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We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates-the amniote egg presenting one "solution" that enabled the conquest of land for reproduction.

Amniote vertebrates share a suite of extra-embryonic membranes that distinguish them from anamniotes. Other than that, however, their reproductive characteristics could not be more different. They differ in basic ectothermic vs endothermic physiology, in that two clades evolved powered flight, and one clade evolved a protective shell. In terms of reproductive strategies, some produce eggs and others give birth to live young, at various degrees of development. Crucially, endotherms provide lengthy parental care, including thermal and food provisioning—whereas ectotherms seldom do. These differences could be expected to manifest themselves in major differences between clades in quantitative reproductive traits. We review the reproductive characteristics, and the distributions of brood sizes, breeding frequencies, offspring sizes and their derivatives (yearly fecundity and biomass production rates) of the four major amniote clades (mammals, birds, turtles and squamates), and several major subclades (birds: Palaeognathae, Galloanserae, Neoaves; mammals: Metatheria and Eutheria). While there are differences between these clades in some of these traits, they generally show similar ranges, distribution shapes and central tendencies across birds, placental mammals and squamates. Marsupials and turtles, however, differ in having smaller offspring, a strategy which subsequently influences other traits.

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.

An evolutionary hypothesis explaining failure of regeneration among vertebrates is presented. Regeneration derives from postembryonic processes present during the life cycles of fish and amphibians that include larval and metamorphic phases with broad organ reorganizations. Developmental programs imprinted in their genomes are re-utilized with variations also in adults for regeneration. When vertebrates colonized land adopting the amniotic egg, some genes driving larval changes, and metamorphosis were lost and new genes evolved, further limiting regeneration. These included neural inhibitors for maintaining complex nervous systems, behavior and various levels of intelligence, and adaptive immune cells. The latter, that in anamniotes are executioners of metamorphic reorganization, became intolerant to embryonic-oncofetal-antigens impeding organ regeneration, a process that requires de-differentiation of adult cells and/or expansion of stem cells where these early antigens are formed. The evolution of terrestrial lifecycles produced vertebrates with complex bodies but no longer capable to regenerate their organs, mainly repaired by regengrow. Efforts of regenerative medicine to improve healing in humans should determine the diverse developmental pathways evolved between anamniotes and amniotes before attempting genetic manipulations such as the introduction of "anamniote regenerative genes" in amniotes. This operation may determine alteration in amniote developmental programs leading to teratomes, cancer, or death.

Amniotes, such as mammals and reptiles, have vision and other senses represented in the pallium, whereas anamniotes, such as amphibians, fish and cyclostomes (including lampreys), which diverged much earlier, were historically thought to process olfactory information predominantly or even exclusively in the pallium. Here, we show that there is a separate visual area with retinotopic representation, and that somatosensory information from the head and trunk is represented in an adjacent area in the lamprey pallial cortex (lateral pallium). These cortical sensory areas flank a non-primary-sensory motor area. Both vision and somatosensation are relayed via the thalamus. These findings suggest that the basic sensorimotor representation of the mammalian neocortex, as well as the sensory thalamocortical relay, had already evolved in the last common ancestor of cyclostomes and gnathostomes around 560 million years ago. This study uses isolated eye–brain preparations to show topographic visual and somatosensory representation in the lamprey pallium, which is similar to the basic sensorimotor representation of the mammalian neocortex.

Evolution selects those adaptive features that increase reproductive probabilities and facilitate survival. Analysing the brain circuits mediating risk-avoidance (e.g. defense) and those allowing reward-seeking (motivated) behaviours in different vertebrates leads to several main conclusions. First, circuits mediating risk-avoidance are similar in all studied vertebrates, where they include amygdala homologues located in the posterior half of the cerebral hemispheres, in close relationship with the chemosensory systems. Second, in all vertebrates, reward-seeking behaviours involve the activity of tegmento-striatal dopaminergic pathways, plus other inputs to the ventral striatum, including amygdalo-striatal glutamatergic projections. Third, output structures in these forebrain circuits for both risk-avoidance and reward-seeking behaviours occupy the caudal and rostral poles of the ventral striato-pallidum, namely the central amygdala and nucleus accumbens-olfactory tubercle respectively. This brain configuration was already present in at least the ancestral amniote, likely also in anamniotes. Finally, social behaviours (sexual, agonistic-territorial, parental) are fundamental for reproduction and survival. Consequently, the so-called socio-sexual brain network that governs these conducts is closely related with brain centres mediating motivation (maybe also risk-avoidance). Central nonapeptidergic circuits are apparently required for endowing social stimuli with rewarding (attractive) properties. More studies in non-mammals are required to further test and expand these ideas.

Spermatogenesis, the production of sperm, is a fundamental part of the reproductive cycle in vertebrates and warrants detailed study. Reptiles exhibit a hybrid pattern of spermatogenesis that has similarities with both anamniotes and amniotes. Researching this interesting transition will help us better understand vertebrate evolution. To this end, we histologically examined the spermatogenic and testicular cycle of Rough Greensnakes (Opheodrys aestivus) from Arkansas. We hypothesized that spermatogenesis in Rough Greensnakes would follow the different spermatogenic stages found in previously published studies on temperate snakes. In addition, we hypothesized that both season and seminiferous tubule diameter would have a significant effect on seminiferous tubule epithelial height. Finally, we hypothesized that Rough Greensnakes would exhibit postnuptial spermatogenesis. We constructed a cell wheel illustrating the chronological development of all germ cell stages for this species and measured semi...

Lungfishes are a group of sarcopterygian fishes currently considered the closest living relatives of tetrapods, and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. Catecholaminergic systems in the brain are among the most carefully analyzed neurotransmitter systems in the brain of most vertebrate groups. Their organization shows major shared characteristics, although traits particular to each vertebrate class have also been found, primarily between anamniotes and amniotes. Given the relevance of lungfishes in evolutionary terms, the present study provides the first comprehensive and detailed map of the catecholaminergic structures in the brain of two representative species of lungfishes, an African lungfish (Protopterus dolloi) and the Australian lungfish (Neoceratodus forsteri), as revealed by immunohistochemistry. Distinct groups of catecholaminergic cells were observed in the olfactory bulb, pallium, and preoptic area of the telencephalon, and the subpallium is devoid of these cells. Hypothalamic and diencephalic groups were detected and, in particular, the dopaminergic nucleus of the periventricular organ was evidenced with dopamine antibodies but not with anti-tyrosine hydroxylase. A well developed mesostriatal system was revealed formed by conspicuous groups of dopamine cells in the midbrain tegmentum and profuse innervation of the subpallium. Comparison of these results with those from other classes of vertebrates shows numerous common traits shared by most groups and also highlights particular features in lungfishes different from actinopterygian fishes that resemble those of amphibians and amniotes.

