Two Pax2/5/8-binding sites in Engrailed2 are required for proper initiation of endogenous mid-hindbrain expression

During early brain development mouse Engrailed2 (En2) is expressed in a bro ad band across most of the mid-hindbrain region. Evidence from gene express ion data, promoter analysis in transgenic mice and mutant phenotype analysi s in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical ro le in regulating En2 mid-hindbrain expression, Previously, we identified tw o Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficie nt to directed reporter gene expression to the early mid-hindbrain region a nd showed that the two Pax2/5/8-binding sites are essential for the mid-hin dbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the con text of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced wi th the bacterial neo gene by homologous recombination in mouse embryonic st em cells. After transmitting the mutation into mice, the neo gene was remov ed by breeding with transgenic mice expressing cre from a CMV promoter. Emb ryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression in distinguishable from that seen in wild type embryos. Furthermore, the mutan ts did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expres sion of the endogenous En2 gene. Interestingly, a comparison of the lacZ RN A and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbr ain region by persisting longer than the mRNA. The transgene expression dir ected by the 1.0 kb enhancer fragment therefore does not mimic the entire b road domain of En2 expression. Taken together, these two studies demonstrat e that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression. (C) 2000 Elsevier Science I reland Ltd. All rights reserved.