Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications.

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.

The genetic basis of modularity in the development and evolution of the vertebrate dentition.

The construction of organisms from units that develop under semi-autonomous genetic control (modules) has been proposed to be an important component of their ability to undergo adaptive phenotypic evolution. The organization of the vertebrate dentition as a system of repeated parts provides an opportunity to study the extent to which phenotypic modules, identified by their evolutionary independence from other such units, are related to modularity in the genetic control of development. The evolutionary history of vertebrates provides numerous examples of both correlated and independent evolution of groups of teeth. The dentition itself appears to be a module of the dermal exoskeleton, from which it has long been under independent genetic control. Region-specific tooth loss has been a common trend in vertebrate evolution. Novel deployment of teeth and reacquisition of lost teeth have also occurred, although less frequently. Tooth shape differences within the dentition may be discontinuous (referred to as heterodonty) or graded. The occurrence of homeotic changes in tooth shape provides evidence for the decoupling of tooth shape and location in the course of evolution. Potential mechanisms for region-specific evolutionary tooth loss are suggested by a number of mouse gene knockouts and human genetic dental anomalies, as well as a comparison between fully-developed and rudimentary teeth in the dentition of rodents. These mechanisms include loss of a tooth-type-specific initiation signal, alterations of the relative strength of inductive and inhibitory signals acting at the time of tooth initiation and the overall reduction in levels of proteins required for the development of all teeth. Ectopic expression of tooth initiation signals provides a potential mechanism for the novel deployment or reacquisition of teeth; a single instance is known of a gene whose ectopic expression in transgenic mice can lead to ectopic teeth. Differences in shape between incisor and molar teeth in the mouse have been proposed to be controlled by the region-specific expression of signalling molecules in the oral epithelium. These molecules induce the expression of transcription factors in the underlying jaw mesenchyme that may act as selectors of tooth type. It is speculated that shifts in the expression domains of the epithelial signalling molecules might be responsible for homeotic changes in tooth shape. The observation that these molecules are regionally restricted in the chicken, whose ancestors were not heterodont, suggests that mammalian heterodonty may have evolved through the use of patterning mechanisms already acting on skeletal elements of the jaws. In general, genetic and morphological approaches identify similar types of modules in the dentition, but the data are not yet sufficient to identify exact correspondences. It is speculated that modularity may be achieved by gene expression differences between teeth or by differences in the time of their development, causing mutations to have cumulative effects on later-developing teeth. The mammalian dentition, for which virtually all of the available developmental genetic data have been collected, represents a small subset of the dental diversity present in vertebrates as a whole. In particular, teleost fishes may have a much more extensive dentition. Extension of research on the genetic control of tooth development to this and other vertebrate groups has great potential to further the understanding of modularity in the dentition.

Dynamic interactions and the evolutionary genetics of dental patterning.

The mammalian dentition is a segmental, or periodically arranged, organ system whose components are arrayed in specific number and in regionally differentiated locations along the linear axes of the jaws. This arrangement evolved from simpler dentitions comprised of many single-cusp teeth of relatively indeterminate number. The different types of mammalian teeth have subsequently evolved as largely independent units. The experimentally documented developmental autonomy of dental primordia shows that the basic dental pattern is established early in embryogenesis. An understanding of how genetic patterning processes may work must be consistent with the different modes of development, and partially independent evolution, of the upper and lower dentition in mammals. The periodic nature of the location, number, and morphological structure of teeth suggests that processes involving the quantitative interaction of diffusible signaling factors may be involved. Several extracellular signaling molecules and their interactions have been identified that may be responsible for locating teeth along the jaws and for the formation of the incisor field. Similarly, the wavelike expression of signaling factors within developing teeth suggests that dynamic interactions among those factors may be responsible for crown patterns. These factors seem to be similar among different tooth types, but the extent to which crown differences can be explained strictly in terms of variation in the parameters of interactions among the same genes, as opposed to tooth-type-specific combinatorial codes of gene expression, is not yet known. There is evidence that combinatorial expression of intracellular transcription factors, including homeobox gene families, may establish domains within the jaws in which different tooth types are able to develop. An evolutionary perspective can be important for our understanding of dental patterning and the designing of appropriate experimental approaches, but dental patterns also raise basic unresolved questions about the nature of the evolutionary assumptions made in developmental genetics.

A monophyletic origin of heart-predominant lactate dehydrogenase (LDH) isozymes of gnathostome vertebrates: evidence from the cDNA sequence of the spiny dogfish (Squalus acanthias) LDH-B.

Duplication of a single lactate dehydrogenase locus early in vertebrate evolution has been proposed to have given rise to Ldh-A and Ldh-B, the encoded isozymes of which predominate in skeletal and heart muscle, respectively. This view has been challenged recently by phylogenetic analyses of LDH sequences. One question that has been raised is whether the heart-predominant isozyme (LDH-B) of cartilaginous fishes is orthologous to that of bony fishes and their derivatives. To address this issue, we determined the complementary DNA sequence of the LDH-B of the chondrichthyan Squalus acanthias. Phylogenetic analysis of this and other LDH isozyme sequences provided strong support for a single origin of LDH-Bs prior to the divergence of cartilaginous and bony fishes.

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata.

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.

Patterning of the mammalian dentition in development and evolution.

