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New radiocarbon dates on biological remains from cave deposits in the Canadian Rocky Mountains range in age from infinite to modern but are largely restricted to the Holocene (9560[+ ou -]40 years B.P. to modern). The consistency in radiocarbon ages is interpreted as resulting from geologic processes associated with deglaciation and may reflect timeframes when cave conditions in some areas of the Canadian Rocky Mountains became suitable for deposition and preservation of biological remains. When considered at taxonomic levels permitted by morphological identification, recovered vertebrate remains from cave deposits are consistent with the modern biota of the region, suggesting early post-Late Glacial Maximum establishment of the modern biota and relative stability of mammal assemblages through the Holocene. Preliminary evaluation of recovered plant remains highlights a potentially underevaluated source of palaeoecological data for higher elevations. Key words: Mammalia, chronology, taphonomy, Holocene, Pleistocene De nouveaux âges au radiocarbone sur des restes biologiques provenant de dépôts de caverne dans les Rocheuses canadiennes vont d'infinis à modernes mais sont pour la plupart restreints à l'Holocène (9560[+ ou -]40 années BP à modernes). La cohérence des âges au radiocarbone est interprétée comme étant le résultat de processus géologiques associés à la déglaciation et pourrait refléter des périodes durant lesquelles les conditions dans les cavernes de certains secteurs des Rocheuses canadiennes se prêtaient au dépôt et à la préservation de restes biologiques. Aux niveaux taxonomiques que permet l'identification morphologique, les restes de vertébrés récupérés de dépôts de caverne concordent avec le biote moderne de la région, ce qui indiquerait un établissement de ce dernier tôt après le dernier maximum glaciaire et une stabilité relative des assemblages mammaliens durant l'Holocène. L'évaluation préliminaire de restes de plantes récupérés fait ressortir une source potentiellement sous-évaluée de données paléoécologiques pour les hautes altitudes. [Traduit par la Rédaction] Mots-clés: mammifères, chronologie, taphonomie, Holocène, Pléistocène, Introduction Quaternary palaeontological deposits preserved in caves are a significant data source for the reconstruction of past biotas (Harris 2005; Jass and George 2010; Harington 2011). Caves are especially significant [...]

The transition between the Devonian and Carboniferous is important for tetrapod vertebrates. By the end of the Devonian the first limbs are present in aquatic animals, and by the mid Carboniferous fully terrestrial tetrapods have diversified. Knowledge of the fin-to-limb transition is sparse because few fossils from the earliest Carboniferous (Tournaisian) are known. Blue Beach Nova Scotia, in addition to a small number of sites in Scotland and Australia, is an exception to this global trend. Previous reports from Blue Beach identified fossils from a variety of Devonian-like and Carboniferous-like tetrapod body forms, making it a valuable site for studying the fin-to-limb transition. Here we report on a new left fibula from Blue Beach, which we attribute to the later occurring Visean-aged (early Carboniferous) Crassigyrinidae. Recent investigations of deposits in Scotland, similar in age to the Tournaisian exposed at Blue Beach, have found Crassigyrinus-like elements as well, reinforcing this 20-million-year lineage extension. Key words: fin-to-limb, Carboniferous, Crassigyrinidae, early tetrapod, vertebrate evolution, Romer's Gap. La transition du Dévonien au Carbonifère est importante en ce qui concerne les vertébrés tétrapodes. À la fin du Dévonien, les premières pattes sont présentes chez des animaux aquatiques, et une diversification de tétrapodes entièrement terrestres a déjà eu lieu au milieu du Carbonifère. Les connaissances relatives au passage de nageoires à des pattes sont limitées parce qu'il existe peu de fossiles connus du tout début du Carbonifère (Tournaisien). Blue Beach, en Nouvelle-Écosse, en plus d'un petit nombre de sites en Écosse et en Australie, constitue une exception à cette tendance planétaire. Des signalements antérieurs de Blue Beach ont relevé des fossiles issus de diverses formes de corps semblables à celles de tétrapodes dévoniens et carbonifères, ce qui en fait un site de grande valeur pour l'étude de la transition des nageoires aux pattes. Nous faisons état d'un nouveau péroné gauche de Blue Beach que nous attribuons aux crassigyrinidés plus tardifs, d'âge viséen (Carbonifère précoce). Des études récentes de dépôts en Écosse d'âge semblable aux dépôts tournaisiens exposés à Blue Beach ont relevé des éléments s'apparentant à Crassigyrinus, ce qui appuie le prolongement de 20 millions d'années de la lignée. [Traduit par la Rédaction] Mots-clés : transition des nageoires aux pattes, Carbonifère, crassigyrinidés, tétrapode précoce, évolution des vertébrés, lacune de Romer., Introduction Crassigyrinus scoticus is an unusual tetrapod first described from the Visean (early Carboniferous) and Namurian (late Carboniferous; Panchen 1985; Panchen and Smithson 1990). The holotype, described by Lydekker (1890), [...]

The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century (1). These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate 'new head' (2,3). A previous study suggests that rudimentary neural crest cells existed in ancestral chordates (4). However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates (5). Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates., Neurogenic placodes contribute to cranial sensory systems that mediate hearing, smell and taste (2). They are considered a vertebrate innovation (6) and the full repertoire, including olfactory, otic, epibranchial and [...]

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts--both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates., In their influential 1983 paper, Gans and Northcutt (1) proposed that early vertebrates evolved from invertebrates principally through innovations in the head. These include the muscularization of the ventrolateral mesoderm, [...]

The construction of the vertebral column has been used as a key anatomical character in defining and diagnosing early tetrapod groups (1). Rhachitomous vertebrae2--in which there is a dorsally placed neural arch and spine, an anteroventrally placed intercentrum and paired, posterodorsally placed pleurocentra--have long been considered the ancestral morphology for tetrapods (13-6). Nonetheless, very little is known about vertebral anatomy in the earliest stem tetrapods, because most specimens remain trapped in surrounding matrix, obscuring important anatomical features (7-9). Here we describe the three-dimensional vertebral architecture of the Late Devonian stem tetrapod Ichthyostega using propagation phase-contrast X-ray synchrotron microtomography. Our scans reveal a diverse array of new morphological, and associated developmental and functional, characteristics, including a possible posterior-to-anterior vertebral ossification sequence and the first evolutionary appearance of ossified sternal elements. One of the most intriguing features relates to the positional relationships between the vertebral elements, with the pleurocentra being unexpectedly sutured or fused to the intercentra that directly succeed them, indicating a 'reverse' rhachitomous design (10). Comparison of Ichthyostega with two other stem tetrapods, Acanthostega (7) and Pederpes (8), shows that reverse rhachitomous vertebrae may be the ancestral condition for limbed vertebrates. This study fundamentally revises our current understanding (11) of vertebral column evolution in the earliest tetrapods and raises questions about the presumed vertebral architecture of tetrapodomorph fish (12,13) and later, more crownward, tetrapods., Jarvik (3,14,15) reassembled the vertebral column of the Devonian stem tetrapod Ichthyostega from 'imperfectly' preserved material. Each vertebral unit was thought to consist of a relatively straight neural spine; paired, [...]

Oligodendrocytes and astrocytes are macroglial cells of the vertebrate central nervous system. These cells have diverse roles in the maintenance of neurological function. In the embryo, the genetic mechanisms that underlie the specification of macroglial precursors in vivo appear strikingly similar to those that regulate the development of the diverse neuron types. The switch from producing neuronal to glial subtype-specific precursors can be modelled as an interplay between region-restricted components and temporal regulators that determine neurogenic or gliogenic phases of development, contributing to glial diversity. Gaining insight into the developmental genetics of macroglia has great potential to improve our understanding of a variety of neurological disorders in humans., Glia make up 10-20% of the cells in the Drosophila nervous system and at least 50% of the cells in the human brain. These findings indicate that glial-cell function is [...]

In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu--Gli complexes to cilia, suggesting a model in which Smo activates Gli by relieving inhibition by SuFu. In support of this model, we find that Hh causes rapid dissociation of the SuFu--Gli complex, thus allowing Gli to enter the nucleus and activate transcription. Activation of protein kinase A (PKA), an inhibitor of Hh signaling, blocks ciliary localization of SuFu--Gli complexes, which in turn prevents their dissociation by signaling. Our results support a simple mechanism in which Hh signals at vertebrate cilia cause dissociation of inactive SuFu--Gli complexes, a process inhibited by PKA. doi/ 10.1083/jcb.201004108

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by A6 desaturation of 18:2n- 6 to produce 18:3n- 6 that is elongated to 20:3n- 6 followed by A5 desaturation. Synthesis of EPA from 18:3n- 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second [DELTA]6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional [DELTA]6/[DELTA]5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22: 5n--3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having [DELTA]4 Fad activity. This is a unique report of a Fad with [DELTA]4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22: 5n--3 followed by [DELTA]4 desaturation, as well as the more complicated pathway as described above. [DELTA]4 desaturase I bifunctional [DELTA]6/[DELTA]5 desaturase | polyunsaturated fatty acid biosynthesis | Siganus canaliculatus | teleost doi/ 10.1073/pnas.1008429107

