Genes involved in late specification of the mandibular arch, the source of the vertebrate jaw, are expressed with similar patterns in the oral regions of chick and lamprey embryos. However, morphological comparisons indicate that apparently orthologous homeobox genes were expressed in different subdivisions of the ectomesenchyme in the two species. Therefore, the homology and gene expression of the oral region are uncoupled during the transition from agnathan to gnathostome; we conclude that a heterotopic shift of tissue interaction was involved in the evolution of the jaw.

The most rostral cephalic crest cells in the chick embryo first populate ubiquitously in the rostroventral head. Before the influx of crest cells, the ventral head ectoderm expresses Fgf8 in two domains that correspond to the future mandibular arch. Bmp4 is expressed rostral and caudal to these domains. The rostral part of the Bmp4 domain develops into the rostral end of the maxillary process that corresponds to the transition between the maxillomandibular and premandibular regions. Thus, the distribution patterns of FGF8 and BMP4 appear to foreshadow the maxillomandibular region in the head ectoderm. In the ectomesenchyme of the pharyngula embryo, expression patterns of some homeobox genes overlap the distribution of their upstream growth factors. Dlx1 and Barx1, the targets of FGF8, are expressed in the mandibular ectomesenchyme, and Msx1, the target of BMP4, in its distal regions. Ectopic applications of FGF8 lead to shifted expression of the target genes as well as repatterning of the craniofacial primordia and of the trigeminal nerve branches. Focal injection of a lipophilic dye, DiI, showed that this shift was at least in part due to the posterior transformation of the original premandibular ectomesenchyme into the mandible, caused by the changed distribution of FGF8 that defines the mandibular region. We conclude that FGF8 in the early ectoderm defines the maxillomandibular region of the prepharyngula embryo, through epithelial-mesenchymal interactions and subsequent upregulation of homeobox genes in the local mesenchyme. BMP4 in the ventral ectoderm appears to limit the anterior expression of Fgf8. Ectopic application of BMP4 consistently diminished part of the mandibular arch.

Evolution of the vertebrate jaw has been reviewed and discussed based on the developmental pattern of the Japanese marine lamprey, Lampetra japonica. Though it never forms a jointed jaw apparatus, the L. japonica embryo exhibits the typical embryonic structure as well as the conserved regulatory gene expression patterns of vertebrates. The lamprey therefore shares the phylotype of vertebrates, the conserved embryonic pattern that appears at pharyngula stage, rather than representing an intermediate evolutionary state. Both gnathostomes and lampreys exhibit a tripartite configuration of the rostral-most crest-derived ectomesenchyme, each part occupying an anatomically equivalent site. Differentiated oral structure becomes apparent in post-pharyngula development. Due to the solid nasohypophyseal plate, the post-optic ectomesenchyme of the lamprey fails to grow rostromedially to form the medial nasal septum as in gnathostomes, but forms the upper lip instead. The gnathostome jaw may thus have arisen through a process of ontogenetic repatterning, in which a heterotopic shift of mesenchyme-epithelial relationships would have been involved. Further identification of shifts in tissue interaction and expression of regulatory genes are necessary to describe the evolution of the jaw fully from the standpoint of evolutionary developmental biology.

In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half ofthe cells integrating with the neomycin phosphotransferase gene were homologous recombinants.

Non-receptor-type tyrosine kinases of the Src family, such as Src, Yes and Fyn, are strongly expressed in the brain and have been suggested to have an important function in the central nervous system. We generated Fyn-deficient mice by inserting the beta-galactosidase gene (lacZ) into the fyn gene. The homozygous Fyn-mutant neonates from homozygous Fyn-deficient parents died because of a suckling problem. Neonates were, however, able to suckle milk normally when the homozygous mother's mammary glands had been activated by suckling of a heterozygous or wild-type pup. In these homozygous pups, the modified glomerular complex of the olfactory bulb, which had been suggested to play a role in perceiving pheromones, was abnormal in shape and reduced in size, and the hippocampal cell-layer was undulated. These results suggest that Fyn may be involved in the initial step of instinctive suckling behaviour in neonates.

Among the transcription factor gene families, Pax genes play important and unique roles in morphological patterning of animal body plans. Of these, Group I Pax genes (Pax1 and Pax9) are expressed in the endodermal pharyngeal pouches in many groups of deuterostomes, and vertebrates seem to have acquired more extensive expression domains in embryos. To understand the evolution of Pax1/Pax9-related genes in basal groups of vertebrates, their cognates were isolated from the Japanese marine lamprey, Lampetra japonica. RT-PCR of larval lamprey cDNA yielded two different fragments containing vertebrate Pax1- and Pax9-like paired domains. The Pax9 orthologue was isolated and named LjPax9. Whole-mount in situ hybridization revealed that this gene was expressed in endodermal pharyngeal pouches, mesenchyme of the velum (the oral pumping apparatus) and the hyoid arch, and the nasohypophysial plate, but not in the somitic mesoderm of the lamprey embryo. These expression patterns could be regarded as a link between the basal chordates and the gnathostomes and are consistent with the phylogenetic position of the lamprey. Especially, the appearance of neural crest seemed to be the basis of velar expression. Homology of the velum and the jaw is also discussed based on the LjPax9 expression in the first pharyngeal pouch and in the velar mesenchyme. We conclude that Pax9 genes have sequentially expanded into new expression domains through evolution as more complicated body plans emerged.

Agnathan cognates of vertebrate homeobox genes, Emx and Dlx, were isolated from embryonic cDNA of a Japanese marine lamprey, Lampetra japonica. Analyses of amino acid sequences indicated that the Dlx cognate was closely related to the common ancestor of gnathostome Dlx1 and Dlx6 groups and termed LjDlx1/6. Southern blot analyses could not rule out the possibility that L. japonica possesses more than one paralog for both LjDlx1/6 and LjEmx, the lamprey cognate of Emx. Expression of LjDlx1/6 was regulated spatially as well as developmentally, and its transcripts were mainly found in the craniofacial and pharyngeal mesenchyme and in the forebrain. The expression pattern of LjEmx changed dramatically during embryogenesis; expression was seen initially in the entire neural tube and mesoderm, which were secondarily downregulated, and secondarily in cranial nerve ganglia and in the craniofacial mesenchyme. No specific expression of LjEmx was seen in the telencephalon. Comparisons of Dlx and Otx gene expression patterns suggested a shared neuromeric pattern of the vertebrate brain. Absence of Emx expression implied that the patterning of the lamprey telencephalon is not based on the tripartite plan that has been presumed in gnathostomes. Expression domains of LjDlx1/6 in the upper lip and of LjEmx in the craniofacial mesenchyme were peculiar features that have not been known in gnathostomes. Such differences in expression pattern may underlie distinct morphogenetic pathway of the mandibular arch between the agnathans and gnathostomes.

The jaw is one of the earliest innovations in vertebrate history. Several recent findings suggest a scenario for jaw evolution as a progression of changes in pharyngeal developmental mechanisms. The lamprey, an extant jawless vertebrate, constitutes a model for the pre-gnathostome ancestry. Comparing expression patterns of regulatory genes between the gnathostome and lamprey embryos may enable us to get a glimpse of the essential changes that were responsible for the evolution of the jaw. We hypothesize that a specific topographical change of inductive tissue interactions to be described here brought about the jaw as an evolutionary novelty.