Origin of the propatagium in non-avian dinosaurs.

Avian wings as organs for aerial locomotion are furnished with a highly specialized musculoskeletal system compared with the forelimbs of other tetrapod vertebrates. Among the specializations, the propatagium, which accompanies a skeletal muscle spanning between the shoulder and wrist on the leading edge of the wing, represents an evolutionary novelty established at a certain point in the lineage toward crown birds. However, because of the rarity of soft-tissue preservation in the fossil record, the evolutionary origin of the avian propatagium has remained elusive. Here we focus on articulated skeletons in the fossil record to show that angles of elbow joints in fossils are indicators of the propatagium in extant lineages of diapsids (crown birds and non-dinosaurian diapsids), and then use this relationship to narrow down the phylogenetic position acquiring the propatagium to the common ancestor of maniraptorans. Our analyses support the hypothesis that the preserved propatagium-like soft tissues in non-avian theropod dinosaurs (oviraptorosaurian Caudipteryx and dromaeosaurian Microraptor) are homologous with the avian propatagium, and indicate that all maniraptoran dinosaurs likely possessed the propatagium even before the origin of flight. On the other hand, the preserved angles of wrist joints in non-avian theropods are significantly greater than those in birds, suggesting that the avian interlocking wing-folding mechanism involving the ulna and radius had not fully evolved in non-avian theropods. Our study underscores that the avian wing was acquired through modifications of preexisting structures including the feather and propatagium.

Lung evolution in vertebrates and the water-to-land transition.

A crucial evolutionary change in vertebrate history was the Palaeozoic (Devonian 419-359 million years ago) water-to-land transition, allowed by key morphological and physiological modifications including the acquisition of lungs. Nonetheless, the origin and early evolution of vertebrate lungs remain highly controversial, particularly whether the ancestral state was paired or unpaired. Due to the rarity of fossil soft tissue preservation, lung evolution can only be traced based on the extant phylogenetic bracket. Here we investigate, for the first time, lung morphology in extensive developmental series of key living lunged osteichthyans using synchrotron x-ray microtomography and histology. Our results shed light on the primitive state of vertebrate lungs as unpaired, evolving to be truly paired in the lineage towards the tetrapods. The water-to-land transition confronted profound physiological challenges and paired lungs were decisive for increasing the surface area and the pulmonary compliance and volume, especially during the air-breathing on land.

All life on Earth started out under water. However, around 400 million years ago some vertebrates, such as fish, started developing limbs and other characteristics that allowed them to explore life on land. One of the most pivotal features to evolve was the lungs, which gave vertebrates the ability to breathe above water. Most land-living vertebrates, including humans, have two lungs which sit on either side of their chest. The lungs extract oxygen from the atmosphere and transfer it to the bloodstream in exchange for carbon dioxide which then gets exhaled out in to the atmosphere. How this important organ first evolved is a hotly debated topic. This is largely because lung tissue does not preserve well in fossils, making it difficult to trace how the lungs of vertebrates changed over the course of evolution. To overcome this barrier, Cupello et al. compared the lungs of living species which are crucial to understand the early stages of the water-to-land transition. This included four species of lunged bony fish which breathe air at the water surface, and a four-legged salamander that lives on land. Cupello et al. used a range of techniques to examine how the lungs of the bony fish and salamander changed shape during development. The results suggested that the lungs of vertebrates started out as a single organ, which became truly paired later in evolution once vertebrates started developing limbs. This anatomical shift increased the surface area available for exchanging oxygen and carbon dioxide so that vertebrates could breathe more easily on land. These findings provide new insights in to how the lung evolved into the paired structure found in most vertebrates alive today. It likely that this transition allowed vertebrates to fully adapt to breathing above water, which may explain why this event only happened once over the course of evolution.

Morphology of Palaeospondylus shows affinity to tetrapod ancestors.

