A new evolutionary scenario for the vertebrate jaw.

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.

Embryology of the lamprey and evolution of the vertebrate jaw: insights from molecular and developmental perspectives.

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.

Identification and expression of the lamprey Pax6 gene: evolutionary origin of the segmented brain of vertebrates.

The Pax6 gene plays a developmental role in various metazoans as the master regulatory gene for eye patterning. Pax6 is also spatially regulated in particular regions of the neural tube. Because the amphioxus has no neuromeres, an understanding of Pax6 expression in the agnathans is crucial for an insight into the origin of neuromerism in the vertebrates. We have isolated a single cognate cDNA of the Pax6 gene, LjPax6, from a Lampetra japonica cDNA library and observed the pattern of its expression using in situ hybridization. Phylogenetic analysis revealed that LjPax6 occurs as an sister group of gnathostome Pax6. In lamprey embryos, LjPax6 is expressed in the eye, the nasohypophysial plate, the oral ectoderm and the brain. In the central nervous system, LjPax6 is expressed in clearly delineated domains in the hindbrain, midbrain and forebrain. We compared the pattern of LjPax6 expression with that of other brain-specific regulatory genes, including LjOtxA, LjPax2/5/8, LjDlx1/6, LjEmx and LjTTF1. Most of the gene expression domains showed conserved pattern, which reflects the situation in the gnathostomes, conforming partly to the neuromeric patterns proposed for the gnathostomes. We conclude that most of the segmented domains of the vertebrate brain were already established in the ancestor common to all vertebrates. Major evolutionary changes in the vertebrate brain may have involved local restriction of cell lineages, leading to the establishment of neuromeres.

Expression of thyroid transcription factor-1 (TTF-1) gene in the ventral forebrain and endostyle of the agnathan vertebrate, Lampetra japonica.

The Thyroid transcription factor-1 (TTF-1) gene belongs to the Nkx-2.1 subfamily and encodes a transcription factor containing an NK-2-type homeodomain. In our study, we isolated and characterized cDNA clones for the TTF-1/Nkx-2.1 orthologue (LjTTF-1) from the agnathan vertebrate Lampetra japonica. Spatial and temporal expression patterns assessed by in situ hybridization revealed the expression of LjTTF-1 in the anterior nerve cord and anteroventral region of the pharynx. The neural expression was subsequently restricted to the ventral diencephalon. The pharyngeal expression, on the other hand, extended posteriorly to the fourth pharyngeal-pouch level and was finally localized in the endostyle anlage. In the differentiated endostyle of ammocoete larvae, the expression of LjTTF-1 was chiefly detected in type 2a, 2b, and 2c cells, which develop adjacent to glandular cells. These expression patterns of LjTTF-1 support the idea that this gene family plays an important role in the development of the rostral brain and endostyle equivalent organs. Furthermore, histological comparisons between TTF-1/Nkx-2.1 expression in the endostyles of ammocoetes and ascidians suggested the possibility that the organogenetic architecture of the endostyle is conserved among chordates.

Isolation of Dlx and Emx gene cognates in an agnathan species, Lampetra japonica, and their expression patterns during embryonic and larval development: conserved and diversified regulatory patterns of homeobox genes in vertebrate head evolution.

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.

Ectodermally derived FGF8 defines the maxillomandibular region in the early chick embryo: epithelial-mesenchymal interactions in the specification of the craniofacial ectomesenchyme.

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.

Pax1/Pax9-Related genes in an agnathan vertebrate, Lampetra japonica: expression pattern of LjPax9 implies sequential evolutionary events toward the gnathostome body plan.

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.

Middle ear defects associated with the double knock out mutation of murine goosecoid and Msx1 genes.

A number of developmental regulatory genes, including homeobox genes, are dynamically expressed in the mammalian cephalic ectomesenchyme during craniofacial morphogenesis. Owing to the vast amount of gene knock out experiments, functions of such genes are now being revealed in the mammalian skeletal patterning process. The murine goosecoid (Gsc) and Msx1 genes are expressed during craniofacial development and each mutant mouse displays intriguing facial abnormalities including those of middle ear ossicles, suggesting that both genes play roles in spatial programming of craniofacial regions. In order to examine whether these genes could function in concert to direct particular craniofacial morphogenesis, double knock out mice were analyzed. The phenotype of the double mutant mice was restricted to the first arch derivatives and was apparently additive of the single gene mutant mice, implying region specific genetic interactions of these homeobox genes expressed in overlapping regions of middle ear forming ectomesenchyme. Our results also suggested that the patterning of distal portions of the malleus depends on the tympanic membrane, for which normal expressions of both the genes are prerequisite.

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.

Developmental morphology of the head mesoderm and reevaluation of segmental theories of the vertebrate head: evidence from embryos of an agnathan vertebrate, Lampetra japonica.