Senescence represents a mechanism to avoid undesired cell proliferation that plays a role in tumor suppression, wound healing and embryonic development. In order to gain insight on the evolution of senescence, we looked at its presence in developing axolotls (urodele amphibians) and in zebrafish (teleost fish), which are both anamniotes. Our data indicate that cellular senescence is present in various developing structures in axolotls (pronephros, olfactory epithelium of nerve fascicles, lateral organs, gums) and in zebrafish (epithelium of the yolk sac and in the lower part of the gut). Senescence was particularly associated with transient structures (pronephros in axolotls and yolk sac in zebrafish) suggesting that it may play a role in the elimination of these tissues. Our data supports the notion that cellular senescence evolved early in vertebrate evolution to influence embryonic development., Summary: We report the presence of senescent cells in several transient structures in developing amphibian and teleost fish, suggesting novel mechanisms of morphogenesis that appeared early in vertebrate evolution.

Amphibians have often been selected as experimental animals in brain research for comparative studies. They are tetrapod anamniotes that represent a key model for understanding the water-to-land transition. In amphibians the basic traits of brain organization in amniotes can be already recognized. In the present chapter we deal with the regional anatomy of the amphibian brain (in the three extant orders) focusing mainly on the chemoarchitecture and genoarchitecture, which serve to define anatomical subdivisions that are otherwise indistinct. The new data are interpreted within the framework of the current neuromeric model that helps comparisons across vertebrates.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

In our review we have completed current knowledge on myotomal myogenesis in model and non-model vertebrate species (fishes, amphibians, reptiles, birds and mammals) at morphological and molecular levels. Data obtained from these studies reveal distinct similarities and differences between amniote and anamniote species. Based on the available data, we decided to present evolutionary implications in vertebrate trunk muscle development. Despite the fact that in all vertebrates muscle fibres are multinucleated, the pathways leading to them vary between vertebrate taxa. In fishes during early myogenesis myoblasts differentiate into multinucleated lamellae or multinucleate myotubes. In amphibians, myoblasts fuse to form multinucleated myotubes or, bypassing fusion, directly differentiate into mononucleated myotubes. Furthermore, mononucleated myotubes were also observed during primary myogenesis in amniotes. The mononucleated state of myogenic cells could be considered as an old phylogenetic, plesiomorphic feature, whereas direct multinuclearity of myotubes has a synapomorphic character. On the other hand, the explanation of this phenomenon could also be linked to the environmental conditions in which animals develop. The similarities observed in vertebrate myogenesis might result from a conservative myogenic programme governed by the Pax3/Pax7 and myogenic regulatory factor (MRF) network, whereas differences in anamniotes and amniotes are established by spatiotemporal pattern expression of MRFs during muscle differentiation and/or environmental conditions.

Genomic GC content can vary locally, and GC-rich regions are usually associated with increased DNA thermostability in thermophilic prokaryotes and warm-blooded eukaryotes. Among vertebrates, fish and amphibians appeared to possess a distinctly less heterogeneous AT/GC organization in their genomes, whereas cytogenetically detectable GC heterogeneity has so far only been documented in mammals and birds. The subject of our study is the gar, an ancient "living fossil" of a basal ray-finned fish lineage, known from the Cretaceous period. We carried out cytogenomic analysis in two gar genera (Atractosteus and Lepisosteus) uncovering a GC chromosomal pattern uncharacteristic for fish. Bioinformatic analysis of the spotted gar (Lepisosteus oculatus) confirmed a GC compartmentalization on GC profiles of linkage groups. This indicates a rather mammalian mode of compositional organization on gar chromosomes. Gars are thus the only analyzed extant ray-finned fishes with a GC compartmentalized genome. Since gars are cold-blooded anamniotes, our results contradict the generally accepted hypothesis that the phylogenomic onset of GC compartmentalization occurred near the origin of amniotes. Ecophysiological findings of other authors indicate a metabolic similarity of gars with mammals. We hypothesize that gars might have undergone convergent evolution with the tetrapod lineages leading to mammals on both metabolic and genomic levels. Their metabolic adaptations might have left footprints in their compositional genome evolution, as proposed by the metabolic rate hypothesis. The genome organization described here in gars sheds new light on the compositional genome evolution in vertebrates generally and contributes to better understanding of the complexities of the mechanisms involved in this process.

The evolution of the process of cornification in amniote epidermis from the general process of keratinization present in simple epithelia of anamniotes took place through the evolution of specialized intermediate filament (α) keratins, keratin-associated proteins (KAPs) and corneous proteins (CPs). The scanty information on the three-dimensional conformation of known KAPs and CPs indicate these proteins contain α-helix, random coiled, or beta sheets with different lengths and organizations. CP genes originated in a chromosome locus indicated as epidermal differentiation complex (EDC), and transformed the epidermal keratinization of anamniotes into the cornified epidermis and skin appendages of amniotes (claws, beaks, and feathers). In particular, peculiar genes encoding for small proteins with a central region of 34 amino acids conformed as beta sheets were originated in the EDC of sauropsids (reptiles and birds). These proteins were traditionally indicated as beta-keratins because they form filaments of 3-4 nm in diameter and show an X-ray beta pattern. Different from other proteins of the EDC, dimers of these corneous beta-proteins associate into long polymers of filamentous proteins utilized in sauropsids skin appendages, such as scales and feathers. Future challenges in this area of research will be the study on gene regulation and expression for these proteins, their origin and evolution in different lineages of sauropsids, and their role in determining the material properties of sauropsid scales and other skin appendages.

