Comparison of Hoxb-1 regulatory regions from different vertebrates identified three related sequence motifs critical for rhombomere 4 (r4) expression in the hindbrain. Functional analysis in transgenic mice and Drosophila embryos demonstrated that the conserved elements are involved in a positive autoregulatory loop dependent on labial (lab) family members. Binding of Hoxb-1 to these elements in vitro requires cofactors, and the motifs closely resemble the consensus binding site for pbx1, a homolog of the Drosophila extradenticle (exd) homoedomain protein. In vitro exd/pbx serves as a Hoxb-1 cofactor in cooperative binding and in Drosophila expression mediated by the r4 enhancer is dependent on both lab and exd. This provides in vivo and in vitro evidence that r4 expression involves direct autoregulation dependent on cooperative interactions of Hoxb-1 with exd/pbx proteins as cofactors.
Within the Hoxb homeobox gene complex, Hoxb-1 is the earliest member expressed in the mesoderm and neuroectoderm of primitive streak and presomite embryos, preceding rhombomere-restricted expression in the hindbrain. Ectopic exposure of embryos to retinoic acid alters spatial aspects of Hox gene expression patterns. However, the role of retinoids in regulating these genes during normal development is unclear. We have now identified two enhancers, 3' of the mouse Hoxb-1 gene, which together reconstruct the early endogenous expression pattern and mediate the early ectopic response to retinoic acid. Furthermore, these regions are functionally conserved in both chicken and pufferfish (Fugu rubripes) Hoxb-1 genes. The enhancer that controls the retinoic acid response, and regulates expression predominantly in neuroectoderm, contains a retinoic acid response element (RARE). Point mutations in the RARE abolish expression in neuroectoderm. Therefore, this RARE is not only involved in the ectopic response to retinoic acid, but is also essential for establishing aspects of the early Hoxb-1 expression pattern.
Correct regulation of the segment.restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxbl will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxbl, Hoxal, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxbl homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxbl involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxbl in maintaining r4 identity may be mediated by the Hoxb2 gene. Segmentation in the developing vertebrate hindbrain generates repeated morphological units, termed rhombomeres. These segmental units are lineage-restricted cellular compartments that provide a means of allocating blocks of cells that have distinct properties (for review, see Lumsden 1990;Wilkinson 1993;Keynes and Krumlauf 1994). Underlying this cellular organization, the patterns of expression of a number of transcription factors, growth factors, tyrosine kinase receptors, and their ligands have boundaries of expression that are tightly linked to specific hindbrain segments (for review, see Keynes and Krumlauf 1994;Lumsden and Krumlauf 1996). Prominent among these are the Hox genes whose expression patterns form an ordered set of overlapping domains that correlate with their gene order along the Present addresses: ~Department
After activation in mesoderm and neuroectoderm, expression of the Hoxb-1 gene is progressively restricted to rhombomere (r) 4 in the hindbrain. Analysis of the chick and mouse Hoxb-1 genes identified positive and negative regulatory regions that cooperate to mediate segment-restricted expression during rhombomere formation. An enhancer generates expression extending into r3 and r5, and a repressor limits this domain to r4. The repressor contains a conserved retinoic acid response element, point mutations in which allow expression to spread into adjacent rhombomeres. Retinoids and their nuclear receptors may therefore participate in sharpening segment-restricted expression of Hoxb-1 during rhombomere boundary formation.
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