Neural expression of the Huntington's disease gene as a chordate evolutionary novelty

Huntington's disease is a progressive neuro-degenerative disorder in humans, which is scharacterized by onset of dementia, muscular ataxia, and death. Huntington's disease is caused by the expansion of the polyglutamine (polyQ) tract in the N-terminus of the HD protein (Huntingtin). CAG expansion is a dominant gain of function mutation that affects striated neurons in the brain (Cattaneo, 2003, News Physiol Sci 18:34). The evolutionary origins of the vertebrate Hd gene are not well understood. In order to address the evolutionary history of the Hd gene, we have cloned and characterized the expression of the Hd gene in two invertebrate deuterostomes, an echinoderm and an ascidian, and have examined the expression patterns in a phylogenetic context. Echinoderms are basal deuterostomes and ascidians are basal chordates; both are useful for understanding the origins of and evolutionary trends in genes important in vertebrates such as the Huntigton's disease gene. Expression of Hd RNA is detected at all stages of development in both the echinoderm and ascidian studied. In the echinoderm Heliocidaris erythrogramma, Hd is expressed in coelomic mesodermal tissue derivatives, but not in the central nervous system. In the ascidian Halocynthia roretzi expression is located in both mesoderm and nervous tissue. We suggest that the primitive deuterostome expression pattern is not neural. Thus, neural expression of the Hd gene in deuterostomes may be a novel feature of the chordate lineage, and the original role(s) of HD in deuterostomes may have been non-neural.     

The evolutionary origin of chordate segmentation: revisiting the enterocoel theory

One of the definitive characteristics of chordates (cephalochordates, vertebrates) is the somites, which are a series of paraxial mesodermal blocks exhibiting segmentation. The presence of somites in the basal chordate amphioxus and in vertebrates, but not in tunicates (the sister group of vertebrates), suggests that the tunicates lost the somites secondarily. Somites are patterned from anterior to posterior during embryogenesis. How such a segmental pattern evolved from deuterostome ancestors is mysterious. The classic enterocoel theory claims that chordate mesoderm evolved from the ancestral deuterostome mesoderm that organizes the trimeric body parts seen in extant hemichordates. Recent progress in molecular embryology has been tremendous, which has enabled us to test this classic theory. In this review, the history of the study on the evolution of the chordate mesoderm is summarized. This is followed by a review of the current understanding of genetic mapping on anterior/posterior (A/P) mesodermal patterning between chordates (cephalochordates, vertebrates) and a direct developing hemichordate (Saccoglossus kowalevskii). Finally, a possible scenario about the evolution of the chordate mesoderm from deuterostome ancestors is discussed.     

The inversion hypothesis: A novel explanation for the contralaterality of the human brain

The brain's wrong-sidedness, or contralaterality, is one of life's great mysteries. Unlike invertebrates, all vertebrates from fish to mammals possess a forebrain with hemispheres innervated from sense organs on the body's opposite side. The vertebrate chambered eye has long been implicated as the cause for this paradox but no credible or testable theory had previously been postulated to support such an idea. Ramon y Cajal, the founder of neurobiology, made such a claim in the early 1900s but failed to provide adequate evidence. Here we show that the eye indeed appears to be the inverting culprit based on this author's entirely original, but untested, concept of a cyclopean origin to vertebrates at a time when single-eyed chordates were evolving to double-eyed agnathan fish.This framework of evidence has been entitled The Inversion Hypothesis and was originally based on the author's observation that the primary cortical sensorimotor map in mammals appears to unfold from a prior longitudinal position which was inverted in all three dimensions to the body itself. It postulates a four-stage development of the vertebrate forebrain from primitive to modern whereby a longitudinal primary bodymap (Protomap) unfolds in a predictable caudal direction. As proposed, the initial establishment of the map in the contralateral hemispheres was consequent on the inverting effect of a single frontal imaging eye as it developed in tandem with the early forebrain in ancient fish.The most conspicuous evidence today of these cyclopean origins is the common fetal abnormality in all vertebrates of cyclopia, the default developmental condition when cyclopia-nulling genes fail to activate. Further, the most exciting and convincing evidence of these origins promises to come from a change in paradigm for explaining the bizarre single circular stain at the anterior snout of primitive agnathan fossils. This circular stain is presently causing much consternation amongst palaeontologists. Unable to ascribe two eyes to these primitive fossils, mainstream palaeontology is bereft of a weltanschauung that allows the interpretation of the circular stain as a single frontal eye. The Inversion Hypothesis offers a very credible solution.     