In contrast to amniotes (reptiles, birds and mammals), anamniotes (fishes and amphibians) can effectively regenerate body appendages such as fins, limbs and tails. Why such a useful capability was progressively lost in amniotes remains unknown. As we have hypothesized recently, one of the reasons for this could be loss of some genes regulating the regeneration in evolution of amniotes. Here, we demonstrate the validity of this hypothesis by showing that genes of small GTPases Ras-dva1 and Ras-dva2, that had been lost in a stepwise manner during evolution of amniotes and disappeared completely in placental mammals, are important for regeneration in anamniotes. Both Ras-dva genes are quickly activated in regenerative wound epithelium and blastema forming in the amputated adult Danio rerio fins and Xenopus laevis tadpoles’ tails and hindlimb buds. Down-regulation of any of two Ras-dva genes in fish and frog resulted in a retardation of regeneration accompanied by down-regulation of the regeneration marker genes. On the other hand, Ras-dva over-expression in tadpoles’ tails restores regeneration capacity during the refractory period when regeneration is blocked due to natural reasons. Thus our data on Ras-dva genes, which were eliminated in amniotes but play role in anamniotes regeneration regulation, satisfy our hypothesis.

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such asXenopus laevis,depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 μN during gastrulation and over 4 μN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 μN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.

Cladistians (Polypteriformes) are currently considered basal to other living ray-finned fishes (actinopterygians), and their brain organization is therefore critical to providing information about the primitive neural characters that existed in the earliest ray-finned fishes. The organization of the serotonergic system in the brain has been carefully analyzed in most vertebrate groups, and in the present study we provide the first detailed information on the distribution of serotonergic cell bodies and fibers in the central nervous system of representative species of the two extant genera of cladistians, i.e. Polypterus senegalus and Erpetoichthys calabaricus, by means of immunohistochemistry against serotonin (5-HT). Distinct groups of immunoreactive cells were detected in the preoptic area, the hypothalamic paraventricular organ, the pineal organ, the pretectal region, the long column of the raphe in the rhombencephalic midline, the spinal cord, and amacrine cells in the inner nuclear layer of the retina. Fiber labeling was widely distributed in all main brain subdivisions but was more abundant in distinct pallial and subpallial areas, the preoptic area, the thalamus, the optic tectum, the tori semicircularis and lateralis, the rhombencephalic reticular formation, the nucleus of the solitary tract, and the dorsal aspect of the spinal cord. Our analysis makes it possible to establish which serotonergic structures characterized the earliest ray-finned fishes, and a comparison of these results with those from other classes of vertebrates, including a segmental analysis to correlate cell populations, reveals that most characteristics, such as the presence of serotonergic cells in the preoptic area and the basal hypothalamus, are preserved in all anamniotes. However, this system seems to be reduced in amniotes, mainly mammals, although important features are shared, such as the presence of serotonergic cells in the pineal organ, the retina, and the raphe nuclei.

Oviparous amniotes produce a large yolky egg that gives rise to a free-living hatchling. Structural characteristics and functional attributes of the egg are best known for birds, which have a large mass of fluid yolk surrounded by an extraembryonic yolk sac. Yolk nutrients are delivered to the embryo via the vascular yolk sac. This developmental pattern and nutrient transport mechanism is thought to be representative of all other lineages of amniotes. Recent discovery of a snake with cellularized yolk organized around a meshwork of blood vessels reveals an additional pattern for yolk mobilization, which may also occur in other squamate reptiles (lizards and snakes). This complex yolk sac raises interesting questions about developmental mechanisms and suggests a possible model for the transition between the egg of anamniotes and that of amniotes.

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.

In the mature mammalian and avian central nervous systems, neuronal destructions are followed by reactive gliosis, but data on other vertebrates are rather controversial. Mammals and birds belong to different amniote groups (Synapsida and Diapsida, respectively), but exhibit common general features in their glial architecture, mainly the predominance of astrocytes. Two vertebrate groups seem to be in special positions of glial evolution: turtles (Testudiniformes) and skates and rays (Batoidea). The purely ependymoglial system of turtles seems to be the simplest one among the extant amniotes. In skates and rays, true astrocytes are preponderant glial elements, in contrast to the other "anamniotes" (and even to reptiles). We investigated stab wounds by the immunohistochemical detection of GFAP in turtles (Trachemys-formerly Pseudemys-scripta elegans), a skate (Raja clavata) and rays (Dasyatis akajei and Torpedo marmorata). Sharks (Scyliorhinus canicula) as ependymoglia-predominated chondrichthyans, and-for positive controls-rats were also studied. In the elasmobranchs, other astroglial markers: glutamine synthetase and S100 protein were also applied. Neither turtles nor elasmobranchs presented considerable astroglial reactions. Critically surveying the former reports on different vertebrates, these results complete the picture that typical post-lesion reactive gliosis is confined to mammals and birds. Analysis of the astroglial systems from phylogenetic perspective suggests that the capability of forming glial demarcation and scar formation evolved independently in mammals and birds. Predominance of astrocytes is a necessary condition but not sufficient for reactive gliosis. The intense glial reactivity of mammals and birds may be attributed to their complex cerebralization.

The complexity of the pallium during evolution has increased dramatically in many different respects. The highest level of complexity is found in mammals, where most of the pallium (cortex) shows a layered organization and neurons are generated during development following an inside-out order, a sequence not observed in other amniotes (birds and reptiles). Species-differences may be related to major neurogenetic events, from the neural progenitors that divide and produce all pallial cells. In mammals, two main types of precursors have been described, primary precursor cells in the ventricular zone (vz; also called radial glial cells or apical progenitors) and secondary precursor cells (called basal or intermediate progenitors) separated from the ventricle surface. Previous studies suggested that pallial neurogenetic cells, and especially the intermediate progenitors, evolved independently in mammalian and sauropsid lineages. In the present study, we examined pallial neurogenesis in the amphibian Xenopus laevis, a representative species of the only group of tetrapods that are anamniotes. The pattern of pallial proliferation during embryonic and larval development was studied, together with a multiple immunohistochemical analysis of putative progenitor cells. We found that there are two phases of progenitor divisions in the developing pallium that, following the radial unit concept from the ventricle to the mantle, finally result in an outside-in order of mature neurons, what seems to be the primitive condition of vertebrates. Gene expressions of key transcription factors that characterize radial glial cells in the vz were demonstrated in Xenopus. In addition, although mitotic cells were corroborated outside the vz, the expression pattern of markers for intermediate progenitors differed from mammals.

