Auto/Cross-Regulation of Hoxb3 Expression in Posterior Hindbrain and Spinal Cord

Abstract

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler.

Hox Genes and Their Candidate Downstream Targets in the Developing Central Nervous System

“…This is, in part, due to the high degree of Hox gene auto-and cross-regulation (Pöpperl et al, 1995;Gould et al, 1997;Maconochie et al, 1997;Zhang et al, 1997a;Packer et al, 1998;Hooiveld et al, 1999;Jacobs et al, 1999;Kwan et al, 2001;Manzanares et al, 2001;Yau et al, 2002). Regulatory interactions between the Hox genes and other homeobox genes are also quite common, as has been observed in the development of the brain for the regulation of Otx.…”

Comparative genomic analysis of vertebrate Hox3 and Hox4 genes

“…Although the zebrafish hoxb3a regulatory sequences include a motif similar to the mouse ERAS/KroxA site, there are three nucleotide changes in the motif suggesting that the zebrafish hoxb3a r5 enhancer may function independently of Krox20. In mice, r6 expression of Hoxb3 is separately controlled by element III (r6 enhancer), which includes a Hox/Pbx binding site (Kwan et al, 2001;Yau et al, 2002). We could not identify an equivalent site in zebrafish, however, the 36 bp CNS 14, which includes a Pbx/Hox consensus sequence (Fig.…”

“…The function is mediated by two Hox binding sites, HS1 and HS2, in an auto and crossregulatory manner (Yau et al, 2002). This r6 enhancer is not conserved within zebrafish, as none of the CNS in our Pipmaker alignment corresponds to the mouse r6 enhancer.…”

The Role ofHoxGenes in Female Reproductive Tract Development, Adult Function, and Fertility

“…There are transformations of sacral and first caudal vertebrae in Hoxa11 knockout mice. In the vertebrate nervous system, the hindbrain or rhombencephalon develops under the regulating of such segmental patterning directed by Hox gene expression as well; regional expression of Hox genes in the hindbrain is thought to confer identity to rhombomeres (Carpenter et al 1993;Mark et al 1993;Goddard et al 1996;Studer et al 1996;Morrison et al 1997;Manzanares et al 1999;Ferretti et al 2000;Yau et al 2002). Mice harboring a Hoxa1 mutation have alteration in hindbrain segmentation, deleting all or part of rhombomere5 (r5).…”