A new calcichordate from the Ordovician of Bohemia and its anatomy, adaptations and relationships

The paper describes a new member of a group of Lower Palaeozoic marine fossils which partly bridge the gap between echinoderms and chordates. Evidence suggests that this group included the ancestors of the vertebrates. Its members are traditionally regarded as primitive echinoderms, but are better seen as primitive chordates with echinoderm affinities. They form a basal subphylum of chordates-the Calcichordata Jefferies 1967. The Calcichordata, in accordance with an early suggestion by Gislén, are probably ancestral to all living chordates. The new calcichordate is named Reticulocarpos hanusi gen et sp. nov. It comes from the Lower Ordovician ?árka Formation (Llanvirn) of ?árka near Prague, Czechoslovakia and is placed in the family Amygdalothecidae Ubaghs 1970. It is important because of its position in the Calcichordata. This group is divided into two very different orders–the Cornuta and the Mitrata. The Cornuta are the more primitive order and gave rise to the Mitrata, which had the structure of giant, calcite-plated tunicate tadpoles. Many features show that the new species is a very advanced cornute, closely related to the stock that gave rise to the mitrates. For this reason it is important in the general history of the chordates, since some primitive mitrate was probably the latest common ancestor of the living chordate subphyla i.e. of tunicates, of amphioxus and its allies and of the vertebrates. Being a mitrate-like cornute, the new species allows the cornutes and mitrates to be compared more confidently than before. Four results are especially important. Firstly it is likely that the stem (=tail) of mitrates is equivalent only to the anterior part of the stem of cornutes. This is significant, because traditional views as to which was the upper surface in mitrates have been based on stem homologies now seen as false. Secondly Reticulocarpos hanusi is adapted to stay up on very soft mud, using only the strength of the mud for support. The mitrates, on the other hand, supported themselves on soft mud by a much more reliable method resembling buoyancy. Thirdly, the new form had paired transpharyngeal eyes which are otherwise known only in mitrates, and which are the earliest type of paired eyes in chordates. Fourthly, it becomes possible to homologize the thecal plates of cornutes with those of mitrates. Reticulocarpos hanusi represents an important phase in chordate evolution dominated by the necessity of staying up on mud by a very precarious method. During this phase many pre-adaptations for swimming were acquired. Primitive mitrates, descended from a very similar form, were probably the first chordates that could swim.     

Molecular phylogeny and divergence times of deuterostome animals

The phylogenetic relationships among deuterostome animals have been debated for many years, and a diversity of hypotheses have been proposed based on both morphological and molecular data. Here we have assembled sequences of 217 nuclear-encoded proteins to address specific questions concerning their relationships and times of origin. We recovered significant support for urochordates as the closest relative of vertebrates with an analysis of 59 proteins (17,400 amino acids) and suggest that the basal position of urochordates found in previous molecular studies may have been the result of long-branch attraction biases. Our results also support Ambulacraria, the pairing of hemichordates with echinoderms (nine proteins; 2,382 amino acids), and Cyclostomata, the pairing of lampreys with hagfish (25 proteins; 6,895 amino acids). In addition, 325 shared proteins (102,110 amino acids) were obtained from the complete genomes of six vertebrates and a urochordate for phylogenetic analysis and divergence time estimation. An evolutionary timescale was estimated using a local (Bayesian) molecular clock method. We found that most major lineages of deuterostomes arose prior to the Cambrian Explosion of fossils (approximately 520 MYA) and that several lineages had originated before periods of global glaciation in the Precambrian.     

