Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Comparative analysis of Hmx expression and the distribution of neuronal somata in the trigeminal ganglion in lamprey and shark: insights into the homology of the trigeminal nerve branches and the evolutionary origin of the vertebrate jaw.

The evolutionary origin of the jaw remains one of the most enigmatic events in vertebrate evolution. The trigeminal nerve is a key component for understanding jaw evolution, as it plays a crucial role as a sensorimotor interface for the effective manipulation of the jaw. This nerve is also found in the lamprey, an extant jawless vertebrate. The trigeminal nerve has three major branches in both the lamprey and jawed vertebrates. Although each of these branches was classically thought to be homologous between these two taxa, this homology is now in doubt. In the present study, we compared expression patterns of Hmx, a candidate genetic marker of the mandibular nerve (rV3, the third branch of the trigeminal nerve in jawed vertebrates), and the distribution of neuronal somata of trigeminal nerve branches in the trigeminal ganglion in lamprey and shark. We first confirmed the conserved expression pattern of Hmx1 in the shark rV3 neuronal somata, which are distributed in the caudal part of the trigeminal ganglion. By contrast, lamprey Hmx genes showed peculiar expression patterns, with expression in the ventrocaudal part of the trigeminal ganglion similar to Hmx1 expression in jawed vertebrates, which labeled the neuronal somata of the second branch. Based on these results, we propose two alternative hypotheses regarding the homology of the trigeminal nerve branches, providing new insights into the evolutionary origin of the vertebrate jaw.

High-quality total RNA extraction from early-stage lamprey embryos.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

Ultrastructure of the lamprey head mesoderm reveals evolution of the vertebrate head.

The cranial muscle is a critical component in the vertebrate head for a predatory lifestyle. However, its evolutionary origin and possible segmental nature during embryogenesis have been controversial. In jawed vertebrates, the presence of pre-otic segments similar to trunk somites has been claimed based on developmental observations. However, evaluating such arguments has been hampered by the paucity of research on jawless vertebrates. Here, we discovered different cellular arrangements in the head mesoderm in lamprey embryos (Lethenteron camtschaticum) using serial block-face scanning electron and laser scanning microscopies. These cell populations were morphologically and molecularly different from somites. Furthermore, genetic comparison among deuterostomes revealed that mesodermal gene expression domains were segregated antero-posteriorly in vertebrates, whereas such segregation was not recognized in invertebrate deuterostome embryos. These findings indicate that the vertebrate head mesoderm evolved from the anteroposterior repatterning of an ancient mesoderm and developmentally diversified before the split of jawless and jawed vertebrates.

Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency.

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.

Thyroid and endostyle development in cyclostomes provides new insights into the evolutionary history of vertebrates.

BACKGROUND: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. RESULTS: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages. CONCLUSIONS: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

Forebrain Architecture and Development in Cyclostomes, with Reference to the Early Morphology and Evolution of the Vertebrate Head.

The vertebrate head and brain are characterized by highly complex morphological patterns. The forebrain, the most anterior division of the brain, is subdivided into the diencephalon, hypothalamus, and telencephalon from the neuromeric subdivision into prosomeres. Importantly, the telencephalon contains the cerebral cortex, which plays a key role in higher order cognitive functions in humans. To elucidate the evolution of the forebrain regionalization, comparative analyses of the brain development between extant jawed and jawless vertebrates are crucial. Cyclostomes - lampreys and hagfishes - are the only extant jawless vertebrates, and diverged from jawed vertebrates (gnathostomes) over 500 million years ago. Previous developmental studies on the cyclostome brain were conducted mainly in lampreys because hagfish embryos were rarely available. Although still scarce, the recent availability of hagfish embryos has propelled comparative studies of brain development and gene expression. By integrating findings with those of cyclostomes and fossil jawless vertebrates, we can depict the morphology, developmental mechanism, and even the evolutionary path of the brain of the last common ancestor of vertebrates. In this review, we summarize the development of the forebrain in cyclostomes and suggest what evolutionary changes each cyclostome lineage underwent during brain evolution. In addition, together with recent advances in the head morphology in fossil vertebrates revealed by CT scanning technology, we discuss how the evolution of craniofacial morphology and the changes of the developmental mechanism of the forebrain towards crown gnathostomes are causally related.

Genetic Mechanism for the Cyclostome Cerebellar Neurons Reveals Early Evolution of the Vertebrate Cerebellum.