Sensory hair cell losses lead to hearing and balance deficits that are permanent for mammals, but temporary for nonmammals because supporting cells in their ears give rise to replacement hair cells. In mice and humans, vestibular supporting cells grow exceptionally large circumferential F-actin belts and their junctions express E-cadherin in patterns that strongly correlate with postnatal declines in regeneration capacity. In contrast, chicken supporting cells retain thin F-actin belts throughout life and express little E-cadherin. To determine whether the junctions in chicken ears might be representative of other ears that also regenerate hair cells, we investigated inner ears from dogfish sharks, zebrafish, bullfrogs, Xenopus, turtles, and the lizard, Anolis. As in chickens, the supporting cells in adult zebrafish, Xenopus, and turtle ears retained thin circumferential F-actin belts and expressed little E-cadherin. Supporting cells in adult sharks and bullfrogs also retained thin belts, but were not tested for E-cadherin. Supporting cells in adult Anolis exhibited wide, but porous webs of F-actin and strong E-cadherin expression. Anolis supporting cells also showed some cell cycle reentry when cultured. The results reveal that the association between thin F-actin belts and low E-cadherin is shared by supporting cells in anamniotes, turtles, and birds, which all can regenerate hair cells. Divergent junctional specializations in supporting cells appear to have arisen independently in Anolis and mammals. The presence of webs of F-actin at the junctions in Anolis appears compatible with supporting cell proliferation, but the solid reinforcement of the F-actin belts in mammals is associated with its absence. J. Comp. Neurol., 521:1430–1448, 2013. © 2012 Wiley Periodicals, Inc.

Vertebrate neck musculature spans the transition zone between head and trunk. The extent to which the cucullaris muscle is a cranial muscle allied with the gill levators of anamniotes or is instead a trunk muscle is an ongoing debate. Novel computed tomography datasets reveal broad conservation of the cucullaris in gnathostomes, including coelacanth and caecilian, two sarcopterygians previously thought to lack it. In chicken, lateral plate mesoderm (LPM) adjacent to occipital somites is a recently identified embryonic source of cervical musculature. We fate-map this mesoderm in the axolotl (Ambystoma mexicanum), which retains external gills, and demonstrate its contribution to posterior gill-levator muscles and the cucullaris. Accordingly, LPM adjacent to the occipital somites should be regarded as posterior cranial mesoderm. The axial position of the head-trunk border in axolotl is congruent between LPM and somitic mesoderm, unlike in chicken and possibly other amniotes. DOI: http://dx.doi.org/10.7554/eLife.09972.001, eLife digest Muscles in the head and trunk (main body) form from different parts of the embryo, and their development uses different genes. Trunk muscles are derived from somites – paired blocks of cells arranged in segments on either side of the midline (which divides the body into left and right halves). By contrast, cells that give rise to head muscles are arranged in a continuous mass. But what about neck muscles? Some studies claim they develop like head muscles; others suggest they are trunk muscles. These studies commonly examine mice or chickens. By examining species that have a more primitive complement of head and neck muscles, Sefton et al. now show that a neck muscle should be considered a kind of head muscle. Gill muscles are definitive head muscles. Sefton et al. found that the cucullaris, a prominent neck muscle in fishes and amphibians, forms from the same mass of cells that gives rise to gill muscles. Moreover, studying muscle development in Mexican axolotls showed that cells that contribute to gill muscles extend into the trunk, which is further back in the embryo than was previously known. Previous studies reported the cucullaris muscle is absent in a “lobe-finned” fish called the coelacanth, which is closely related to four-limbed animals. However, by using a technique called micro-computed tomography to visualize the neck muscles of this fish, Sefton et al. show that the cucullaris muscle is present and connects the rear-most gill to the shoulder. The finding that neck muscles form like head muscles in the axolotl confirms a previous claim that was based on studies of bird embryos. A future challenge is to understand the molecular and genetic mechanisms that establish the boundary between head and trunk muscles, and work out how those mechanisms might have influenced how the neck evolved. DOI: http://dx.doi.org/10.7554/eLife.09972.002

The axial skeleton is a defining feature of vertebrates and is patterned during somitogenesis. Cyclically expressed members of the notch and other signaling pathways, described as the ‘segmentation clock’, regulate the formation of somite boundaries. Comparisons among vertebrate model systems have revealed fundamental shifts in the regulation of expression among critical genes in the notch pathway. However, insights into the evolution of these expression differences have been limited by the lack of information from non-avian reptiles. We analyzed the segmentation clock of the first Lepidosaurian reptile sequenced, the green anole lizard, Anolis carolinensis, for comparison with avian and mammalian models. Using genomic sequence, RNA-Seq transcriptomic data, and in situ hybridization analysis of somite-stage embryos, we carried out comparative analyses of key genes and found that the anole segmentation clock displays features common to both amniote and anamniote vertebrates. Shared features with anamniotes, represented by Xenopus laevis and Danio rerio, include an absence of lunatic fringe (lfng) expression within the presomitic mesoderm (PSM), a hes6a gradient in the PSM not observed in the chicken or mouse, and EGF repeat structure of the divergent notch ligand, dll3. The anole and mouse share cycling expression of dll1 ligand in the PSM. To gain insight from an Archosaurian reptile, we analysed LFNG and DLL1 expressions in the American alligator. LFNG expression was absent in the alligator PSM, like the anole but unlike the chicken. In contrast, DLL1 expression does not cycle in the PSM of the alligator, similar to the chicken but unlike the anole. Thus, our analysis yields novel insights into features of the segmentation clock that are evolutionarily basal to amniotes versus those that are specific to mammals, Lepidosaurian reptiles, or Archosaurian reptiles.

Networked gene activities control the evolutionarily conserved histogenetic organization of the central nervous system of vertebrates. Genoarchitectonic studies contribute to the analysis of each morphogenetic field by identifying distinct progenitor domains and corresponding derivatives whose pattern of gene expression shows a unique combinatory code. Previous studies in the pretectal region (caudal diencephalon) have defined three anteroposterior genoarchitectonic domains that are conserved in birds and mammals. Here, we have studied the embryonic pretectal genoarchitecture in the amphibian Xenopus laevis, in order to determine whether it is possible to define a comparable anteroposterior tripartition of the amphibian pretectal area. The expression patterns of 14 genes mapped from early embryonic stages to metamorphic climax allowed us to define the boundaries of the pretectum, the expected precommissural, juxtacommissural, and commissural anteroposterior domains, and some dorsoventral subdivisions. Taken together, our data provide evidence for a conserved pattern of pretectal domains and subdomains, shared by amniotes and amphibian anamniotes (tetrapods), understandable as part of a general Bauplan in vertebrates. J. Comp. Neurol. 519:1024‐1050, 2011.