Inductive patterning of the embryonic brain in Drosophila

In vertebrates (deuterostomes), brain patterning depends on signals from adjacent tissues. For example, holoprosencephaly, the most common brain anomaly in humans, results from defects in signaling between the embryonic prechordal plate (consisting of the dorsal foregut endoderm and mesoderm) and the brain. I have examined whether a similar mechanism of brain development occurs in the protostome Drosophila, and find that the foregut and mesoderm act to pattern the fly embryonic brain. When the foregut and mesoderm of Drosophila are ablated, brain patterning is disrupted. The loss of Hedgehog expressed in the foregut appears to mediate this effect, as it does in vertebrates. One mechanism whereby these defects occur is a disruption of normal apoptosis in the brain. These data argue that the last common ancestor of protostomes and deuterostomes had a prototype of the brains present in modern animals, and also suggest that the foregut and mesoderm contributed to the patterning of this 'proto-brain'. They also argue that the foreguts of protostomes and deuterostomes, which have traditionally been assigned to different germ layers, are actually homologous.     

High-mobility-group family genes from Lampetra japonica reveal their early origin and molecular evolution in the vertebrate lineage

High-mobility group family (HMG) genes are ubiquitous in vertebrates, including mammals, birds, amphibians and fishes. To elucidate the molecular phylogeny of the HMG genes in the primitive vertebrate, we have cloned three homologues of HMG-box genes, called Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX, from a cDNA library generated from lymphocyte-like cells of the Japanese lamprey (Lampetra japonica), an Agnathan that occupies a critical phylogenetic position between invertebrates and vertebrates. The open reading frames of Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX contained 627 bp, 585 bp and 678 bp, respectively. The analysis of the deduced amino acid sequences indicated that these three putative Lj-HMGB proteins contain four domains: HMG-box A, HMG-box B, an acidic carboxyl-terminal tail and a linker. A phylogenetic analysis revealed that the Lj-HMGB proteins fall outside the vertebrate clade; Lj-HMGBX is descended from a gene ancestral to the mammalian HMGB1/2/3. This discovery implies that there was a gene duplication event in the HMGB1/2/3 gene family that occurred after the divergence of the vertebrates (Cyclostomata) from the Cephalochordata and Urochordata at least 450 million years ago (MYA). The Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX genes were detected in most tissues of the lamprey by RT-PCR. Our findings provide insight into the phylogeny of the HMGB genes in vertebrates.     

The urbilaterian brain: developmental insights into the evolutionary origin of the brain in insects and vertebrates

Classical phylogenetic, neuroanatomical and neuroembryological studies propose an independent evolutionary origin of the brains of insects and vertebrates. Contrasting with this, data from three sets of molecular and genetic analyses indicate that the developmental program of brains of insects and vertebrates might be highly conserved and suggest a monophyletic origin of the brain of protostomes and deuterostomes. First, recent results of molecular phylogeny imply that none of the currently living animals correspond to evolutionary intermediates between protostomes and deuterostomes, thus making it impossible to infer the morphological organization of an ancestral bilaterian brain from living specimens. Second, recent molecular genetic evidence provides support for the body axis inversion hypothesis, which implies that a dorsoventral inversion of the body axis occurred in protostomes versus deuterostomes, leading to the inverted location of neurogenic regions in these animal groups. Third, recent developmental genetic analyses are uncovering the existence of structurally and functionally homologous genes that have comparable and interchangeable functions in early brain development in insect and vertebrate model systems. Thus, development of the anteriormost part of the embryonic brain in both insects and vertebrates depends upon the otd/Otx and ems/Emx genes; development of the posterior part of the embryonic brain in both insects and vertebrates involves homologous control genes of the Hox cluster. These findings, which demonstrate the conserved expression and function of key patterning genes involved in embryonic brain development in insects and vertebrates support the hypothesis that the brains of protostomes and deuterostomes are of monophyletic, urbilaterian origin.     

