Nolte Christof, Rastegar Mojgan, Amores Angel, Bouchard Maxime, Grote David, Maas Richard, Kovacs Erzsebet Nagy, Postlethwait John, Rambaldi Isabel, Rowan Sheldon, Yan Yi-Lin, Zhang Feng, Featherstone Mark
McGill Cancer Centre, McGill University, 3655 Promenade Sir-William-Osler, Montreal, QC, Canada H3G 1Y6.
Dev Biol. 2006 Nov 15;299(2):582-93. doi: 10.1016/j.ydbio.2006.08.061. Epub 2006 Aug 30.
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 Sey(Neu) 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.
神经管的前后（AP）模式和背腹（DV）模式分别部分受HOX和PAX转录因子的控制。我们曾报道过Hoxd4的一个神经增强子，它在后脑中将表达定向于中枢神经系统（CNS）并具有正确的前边界。与斑马鱼的直系同源增强子相比，发现了七个保守足迹，包括一个必需的视黄酸反应元件（RARE）以及相邻的D、E和F位点。虽然胚胎中枢神经系统中的增强子功能会因DNA旋转半圈使RARE与D - E - F位点分离而被破坏，但旋转一整圈则可挽救该功能，这表明DNA结合的类视黄醇受体与识别D - E - F位点的因子之间存在立体特异性限制。位点D或E突变后，Hoxd4前表达边界的DV轨迹发生改变，这暗示了在DV轴上起作用的转录调节因子。我们发现PAX6在体外能特异性结合位点D和E，并利用染色质免疫沉淀法证明在小鼠胚胎中PAX6被招募到Hoxd4神经增强子上。在胚胎第8天，Pax6突变的Sey(Neu)动物中枢神经系统中Hoxd4的表达降低。此外，pax6a和/或pax6b活性降低的阶段匹配斑马鱼胚胎表现出菱形脑节边界畸形以及hoxd4a表达边界前移。这些结果揭示了Pax6在脊椎动物Hoxd4直系同源基因的AP限制表达中具有进化保守作用。
