Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20.
The hindbrain is a segmented structure divided into repeating metameric units termed rhombomeres (r). The Hox family, vertebrate homologs of the Drosophila HOM-C homeotic selector genes, are expressed in rhombomere-restricted patterns and are believed to participate in regulating segmental identities. Krox-20, a zinc finger gene, has a highly conserved pattern of expression in r3 and r5 and is functionally required for their maintenance in mouse embryos. Krox-20 has been shown to directly regulate the Hoxb-2 gene and we wanted to determine if it was involved in regulating multiple Hox genes as a part of its functional role. Hoxa-2 is the only known paralog of Hoxb-2, and we examined the patterns of expression of the mouse Hoxa-2 gene with particular focus on r3 and r5 in wild type and Krox-20-/- mutant embryos. There was a clear loss of expression in r3, which indicated that Hoxa-2 was downstream of Krox-20. Using transgenic analysis with E. coli lacZ reporter genes we have identified and mapped an r3/r5 enhancer in the 5' flanking region of the Hoxa-2 gene. Deletion analysis narrowed this region to an 809 bp Bg/II fragment, and in vitro binding and competition assays with bacterially expressed Krox-20 protein identified two sites within the enhancer. Mutation of these Krox-20 sites in the regulatory region specifically abolished r3/r5 activity, but did not affect neural crest and mesodermal components. This indicated that the two Krox-20 sites are required in vivo for enhancer function. Furthermore, ectopic expression of Krox-20 in r4 was able to transactivate the Hoxa 2/lacZ reporter in this rhombomere. Together our findings suggest that Krox-20 directly participates in the transcriptional regulation of Hoxa-2 during hindbrain segmentation, and is responsible for the upregulation of the r3 and r5 domains of expression of both vertebrate group 2 Hox paralogs. Therefore, the segmental phenotypes in the Krox-20 mutants are likely to reflect the role of Krox-20 in directly regulating multiple Hox genes.
Nonchev
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Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, UK.
Vesque
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C
Maconochie
M
M
Seitanidou
T
T
Ariza-McNaughton
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L
Frain
M
M
Marshall
H
H
Sham
M H
MH
Krumlauf
R
R
Charnay
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P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D051767:Early Growth Response Protein 2; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005801:Genes, Homeobox; D006720:Homozygote; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D015345:Oligonucleotide Probes; D010641:Phenotype; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D012249:Rhombencephalon / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism*; D014714:Vertebrates; D016335:Zinc Fingers; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D051767:Early Growth Response Protein 2; C497327:Egr2 protein, mouse; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase