The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes.

This paper reports the cloning of the fourth major murine homeogene complex, HOX-5. The partial characterization of this gene cluster revealed the presence of two novel genes (Hox-5.2, Hox-5.3) located at the 5' extremity of this complex. In situ hybridization experiments showed that these two genes are transcribed in very posterior domains during embryonic and foetal development. We also show that Hox-1.6, the gene located at the 3' most position in the HOX-1 complex, has a very anterior expression boundary during early development. These results clearly support the recently proposed hypothesis that the expression of murine Antp-like homeobox-containing genes along the antero-posterior developing body axis follows a positional hierarchy which reflects their respective physical positions within the HOX clusters, similar to that which is found for the Drosophila homeotic genes. Such a structural and functional organization is likely conserved in most vertebrates. Moreover, on the basis of sequence comparisons, we propose that the ordering of homeobox-containing genes within clusters has been conserved between Drosophila and the house mouse. Thus, very different body plans might be achieved, both in insects and vertebrates, by evolutionarily conserved gene networks possibly displaying similar regulatory interactions.     

Conserved expression of<Emphasis Type="Italic"> Hoxa1</Emphasis> in neurons at the ventral forebrain/midbrain boundary of vertebrates

The previously described expression patterns of zebrafish and mouse Hoxa1 genes are seemingly very disparate, with mouse Hoxa1 expressed in the gastrula stage hindbrain and the orthologous zebrafish hoxa1a gene expressed in cell clusters within the ventral forebrain and midbrain. To investigate the evolution of Hox gene deployment within the vertebrate CNS, we have performed a comparative expression analysis of Hoxa1 orthologs in a range of vertebrate species, comprising representatives from the two major lineages of vertebrates (actinopterygians and sarcopterygians). We find that fore/midbrain expression of hoxa1a is conserved within the teleosts, as it is shared by the ostariophysan teleost zebrafish (Danio rerio) and the distantly related acanthopterygian teleost medaka (Oryzias latipes). Furthermore, we find that in addition to the described gastrula stage hindbrain expression of mouse Hoxa1, there is a previously unreported neurula stage expression domain, again located more anteriorly at the ventral fore/midbrain boundary. A two-phase expression profile in early hindbrain and later fore/midbrain is shared by the other tetrapod model organisms chick and Xenopus. We show that the anterior Hoxa1 expression domain is localized to the anterior terminus of the medial longitudinal fasciculus (MLF) in mouse, chick, and zebrafish. These findings suggest that anterior expression of Hoxa1 is a primitive characteristic that is shared by the two major vertebrate lineages. We conclude that Hox gene expression within the vertebrate CNS is not confined exclusively to the segmented hindbrain and spinal cord, but rather that a presumptive fore/midbrain expression domain arose early in vertebrate origins and has been conserved for at least 400 million years.     

Evolution of Conserved Non-Coding Sequences Within the Vertebrate Hox Clusters Through the Two-Round Whole Genome Duplications Revealed by Phylogenetic Footprinting Analysis

As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist in vertebrate genomes. The paralogous genes in the Hox clusters show similar expression patterns, implying shared regulatory mechanisms for expression of these genes. Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that control these paralogous gene expression are still poorly understood. Toward solving this problem, the authors searched conserved non-coding sequences (CNSs), which are candidates of cis-regulatory elements. When comparing orthologous Hox clusters of 19 vertebrate species, 208 intergenic conserved regions were found. The authors then searched for CNSs that were conserved not only between orthologous clusters but also among the four paralogous Hox clusters. The authors found three regions that are conserved among all the four clusters and eight regions that are conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in the paralogous Hox clusters, and nine of them were newly found in this study. One of these novel regions bears a RARE motif. These CNSs are candidates for gene expression regulatory regions among paralogous Hox clusters. The authors also compared vertebrate CNSs with amphioxus CNSs within the Hox cluster, and found that two CNSs in the HoxA and HoxB clusters retain homology with amphioxus CNSs through the two-round whole genome duplications.     

Conservation and diversity in the cis-regulatory networks that integrate information controlling expression of Hoxa2 in hindbrain and cranial neural crest cells in vertebrates

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components.     

Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo

In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.     

Expression patterns of mouse Hox genes: clues to an understanding of developmental and evolutionary strategies.

Expression patterns of Antennapedia-like homeogenes in the mouse embryo show many similarities o those of their homologues in Drosophila. It is argued here that homeogenes may regulate development of the body plan in mouse by mechanisms similar to those used in Drosophila. In particular, they may differentially specify positional address of cell groups within lineage compartments along the body axes. In vertebrates, a single ancestral homeogene cluster has become duplicated to give four separate clusters. Comparisons of homeogene expression patterns between different clusters of the mouse suggest ways in which duplication has permitted development of a more complex body plan. Cluster duplication may therefore have provided a selective advantage during vertebrate evolution.