The testes of Hemidactylus turcicus are composed of seminiferous tubules lined with continuous germinal epithelia in which multiple germ cell morphologies can be found during the active months of sperm development. Spermatogenesis is quiescent during September, with only spermatogonia A and spermatogonia B present in the seminiferous epithelia and minimal mitotic activity is observed. Recrudescence begins in October and the early stages of spermatogenesis progress through November. The onset of spermiation is observed in December and continues through August with the heaviest sperm release occurring in June and July. Multiple generations of late elongated spermatids are found in association with early mitotic and meiotic cells during the months of December–August. This temporal germ cell development strategy is similar to that described in other squamates and anamniotes and is different from the spatial development exhibited by birds and mammals, in which germ cell populations collectively progre...

Ground Skink (Scincella lateralis) testes were examined histologically to determine the testicular organization and germ cell development strategy employed during spermatogenesis. Testicular tissues were collected from 19 ground skinks from Aiken County, South Carolina during the months of March-June, August, and October. The testes consisted of seminiferous tubules lined with germinal epithelia in which germ cells matured in close association with Sertoli cells. As germ cells matured, they migrated away from the basal lamina of the epithelia towards the lumina of the seminiferous tubules. The testes were spermatogenically active during the months of March, April, May, June, and October (largest seminiferous tubule diameters and epithelial heights), but entered a quiescent period in August (smallest seminiferous tubule diameter and epithelial height) where only spermatogonia type A and B and early spermatocytes were present in low numbers within the seminiferous epithelium. Although the testicular organization was similar to other amniotes, a temporal germ cell development strategy was employed during spermatogenesis within Ground Skinks, similar to that of anamniotes. Thus, this skink's germ cell development strategy, which also has been recently reported in all other major reptilian clades, may represent an evolutionary intermediate in terms of testicular organization between anamniotes and birds and mammals.

Some animals, notably birds, preferentially approach and capture food items in their right visual field. However, this lateralised behaviour has not been studied extensively in anamniotes. Here we test eye preference during feeding for a fish, (Perccottus glenii; Teleostei, Perciformes), a newt, (Pleurodeles walti; Amphibia, Caudata), and a frog, (Xenopus laevis; Amphibia, Anura) using a test chamber that assesses reaction to visual stimuli while blocking olfactory and mechanical input. Both the fish and the newt showed right preferences in reactions to food items, but the frog did not. Our data extend our knowledge of the lateralised behaviours of vertebrates and are the first record of lateralised prey capture in a caudate amphibian. This finding dates back the history of the common pattern for visual lateralisation in vertebrates to Devonian, when the fish and quadruped lineages diverged.

The Lower Permian fossiliferous infills of the Dolese Brothers Limestone Quarry, near Richards Spur, Oklahoma, have preserved the most diverse assemblage of Paleozoic terrestrial vertebrates, including small-bodied reptiles and lepospondyl anamniotes. Many of these taxa were previously known only from fragmentary remains, predominantly dentigerous jaw elements and numerous isolated skeletal elements. The recent discovery of articulated skulls and skeletons of small reptiles permits the recognition that dentigerous elements, previously assigned at this locality to the anamniote lepospondyl Euryodus primus, belong to a new captorhinid eureptile, Opisthodontosaurus carrolli gen. et sp. nov. This mistaken identity points to a dramatic level of convergence in mandibular and dental anatomy in two distantly related and disparate clades of terrestrial tetrapods and sheds light on the earliest instance of durophagy in eureptiles.

Somites are a common feature of the phylotypic stage of embryos of all higher chordates. In amniote species like mouse and chick, somite development has been the subject of intense research over many decades, giving insight into the morphological and molecular processes leading to somite compartmentalization and subsequent differentiation. In anamniotes, somite development is much less understood. Except for recent data from zebrafish, and morphological studies in Xenopus, very little is known about the formation of somite compartments and the differentiation of somite derivatives in anamniotes. Here, we give a brief overview on the development of myotome, sclerotome and dermomyotome in various anamniote organisms, and point out the different mechanisms of somite development between anamniotes and the established amniote model systems.

Gribbins, K.M., Happ, C.S. and Sever, D.M. 2005. Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis. — Acta Zoologica (Stockholm) 86: 223-230 The germ cell development strategy during spermatogenesis was investigated in the black swamp snake (Seminatrix pygaea). Testicular tissues were collected, embedded in plastic, sectioned by ultramicrotome, and stained with methylene blue and basic fuchsin. Black swamp snakes have a postnuptial pattern of development, where spermatogenesis occurs from May to July and spermiation is completed by October. Though spatial relationships are seen between germ cells within the seminiferous epithelium during specific months, accumulation of spermatogonia and spermatocytes early in spermatogenesis and the depletion of spermatocytes and accumulation of spermatids late in spermatogenesis prevent consistent cellular associations. This temporal germ cell development within an amniotic testis is consistent with that seen in other recently studied temperate reptiles (slider turtle and wall lizard). These reptiles' temporal development is more similar to the developmental strategy found in anamniotes than the spatial germ cell development that characterizes birds and mammals. Our findings also imply that a third germ cell development strategy may exist in temperate breeding reptiles. Because of the phylogenetic position of reptiles between anamniotes and other terrestrial amniotes, this common germ cell development strategy shared by temperate reptiles representing different orders may have significant implications as far as the evolution of sperm development within vertebrates.

We used two developmental transcription factors, x-Eomes (T-box family) and x-Lhx5 (LIM-homeodomain family), to follow the origin and development of the olfactory bulbs in Xenopus. During embryonic and larval development, x-Eomes and x-Lhx5 were expressed in highly similar patterns, in the lateral and latero-ventral wall of the pallium. In adults, both markers were strongly and specifically expressed in mitral cells, i.e., in the projection neurons of the main and accessory olfactory bulbs. These results demonstrate the pallial origin of the olfactory projecting cells in Xenopus. Combined with previous results suggesting a subpallial origin for olfactory interneurons, these findings emphasize the dual origin of different neuronal populations in the bulbs of anamniotes, and suggest that this organization is a shared feature of tetrapods.

