Revisiting the origin of the vertebrate Hox14 by including its relict sarcopterygian members

Bilaterian Hox genes play pivotal roles in the specification of positional identities along the anteroposterior axis. Particularly in vertebrates, their regulation is tightly coordinated by tandem arrays of genes [paralogy groups (PGs)] in four gene clusters (HoxA-D). Traditionally, the uninterrupted Hox cluster (Hox1-14) of the invertebrate chordate amphioxus was regarded as an archetype of the vertebrate Hox clusters. In contrast to Hox1-13 that are globally regulated by the "Hox code" and are often phylogenetically conserved, vertebrate Hox14 members were only recently revealed to be present in an African lungfish, a coelacanth, chondrichthyans and a lamprey, and decoupled from the Hox code. In this study we performed a PCR-based search of Hox14 members from diverse vertebrates, and identified one in the Australian lungfish, Neoceratodus forsteri. Based on a molecular phylogenetic analysis, this gene was designated NfHoxA14. Our real-time RT-PCR suggested its hindgut-associated expression, previously observed also in cloudy catshark HoxD14 and lamprey Hox14α. It is likely that this altered expression scheme was established before the Hox cluster quadruplication, probably at the base of extant vertebrates. To investigate the origin of vertebrate Hox14, by including this sarcopterygian Hox14 member, we performed focused phylogenetic analyses on its relationship with other vertebrate posterior Hox PGs (Hox9-13) as well as amphioxus posterior Hox genes. Our results confirmed the hypotheses previously proposed by other studies that vertebrate Hox14 does not have any amphioxus ortholog, and that none of 1-to-1 pairs of vertebrate and amphioxus posterior Hox genes, based on their relative location in the clusters, is orthologous.     

Further evidence for paternal DNA transmission in gynogenetic grass carp

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species. If gynogenetic progenies are to inherit paternal partial genomic DNA, this will increase genetic variation and will provide a useful outcome for breeding. In this study, we investigated the genetic variation in homeobox(Hox) gene clusters(Hox A4 a, Hox A9 a, Hox A11 b,Hox B1 b, Hox C4 a, Hox C6 b, and Hox D10 a) among koi carp(Cyprinus carpio haematopterus, KOC; the stimulation sperm source), grass carp(Ctenopharyngodon idellus), and gynogenetic grass carp(GGC). We found paternal DNA(a special DNA fragment and Hox C6 b) derived from KOC and a recombinant gene belonging to Hox C6 b in GGC. We are the first to report the recombinant Hox C6 b in GGC. Our study provides further evidence for paternal DNA transmission to gynogenetic progenies,which is a finding with great significance for the genetic breeding of fish.     

No more than 14: the end of the amphioxus Hox cluster

The Hox gene cluster has been a key paradigm for a generation of developmental and evolutionary biologists. Since its discovery in the mid-1980's, the identification, genomic organization, expression, colinearity, and regulation of Hox genes have been immediate targets for study in any new model organism, and metazoan genome projects always refer to the structure of the particular Hox cluster(s). Since the early 1990's, it has been dogma that vertebrate Hox clusters are composed of thirteen paralogous groups. Nonetheless, we showed that in the otherwise prototypical cephalochordate amphioxus (Branchiostoma floridae), the Hox cluster contains a fourteenth Hox gene, and very recently, a 14(th) Hox paralogous group has been found in the coelacanth and the horn shark, suggesting that the amphioxus cluster was anticipating the finding of Hox 14 in some vertebrate lineages. In view of the pivotal place that amphioxus occupies in vertebrate evolution, we thought it of considerable interest to establish the limits of its Hox gene cluster, namely resolution of whether more Hox genes are present in the amphioxus cluster (e.g., Hox 15). Using two strategies, here we report the completion and characterization of the Hox gene content of the single amphioxus Hox cluster, which encompasses 650 kb from Hox1 to Evx. Our data have important implications for the primordial Hox gene cluster of chordates: the prototypical nature of the single amphioxus Hox cluster makes it unlikely that additional paralogous groups will be found in any chordate lineage. We suggest that 14 is the end.     

