The cerebral cortex develops from the dorsal telencephalon, at the anterior end of the neural tube. Neurons are generated by cell division at the inner surface of the telencephalic wall (in the ventricular zone) and migrate towards its outer surface, where they complete their differentiation. Recent studies have suggested that the transcription factor Pax6 is important for regulation of cell proliferation, migration and differentiation at various sites in the CNS. This gene is widely expressed from neural plate stage in the developing CNS, including the embryonic cerebral cortex, where it is required for radial glial cell development and neuronal migration. We report new findings indicating that, in the absence of Pax6, proliferative rates in the early embryonic cortex are increased and the differentiation of many cortical cells is defective. A major question concerns the degree to which cortical defects in the absence of Pax6 are a direct consequence of losing the gene function from defective cells themselves, rather than being secondary to abnormalities in other cells. Cortical defects in the absence of Pax6 become much more pronounced later in cortical development, and we propose that many result from a compounding of abnormalities in proliferation and differentiation that first appear at the onset of corticogenesis.

Pax-6 is one of the earliest regulatory genes to be expressed in the diencephalon. We tested whether normal Pax-6 protein is required for early diencephalic development by examining morphology, precursor proliferation and patterns of regulatory gene expression in the embryonic diencephalon of Small-eye mice (Pax-6 mutants). In Small-eye mice, diencephalic morphology was abnormal at all the embryonic ages studied (days 10.5, 12.5 and 14.5). Regional differences in diencephalic cell density were lost, the diencephalon/mesencephalon boundary was unclear and the third ventricle was enlarged. We estimated diencephalic proliferative rates after labelling with bromodeoxyuridine and found that they were abnormally low in mutants aged embryonic day 10.5. In older mutants, the diencephalon contained fewer cells than normal. In wild-type E14.5 diencephalon, Pax-6, Dlx-2 and Wnt-3 are expressed in discrete regions along the rostrocaudal and dorsoventral axes. In situ hybridizations for these genes in E14.5 Small-eye mice revealed discrete zones of diencephalic expression that had similar relative positions to those in wild-type mice. Some differences of detail in their expression were seen: Pax-6 had an expanded rostral domain of expression and an abnormally indistinct caudal boundary; Dlx-2 had a diffuse, rather than a sharp, caudal boundary of expression; the normally high dorsal midline expression of Wnt-3 was lost. We conclude that normal expression of Pax-6 is required for the correct regulation of diencephalic precursor proliferation. Pax-6 may also control some aspects of diencephalic differentiation, but its mutation in Small-eye mice does not preclude the development of a degree of diencephalic regionalization resembling that in normal mice.

The transcription factor Pax6 is widely expressed throughout the developing nervous system, including most alar regions of the newly formed murine diencephalon. Later in embryogenesis its diencephalic expression becomes more restricted. It persists in the developing anterior thalamus (conventionally termed “ventral” thalamus) and pretectum but is downregulated in the body of the posterior (dorsal) thalamus. At the time of this downregulation, the dorsal thalamus forms its major axonal efferent pathway via the ventral telencephalon to the cerebral cortex. This pathway is absent in mice lacking functional Pax6 (small eye homozygotes: Sey/Sey). We tested whether the mechanism underlying this defect includes abnormalities of the dorsal thalamus itself. We exploited a new transgenic mouse ubiquitously expressing green fluorescent protein tagged with tau, in which axonal tracts are clearly visible, and co-cultured dorsal thalamic explants from Pax6(+/+)or Pax6(Sey/Sey)embryos carrying the transgene with wild-type tissues from other regions of the forebrain. Whereas Pax6(+/+)thalamic explants produced strong innervation of wild-type ventral telencephalic explants in a pattern that mimicked the thalamocortical tract in vivo, Pax6(Sey)(/Sey) explants did not, indicating a defect in the ability of mutant dorsal thalamic cells to respond to signals normally present in ventral telencephalon. Pax6(Sey)(/Sey) embryos also showed early alterations in the expression of regulatory genes in the region destined to become dorsal thalamus. Whereas in normal mice Nkx2.2 and Lim1/Lhx1 are expressed ventral to this region, in the mutants their expression domains are throughout it, suggesting that a primary action of Pax6 is to generate correct dorsoventral patterning in the diencephalon. Our results suggest that normal thalamocortical development requires the actions of Pax6 within the dorsal thalamus itself.

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