The formation of primitive (embryonic) blood in vertebrates is mediated by spatio-temporally restricted signaling between different tissue layers. In Xenopus, in which primitive blood originates in the ventral blood island, this involves the secretion of bone morphogenetic protein (BMP) ligands by the ectoderm that signal to the underlying mesoderm during gastrulation. Using novel transgenic reporter lines, we report that the canonical Wnt/[beta]-catenin pathway is also activated in the blood islands in Xenopus. Furthermore, Wnt-reporter activity was also detected in the blood islands of the mouse yolk sac. By using morpholino-mediated depletion in Xenopus, we identified Wnt4 as the ligand that is expressed in the mesoderm of the ventral blood island and is essential for the expression of hematopoietic and erythroid marker genes. Injection of an inducible Wnt-interfering construct further showed that, during gastrulation, Wnt/[beta]-catenin signaling is required both in the mesoderm and in the overlying ectoderm for the formation of the ventral blood island. Using recombination assays with embryonic explants, we document that ectodermal BMP4 expression is dependent on Wnt4 signals from the mesoderm. Our results thus reveal a unique role for Wnt4-mediated canonical signaling in the formation and maintenance of the ventral blood island in Xenopus. Xenopus | ventral blood islands | Wnt reporter | bone morphogenetic protein I destabilized GFP www.pnas.org/cgi/doi/ 10.1073/pnas.1007725107

Natural selection often promotes evolutionary innovation by coopting preexisting genes for new functions, and this process may be greatly facilitated by gene duplication. Here we report an example of cooptive convergence where paralogous members of the globin gene superfamily independently evolved a specialized [O.sub.2] transport function in the two deepest branches of the vertebrate family tree. Specifically, phylogenetic evidence demonstrates that erythroid-specific [O.sub.2] transport hemoglobins evolved independently from different ancestral precursor proteins in jawed vertebrates (gnathostomes) and jawless fish (cyclostomes, represented by lamprey and hagfish). A comprehensive phylogenetic analysis of the vertebrate globin gene superfamily revealed that the erythroid hemoglobins of cyclostomes are orthologous to the cytoglobin protein of gnathostome vertebrates, a hexacoordinate globin that has no [O.sub.2] transport function and that is predominantly expressed in fibroblasts and related cell types. The phylogeny reconstruction also revealed that vertebrate-specific globins are grouped into four main clades: (i) cyclostome hemoglobin + cytoglobin, (ii) myoglobin + globin E, (iii) globin Y, and (iv) the [alpha]- and [beta]-chain hemoglobins of gnathostomes. In the hemoglobins of gnathostomes and cyclostomes, multisubunit quaternary structures provide the basis for cooperative [O.sub.2] binding and allosteric regulation by coupling the effects of ligand binding at individual subunits with interactions between subunits. However, differences in numerous structural details belie their independent origins. This example of convergent evolution of protein function provides an impressive demonstration of the ability of natural selection to cobble together complex design solutions by tinkering with different variations of the same basic protein scaffold. cytoglobin | gene family evolution | globin | hagfish | lamprey doi/ 10.1073/pnas.1006756107

Expression of Sonic Hedgehog (Shh) in the posterior mesenchyme of the developing limb bud regulates patterning and growth of the developing limb by activation of the Hedgehog (Hh) signaling pathway. Through the analysis of Shh and Hh signaling target genes, it has been shown that activation in the limb bud mesoderm is required for normal limb development to occur, in contrast, it has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. However, recent array-based data identified both the components necessary to activate the Hh signaling pathway and targets of this pathway in the limb bud ectoderm. Using immunohistochemistry and various methods of detection for targets of Hh signaling, we found that SHH protein and targets of Hh signaling are present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning, we removed Smo, an essential component of the Hh signaling pathway, from the apical ectodermal ridge (AER). Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional postaxial cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development. limb patterning | polydactyly | Shh signaling www.pnas.org/cgi/doi/10.1073/pnas.0912818107

The Metabolic Theory of Ecology (MTE) posits that the temperature-dependent kinetics of metabolism shape broad-scale patterns of biodiversity. Here we test whether the MTE accounts for patterns of diversity using 102 elevational diversity gradients of reptiles and amphibians. In particular, we examined the support for the two key predictions of the MTE: that the reciprocal of absolute temperature (1/k T) and diversity are linearly related and that the slope of that relationship is -0.65. We also tested two underlying assumptions of the MTE in cases with appropriate data, namely, that abundance is invariant among samples, and that behavioral thermoregulation influences the MTE predictions. We found that few studies supported the predictions of the MTE for the relationship between environmental temperature and elevational diversity using previous methods on individual gradients and using metaanalysis. The predominant relationship was curvilinear, and the slopes were steeper than predicted. In analyses of individual gradients, only 6% followed the MTE predictions in the strictest application, and 25% in the broadest. We found violations of the assumption of invariant abundances in all five test cases. All four herpetofaunal groups, regardless of behavioral thermoregulatory abilities, demonstrated poor fits to the MTE predictions. Even when arid gradients are removed, ameliorating the potential effects of water limitation, the MTE did not account for herpetofaunal elevational diversity. We conclude that an interplay of factors shapes elevational diversity gradients rather than the simple kinetics of biochemical reactions. Key words: abundance; amphibians; mountains; reptiles; species richness; temperature.

Synopsis Vertebrates inhabit and communicate acoustically in most natural environments. We review the influence of environmental factors on the hearing sensitivity of terrestrial vertebrates, and on the anatomy and mechanics of the middle ears. Evidence suggests that both biotic and abiotic environmental factors affect the evolution of bandwidth and frequency of peak sensitivity of the hearing spectrum. Relevant abiotic factors include medium type, temperature, and noise produced by nonliving sources. Biotic factors include heterospecific, conspecific, or self-produced sounds that animals are selected to recognize, and acoustic interference by sounds that other animals generate. Within each class of tetrapods, the size of the middle ear structures correlates directly to body size and inversely to frequency of peak sensitivity. Adaptation to the underwater medium in cetaceans involved reorganization of the middle ear for novel acoustic pathways, whereas adaptation to subterranean life in several mammals resulted in hypertrophy of the middle ear ossicles to enhance their inertial mass for detection of seismic vibrations. The comparative approach has revealed a number of generalities about the effect of environmental factors on hearing performance and middle ear structure across species. The current taxonomic sampling of the major tetrapod groups is still highly unbalanced and incomplete. Future expansion of the comparative evidence should continue to reveal general patterns and novel mechanisms. doi: 10.1093/icb/icp088

The uneven distribution of species richness is a fundamental and unexplained pattern of vertebrate biodiversity. Although species richness in groups like mammals, birds, or teleost fishes is often attributed to accelerated cladogenesis, we lack a quantitative conceptual framework for identifying and comparing the exceptional changes of tempo in vertebrate evolutionary history. We develop MEDUSA, a stepwise approach based upon the Akaike information criterion for detecting multiple shifts in birth and death rates on an incompletely resolved phylogeny. We apply MEDUSA incompletely to a diversity tree summarizing both evolutionary relationships and species richness of 44 major clades of jawed vertebrates. We identify 9 major changes in the tempo of gnathostome diversification; the most significant of these lies at the base of a clade that includes most of the coral-reef associated fishes as well as cichlids and perches. Rate increases also underlie several well recognized tetrapod radiations, including most modern birds, lizards and snakes, ostariophysan fishes, and most eutherian mammals. In addition, we find that large sections of the vertebrate tree exhibit nearly equal rates of origination and extinction, providing some of the first evidence from molecular data for the importance of faunal turnover in shaping biodiversity. Together, these results reveal living vertebrate biodiversity to be the product of volatile turnover punctuated by 6 accelerations responsible for >85% of all species as well as 3 slowdowns that have produced 'living fossils.' In addition, by revealing the timing of the exceptional pulses of vertebrate diversification as well as the clades that experience them, our diversity tree provides a framework for evaluating particular causal hypotheses of vertebrate radiations. evolutionary radiation | macroevolution | phylogeny

Eukaryotic gene expression is commonly controlled at the level of RNA polymerase II (RNAPII) pausing subsequent to transcription initiation. Transcription elongation is stimulated by the positive transcription elongation factor b (P-TEFb) kinase, which is suppressed within the 7SK small nuclear ribonucleoprotein (7SK snRNP). However, the biogenesis and functional significance of 7SK snRNP remain poorly understood. Here, we report that LARP7, BCDIN3, and the noncoding 7SK small nuclear RNA (7SK) are vital for the formation and stability of a cell stress-resistant core 7SK snRNP. Our functional studies demonstrate that 7SK snRNP is not only critical for controlling transcription elongation, but also for regulating alternative splicing of pre-mRNAs. Using a transient expression splicing assay, we find that 7SK snRNP disintegration promotes inclusion of an alternative exon via the increased occupancy of P-TEFb, Ser2-phosphorylated (Ser2-P) RNAPII, and the splicing factor SF2/ASF at the minigene. Importantly, knockdown of larp7 or bcdin3 orthologues in zebrafish embryos destabilizes 7SK and causes severe developmental defects and aberrant splicing of analyzed transcripts. These findings reveal a key role for P-TEFb in coupling transcription elongation with alternative splicing, and suggest that maintaining core 7SK snRNP is essential for vertebrate development. LARP7 | BCDIN3 | MePCE | HEXIM1 | SF2/ASF

