IDENTIFICATION OF AN EVOLUTIONARILY CONSERVED 110 BASE-PAIR CIS-ACTING REGULATORY SEQUENCE THAT GOVERNS WNT-1 EXPRESSION IN THE MURINE NEURAL PLATE

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at e arly somite stages. Wnt-1 encodes a signal that is expressed in the de veloping midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located d ownstream of the Wnt-1 coding sequence which is necessary and sufficie nt for Wnt-1 expression in vivo. Using a transgenic mouse reporter ass ay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lac Z reporter in the approximate Wnt-1 pattern at neural plate stages. Mu ltimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(-/-) embryos. The possibilit y that this region represents an evolutionarily conserved regulatory m odule is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes ). These sequences are capable of appropriate temporal and spatial act ivation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evid ence has implicated Pax transcription factors in the regulation of Wnt -1. Although Pax-2 binds to the 110 base pair murine regulatory elemen t in vitro, the location of the binding sites could not be precisely e stablished and mutation of two putative low affinity sites did not abo lish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unl ikely that Pax proteins regulate Wnt-1 by direct interactions with thi s cis-acting regulatory region. Our analysis of the 110 base pair mini mal regulatory element suggests that Wnt-1 regulation is complex, invo lving different regulatory interactions for activation and the later m aintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.