Palaeospondylus gunni, from the Middle Devonian period, is one of the most enigmatic fossil vertebrates, and its phylogenetic position has remained unclear since its discovery in Scotland in 1890 (ref. 1). The fossil's strange set of morphological features has made comparisons with known vertebrate morphotype diversity difficult. Here we use synchrotron radiation X-ray micro-computed tomography to show that Palaeospondylus was a sarcopterygian, and most probably a stem-tetrapod. The skeleton of Palaeospondylus consisted solely of endoskeletal elements in which hypertrophied chondrocyte cell lacunae, osteoids and a small fraction of perichondral bones developed. Despite the complete lack of teeth and dermal bones, the neurocranium of Palaeospondylus resembles those of stem-tetrapod Eusthenopteron2 and Panderichthys3, and phylogenetic analyses place Palaeospondylus in between them. Because the unique features of Palaeospondylus, such as the cartilaginous skeleton and the absence of paired appendages, are present in the larva of crown tetrapods, our study highlights an unanticipated heterochronic evolution at the root of tetrapods.

Mammalian face as an evolutionary novelty.

The anterior end of the mammalian face is characteristically composed of a semimotile nose, not the upper jaw as in other tetrapods. Thus, the therian nose is covered ventrolaterally by the "premaxilla," and the osteocranium possesses only a single nasal aperture because of the absence of medial bony elements. This stands in contrast to those in other tetrapods in whom the premaxilla covers the rostral terminus of the snout, providing a key to understanding the evolution of the mammalian face. Here, we show that the premaxilla in therian mammals (placentals and marsupials) is not entirely homologous to those in other amniotes; the therian premaxilla is a composite of the septomaxilla and the palatine remnant of the premaxilla of nontherian amniotes (including monotremes). By comparing topographical relationships of craniofacial primordia and nerve supplies in various tetrapod embryos, we found that the therian premaxilla is predominantly of the maxillary prominence origin and associated with mandibular arch. The rostral-most part of the upper jaw in nonmammalian tetrapods corresponds to the motile nose in therian mammals. During development, experimental inhibition of primordial growth demonstrated that the entire mammalian upper jaw mostly originates from the maxillary prominence, unlike other amniotes. Consistently, cell lineage tracing in transgenic mice revealed a mammalian-specific rostral growth of the maxillary prominence. We conclude that the mammalian-specific face, the muzzle, is an evolutionary novelty obtained by overriding ancestral developmental constraints to establish a novel topographical framework in craniofacial mesenchyme.

Twins at Conspicuously Different Developmental Stages in a Turtle Egg.

One-egg twins, in general, initiate embryonic development at the same time, and their developmental stages proceed in parallel. Here we report a rare case of the embryonic development of the red-eared slider turtle, Trachemys scripta, in which twins at conspicuously different developmental stages developed on a single yolk. One of the twins appeared to have developed at the normal developmental rate, whereas the development of the other was markedly delayed, despite the absence of any overt anomalies. This observation suggests uncoupled or fully independent differential regulation of embryonic development from either a single or, more likely, two distinct pluripotent blastoderms sharing the same yolk and same environmental conditions.

Evolution of the muscular system in tetrapod limbs.

While skeletal evolution has been extensively studied, the evolution of limb muscles and brachial plexus has received less attention. In this review, we focus on the tempo and mode of evolution of forelimb muscles in the vertebrate history, and on the developmental mechanisms that have affected the evolution of their morphology. Tetrapod limb muscles develop from diffuse migrating cells derived from dermomyotomes, and the limb-innervating nerves lose their segmental patterns to form the brachial plexus distally. Despite such seemingly disorganized developmental processes, limb muscle homology has been highly conserved in tetrapod evolution, with the apparent exception of the mammalian diaphragm. The limb mesenchyme of lateral plate mesoderm likely plays a pivotal role in the subdivision of the myogenic cell population into individual muscles through the formation of interstitial muscle connective tissues. Interactions with tendons and motoneuron axons are involved in the early and late phases of limb muscle morphogenesis, respectively. The mechanism underlying the recurrent generation of limb muscle homology likely resides in these developmental processes, which should be studied from an evolutionary perspective in the future.

