Otx2 is a paired-type homeobox gene expressed at every step of tetrapod head development – in the epiblast (EP), the distal anterior visceral endoderm, the anterior mesendoderm, the anterior neuroectoderm (AN),the entire forebrain and midbrain, and the cephalic mesenchyme. However,because Otx2 is essential for mouse head development, it is difficult to distinguish the functions that it performs at these different stages. In two papers in this issue, Aizawa and colleagues describe the enhancers responsible for Otx2 expression in the EP and the AN(p. 3307), and in the forebrain and midbrain (p. 3319). The EP enhancer is active in the EP at E5.5 and the AN enhancer is active in the AN from E7.0 until E8.5. By mutating the AN enhancer in vivo, the researchers found that it is required for the maintenance of the AN during development. Unexpectedly, although the AN gives rise to the forebrain and midbrain, Otx2 expression in the forebrain and midbrain is not regulated by the AN enhancer, but by two other distinct enhancers: FM and FM2. In their second paper, the researchers show that this switch in enhancer activity occurs between E8.0 and E8.5, corresponding to the stage at which the boundary between the midbrain and the hindbrain is established. Furthermore, the development of these regions is regulated differentially in a dose-dependent manner; higher levels of Otx2 appear to be required for the development of more posterior portions of the forebrain/midbrain. Aizawa and colleagues maintain that these results represent a starting point for finding the many unknown factors that regulate these enhancers. They also consider the conservation of these enhancer sequences across mouse, human, Xenopusand zebrafish, and discuss the future work required to determine the phylogenetic significance of Otx2 enhancer organisation.