For the vast majority of the genes that we tested by ChIP-PCR, we were able to validate Arx promoter binding not only in transfected N2a cells but also using mouse embryonic brains immunoprecipitates . Similarly, we confirmed Arx fixation to Zic3, and yet this gene was below our threshold of selection as it was positive in only one experiment in transfected cells and one experiment in embryonic brain. This observation suggests that some genes we identified in only one or two replicates of ChIP experiments may be true targets of Arx and that the number of genes we selected as positive in all three replicates may be slightly underestimated. We then showed that quantitative Abmole Company VE-822 expression levels of putative targets could be significantly altered by the ectopic expression of Arx in N2a cells or its knock-down in ventral telencephalon. In total, we found that approximately one quarter of Arx putative targets were deregulated following increasing or decreasing Arx levels in neuroblastoma cells or embryonic brain. However, this number is probably underestimated as Arx is normally not expressed in N2a cells and may thus lack binding partners and/or cofactors necessary to regulate the expression of certain genes. For example, we noticed that among the 159 ChIP-positive genes that showed gene expression changes in mutant brain, 135 genes did not show any mRNA change in Arx-transfected N2a cells. In addition, Fulp and Colasante have focused on ventral telencephalon for their gene expression experiments , thus probably leaving out genes involved in neuroblast proliferation and/or radial migration. Similarly, as we selected only one stage of development in mouse in our ChIP experiments, it is likely that we missed several genes involved in earlier steps such as brain patterning or on the contrary later steps such as synaptogenesis and connectivity. Some changes in gene expression may also be too low to be detected in microarrays experiments. It is for example the case of Gabrb3, Lmo3 or Cdh2 that show a change of expression in qRT-PCR experiments following Arx expression in N2a cells but not on microarrays. In conclusion, this study presents the first global analysis of Arx direct transcriptional targets.