The T-box genes Tbx4 and Tbx5 have been shown to have key functions in the specification of the identity of the vertebrate forelimb (Tbx5) and hindlimb (Tbx4). Here we show that in zebrafish, Tbx5 has an additional early function that precedes the formation of the limb bud itself. Functional knockdown of zebrafish tbx5 through the use of an antisense oligonucleotide resulted in a failure to initiate fin bud formation, leading to the complete loss of pectoral fins. The function of the tbx5 gene in the development of zebrafish forelimbs seems to involve the directed migration of individual lateral-plate mesodermal cells into the future limb-bud-producing region. The primary defect seen in the tbx5-knockdown phenotype is similar to the primary defects described in known T-box-gene mutants such as the spadetail mutant of zebrafish and the Brachyury mutant of the mouse, which both similarly exhibit an altered migration of mesodermal cells. A common function for many of the T-box genes might therefore be in mediating the proper migration and/or changes in adhesive properties of early embryonic cells.

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.

No abstract

In any comparative studies striving to understand the similarities and differences of the living organisms at the molecular genetic level, the crucial first step is to establish the homology (orthology and paralogy) of genes between different organisms. Determination of the homology of genes becomes complicated when the genes have undergone a rapid divergence in sequence or when the involved genes are members of a gene family that has experienced a differential gain or loss of its constituents in different taxonomic groups. Organisms with duplicated genomes such as teleost fishes might have been especially prone to these problems because the functional redundancies provided by the duplicate copies of genes would have allowed a rapid divergence or loss of genes during evolution. In this study, we will demonstrate that much of the ambiguities in the determination of the homology between fish and tetrapod genes resulting from the problems like these can be eliminated by complementing the sequence-based phylogenies with nonsequence information, such as the exon-intron structure of a gene or the composition of a gene's genomic neighbors. We will use the Tbx6/16 subfamily genes of zebrafish (tbx6, tbx16, tbx24, and mga genes), which have been well known for the ambiguity of their evolutionary relationships to the Tbx6/16 subfamily genes of tetrapods, as an illustrative example. We will show that, despite the similarity of sequence and expression to the tetrapod Tbx6 genes, zebrafish tbx6 gene is actually a novel T-box gene more closely related to the tetrapod Tbx16 genes, whereas the zebrafish tbx24 gene, hitherto considered to be a novel gene due to the high level of sequence divergence, is actually an ortholog of tetrapod Tbx6 genes. We will also show that, after their initial appearance by the multiplication of a common ancestral gene at the beginning of vertebrate evolution, the Tbx6/16 subfamily of vertebrate T-box genes might have experienced differential losses of member genes in different vertebrate groups and gradual pooling of member gene's functions in surviving members, which might have prevented the revelation of the true identity of member genes by way of the comparison of sequence and function.

Despite mounting evidence that key developmental regulator genes are involved in significant macroevolutionary changes, there have been few studies demonstrating the functional significance of variation in such genes for the generation of population-level variation. In this study we examined and compared the expression domains of three Hox gene homeobox sequences in embryos derived from two morphologically distinct populations of the threespine stickleback, Gasterosteus aculeatus. We found within-population variation in the location of anterior limits, particularly in more 5' Hox genes whose anterior expression domains showed graded distributions of transcripts over several somites. However, despite considerable and statistically significant differences in the anteroposterior pattern of the axial and median skeletons between the two stickleback populations, this phenotypic variation was not found to be correlated with any of the variation in Hox gene expression. The possible functional significance of the combinatorial Hox code in fish species is discussed with respect to the buffering of development in fluctuating environments, and it is argued that population and quantitative genetic perspectives should also be taken into account in considering the function and evolution of Hox genes.