Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

Expression of somite segmentation genes in amphioxus: a clock without a wavefront?

In the basal chordate amphioxus (Branchiostoma), somites extend the full length of the body. The anteriormost somites segment during the gastrula and neurula stages from dorsolateral grooves of the archenteron. The remaining ones pinch off, one at a time, from the tail bud. These posterior somites appear to be homologous to those of vertebrates, even though the latter pinch off from the anterior end of bands of presomitic mesoderm rather than directly from the tail bud. To gain insights into the evolution of mesodermal segmentation in chordates, we determined the expression of ten genes in nascent amphioxus somites. Five (Uncx4.1, NeuroD/atonal-related, IrxA, Pcdhdelta2-17/18, and Hey1) are expressed in stripes in the dorsolateral mesoderm at the gastrula stage and in the tail bud while three (Paraxis, Lcx, and Axin) are expressed in the posterior mesendoderm at the gastrula and neurula stages and in the tail bud at later stages. Expression of two genes (Pbx and OligA) suggests roles in the anterior somites that may be unrelated to initial segmentation. Together with previous data, our results indicate that, with the exception that Engrailed is only segmentally expressed in the anterior somites, the genetic mechanisms controlling formation of both the anterior and posterior somites are probably largely identical. Thus, the fundamental pathways for mesodermal segmentation involving Notch-Delta, Wnt/beta-catenin, and Fgf signaling were already in place in the common ancestor of amphioxus and vertebrates although budding of somites from bands of presomitic mesoderm exhibiting waves of expression of Notch, Wnt, and Fgf target genes was likely a vertebrate novelty. Given the conservation of segmentation gene expression between amphioxus and vertebrate somites, we propose that the clock mechanism may have been established in the basal chordate, while the wavefront evolved later in the vertebrate lineage.     

Segmentation in vertebrates: a molecular clock linked to periodic somite formation]

In the vertebrate embryo, somites constitute the basis of the segmental body pattern. They give rise to the axial skeleton, the dermis of the back and all striated muscles of the body. In the chick embryo, a pair of somites buds off, in a highly coordinated fashion, every 90 minutes, from the cranial end of the presomitic mesoderm (PSM) while new mesenchymal cells enter the paraxial mesoderm as a consequence of gastrulation. The processes leading to the segmentation of the somite are not yet understood. We have identified and characterised c-hairy1, an avian homologue of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm where its mRNA exhibits a cyclic posterior-to-anterior wave of expression whose periodicity corresponds to the formation time of one somite (90 min). Fate mapping of the rostral half of the PSM using the quail-chick chimera technique supports a model of cryptic segmentation within the presomitic mesoderm, and indicates that c-hairy1 expression dynamics are not due to massive cell displacement. Analysis of in vitro cultures of isolated presomitic mesoderm demonstrates that rhythmic c-hairy1 mRNA production and degradation is an autonomous property of the paraxial mesoderm. Rather than resulting from the caudal-to-rostral propagation of an activating signal, it arises from pulses of c-hairy1 expression that are coordinated in time and space. Blocking protein synthesis does not alter the propagation of c-hairy1 expression, indicating that negative autoregulation of c-hairy1 expression is unlikely to control its periodic expression. Most of the segmentation models proposed for somite formation rely on the existence of an internal clock coordinating the cells to segment together to form a somite. These results provide the first molecular evidence of a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.     

Dynamic expression of lunatic fringe suggests a link between notch signaling and an autonomous cellular oscillator driving somite segmentation

The metameric organization of the vertebrate trunk is a characteristic feature of all members of this phylum. The origin of this metamerism can be traced to the division of paraxial mesoderm into individual units, termed somites, during embryonic development. Despite the identification of somites as the first overt sign of segmentation in vertebrates well over 100 years ago, the mechanism(s) underlying somite formation remain poorly understood. Recently, however, several genes have been identified which play prominent roles in orchestrating segmentation, including the novel secreted factor lunatic fringe. To gain further insight into the mechanism by which lunatic fringe controls somite development, we have conducted a thorough analysis of lunatic fringe expression in the unsegmented paraxial mesoderm of chick embryos. Here we report that lunatic fringe is expressed predominantly in somite -II, where somite I corresponds to the most recently formed somite and somite -I corresponds to the group of cells which will form the next somite. In addition, we show that lunatic fringe is expressed in a highly dynamic manner in the chick segmental plate prior to somite formation and that lunatic fringe expression cycles autonomously with a periodicity of somite formation. Moreover, the murine ortholog of lunatic fringe undergoes a similar cycling expression pattern in the presomitic mesoderm of somite stage mouse embryos. The demonstration of a dynamic periodic expression pattern suggests that lunatic fringe may function to integrate notch signaling to a cellular oscillator controlling somite segmentation.     