The neural crest and evolution of the head/trunk interface in vertebrates.

The migration and distribution patterns of neural crest (NC) cells reflect the distinct embryonic environments of the head and trunk: cephalic NC cells migrate predominantly along the dorsolateral pathway to populate the craniofacial and pharyngeal regions, whereas trunk crest cells migrate along the ventrolateral pathways to form the dorsal root ganglia. These two patterns thus reflect the branchiomeric and somitomeric architecture, respectively, of the vertebrate body plan. The so-called vagal NC occupies a postotic, intermediate level between the head and trunk NC. This level of NC gives rise to both trunk- and cephalic-type (circumpharyngeal) NC cells. The anatomical pattern of the amphioxus, a basal chordate, suggests that somites and pharyngeal gills coexist along an extensive length of the body axis, indicating that the embryonic environment is similar to that of vertebrate vagal NC cells and may have been ancestral for vertebrates. The amniote-like condition in which the cephalic and trunk domains are distinctly separated would have been brought about, in part, by anteroposterior reduction of the pharyngeal domain.

Anatomical integration of the sacral-hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods.

Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral-hindlimb unit along the anteroposterior axis.

Outflow tract septation and the aortic arch system in reptiles: lessons for understanding the mammalian heart.

BACKGROUND: Cardiac outflow tract patterning and cell contribution are studied using an evo-devo approach to reveal insight into the development of aorto-pulmonary septation. RESULTS: We studied embryonic stages of reptile hearts (lizard, turtle and crocodile) and compared these to avian and mammalian development. Immunohistochemistry allowed us to indicate where the essential cell components in the outflow tract and aortic sac were deployed, more specifically endocardial, neural crest and second heart field cells. The neural crest-derived aorto-pulmonary septum separates the pulmonary trunk from both aortae in reptiles, presenting with a left visceral and a right systemic aorta arising from the unseptated ventricle. Second heart field-derived cells function as flow dividers between both aortae and between the two pulmonary arteries. In birds, the left visceral aorta disappears early in development, while the right systemic aorta persists. This leads to a fusion of the aorto-pulmonary septum and the aortic flow divider (second heart field population) forming an avian aorto-pulmonary septal complex. In mammals, there is also a second heart field-derived aortic flow divider, albeit at a more distal site, while the aorto-pulmonary septum separates the aortic trunk from the pulmonary trunk. As in birds there is fusion with second heart field-derived cells albeit from the pulmonary flow divider as the right 6th pharyngeal arch artery disappears, resulting in a mammalian aorto-pulmonary septal complex. In crocodiles, birds and mammals, the main septal and parietal endocardial cushions receive neural crest cells that are functional in fusion and myocardialization of the outflow tract septum. Longer-lasting septation in crocodiles demonstrates a heterochrony in development. In other reptiles with no indication of incursion of neural crest cells, there is either no myocardialized outflow tract septum (lizard) or it is vestigial (turtle). Crocodiles are unique in bearing a central shunt, the foramen of Panizza, between the roots of both aortae. Finally, the soft-shell turtle investigated here exhibits a spongy histology of the developing carotid arteries supposedly related to regulation of blood flow during pharyngeal excretion in this species. CONCLUSIONS: This is the first time that is shown that an interplay of second heart field-derived flow dividers with a neural crest-derived cell population is a variable but common, denominator across all species studied for vascular patterning and outflow tract septation. The observed differences in normal development of reptiles may have impact on the understanding of development of human congenital outflow tract malformations.

Palaeospondylus as a primitive hagfish.

