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 BglII 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.link_to_OA_fulltex

The role of kreisler in segmentation during hindbrain development

The mouse kreisler gene is expressed in rhombomeres (r) 5 and 6 during neural development and kreisler mutants have patterning defects in the hindbrain that are not fully understood. Here we analyzed this phenotype with a combination of genetic, molecular, and cellular marking techniques. Using Hox/lacZ transgenic mice as reporter lines and by analyzing Eph/ephrin expression, we have found that while r5 fails to form in these mice, r6 is present. This shows that kreisler has an early role in the formation of r5. We also observed patterning defects in r3 and r4 that are outside the normal domain of kreisler expression. In both heterozygous and homozygous kreisler embryos some r5 markers are induced in r3, suggesting that there is a partial change in r3 identity that is not dependent upon the loss of r5. To investigate the cellular character of r6 in kreisler embryos we performed heterotopic grafting experiments in the mouse hindbrain to monitor its mixing properties. Control experiments revealed that cells from even- or odd- numbered segments only mixed freely with themselves, but not with cells of opposite character. Transposition of cells from the r6 territory of kreisler mutants reveals that they adopt mature r6 characteristics, as they freely mix only with cells from even-numbered rhombomeres. Analysis of Phox2b expression shows that some aspects of later neurogenesis in r6 are altered, which may be associated with the additional roles of kreisler in regulating segmental identity. Together these results suggest that the formation of r6 has not been affected in kreisler mutants. This analysis has revealed phenotypic and mechanistic differences between kreisler and its zebrafish equivalent valentino. While valentino is believed to subdivide preexisting segmental units, in the mouse kreisler specifies a particular segment. The formation of r6 independent of r5 argues against a role of kreisler in prorhombomeric segmentation of the mouse hindbrain. We conclude that the mouse kreisler gene regulates multiple steps in segmental patterning involving both the formation of segments and their A-P identity.link_to_subscribed_fulltex