Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity along the antero-posterior axis.
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.
Nolte
Christof
C
McGill Cancer Centre, McGill University, 3655 Promenade Sir-William-Osler, Montreal, QC, Canada H3G 1Y6.
Rastegar
Mojgan
M
Amores
Angel
A
Bouchard
Maxime
M
Grote
David
D
Maas
Richard
R
Kovacs
Erzsebet Nagy
EN
Postlethwait
John
J
Rambaldi
Isabel
I
Rowan
Sheldon
S
Yan
Yi-Lin
YL
Zhang
Feng
F
Featherstone
Mark
M
D000818:Animals; D001483:Base Sequence; D019521:Body Patterning; D002460:Cell Line; D002490:Central Nervous System / Q000196:embryology* / Q000378:metabolism; D017124:Conserved Sequence; D004742:Enhancer Elements, Genetic; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012249:Rhombencephalon / Q000196:embryology / Q000378:metabolism; D013237:Stereoisomerism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D005136:Eye Proteins; D018398:Homeodomain Proteins; C489520:Hoxd4 protein, mouse; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D014157:Transcription Factors