Due to the peculiar morphology of its preotic head, lampreys have long been treated as an intermediate animal which links amphioxus and gnathostomes. To reevaluate the segmental theory of classical comparative embryology, mesodermal development was observed in embryos of a lamprey, Lampetra japonica, by scanning electron microscopy and immunohistochemistry. Signs of segmentation are visible in future postotic somites at an early neurula stage, whereas the rostral mesoderm is unsegmented and rostromedially confluent with the prechordal plate. The premandibular and mandibular mesoderm develop from the prechordal plate in a caudal to rostral direction and can be called the preaxial mesoderm as opposed to the caudally developing gastral mesoderm. With the exception of the premandibular mesoderm, the head mesodermal sheet is secondarily regionalized by the otocyst and pharyngeal pouches into the mandibular mesoderm, hyoid mesoderm, and somite 0. The head mesodermal components never develop into cephalic myotomes, but the latter develop only from postotic somites. These results show that the lamprey embryo shows a typical vertebrate phylotype and that the basic mesodermal configuration of vertebrates already existed prior to the split of agnatha-gnathostomata; lamprey does not represent an intermediate state between amphioxus and gnathostomes. Unlike interpretations of theories of head segmentation that the mesodermal segments are primarily equivalent along the axis, there is no evidence in vertebrate embryos for the presence of preotic myotomes. We conclude that mesomere-based theories of head metamerism are inappropriate and that the formulated vertebrate head should possess the distinction between primarily unsegmented head mesoderm which includes preaxial components at least in part and somites in the trunk which are shared in all the known vertebrate embryos as the vertebrate phylotype.

Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw.

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning.

Otx cognates in a lamprey, Lampetra japonica.

Gnathostomes have two lineages of Otx genes, Otx1 and Otx2, as cognates of a Drosophila head gap gene, orthodenticle. Previous studies with mutant mice have demonstrated that they play essential roles in the development of rostral head. To shed lights on the evolution of the rostral head in vertebrates we isolated their cognates in the Japanese marine lamprey, Lampetra japonica. The lamprey genome appeared to have two Otx cognantes, LjOtxA and LjOtxB. Phylogenetic analyses suggest that LjOtxA clusters with gnathostome Otx2 genes, but LjOtxB does not belong to either the Otx1 or Otx2 lineage. LjOtxA was expressed in the forebrain and midbrain with the caudal limit possibly at the midbrain/hindbrain junction as gnathostome Otx cognates are, but LjOtxB was not expressed in the brain. No Otx1 or Otx2 cognates are known in gnathostomes that are not expressed in the brain. Both LjOtxA and LjOtxB were expressed in the olfactory placode, epiphysis, optic stalks, and lower and upper lips. LjOtxB was also expressed in the eyes, where no LjOtxA transcripts were detected. Thus, Otx1 and Otx2 functions for the development of forebrain and midbrain in gnathostomes appear to be shouldered by LjOtxA alone in the lamprey. LjOtxB may have diverged from the stem of the Otx1 and Otx2 lineages and evolved independently.

Region-specific expression of murine Hox genes implies the Hox code-mediated patterning of the digestive tract.

BACKGROUND: Hox genes encode transcription factors which are involved in the establishment of regional identities along the anteroposterior (AP) body axis. To elucidate the AP patterning of the digestive tract, we have systematically examined the expression patterns of Hox genes belonging to paralogue groups 6, 7, 8 and 9 by whole-mount in situ hybridization and by section in situ hybridization analyses. RESULTS: The expression patterns of these genes showed co-linearity along the wall of the digestive tract, thereby yielding the Hox code of the gut. The expression boundaries of the Hox genes at later stages (12.5 d.p.c.) corresponded to the morphological boundaries of individual gut subdomains. CONCLUSIONS: The visceral mesoderm-restricted expression suggested that the Hox code primarily functions in the mesenchymal specification which eventually leads to the regional differentiation of gut subdomains as the result of epithelial-mesenchymal interactions. Overlapping expression patterns were found among the paralogous Hox genes, indicating that the paralogues may have redundant functions in the specification of the gut.

Stereotyped axonal bundle formation and neuromeric patterns in embryos of a cyclostome, Lampetra japonica.