Formation of the head-trunk boundary in the animal body plan: an evolutionary perspective

Gene expression analyses and anatomical studies suggest that the body plans of protostomes and deuterostomes are phylogenetically related. In the central nervous system (CNS), arthropods and vertebrates (as well as their closest related phyla the urochordates and cephalochordates) share a nerve cord with rostral specification: the cerebral neuromeres in Drosophila, cerebral sensory vesicle of ascidians and lancelets and the large brain of craniates. Homologous genes, in particular of the otd/Otx and Hox families, are at play in these species to specify the anterior and posterior CNS territories, respectively. In contrast, homologies in the establishment of boundary regions like those separating head and trunk structures in arthropods or mid- and hindbrain domains in chordates are still unclear. We compare in these species the formation, properties and molecular characteristics of these boundaries during embryonic development. We also discuss recent findings suggesting that insects and vertebrates might have co-opted factors of related families to control the formation of these boundary regions, the evolution of which would then appear dramatically different from that of the anterior and posterior CNS domains.     

Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives

Deuterostomes are a monophyletic group of animals that include the vertebrates, invertebrate chordates, ambulacrarians and xenoturbellids. Fossil representatives from most major deuterostome groups, including some phylum-level crown groups, are found in the Lower Cambrian, suggesting that evolutionary divergence occurred in the Late Precambrian, in agreement with some molecular clock estimates. Molecular phylogenies, larval morphology and the adult heart/kidney complex all support echinoderms and hemichordates as a sister grouping (Ambulacraria). Xenoturbellids are a relatively newly discovered phylum of worm-like deuterostomes that lacks a fossil record, but molecular evidence suggests that these animals are a sister group to the Ambulacraria. Within the chordates, cephalochordates share large stretches of chromosomal synteny with the vertebrates, have a complete Hox complex and are sister group to the vertebrates based on ribosomal and mitochondrial gene evidence. In contrast, tunicates have a highly derived adult body plan and are sister group to the vertebrates based on the analyses of concatenated genomic sequences. Cephalochordates and hemichordates share gill slits and an acellular cartilage, suggesting that the ancestral deuterostome also shared these features. Gene network data suggest that the deuterostome ancestor had an anterior-posterior body axis specified by Hox and Wnt genes, a dorsoventral axis specified by a BMP/chordin gradient, and was bilaterally symmetrical with left-right asymmetry determined by expression of nodal.     

Midline signaling and evolution of the forebrain in chordates: a focus on the lamprey Hedgehog case

Lampreys are agnathans (vertebrates without jaws). They occupy a key phylogenetic position in the emergence of novelties and in the diversification of morphology at the dawn of vertebrates. We have used lampreys to investigate the possibility that embryonic midline signaling systems have been a driving force for the evolution of the forebrain in vertebrates. We have focused on Sonic Hedgehog/Hedgehog (Shh/Hh) signaling. In this article, we first review and summarize our recent work on the comparative analysis of embryonic expression patterns for Shh/Hh, together with Fgf8 (fibroblast growth factor 8) and Wnt (wingless-Int) pathway components, in the embryonic lamprey forebrain. Comparison with nonvertebrate chordates on one hand, and jawed vertebrates on the other hand, shows that these morphogens/growth factors acquired new expression domains in the most rostral part of the neural tube in lampreys compared to nonvertebrate chordates, and in jawed vertebrates compared to lampreys. These data are consistent with the idea that changes in Shh, Fgf8 or Wnt signaling in the course of evolution have been instrumental for the emergence and diversification of the telencephalon, a part of the forebrain that is unique to vertebrates. We have then used comparative genomics on Shh/Hh loci to identify commonalities and differences in noncoding regulatory sequences across species and phyla. Conserved noncoding elements (CNEs) can be detected in lamprey Hh introns, even though they display unique structural features and need adjustments of parameters used for in silico alignments to be detected, because of lamprey-specific properties of the genome. The data also show conservation of a ventral midline enhancer located in Shh/Hh intron 2 of all chordates, the very species which possess a notochord and a floor plate, but not in earlier emerged deuterostomes or protostomes. These findings exemplify how the Shh/Hh locus is one of the best loci to study genome evolution with regards to developmental events.     

