. On the molecular level, this might result from the ventral expansion of Emx2, Pax6 and Ngn2 expression territories. As a consequence, the expression of several ventral markers, such as Fgf8, Mash1, Dlx1, and most importantly, Nkx2.1 is regionally down-regulated or completely abolished. Interestingly, Shh and Wnt expression seems to be preserved in the mutant, suggesting that the observed perturbation is independent of these signaling pathways. Our findings suggest that Sp8 might have a critical role in the maintenance of gene activity at the mPSB, since early marker expression is not affected. Interestingly, both Sp8 and Fgf8 are expressed in the septum anlage at early developmental stages. Recent findings demonstrate that FGF signaling is acting downstream of Shh to propagate ventral telencephalic cell types and to promote their survival . Our study reveals that, while preserved in the midline and septum anlage until E10.5, the expression of Fgf8 (in the septum) and Nkx2.1 (in the septum and rostral MGE) is completely abolished at E12.5. Moreover, a recent report provides evidence that Fgf8 may regulate Nkx2.1 expression, interfering with the axial patterning of the telencephalon , therefore suggesting that the midline defect in cKO could be mediated through Fgf8.
It was recently found that Sp8 is down-regulated in the medial telencephalon of Fgf8 hypomorphic and conditional mutants . Strikingly, it was demonstrated by three independent studies that Sp8 might positively regulate Fgf8 expression in the mouse AER [12,13], and in the zebrafish pectoral fin . Conversely, the loss of Sp8 in the MHB provokes an expansion of the Fgf8 expression domain . Therefore, an attractive interpretation of the defect at the medial wall might be that a context-dependent bidirectional interaction between Fgf8 and Sp8 is responsible for the correct patterning of the ventro-medial telencephalon. Recently, FGF signaling was shown to be crucial for the genesis of the SE . In the absence of late Fgf8 activity in Sp8cKO, the septal territory abnormally expresses dorsal markers, and therefore, might acquire pallial properties (Figure 8), supporting the idea that Fgf8 and Sp8 may also be required for sustaining ventral cellular identity of septal precursors.
Although Sp8 is expressed in the dLGE, the patterning at the PSB is not disturbed. Two possibilities might be envisioned: first, the D/V patterning at the PSB is established before E9, 5; and second, functional redundancy may exist between Sp8 and the closely related transcription factor Sp9  in the ventral telencephalon (Additional data file 1).
Sp8 affects cortical arealization along the A/P axis
We recently have shown that Sp8 knockout mice display a patterning defect at the MHB . In the present study we demonstrate that, in addition to its role in the formation of a normal mPSB, Sp8 is necessary for the molecular arealization of the cerebral cortex along the A/P axis. The arealization of the early cortical primordium is dependent on the regionalized expression of ligands belonging to the FGF, WNT/BMP and epidermal growth factor signaling pathways, produced by the anterior neural ridge (ANR), cortical hem, roof plate and antihem, respectively [1,3]. Such ligands are assumed to control the graded expression of transcription factors, encoding positional pattern, and specific for distinct cortical fields. So far, only a few regionally enriched transcription factors have been shown to be critical for this process. For instance, in mice where either Pax6 or Emx2 is not functional, the corresponding rostral and caudal cortical regions, where these genes display highest expression, appear malformed and cortical areas are displaced in opposite directions [2,46].
Our findings suggest that the inactivation of Sp8 in the forebrain causes a prominent caudalization of the molecular properties of the cortical neuroepithelium, as highlighted by the ectopic rostral expansion of the expression domains of the regionally enriched marker genes Coup-TF1, EphrinA5 and EphA7. Furthermore, Sp8cKO cortices show an enhanced Emx2 and a reciprocally down-regulated Pax6 expression gradient. This supports the notion that the genetic interplay between Pax6 and Emx2 is controlling the establishment of their normal expression gradients . However, the loss of Emx2 or Pax6 function does not affect the expression of Sp8, suggesting that Sp8 acts upstream of these genes (Additional data file 1).
FGF signaling from the ANR plays a crucial role in the patterning along the cortical A/P axis . Evidence has been presented that Emx2 might indirectly control cortical arealization, through the regulation of Fgf8 . Recent data, however, challenged such a view by demonstrating that Emx2 may operate directly, and independent of, Fgf8 to specify cortical areas . In addition, it was shown that thalamocortical connectivity is affected only in the absence of proper Emx2 function , and conversely does not change in Fgf8 hypomorphic cortices in vivo . In good agreement with these findings we observe defects in thalamocortical projections in Sp8cKO cortices. Notably, the early FGF signaling from the ANR does not seem to be affected in the Sp8cKO forebrain until at least E10.5. Taken together, our findings strongly suggest leading roles for Emx2 and Sp8 in cortical arealization.
In agreement with findings in Drosophila, we show a direct interaction between Sp8 and Emx2 proteins in vitro, indicating a conservation of this regulatory pathway .
Sp8 plays a critical role in cortical neurogenesis
Enhanced apoptosis was detected in the limbs  and basal telencephalon in Sp8 loss-of-function mice . We show here that the forebrain hypoplasia in Sp8cKO mice is primarily due to cell death, affecting both early and late progenitor pools of dorsal and ventral telencephalon.
We further found that when the function of Sp8 is abolished, the preplate splitting is defective, and the MZ contains more Reelin+ cells , possibly due to the enhanced Emx2 expression in the mutant cortex. The basic lamination of the cortex is not compromised in cKO. However, the specification of particular neuronal subtypes (such as ER81-, RzR-β-, and Cux1-positive) is affected. Similarly, conditional ablation of Sp8 in the basal telencephalon results in misspecification of a subset of interneurons .