Ichthyostega and Aconthostega are the earliest tetrapods known from multiple near-complete skeletons, with Acanthostega generally considered the more primitive. New material indicates differing ontogenetic trajectories for their forelimbs: In Ichthyostega, the pattern of muscle attachment processes on small humeri (upper arm bones) resembles that in 'fish' members of the tetrapod stem group such as Tiktaalik, whereas large humeri approach (but fair to attain) the tetrapod crown-group condition; in Aconthostega, both smart and large humeri exhibit the crown-group pattern. We infer that Ichthyostega underwent greater locomotory terrestrialization during ontogeny. The newly recognized primitive characteristics also suggest that Ichthyostega could be phylogenetically more basal than Acanthostega.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase I[epsilon]/[delta] (CKI[epsilon]/[delta]) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKI[epsilon]/[delta]. Moreover, expression of either Drosophila DBT or the vertebrate CKI[delta] kinase carrying the Drosophila [dbt.sup.S] or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKI[delta] carrying the [dbt.sup.L] mutation does not lengthen circadian rhythms, while Drosophila [DBT.sup.L] does. Different effects of the [dbt.sup.S] and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKI[delta] and of the functions affected by their period-shortening mutations.

Because of its basal position on the phylogenetic tree of vertebrates, the lamprey embryo would be expected to exhibit segmental head mesoderm. Recent observations, however, show that the lamprey does not have any somite-like segments in the head. Coelomic head cavities that are most conspicuous in elasmobranch embryos, do not appear to represent universal vertebrate traits. From the perspective of generative constraint, segmental structures in the vertebrate body can be classified into primary segments, which arise as segmental embryonic primordia such as somites and pharyngeal pouches, and secondary segments whose patterns are determined by the presence of primary segments. Secondary segments include neural crest derivatives and epibranchial placodes that are not initially segmented. The head mesoderm of vertebrates is secondarily regionalized into several domains that do not impose any secondary segmental patterns on other structures. Thus, the vertebrate head is characterized by a lack of segmental generative constraint in its mesoderm. Classical segmental theories are now refuted because they attempted to equate the vertebrate head with that of the amphioxus, whose rostral somites are considered primary segments, which are absent from vertebrates.

In most versions of theories of the segmentation of the vertebrate head, a premandibular segment is present rostral to the jaw-forming mandibular segment. These theories posit that in ancient fishes this segment included a gill and a gill-supporting skeleton, which then was modified to support the anterior brain. However, we find no recent evidence for existence of such a premandibular segment. Rather, new findings from studies of fate mapping and gene expression show that the 'premandibular' territory is in fact the maxillary region of the mandibular arch. A signaling cascade, beginning with dorsal midline mesoderm in the gastrula and relayed through neural ectoderm and then oral ectoderm, greatly expands the skeletal derivatives of maxillary neural crest in a manner fully consistent with the Gans--Northcutt theory of the vertebrate new head.

Segmentation of the vertebrate head emerges out of earlier processes that establish the anterior-posterior (A-P) axis. Recent genetic studies and comparisons across species have led to a better understanding of the links between A-P patterning and segmentation. These point to similar signals acting on both head and trunk, such as retinoic acid and fibroblast growth factors. These form interacting networks of diffusible morphogen gradients that pattern both hindbrain rhombomeres and mesodermal somites. New computational models, particularly for retinoic acid, have revealed how morphogen gradients are established and made robust to changes in signaling levels. However, the orientations of these gradients, as well as how they interact to generate segments, differ remarkably between germ layers and body regions. Thus, the vertebrate head is, in part, built through modifications of the same processes that link A-P patterning and segmentation in the trunk, but fundamental differences in how these processes are deployed lend further doubt to the notion that head and trunk segments are homologous.

