Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development

The products of Hox genes function in assigning positional identity along the anterior–posterior body axis during animal development. In mouse embryos, Hox genes located at the 3′ end of HoxA and HoxB complexes are expressed in nested patterns in the progenitors of the secondary heart field during early cardiogenesis and the combined activities of both of these clusters are required for proper looping of the heart. Using Hox bacterial artificial chromosomes (BACs), transposon reporters, and transgenic analyses in mice, we present the identification of several novel enhancers flanking the HoxB complex which can work over a long range to mediate dynamic reporter expression in the endoderm and embryonic heart during development. These enhancers respond to exogenously added retinoic acid and we have identified two retinoic acid response elements (RAREs) within these control modules that play a role in potentiating their regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these control modules, spread throughout the flanking intergenic region, have regulatory activities that overlap with other local enhancers. This suggests that they function as shadow enhancers to modulate the expression of genes from the HoxB complex during cardiac development. Regulatory analysis of the HoxA complex reveals that it also has enhancers in the 3′ flanking region which contain RAREs and have the potential to modulate expression in endoderm and heart tissues. Together, the similarities in their location, enhancer output, and dependence on retinoid signaling suggest that a conserved cis-regulatory cassette located in the 3′ proximal regions adjacent to the HoxA and HoxB complexes evolved to modulate Hox gene expression during mammalian cardiac and endoderm development. This suggests a common regulatory mechanism, whereby the conserved control modules act over a long range on multiple Hox genes to generate nested patterns of HoxA and HoxB expression during cardiogenesis.     

Long-range regulation by shared retinoic acid response elements modulates dynamic expression of posterior Hoxb genes in CNS development

Retinoic acid (RA) signaling plays an important role in determining the anterior boundary of Hox gene expression in the neural tube during embryogenesis. In particular, RA signaling is implicated in a rostral expansion of the neural expression domain of 5? Hoxb genes (Hoxb9–Hoxb5) in mice. However, underlying mechanisms for this gene regulation have remained elusive due to the lack of RA responsive element (RARE) in the 5? half of the HoxB cluster. To identify cis-regulatory elements required for the rostral expansion, we developed a recombineering technology to serially label multiple genes with different reporters in a single bacterial artificial chromosome (BAC) vector containing the mouse HoxB cluster. This allowed us to simultaneously monitor the expression of multiple genes. In contrast to plasmid-based reporters, transgenic BAC reporters faithfully recapitulated endogenous gene expression patterns of the Hoxb genes including the rostral expansion. Combined inactivation of two RAREs, DE-RARE and ENE-RARE, in the BAC completely abolished the rostral expansion of the 5? Hoxb genes. Knock-out of endogenous DE-RARE lead to significantly reduced expression of multiple Hoxb genes and attenuated Hox gene response to exogenous RA treatment in utero. Regulatory potential of DE-RARE was further demonstrated by its ability to anteriorize 5? Hoxa gene expression in the neural tube when inserted into a HoxA BAC reporter. Our data demonstrate that multiple RAREs cooperate to remotely regulate 5? Hoxb genes during CNS development, providing a new insight into the mechanisms for gene regulation within the Hox clusters.     

Combined function of HoxA and HoxB clusters in neural crest cells

The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity.     

Evolutionary Constraints Favor a Biophysical Model Explaining Hox Gene Collinearity

The Hox gene collinearity enigma has often been approached using models based on biomolecular mechanisms. The biophysical model is an alternative approach based on the hypothesis that collinearity is caused by physical forces pulling the Hox genes from a territory where they are inactive to a distinct spatial domain where they are activated in a step by step manner. Such Hox gene translocations have recently been observed in support of the biophysical model. Genetic engineering experiments, performed on embryonic mice, gave rise to several unexpected mutant expressions that the biomolecular models cannot predict. On the contrary, the biophysical model offers convincing explanation. Evolutionary constraints consolidate the Hox clusters and as a result, denser and well organized clusters may create more efficient physical forces and a more emphatic manifestation of gene collinearity. This is demonstrated by stochastic modeling with white noise perturbing the expression of Hox genes. As study cases the genomes of mouse and amphioxus are used. The results support the working hypothesis that vertebrates have adopted their comparably more compact Hox clustering as a tool needed to develop more complex body structures. Several experiments are proposed in order to test further the physical forces hypothesis.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

The duplication of the <Emphasis Type="Italic">Hox</Emphasis> gene clusters in teleost fishes

Higher teleost fishes, including zebrafish and fugu, have duplicated their Hox genes relative to the gene inventory of other gnathostome lineages. The most widely accepted theory contends that the duplicate Hox clusters orginated synchronously during a single genome duplication event in the early history of ray-finned fishes. In this contribution we collect and re-evaluate all publicly available sequence information. In particular, we show that the short Hox gene fragments from published PCR surveys of the killifish Fundulus heteroclitus, the medaka Oryzias latipes and the goldfish Carassius auratus can be used to determine with little ambiguity not only their paralog group but also their membership in a particular cluster. Together with a survey of the genomic sequence data from the pufferfish Tetraodon nigroviridis we show that at least percomorpha, and possibly all eutelosts, share a system of 7 or 8 orthologous Hox gene clusters. There is little doubt about the orthology of the two teleost duplicates of the HoxA and HoxB clusters. A careful analysis of both the coding sequence of Hox genes and of conserved non-coding sequences provides additional support for the “duplication early” hypothesis that the Hox clusters in teleosts are derived from eight ancestral clusters by means of subsequent gene loss; the data remain ambiguous, however, in particular for the HoxC clusters. Assuming the “duplication early” hypothesis we use the new evidence on the Hox gene complements to determine the phylogenetic positions of gene-loss events in the wake of the cluster duplication. Surprisingly, we find that the resolution of redundancy seems to be a slow process that is still ongoing. A few suggestions on which additional sequence data would be most informative for resolving the history of the teleostean Hox genes are discussed. Supplemental material is available at http://www.bioinf.uni-leipzig.de/Publications/SUPPLEMENTS/04-006/.     