BACKGROUND: The taxonomic position of the Middle Devonian fish-like animal Palaeospondylus has remained enigmatic, due mainly to the inability to identify homologous cranial elements. This animal has been classified into nearly all of the major vertebrate taxa over a century of heuristic taxonomic research, despite the lack of conclusive morphological evidence. RESULTS: Here we report the first comparative morphological analysis of hagfish embryos and Palaeospondylus, and a hitherto overlooked resemblance in the chondrocranial elements of these animals; i.e., congruence in the arrangement of the nasal capsule, neurocranium and mandibular arch-derived velar bar. The large ventral skeletal complex of Palaeospondylus is identified as a cyclostome-specific lingual apparatus. Importantly, the overall morphological pattern of the Palaeospondylus cranium coincides well with the cyclostome pattern of craniofacial development, which is not shared with that of crown gnathostomes. Previously, the presence of the vertebral column in Palaeospondylus made its assignment problematic, but the recent identification of this vertebral element in hagfish is consistent with an affinity between this group and Palaeospondylus. CONCLUSION: These lines of evidence support the hagfish affinity of Palaeospondylus. Moreover, based on the less specialized features in its cranial morphology, we conclude that Palaeospondylus is likely a stem hagfish.

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.

Developmental mechanisms of the tympanic membrane in mammals and non-mammalian amniotes.

The tympanic membrane is a thin layer that originates from the ectoderm, endoderm, and mesenchyme. Molecular-genetic investigations have revealed that interaction between epithelial and mesenchymal cells in the pharyngeal arches is essential for development of the tympanic membrane. We have recently reported that developmental mechanisms underlying the tympanic membrane seem to be different between mouse and chicken, suggesting that the tympanic membrane evolved independently in mammals and non-mammalian amniotes. In this review, we summarize previous studies of tympanic membrane formation in the mouse. We also discuss its formation in amniotes from an evolutionary point of view.

Expansion of the neck reconstituted the shoulder-diaphragm in amniote evolution.

The neck acquired flexibility through modifications of the head-trunk interface in vertebrate evolution. Although developmental programs for the neck musculoskeletal system have attracted the attention of evolutionary developmental biologists, how the heart, shoulder and surrounding tissues are modified during development has remained unclear. Here we show, through observation of the lateral plate mesoderm at cranial somite levels in chicken-quail chimeras, that the deep part of the lateral body wall is moved concomitant with the caudal transposition of the heart, resulting in the infolding of the expanded cervical lateral body wall into the thorax. Judging from the brachial plexus pattern, an equivalent infolding also appears to take place in mammalian and turtle embryos. In mammals, this infolding process is particularly important because it separates the diaphragm from the shoulder muscle mass. In turtles, the expansion of the cervical lateral body wall affects morphogenesis of the shoulder. Our findings highlight the cellular expansion in developing amniote necks that incidentally brought about the novel adaptive traits.

Evolution of the vertebrate skeleton: morphology, embryology, and development.

Two major skeletal systems-the endoskeleton and exoskeleton-are recognized in vertebrate evolution. Here, we propose that these two systems are distinguished primarily by their relative positions, not by differences in embryonic histogenesis or cell lineage of origin. Comparative embryologic analyses have shown that both types of skeleton have changed their mode of histogenesis during evolution. Although exoskeletons were thought to arise exclusively from the neural crest, recent experiments in teleosts have shown that exoskeletons in the trunk are mesodermal in origin. The enameloid and dentine-coated postcranial exoskeleton seen in many vertebrates does not appear to represent an ancestral condition, as previously hypothesized, but rather a derived condition, in which the enameloid and dentine tissues became accreted to bones. Recent data from placoderm fossils are compatible with this scenario. In contrast, the skull contains neural crest-derived bones in its rostral part. Recent developmental studies suggest that the boundary between neural crest- and mesoderm-derived bones may not be consistent throughout evolution. Rather, the relative positions of bony elements may be conserved, and homologies of bony elements have been retained, with opportunistic changes in the mechanisms and cell lineages of development.

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids.

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.

The evolutionary origin of the turtle shell and its dependence on the axial arrest of the embryonic rib cage.