The morphology of the vertebrate head is extremely complex and comprises numerous iterative structures that arise from each of the embryonic germ layers. The search for a fundamental plan uniting all of these serial structures spans ~200 years. The earliest attempt to identify a common plan was J. W. Goethe's vertebral theory of skull organization, in which the skull was interpreted as being formed by a series of trunk vertebrae. This theory was rejected by T. H. Huxley in the 1858 Croonian Lecture and was replaced by the segmented mesodermal model of Francis Balfour, which was elaborated subsequently by A. Marshall, Gavin de Beer, and Edwin Goodrich. This model assumes that the head of the earliest vertebrates consisted of eight segments. It further assumes that each segment contained dorsal muscles arising from the somitic mesoderm, and ventral muscles arising from lateral plate mesoderm, except for the first segment, which lacked ventral muscles derived from the lateral plate mesoderm. The muscles of each head segment were believed to be innervated by two pairs of cranial nerves, homologous to the dorsal and ventral spinal nerves of lampreys. The validity of this theory, known as the Goodrich model, came into question, however, after the discovery that the branchiomeric muscles associated with each pharyngeal arch do not arise from lateral plate mesoderm, as initially proposed by Marshall and subsequently accepted by Goodrich and de Beer, but, rather, arise from paraxial mesoderm. Furthermore, segmentation of the brain into some 14 neuromeres cannot be accommodated by any model involving eight segments. Finally, there is also clear evidence that at least one, if not two, additional series of placodally derived sensory nerves occurs in the head and has no counterpart in the trunk. At present, there is no theory of segmentation that can account for all cephalic iterative structures.

The fossil record has been an invaluable aid for reconstructing the major events of vertebrate evolution. There is no comparable record for protochordates, however, which severely limits our knowledge of their ancestral morphology, habits, and mode of life. The alternative is inference based on an interpretation of living protochordates but this is fraught with problems, not least being our own biases of what we think an ancestral chordate ought to look like. Relevant to the present symposium is the problem of head/trunk relationships and whether or not the myotomes of the trunk originally extended into the head in vertebrates. I will review what is currently known of patterns of innervation in tunicates and amphioxus in relation to Romer's somaticovisceral concept of the vertebrate body to show how little progress has been made in resolving this problem. There are, in contrast, surprisingly good prospects for solving some other puzzles concerning chordate origins. Dorsoventral inversion provides a good example. A consensus is now emerging, based largely on molecular data from hemichordates that casts new light on the asymmetry of the head in amphioxus. Specifically, the morphogenetic growth process that reestablishes symmetry in late-stage larvae can now be seen, at least in part, as a recapitulation of past evolutionary events, and this has important implications for the origin and basic organization of the brain.

Classic theories of vertebrate head segmentation clearly exemplify the idealistic nature of comparative embryology prior to the 20th century. Comparative embryology aimed at recognizing the basic, primary structure that is shared by all vertebrates, either as an archetype or an ancestral developmental pattern. Modern evolutionary developmental (Evo-Devo) studies are also based on comparison, and therefore have a tendency to reduce complex embryonic anatomy into overly simplified patterns. Here again, a basic segmental plan for the head has been sought among chordates. We convened a symposium that brought together leading researchers dealing with this problem, in a number of different evolutionary and developmental contexts. Here we give an overview of the outcome and the status of the field in this modern era of Evo-Devo. We emphasize the fact that the head segmentation problem is not fully resolved, and we discuss new directions in the search for hints for a way out of this maze.

The phylogenetic relationships of Ig light chain (IGL) genes are difficult to resolve, because these genes are short and evolve relatively fast. Here, we classify the IGL sequences from 12 tetrapod species into three distinct groups ([kappa], [lambda], and [sigma] isotypes) using conserved amino acid residues, recombination signal sequences, and genomic organization of IGL genes as cladistic markers. From the distribution of the markers we conclude that the earliest extant tetrapods, the amphibians, possess three IGL isotypes: [kappa], [lambda], and [sigma]. Of these, two ([kappa] and [lambda]) are also found in reptiles and some mammals. The [lambda] isotype is found in all tetrapods tested to date, whereas the [kappa] isotype seems to have been lost at least in some birds and in the microbat. Conservation of the cladistic molecular markers suggests that they are associated with functional specialization of the three IGL isotypes. The genomic maps of IGL loci reveal multiple gene rearrangements that occurred in the evolution of tetrapod species. These rearrangements have resulted in interspecific variation of the genomic lengths of the IGL loci and the number and order of IGL constituent genes, but the overall organization of the IGL loci has not changed. cladistic molecular markers | immunoglobulin [kappa], [lambda], and [sigma] chain genes | genomic organization | recombination signal sequence

