During embryonic development, the presumptive GABAergic rostral thalamus (rTh) and glutamatergic caudal thalamus (cTh) are induced by Sonic hedgehog (Shh) signaling from the zona limitans intrathalamica (ZLI) at the rostral border of the thalamic primordium. We found that these inductions are limited to the neuroepithelium between the ZLI and the forebrain-midbrain boundary, suggesting a prepattern that limits thalamic competence. We hypothesized that this prepattern is established by the overlapping expression of two transcription factors: Iroquois-related homeobox gene 3 (Irx3) posterior to the ZLI, and paired box gene 6 (Pax6) anterior to the forebrain-midbrain boundary. Consistent with this assumption, we show that misexpression of Irx3 in the prethalamus or telencephalon results in ectopic induction of thalamic markers in response to Shh, that it functions as a transcriptional repressor in this context, and that antagonizing its function in the diencephalon attenuates thalamic specification. Similarly, misexpression of Pax6 in the midbrain together with Shh pathway activation results in ectopic induction of cTh markers in clusters of cells that fail to integrate into tectal layers and of atypical long-range projections, whereas antagonizing Pax6 function in the thalamus disrupts cTh formation. However, rTh markers are negatively regulated by Pax6, which itself is down-regulated by Shh from the ZLI in this area. Our results demonstrate that the combinatorial expression of Irx3 and Pax6 endows cells with the competence for cTh formation, whereas Shh-mediated down-regulation of Pax6 is required for rTh formation. Thus, thalamus induction and patterning depends both on a prepattern of Irx3 and Pax6 expression that establishes differential cellular competence and on Shh signaling from the ZLI organizer.cell signaling | neural development

The regionalisation of the nervous system begins early in embryogenesis, concomitant with the establishment of the anteroposterior (AP) and dorsoventral (DV) body axes. The molecular mechanisms that drive axis induction appear to be conserved throughout the animal kingdom and may be phylogenetically older than the emergence of bilateral symmetry. As a result of this process, groups of patterning genes that are equally well conserved are expressed at specific AP and DV coordinates of the embryo. In the emerging nervous system of vertebrate embryos, this initial pattern is refined by local signalling centres, secondary organisers, that regulate patterning, proliferation, and axonal pathfinding in adjacent neuroepithelium. The main secondary organisers for the AP neuraxis are the midbrain-hindbrain boundary, zona limitans intrathalamica, and anterior neural ridge and for the DV neuraxis the notochord, floor plate, and roof plate. A search for homologous secondary organisers in nonvertebrate lineages has led to controversy over their phylogenetic origins. Based on a recent study in hemichordates, it has been suggested that the AP secondary organisers evolved at the base of the deuterostome superphylum, earlier than previously thought. According to this view, the lack of signalling centres in some deuterostome lineages is likely to reflect a secondary loss due to adaptive processes. We propose that the relative evolutionary flexibility of secondary organisers has contributed to a broader morphological complexity of nervous systems in different clades.