Coordinated expression of Hoxa2, Hoxd1 and Pax6 proteins were found to coincide with the three developmental stages of the diencephalon, as described for the mouse brain. In the first stage (embryonic day (E) 10-12) Hoxa2, Hoxd1 and Pax6 (an early marker gene of the diencephalon) were expressed as early as E10.5 in prosomeres (p), p2 and p3. All three proteins continue to exhibit overlapping domains of expression at E12.5-13 (beginning of the second stage) when the primitive dense cell layer begins to differentiate into the internal germinal, external germinal and mantle layers. Towards the end of the second stage (E15), Pax6 expression was down-regulated whereas Hoxa2 and Hoxd1 continued to exhibit overlapping domains of expression for both protein and mRNA. Hoxd1 expression decreased significantly in the third stage of diencephalic development (E16-postnatal) such that only Hoxa2 expression persisted in the diencephalon of newborn mice. The temporal and spatial expression of these three proteins imply that coordinated waves of Hoxa2, Hoxd1 and Pax6 expression may be required to provide positional information for the specification of the diencephalon.