Comparative studies on the src proto-oncogene and its gene product pp60c-src in normal and neoplastic tissues of lower vertebrates

1. Oncogenes appear to play an important role in the physiology of transformed as well as of normal cells. 2. The src oncogene is by far the most investigated oncogene in birds and mammals with respect to the biochemical characteristics of its tyrosine kinase activity, although the specific function of this enzymatic activity still remains to be uncovered. 3. Systematic studies on the src related kinase activity in lower animals are lacking. 4. To contribute to a better understanding of the function of the c-src gene, we performed a comparative study on lower chordates. 5. We were able to demonstrate the presence of c-src related sequences in Acrania, Cyclostomata, cartilagenous and bony fish. 6. By performing the pp60src-specific immune complex assay we detected a tyrosine specific kinase activity, that shows the same biochemical properties as the pp60c-src from higher vertebrates. 7. The level of kinase activity is regulated in an organ specific manner. 8. In lymphocystis tumors of flat-fish and in stomatopapilloma of freshwater eels a considerable amount of pp60c-src kinase activity was found, which, however, never exceeded the levels found in the normal brain.     

Comparison of Pax1/9 locus reveals 500-Myr-old syntenic block and evolutionary conserved noncoding regions

Identification of conserved genomic regions within and between different genomes is crucial when studying genome evolution. Here, we described regions of strong synteny conservation between vertebrate deuterostomes (tetrapods and teleosts) and invertebrate deuterostomes (amphioxus and sea urchin). The shared gene contents across phylogenetically distant species demonstrate that the conservation of the regions stemmed from an ancestral segment instead of a series of independent convergent events. Comparison of the syntenic regions allows us to postulate the primitive gene organization in the last common ancestor of deuterostomes and the evolutionary events that occurred to the 3 distinct lineages of sea urchin, amphioxus, and vertebrates after their separation. In addition, alignment of the syntenic regions led to the identification of 8 noncoding evolutionarily conserved regions shared between amphioxus and vertebrates. To our knowledge, this is the first report of conserved noncoding sequences shared by vertebrates and nonvertebrates. These noncoding sequences have high possibility of being elements that regulate neighboring genes. They are likely to be a factor in the maintenance of conserved synteny over long phylogenetic distance in different deuterostome lineages.     

The new head hypothesis revisited

In 1983, a new theory, the New Head Hypothesis, was generated within the context of the Tunicate Hypothesis of deuterostome evolution. The New Head Hypothesis comprised four claims: (1) neural crest, neurogenic placodes, and muscularized hypomere are unique to vertebrates, (2) the structures derived from these tissues allowed a shift from filter feeding to active predation, (3) the rostral head of vertebrates is a neomorphic unit, and (4) neural crest and neurogenic placodes evolved from the epidermal nerve plexus of ancestral deuterostomes. These claims are re-examined within the context of evolutionary developmental biology. The first may or may not be valid, depending on whether protochordates have these tissues in rudimentary form. Regarding the second, clearly, the elaboration of these tissues in vertebrates is correlated with a shift from filter feeding to active predation. The third claim is clarified, i.e., that the elaboration of the alar portion of the rostral brain and the development of olfactory organs and their associated connective tissues represent a neomorphic unit, which appears to be valid. The fourth is rejected. When the origin of neural crest and neurogenic placodes is examined within the context of developmental biology, it appears they evolved due to the rearrangement of germ layers in the blastulae of the deuterostomes that gave rise to chordates. Deuterostome evolution and the origin of vertebrates are also re-examined in the context of new data from developmental biology and taxonomy. The Tunicate Hypothesis is rejected, and a new version of the Dipleurula Hypothesis is presented.     

Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.     

Is retinoic acid genetic machinery a chordate innovation?

Development of many chordate features depends on retinoic acid (RA). Because the action of RA during development seems to be restricted to chordates, it had been previously proposed that the "invention" of RA genetic machinery, including RA-binding nuclear hormone receptors (Rars), and the RA-synthesizing and RA-degrading enzymes Aldh1a (Raldh) and Cyp26, respectively, was an important step for the origin of developmental mechanisms leading to the chordate body plan. We tested this hypothesis by conducting an exhaustive survey of the RA machinery in genomic databases for twelve deuterostomes. We reconstructed the evolution of these genes in deuterostomes and showed for the first time that RA genetic machinery--that is Aldh1a, Cyp26, and Rar orthologs--is present in nonchordate deuterostomes. This finding implies that RA genetic machinery was already present during early deuterostome evolution, and therefore, is not a chordate innovation. This new evolutionary viewpoint argues against the hypothesis that the acquisition of gene families underlying RA metabolism and signaling was a key event for the origin of chordates. We propose a new hypothesis in which lineage-specific duplication and loss of RA machinery genes could be related to the morphological radiation of deuterostomes.     

Retinoic acid signaling and the evolution of chordates

In chordates, which comprise urochordates, cephalochordates and vertebrates, the vitamin A-derived morphogen retinoic acid (RA) has a pivotal role during development. Altering levels of endogenous RA signaling during early embryology leads to severe malformations, mainly due to incorrect positional codes specifying the embryonic anteroposterior body axis. In this review, we present our current understanding of the RA signaling pathway and its roles during chordate development. In particular, we focus on the conserved roles of RA and its downstream mediators, the Hox genes, in conveying positional patterning information to different embryonic tissues, such as the endoderm and the central nervous system. We find that some of the control mechanisms governing RA-mediated patterning are well conserved between vertebrates and invertebrate chordates, such as the cephalochordate amphioxus. In contrast, outside the chordates, evidence for roles of RA signaling is scarce and the evolutionary origin of the RA pathway itself thus remains elusive. In sum, to fully understand the evolutionary history of the RA pathway, future research should focus on identification and study of components of the RA signaling cascade in non-chordate deuterostomes (such as hemichordates and echinoderms) and other invertebrates, such as insects, mollusks and cnidarians.     

Evolution of deuterostomy – and origin of the chordates

The chordates are usually characterized as bilaterians showing deuterostomy, i.e. the mouth developing as a new opening between the archenteron and the ectoderm, serial gill pores/slits, and the complex of chorda and neural tube. Both numerous molecular studies and studies of morphology and embryology demonstrate that the neural tube must be considered homologous to the ventral nerve cord(s) of the protostomes, but the origin of the ‘new’ mouth of the deuterostomes has remained enigmatic. However, deuterostomy is known to occur in several protostomian groups, such as the chaetognaths and representatives of annelids, molluscs, arthropods and priapulans. This raises the question whether the deuterostomian mouth is in fact homologous with that of the protostomes, viz. the anterior opening of the ancestral blastopore divided through lateral blastopore fusion, i.e. amphistomy. A few studies of gene expression show identical expression patterns around mouth and anus in protostomes and deuterostomes. Closer studies of the embryology of ascidians and vertebrates show that the mouth/stomodaeum differentiates from the anterior edge of the neural plate. Together this indicates that the chordate mouth has moved to the anterior edge of the blastopore, so that the anterior loop of the ancestral circumblastoporal nerve cord, which is narrow in the protostomes, has become indistinguishable. In the vertebrates, the mouth has moved further around the anterior pole to the ‘ventral’ side. The conclusion must be that the chordate mouth (and that of the deuterostomes in general) is homologous to the protostomian mouth and that the latest common ancestor of protostomes and deuterostomes developed through amphistomy, as suggested by the trochaea theory.