Early embryonic development of the nervous system of a lamprey, Lampetra japonica, was studied by using immunohistochemical techniques and by scanning electron microscopy. The earliest appearance of axons was detected at Tahara's stage 21-, when dorsolateral and ventral longitudinal fasciculi were present in the hindbrain and spinal cord regions. The branchiomeric nerve roots began to appear at stage 22; the fibers were joined to the dorsolateral fasciculus proximally and also extended distally into each pharyngeal arch. The anterior neural tube was divided into several neuromeres: the mid-hindbrain sulcus became apparent first, then the portion rostral to this sulcus was subdivided into two portions by the syn-parencephalic boundary. In the hindbrain around stage 23, rhombomeres developed transiently, of which, rhombomere 4 was the most distinctive. Putative crest cells forming the octavofacial nerve root anlage were selectively adhering to rhombomere 4, whereas no crest cells were found on rhombomere 3. The assignment of the crest-derived nerve anlage to rhombomeres is conserved between gnathostomes and L. japonica. The neuromerical scheme of the neural tube of L. japonica is also mostly in accordance with that in gnathostomes, sharing the basic developmental patterning of axon bundles at early developmental stages. The most distinct difference between these two groups is the topographical relationships between the hindbrain neuraxis and pharyngeal arches, as well as the otic placode.

Rostral truncation of a cyclostome, Lampetra japonica, induced by all-trans retinoic acid defines the head/trunk interface of the vertebrate body.

The effect of all-trans retinoic acid on embryogenesis was studied in a cyclostome, Lampetra japonica. Treatment with 0.05-0.5 microM retinoic acid on early gastrula and early neurula resulted in loss of the pharynx and in the rostral truncation of the neural tube. The mouth, pharynx, esophagus, heart, endostyle, and rostral brain were missing with graded severity. In the severest case, the embryo consisted only of trunk segments, especially myotomes that extended to the rostral end of the axis. The effect appeared to be dose- and stage-dependent: Rostral pharyngeal arches were more vulnerable to a lower amount of retinoic acid, and earlier treatment resulted in severer defects. The initial protrusion of the anterior axis started equally in control and retinoic acid-treated embryos, implying that the head morphogenesis is omitted in treated embryos. By identifying the number of myotomes based on the differentiation of hypobranchial muscles, there seemed to be no myotomes lost by retinoic acid-induced truncation. The rostral truncation, therefore, was not simply a limitation of the anterior axis but was restricted to the ventral portion; only the branchial arches disappeared with normally developing myotomes dorsally. The absent region can be defined as the vertebrate head in a morphological sense, including the branchiomeric and preotic paraxial regions as well as the heart. The results suggest the presence of distinct programs between somitomeric and branchiomeric portions of the body, providing a developmental basis for the dual-metamerical body plan of vertebrates.

Pax-6 is involved in the specification of hindbrain motor neuron subtype.

Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2 exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos. By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.

Peripheral development of cranial nerves in a cyclostome, Lampetra japonica: morphological distribution of nerve branches and the vertebrate body plan.

The development of peripheral nerves was studied in a Japanese marine lamprey, Lampetra japonica, in whole-mount and sectioned embryos from hatching until the earliest ammocoete. Nerve fibers were immunohistochemically stained with a monoclonal antibody against acetylated tubulin. Branchiomeric nerves first developed in a simple metamerical pattern, each associated with a single pharyngeal arch. Of those, the ophthalmicus profundus, maxillomandibular, and facial nerves later developed a highly modified branching pattern, whereas postotic nerves were less specialized and showed the stereotypical branching pattern of post-trematic nerves. The early distribution of melanocytes in myotome-free space largely overlapped with the morphology of the cranial nerve and ganglion anlage, and resembled the cephalic crest cell distribution pattern in the early chick embryo. It was suggested that the cephalic crest cell distribution, which is also inhibited by myotomes in the lamprey, would be the common basis for branchiomeric nerve patterning. In later development of the lamprey embryo, myotomes 1 through 3, which had originated in the postotic region, grew rostrally into the preotic region, laterally covering all of the branchiomeric nerves. This results in a deep position of the cranial nerves, which is not observed in gnathostomes.

Developmental patterning and evolution of the mammalian viscerocranium: genetic insights into comparative morphology.

The vertebrate cranium is generally classified into the neurocranium and the viscerocranium. The latter is derived from the neural crest and so is the prechordal portion of the neurocranium. A view we favor considers the prechordal neurocranium as the premandibular component of the viscerocranium, and the vertebrate skull to consist of the neural crest-derived viscerocranium and the mesodermal neurocranium. Of these developmental units, only the viscerocranium appears to have completely segmented metamerical organization. The Hox code which is known to function in specification of the viscerocranium does not extend rostrally into the mandibular and premandibular segments. By genetic manipulation of rostrally expressed non-Hox homeobox genes, the patterning mechanism of the head is now demonstrated to be more complicated than isomorphic registration of the Hox code to pharyngeal arches. The phenotype by haplo-insufficiency of Otx2 gene, in particular, implies the premandibular cranium shares a common specification mechanism with the mandibular arch. Our interpretation of the metamerical plan of the viscerocranium offers a new scheme of molecular codes associated with the vertebrate head evolution.