Fumiaki Sugahara

Evolution of the Turtle Body Plan by the Folding and Creation of New Muscle Connections

Shelling Turtles In almost all vertebrates, the shoulder girdle (scapula) lies outside the ribs. The turtle is unique in that the carapace, the dorsal part of the shell, which is formed from the ribs, encapsulates the scapula. To understand the origin of the turtle-specific body plan, Nagashima et al. (p. 193; see the cover; see the Perspective by Rieppel) compared chicken, mouse, and the Chinese soft shelled-turtle, Pelodiscus sinensis. Modern embryos were studied via whole-mount immunostaining, three-dimensional reconstructions, and with markers for early skeletal precursors and compared with previously reported fossils. Initially, embryos of the three animals share a common developmental pattern, one that is likely to have been shared with their last common ancestor. This pattern, however, is modified in the turtle by a specific folding of its body wall during embryogenesis. This folding preserves some of the connectivity between skeletal and muscle elements but also produces new connections. The turtle body plan, unique among amniotes, is based on the folding of an ancestral pattern during embryogenesis. The turtle shell offers a fascinating case study of vertebrate evolution, based on the modification of a common body plan. The carapace is formed from ribs, which encapsulate the scapula; this stands in contrast to the typical amniote body plan and serves as a key to understanding turtle evolution. Comparative analyses of musculoskeletal development between the Chinese soft-shelled turtle and other amniotes revealed that initial turtle development conforms to the amniote pattern; however, during embryogenesis, lateral rib growth results in a shift of elements. In addition, some limb muscles establish new turtle-specific attachments associated with carapace formation. We propose that the evolutionary origin of the turtle body plan results from heterotopy based on folding and novel connectivities.

Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates.

In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes.

Involvement of Hedgehog and FGF signalling in the lamprey telencephalon: evolution of regionalization and dorsoventral patterning of the vertebrate forebrain

Dorsoventral (DV) specification is a crucial step for the development of the vertebrate telencephalon. Clarifying the origin of this mechanism will lead to a better understanding of vertebrate central nervous system (CNS) evolution. Based on the lamprey, a sister group of the gnathostomes (jawed vertebrates), we identified three lamprey Hedgehog (Hh) homologues, which are thought to play central signalling roles in telencephalon patterning. However, unlike in gnathostomes, none of these genes, nor Lhx6/7/8, a marker for the migrating interneuron subtype, was expressed in the ventral telencephalon, consistent with the reported absence of the medial ganglionic eminence (MGE) in this animal. Homologues of Gsh2, Isl1/2 and Sp8, which are involved in the patterning of the lateral ganglionic eminence (LGE) of gnathostomes, were expressed in the lamprey subpallium, as in gnathostomes. Hh signalling is necessary for induction of the subpallium identity in the gnathostome telencephalon. When Hh signalling was inhibited, the ventral identity was disrupted in the lamprey, suggesting that prechordal mesoderm-derived Hh signalling might be involved in the DV patterning of the telencephalon. By blocking fibroblast growth factor (FGF) signalling, the ventral telencephalon was suppressed in the lamprey, as in gnathostomes. We conclude that Hh- and FGF-dependent DV patterning, together with the resultant LGE identity, are likely to have been established in a common ancestor before the divergence of cyclostomes and gnathostomes. Later, gnathostomes would have acquired a novel Hh expression domain corresponding to the MGE, leading to the obtainment of cortical interneurons.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Developmental and evolutionary significance of the mandibular arch and prechordal⁄premandibular cranium in vertebrates: revising the heterotopy scenario of gnathostome jaw evolution

The cephalic neural crest produces streams of migrating cells that populate pharyngeal arches and a more rostral, premandibular domain, to give rise to an extensive ectomesenchyme in the embryonic vertebrate head. The crest cells forming the trigeminal stream are the major source of the craniofacial skeleton; however, there is no clear distinction between the mandibular arch and the premandibular domain in this ectomesenchyme. The question regarding the evolution of the gnathostome jaw is, in part, a question about the differentiation of the mandibular arch, the rostralmost component of the pharynx, and in part a question about the developmental fate of the premandibular domain. We address the developmental definition of the mandibular arch in connection with the developmental origin of the trabeculae, paired cartilaginous elements generally believed to develop in the premandibular domain, and also of enigmatic cartilaginous elements called polar cartilages. Based on comparative embryology, we propose that the mandibular arch ectomesenchyme in gnathostomes can be defined as a Dlx1‐positive domain, and that the polar cartilages, which develop from the Dlx1‐negative premandibular ectomesenchyme, would represent merely posterior parts of the trabeculae. We also show, in the lamprey embryo, early migration of mandibular arch mesenchyme into the premandibular domain, and propose an updated version of the heterotopy theory on the origin of the jaw.