The mammalian dentition is a segmented organ system with shape differences among its serially homologous elements (individual teeth). It is believed to have evolved from simpler precursors with greater similarities in shape among teeth, and a wealth of descriptive data exist on changes to the dentition that have occurred within mammals. Recent progress has been made in determining the genetic basis of the processes that form an individual tooth, but patterning of the dentition as a whole (i.e. the number, location and shape of the teeth) is less well understood. In contrast to similarly organized systems, such as the vertebral column and limb, Hox genes are not involved in specifying differences among elements. Nevertheless, recent work on a variety of systems is providing clues to the transcription factors and extracellular signalling molecules involved.

Relationship between the genomic organization and the overlapping embryonic expression patterns of the zebrafish dlx genes.

To understand the relationship between the expression and the genomic organization of the zebrafish dlx genes, we have determined the genomic structure of the dlx2 and dlx4 loci. This led to the identification of the zebrafish dlx1 and dlx6 genes, which are closely linked to dlx2 and dlx4, respectively. Therefore, the inverted convergent configuration of Dlx genes is conserved among vertebrates. Analysis of the expression patterns of dlx1 and dlx6 showed striking similarities to those of dlx2 and dlx4, respectively, the genes to which they are linked. Furthermore, the expression patterns of dlx3 and dlx7, which likely constitute a third pair of convergently transcribed genes, are indistinguishable. Thus, the overlapping expression patterns of linked Dlx genes during embryonic development suggest that they share cis-acting sequences that control their spatiotemporal expression. The evolutionary conservation of the genomic organization and combinatorial expression of Dlx genes in distantly related vertebrates suggest tight control mechanisms that are essential for their function during development.

Genomic analysis of a new mammalian distal-less gene: Dlx7.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

The evolution of the vertebrate Dlx gene family.

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.

Numerous members of the Sox family of HMG box-containing genes are expressed in developing mouse teeth.

We used RT-PCR to detect the expression in mouse molar and incisor tooth germs of 14 of the 19 known members of the Sox family of HMG box-containing transcription factors. These sequences fell into all 6 of the main subdivisions of the Sox family. In general, the relative transcript abundance of the different Sox genes is similar between molar and incisor tooth germs, although 3 low-abundance transcripts were found in only a single tooth type. The expression of Sox genes during tooth development has not been reported previously and further experiments will be required to determine their role in this process.

The cDNA sequence of the lactate dehydrogenase-A of the spiny dogfish (Squalus acanthias): corrections to the amino acid sequence and an analysis of the phylogeny of vertebrate lactate dehydrogenases.

The cDNA sequence of the lactate dehydrogenase-A (LDH-A) of the spiny dogfish was determined. The deduced amino acid sequence differed from a previously determined protein sequence by 5%. Separate maximum parsimony analyses of the two sequences along with LDHs of other vertebrates resulted in shorter trees with the sequence presented here, as well as fewer equally parsimonious trees. The new sequence also indicates a greater conservation of length among vertebrate LDHs than was previously suspected. Analyses of the phylogeny of vertebrate LDHs resulted in a monophyletic grouping of LDH-As, from within which mammalian LDH-C is derived. The phylogeny of LDH-As did not exactly match the phylogeny of the organisms, raising the possibility of multiple origins and losses of a muscle-predominant gene. LDH-Bs appear to have shared a single origin.

Evidence from 18S ribosomal RNA sequences that lampreys and hagfishes form a natural group.

Lampreys and hagfishes (cyclostomes) traditionally were considered to be a natural (monophyletic) group. Recently, the consensus of opinion, based largely on morphological analyses, has shifted to a view that lampreys are more closely related to jawed vertebrates (gnathostomes) than to hagfishes. Phylogenetic comparisons of 18S ribosomal RNA sequences from two hagfishes, two lampreys, a tunicate, a lancelet, and a number of gnathostomes support the monophyly of the cyclostomes. These data force a reassessment of several features of early vertebrate evolution.

Evolutionary implications of the cDNA sequence of the single lactate dehydrogenase of a lamprey.

All vertebrates other than lampreys exhibit multiple loci encoding lactate dehydrogenase +ADL-LDH; (S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27+BD. Of these loci, Ldh-A is expressed predominantly in muscle, Ldh-B is expressed predominantly in heart, and Ldh-C (where present) exhibits different tissue-restricted patterns of expression depending on the taxon. To examine the relationship of the single LDH of lampreys to other vertebrate LDHs, we have determined the cDNA sequence of the LDH of the sea lamprey Petromyzon marinus and compared it to previously published sequences from bacteria, plants, and vertebrates. The lamprey sequence exhibits a mixture of features of both LDH-A and LDH-B at the amino acid level that may account for its intermediate kinetic properties. Both distance and maximum parsimony analyses strongly reject a relationship of lamprey LDH with mammalian LDH-C but do not significantly distinguish among remaining alternative phylogenetic hypotheses. Evolutionary parsimony analyses suggest that the lamprey LDH is related to Ldh-A and that the single locus condition has arisen as a result of the loss of Ldh-B (prior to the appearance of Ldh-C). The collection of LDH sequences for further studies of the evolution of the vertebrate LDH gene family will be facilitated by the PCR approach that we have used to obtain the lamprey sequence.