Anterior boundaries of Hox gene expression in mesoderm-derived structures correlate with the linear gene order along the chromosome

The developmental expression patterns of four genes, Hox 1.1, Hox 1.2, Hox 1.3 and Hox 3.1, were examined by in situ hybridization to serial embryonic sections. The three genes of the Hox 1 cluster, used in this study, map to adjacent positions along chromosome 6, whereas the Hox 3.1 gene maps to the Hox 3 cluster on chromosome 15. The anterior expression limits in segmented mesoderm varied among the four genes examined. Interestingly, a linear correlation exists between the position of the gene along the chromosome and the extent of anterior expression. Genes that are expressed more posterior are also more restricted in their expression in other mesoderm-derived tissues. The order of expression anterior to posterior was determined as: Hox 1.3, Hox 1.2, Hox 1.1 and Hox 3.1. Similarly, genes of the Drosophila Antennapedia and Bithorax complex specifying segment identity also exhibit anterior expression boundaries that correlate with gene position. The data suggest that Hox genes may specify positional information along the anterior-posterior axis during the formation of the body plan.     

Differential evolution of the 13 Atlantic salmon Hox clusters

Hox cluster organization represents a valuable marker to study the effects of recent genome duplication in salmonid fish (25-100 Mya). Using polymerase chain reaction amplification of cDNAs, BAC library screening, and genome walking, we reconstructed 13 Hox clusters in the Atlantic salmon containing 118 Hox genes including 8 pseudogenes. Hox paralogs resulting from the genome duplication preceding the radiation of ray-finned fish have been much better preserved in salmon than in other model teleosts. The last genome duplication in the salmon lineage has been followed by the loss of 1 of the 4 HoxA clusters. Four rounds of genome duplication after the vertebrate ancestor salmon Hox clusters display the main organizational features of vertebrate Hox clusters, with Hox genes exclusively that are densely packed in the same orientation. Recently, duplicated Hox clusters have engaged a process of divergence, with several cases of pseudogenization or asymmetrical evolution of Hox gene duplicates, and a marked erosion of identity in noncoding sequences. Strikingly, the level of divergence attained strongly depends on the Hox cluster pairs rather than on the Hox genes within each cluster. It is particularly high between both HoxBb clusters and both HoxDa clusters, whereas both HoxBa clusters remained virtually identical. Positive selection on the Hox protein-coding sequences could not be detected.     

PcG蛋白复合体对Hox基因调节的研究

PcG蛋白广泛参与到生长、发育、增殖、分化以及肿瘤发生等重要过程.而目前为止对PcG蛋白的靶基因研究最透彻的就是Hox家族. Hox基因存在于一个高度保守的基因簇内,在调控维持正常发育及肿瘤发生中有重要作用.一般认为,PcG蛋白复合物对Hox基因进行以组蛋白表观修饰为主的沉默作用,指导Hox基因适时适地发挥功能. 同时,这个过程还需要DNA连接蛋白、ncRNA等分子的辅助.本文对Hox基因和PcG蛋白的组成和功能进行介绍,并重点归纳总结了对二者关系的经典和最新认识.     

Hox clusters of the bichir (Actinopterygii, Polypterus senegalus) highlight unique patterns of sequence evolution in gnathostome phylogeny

Teleost fishes have extra Hox gene clusters owing to shared or lineage-specific genome duplication events in rayfinned fish (actinopterygian) phylogeny. Hence, extrapolating between genome function of teleosts and human or even between different fish species is difficult. We have sequenced and analyzed Hox gene clusters of the Senegal bichir (Polypterus senegalus), an extant representative of the most basal actinopterygian lineage. Bichir possesses four Hox gene clusters (A, B, C, D); phylogenetic analysis supports their orthology to the four Hox gene clusters of the gnathostome ancestor. We have generated a comprehensive database of conserved Hox noncoding sequences that include cartilaginous, lobe-finned, and ray-finned fishes (bichir and teleosts). Our analysis identified putative and known Hox cis-regulatory sequences with differing depths of conservation in Gnathostoma. We found that although bichir possesses four Hox gene clusters, its pattern of conservation of noncoding sequences is mosaic between outgroups, such as human, coelacanth, and shark, with four Hox gene clusters and teleosts, such as zebrafish and pufferfish, with seven or eight Hox gene clusters. Notably, bichir Hox gene clusters have been invaded by DNA transposons and this trend is further exemplified in teleosts, suggesting an as yet unrecognized mechanism of genome evolution that may explain Hox cluster plasticity in actinopterygians. Taken together, our results suggest that actinopterygian Hox gene clusters experienced a reduction in selective constraints that surprisingly predates the teleost-specific genome duplication.     