The opioid peptides and receptors have prominent roles in pain transmission and reward mechanisms in mammals. The evolution of the opioid receptors has so far been little studied, with only a few reports on species other than tetrapods. We have investigated species representing a broader range of vertebrates and found that the four opioid receptor types (delta, kappa, mu, and NOP) are present in most of the species. The gene relationships were deduced by using both phylogenetic analyses and chromosomal location relative to 20 neighboring gene families in databases of assembled genomes. The combined results show that the vertebrate opioid receptor gene family arose by quadruplication of a large chromosomal block containing at least 14 other gene families. The quadruplication seems to coincide with, and, therefore, probably resulted from, the two proposed genome duplications in early vertebrate evolution. We conclude that the quartet of opioid receptors was already present at the origin of jawed vertebrates [approximately equal to] 450 million years ago. A few additional opioid receptor gene duplications have occurred in bony fishes. Interestingly, the ancestral receptor gene duplications coincide with the origin of the four opioid peptide precursor genes. Thus, the complete vertebrate opioid system was already established in the first jawed vertebrates. chromosome | G protein-coupled receptor | gene duplication

Steroid hormones are essential for the normal function of most organ systems in vertebrates. Reproductive activities in females and males, such as the differentiation, growth and maintenance of the reproductive system, require signaling by sex steroid hormones. Although extensively studied in mammals and a few fish and bird species, the evolution and molecular mechanisms associated with the nuclear steroid hormone receptors are still poorly understood in amphibians and reptiles. Given our interest in environmental signaling of sex determination as well as a major interest in environmental contaminants that can mimic steroid hormone signaling, we have established an approach to study the molecular function (ligand binding and trans-activation) of steroid hormone receptors cloned from reptiles. This approach involves molecular cloning and sequencing of steroid hormone receptors, phylogenic analysis and in vitro transactivation assays using endogenous or exogenous ligands. Comparing the in vitro trans-activation induced by different ligands with receptors cloned from different species would develop additional functional relationships (classification) among steroid hormone receptors. This approach can provide insight into understanding why each species could have different responses to exogenous ligands. Further, we have developed a novel and less invasive approach to obtaining mRNA for molecular cloning and sequencing of steroid hormone receptors in reptiles and other non-mammalian species, using blood cells as a source of genetic material. For example, white blood cells (WBCs) and red blood cells (RBCs) of the American alligator both express steroid hormone receptors and have adequate amounts of mRNA for molecular cloning. This approach would allow us to analyze components of endocrine function of steroid hormones without sacrificing animals. Especially in endangered species, this approach could provide an understanding of endocrine functions, elucidate the phylogenic relationships of various receptors in vitro, such as the steroid hormone receptors, and determine possible effects of environmental contaminants in a minimally invasive manner.

Vertebrate vision is mediated by five groups of visual pigments, each absorbing a specific wavelength of light between ultraviolet and red. Despite extensive mutagenesis analyses, the mechanisms by which contemporary pigments absorb variable wavelengths of light are poorly understood. We show that the molecular basis of the spectral tuning of contemporary visual pigments can be illuminated only by mutagenesis analyses using ancestral pigments. Following this new principle, we derive the 'five-sites' rule that explains the absorption spectra of red and green (M/LWS) pigments that range from 510 to 560 nm. Our findings demonstrate that the evolutionary method should be used in elucidating the mechanisms of spectral tuning of four other pigment groups and, for that matter, functional differentiations of any other proteins.

The [approximately equal to]28,300 species of tetrapods (four-limbed vertebrates) almost exclusively have perennial life spans. Here, we report the discovery of a remarkable annual tetrapod from the arid southwest of Madagascar: the chameleon Furcifer labordi, with a posthatching life span of just 4-5 months. At the start of the active season (November), an age cohort of hatchlings emerges; larger juveniles or adults are not present. These hatchlings grow rapidly, reach sexual maturity in less than 2 months, and reproduce in January-February. After reproduction, senescence appears, and the active season concludes with population-wide adult death. Consequently, during the dry season, the entire population is represented by developing eggs that incubate for 8-9 months before synchronously hatching at the onset of the following rainy season. Remarkably, this chameleon spends more of its short annual life cycle inside the egg than outside of it. Our review of tetrapod longevity (>1,700 species) finds no others with such a short life span. These findings suggest that the notorious rapid death of chameleons in captivity may, for some species, actually represent the natural adult life span. Consequently, a new appraisal may be warranted concerning the viability of chameleon breeding programs, which could have special significance for species of conservation concern. Additionally, because F. labordi is closely related to other perennial species, this chameleon group may prove also to be especially well suited for comparative studies that focus on life history evolution and the ecological, genetic, and/or hormonal determinants of aging, longevity, and senescence. Madagascar | lizard | longevity | semelparity | senescence

