Position-specific activity of the Hox1.1 promoter in transgenic mice

During development, positional values have to be assigned to groups of cells. The murine Hox genes are a class of genes that are predicted to be involved at some stage in this process. During embryogenesis they are expressed in distinct overlapping region- and stage-specific patterns and therefore must be regulated in response to positional information. In this study, we have analysed the activity of Hox1.1 promoter sequences in transgenic mice. The use of lacZ as a marker allows a detailed analysis of expression at the single cell level during early embryonic development. We show that 3.6 kbp of promoter and 1.7 kbp of 3' sequences provide sufficient regulatory information to express a transgene in a spatial and temporal manner indistinguishable from the endogenous Hox1.1 gene during the period of development when Hox1.1 expression is established. The activation occurs in a strict order in specific ectodermal and mesodermal domains. Within each of these domains the transgene is activated over a period of four hours apparently randomly in single cells. In a following second period, Hox1.1 and transgene expression patterns diverge. In this period, transgene expression persists in many mesodermally derived cells that do not express Hox1.1 indicating the absence of a negative regulatory element in the transgene. The anterior boundary of transgene expression is identical to that of Hox1.1. However, no posterior boundary of transgene expression is set, suggesting that a separate element absent from the transgene specifies this boundary.     

Swim bladder and posterior lateral line nerve of the nurseryfish, Kurtus gulliveri (Perciformes: Kurtidae)

The morphology of the swim bladder and inner ear of the nurseryfish, Kurtus gulliveri, appear adapted for enhanced pressure wave reception. The saccule is enlarged and surrounded by very thin bone and two large fontanelles that would present reduced resistance to pressure waves. The swim bladder is elaborate, with six dorsolaterally projecting pairs of lobes that are tightly encased in ribs and an additional caudally projecting pair of lobes encased in the first hemal spine. The ribs and musculature surrounding the swim bladder laterally are very thin, so that four or five "rib windows" are readily apparent on back-lit specimens. This swim bladder-rib configuration would also present reduced resistance to pressure waves to enhance function as a peripheral auditory structure. However, high-resolution X-ray computed tomography and dissection reveal no anterior projections of the swim bladder that could serve as a mechanical coupling to the inner ear. The posterior lateral line nerve is well developed and lies directly over the tips of the ribs encasing the swim bladder lobes. This nerve is not, however, associated with a lateral line canal and a lateral line canal is absent on most of the body. We hypothesize that the posterior lateral line nerve transmits mechanosensory information from the swim bladder.     

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.     

The mouse Hoxd13(spdh) mutation, a polyalanine expansion similar to human type II synpolydactyly (SPD), disrupts the function but not the expression of other Hoxd genes.

Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have studied the genetics of this condition, by using several engineered Hoxd alleles, as well as by looking at the expression of Hox and other marker genes. We show that the mutated SPDH protein induces a gain-of-function phenotype, likely by behaving as a dominant negative over other Hox genes. The mutation, however, seems to act independently from Hoxa13 and doesn't appear to affect Hox gene expression, except for a slight reduction of the HOXD13 protein itself. Developmental studies indicate that the morphological effect is mostly due to a severe retardation in the growth and ossification of the bony elements, in agreement with a general impairment in the function of posterior Hoxd genes.     

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.     

Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.     

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.     

A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb

Posterior prevalence is the general property attributed to HOX proteins describing the dominant effect of more posterior HOX proteins over the function of anterior orthologs in common areas of expression. To explore the HOX group 13 protein domains required for this property, we used the mouse Prx-1 promoter to drive transgenic expression of Hox constructs throughout the entire limb bud during development. This system allowed us to conclusively demonstrate a hierarchy of Hox function in developing limbs. Furthermore, by substituting the HOXD11 or HOXA9 homeodomain for that of HOXD13, we show that a HOXD13 homeodomain is not necessary for posterior prevalence. Proximal expression of these chimeric proteins unexpectedly caused defects consistent with wild-type HOXD13 mediated posterior prevalence. Moreover, group 13 non-homeodomain residues appear to confer the property as proximal expression of HOXA9 containing the HOXD13 homeodomain did not result in limb reductions characteristic of HOXD13. These data are most compatible with models of posterior prevalence based on protein-protein interactions and support examination of the N-terminal non-homeodomain regions of Hox group 13 proteins as necessary agents for posterior prevalence.     

Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra

Growth differentiation factor 11 (GDF11) is one of the significant genes that control skeletal formation. Knockout of GDF11 function causes abnormal patterning of the anterior/posterior axial skeleton. The mRNA of GDF11 is initially translated to a precursor protein that undergoes a proteolytic cleavage to generate the C-terminal peptide or mature GDF11, and the N-terminal peptide named GDF11 propeptide. The propeptide can antagonize GDF11 activity in vitro. To investigate the effects of GDF11 propeptide on GDF11 function in vivo, we generated transgenic mice that over-express the propeptide cDNA in skeletal tissue. The transgenic mice showed formation of extra ribs on the seventh cervical vertebra (C7) as a result of transformation of the C7 vertebra into a thoracic vertebra. The GDF11 propeptide transgene mRNA was detected in tail tissue in embryos and was highly expressed in tail and calvaria bones after birth. A high frequency of C7 rib formation was noticed in the transgenic mouse line with a high level of transgene expression. The anterior boundaries of Hoxa-4 and Hoxa-5 mRNA in situ expressions showed cranial shifts from their normal prevertebra locations in transgenic embryos. These results demonstrated significant effects of GDF11 propeptide transgene on vertebral formation, which are likely occurring through depressing GDF11 function and altered locations of Hoxa-4 and Hoxa-5 expression.     

Hox and ParaHox genes in Nemertodermatida, a basal bilaterian clade

Molecular evidence suggests that Acoelomorpha, a proposed phylum composed of acoel and Nemertodermatida flatworms, are the most basal bilaterian animals. Hox and ParaHox gene complements characterised so far in acoels consist of a small set of genes, comprising representatives of anterior, central and posterior genes, altogether Hox and ParaHox, but no PG3-Xlox representatives have been reported. It has been proposed that this might be the ancestral Hox repertoire in basal bilaterians. However, no studies of the other members of the group, the Nemertodermatida, have been done. In order to get a more complete picture of the basal bilaterian Hox and ParaHox complement, we have analysed the Hox/ParaHox complement of the nemertodermatid Nemertoderma westbladi. We have found representatives of two central and one posterior Hox genes, as well as an Xlox and a Caudal ParaHox gene. From our data we conclude that a PG3-Xlox gene was present in the ancestor of bilaterians. These findings support the speculation that basal bilaterians already had the beginnings of the extended central Hox set, driving back gene duplications in the central part of the Hox cluster deeper in phylogeny than previously suggested.