Homeobox B3 promotes capillary morphogenesis and angiogenesis

Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin alphavbeta3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.     

Characterization of Hox genes in the bichir,Polypterus palmas

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred.We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.     

Unexpectedly large number of conserved noncoding regions within the ancestral chordate Hox cluster

The single amphioxus Hox cluster contains 15 genes and may well resemble the ancestral chordate Hox cluster. We have sequenced the Hox genomic complement of the European amphioxus Branchiostoma lanceolatum and compared it to the American species, Branchiostoma floridae, by phylogenetic footprinting to gain insights into the evolution of Hox gene regulation in chordates. We found that Hox intergenic regions are largely conserved between the two amphioxus species, especially in the case of genes located at the 3' of the cluster, a trend previously observed in vertebrates. We further compared the amphioxus Hox cluster with the human HoxA, HoxB, HoxC, and HoxD clusters, finding several conserved noncoding regions, both in intergenic and intronic regions. This suggests that the regulation of Hox genes is highly conserved across chordates, consistent with the similar Hox expression patterns in vertebrates and amphioxus.     

egl-27 generates anteroposterior patterns of cell fusion in C. elegans by regulating Hox gene expression and Hox protein function.

Hox genes pattern the fates of the ventral ectodermal Pn.p cells that lie along the anteroposterior (A/P) body axis of C. elegans. In these cells, the Hox genes are expressed in sequential overlapping domains where they control the ability of each Pn.p cell to fuse with the surrounding syncytial epidermis. The activities of Hox proteins are sex-specific in this tissue, resulting in sex-specific patterns of cell fusion: in hermaphrodites, the mid-body cells remain unfused, whereas in males, alternating domains of syncytial and unfused cells develop. We have found that the gene egl-27, which encodes a C. elegans homologue of a chromatin regulatory factor, specifies these patterns by regulating both Hox gene expression and Hox protein function. In egl-27 mutants, the expression domains of Hox genes in these cells are shifted posteriorly, suggesting that egl-27 influences A/P positional information. In addition, egl-27 controls Hox protein function in the Pn.p cells in two ways: in hermaphrodites it inhibits MAB-5 activity, whereas in males it permits a combinatorial interaction between LIN-39 and MAB-5. Thus, by selectively modifying the activities of Hox proteins, egl-27 elaborates a simple Hox expression pattern into complex patterns of cell fates. Taken together, these results implicate egl-27 in the diversification of cell fates along the A/P axis and suggest that chromatin reorganization is necessary for controlling Hox gene expression and Hox protein function.     

Hox patterning of the vertebrate rib cage

Unlike the rest of the axial skeleton, which develops solely from somitic mesoderm, patterning of the rib cage is complicated by its derivation from two distinct tissues. The thoracic skeleton is derived from both somitic mesoderm, which forms the vertebral bodies and ribs, and from lateral plate mesoderm, which forms the sternum. By generating mouse mutants in Hox5, Hox6 and Hox9 paralogous group genes, along with a dissection of the Hox10 and Hox11 group mutants, several important conclusions regarding the nature of the ;Hox code' in rib cage and axial skeleton development are revealed. First, axial patterning is consistently coded by the unique and redundant functions of Hox paralogous groups throughout the axial skeleton. Loss of paralogous function leads to anterior homeotic transformations of colinear regions throughout the somite-derived axial skeleton. In the thoracic region, Hox genes pattern the lateral plate-derived sternum in a non-colinear manner, independent from the patterning of the somite-derived vertebrae and vertebral ribs. Finally, between adjacent sets of paralogous mutants, the regions of vertebral phenotypes overlap considerably; however, each paralogous group imparts unique morphologies within these regions. In all cases examined, the next-most posterior Hox paralogous group does not prevent the function of the more-anterior Hox group in axial patterning. Thus, the ;Hox code' in somitic mesoderm is the result of the distinct, graded effects of two or more Hox paralogous groups functioning in any anteroposterior location.