Current models in biological psychiatry focus on a handful of model species, and the majority of work relies on data generated in rodents. However, in the same sense that a comparative approach to neuroanatomy allows for the idenfication of patterns of brain organization, the inclusion of other species and an adoption of comparative viewpoints in behavioral neuroscience could also lead to increases in knowledge relevant to biological psychiatry. Specifically, this approach could help to identify conserved features of brain structure and behavior, as well as to understand how variation in gene expression or developmental trajectories relates to variation in brain and behavior pertinent to psychiatric disorders. To achieve this goal, the current focus on mammalian species must be expanded to include other species, including non-mammalian taxa. In this article, we review behavioral neuroscientific experiments in non-mammalian species, including traditional 'model organisms' (zebrafish, Drosophila and C. elegans) as well as in other species which can be used as 'reference'. The application of these domains in biological psychiatry and their translational relevance is considered.

The basic architecture of the mammalian neocortex is remarkably similar across species. Pallial structures in the reptilian brain are considered amniote precursors of mammalian neocortex, whereas pallia of anamniotes ("lower" vertebrates) have been deemed largely insignificant with respect to homology. Here, we examine the cytoarchitecture of the lateral pallium in the lamprey, the phylogenetically oldest group of extant vertebrates. We reveal a three-layered structure with similar excitatory cell types as in the mammalian cortex and GABAergic interneurons. The ventral parts are sensory areas receiving monosynaptic thalamic input that can be activated from the optic nerve, whereas the dorsal parts contain motor areas with efferent projections to the brainstem, receiving oligosynaptic thalamic input. Both regions receive monosynaptic olfactory input. This three-layered "primordial" lamprey lateral pallium has evolved most features of the three-layered reptilian cortices and is thereby a precursor of the six-layered "neo" cortex with a long-standing evolutionary precedent (some 500 million years ago).

A comparative analysis of catecholaminergic systems in the brain and spinal cord of vertebrates forces to reconsider several aspects of the organization of catecholamine systems. Evidence has been provided for the existence of extensive, putatively catecholaminergic cell groups in the spinal cord, the pretectum, the habenular region, and cortical and subcortical telencephalic areas. Moreover, putatively dopamine- and noradrenaline-accumulating cells have been demonstrated in the hypothalamic periventricular organ of almost every non-mammalian vertebrate studied. In contrast with the classical idea that the evolution of catecholamine systems is marked by an increase in complexity going from anamniotes to amniotes, it is now evident that the brains of anamniotes contain catecholaminergic cell groups, of which the counterparts in amniotes have lost the capacity to produce catecholamines. Moreover, a segmental approach in studying the organization of catecholaminergic systems is advocated. Such an approach has recently led to the conclusion that the chemoarchitecture and connections of the basal ganglia of anamniote and amniote tetrapods are largely comparable. This review has also brought together data about the distribution of receptors and catecholaminergic fibers as well as data about developmental aspects. From these data it has become clear that there is a good match between catecholaminergic fibers and receptors, but, at many places, volume transmission seems to play an important role. Finally, although the available data are still limited, striking differences are observed in the spatiotemporal sequence of appearance of catecholaminergic cell groups, in particular those in the retina and olfactory bulb.

We analyzed the medaka optic tectum (OT) morphogenesis by using 5-bromo-2’deoxyuridine (BrdU) immunohistochemistry (with a new method we developed for pulselabeling embryos) and in situ hybridization with three probes, two for recently cloned homeobox genes (Ol-Prx3 [Paired-Related-Homeobox3] and Ol-Gsh1 [Genetic-ScreenHomeobox1]) and one for Ol-tailless. The tectal anlage first appears as a sheet of proliferating cells expressing Ol-Gsh1 and Ol-tailless but not Ol-Prx3. Cells subsequently cease to proliferate in a superficial and rostral zone and begin to express Ol-Prx3. When tectal lamination begins, the proliferative zone (mpz) becomes restricted to a crescent at the OT medial, caudal, and lateral margin. This mpz functions throughout the fish’s entire life. It produces cells that are added at the OT’s edge as radial rows, spanning every layer of the OT. The cells of the mpz continue to express Ol-tailless in the adult, whereas Ol-Gsh1 expression is turned off. When superficial layers form, Ol-Prx3 expression becomes restricted to the underlying deep layer, where it persists in the adult. Ol-Prx3 seems to be a marker for the differentiation of a subset of deep cells and allows analysis of tectal lamination, whereas Ol-tailless and Ol-Gsh1 could be involved in the control of tectal cell proliferation. This study constitutes a first step toward molecular approach to OT development in anamniotes. We compare and discuss the expression patterns of the homologs of the genes studied, and more generally the morphogenetic patterns of the medaka tectum, with those encountered in other cortical structures and in other vertebrate groups. J. Comp. Neurol. 413:385‐404, 1999. r 1999 Wiley-Liss, Inc.

Some anamniotic aquatic vertebrates lay eggs in a terrestrial habitat that is hostile to the survival of hatchings or larvae. These terrestrial eggs are ready and able to hatch at a particular developmental time, but do not hatch until presented with suitable conditions for aquatic larval survival. Beyond this time, hatching is possible whenever aquatic conditions occur. The duration of extended terrestrial incubation is dependent on the availability of energy for metabolism from the yolk. Extended incubation is useful for anamniotic eggs laid in terrestrial habitats where conditions suitable for larval survival arrive with unpredictable or variable timing. Examples of anamniotes with delayed hatching and extended terrestrial incubation can be found among teleost fishes, anurans, and caudate amphibians. This paper characterizes the embryonic period, compares this mode with other forms of developmental plasticity in anamniotes, evaluates the constraints and advantages of this life history mode, and examines how some fishes and amphibians are able to obtain the benefits of terrestriality for their eggs when the timing of the return to aquatic conditions is not entirely predictable.

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups.