The vertebrate cerebellum arises at the dorsal part of rhombomere 1, induced by signals from the isthmic organizer. Two major cerebellar neuronal subtypes, granule cells (excitatory) and Purkinje cells (inhibitory), are generated from the anterior rhombic lip and the ventricular zone, respectively. This regionalization and the way it develops are shared in all extant jawed vertebrates (gnathostomes). However, very little is known about early evolution of the cerebellum. The lamprey, an extant jawless vertebrate lineage or cyclostome, possesses an undifferentiated, plate-like cerebellum, whereas the hagfish, another cyclostome lineage, is thought to lack a cerebellum proper. In this study, we found that hagfish Atoh1 and Wnt1 genes are co-expressed in the rhombic lip, and Ptf1a is expressed ventrally to them, confirming the existence of r1's rhombic lip and the ventricular zone in cyclostomes. In later stages, lamprey Atoh1 is downregulated in the posterior r1, in which the NeuroD increases, similar to the differentiation process of cerebellar granule cells in gnathostomes. Also, a continuous Atoh1-positive domain in the rostral r1 is reminiscent of the primordium of valvula cerebelli of ray-finned fishes. Lastly, we detected a GAD-positive domain adjacent to the Ptf1a-positive ventricular zone in lampreys, suggesting that the Ptf1a-positive cells differentiate into some GABAergic inhibitory neurons such as Purkinje and other inhibitory neurons like in gnathostomes. Altogether, we conclude that the ancestral genetic programs for the formation of a distinct cerebellum were established in the last common ancestor of vertebrates.

FGF- and SHH-based molecular signals regulate barbel and craniofacial development in catfish.

BACKGROUND: Catfish (Siluriformes) are characterized by unique morphologies, including enlarged jaws with movable barbels and taste buds covering the entire body surface. Evolution of these characteristics was a crucial step in their adaptive radiation to freshwater environments. However, the developmental processes of the catfish craniofacial region and taste buds remain to be elucidated; moreover, little is known about the molecular mechanisms underlying the morphogenesis of these structures. RESULTS: In Amur catfish (Silurus asotus), three pairs of barbel primordia are formed by 2 days post-fertilization (dpf). Innervation of the peripheral nerves and formation of muscle precursors are also established during early development. Taste buds from the oral region to the body trunk are formed by 4 dpf. We then isolated catfish cognates Shh (SaShh) and Fgf8 (SaFgf8), which are expressed in maxillary barbel primordium at 1-2 dpf. Further, SHH signal inhibition induces reduction of mandibular barbels with abnormal morphology of skeletal elements, whereas it causes no apparent abnormality in the trigeminal and facial nerve morphology. We also found that mandibular barbel lengths and number of taste buds are reduced by FGF inhibition, as seen in SHH signal inhibition. However, unlike with SHH inhibition, the abnormal morphology of the trigeminal and facial nerves was observed in FGF signal-inhibited embryos. CONCLUSION: The developmental processes of Amur catfish are consistent with those reported for other catfish species. Thus, developmental aspects of craniofacial structures and taste buds may be conserved in Siluriformes. Our findings also suggest that SHH signaling plays a crucial role in the formation of barbels and taste buds, without affecting nerve projection, while FGF signaling is required for the development of barbels, taste buds, and branchial nerves. Thus, SHH and FGF signaling plays key roles in the ontogenesis and evolution of some catfish-specific characteristics.

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates.

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)-a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species-a hagfish, a lamprey and a shark-encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys.

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

Reconstructing the ancestral vertebrate brain.

Highly complicated morphologies and sophisticated functions of vertebrate brains have been established through evolution. However, the origin and early evolutionary history of the brain remain elusive, owing to lack of information regarding the brain architecture of extant and fossil species of jawless vertebrates (agnathans). Comparative analyses of the brain of less studied cyclostomes (only extant agnathan group, consisting of lampreys and hagfish) with the well-known sister group of jawed vertebrates (gnathostomes) are the only tools we have available to illustrate the ancestral architecture of the vertebrate brain. Previous developmental studies had shown that the lamprey lacked well-established brain compartments that are present in gnathostomes, such as the medial ganglionic eminence and the rhombic lip. The most accepted scenario suggested that cyclostomes had fewer compartments than that of the gnathostome brain and that gnathostomes thus evolved by a stepwise addition of innovations on its developmental sequence. However, recent studies have revealed that these compartments are present in hagfish embryos, indicating that these brain regions have been acquired before the split of cyclostomes and gnathostomes. By comparing two cyclostome lineages and gnathostomes, it has become possible to speculate about a more complex ancestral state of the brain, excluding derived traits in either of the lineages. In this review, we summarize recent studies on the brain development of the lamprey and hagfish. Then, we attempt to reconstruct the possible brain architecture of the last common ancestor of vertebrates. Finally, we discuss how the developmental plan of the vertebrate brain has been modified independently in different vertebrate lineages.