Cartilaginous fishes are the oldest living phylogenetic group of jawed vertebrates. Here, we demonstrate the value of cartilaginous fish sequences in reconstructing the evolutionary history of vertebrate genomes by sequencing the protocadherin cluster in the relatively small genome (910 Mb) of the elephant shark (Callorhinchus milii). Human and coelacanth contain a single protocadherin cluster with 53 and 49 genes, respectively, that are organized in three subclusters, Pcdh[alpha], Pcdh[beta], and Pcdh[gamma] whereas the duplicated protocadherin clusters in fugu and zebrafish contain >77 and 107 genes, respectively, that are organized in Pcdh[alpha] and Pcdh[gamma] subclusters. By contrast, the elephant shark contains a single protocadherin cluster with 47 genes organized in four subclusters (Pcdh[delta], Pcdh[epsilon], Pcdh[micro], and Pcdhv). By comparison with elephant shark sequences, we discovered a Pcdh[delta] subcluster in teleost fishes, coelacanth, Xenopus, and chicken. Our results suggest that the protocadherin cluster in the ancestral jawed vertebrate contained more subclusters than modern vertebrates, and the evolution of the protocadherin cluster is characterized by lineagespecific differential loss of entire subclusters of genes. In contrast to teleost fish and mammalian protocadherin genes that have undergone gene conversion events, elephant shark protocadherin genes have experienced very little gene conversion. The syntenic block of genes in the elephant shark protocadherin locus is well conserved in human but disrupted in fugu. Thus, the elephant shark genome appears to be less prone to rearrangements compared with teleost fish genomes. The small and 'stable' genome of the elephant shark is a valuable reference for understanding the evolution of vertebrate genomes. ancestral jawed vertebrate | clustered protocadherins | cartilaginous fish | Callorhinchus milii | gene conversion

The sensing of blood gas tensions and/or pH is an evolutionarily conserved, homeostatic mechanism, observable in almost all species studied from invertebrates to man. In vertebrates, a shift from the peripheral [O.sub.2]-oriented sensing in fish, to the central C[O.sub.2]/pH sensing in most tetrapods reflects the specific behavioral requirements of these two groups whereby, in teleost fish, a highly [O.sub.2]-oriented control of breathing matches the ever-changing and low oxygen levels in water, whilst the transition to air-breathing increased the importance of acid-base regulation and [O.sub.2]-related drive, although retained, became relatively less important. The South American lungfish and tetrapods are probably sister groups, a conclusion backed up by many similar features of respiratory control. For example, the relative roles of peripheral and central chemoreceptors are present both in the lungfish and in land vertebrates. In both groups, the central C[O.sub.2]/pH receptors dominate the ventilatory response to hypercarbia (60-80%), while the peripheral C[O.sub.2]/pH receptors account for 20-30%. Some basic components of respiratory control have changed little during evolution. This review presents studies that reflect the current trends in the field of chemoreceptor function, and several laboratories are involved. An exhaustive review on the previous literature, however, is beyond the intended scope of the article. Rather, we present examples of current trends in respiratory function in vertebrates, ranging from fish to humans, and focus on both [O.sub.2] sensing and C[O.sub.2] sensing. As well, we consider the impact of chronic levels of hypoxia--a physiological condition in fish and in land vertebrates resident at high elevations or suffering from one of the many cardiorespiratory disease states that predispose an animal to impaired ventilation or cardiac output. This provides a basis for a comparative physiology that is informative about the evolution of respiratory functions in vertebrates and about human disease. Currently, most detail is known for mammals, for which molecular biology and respiratory physiology have combined in the discovery of the mechanisms underlying the responses of respiratory chemoreceptors. Our review includes new data on nonmammalian vertebrates, which stresses that some chemoreceptor sites are of ancient origin.

Mammalian adult neurogenesis are examined and compared with fishes, reptiles, amphibians and birds. Non-mammalian vertebrate species have provided comparative material for extracting core mechanisms for adult neural stem cell maintenance and fate.

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter 1 (Ctr1) protein is a critical router of FGF signals during early embryogenesis. Ctr1 both promotes the differentiation and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr1 involves the activation of the Ras-MAP kinase cascade and appears to be independent of its role in copper transport. Mouse embryonic stem (ES) cells deficient for Ctrl ([Ctr1.sup.-1-]) retain characteristics of pluripotency under conditions that favor differentiation in wild-type ES cells, indicating a conserved role for Ctr1 during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr1 functions as a key regulator of the differentiation capacity of both stem and progenitor cell populations. Xenopus | FGF | pluripotent | ERK | Laloo

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual MOiler cells, which are radial glial cells spanning the entire retinal thickness. Muller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Muller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Muller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells. fiberoptic plate | glial cells | refractive index | light guides | optical trap