Because our knowledge of cholinergic systems in the brains of amphibians is limited, the present study aimed to provide detailed information on the distribution of cholinergic cell bodies and fibers as revealed by immunohistochemistry with antibodies directed against the enzyme choline acetyltransferase (ChAT). To determine general and derived features of the cholinergic systems within the class of Amphibia, both anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians were studied. Distinct groups of ChAT-immunoreactive cell bodies were observed in the basal telencephalon, hypothalamus, habenula, isthmic nucleus, isthmic reticular formation, cranial nerve motor nuclei, and spinal cord. Prominent plexuses of cholinergic fibers were found in the olfactory bulb, pallium, basal telencephalon, ventral thalamus, tectum, and nucleus interpeduncularis. Comparison of these results with those obtained in other vertebrates, including a segmental approach to correlate cell populations, reveals that the cholinergic systems in amphibians share many features with amniotes. Thus, cholinergic pedunculopontine and laterodorsal tegmental nuclei could be identified in the amphibian brain. The finding of weakly immunoreactive cells in the striatum of Rana, which is in contrast with the condition found in Xenopus, Pleurodeles, and other anamniotes studied so far, has revived the notion that basal ganglia organization is more preserved during evolution than previously thought. J. Comp. Neurol. 382:499-534, 1997. © 1997 Wiley-Liss Inc.

In common with the embryos of other anamniotes, young of the Australian lungfish, Neoceratodus forsteri, have ciliated cells in the epidermis. These first appear at stage 28, ˜ 10 days before hatching, and develop progressively to a peak in numbers and in activity at stage 44, just after hatching. After this point, ciliary action in the epidermal cells slowly declines, and cilia disappear completely from the outer surface of the hatchling by stage 52. Cilia are lost earlier from the oral epithelium, between stages 45 and 46, and from the epithelium covering the gills and lining the operculum at stage 51, although they are retained in the nares and in the cavity of the olfactory organ. To assess possible functions for the ciliated epidermis in lungfish hatchlings, the presence of cilia in the epidermis of young N. forsteri is compared with landmarks of development. The ciliated epidermal cells are not associated with movements of the embryo within the egg capsule, nor are they a part of a feeding mechanism. They are not related to oxygen uptake. The ciliated epidermis appears to function as a mechanism for clearing the animal of particles and settling organisms before hatching, when the egg membranes have developed holes, and after hatching, when the young fish is living among the submerged rootlets of trees growing on the river bank or in dense stands of aquatic plants. The function of a ciliated epidermis in N. forsteri hatchlings in relation to microhabitat is discussed. © 1996 Wiley-Liss, Inc.

Previous studies have shown that Agr genes, which encode thioredoxin domain-containing secreted proteins, play a critical role in limb regeneration in salamanders. To determine the evolutionary conservation of Agr function, it is important to examine whether Agrs play a similar role in species with a different type of regeneration. Here, we refined the phylogeny of Agrs, revealing three subfamilies: Ag1, Agr2 and Agr3. Importantly, we established that Ag1 was lost in higher vertebrates, which correlates with their decreased regeneration ability. In Xenopus laevis tadpoles (anamniotes), which have all three Agr subfamilies and a high regenerating capacity, Agrs were activated in the stumps of tails and hindlimb buds that were amputated at stage 52. However, Agrs were not up-regulated when the hindlimb buds were amputated at stage 57, the stage at which their regeneration capacity is lost. Our findings indicate the general importance of Agrs for body appendages regeneration in amphibians.

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologues (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogues and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes.

This note presents a mechanistic explanation of the transition between the morphology of cephalochordates to that of amniotes. By a careful study of the morphogenetic movements which occur during the early stages of development of a typical amniote (a chicken embryo), we are able to show that the formation of a vertebrate body follows a sequence: first, formation of dorsal folds, then head and heart as dorsal and ventral folds, and finally another dorsal fold, which eventually builds up the chorion. This order has a physical origin linked to the velocity field of the tissue flow. These folds form at right angles to the flow direction, and the topology of the chordates flow is hyperbolic. This mechanism explains the differences between the successive bauplans, by the cumulate forward and backward movement of the flow. Eventually, the entire phenomenon can be described as a self-organized system of Russian dolls, by which the heart finds itself inside the embryo, and the embryo itself inside the chorion. In addition, the phenomenon has a mirror symmetry in the anterior and in the posterior part, thereby explaining naturally the existence of animals having a caudal heart.

Actin-myosin contraction has been shown to play a major role in early morphogenetic movements in Drosophila (fly) and Xenopus (frog) [1,2]. However, the specific role of actomyosin contractility in amniote embryos (reptiles, birds, and mammals) during primitive streak (PS) formation, the “organizing center” for gastrulation (formation of three primary germ layers), is not known. Current theories regarding primitive streak formation in higher order amniotes center around cell-cell intercalation or chemotactic cell movement [3,4]. We hypothesize that contraction via actin-myosin (AM) filaments is conserved from anamniotes and drives formation of the PS and the associated morphogenetic cell movements.Copyright © 2010 by ASME

Quantitative analyses based upon the superimposition of phylogenetic and reproductive data have revealed that viviparity has originated on at least 132 independent occasions among vertebrates, with 98 of these origins having occurred among reptiles. The viviparous lineages have given rise to at least 24 matrotrophic clades, all but four of which are anamniotes. Traditional scenarios assume progressive, gradualistic evolution from oviparity to lecithotrophic viviparity to matrotrophic viviparity. However, mammalian evidence indicates that matrotrophy can precede the evolution of viviparity. Moreover, data on reptiles seem to be consistent with a punctuated equilibrium model for viviparity and a saltatory model for incipient matrotrophy and placentation. Among the specializations for fetal nutrition, strong convergence is evident at organismal, organological, and cytological levels. Examples include yolk sac placentation, trophotaeniae, and adaptations for embryonic cannibalism. Certain lizards of the genera Mabuya and Chalcides have converged strongly on eutherian mammals with respect to morphology of the chorioallantoic placenta. Placental specializations that have evolved independently in some eutherians and matrotrophic lizards include placentomes, giant binucleate cells, deciduate maternal tissue, and chorionic areolae.