Turtles are characterized by their possession of a shell with dorsal and ventral moieties: the carapace and the plastron, respectively. In this review, we try to provide answers to the question of the evolutionary origin of the carapace, by revising morphological, developmental, and paleontological comparative analyses. The turtle carapace is formed through modification of the thoracic ribs and vertebrae, which undergo extensive ossification to form a solid bony structure. Except for peripheral dermal elements, there are no signs of exoskeletal components ontogenetically added to the costal and neural bones, and thus the carapace is predominantly of endoskeletal nature. Due to the axial arrest of turtle rib growth, the axial part of the embryo expands laterally and the shoulder girdle becomes encapsulated in the rib cage, together with the inward folding of the lateral body wall in the late phase of embryogenesis. Along the line of this folding develops a ridge called the carapacial ridge (CR), a turtle-specific embryonic structure. The CR functions in the marginal growth of the carapacial primordium, in which Wnt signaling pathway might play a crucial role. Both paleontological and genomic evidence suggest that the axial arrest is the first step toward acquisition of the turtle body plan, which is estimated to have taken place after the divergence of a clade including turtles from archosaurs. The developmental relationship between the CR and the axial arrest remains a central issue to be solved in future.

Evolution and development of ventricular septation in the amniote heart.

During cardiogenesis the epicardium, covering the surface of the myocardial tube, has been ascribed several functions essential for normal heart development of vertebrates from lampreys to mammals. We investigated a novel function of the epicardium in ventricular development in species with partial and complete septation. These species include reptiles, birds and mammals. Adult turtles, lizards and snakes have a complex ventricle with three cava, partially separated by the horizontal and vertical septa. The crocodilians, birds and mammals with origins some 100 million years apart, however, have a left and right ventricle that are completely separated, being a clear example of convergent evolution. In specific embryonic stages these species show similarities in development, prompting us to investigate the mechanisms underlying epicardial involvement. The primitive ventricle of early embryos becomes septated by folding and fusion of the anterior ventricular wall, trapping epicardium in its core. This folding septum develops as the horizontal septum in reptiles and the anterior part of the interventricular septum in the other taxa. The mechanism of folding is confirmed using DiI tattoos of the ventricular surface. Trapping of epicardium-derived cells is studied by transplanting embryonic quail pro-epicardial organ into chicken hosts. The effect of decreased epicardium involvement is studied in knock-out mice, and pro-epicardium ablated chicken, resulting in diminished and even absent septum formation. Proper folding followed by diminished ventricular fusion may explain the deep interventricular cleft observed in elephants. The vertical septum, although indistinct in most reptiles except in crocodilians and pythonidsis apparently homologous to the inlet septum. Eventually the various septal components merge to form the completely septated heart. In our attempt to discover homologies between the various septum components we aim to elucidate the evolution and development of this part of the vertebrate heart as well as understand the etiology of septal defects in human congenital heart malformations.

Comparative analysis of pleurodiran and cryptodiran turtle embryos depicts the molecular ground pattern of the turtle carapacial ridge.

The turtle shell is a wonderful example of a genuine morphological novelty, since it has no counterpart in any other extant vertebrate lineages. The evolutionary origin of the shell is a question that has fascinated evolutionary biologists for over two centuries and it still remains a mystery. One of the turtle innovations associated with the shell is the carapacial ridge (CR), a bulge that appears at both sides of the dorsal lateral trunk of the turtle embryo and that probably controls the formation of the carapace, the dorsal moiety of the shell. Although from the beginning of this century modern genetic techniques have been applied to resolve the evolutionary developmental origin of the CR, the use of different models with, in principle, dissimilar results has hampered the establishment of a common mechanism for the origin of the shell. Although modern turtles are divided into two major groups, Cryptodira (or hidden-necked turtles) and Pleurodira (or side-necked turtles), molecular developmental studies have been carried out mostly using cryptodiran models. In this study, we revisit the past data obtained from cryptodiran turtles in order to reconcile the different results. We also analyze the histological anatomy and the expression pattern of main CR factors in a pleurodiran turtle, the red-bellied short-necked turtle Emydura subglobosa. We suggest that the turtle shell probably originated concomitantly with the co-option of the canonical Wnt signaling pathway into the CR in the last common ancestor of the turtle.