Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity along the antero-posterior axis

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The antero-posterior (AP) and dorso-ventral (DV) patterning of the neural tube is controlled in part by HOX and PAX transcription factors, respectively. We have reported on a neural enhancer of Hoxd4 that directs expression in the CNS with the correct anterior border in the hindbrain. Comparison to the orthologous enhancer of zebrafish revealed seven conserved footprints including an obligatory retinoic acid response element (RARE), and adjacent sites D, E and F. Whereas enhancer function in the embryonic CNS is destroyed by separation of the RARE from sites D–E–F by a half turn of DNA, it is rescued by one full turn, suggesting stereospecific constraints between DNA-bound retinoid receptors and the factor(s) recognizing sites D–E–F. Alterations in the DV trajectory of the Hoxd4 anterior expression border following mutation of site D or E implicated transcriptional regulators active across the DV axis. We show that PAX6 specifically binds sites D and E in vitro, and use chromatin immunoprecipitation to demonstrate recruitment of PAX6 to the Hoxd4 neural enhancer in mouse embryos. Hoxd4 expression throughout the CNS is reduced in Pax6 mutant SeyNeu animals on embryonic day 8. Additionally, stage-matched zebrafish embryos having decreased pax6a and/or pax6b activity display malformed rhombomere boundaries and an anteriorized hoxd4a expression border. These results reveal an evolutionarily conserved role for Pax6 in AP-restricted expression of vertebrate Hoxd4 orthologs.