Body wall development in lamprey and a new perspective on the origin of vertebrate paired fins

Classical hypotheses regarding the evolutionary origin of paired appendages propose transformation of precursor structures (gill arches and lateral fin folds) into paired fins. During development, gnathostome paired appendages form as outgrowths of body wall somatopleure, a tissue composed of somatic lateral plate mesoderm (LPM) and overlying ectoderm. In amniotes, LPM contributes connective tissue to abaxial musculature and forms ventrolateral dermis of the interlimb body wall. The phylogenetic distribution of this character is uncertain because lineage analyses of LPM have not been generated in anamniotes. We focus on the evolutionary history of the somatopleure to gain insight into the tissue context in which paired fins first appeared. Lampreys diverged from other vertebrates before the acquisition of paired fins and provide a model for investigating the preappendicular condition. We present vital dye fate maps that suggest the somatopleure is eliminated in lamprey as the LPM is separated from the ectoderm and sequestered to the coelomic linings during myotome extension. We also examine the distribution of postcranial mesoderm in catshark and axolotl. In contrast to lamprey, our findings support an LPM contribution to the trunk body wall of these taxa, which is similar to published data for amniotes. Collectively, these data lead us to hypothesize that a persistent somatopleure in the lateral body wall is a gnathostome synapomorphy, and the redistribution of LPM was a key step in generating the novel developmental module that ultimately produced paired fins. These embryological criteria can refocus arguments on paired fin origins and generate hypotheses testable by comparative studies on the source, sequence, and extent of genetic redeployment.

Compartments in the lamprey embryonic brain as revealed by regulatory gene expression and the distribution of reticulospinal neurons

The vertebrate neural tube consists of a series of neuromeres along its anteroposterior axis. Between amphioxus that possesses no neuromeres and gnathostomes, the lamprey occupies a critical position in the phylogeny for the origin of the segmented brain. To clarify the rhombomeric configuration of the Japanese lamprey, Lampetra japonica, we injected rhodamine- and fluorescein-labeled dextrans into the larval spinal cord, and retrogradely labeled the reticulospinal neurons. We also isolated prosomere marker genes from the embryonic cDNA library of L. japonica, and performed in situ hybridization on the embryonic brain. Of the genes examined, LjOtxA, LjPax6, LjPax2/5/8, LjDlx1/6, and LjTTF-1 were expressed in clearly demarcated polygonal domains. In the telencephalon, LjDlx1/6, LjPax6, and a putative paralogue of LjEmx were expressed in different domains; the LjEmx paralogue was expressed in the dorsal region, and LjDlx1/6 and LjPax6 in a complimentary fashion of the middle part. These expression patterns implied existence of a tripartite configuration of the lamprey telencephalon similar to that in gnathostomes. All these evidences strongly suggest that the segmental and compartmental architecture of the vertebrate brain was already established before the divergence of agnathans and gnathostomes.

Evolution of the regionalization and patterning of the vertebrate telencephalon: what can we learn from cyclostomes?

The telencephalon, the most anterior part of the vertebrate central nervous system (CNS), is a highly diversified region of the vertebrate body. Its evolutionary origin remains elusive, especially with regard to the ancestral state of its architecture as well as the origin of telencephalon-specific neuron subtypes. Cyclostomes (lampreys and hagfish), the sister group of the gnathostomes (jawed vertebrates), serve as valuable models for studying the evolution of the vertebrate CNS. Here, we summarize recent studies on the development of the telencephalon in the lamprey. By comparing detailed developmental studies in mammals, we illustrate a possible ancestral developmental plan underlying the diversification of the vertebrate telencephalon and propose possible approaches for understanding the early evolution of the telencephalon.

Origin of the unique morphology of the shoulder girdle in turtles

The shoulder girdle of turtles has a triradiate morphology. Although its dorsal process represents the scapular blade, the skeletal identities of the two ventral processes remain uncertain. To elucidate the question, developmental patterns of the girdles were compared between Chinese soft‐shelled turtles, chickens, and mice. Despite the morphological diversity of adults, the initial primordia of the shoulder girdles showed similar morphological patterns. The ventral two processes developed from the anlagen comparable to those of the acromion and the coracoid in other amniotes. The developmental pattern of the acromion is very similar among embryos, whereas that of the coracoid in mammals differs from that in non‐mammals, implying that coracoids are not homologous between non‐mammals and mammals. Therefore, amniotes have retained the ancestral pattern of the girdle anlage, and the shoulder girdle of turtles has been achieved through a transformation of the pattern in the late ontogenic period.

Developmental origin of the clavicle, and its implications for the evolution of the neck and the paired appendages in vertebrates

In fish, the pectoral appendage is adjacent to the head, but during vertebrate evolution a long neck region emerged via caudal relocation of the pectoral appendage. The pectoral appendage is comprised of endochondral portions, such as the humerus and the scapula, and a dermal portion, such as the clavicle, that contributes to the shoulder girdle. In the search for clues to the mechanism of the caudal relocation of the pectoral appendage, the cell lineage of the rostral lateral plate mesoderm was analyzed in chickens. It was found that, despite the long neck region in chickens, the origin of the clavicle attached to the head mesoderm ranged between 1 and 14 somite levels. Because the pectoral limb bud and the endochondral pectoral appendage developed on 15–20 and 15–24 somite levels, respectively, the clavicle‐forming region corresponds to the embryonic neck, which suggests that the relocation would have been executed by the expansion of the source of the clavicle. The rostral portion of the clavicle‐forming region overlaps the source of the cucullaris muscle, embraces the pharyngeal arches caudally, and can be experimentally replaced with the head mesoderm to form the cucullaris muscle, which implies that the mesodermal portion could have been the head mesoderm and that the clavicle would have developed at the head/trunk boundary. The link between the head mesoderm and the presumptive clavicle appears to have been the developmental constraint needed to create the evolutionarily conserved musculoskeletal connectivities characterizing the gnathostome neck. In this sense, the dermal girdle of the ganathostomes would represent the wall of the branchial chamber into which the endochondral pectoral appendage appears to have attached since its appearance in evolution.