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.

On the vagal cardiac nerves, with special reference to the early evolution of the head-trunk interface.

The vagus nerve, or the tenth cranial nerve, innervates the heart in addition to other visceral organs, including the posterior visceral arches. In amniotes, the anterior and posterior cardiac branches arise from the branchial and intestinal portions of the vagus nerve to innervate the arterial and venous poles of the heart, respectively. The evolution of this innervation pattern has yet to be elucidated, due mainly to the lack of morphological data on the vagus in basal vertebrates. To investigate this topic, we observed the vagus nerves of the lamprey (Lethenteron japonicum), elephant shark (Callorhinchus milii), and mouse (Mus musculus), focusing on the embryonic patterns of the vagal branches in the venous pole. In the lamprey, no vagus branch was found in the venous pole throughout development, whereas the arterial pole was innervated by a branch from the branchial portion. In contrast, the vagus innervated the arterial and venous poles in the mouse and elephant shark. Based on the morphological patterns of these branches, the venous vagal branches of the mouse and elephant shark appear to belong to the intestinal part of the vagus, implying that the cardiac nerve pattern is conserved among crown gnathostomes. Furthermore, we found a topographical shift of the structures adjacent to the venous pole (i.e., the hypoglossal nerve and pronephros) between the extant gnathostomes and lamprey. Phylogenetically, the lamprey morphology is likely to be the ancestral condition for vertebrates, suggesting that the evolution of the venous branch occurred early in the gnathostome lineage, in parallel with the remodeling of the head-trunk interfacial domain during the acquisition of the neck. J. Morphol. 277:1146-1158, 2016. © 2016 Wiley Periodicals, Inc.

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.

Reevaluating Emx gene phylogeny: homopolymeric amino acid tracts as a potential factor obscuring orthology signals in cyclostome genes.

BACKGROUND: Vertebrate Emx genes, retained as multiple copies, are expressed in a nested pattern in the early embryonic forebrain and required for its regionalization. This pattern seems to have originated in a vertebrate common ancestor; however, a previous analysis, reporting two lamprey Emx genes, claimed independent Emx gene duplications in both cyclostome (extant jawless fish) and gnathostome (jawed vertebrate) lineages after their divergence. This scenario is neither parsimonious nor consistent with the hypothesis that genome expansion occurred before the cyclostome-gnathostome split, which is supported by recent genome-wide analyses. RESULTS: We isolated and sequenced cDNA of two hagfish Emx genes and performed intensive molecular phylogenetic analyses, including the hagfish and/or lamprey Emx genes. The lamprey genes tended to attract each other in inferred phylogenetic trees, an effect that tended to be relaxed on inclusion of the hagfish genes. The results of these analyses suggest that cyclostome EmxB is orthologous to gnathostome Emx2, which was also supported by conserved synteny. Homopolymeric amino acid (HPAA) tracts represent a remarkable feature of the lamprey Emx sequences, and a comparative genome-wide scan revealed that lamprey proteins exhibit a unique pattern of HPAA tract accumulation. CONCLUSIONS: Our analysis, including hagfish Emx genes, suggests that gene duplications gave rise to Emx1, -2 and -3 before the cyclostome-gnathostome split. We propose that independent HPAA tract accumulations in multiple ancient duplicates, as identified in lamprey Emx gene products, may have led to erroneous identification of gene duplication in the lamprey lineage. Overall, our reanalysis favors the scenario that the nested Emx expression pattern in mouse and lamprey shares a common origin.

On the homology of the shoulder girdle in turtles.

The shoulder girdle in turtles is encapsulated in the shell and has a triradiate morphology. Due to its unique configuration among amniotes, many theories have been proposed about the skeletal identities of the projections for the past two centuries. Although the dorsal ramus represents the scapular blade, the ventral two rami remain uncertain. In particular, the ventrorostral process has been compared to a clavicle, an acromion, and a procoracoid based on its morphology, its connectivity to the rest of the skeleton and to muscles, as well as with its ossification center, cell lineage, and gene expression. In making these comparisons, the shoulder girdle skeleton of anurans has often been used as a reference. This review traces the history of the debate on the homology of the shoulder girdle in turtles. And based on the integrative aspects of developmental biology, comparative morphology, and paleontology, we suggest acromion and procoracoid identities for the two ventral processes.

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.

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.