Genetic Interactions Between Shox2 and Hox Genes During the Regional Growth and Development of the Mouse Limb

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb.     

Multiple Chromosomal Rearrangements Structured the Ancestral Vertebrate Hox-Bearing Protochromosomes

While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion—such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model) occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes (the Hox “paralogon”) and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.     

Additional duplicated Hox genes in the earthworm: Perionyx excavatus Hox genes consist of eleven paralog groups

Annelida is a lophotrochozoan phylum whose members have a high degree of diversity in body plan morphology, reproductive strategies and ecological niches among others.Of the two traditional classes pertaining to the phylum Annelida (Polychaete and Clitellata), the structure and function of the Hox genes has not been clearly defined within the Oligochaeta class. Using a PCR-based survey, we were able to identify five new Hox genes from the earthworm Perionyx excavatus: a Hox3 gene (Pex-Hox3b), two Dfd genes (Pex-Lox6 and Pex-Lox18), and two posterior genes (Pex-post1 and -post2a). Our result suggests that the eleven earthworm Hox genes contain at least four paralog groups (PG) that have duplicated. We found the clitellates-diagnostic signature residues and annelid signature motif. Also, we show by semi-quantitative RT-PCR that duplicated Hox gene orthologs are differentially expressed in six different anterior-posterior body regions. These results provide essential data for comparative evolution of the Hox cluster within the Annelida.     

Why are Hox genes clustered?

The evolutionarily conserved genomic organization of the Hox genes has been a puzzle ever since it was discovered that their order along the chromosome is similar to the order of their functional domains along the antero-posterior axis. Why has this colinearity been maintained throughout evolution? A close look at regulatory sequences from the mouse Hox clusters^(1,2) suggests that enhancer sharing between adjacent Hox genes may be one reason. Moreover, characterizing the activity of one of these mouse enhancers in Drosophila⁽²⁾ illustrates that despite many similarities, not all Hox clusters are built in the same way.     

More constraint on ParaHox than Hox gene families in early metazoan evolution

Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early evolution, we performed multiple systematic comparative phylogenetic analyses, which identified derived sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx-Pdx/Xlox-PG2-PG3] families in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario whereby H/P genes originated from a [Gsx-Pdx/Xlox-PG2-PG3]-related ProtoHox gene, the «posterior» and «anterior» H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX clusters in the Cnidaria-Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as neurogenesis rather than axis specification.     

Hoxc Gene Collinear Expression and Epigenetic Modifications Established during Embryogenesis Are Maintained until after Birth

The Hox genes, which are organized into clusters on different chromosomes, are key regulators of embryonic anterior-posterior (A-P) body pattern formation and are expressed at specific times and in specific positions in developing vertebrate embryos. Previously, we have shown that histone methylation patterns are closely correlated with collinear Hox gene expression patterns along the A-P axis of E14.5 mouse embryos. Since histone modification is thought to play a crucial mechanistic role in the highly coordinated pattern of collinear Hox gene expression, we examined the maintenance of the spatial collinear expression pattern of Hoxc genes and the corresponding histone modifications during embryogenesis and in early postnatal mice. Hox expression patterns and histone modifications were analyzed by semi-quantitative RT-PCR and chromatin immunoprecipitation (ChIP)-PCR analyses, respectively. The spatiotemporal expression patterns of Hoxc genes in a cluster were maintained until the early postnatal stage (from E8.5 through P5). Examination of histone modifications in E14.5 and P5 tissues revealed that level of H3K27me3 is only a weak correlation with collinear Hoxc gene expression in the trunk regions although diminished in general, however the enrichment of H3K4me3 is strongly correlated with the gene expression in both stages. In summary, the initial spatiotemporal collinear expression pattern of Hoxc genes and epigenetic modifications are maintained after birth, likely contributing to the establishment of the gene expression code for position in the anatomic body axis throughout the entire life of the organism.     

Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis

The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5′ chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.     

A regulatory 'landscape effect' over the HoxD cluster

Faithful expression of Hox genes in both time and space is essential for proper patterning of the primary body axis. Transgenic approaches in vertebrates have suggested that this collinear activation process is regulated in a largely gene cluster-autonomous manner. In contrast, more recently co-opted expression specificities, required in other embryonic structures, depend upon long-range enhancer sequences acting from outside the gene clusters. This regulatory dichotomy was recently questioned, since gene activation along the trunk seems to be partially regulated by signals located outside of the cluster. We investigated these alternative regulatory strategies by engineering a large inversion that precisely separates the murine HoxD complex from its centromeric neighborhood. Mutant animals displayed posterior transformations along with subtle deregulations of Hoxd genes, indicating an impact of the centromeric landscape on the fine-tuning of Hoxd gene expression. Proximal limbs were also affected, suggesting that this ‘landscape effect’ is generic and impacts upon regulatory mechanisms of various qualities and evolutionary origins.