Development of the head and trunk mesoderm in the dogfish,Scyliorhinus torazame: II. Comparison of gene expression between the head mesoderm and somites with reference to the origin of the vertebrate head

The vertebrate mesoderm differs distinctly between the head and trunk, and the evolutionary origin of the head mesoderm remains enigmatic. Although the presence of somite‐like segmentation in the head mesoderm of model animals is generally denied at molecular developmental levels, the appearance of head cavities in elasmobranch embryos has not been explained, and the possibility that they may represent vestigial head somites once present in an amphioxus‐like ancestor has not been ruled out entirely. To examine whether the head cavities in the shark embryo exhibit any molecular signatures reminiscent of trunk somites, we isolated several developmentally key genes, including Pax1, Pax3, Pax7, Pax9, Myf5, Sonic hedgehog, and Patched2, which are involved in myogenic and chondrogenic differentiation in somites, and Pitx2, Tbx1, and Engrailed2, which are related to the patterning of the head mesoderm, from an elasmobranch species, Scyliorhinus torazame. Observation of the expression patterns of these genes revealed that most were expressed in patterns that resembled those found in amniote embryos. In addition, the head cavities did not exhibit an overt similarity to somites; that is, the similarity was no greater than that of the unsegmented head mesoderm in other vertebrates. Moreover, the shark head mesoderm showed an amniote‐like somatic/visceral distinction according to the expression of Pitx2, Tbx1, and Engrailed2. We conclude that the head cavities do not represent a manifestation of ancestral head somites; rather, they are more likely to represent a derived trait obtained in the lineage of gnathostomes.     

A transgenic Tbx6;CreERT2 line for inducible gene manipulation in the presomitic mesoderm

The rhythmic segmentation process of the presomitic mesoderm (PSM) orchestrates the formation of somites, the fundamental units for the vertebrate axial body plan. To aid the investigation of molecular components governing the conversion from PSM into somites, we generated a transgenic mouse line that expresses a tamoxifen (tmx) inducible CreER(T2) under the control of a 2.5 kb enhancer element of Tbx6, a gene essential for PSM formation and somite patterning. Combined with Cre reporters, this Tbx6;CreER(T2) line displays robust tmx-inducible Cre activity in the PSM at various embryonic stages. This tool should be useful for studying gene function during somitogenesis by either conditional inactivation or mis-expression, and potentially coupled with cell marking.     

The single amphioxus Mox gene: insights into the functional evolution of Mox genes,somites, and the asymmetry of amphioxus somitogenesis

Mox genes are members of the "extended" Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate-vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis.     

Amphioxus and the evolution of head segmentation

Whether or not the vertebrate head is fundamentally segmented has been controversial for over 150 years. Beginning in the late 19th century, segmentalist theories proposed that the vertebrate head evolved from an amphioxus-like ancestor in which mesodermal somites extended the full length of the body with remnants of segmentation persisting as the mesodermal head cavities of sharks and lampreys. Antisegmentalists generally argued either that the vertebrate ancestors never had any mesodermal segmentation anteriorly or that they lost it before the origin of the vertebrates; in either case, the earliest vertebrates had an unsegmented head and the embryonic cranial mesoderm of vertebrates is at best pseudo-segmented, evolving independently of any pre-vertebrate segmental pattern. Recent morphologic studies have generally confirmed the accuracy of the major classical studies of head development in lampreys and sharks, yet disagree with their theoretical conclusions regarding the evolution of head segmentation. Studies of developmental genes in amphioxus and vertebrates, which have demonstrated conservation of the mechanisms of anterior-posterior patterning in the two groups, have shed new light on this controversy. Most pertinently, some homologs of genes expressed in the anterior amphioxus somites, which form as outpocketings of the gut, are also expressed in the walls of the head cavities of lampreys, which form similarly, and in their major derivatives (the velar muscles) as well as in the eye and jaw muscles of bony gnathostomes, which derive from unsegmented head mesoderm. These muscles share gene expression with the corresponding muscles of the shark, which derive from the walls of head cavities that form, not as outpocketings of the gut, but as secondary cavities within solid blocks of tissue. While molecular data that can be compared across all the relevant taxa remain limited, they are consistent with an evolutionary scenario in which the cranial paraxial mesoderm of the lamprey and shark evolved from the anterior somites of an amphioxus-like ancestor. Although, bony vertebrates have lost the mesodermal head segments present in the shark and lamprey, their remnants persist in the muscles of the eye and jaw.     

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.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.