Differences in Krox20-dependent regulation of Hoxa2 and Hoxb2 during hindbrain development

During hindbrain development, segmental regulation of the paralogous Hoxa2 and Hoxb2 genes in rhombomeres (r) 3 and 5 involves Krox20-dependent enhancers that have been conserved during the duplication of the vertebrate Hox clusters from a common ancestor. Examining these evolutionarily related control regions could provide important insight into the degree to which the basic Krox20-dependent mechanisms, cis-regulatory components, and their organization have been conserved. Toward this goal we have performed a detailed functional analysis of a mouse Hoxa2 enhancer capable of directing reporter expression in r3 and r5. The combined activities of five separate cis-regions, in addition to the conserved Krox20 binding sites, are involved in mediating enhancer function. A CTTT (BoxA) motif adjacent to the Krox20 binding sites is important for r3/r5 activity. The BoxA motif is similar to one (Box1) found in the Hoxb2 enhancer and indicates that the close proximity of these Box motifs to Krox20 sites is a common feature of Krox20 targets in vivo. Two other rhombomeric elements (RE1 and RE3) are essential for r3/r5 activity and share common TCT motifs, indicating that they interact with a similar cofactor(s). TCT motifs are also found in the Hoxb2 enhancer, suggesting that they may be another common feature of Krox20-dependent control regions. The two remaining Hoxa2 cis-elements, RE2 and RE4, are not conserved in the Hoxb2 enhancer and define differences in some of components that can contribute to the Krox20-dependent activities of these enhancers. Furthermore, analysis of regulatory activities of these enhancers in a Krox20 mutant background has uncovered differences in their degree of dependence upon Krox20 for segmental expression. Together, this work has revealed a surprising degree of complexity in the number of cis-elements and regulatory components that contribute to segmental expression mediated by Krox20 and sheds light on the diversity and evolution of Krox20 target sites and Hox regulatory elements in vertebrates.     

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.     

Novel interactions between vertebrate Hox genes

Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.     

Independent regulation of initiation and maintenance phases of Hoxa3 expression in the vertebrate hindbrain involve auto- and cross-regulatory mechanisms

During development of the vertebrate hindbrain, Hox genes play multiple roles in the segmental processes that regulate anteroposterior (AP) patterning. Paralogous Hox genes, such as Hoxa3, Hoxb3 and Hoxd3, generally have very similar patterns of expression, and gene targeting experiments have shown that members of paralogy group 3 can functionally compensate for each other. Hence, distinct functions for individual members of this family may primarily depend upon differences in their expression domains. The earliest domains of expression of the Hoxa3 and Hoxb3 genes in hindbrain rhombomeric (r) segments are transiently regulated by kreisler, a conserved Maf b-Zip protein, but the mechanisms that maintain expression in later stages are unknown. In this study, we have compared the segmental expression and regulation of Hoxa3 and Hoxb3 in mouse and chick embryos to investigate how they are controlled after initial activation. We found that the patterns of Hoxa3 and Hoxb3 expression in r5 and r6 in later stages during mouse and chick hindbrain development were differentially regulated. Hoxa3 expression was maintained in r5 and r6, while Hoxb3 was downregulated. Regulatory comparisons of cis-elements from the chick and mouse Hoxa3 locus in both transgenic mouse and chick embryos have identified a conserved enhancer that mediates the late phase of Hoxa3 expression through a conserved auto/cross-regulatory loop. This block of similarity is also present in the human and horn shark loci, and contains two bipartite Hox/Pbx-binding sites that are necessary for its in vivo activity in the hindbrain. These HOX/PBC sites are positioned near a conserved kreisler-binding site (KrA) that is involved in activating early expression in r5 and r6, but their activity is independent of kreisler. This work demonstrates that separate elements are involved in initiating and maintaining Hoxa3 expression during hindbrain segmentation, and that it is regulated in a manner different from Hoxb3 in later stages. Together, these findings add further strength to the emerging importance of positive auto- and cross-regulatory interactions between Hox genes as a general mechanism for maintaining their correct spatial patterns in the vertebrate nervous system.     

Pbx raises the DNA binding specificity but not the selectivity of antennapedia Hox proteins.

We have used a binding site selection strategy to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site, TGATTGATTTGAT. An inhibitory domain located N terminal of the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. The affinities of these proteins as heterodimeric partners with Pbx1 for the selected optimal binding site are similar. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx.     

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity. In this study, the functions of four potential homeodomain binding TAAT sites in this enhancer were studied using transgenic mice and in vitro protein-DNA interactions. Transgenic studies indicate that the four TAAT sites are not redundant and that only the B-TAAT site is critical for AER enhancer activity. The expression patterns of Msx2 and Dlx5 genes in the AER suggest that they might be involved in the regulation of Msx2. In support of this hypothesis, we found that Msx2 and Dlx5 can bind to the B-TAAT site as well as to a fragment containing the D- and E-TAAT sites in the Msx2 AER enhancer sequences. (c)2002 Elsevier Science (USA).     

Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Auto/cross-regulation of Hoxb3 expression in posterior hindbrain and spinal cord

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.     

Hoxb-5 is expressed in gill arch 5 during pharyngeal arch development of flounder Paralichthys olivaceus embryos.

Hox genes are expressed in domains with clear anterior borders exhibiting 3'-->5' hierarchy in hindbrain and in the pharyngeal area commonly in vertebrate embryos. Teleost embryos form seven pharyngeal arches, the mandibular arch, hyoid arch and the gill arches 1-5. We previously reported that, in Japanese flounder (Paralichthys olivaceus) embryos, Hoxd-4 is expressed from rhombomere 7 to the spinal cord in the central nervous system and at gill arches 2-5. At present, the hierarchy of Hox genes at gill arches 3-5 of teleost fish is unclear. Here, we investigated the expression domains of Hoxb-5 in the flounder embryo by whole-mount in situ hybridization to gain insight into the Hox code at gill arches. The initial signal indicating Hoxb-5 expression was identified in the spinal cord at hatching, corresponding with the prim-5 stage of zebrafish. Then, intense signals were detected from the anterior part of the spinal cord and from the posterior part of the pharyngeal area at 36 h after hatching. By serially sectioning the hybridized embryos, it was found that signal in the pharyngeal area came from the most posterior gill arch 5. Therefore, it is speculated that Hoxb-5 functions in regional identification of gill arch 5 in this teleost.     

The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.