The axial motor column has undergone a major reorganization during the evolution of vertebrates. In aquatic anamniotes including lampreys, goldfish, and mudpuppies, epaxial and hypaxial motoneurons are intermingled in the column. In contrast, epaxial and hypaxial motoneurons are spatially segregated in water snakes, rats, and monkeys, apparently as a consequence of an isomorphic mapping of motoneuron location onto the position of innervated muscle in the embryonic myotome. The presence of these two very different arrangements of motoneurons requires a major restructuring of the motor column during vertebrate evolution. The time of this reorganization is unknown. All amniotes studied to date have an epaxial/hypaxial segregation, and all anamniotes do not, suggesting that the map arose with the origin of amniotes. All the anamniotes examined previously were permanently aquatic, however, and the map might therefore be associated with terrestrial locomotion. If so, we would expect terrestrial anamniotes to have an arrangement of motoneurons like that in amniotes. We studied the organization of motoneurons innervating the trunk muscles of postmetamorphic, terrestrial tiger salamanders and asked whether their motor columns are more like those of amniotes or those of aquatic anamniotes. The motor column in tiger salamanders is similar to that seen in aquatic anamniotes and very like that in mudpuppies--permanently aquatic salamanders. There are several classes of motoneurons with morphological similarities to the primary and secondary motoneurons characteristic of aquatic anamniotes. Epaxial and hypaxial motoneurons show no obvious morphological differences and occupy extensively overlapping positions in the motor column. The only epaxial/hypaxial distinction is the presence of a few, small, relatively undifferentiated motoneurons located subadjacent to the ependymal layer. These motoneurons are filled only by horseradish peroxidase (HRP) applied to hypaxial nerves. They are probably newly born motoneurons, and their presence suggests continued addition of motoneurons, even in adult salamanders. We conclude that the epaxial/hypaxial segregation seen in amniotes is not necessarily associated with terrestrial locomotion. The segregation and the topographic map it reflects may have arisen in conjunction with the origin of amniotes. If they instead arose prior to the origin of extant amphibians, they must have been secondarily lost in those salamanders studied to date. An examination of the motor column of other amphibians should help to resolve this issue.

The vertebrate brain is highly complex with millions to billions of neurons. During development, the neural plate border region gives rise to the neural crest, cranial placodes and, in anamniotes, to Rohon-Beard sensory neurons, whereas the boundary region of the midbrain and hindbrain develops organizer properties. Comparisons of developmental gene expression and neuroanatomy between vertebrates and the basal chordate amphioxus, which has only thousands of neurons and lacks a neural crest, most placodes and a midbrain-hindbrain organizer, indicate that these vertebrate features were built on a foundation already present in the ancestral chordate. Recent advances in genomics have provided insights into the elaboration of the molecular toolkit at the invertebrate-vertebrate transition that may have facilitated the evolution of these vertebrate characteristics.

To gain more insight into the dopaminergic system of amphibians and the evolution of catecholaminergic systems in vertebrates in general, the distribution of dopamine and tyrosine hydroxylase immunoreactivity was studied in the brains of the anuran Rana ridibunda and the urodele Pleurodeles waltlii. In both species, dopamine-immunoreactive (DAi) cell bodies were observed in the olfactory bulb, the preoptic area, the suprachiasmatic nucleus, the nucleus of the periventricular organ and its accompanying cells, the nucleus of the posterior tubercle, the pretectal area, the midbrain tegmentum, around the solitary tract, in the ependymal and subependymal layers along the midline of the caudal rhombencephalon, and ventral to the central canal of the spinal cord. Tyrosine hydroxylase (TH) immunohistochemistry revealed a similar pattern, although some differences were noted. For example, with the TH antibodies, additional cell bodies were stained in the internal granular layer of the olfactory bulb and in the isthmal region, whereas the same antibodies failed to stain the liquor contacting cells in the nucleus of the periventricular organ. Both antisera revealed an almost identical distribution of fibers in the two amphibian species. Remarkable differences were observed in the forebrain. Whereas the nucleus accumbens in Rana contains the densest DAi plexus, in Pleurodeles the dopaminergic innervation of the striatum prevails. Moreover, cortical structures of the newt contain numerous DAi fibers, whereas the corresponding structures in the frog are devoid of immunoreactivity. The dopaminergic system in amphibians appears to share many features not only with other anamniotes but also with amniotes.

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore- and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step-by-step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore- and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.

The dorsomedial telencephalon of lepidosirenid lungfishes has been interpreted in two divergent ways: earlier investigators regarded it as a subpallial (septal) structure; more recently, it has been reinterpreted as the medial pallium (hippocampus). To resolve this question, we identified parameters that are conclusive in their association with either the medial pallium or the septum in anamniotes. The present study examines the position of ependymal thickenings and the distribution of acetylcholinesterase (AchE) in the cerebral hemispheres of the African lungfish Protopterus, the Australian lungfish Neoceratodus, and the amphibian species Xenopus and Ambystoma. In addition, projections from the hypothalamus (paraventricular organ) to the telencephalon are investigated in Protopterus. Ependymal specializations are located dorsally and ventrally in the lateral ventricles of amphibians, but laterally and medially in lungfishes. In Protopterus, the paraventricular organ projects to the medial telencephalic hemisphere, but not to the dorsal roof. High levels of AchE are present in restricted neuropil regions of the medial hemisphere and in the ventral and ventrolateral telencephalon, but they are lacking in the dorsal roof. Intensely AchE-stained neuronal cell bodies are located in the ventral telencephalon (rostrally) and the dorsomedial telencephalon (at mid-level). In Neoceratodus, AchE staining is pronounced in the septal area, but absent in the pallium. The terminal nerve proper lacks AchE stain in Protopterus; nerve fibres of the preoptic nerve are AchE-positive in both lungfish species. In Xenopus, AchE staining of fibers and terminals is restricted to the subpallium (medial septum, tuberculum olfactorium, striatum, nucleus accumbens, and medial amygdala); cell bodies are AchE positive in parts of the subpallium and rostral pallium. Comparison of cytological, histochemical, and "connectional" parameters substantiates the interpretation that the dorsomedial telencephalon of lungfishes represents a subpallial, but not a "medial pallial" structure. The dorsomedial part of the lepidosirenid telencephalon corresponds to the septum in the most plesiomorphic living lungfish, Neoceratodus forsteri, but it differs considerably from the dorsomedial telencephalon (medial pallium) in amphibians.